Wehby et. al., 2012

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STUD Y PRO T O C O L Open Access
Oral cleft prevention program (OCPP)
George L Wehby
1
, Norman Goco
7
, Danilo Moretti-Ferreira
2
, Temis Felix
4
, Antonio Richieri-Costa
3
, Carla Padovani
5
,
Fernanda Queiros
5
, Camilla Vila Nova Guimaraes
5
, Rui Pereira
6
, Steve Litavecz
7
, Tyler Hartwell
7
,
Hrishikesh Chakraborty
7
, Lorette Javois
8
and Jeffrey C Murray
1*
Abstract
Background: Oral clefts are one of the most common birth defects with significant medical, psychosocial, and
economic ramifications. Oral clefts have a complex etiology with genetic and environmental risk factors. There are
suggestive results for decreased risks of cleft occurrence and recurrence with folic acid supplements taken at
preconception and during pregnancy with a stronger evidence for higher than lower doses in preventing
recurrence. Yet previous studies have suffered from considerable design limitations particularly non-randomization
into treatment. There is also well-documented effe ctiveness for folic acid in preventing neural tube defect
occurrence at 0.4 mg and recurrence with 4 mg. Given the substantial burden of clefting on the individual and the
family and the supportive data for the effectiveness of folic acid supplementation as well as its low cost, a
randomized clinical trial of the effectiveness of high versus low dose folic acid for prevention of cleft recurrence is
warranted.
Methods/design: This study will assess the effect of 4 mg and 0.4 mg doses of folic acid, taken on a daily basis
during preconception and up to 3 months of pregnancy by women who are at risk of having a child with
nonsyndromic cleft lip with/without palate (NSCL/P), on the recurrence of NSCL/P. The total sample will include
about 6,000 women (that either have NSCL/P or that have at least one child with NSCL/P) randomly assigned to
the 4 mg and the 0.4 mg folic acid study groups. The study will also compare the recurrence rates of NSCL/P in the
total sample of subjects, as well as the two study groups (4mg, 0.4 mg) to that of a historical control group.
The study has been approved by IRBs (ethics committees) of all involved sites. Results will be disseminated through
publications and presentations at scientific meetings.
Discussion: The costs related to oral clefts are high, including long term psychological and socio-economic effects.
This study provides an opportunity for huge savings in not only money but the overall quality of life. This may help
establish more specific clinical guidelines for oral cleft prevention so that the intervention can be better tailored for
at-risk women.
ClinicalTrials.gov Identifier: NCT00397917
Keywords: Oral clefts, Cleft lip, Cleft palate, Craniofacial anomalies, Congenital anomalies, Birth defects, Folic acid,
Vitamins, Prevention
* Correspondence: jeff-murray@uiowa.edu
1
University of Iowa, Iowa City, IA, USA
Full list of author information is available at the end of the article
© 2012 Wehby et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Wehby et al. BMC Pediatrics 2012, 12:184
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Background
Study objectives and significance
Statement of the problem
Craniofacial anomalies comprise a significant compo-
nent of mor bid human birth defects. They require surgi-
cal, nutritional, dental, spe ech, medical, and behavioral
interventions and impose a substantial economic bur-
den. Clefts of the lip and palate affect about 1/700 births
with wide variability related to geographic or igin [1] and
socioeconomic status [2]. The complex etiology of cleft
lip with or without cleft palate (oral clefts) affords ample
opportunities to identify environmental and gene-
environment interactions and to establish programs for
prevention.
Numerous studies have looked at health inequalities
and group differences on worldwide populations, often
under the dire ctorate of the World Health Organization
(WHO) [3,4]. Mechanisms for measuring the burden of
these diseases are controversial, but some standard
methodologies are beginning to emerge [5]. Studies in
Latin America suggest that health expenditures in total
are currently less than one-tenth per capita of what they
are in developed nations [6]. As countries become more
developed, these expenditures will increase and begin to
address complex problems such as birth defects, which
currently remain largely untreated and ignored in the in-
digent populations of South America. Although at the
present time, birth defect s, and cleft lip and palate in
particular, are not substantial contributors to the overall
global burden of disease [7], it is clear from predictive
models that over the next 20 years birth defects will play
an increasingly important role in the burden of disease.
Birth defe cts will eventually supplant infectious diseases
and prematurity as a single major cause of morbidity
and mortality in the first month of life, as has become
evident in Western developed populations .
An effect of vitamin supplementation on the incidence
of cleft lip and palate has been hypothesized for over 40
years [8]. A number of subsequent studies reviewe d by
Czeizel [9] and Munger [10] have continued to suggest
that folic acid and/or other micronutrients or vitamins,
including vitamin A and vitamin B6, may be important
in the etiology of clefts as well. These studies strongly
support further investigations of the role of vitamins and
other environmental components in clefting and compel
the determination as to whether interventional strategies
can result in decreases.
Genetics and epidemiology
CL/P affects about 1/700 births with wide variability across
geographic origin [1] and socioeconomic status [2]. In ge-
neral, Asian or Amerindian populations have the highest
birth prevalence, often as high as 1/500, with European
derived populations intermediate at about 1/1000, and
African-derived populations the lowest at 1/2500. In South
America, the incidence of CL/P is 1/1150 [11] and the re-
currence rate among sibs is about 4%, similar to sibling
risks in other populations [12].
CL/P is thought to result from a complex interplay of
genetic and environmental factors. In huma ns, a finely
choreographed cascade of gene expression, cell migra-
tion, cell transformation and apoptosis between 14 and
60 days post conception creates the soft and hard tissues
of the face from the originating oropharyngeal membrane.
By 48 days the upper lip is continuous and by 60 days pa-
latal shelf fusion completes facial embryogenesis [13]. Dis-
ruption of any of the tightly regulated processes occurring
in this time frame by environmental and/or genetic ab-
normalities may then predispose to cleft lip and/or pa-
late. Clefts can be divided into nonsyndromic (NS) and
syndromic oral clefts. In NS oral clefts, affected indivi-
duals have no other physical or developmental anoma-
lies [14]. Most studies suggest that about 70% of cases
are NS [15]. The remaining 30% of cases are consid-
ered syndromic. The etiology of syndromic cases
includes chromosomal syndromes, Mendelian disorders,
teratogens (e.g., phenytoin or alcohol), and uncatego-
rized syndromes with more than 400 forms reported in
Online Mendelian Inherited Diseases in Man (OMIM).
Facial clefts also have been divided into those that
affect the lip only or the lip and palate together from
those that affect the palate only. This division is based
on both genetic (recurrences are almost exclusively
limited to a single group in first degree relatives) and
embryologic (the anterior or hard palate forms se-
parately from the posterior or soft palate). In this
study, we will include non-syndromic CL/P cases
only (i.e. clefts o f the lip or the lip and palate to-
gether) to avoid the confounding effects of cleft palate
only, recognizing that there may be overlap in etiology and
recurrence prevention.
Fogh-Andersen [16] in Denmark first proposed genetic
factors in clefting, which have subsequently been con-
firmed by segregation analysis [17] and twin studies [18].
The similarities in predicted mechanisms, genes and en-
vironmental contributions across populations including
European/North American [17], Asian [2] and South
American [19,20] make it likely that the effects of re cur-
rence prevention observed in one region can be extrapo-
lated to other regions. This is especially relevant for
Brazil (where this study will take place) and the US, as
there are varying degrees of admixture with Europeans,
Native Americans and Africans [21] but significant over-
laps with US populations. Bolsterin g this argument for
cross ethnic similarities are data showing that NTD pre-
vention by folic acid is similar in China, Chile and the
US (see below). A few specific genetic contributors to
cleft etiology have begun to be identified including
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TGFA, D4S192, MSX1, TGFB3, and RARA, and IRF6
[22-34] but the majority remain unexplained.
Environmental studies
An environmental component to clefting was recognized
when Warkany [35] associated nutritional deficiencies
with cleft palate [reviewed in Murray [14]]. Nutritional
factors continue to be supported [36] as does low socio-
economic status [2]. Recognized teratogens that cause
clefts include rare exposures, such as phenytoin, valproic
acid and thalidomide, and also common environmental
exposures, such a s matern al alcohol or cigarette use and
herbicides such as dioxin [37]. There is strong support
for an increased risk of CL/P with maternal smoking
[38-41]. Two meta-analyses have estimated increased
odds by a factor of 1.3 for CL/P occurrence with mater-
nal smoking [42,43]. Maternal alcohol consumption has
also been reported to increase the risk of clefting [44-46].
Exposures to drugs such as anticonvulsants [47], Benzo-
diazepines [48], and corticosteroids [49,50] have also
shown an increased risk for oral clefts.
Other epidemiological studies also support a role for
environmental factors in clefting, especially in regions of
low socioeconomic status (SES). In the Philippines, three
studies [2,51,52] all report incidences of CL/P of 2/1000
in indigent populations while complementary studies
show an incidence of 1.2/1000 in native Filipinos living
in areas of higher SES, including Manila [52]; Hawaii
[53] and California [54]. In South America, heteroge-
neity of birth defect rates also suggests environmental
etiologies [55] with altitude noted as a particular risk
factor [56]. Thus, nutritional or toxic environmental
exposures may contribute directly to as much as one-
third of cleft cases, and etiologies will be most identifi-
able and preventable in indigent populations.
Gene-environment interaction
The development of single nucleotide polymorphisms
(SNPs) provides the opportunity to develop assays that
are gene specific and often functionally relevant. Identifi-
cation of SNPs is available both through genome wide
efforts including the recently completed HapMap project
[57]. Studies of gene-environment interactions ca n ex-
ploit advances in methods development [58] and for CL/P
present some interesting data. Interaction between smo-
king and TGFA ha s been reported primarily for CPO
[59-61], but not in other studies [45,62-64]. Interac-
tions between smoking and MSX1, TGFB3, NAT1,
NAT2, CYP1A1, GST T1, GSTM1, and EPHX1 h ave
all been studied [45,65-69], with some suggestive b ut
generally modest effe cts.
Interactions between vitamin use and the folate meta-
bolic pathway have also been intensively studied. Folate
plays a pivotal role in DNA synthesis and methylation,
and contributes to both development and gene expres-
sion. Metabolic forms of folate are involved in synth esis
of nucle otides and in the methionine cycle, which gene-
rates methyl groups that are essential for DNA methyla-
tion. Genes that code for folate metabolizing enzymes,
such as Methylenetetrahydrofolate reductase (MTHFR),
are optimal candidates for gene-folic acid interaction
studies given the suggestive results of the role of folic
acid supplementation in incidence and recurrence of
oral clefting. Specific alleles in these genes , such as the
C677T of MTHFR, may modify the effects of folic acid
supplementation. Genes that are good candidates for
consideration include MTHFR, MTHFD, MTR, MTRR,
RFC1, GCP2, CBS, BHMT, BHMT2 and TS.
There are numerous and often contradictory studies
for the MTHFR C677T variant [33,70-73]. Changes in
serum/red blood cell folate and homocysteine with
increased dietary folate consumption after low folate
consumption have been reported to vary by MTHFR 677
status [74]. A potential interaction between vitamin use
and RFC1 has also been suggested [75,76], though no
evidence has been observed in a recent study [77]. In
summary, there is as yet little consensus among the
many studies of interaction between vitamin/folic acid
use and genetic factors in the etiology of CL/P.
Observational studies of multivitamin and folic acid use
and clefts
Several case–control observational studies have reported
a protective effect of pericon ceptional use of mu ltivita-
mins and folic acid on occurrence of clefts [see Botto
et al. [78] for a review]. The estimated decrease in CL/P
risk with supplements containing folic acid has ranged
from 18% [79] to 50% [80,81]. Studies of multivitamin
use without specification of folic acid content have
reported risk reductions of 30% [82], 40% [83,84], and
50% [85 ]. The smaller risk reductions were generally not
statistically significant. Only one observational study
[86] has reported an increased risk for CL/P with folic
acid supplements (by 30% but insignificant) yet their
control group included only children with birth defects
outside of midline defects, which might be interpreted
as a protective severity reduction effect of folic acid for
those anomalies.
Other studies of micronutrient and folate exposures
have also suggested a ssociations with oral clefts in
humans. Dietary folate intake was reported to be asso-
ciated with oral cleft risk by up to 70% increase with
0.18mg compared to 0.35mg of daily folate intake [87].
Low maternal B6 [10] measured after pregnancy was
reported to increase the risk of CL/P, particularly in
cases with low serum folate. Post pregnancy low B12
levels and low infant serum folate have also been linked
to increased oral cleft risk [81]. B1 and B6 deficiencies in
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maternal diet were also associated with an increased risk
of oral cleft s [88] as were myo-in ositol and zinc [89,90].
Exposure to folic acid antagonists such as antiepileptic
drugs and dihydrofolate reducta se inhibitors was
reported to double the odds for oral clefts [91]. An imal
studies also provide support for anti-teratogenic effects
of prenatal folic acid supplementation and dietary folate.
Peer e t al. [92] showed a 69% reduction in cortisone
induced cleft palate occurrence in mice with folic acid
injection; there was an 82% reduction with a combined
treatment of folic acid and B6. Folic acid supplementa-
tion wa s also shown to decrease the frequency of reti-
noic acid induced cleft palate in mice by up to 92%, with
sugges ted ad ditive effects with methionine [93]. Procar-
bazine induced cleft palate wa s also re ported to de-
crease with folic acid supplementation in rats [94-96]
with potential dose and gender dependent effec ts of
folic acid. Supplementing m ice who have a higher risk
of spontaneously occurring oral clefts (A/WySn Mice)
with folinic acid, a metabolic form of folic acid, was
reported to de crease the frequency of CL/P by up to
75% [97]. Adequate dietary folate de crea sed the terato-
genic effect s of methanol in mice b y about 74% [98],
and very low folic acid diets have been shown to delay
thesecondarypalateclosureinmice[99].Supplementa-
tion with 5 mg/day dose of folic acid was reported to de-
crease the occurrence of cleft palate in dogs by 76%
[100]. These studies also provide suggestive results for a
potential role o f folic acid and possibly other micronu-
trients in oral cleft etiology/prevention.
Folic acid fortification and oral clefts
A few countries have introduced folic acid fortification
of grain and flour given the strong evidence for a pre-
ventive effect of folic acid on neural tube defects
(NTDs). Indeed, this evidence and its subsequent appli-
cation to populations is one of the major public health
successes in the field of birth defects (see below). Unlike
the case for NTDs, there is no converging evidence for
significant changes in birth prevalence for oral clefts
post folic acid fortification. In the United States, where
folic acid fortification of grain products was mandated
on January 1, 1998, thre e studies have generally found
non-significant reductions of 3% [101], 5% [102], and
12% [103] in CL/P prevalence post fortification. A sig-
nificant 12% reduction in CPO prevalence has been
reported [102].
A slight non-significant increase in the prevalence of
oral clefts has been reported after two years of fortifica-
tion of cereal grain products (1998 through 2000) com-
pared to pre-fortification (1994–1997) period in Ontario,
Canada [104]. Also, in a preliminary evaluation of the
effects of fortifying wheat flour with folic acid in Chile
starting 2000, Castilla et al. [105] reported no significant
changes in prevalence of oral cleft s through the end of
2001 compared to 1999, while significant change of 31%
was shown for NTDs. Longer period s may be required
for a more comprehensive evaluation of potential
changes in prevalence of oral clefts post fortification, yet
given the e vidence of NTD reduction of up to 50% in
similar periods [e.g.[102,106-108], see Mills and Signore
[109] for a review], these results suggest that low doses
of folic acid may be inadequate to even prevent primary
occurrence of oral clefts. Further, these studies of preva-
lence changes over time also suffer from limitations in-
cluding potential confounding by other simultaneously
changing contributory factors and the lack of well-
matched control groups.
In Brazil, fortification of wheat and corn flour with
folic acid be came mandatory in June 2004. Flour is forti-
fied at a dose of 150 μg/100 g, which is lower than that
used in Chile (220 μg/100 g of flour) and comparable to
the dose used in the United States (average of 150 μg/
100 g of cereal grain product). Assuming that women
consume about 200 g of fortif ied flour on average per
day, the daily intake of folic acid would be expected to
increase by about 300 μg.
Interventional studies of folic acid and neural tube defects
(NTDs)
Several clinical trials have evaluated the effectiveness of
folic acid supplementation at high or low doses in pre-
vention of NTDs. These studies have provided strong
evidence for a large preventive effect of folic acid on
both recurrence and occurrence of NTDs. The strongest
evidence for a preventive effect of high dose folic acid
supplementation on recurrence of NTDs comes from
the Medical Research Councial (MRC) 1991 double-
blinded randomized study, where women with a pre-
vious child with NTD were randomly assigned to groups
of 4 mg folic acid, other vitamins, vitamins with 4 mg
folic acid, and placebo, taken daily at preconception and
throughout the first trimester of pregnancy. The folic
acid groups had a lower relative risk of NTDs in off-
spring of 0.28 compared to the other groups, indicating
a 78% decrease in recurrence risk of NTDs. No signifi-
cant decreases in NTD recurrence were observed in the
group receiving other vitamins. This indicates that pre-
ventive effects are due the folic acid component, though
the study was not powered enough to detect potential
interactive effects between folic acid and the other
vitamins.
Multivitamin supplementation with a 0.8 mg folic acid
at preconception and through at least two months post
conception was also shown to lower the risk of first oc-
currence of NTDs by up to 100% in a randomized cli-
nical trial in Hungary using a sample of women with no
history of NTD in offspring [110]. This same study
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showed no decrease in the occurrence of CL/P thoug h
the overall rate of non-NTD other genetic syndromes
was also reported to decrease. As a confirmatory study
applying a two-cohort controlled design in Hungary,
with the interventional group receiving the same folic
acid containing multivitamin as Czeizel [110] also found
a significant decrease in NTD occurrence by up to 89%
and in cardiovascular defects (40%), but no significant
decrease for oral clefts.
Intake of 0.4 mg folic acid beginning before concep-
tion and continuing in the first trimester of pregnancy
was shown [111] to decrease the occurrence of NTDs in
China by up to 79 percent in a sample from the north-
ern area with higher baseline rates of NTDs (5/1000
births) and by 16 percent in the Southern region sample
(baseline rates of 1 per 1000). In the groups with >80%
compliance with folic acid, there were 85% and 41%
reductions in NTD occurrence in the Northern and
Southern samples respectively. These results strongly in-
dicate that the preventive effects on recurrence and oc-
currence of NTDs are due to the folic acid component
rather than the other vitamins, thoug h interactive effects
have not been thoroughly evaluated. The NTD research
provides a model for developing clinical trials aimed at
assessing preventive effects of folic acid on recurrence
and occurrence of oral clefts of direct relevance for cli-
nical practice.
A connection between NTDs and CL/P can be sup-
ported by their similar time of occurrence during em-
bryogenesis, their status as defects involving the midline
of the embryo, their near identical population genetic
characteristics (variable by geographic origin but with
near identical recurrence risks and very similar birth pre-
valances overall), evidence of similar gene/environment
contributions and the failure to identify any major genetic
factor for either. The mechanisms by which folic acid
might prevent NTDs or other birth defects remain unex-
plained. It might be secondary to the need to overcome
pharmacogenetic deficiencies in women who require
higher baseline intakes to reach therapeutic levels. A re-
cently proposed mechanism relates to antibodies to the
folic acid receptor [112]. The role of antibodies to the fol-
ate receptor has yet to be confirmed but could explain
why some women respond to high doses of folic acid as
this may be required to titer the effects of antibody bound
to receptors. The pharmacologic rescue by high dose folic
acid has been reported in a rat model where folate recep-
tor antibodies induced intracellular folate deficiency asso-
ciated with birth defects [113].
Interventional studies of folic acid and oral cleft recurrence
Only a handful of inter ventional studies have been con-
ducted over the last 48 years to study the effect of folic
acid supplementation on recurrence of oral clefts in
mothers with a child with CL/P. The decrease in cleft re-
currence among the folic acid groups reported in these
studies, independent of statistical significance, ranges
from 24 to 100% and is summarized in Table 1.
Conway [8] reported no recurrent cleft cases among
59 births to mothers with history of cleft lip and/or cleft
palate in previous births. They received a multivitamin
that included 0.5 mg of folic acid and the recurrence
rate in a group of 78 births to mothers who did not re-
ceive the supplement was 5.1%. Peer et al. [114] reported
a 53% reduction in the recurrence of cleft lip and/or
cleft palate in a group of 176 women who received a
multivitamin in addition 5 mg folic acid and 10 mg vita-
min B6 during the first pregnancy trimester, compared
to a control group of 418 mothers (p=0.1). In an
extended study of Peer et al. [114] with more supple-
mented women, Briggs [115] reported a 35% reduction
in recurrence of cleft lip and/or cleft palate (p=0.2), but
a 65% reduction in CL/P recurrence (p=0.06). Tolarova
[116] reported an 84% reduction in recurrence of CL/P
in a group of 80 women who received a multivitamin
and 10 mg of folic acid during three months before con-
ception and throughout the first three months of preg-
nancy (p=0.02), compared to a control group of 202
women. Using data on a larger sample that included
women with CL/P (40% of intervened sample) and
mothers of a child with CL/P, and the same intervention
as Tolarova [116] and Tolarova and Harris [117] reported
a 66% reduction in recurrence of CL/P (p=0.03) The ave-
rage reduction effect when combining the unique data
samples of these studies [8,115,117], is 54.1% (47% when
Conway (1958) is excluded), and 66.5% for CL/P recur-
rence combining Tolarova and Harris [117] and the CL/P
sample of Briggs [115]. These calculations are inappro-
priate from a confirmatory side given the array of inter-
ventions and populations used, but from an exploratory
perspective, may be helpful for gauging expected treat-
ment effects of folic acid to form hypotheses in clinical
trials. Our study hypothesis of 50% reduction is based on
this result, the case–control observational study results,
many of which also suggested similar effects, and the re-
sult of MRC study which showed a 78% in recurrence of
NTDs [118].
The results of these studies are suggestive of potential
preventive effects of high dose folic acid on cleft recur-
rence. The data from the Hungarian trials [79,110] also
support the notion of lack of preventive effects of low
doses of folic acid on occurrence of oral clefts. The
NTD model showing pre ventive effects of high and low
dose folic acid on recurrence and occurrence res pect-
ively, and the suggestive results from interventional
studies and observation al studies [119] for preventive
effects of high doses on recurrence and occurrence of
oral clefts strongly indicate that large doses of folic acid
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are best suited for evaluation in randomized clinical
trials of recurrence.
While many of these studies used different doses of
folic acid than what we are using, did not have a control
group that also received 0.4 mg, and in some cases also
included other multivitamins in the supplementation,
the possible reduction in clefting rates nonetheless spans
the range which we are attempting to demonstrate in
this inte rvention. Other micronutrients could also be
considered (B6, Zinc) but the case for folic acid alone is
far more compelling.
Objectives and significance of proposed study
The studies reviewed above are suggestive of protective
effects of folic acid supplementation on cleft risks, but
all suffered from data and design limitations. The inter-
ventional studies for human recurrence have serious
flaws , particularly in lacking a real control group gene-
rated by a randomization and in using combined inter-
ventions that do not allow for isolating the effects of
folic acid [120]. Control subjects in these studies were
usually women who either refused or did not comply
with the intervention [116,117] or are otherwise poorly
defined. This increases the chance of biases due to self
or researc her selection of treatment, which confound the
study results and introduces outcome differences that
are not necessarily generated by the intervention. The
different supplements and folic acid dose combinations
make it hard to compare results and to attribute effects
specifical ly. The sample sizes employed in these studies
were also small and overall only borderline statistical sig-
nificance was observed.
Observational case–control studies also have inherent
problems, including non-random self or provider selec-
tion of supp lement use. This is in part determined by
perceived health risks that may also affect the risk for
clefts. For example, women with unfavorable pregnancy
histories, health problems or a family history of birth
defects may use more folic acid but may also have a
greater risk of poor pregnancy outcomes including CL/P.
Further confounding results from the lack of data on
health behaviors that may be correlated with both supple-
ment use and oral cleft occurrence. Other limitations in-
clude potential bias in self-reported use of supplements
and the lack of a well defined regimen for vitamin content,
dose, and time of use. While helpful for exploratory re-
search purposes, observational studies are not sufficient
for answering the research hypothesis posed in this study.
Only a double-blinded, randomized experimental study,
with sufficient sample sizes, can provide the opportunity
to clearly address our hypothesis. The objective of this
study is to assess the effect of folic acid supplementation
of 4 mg/day taken at preconception and throughout the
first three months of pregnancy on reducing the recur-
rence of CL/P compared to a 0.4 mg/day standard dose
among women born with CL/P or mothers of children
with CL/P using a double blinded randomized clinical trial
design.
This study has the ability to identify, for the first time,
the true preventive effects of folic acid on recurrence of
oral clefts, using a dose that has proved effective in pre-
venting recurrences of NTDs [118]. The double blind
randomized design will cleanly separate the effect of the
intervention from those confounding effects that have pla-
gued previous interventional and observational studies.
This study provides the chance to evaluate a treatment
that if successful, will determine the standard of care for
high risk women in the United States and abroad.
Methods/design
Study design
Objectives
The overall goal of this randomized, double-blinded
study is to reduce the recurrence of non-syndromic cleft
lip with or without cleft palate (NSCL/P) in a high-risk
group of women suppleme nted with folic acid from pre-
conception and continuing throughout the first three
months of pregnancy.
Table 1 Summary of non-randomized interventional human trials for cleft recurrence*
Study Intervention Supplemented group Control group Reduction
# births RR (%) # births RR (%)
Conway (1958) MV and 0.5mg FA 59 0.0 78 6.4 100% (p=0.1)
Peer et al. (1964) MV and 5mg FA and 10mg B6 176 2.2 418 4.7 53% (p=0.1)
Briggs 1976 [extension of Peer et al. (1964)] MV and 5mg FA and 10mg B6 228 3.1 417 4.8 35% (p=0.2)
Tolarova (1982) MV and 10mg FA 84 1.2 206 7.3 84% (p=0.02)
Tolarova and Harris (1995) MV and 10mg FA 211 1.4 1824 4.2 66% (p=0.03)
Total** 498 2.01 2319 4.38 54.1%
MV=multivitamin; FA=Folic acid; RR=Recurrence Rate.
* Conway [8], Peer et al.[114] and Briggs [115] included mothers to children with cleft lip and/or cleft palate. Tolarova [116] included mothers to children with
cleft lip with/without cleft palate (CL/P). Tolarova and Harris [117] included women with CL/P or mothers to a child with CL/P.
** The total recurrence for treated and control groups is based on total number of affected births divided by total group size (or proportion of each study’s
recurrence relative to its sample size). Peer et al. [114] and Tolarova [116] are excluded as these data were included in other studies.
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Design
Subjects will be randomly a ssigned to 4 mg or 0.4 mg
pills of folic acid (identical in appearance). A single pill
of 4 mg or 0.4 mg of folic acid will be taken daily until
pregnancy is documented. Once pregnancy is documen-
ted, the trial drug will be continued until 3 months after
the last menstrual period. At this point, the trial drug
will be discontinued and women will take routine pre-
natal vitamins until delivery following routine obstetric
care. At the end of the study, recurrence will be assessed
and compared between groups. The total sample recur-
rence rate as well as recurrence rates that are specific to
the 0.4 mg and 4 mg folic acid groups will also be com-
pared to the recurrence rates of oral clefts in a historical
control group.
Primary hypothesis/primary outcome
The primary hypothesis is that folic acid supplementa-
tion of 4 mg/day at preconception and during the first
three months of pregnancy will decrease the recurrence
of NSCL/P in a high risk (history of NSCL/P in mother
or child) group of women when compared to women
taking 0.4 mg per day of folic acid. The primary out-
come measure will be the difference in NSCL/P recur-
rence rates between the two groups and the a ssociated
confidence inter val. The study is powered to detect a
50% reduction at a 0.8 power.
Secondary hypotheses
The following secondary hypotheses will be evaluated:
An increase in serum and/or red cell folate levels in all
women following daily supplementation with folic acid.
When compared to the 0.4 mg supplemented groups,
the 4 mg per day group will have the following:
– Greater increases in serum and/or red cell folate.
– Decreases in the recurrence of NSCL/P in each of
the following subgroups:
 Women with NSCL/P themselves
 Women with one or more children with NSCL/P
– A decrease in the severity of NSCL/P in offspring of
trial mothers.
– No increase in twinning rates.
– No increase in miscarriage rates.
– No increase in rates of preeclampsia.
– No increase in rates of other birth defects.
– No decrease in birth weight
– No decrease in gestational age
A decrease in recurrence of NSCL/P in the group of
study subjects compared to an historical control group
obtained from the populations served by the
craniofacial clinics participating in the study.
A greater decrease in the recurrence of NSCL/P in the
group of study subjects receiving 4 mg of folic acid
when compared to the historical control group relativ e
to that of the group receiving 0.4 mg when compared
to the same control group.
Under separate funding we will examine the role of
genes involved in folate metabolism on folate, B12 and
homocysteine levels, as well as potential interaction be-
tween these genes and the supplementation dose. This
project will also establish a resource to examine the
long-term outco mes of infants exposed to high dose folic
acid in utero.
Historical control group
The recurrence rates in this study will be compared to
that of an historical control group. The rationale for
using this group relates to the inability to use a placebo
control in this study due to ethical considerations. A
comparison with an historical control group will allow
the assessment of the combi ned effect of a minimum
dose of folic acid of 0.4 mg and a maximum dose of 4
mg by comparing the NSCL/P recurrence rate in the
total study group to that of a group of women who did
not receive the folic acid dose of 0.4 mg per day. The ef-
fect of each of the two folic acid doses on the recurrence
risk of N SCL/P could also be evaluated by comparing
the NSCL/P rate in each of the two study groups
(0.4 mg and 4 mg folic acid) to that of the historical
control group.
The historical control will include women with NSCL/
P or mothers of children with NSCL/P who are regis-
tered at the craniofacial clinics enrolled in the study.
Those clinics have been involved in the provision of ser-
vices to subjects with clefts for an extensive period, thus
rates of cleft re currence can be calculated over an
extended time course providing not only cross-sectional
data but also trends data adding to the validity of the
historical control approach. Such data will reveal any
changes in recurrence rates that might have occurred
over time.
Taking into consideration the traditional internal valid-
ity challenges associated with using historical controls ,
no major historical events that could affect recurrence
rates of NSCL/P in this population are expected to have
occurred during the years immediately preceding this
study.
Since the two randomized study groups (0.4 mg and
4 mg folic acid) will be compared to the same group
of historical controls, any differences in the relative
decreases of NSCL/P recurrence rates that may be
observed between each of the study groups and the
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historical control group can be safely attribute d to the
effect of administered folic acid.
Pilot studies
Initial pilot study and subsequent recruitment at
Bauru With funding from NICHD’s Global Network for
Women’s and Children’s Health Research, study enroll-
ment was initiated in Bauru in January 2004. This initial
pilot aimed at first recruiting about 500 subjects, identi-
fied through the records of the Hospital de Reabilitação
de Anomalias Craniofaciais (HRAC) clinic, and located
within 100 Km of Bauru, using a field-ba sed strategy for
recruitment and follow-up. Letters were mailed to 526
women inviting them to enrollment meetings in local
communities; 181 women attended the enrollment meet-
ings, and 134 women were enrolled (96% of those eli-
gible). Several factors contributed to low attendance at
these enrollment meetings . We found that 23% of the
500 subjects had either moved out of the catchment area
or could not be loca ted and that 12% had had a tubal
ligation. The inconvenient recruitment and follow up
strategies required multiple visits and poor compliance
with these wa s amplified by poverty.
An extension of this pilot study to re-contact potential
subjects from the first sample who did not atten d initial
enrollment meetings was carried out in June, 2004.
About 280 subjects were re-invited to participate in the
study; 100 women attended enrollment meetings and
about 70 of them were enrolled in the study. During this
second phase, we used 30 social workers from local public
health offices to assist in contacting and locating subjects.
The overall rate of attendance at enrollment meetings
among potential subjects was more than 50%, and the rate
of enrollment among eligible subjects was more than 85%.
After this first pilot phase, we introduced some changes
into recruitment strategies, including more flexibility in
recruitment timelines, adding more social workers, and in-
creasing the number of attempts of contacting subjects
prior to enrollment meetings. We also developed a practical
step by step recruitment decision tree to accommodate
most recruitment scenarios. In November 2004, a third
phase of recruitment was conducted in areas outside Bauru,
targeting about 1,100 subjects and 285 were enrolled.
As of November 30, 2006, about 355 subjects are ac-
tively participating at the Bauru site and taking folic acid
pills. To date, there have been 62 pregnancies in the
Bauru sample; 3 resulting in miscarriages, 49 delivered
and 9 are ongoing.
Revision of subject recruitment/follow-Up strategies
and pilot at clinic sites We identified some limitations
in the strategies originally developed to identify, recruit,
and follow up subjects at the Bauru site.
Inaccurate and outdated contact information with high
population mobility decreased the number of available
cases. 18% of potential subjects were untraceable.
Inviting subjects to attend prescheduled meetings for
screening and enrollment lowered subject motivation
to participate due to extra effort on their behalf, further
intensified by the high prevalence of poverty in the
targeted population.
The follow-up strategy also seemed intensive and
burdensome by requiring subjects to attend follow-up
visits and provide blood samples on a bimonthly ba sis,
without providing any real direct incentives (besides
potential benefits of the study pills). This also lowered
compliance with the follow up schedule.
It became clear that the required sample size could not
be secured through the Bauru site alone.
In order to address these limitations, we developed
new sampling, recruitment, and follow up strategies
using a clinic model. This strategy focuses on women
who still attend the clinic for their own craniofacial
care or for their children’s care, but can also include
women who no longer attend the clinic regularly. We
identified six new clinics in Brazil as candidates to join
the study and pilot test these new strategies. Three of
these clinics, Hospital de Clinicas de Porto Alegre (HCPA)
in Porto Alegre, Hospital Santo Antônio- Centrinho-
Obras Sociais Irmã Dulce (OSID) in Salvador, and Insti-
tuto Materno Infantil Prof. Fernando Figueira-CADEFI/
IMIP (IMIP) in Recife formally joined the study. HCPA
and OSID were able to start the pilot test studies. Re-
cife wa s put on hold until additional funding could be
secured.
The new recruitment strategies implemented at Porto
Alegre and Salvador involve identifying potential sub-
jects from subjects who attend the clinic for care. Regis-
tered subjects who are not scheduled to visit the clinic
may still be invited by phone or mail to participate in
the study. Initial screening and enrollment procedures
occur as soon as a subject agrees to participate, often
during first contact with subject, eliminating the need
for extra visits for enrollment. Study pills are sent every
two months to subjects by mail and health and preg-
nancy checkups are conducted mostly through phone
rather than in person. In-person follow-ups are currently
limited to the first two months after enrollment, to
measure post supplementation folate and B12 levels, and
every six months thereafter or at pregnancy. These pro-
cedures will be further eased in the proposed continu-
ation. Food vouchers are being provided at enrollment
and at every third completed follow-up.
This clinic-based strategy is expected to ensure a con-
tinuous increase in the number of study subjects and to
enhance efficiency. Inaccuracies of contact information
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inherent in clinical registries can be avoided by this ap-
proach using in person contact. Further, the groups of
subjects recruited through this approach may be more
motivated to participate in prevention research given
that the majority are still obtaining treatment either for
themselves or their children and may thus perceive a
higher burden of CL/P compared to groups with com-
pleted treatments. Reducing the frequency of blood col-
lection and of in-person attendance by mailing pills and
conducting follow up by phone also make subjects more
willing to maintain active participation after enrollment.
These strategies also require fewer personnel since most
activities can be implemented at the clinic’s research
unit by the clinical coordinators.
Subject recruitment began in May 2005 at Porto Ale-
gre site. Through November 30 2006, 137 women have
been screened and 103 enrolled. Recruitment activities
began in Salvador in December 2005. Through November
30, 2006, 174 women have been screened and 106 en-
rolled. Recruitment and follow-up strategies have been
successful with no significant problems. Mailing the pills
and conducting follow-ups by telephone proved easy and
effective.
Study population and procedures
Study site and populations
The study will be conducted at four craniofacial sites in
Brazil including Hospital de Reabilitação de An omalias
Craniofaciais (HRAC) in Bauru (state of Sao Paulo),
Hospital Santo Antônio- Centrinho: Obras Sociais Irmã
Dulce in Salvador (state of Bahia), Hospital de Clínicas
de Porto Alegre (HCPA) in Porto Alegre (state of Rio
Grande do Sul), and Instituto Materno Infantil Prof.
Fernando Figueira-CADEFI/IMIP (IMIP) in Recife (State
of Pernambuco). All of these clinics have had a long ex-
perience in providing care to patients with oral clefts.
Inclusion and exclusion criteria
Inclusion criteria In this study, NSCL/P is defined as
all cases of unilateral or bilateral clefts of the lip with or
without cleft palate excluding the following: cases with
recognized syndromes, cases with a chromosome abnor-
mality, cases with one or more other major structural
anomaly, cases with cognitive delay (IQ or equivalent
less than 80), or cases exp osed to recognized teratogens
in utero (phenytoin or valproic acid). Cases with cleft
palate only will not be included in the study as specified
in Chapter 1. The following define the inclusion criteria
for the study:
1. Women with NSCL/P, who are 16 to 45 years of age
(after 45 fecundity decreases substantially) and who
attend or have attended the craniofacial clinic for
their care.
2. Women (age 16 to 45 years of age) who have at least
one natural child of any age with NSCL/P who
receives (received) care at the participating
craniofacial clinics.
3. All women must reside in the catchment area of the
study, which includes the state where the clinic is
located and surrounding states.
Exclusion criteria
1. Having a first degree relative (that is a parent,
sibling or child) who has cleft palate only.
2. Cases resulting from consanguineous couples (first,
second, and third degree, i.e., first cousins or
closer).
3. Couples where at least one of the two is definitely
sterilized.
4. Women who are using intrauterine devices
5. Women who are using injectible contraceptives
6. Women on anti-epileptic drugs (The metabolism of
anti-epileptic drugs requires a great deal of folic
acid. Therefore, it is very difficult to verify the effect
of folic acid on the prevention of clefting among
these women. In order to avoid this situation, such
women will not be included in this study).
7. Women on drugs that contain benzodiazepines
8. Women who are pregnant. They will be re-
contacted later at an appropriate time for
participation in the study.
9. Women who are planning to move outside of the
catchment area of the study within the next year.
10. Women who have B12 deficiency (B12 level is
below 174 pg/ml or 134.328 pmol/L).
11. Women who are allergic to folic acid.
Sampling, recruitment, and screening procedures
Sampling Potential study subjects include a conve-
nience sample of women with NSCL/P and/or mothers
of at least one child with NSCL/P who currently attend
or have attended in the past the study clinics for the ir
own care or for the care of their children during the
study period and who meet the inclusion criteria (age
and residence) listed above. Potential subjects will be
invited to participate. The majority of potential subjects
(about 70%) are expected to be mothers of children with
NSCL/P and the res t (30%) to be women who them-
selves have NSCL/P. Based on published literature and
review of CL/P pedigrees at HRAC clinic in Bauru, the
recurrence rates in the two subject groups of women
with NSCL/P and mothers to a child with N SCL/P were
estimated to be about 7 and 4 percent respe ctively.
While women up to 45 years of age are potentially eli-
gible to participate, the sampling frame will be limited,
when possible (e.g. when women’s age is known a priori
from the clinic’s records), to women who are up to 40
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years of age. The rationale for this sampling frame is
that chances of pregnancy and propensities to have chil-
dren decrease significantly after 40 years of age. Yet,
due to potential inaccuracies in the age of women in the
clinic’s re cords used for sampling, women who are
invited to participate in the study and who are between
40 and 45 years of age will still be eligible to participate
in the study on age basis in order to keep a positive en-
vironment for implementation of the study in the local
community.
All potential subjects are identified from the clinics’
medical records and registries in addition to activ e sur-
veillance of all patients who attend the clinics for care.
The study staff will search patient records to identify po-
tentially eligible subjects based on the primary informa-
tion that are available in these records including CL/P
diagnosis, women’s age, and residence. All patient chart s
from the past 10 years will be reviewed to identify
women who themselves or their children have been trea-
ted at the clinic. Study staff will search the records of
subjects who will attend the clinic to also identify poten-
tial subjects on a weekly basis. The study will be pro-
moted to all the staff of these craniofacial clinics so that
they also can assist in identifying and referring subjects
to the study and will be advertised in local media.
Recruitment Two models of recruitment will be applied:
1. Outreach model: This model uses a field-based
strategy to recruit potential subjects most of whom
are no longer active patients at HRAC in Bauru. This
model will be implemented by the Bauru study site,
given the density of potential subjects in the
proximate region who have been treated at HRAC.
Field visits to conduct group recruitment meetings
with potential subjects at local community facilities
are the primary recruitment method in this model.
2. Clinic-based model: This model will use the
craniofacial clinics as the sites for recruitment of
subjects. Recruitment will be focused primarily on
potential subjects attending the clinic during the
study peri od. Additional recruitment activities will
include identifying potential subje cts from the clinic
registry that are not active patients. This model will
be implemented at all participating craniofacial
clinics , including HRAC in Bauru.
Introduction to study and screening Potential subjects
will be contacted to verify the age and NSCL/P, present
the study, and assess their interest to participate and
willingness to be screened to determine final eligibility.
Subjects who show interest will be offered to undergo
immediate screening to confirm their eligibility.
Outreach model The study staff will search patient
records in the HRAC database to identify potentially eli-
gible subjects based upon inclusion criteria in 3.2.1. The
study staff will mail letters of invitation to identified po-
tentially eligible subjects for attendance at prescheduled
recruitment meetings. These meetings will take place at
public facilities (e.g. health centers, schools, churches, or
other public facilities) as coordinated with local commu-
nity leaders. The study staff will follow-up with phone
calls to check on intent to attend these meetings. In
some cases, the local network of social workers may be
used to visit invited subjects and confirm their intention
to attend these meetings. This is especially helpful for
contact with potential subjects with incorrect contact in-
formation in the HRAC database. During the first meet-
ing, study staff will explain the purpose of the study, the
study intervention, subject responsibility (e.g. com-
pliance with study drug, maintaining follow-up), and de-
sign of recruitment, enrollment, and follow-up procedures.
The study staff will offer to answer any questions the sub-
ject may have regarding the study, and will provide the
subject with a study summary brochure. The study staff
will screen those interested and enroll them if eligible.
Clinic-based model The Clinical Coordinator will
search patient records to identify a potential subjects
that meet the inclusion criteria of NSCL/P and age,
and will check the appointment schedule at the clinic
to see if they may visit the clinic within the next six
months. Subjects scheduled to visit the clinic in the
next six months will be first contacted to participate in
the study during their visit to the clinic. Potential subjects
unscheduled to visit the clinic in the next six months and/
or who no longer attend the clinic will be contacted via in-
vitation letters and/or phone and invited to participate.
After verifying the po tential eligibility status based on
age and NSCL/P, the Clinical Coordinator will indivi-
dually present the study to each potential subject. This
first contact may take place at the clinic or via phone,
and involves explaining the purpose of the study, the
study intervention, subject responsibility (e.g. compliance
with study drug, maintaining follow-up), and design of re-
cruitment, enrollment, and follow-up procedures. The
Clinical Coordinator will offer to answer any questions
the subject may have regarding the study, and will provide
the subject with a study summary brochure.
After presenting the study to the potential subject, the
Clinical Coordinator will ask her whether she might be
interested in participating in the study. Subjects who
show interest will be offered to undergo immediate
screening to confirm their eligibility. Subjects who show
interest but are hesitant to be screened and enrolled will
be given more time to consider their participation.
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The Clinical Coordinator will a sk subjects who are un-
willing to participate about their reasons for not partici-
pating and whether any accommodations could be made
to help them participate. This will be done in a non-
coercive manne r in order to inquire about information
that may help to improve the study design and proce-
dures. No further attempt will be made to recruit sub-
jects who remain unwilling to participate.
Informed consent
Once screened, the Clinical Coordinator will provide the
informed consent in the local language at the participat-
ing site to eligible subjects who are willing to be enrolled
into the study. After reading the informed consent to
the subject and answering any questions she may have,
the Clinical Coordinator will ask her to sign the
informed consent. If the subject is illiterate, confirmation
will be obtained as a thumbprint in the presence of a
witness. Women will be assured that refusal to partici-
pate in the study will in no way affect further treatment
or care at the clinic.
Enrollment and baseline data collection
Once screened, the Clinical Coordinator will enroll eli-
gible subjects who provide informed consent. The Cli-
nical Coordinator will complete the enrollment form,
and will obtain a blood sample (about 8 ml) from the
subject for ba seline measures of RBC, hemoglobin, B12,
and serum/RBC folate.
Blood samples will be analyzed at a maximum of 2 weeks.
Enrollment of subjects with normal B12 levels will be con-
firmed. Subjects found to be B12 deficient will be referred
to a hematologist for treatment, and will be excluded from
participation.
The Clinical Coordinator will randomize each con-
firmed subject into the 0.4 and the 4 mg folic acid
groups (see section 3.6) and will dispense the folic acid
pillbox with the relevant pillbox ID number. Study pills
will be sent to randomized subjects by mail in most
cases; subjects with unreliable addresses or who fre-
quently attend the clinic for care receive their pills at the
clinic. When delivered by mail, the clinical coordinator
will contact the subject by phone to confirm their re-
ceipt of the pillbox.
Randomization procedures
Study subjects will be randomized to either 4mg or
0.4mg of folic acid. The study will be double-blinde d;
subjects, investigators and research staff will be blinded
to the randomization assignments.
The Data Center will prepare the randomization se-
quence for the study using permuted blocks of random
size. Randomization occurs at the subject level and is
stratified by study site to ensure a balanced site
representation in both treatment groups. The randomization
will not be stratified according to risk group (mother with
oral cleft or mother of child with oral cleft) or other fac-
tors due to the large sample size. As shown in [121], strati-
fication becomes irrelevant after the sample rises above
200 subjects. Since randomization occurs at the mother’s
level, the sample allocation should be very well balanced
with respect to the risk group and other factors.
The randomization sequence will link the treatment
assignment (0.4mg or 4mg) to a sequential list of serial
numbers to be used for the study pill boxes. The Data
Center will generate the randomization sequence. The
Data Center will supervise the labeling of the boxes with
the serial number. In addition, the boxes will be num-
bered in order of dispensing (1, 2, 3, etc.3). The serial
and dispensing order numbers will be recorded on the
study visit/follow-up form to track dispens ing.
The random assignment will be accomplished by
assigning the next serial number in the randomization
list as the enrollment of each new subject is confirmed.
Once confirmed, the study subject ID is recorded in a
computerized randomization log (that will include
randomization order, serial number of assigned pill box
and the study subject ID). The Clinical Coordinators will
affix the study subject ID labels to the boxes of each
subject with the serial number assigned to her.
The Data Center will maintain the randomiz ation se-
quence at RTI headquarters in NC. Revealing the indi-
vidual assignment will be highly restricted and will only
be done if deemed clinically necessary. Only a physician
attending to the study subject may request revealing the
treatment assignment for a clinical purpose. The Co-
Principal Investigator will review this request and evaluate
the necessity of revealing the treatment assignment for
the given purpose. The individual assignment will be
revealed only after the Co-Principal Investigator approves
the request. The Manual of Operations details the proce-
dures for requesting the individual assignment from the
Data Center.
Study treatments
The study drugs are folic acid in concentrations of 4mg
(high dose) and 0.4mg (low dose). The study drugs are
supplied in tablet form, manufactured by ATIVUS
Pharmaceutical Industries in Sao Paulo, Brazil. Quality
control assessments of samples of production batches
will be performed for all manufacturers in an inde pend-
ent quality control lab in the United States (Celsis La-
boratory Group, Saint Louis, US). All pills, regardless of
their folic acid con centration, will be identical in size,
shape, and color and will be provided in identical packa-
ging. Patients enrolled in the study will be instructed to
ingest 1 tablet daily from enrollment until 3 months
gestation.
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The manufacturers will package the folic acid pills in
boxes containing 5 blister packs of 14 pills each (2 rows of
7 pills), for a total of 70 pills per box. Each box will contain
an information sheet that will include all recommendations
of the country’s drug monitoring agencies (ANVISA for
Brazil) regarding information about the drug, yet will not
reveal in any way the actual folic acid content. The manu-
facturer will also label each of the pill boxes with the follow-
ing information, in addition to any other item that is
recommended by the monitoring agency:
Manufacturing and expiration dates (including only the
month and the year of production which will be
common for the two doses of folic acid)
Description of the drug (folic acid)
Identification of the manufacturer
Statement about the use of the drug (“this product is
solely intended for research use and shall not be
commercialized”)
Patient instructions for taking pills (or if a daily dose is
missed)
Clearance number of monitoring agency
Clinic ’s logo
The manufacturers will also label the blister packs
with a production lot number. Each production run of
pills will use several lot numbers for each dose. The lot
numbers pertaining to each dose will be sent from the
manufacturer directly to RTI after each production run.
After each production, RTI staff will supervise labeling
the pillboxes with a serial number (##### - ##, a seven
digit number with 5 digit base number and 2 check
digits). A local staff independent of the study may be
used to label the pill boxes.
There will not be any information on the pill box or
blister pack that may indicate to the subject the folic
acid content of the pills.
A sample of pills from the boxes will be selected from
each production lot to be sent out for independent assay
of folate content to assure manufacturing compliance.
Follow-up of study subjects
Subjects will be followed up every 2 month s (app roxi-
mately 8 weeks) post enrollment to deliver the study
pills and evaluate their health status, preg nancy occur-
rence, and compliance with study pills. The study pills
will be delivered to subjects through express mail when
no in-person follow-ups are scheduled; subjects will be
contacted by phone in this case to complete the follow-
up data form and ensure that pills have been received.
Periodic in-person follow-ups will be conducted every
six months if subjects are available; otherwise a phone
and mail follow-up will be conducted. During each fol-
low-up, the subjects will be advised of the date and time
of the ir next appointment. Within two weeks prior to
each in-person follow-up, the Clinical Coordinator will
contact the subject by mail or telephone to remind her
of the follow-up date.
If subjects report menstruation delays of 14 days or
more during the follow-up and pregnancy has not been
confirmed, the study staff will advise the subject to take
a pregnancy test either at the study clinic, at an outside
clinic, or at home if subject prefers (subjects will be
reimbursed for pregnancy tests outside of the study
clinic).
Subjects who are confirmed to be pregnant will be
advised as when to stop taking the study pill so that the
intervention is limited to the first trimester of preg-
nancy. Gestational age will be calculated based on the
last menstrual period. Pregnant subjects will be referred
to prenatal care as soon as pregnancy is confirmed.
Pregnant subjects will be monitored by their local pre-
natal care providers. Participating subjects might receive
care from various prenatal care providers during the
pregnancy period and may not have a single prenatal
care provider. Most women usually seek prenatal care at
public health clinics, where they may get assigned a dif-
ferent provider at each visit. Thus it might be hard to
maintain contact between the Clinical Coordinator and
the prenatal care providers of the subjects. The Clinical
Coordinator will attempt to initiate an d maintain con-
tact with identified prenatal care providers to follow up
on pregnancy progress. The Clinical Coordinator will
also maintain periodic direct contact through phone
with the subje ct to check on her pregnancy progress.
Upon delivery, the Clinical Coordinator will follow with
the subject and her physician to check on the occurrence
of NSCL/P and other birth defects in the infant. Research
staff may also access prenatal care records of subjects,
pending approval of subject prenatal care providers, to ab-
stract significant health and care events that occurred du-
ring pregnancy and that required physician care.
The Clinical Coordinator or Co-Prinicipal Investigator
will also evaluate in person the infant for the presence
clefting and other anomalies either through an appoint-
ment at the clinic or at subject’s home as convenient to
the subject. When a birth defect is identified, research
team members will carry out a complete physical exam
and confirm all oral cleft diagnoses. The Co-Principal In-
vestigator may also evaluate the infant along with the
Clinical Coordinator or independently.
Gestational loss will be reviewed by one of the research
team members (Clinical Coordinator, Co-Principal Inves-
tigator, and/or geneticists of the clinic). When available,
miscarriage material will be examined. This examination
will may include macroscopic analysis (presence or ab-
sence of NSCL/P; other apparent malformations), photo-
graphic documentation, and cytogenetic analysis in the
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event of malformations depending on the capacity of the
clinic site in doing these investigations.
After completion of a pregnancy outcome, subjects will
be asked about their willingness to resume participation in
the study at an appropriate time following the end of preg-
nancy. Subjects who are willing to resume participation
will be assigned to the same treatment group as their pre-
vious randomization assignment, and the standard enroll-
ment procedures will be followed.
Blood collection
Folate (Serum and RBC) levels will be assessed, at base-
line (prior to supplementation), every 12 months after
enrollment, and at pregnancy. B12 levels will be assessed
at baseline and subsequently every year to monitor for
B12 deficiency. Folate and B12 tests may be conducted
at the own laboratory facilities of the clinic/hospital
when available or alternatively at local laboratories deter-
mined by the Co-Principal Investigator. The Clinical Co-
ordinator and Co- Principal Investigator will monitor
B12 levels and will refer subjects with B12 defi ciency to
the hematology service for treatment. R andomized sub-
jects found to be B12 deficient may be temporarily or
permanently suspended from the study drug based upon
recommendations of the hematology referral.
Based on clinical and research experience, few subjects
(perhaps none) are expected to ask for their blood results.
If requested prior to the end of the study, an independent
physician serving in the clinic will provide confidential
results to the subject. The subject will also have the op-
portunity to discuss what her results mean (in terms of
being within normal range or not) with the physician in-
dependently from other study subjects and research unit
staff. This procedure will prevent sharing the results with
other study participants as well as with any member of the
research team. Since no data is available on what these fol-
ate measurements will actually mean in terms of the levels
of folic acid supplementation that are provided, it would
be impossible for any single woman to interpret these data
points in a way that could result in her un-blinding.
Therefore, any disclosure of folate test results under these
specific circumstances will not affect the blinding of study
subjects or of research unit personnel.
Management and retention of study subjects
All subjects will be provided with a toll-free phone num-
ber for the study that they could call at regular office
hours or anytime in case of emergency for any que stions
they may have or issues they would like to report (preg-
nancy, illness, concerns, etc.).
The study will maintain periodic phone contacts with
pregnant subjects to inquire on pregnancy progress and
remain updated on subject’s residence. Pregnant subjects
will be followed by their prenatal care provider. With
subject approval, contact between the study staff and the
prenatal care providers will allow follow up of the preg-
nancy and complications.
Incentives and reimbursements
Incentives, in the form of 30 Reais value food vouchers,
will be provided to subjects participating in the study at
enrollment and every six months thereafter to strengthen
retention and motivation. All subjects will be reimbursed
for transportation costs incurred to attend follow-up visits
at the clinic. Postage costs for the returning of used pill-
boxes will be paid by the project.
As a substantial percentage of potential study subjects
are economically disadvantaged, providing food vouchers
is expected to help minimize the indirect burden of par-
ticipation in the study, such as time lost from work or
household production due to study requirements.
Protocol violations
A protocol violation must be reported to the Data Cen-
ter within one week of its discovery. The Clinical Coor-
dinator or the Co- Principal Investigator will complete
the Protocol Violation Form. The Co-Principal Investi-
gator will sign the form and submit it to the Data Cen-
ter. Each protocol violation requires a completed form.
Possible protocol violations that require reporting to
the Data Center include the following:
1. Informed consent was not obtained before
randomization.
2. Subject did not meet inclusion criteria.
3. Subject did meet criteria requiring exclusion.
4. Study medication was never initiated for the patient.
5. Subject was permanently discontinued from study
medication (e.g., early termination of study
medication).
6. The wrong folic acid dose was given to the subject.
7. The treatment assignment of the subject was
revealed to study personnel or to the subject either
by request or inadvertent means.
8. Failure to take at least 50 pills in 2 months (A
compliance rate of at least 80% is required).
9. Study subject withdrew consent.
10. The study subject became permanently lost to
follow-up.
In situations where it is not clear whether a form is
required, the Research Unit personnel should contact
the Data Center staff to find out if a Protocol Violation
Form is necessary.
If subjects are enrolled and later found to have been
ineligible at the time of enrollment (violations 2 and 3,
above), a decision to continue or terminate the study
medication will be made depending on the part icular
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inclusion/exclusion criteria violated as stated in the
MOO. Except in specific situations listed in the MOO
(such as when subject is perma nently sterilized, has B12
deficiency and is recommended to be fully withdrawn
from the study, takes epileptic drugs), subject’s enrol l-
ment will not be terminated in order to not violate the
intention-to-treat principle. The medical status of a
woman who discontinues the study medication will be
monitored for 2 months following termination.
Study interventions (Treatments)
Intervention (Treatment) descriptions
Eligible women with NSCL/P or with at least one child
with NSCL/P will be randomly assigned to one of two
treatment groups: one taking 4 mg and the other taking
0.4 mg of identically appearing folic acid pills. Subjects
will take the folic acid preconceptionally and up to three
months of gestation.
Delivery of interventions (Treatments)
An 8-week supply of study pills will be dispensed to sub-
jects upon enrollment and at regular 8 week intervals to
ensure unin terrupted dosing during the preconception
and prenatal periods of participation. Study pills may be
dispensed during in-person visit s or delivered to subjects
through express mail or in person visits to the clini c or
subject’s home. In all sites, the pillbox provided for the
previous supplementation period will be obtained from
the subject to determine pill compliance.
Subjects who are confirmed to be pregnant will be
advised as when to stop taking the study pills so that
the intervention is limited to the first three months of ges-
tation. If a subject experiences an adverse event, the study
medication may be discontinued either temporarily or
permanently after review of the principal or co-principal
investigator and depending on the recommendation of the
physician attending the subject when applicable.
Control group
The control group in this study will be the 0.4-mg folic
acid group. A placebo control group is not used, as a
low dose of folic acid is already recommended as a
standard vitamin therapy for women during preconcep-
tion and pregnancy period.
Related risks
Potential risks involved in this study include those of blood
drawing and interview participation, which are felt to be
minimal. Folic acid supplementation has overall three
aspects that have already been discussed in the literature:
1. The use of folic acid may mask vitamin B12
deficiency in cases of pernicious anemia and cause
irreversible neurological damage. In order to avoid
this effect, a B12 assay on every subject will be
performed prior to folic acid administration, and
every year thereafter. Complete blood tests will be
performed on collected samples at enrollment to
help detect anemia. Current exposure to folic acid
through fortification in the US has been reported not
to increase the risk of masking anemia [122]. This
does not imply though that high doses of folic acid
do not necessarily increase the masking risk, mainly
for cases with B12-deficiency related neuropathy
with no anemia. Yet due to the small percentage of
subjects expected to have B12 deficiency, the
assessment of B12 levels on a yearly basis after
supplementation provides an appropriate strategy to
monitor subject safety related to this issue. The
added cost compared to added benefit of more
frequent blood sampling to measure B12 (or other
analytes) is considered high in terms of increasing
the burden on subjects and lowering motiv ation that
it should be avoided.
2. It has been suggested in the literature, though not
confirmed, that high folic acid doses, such as the one
used in the present study, may increase the risk of
spontaneous abortions (expected spontaneous
abortion rate = 15%). The numbers of spontaneous
abortions will be reported and the Data Safety
Monitoring Board (DSMB) will determine whether
these exceed the threshold for an adverse event.
3. It has been suggested in the literature, though not
confirmed, that folic acid increases twinning. Should
a pregnancy result in twins, twin pregnancie s in
general have an increased risk of premature delivery
with an intendant risk of increased mortality and
morbidity for the infant, and also an increased risk of
maternal complications including pre-eclampsia.
Therefore, information on this matter will be collected,
and the DSMB will evaluate whether the rate of
twinning exceeds the threshold for an adverse event.
Measurement methods
Description of data forms
Table 2 describes the data forms that will be used for
this study:
Description of biological measures
The following laboratory measures will be collected from
the subjects found in Table 3.
The following physical exam measures of babies born
to subjects will be abstracted from medical re cords:
Birth defects
Birth weight
Length
Head circ umference (when available)
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The Clinical Coordinator will also obtain and docu-
ment information on the presence of craniofacial malfor-
mations includin g clefts or other birth defects in babies
born through personal evalua tion of the infant, contact
with the subje ct’s doctor after delivery, or abstraction
from the medical re cord of the infant, but the clinical
coordinator will make every attempt to evaluate in per-
son every infant born into the study. Information about
any malformations detected during the prenatal period
through ultrasound testing will also be obtained from
contact with the subject, her prenatal care provider, or
through abstraction from the prenatal care record.
Schedule of data collection
Data will be collected during the various stages of sub-
ject sampling, screening, enrollment, follow-up, preg-
nancy occurrence and progress, and termination from
study. General contact information (name, telephone,
number, etc.) will be documented prior to introducing
the potential subject to the study when possible. This in-
formation (telephone, residence, name, etc.) will be veri-
fied upon initial contact with the subject. Data will also
be documented when screening willing subjects, which
will occur either directly or within a week period after
first contact with the subject. Data will also be collected
during enrollment of eligible subjects, which will occur
either directly or within a week period after screening,
and during each bi-monthly follow-up. Upon occurrence
of pregnancy, data will be collected through contact with
subject and/or subject prenatal care provider and/or
through abstraction from the medical record. The sub-
ject will be contacted at an appropriate time after preg-
nancy resolution, regardless of the pregnancy outcome,
to obtain information on study outcomes and check on
her willingness to resume participation in the study.
Standard enrollment, data collection, and follow-up pro-
cedures will be followed with study subjects who agree
to resume participation in the study. A study termin-
ation form will be completed with subjects who wish not
to resume participation in the study, and a follow-up
visit is conducted after two months, whenever possible,
to check on pregnancy occurrence and source of pre-
natal care if subject is pregnant and obtain permission
to contact her provider to inquire on her pregnancy.
Tables 4 and 5 below include schedules of data collec-
tion, procedures performed, data forms completed, and
laboratory tests conducted before and after detection of
a delay in menstruation respectively.
Administration of data forms
Research staff will administer and complete the data col-
lection forms being used for this study using pencil and
paper format in primary subject language (Portuguese).
Subjects will also self-complete mailed follow-up forms
in the outreach model. Research staff will transcribe
completed data forms to the study database.
Collection of biological samples/shipping
Each blood sample will con sist of 4–12 millimeters of
peripheral venous blood collected from the subject.
Blood will be collected by the Clinical Coordinator or
other qualified staff in vacuum tubes either containing
anti-coagulant (1.5 ml) or not. All blood collection tubes
will be identified by date, name, and subject ID number.
Blood samples will be delivered to the laboratory to be
analyzed. Blood samples obtained during follow-up outside
of the clinic (such as at subject home) will be stored in
portable freezers before being delivered to the laboratory.
Laboratory analysis
See Section 3
Primary and secondary outcome measures
Primary outcome measure The primary outcome
assessed is a measure of recurrences of NSCL/P in off-
spring of the trial mothers. All live born children born
of at least 500g or gestation of 24 weeks or beyond with
NSCL/P (only Tessier X clefts included) will be identi-
fied by case finding and verified by in person clinical
exam. For the classification of cleft lip and cleft palate
malformations, the following schemata taken from the
Division of Plastic Surgery and Burns of the Department
of Surgery of the University of Sao Paulo Medical
School, Brazil will be used in Table 6.
Secondary outcome measures Table 7 lists the se cond-
ary outcome mea sures to be recorded
Training
Training study personnel in data collection
The aims and objectives of the study and the study protocol
procedures will be provided to all research staff at training.
Research staff will be evaluated at training via the use of a
posttest. For those who demonstrate lack of proficiency,
additional training and mentoring will be provided. Subse-
quently, individual training will be offered separately.
Clinical Coordinators and other local staff will be
trained by the Co- Principal Investigators, core research
staff including staff who were involved in the pilot study,
and staff from Iowa’s research unit and RTI. Training
will include (1) conducting recruitment, interviews,
completion of data collection forms; (2) delivery of inter-
vention; (3) blood collection; (4) ethical conduct of re-
search. Future study personnel to be hired for the main
study will receive similar training with input from the
pilot study staff. In case of turnover, the newly hired staff
will also receive similar training. Any drivers hired for
the study should hold appropriate driving licenses for
participating country. Train ing in data entry and
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Table 2 Data forms
Form # Form title Description Completed by
FA00 Sampling and Initial
Recruitment Log Form
Documents the outcomes of searching the clinical
records for potentially eligible subjects and of initial
contacts with potentially eligible subjects including
their interest in participation in the study.
Clinical Coordinator
FA01 Contact Form Documents contact information for each potentially
eligible subject and updated contact information for
participating subjects.
Information extracted initially from clinical records
and reviewed/updated with potentially eligible
subjects and participating subjects by the Clinical
Coordinator.
FA02 Screening Form Documents eligibility for participation for each study
subject selected.
Clinical Coordinator
FA03 Enrollment Form Documents marital status, information about children,
and smoking, alcohol use, menstruation,
contraception and gynecological care of the study
subject.
Clinical Coordinator
FA04 Enrollment Confirmation
Form
Records blood test results, and study medication
delivery to the subject, and plans for follow-up visit.
This form may not need to be completed with the
subject at some sites as all the information will be
available to the Clinical Coordinator.
Clinical Coordinator.
FA05 Follow-up Form Confirms address information, documents
menstruation, administration of study medication,
and plans for next follow-up visit.
Clinical Coordinator
FA06 Initial Prenatal Contact
Form (With Study
Subject)
Documents date of last menstruation, date of
pregnancy confirmation, and information about the
study subject’s doctor and source of prenatal care.
Clinical Coordinator
FA07 Prenatal Form (With
Doctor)
Documents via periodic contacts with the subject
prenatal care provider, or via abstraction from
prenatal care records, information about the study
subject’s pregnancy, prenatal vitamins, illness,
ultrasound results, etc.
Co-Principal Investigator (initial contact),Clinical
Coordinator
FA08 Study Continuation Post
Miscarriage/Stillbirth Form
(With Study Subject)
Collects data from the subject about her readiness to
resume participation in the study after a miscarriage/
stillbirth occurrence and arrange for the date and
time of the next visit if she is willing to continue in
the study.
Clinical Coordinator
FA09&10 Miscarriage/Stillbirth Form
(With Doctor)
Documents the miscarriage or stillbirth date and age,
as well assessment of miscarriage/still birth product if
available including the presence or absence of
clefting and other malformations, etc.
Co-Principal Investigator, Clinical Coordinator
FA11 Delivery Form (With
Subject)
Documents date of delivery, birth weight and sex of
the infant, mother’s smoking and drinking habits
while pregnant, and any complications and
malformations associated with the baby.
Clinical Coordinator
FA12 Delivery Form (With
Doctor)
Documents any malformations that may be
associated with the baby including craniofacial
malformations and abnormal delivery events. Filled
with the subject’s doctor (if possible) or abstraction
from the medical records of the subject.
Clinical Coordinator
FA13 Phone Call Form Documents telephone calls made to the study office
by the study subjects, the reason for each call, and
the action taken.
(Clinical Coordinator, Co-Principal Investigator)
FA14 Study Termination Form Documents withdrawal from the study by a study
subject, the reason(s) for termination and approval (or
not) for a two-month post termination follow-up.
Clinical Coordinator
FA15 Post Termination Follow-
up Form
Confirms address information, documents
menstruation and whether or not the former study
subject has gotten pregnant.
Clinical Coordinator
FA16 Laboratory Form Documents the date of blood collection, date of
analysis, and results of the blood testing.
Clinical Coordinator
FA17 Adverse Events Form Documents all information related to any adverse
event encountered during the course of the study.
Clinical Coordinator/Co-Principal Investigator
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transmission and in using the DMS will be provided by
Data Cen ter (RTI) staff.
Job descriptions of study personnel
a) Principal Investigator: Provides study design
guidance, administrative oversight, and budget/
finance review for the entire project. His background
is in clinical genetics, pediatrics, molecular biology,
and the etiology of cleft lip and palate.
Co- Principal Investigator: Will oversee clinical case
definition, provide access to subjects through the
clinic, develop protocol and MOO, direct the study
personnel, be responsible for translations and oversee
the laboratory analysis of blood samples.
Study Coordinator (Iowa): Will be responsible for
coordination with RTI, Iowa, and the study sites in
Brazil. He will be involved in the de velopment of the
protocol, MOO and data forms.
1. Clinical Coordinators/Study Nurses (local units—
Brazil): Will be responsible for ensuring the proper
conduct of the study, maintaining master files of
the protocol and the MOO, acting as the liaison
between study subjects and the clinic, conducting
follow-up of subjects including dispensing the
study pills and collection of blood samples,
monitoring pregnancies that occur in the subject
group, maintaining an inventory of dispensed and
stored drugs, distributing and receiving back folic
acid pill boxes, keeping the warehouse supplied
with all biological material needed, coordinating
blood analysis with the clinic laboratory, checking
laboratory data (principally to monitor B12
deficiency), printing questionnaires to be
administered to study subjects, keying in
questionnaire data to the computer, performing
the re-keying of a pre-determined percentage of
data forms, entering free-text data in the main
computer in English, editing all data entry done,
and communicating with RTI for correction and
transmission of entered data.
b)Data entry clerk (outreach model only): Keys in data
to the DMS. Check the transcription of questionnaire
data to computer files. Perform the re-keying of a
pre-determined percentage of data forms. Enter free-
text data in the main computer in Engli sh. Edits all
data entry done.
c) Administrative assistant: Responsible for
coordinating correspondence as well meetings with
Table 2 Data forms (Continued)
FA18 Protocol Violations Form Documents all information related to any protocol
violation encountered during the course of the study.
Clinical Coordinator/Co-Principal Investigator
FA19 Pregnancy Confirmation
Form
Documents the results of a performed pregnancy test
after randomization and related schedule of study
folic acid supplementation
Clinical Coordinator
FA20 Prenatal Form (With
Subject)
Documents via periodic contacts with the subject,
information about pregnancy progress, prenatal
vitamins, illness, ultrasound results, smoking/alcohol
etc.
Clinical Coordinator
FA21 and
FA21
Suppl-
ement
Delivery Form (In-Person
Evaluation of Live Birth
Documents the presence or absence of
malformations and/or complications with birth upon
in-person evaluation of the live born by the Clinical
Coordinator.
Clinical Coordinator or Co-Principal Investigator and
clinical geneticist (or pediatrician)
FA22
(Outreach-
model
only)
Pre-Contact Form Documents the outcome of each attempt to contact
subject by the study staff or voluntary professionals
assisting on the study.
A study team member, Social worker, Parent
Coordinator
Table 3 Laboratory measures
Laboratory
measure
Timing Aim Performed by
Complete
blood count
At baseline, every 12 months post initiation of
supplementation, and at pregnancy confirmation.
Anemia Laboratory of study clinic
Pregnancy test Whenever menstrual delay of 14 days or more is detected Pregnancy Laboratory of study clinic or an
outside affiliated laboratory
Folate analysis At baseline, every 12 months post initiation of
supplementation, and at pregnancy confirmation.
Measure serum/red cell
folate concentration
Laboratory of study clinic or an
outside affiliated laboratory
Vitamin B12
analysis
At baseline and every 12 months thereafter Detect pernicious anemia Laboratory of study clinic or an
outside affiliated laboratory
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foreign collaborators; and, elaborating and translating
all written materials in English (protocols, manuals,
informed consent, etc.).
d)Local IT staff: Responsible for maintaining maintain
database, troubleshooting, software and hardware
problems, and interface with RTI and data
management.
e) Drivers (outreach model only): Drive study personnel
safely and efficiently to their destinati ons; ensures the
safety of the vehicle, passengers and material
transported.
f) Other staff: The clinics may utilize adjunct personnel
on an as needed basis such as health science students
who are interested in gai ning research experience to
assist in research and data management activities
after receiving proper training.
Training materials
Training materials will include the protocol, the manual
of operations (MOO), hand-outs, overheads, slides, role-
playing , case-discussions, data forms, and phlebotomy
equipment (if study staff will be involved in blood
collection ).
Certification of study personnel
All study personnel und ergoing training will be certified
to work on the study once they have completed the
training, passed the certification exam, and satisfactorily
mastered the components of the study.
Maintenance of training and certification
Maintenance of training will take place at an annual
meeting of all staff in which reinforcement of protocols,
changes in methods, and problems encountered will be
discussed. Refresher training sessions will also be pro-
vided during group meetings combing all sites on an a s
needed basis.
Training in ethical issues
All research staff must receive training in ethical an d re-
sponsible conduct of researc h. Study personnel can take
the English version of the “Human Participants
Table 4 Schedule A (Data collection before any detection of a menstruation delay)
Task Timing Procedure Forms
Abstraction from clinical record Sampling phase Search clinical records to identify potentially
eligible subjects
FA00
Introduction to study First contact with subject (in
person or by phone)
-Verify potential eligibility FA00
-Verify subject address and contact information FA01
-Introduce potential subject to the study
-Explain study purpose and subject responsibilities
-Answer any questions that potential subject may
have
-Check willingness of potential subject to
participate
Screening Directly or within a week
period after introduction into
study
Screen subject for eligibility FA02
Enrollment Directly or within a week
period after screening
-Obtain informed consent FA03
-Obtain blood sample from subject
-Inform subject of process of study drug
dispensing and next follow-up
Verification of B12 and pregnancy test results,
randomization, dispensing study pills, and baseline
contact
Within 1–2 weeks days after
enrollment
-Verify blood test results (mainly B12 levels) FA04
Randomize subjects who have no B12 deficiency
and are not pregnant and dispense folic acid pills
Refer subjects who have B12 deficiency to
hematology service
Follow-up onward Every 2 months (about 8
weeks)
-Dispense and confirm receiving folic acid pills FA05
-Complete follow-up visit form
Follow-up when blood sampling is due (Once every 12 months after
enrollment.
-Schedule an in person follow-up for blood
sampling
FA05
-Dispense folic acid pills FA16
-Complete follow-up visit form
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Table 5 Schedule B (Data collection after detection of menstruation delay or pregnancy)
Task Timing Procedure Forms
Verification of pregnancy
and measuring folate levels
(if pregnant).
- Whenever delay in menstruation of 14 days or
more or pregnancy are identified at either at bi-
monthly follow-up or contact with subject between
follow-ups
- Request that subject completes a pregnancy
test within a week after detection of
menstruation delay if pregnancy is not
confirmed (For clinic based model, pregnancy
can be tested for at study clinic).
- FA19 to
document
pregnancy
test result
- Schedule an in-person follow-up within a week
after confirming pregnancy (if subject is not in
clinic when first confirmed). An in-person follow-
up in this case is only required if pregnancy
occurs before the end of first year post initiation
of supplementation and blood sampling is
required for folate measurement.
- FA16 to
document
folate level
Obtain blood samples for a folate testing (if
applicable).
- FA06 (if
pregnancy is
confirmed)
- If pregnancy is confirmed, calculate gestational
age and advise subject as when to stop taking
the study pills
- If subject is not pregnant, then proceed with
the regular follow-up and supplementation
schedule for non-pregnant subjects
Prenatal follow-up with
subject
Bimonthly follow-up during pregnancy Contact subject to inquire on pregnancy
progress and subject health status
FA07
supplement
Prenatal follow-up with
doctor
Monthly follow-up during pregnancy Contact subject’s prenatal care provider or
abstract from prenatal care records to monitor
pregnancy events
FA07
Follow-up after
miscarriage/stillbirth
occurrence
Within 60 days of Miscarriage/Stillbirth - Check willingness of subject to resume
participation in the study
- FA08
- Contact subject’s doctor (if possible) to obtain
information about miscarriage/stillbirth event
and material
- FA09&10
- Check the presence of clefting - FA13
- Photographic documentation
- Complete study termination form if subject is
unwilling to continue in the study
Follow up after delivery
(live birth)
Within 60 days of delivery date - Check delivery events, maternal health behavior
during pregnancy, and infant health measures
with study subject; check subject’s readiness to
resume enrollment in the study
- FA11
- Evaluate the infant in person to check for
presence of craniofacial or other malformations.
- FA12
- Complete study termination form if subject is
unwilling to continue in the study
- FA21 and
FA21
Supplement
- Contact subject’s doctor (if possible) or review
medical records to check the presence of
craniofacial malformations including clefting and
other defects as well as delivery complications; -
Photographic documentation
- FA13
Post-termination follow up Two months after termination from study Determine whether the subject became
pregnant and request consent to contact her
prenatal care provider
- FA14
- Screening and enrollment After subject agrees to resume enrollment in the
study
- Follow standard screening, enrollment
procedures listed above
- FA02
- Verification of B12 and
pregnancy, dispensing
study pills, and Baseline
contact
- Subject remains in the original randomization
group
- FA03
- FA04
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Protection Education for Resear ch Teams” online course,
sponsored by the National Institutes of Health (http://
cme.cancer.gov/c01/n ih_intro_01.htm). Alternatively, a
special ethics training session based on the Research
Ethics Training Curriculum created by Family Health
International (www.fhi.org) may be substituted. This
training is available in Portuguese at the fo llowing web-
site: http://www.fhi.org/sp/RH/Training/trainmat/ethicscurr/
RETCPo/index.htm.
Data collection and management
Overview
The Data Center maintains the central database for the
study and works with the Research Unit and the Na-
tional Institute of Child Health and Human Develop-
ment (NICHD) when analyzing and publishing data
from the study.
Facilities
Computer hardware and software The study clinics
will be equipped with desktop personal computers which
will be used for entry of questionnaire data, keeping the
study pills inventory, counts of unused pills, blood ana-
lysis results, and other study files. Desktops will be
equipped with data back-up and transmission software.
Data collected by each clinic site will be transmitted to
the Data Center over the Internet using software pro-
vided by RTI.
Laboratory equip ment The laboratory at each hospital
with which the clinic is affiliated will be used for analysis
of subject blood sampl es if possible. These laboratories
will have to be equipped with appropriate equipment to
be able perform measures of folate and B12 levels. In
case of inability to do B12 and folate analyses at the
clinic laboratory, local laboratories will be identified and
contracted to perform the analysis.
Data entry
Each study site is responsible for entering and updating
all subject records into the study databa se. The Clinical
Coordinator an d/or data entry clerk will be primar ily re-
sponsible for this task. A second keying of a percentage
Table 6 Classification of cleft lip and cleft palate
malformations:
Group I Pre-incisor foramen clefts (Clefts lying anterior to the
incisor foramen). Clefts of the lip with or without an
alveolar cleft.
a) Unilateral
Right Total – when they
reach the alveolar
arcade
b) Bilateral Partial
1) Total}On one
or both sides
2) Partial
c) Median 1) Total
2) Partial
Group II. Trans – incisor
foramen clefts
(Clefts of the lip,
alveolus and palate)
a) Unilateral Right
Left
b) Bilateral
Group III. Post-incisor
foramen clefts
(Clefts lying
posterior to
the incisor foramen)
1) Total
2) Partial
Group IV. Rare facial clefts
Taken from Division of Plastic Surgery and Burns of the Department of Surgery of
the University of Sao Paulo Medical School, Brazil (Spina et al., 1972).
Table 7 Secondary outcome measures
Secondary outcome Measurement
Serum/red cell folate levels Measured using appropriate equipment (e.g. the Elecsys 2010 (Roche) or Immulite 2000) and
reported in ng/ml.
Recurrence and severity of CL/P A severity score will be generated based on Table 6 above and the classification reported in
Additional file 1
Occurrence of twinning Greater than 1 fetus per current pregnancy
Occurrence of a miscarriage/spontaneous
abortion
Fetal loss before 20 weeks of pregnancy
Incidence of Preeclampsia As reported according to local obstetric standards
Occurrence of other birth defects As diagnosed by clinical exam.
Birth Weight of the baby As measured at birth (in grams)
Gestational age at birth As determined by the obstetrician (in weeks-days)
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of the completed data forms for verification will be per-
formed. The percentage of the data forms to be re-keyed
will be relatively high at the beginning and will start de-
clining as the study progresses due to acquiring greater
experience and famil iarity with data entry system. The
Clinical Coordinator (data entry clerk) will resolve in-
consistencies before transmission to the Data Center.
The completed data forms will be entered onto data
entry screens in Portuguese. Responses with free text
will be translated from the local language into English
by the Clinical Coordinator and keyed in English.
Data editing and error resolution
The Research Unit, in collaboration with the Data Cen-
ter, will develop the data entry programs. The data entry
programs will include the following edit features:
Field checks (i.e., only numeric data are allowed in
numeric fields)
Required data item checks (i.e., data entry cannot
proceed until a legitimate value is entered into that
field)
Range checks and/or valid value checks
Checks for legitimate data variables
Within form logical consistency checks (e.g., age and
date of birth are consistent)
Logical branching algorithms (e.g., the program skips
over sections if prompted by a previous entry)
Check digit verification on the ID number to help
ensure accuracy
Any restrictions that are overridden during data entry
will be documented by the DMS in an audit trail.
On a routine basis, the Research Unit study sites will
run edit checks that include the following features:
Eligibility Checks: To check inconsistencies or apparent
protocol violations on the eligibility forms.
Date/Gestation Edits: To check inconsistencies between
the delivery gestation calculated and other date
inconsistencies (e.g., between birth date/time and
delivery date/time).
Maternal and Baby Outcome Data: To check
inconsistencies between delivery data and data
recorded for the neonate.
Screening Log Edits: To check inconsistencies on the
screening logs and between the logs and the database
Compliance Data: To check consistency within the
compliance and drug dispensing recorded on the study
visit forms.
Laboratory Value Checks: To check for unusual or
missing lab values on the enrollment form and on the
study visit forms.
Missing/Overdue Forms
A special program will be run tha t checks for serious
problems in identification of the subjects, such as dupli-
cate stud y identification numbers.
As the Data Center re ceives transmissions from the
Research Unit study sites, edit checks similar to those
describe above are performed. In addition, further
consistency checks are performed. Such checks include:
identification of duplicate or missing forms; performing
comparisons that requ ire computations or variable co-
ding; and checking values across multiple records.
The Data Center generates and examines the edit
check reports. Any edit with a message that explains the
problem satisfactorily is marked as OK. The Data Center
notifies the Research Unit study site of any unexplained
edits or errors and requests clarification.
Any corrections to the database in the study sites are
accompanied by notes written directly into the database
as part of an audit trail. The initials of the research staff
making these changes are automatically attached to the
notes.
Transmission of data
Within country At each clinic site, the study office will
house the Research Unit database for the study on a desig-
nated personal desktop computer or a server if available.
Study databases will be maintained separate for each study
site.
To data co ordinating center Data files will be transmit-
ted from the Research Unit offices at the clinic sites to
the Data Center (RTI) using the transmission software
provided. Transmissions from the server are set to occur
routinely. The Data Center will notify the Research Unit
when data files that were expected are not received.
Security
Research Unit personnel at each study site will keep all
completed data forms in a locked facility. Only authorized
research personnel will have access to the data forms of
the study.
All data files transferred to the Data Center will be
stripped of personal identifiers. Any printed material
from the data files will be maintained in a locked archive.
The study database will be password protected. Only
authorized research personnel will have access to the re-
search database. Data transferred to the Data Center will
be password protected, and access will be limited to
authorized personnel only.
Database construction
The Data Center creates a final database after the com-
pletion of the final phase of data collection. The Data
Center, in collaboration with the Research Unit, and
NIDCR will develop the specifications for this databa se
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during the final phase of data collection. The Data Cen-
ter will deliver one copy of this edited, fully do cumented
database to the Principal Investigator. The data are
delivered in a format that will allow ease of import to
frequently used statistical packages. If formatting is
requested for a particular statistical package, the Data
Center will make every effort to satisfy the request.
Monitoring data collection
The Data Center will use RTI software to monitor data
collection and data processing activities. If data are not
received when expected, the Data Center will promptly
follow up with the Research Unit. Regular reports (as out-
lined below) will be produced on the progress of data col-
lection and database development for the use of the Data
Center, the Research Unit, and NICHD. These reports will
include enrollment reports, forms received, delinquencies,
missing and invalid data, and participants due for follow
up. The Data Center will also monitor their own data edit-
ing efforts to ensure that all failed edits are resolved.
The Data Center will carry out edits that may include
Redoing all the keying edits as a double-check,
Looking for duplicate or missing forms,
Performing comparisons that require computations or
variable codes, and
Checking values across multiple records.
Reports
The Data Center will collaborate with the Research Unit
in the development of any progress reports throughout
the course of the study. Periodic reports are expected to
be submitted to the DSMB, the Data Center, and NIDCR.
Routine reports for local use (e.g., Monitoring
reports) The Research Units at the study sites will gen-
erate routine reports for local monitoring. Reports may
include enrollment, study visit tracking (scheduling,
missed visits and contacts), laboratory results, and miss-
ing data forms. The research unit at University of Iowa
and the Data Center will work with the local Research
Units to create other reports deemed necessary for the
proper monitoring of study implementation.
Data safety and monitoring board The Data Safety
and Management Board (DSMB) will periodically eva-
luate the progress of study and the safety and efficacy
of the intervention. The Data Center collaborates with
the Research Unit to submit to DSMB study reports
every 6 months including serious protocol violations
and adverse events, on a schedule agreed upon by the
DSMB (this is typically once every six months). A
detailed description of the responsibilities, organization,
and functioning of the DSMB is provided in the
Charter for the Data and Safety Monitoring Board for
the Oral Cleft Prevention Trial.
Data center Ongoing data reports including progress of
subject enrollment are transmitt ed to Data Center. The
reports include subje ct recruitment, data verification
rates , protocol violations, and adverse events. The fre-
quency of data transmission is decided in consultation
with the Data Center.
NIDCR The Research Unit will submit an annual pro-
gress report to NIDCR and, thus, will provide study and
site performance information as stipulated. These annual
continuation reports must be submitted 2 months before
the end of each project year and will also be sent to the
local IRB.
Adverse events The following require ments apply to
events involving the mother, the fetus or newborn child,
and any other person who may ingest the study
medication.
The Co-Principal Investigators or Clinical Coordina-
tors must fax or e-mail an adverse event form in the sta-
ted timefra mes to the Data Center (please see MOO
Section 14.2 Method of Reporting for complete instruc-
tions). The Data Center will forward the report to the
Principal Investigator, the NIDCR Program Official and
Project Officer, and any additional assigned contacts. An
adverse report is required for the following events:
All Fatal or Life Threatening events, whether or not
there is any possible association with use of the study
medication or participation in the study, must be
reported within 24 hours.
Events that are both Serious and Unexpected, whether
or not there is any possible association with the use of
the study medication or study participation, must be
reported within 7 calendar days.
Other adverse e vents that are deemed reportable by the
clinic staff must be reported within 7 calendar days.
The adverse event reporting form will monitor the fol-
lowing specific events:
Miscarriages
Twinning
Death of mother
Death of infant (if occurring in the neonatal period)
Overdose of pills by mother
Ingestion of pills by anyone other than the mother
Illness in mother requiring hospitalization or medical
treatment
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Illness of infant (if occurring in the neonatal period)
Occurrence of Preeclampsia.
The Research Unit foreign site IRB will be notified by
the Co-Principal Investigator, in accordance with the
IRB’s policy for reporting adverse events.
The Data Center will inform the DSMB, at its regular
meetings, of any adverse events experienced by study
subjects. The Principal Investigator and the Data Center
will also notify the IRBs at the University of Iowa and
RTI according to the requirements and time frames of
these local IRBs.
Statistical analysis
Analysis plan
An intention to treat analysis will be first applied to test
the primary and secondary outcomes of the study, where
each randomized subject that contributes an informative
observation to these outcomes (e.g. a birth) will be
included in the analysis, regardle ss of compliance with
study pills or follow-up schedules. Accordingly, rando-
mized subjects will not be purposefully excluded from
the study except if they become permanently sterilized
or in the unexpected cases where they may no longer
take folic acid due to a significant health problem (e.g.
allergy to folic acid). The first analysis for the primary
hypothesis will exclude subsequent births for subjects
who continue to participate in the study after giving
birth in the study as these multiple observations will be
correlated yet these will be included in secondary ana-
lyses that account for multiple observation correlation as
further described below.
Due to the randomized design of the study and the
large sample sizes, the two treatment groups are
expected to be equivalent for factors that may affect re-
currence of CL/P and the other secondary outcom es.
Therefore, the effects of the treatment dose on study out-
comes will first be analyzed using simple bivariate tests
such as Fisher’s exact test for rate differences (e.g. CL/P
recurrence) and means test for continuous outcomes
(e.g. folate levels). The primary estimated effect of the 4
mg folic acid dose compared to 0.4 mg on CL/P recur-
rence will reflect an average estimate across the two risk
groups of women with CL/P and mothers of children with
CL/P, but we will also evaluate potential interaction be-
tween the dose effect and risk group (such as by using a
chi-square test for a common odds ratio across the two
groups) and test for the effect of the intervention sepa-
rately in the two risk groups (though at reduced power).
As secondary analyses, we will check for potential se-
lection bias in the sample of subje cts who contribute in-
formative data for analysis (e.g. births or folate levels)
due to non-random choice s of subjects to participate in
the study or to get pregnant and/or give birth (mainly
relevant for the primary hypothesis), resulting in inform-
ative sample censoring. The issue of concern is not a
biased estimate of the intervention effect in the used
data samples (randomization solves this problem), but
whether these effects are related to any non-randomness
in the nature of the used sample. Given the limited avail-
ability of data on important characteristics for potential
subjects who do not respond to the invitation to partici-
pate (refuse to participate, not located, etc.) across the
sites, we will analyze dropouts and absence of inform-
ative births as potential processes for sample selection
bias. We will estimate separate models using logistic re-
gression for propensity to continue enrollment in the
study versus dropping out and for giving birth versus
dropping out or participation without giving birth. This
will be done as a function of characteristics that may
affect these propensities such as age, marital status,
schooling level, employment status, fertility preferences
(child wantedness, contraceptive use), cleft risk group
(woman with a cleft or mother to a child with a cleft),
previous fertility history (e.g. number of previous chil-
dren), and area of residence. Data is collected on all
these characteristics at enrollment. The propensit ies will
be used as inverse probability weights for the analyses of
effects of intervention on study outcomes. The propen-
sities for giving birth will apply to the outcomes mea-
sured among study births (cleft recurrence and severity,
birthweight , gestational age, etc.) while those for main-
taining enrollment will apply to outcomes measured at
the subject level (e.g. folate levels). For pregna ncy-level
outcomes, such as miscarriage, twinning, and pre-
eclampsia, propensities for pregnancy (in reference to
dropping out or participating without getting pregnant)
can be estimated.
Secondary analyses of intervention effects by level of
compliance with study pills will be conducted. In addition
to the bivariate analyses of intervention effects on study
outcomes, we will also employ regression analyses (logistic
regression for binary outcomes) that adjust for known and
measured risk factors, in order to account for any differ -
ences between the two treatment groups due to inadequate
randomization, though this is considered unlikely. Several
behavioral and environmental factors may affect the recur -
rence of NSCL/P and will thus be controlled for including
smoking, alcohol, age, other vitamin use, age, poverty
(instrumented by education, employment), and birth order,
in addition to other factors such as the subject risk group
(women with cleft or mother to a child with cleft). Inter-
action effects between the study intervention and these
characteristics can be evaluated in these analyses as well.
Correlation between subjects recruited at the same sites will
be accounted for by either including indicator variables to
represent the recruiting site or by using a Generalized Esti-
mating Equations (GEE) or a Hierarchical (Mixed) model,
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which also can be used to account for within-subject correl-
ation in case of multiple observations per subject.
Power analysis
Table 8 presents estimates of prospective number of
births in the two study groups required for 70 and 80%
statistical power and 30, 50, and 70% reductions in CL/P
recurrence for alternative recurrenc e rates of 3, 5, and
7% in the 0.4 mg group. The 30% reduction represents a
lower effect that is still very clinically significant, while
the 70% represents an effect similar to that observed for
NTD recurrence [118]. The recurrence rates cover most
of those reported in the literature and we use the 5%
rate as a primary reference for our total suggested sam-
ple size given that it represents a proportional estimate
of the 4 and 7% recurrence rates assuming that 30% of
the study subjects will be women with clefts and 70%
will be mothers to children with clefts. The power calcu-
lations are based on a Type I error probability of 5%. We
use a one sided test as the recurrence rate in the 4 mg is
not expected to exceed that of the 0.4 mg group given
the converging evidence of protective effects of folic
acid; a Fisher’s exact test is used.
For a 50% reduction in a baseline rate of 5% recur-
rence, a total of 1582 births distributed equally across
the two treatment groups are required. This number of
births also provides acceptable power (0.75) to detect a
60% decrease in a 3% baseline recurrence rate, or a 40%
decrease in a 7% baseline risk. If the 4 mg dose shows a
preventive effect similar to that seen for NTDs (70%),
the required numbers of births are halved regardless of
the baseline recurrence risk (range from 524 to 1254
births for 80% power). The numbers of births required
to observe a 30% reduction are about three times as
large those for a 50% reduction.
This study may also allow evaluating secondary out-
comes. The effect of the intervention on severity of cleft-
ing will be assessed. We will also stratify our analysis to
examine differences in outcomes in the women born
with CL/P independently from women who have a child
with CL/P. The data from previous studies suggest a
similar affe ct of folic acid supplementation in mothers
with CL/P when compared to mothers who had a child
with CL/P but as described below we will examine the
groups to gether and separately.
Table 9 lists a few plausible effects and changes be-
tween the 4 and 0.4 mg groups and the total number of
births needed to observe those effects with 80% power
(type I error of 5%). Each of these (and others) will be
studied as secondary outcomes. The DSMB will also
monitor these outcomes to determine if significant unto-
ward side effects are occurring (incre ases in miscarriage
rates for example) but this is done independently of the
research staff. All power simulations are based on a two-
sided test except for the severity measure which is based
on a one-sided test given that severity is expected to de-
crease with the higher dose.
Sample size
In order to estimate the numbers of subjects and subject
participation years required to obtain these numbers of
births given that subjects are enrolled at pre-conception,
Table 8 Power analysis for primary hypothesis
Baseline recurrence rate % Reduction Power Total births Total subject years
0.07 70 0.8 524 5475
0.7 418 4368
50 0.8 1114 11641
0.7 874 9133
30 0.8 3324 34734
0.7 2574 26897
0.05 70 0.8 742 7753
0.7 590 6165
50 0.8 1582 16531
0.7 1240 12957
30 0.8 4734 49467
0.7 3666 38307
0.03 70 0.8 1254 13103
0.7 996 10408
50 0.8 2676 27962
0.7 2096 21902
30 0.8 8024 83845
0.7 6210 64890
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we use a birth rate of 0.0957 births per subje ct-year,
which is the rate observed to date in the ongoing study.
Subject participation years are included in the fifth co-
lumn of Table 8.
For the primary hypothesis, a total of about 16,531
subject-years are potentially required to provide the
1,582 births. Doubling the birth rate (0.19) would de-
crease the number of required subject years for all sce-
narios by half; about 8,266 total subject years will be
needed to obtain 1,582 births. We believe the 0.0957 rate
is conservative and underestimates the birth rate likely
to be observed in this study due to the short period on
which it is based and given that at lea st half of the sub-
jects en rolled to date have expressed a desire to have
(more) children. Any increase in the birth rate will result
in a proportional decrease in the total required subject
years.
Our estimate of the targeted sample size of subjects
uses the estimate of 16,531 subject participation years as
the lower bound for targeted subject years and takes into
account the recruitment capacities of the participating
sites. We will enroll about 21 subjects per week across
the 4 sites using the clinic-based model (6 subjects in
each of HRAC, Recife, and Salvador, and 3 in Porto Ale-
gre) during the first four years, which are expe cted to
provide about 12,035 subject-years, and will enroll about
1000 subjects by the end of the first year using the out-
reach model at the Bauru site, which are expected to
provide about 4,250 subject years. Follow-up of subjects
is scheduled to be completed about nine months after
the end of recruitment. The 550 subjects currently par-
ticipating in the study at Porto Alegre, Salvador, and
Bauru are expected to add about 2,612 subject years if
they maintain their participation in the con tinued phase.
This is independent of the number of births that this
group already had (50 births) which by itself lowers the
sample size (1582 births) and the subject years required
for the continued phase. Therefore, a total of about
18,897 “birth risk” years are targeted in the continued
phase to ensure that the minimum estimate of 16,531
subject years is reached. About 5,368 new subjects are
thus expected to be enrolled in the study including
1,000 subjects at the Bauru site using the outreach
model and 4,368 subjects across the study clinics using
the clinic-based model (1,092 subjects per year). Table 10
provides a summary of new subject recruitment and
subject years.
Interim analysis and study monitoring
The DCC will conduct analyses of screening, recruit-
ment, and participation rates as well as analysis of com-
pliance, every quarter of the study. The DCC will
compare screening, recruitment, participation, and com-
pliance to the projected rates. If the observed rates are
below the expected rates by 20% for any study site, the
study team will examine the causes of the discrepancy
and develop actions to improve these rates. The DCC
will examine the characteristics of dropouts and non-
compliers with study procedures and reasons for non-
participation when possible. For data quality, error rates
will be reviewed from double keying. The DCC will
check lab data for consistency of collected information.
The birth rates among study subjects will be estimated
periodically to determine the number of subjects to be
recruited. The DCC will also monitor the generate
reports on study adherence and adverse events. A sum-
mary monitoring report will be provided to the Data Safety
and Monitoring Board (DSMB) for their semiannual
meetings.
To examine the efficacy of the intervention, we
propose two interim analyses at 40% and 70% of the tar-
geted number of births and a final analysis. The corre-
sponding alpha boundaries are 0.002, 0.017, and 0.031
for a one sided test and unequal spacing of analyses using
O’Brien-Fleming spending boundaries [123]. For the pri-
mary hypothesis of this study, the power remains at ap-
proximately 80% and the targeted sample size remains
sufficient to allow the proposed interim analysis.
Table 9 Power to evaluate secondary outcomes
Secondary outcome Total births
Birthweight (difference of 100 gm)* 928
Birthweight (difference of 200 gm)* 234
Gestational age (difference of 2 days* 2484
Gestational age (difference of 4 days* 622
Pre-eclampsia-Change by 40% from 10% baseline rate 1540
Pre-eclampsia-Change by 50% from 10% baseline rate 948
Severity (50% change from cleft lip with palate to cleft lip only in affected cases with 25% overall cleft prevention) 680
Miscarriage (30% change from 15% baseline rate) 1629
Miscarriage (50% change from 15% baseline rate) 562
* Assumes SD of 543 grams (birthweight) and 17.8 days (gestational age) as estimated in our studies in these populations.
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All interim analysis reports will be provided to the
DSMB for their semiannual meetings. Data will be pro-
vided on all the monitored adverse events that are listed
in the protocol. The DSMB will review the adverse
events by treatment groups, the observed enrollment
rate, and the required number of subjects based on
observed birth rate at each meeting and make decisions
about study continuation. The DSMB will review the
rates of primar y outcome by treatment groups at each
interim look and make decisions about study conti-
nuation. The magnitude of any observed rates of pri-
mary outcome differences by treatment status and what
to consider a "substantial" difference will be revisited at
each interim looks.
In addition, conditional power/stochastic curtailment
method [124] may be used if requested by the DSMB
due to small observed differences in primar y outcome
rates between treatments. This analysis involves calcula-
ting the conditional probability of rejecting the null hy-
pothesis given a specific alternative and the sample size
at the time of the analysis. A very small probability sug-
gests that the null effect may be inevitable.
Sample size and spending boundary estimations were
performed using the East version: 4.0.1.50 by Cytel Soft-
ware Corporation.
Quality control
The quality control plan will involve establishing a com-
petitive set of selection criteria of study personnel, ad-
equate training and certification of training, adequate
on-site monitoring of study progress, periodic and on
as-needed basis conduction of site visit s by principal
investigator, NICHD Staff Science Collaborator, and
the Data Center, and appropriate provision of guidance
and feedback to study personnel upon occurrence of
protocol violations. The quality control process also
includes the following: verification of data entry, estab-
lishing a toll-free phone line for subjects to call for
questions, monitoring quality of folic acid pills, provi-
ding adequate and continu ous guidance to subjects
regarding proper intake and handling of pills , and
continuous and adequate reporting of adverse events
and protocol violations.
Selection of study personnel
Selection criteria to be utilized in the recru itment and
hiring of study personnel include (1) appropriate educa-
tional level, (2) high technical competence, (3) good
communication skills, (4) a commitment to and interest
in the study design, and (5) quality related professional
experience.
Job descriptions
Please see Section for job descriptions. The table below
lists selection criteria for each position.
Training procedures
All study personnel will receive training as outlined in
Table 11.
Certification procedures
All the study personnel will undergo training before data
collection commences. The competence of the trainees
will be certified at the end of the course and via a post-
training test. The Co-Principal Investigators will be re-
sponsible for certification.
On-site monitoring
At each site, the research will meet on a periodic basis
as determined locally to review ongoing activities and
assurance of quality control.
Site visits
Site visits will be conducted by the Principal Investigator,
NIDCR, and the Data Coordinating Center to monitor
the quality of study implementation by the Research
Units. Site visits will be conducted at least once a year
and on an as needed basis (if there are study implemen-
tation concerns that seem to require immediate eva-
luation and/or intervention), in consultation with the
local IRBs and will include the participation of the
Table 10 Estimates of new enrollment and subject years
Site Total of new enrollees Total subject years from new recruitment
Bauru – Outreach Model 1000 4250
Bauru – HRAC clinic 1248 3439
Salvador 1248 3439
Recife 1248 3439
Porto Alegre 624 1719
Total 5368 16281
Note: The table shows the total number of new subjects estimated to be enrolled by March 2011 and the total subject years estimated to be provide d by this
recruitment by December 2011.
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Principal Investigator, NIDCR Program Official and/or
Project Officer, and the Data.
Coordinating Center Principal Investigator or his/her
designee. The NIDCR, the Data Coor dinating Center,
and the U.S. and foreign Research Units will collaborate
in the planning and implementation of each site visit.
The purpose of a site visit is to monitor study proce-
dures including those related to the protection of human
subjects, recruitment and eligibility, data collection and
management, the protocol violations, and adverse e vent
reporting.
Methods for monitoring include chart reviews, inter-
views with research staff, and facility observation. The site
visit will include a feedback session to review results and
suggest recommendations for improvement. The Data Co-
ordinating Center will submit a report to NIDCR that
documents the assessment, results, and feedback given to
the Research Units, as well as its recommendations.
Feedback for protocol violations
The Co-Principal Investigators will provide guidance
and feedback to the appropriate study personnel on any
protocol violations. A list of these is provided in Section
4.6. In some cases, retraining or retesting may be indi-
cated. Protocol violations will also be re viewed at weekly
staff meetings.
Quality control of study pills and randomization
The folic acid pills for the ongoing study are manufac-
tured by ATIVUS Pharmaceutical Industries in Brazil
(http://www.ativus.com.br/). The manufacturer will con-
duct quality control tests for each batch of pills includ-
ing identification test of the raw folic acid material used
in the pills (prior to production), assay tests for the folic
acid dosage in the pill s, content uniformity tests (to
ensure that the dosage is consistent across pills), and
dissolution tests to ensure that the pills dissolve ad-
equately in a standard time. The manufacturer will pro-
vide these results to the investigators before labeling and
shipping the pills to the sites. If the results are not
within the acceptable range, the manufacturer will re-
produce and retest new batches of pills. Only batches
with documented test results that are within acceptable
ranges will be shipped to the sites.
The manufacturer will label the blister packs with lot
numbers, enclose the blister packs in boxes, and place
the 0.4 and 4 mg boxes in separate containers. An inde-
pendent contractor will label the pillboxes, at the manu-
facturer’s facility, with the serial numbers that are
matched to randomization assignment. The labeling will
be conducted under RTI’s supervis ion (see section 4.1.1).
The accuracy of the labeling will be independently
tested, before sending the pills to the sites, through ran-
domly selecting and sending to RTI a sample of boxes
that are labeled with the serial numbers. RTI will com-
pare the lot numbers on the blister packs to the serial
numbers on the pillboxes to ensure that serial numbers
and lot numbers match corre ctly. Further, the pills from
this sample will be sent to an independent laboratory in
the US to conduct assay content tests to ensure that the
folic acid dose matches the treatment assignment of the
lot numbers (0.4 versus 4 mg). These steps will identify
errors in labeling the blister packs and in labeling with
serial numbers. The latter can result due to incorrect
packaging the pillboxes into the 0.4 and 4mg containers
by the manufacturer or due to inaccurate serial number
labeling by the contractor.
Different procedures will be used to address labeling
errors depending on the error type. Err ors in serial num-
ber labeling will require verifying the lot numbers inside
Table 11 Staff selection criteria
Job title Selection criteria
Brazil Research Unit Staff
Clinical Coordinator (or Study
Nurse)
Higher or intermediate schooling level*, high communication skills that allow for conducting successful interviews
with subjects, quality skills in drawing blood samples (if needed), as well as acceptable computer knowledge/
familiarity that enable them to manage laboratory and patient compliance data.
Data entry person (outreach
model only)
Intermediate schooling level*, good skills in using basic computer packages such as Word and/or Excel, as well an
acceptable speed of data entry
Drivers (outreach model only) Fundamental schooling level*, having a driver’s license and showing a mature attitude and responsibility towards
driving.
Administrative Assistant Higher schooling level*, high communication skills that allow coordination of correspondence and meetings with
the Principal Investigator and other related personnel, as well as having a very good knowledge of written and
spoken English to be able to translate both written and spoken English to Portuguese and vice versa.
US Staff
Study Coordinator PhD or equivalent degree in a health care related field, good experience in project management and research
conduct, as well as strong communication skills and commitment to responsible care.
*The Brazilian Educational System is divided into three levels: fundamental, intermediate and higher education. Fundamental school extends over eight grades.
Intermediate school extends over at least three grades, and may include professional training. It thus may last for four or five years and provides professional
qualification. Higher education is composed of two different levels: undergraduate (e.g., full-nurse) and graduate. Additionally, professional training may be
provided to students who have only completed fundamental school.
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each pillbox to ensure that they match the assigned ser-
ial number and relabeling those that do not match. All
the pillboxes will be rechecked when this error occurs.
Errors in blister pack labeling will require production
and labeling of new pills, which will be test using the
procedures described above.
Pills will not be sent to the sites before all the quality
control procedures are completed. This necessitates that
pill orders are placed at least 5 months before the
intended date of shipping to the sites.
Sustainability
Dissemination plans for research findings
Significant findings from the analyzed data of either the
primary or secondary hypotheses will be discussed by
the investiga tors. When confirmed, the data will be pre-
sented at national and international meetings in abstract
or meeting report formats. Formal publications will be
developed from these findings and submitted to appro-
priate peer reviewed journals. Methodologies such as
study design may also be reported.
The ability to obtain high dose (4 mg) folic acid pills
at reasonable costs will allow women who are at risk of
having a child with NSCL/P, and who are concerned
about preventing such recurrence, to benefit from the
outcome of this medication, when demonstrated, with-
out facing a financial burden that may hinder this
process. In fact, it is estimated that the cost of obtaining
high dose folic acid pills by a woman per year falls in the
range of 3 to 4 U.S. dollars, which implies that even
women who suffer from real poverty may be able to pur-
chase the medications. However, in cases of extreme
poverty, where women cannot afford to get folic acid
even at such a low cost, local governments and private
health organization s (such as the participating clinics)
will be highly encouraged to provide the medication to
those women at no cost .
Processes and equipment within country
Equipment will be retained by the host site including
computer hardware and software. Any other purchased
equipment will be retained at the host site as well.
Plans for use of trained personnel
The Clinical Coordinators participating in the study may
be retained in a formal program aimed at prevention if
the primary hypothesis is realized. Funding for contin-
ued support will come from existing mechanisms of care
provided to families of children with clefts including fe-
deral and private fundin g. When continuing research
questions are under evaluation, grant support will be
sought to provide an ongoing base for investigative
work. A national program of vitamin supplementation
will be encouraged and sought after the study is com-
pleted if results suggest it is warranted.
Study organization
Duties of the research unit
The Research Unit, which consists of a joint collaboration
between the local research units and University of Iowa
research teams, will be responsible for the following:
1. Initial and ongoing training and certification of the
study personnel as outlined in Section 6;
2. Maintaining compliance and follow up rates of at
least 80 percent;
3. Disseminating and applying research findings to
benefit the study populations, including providing
information about related plans on an annual basis
(see Section 10.1);
4. Implementing the study protocol and MOO;
5. Managing the process of ongoing reporting to the
IRBs, NIDCR, and RTI;
6. Processing and stori ng the samples collected in the
field at each maternal visit, including preparation for
folate and storage of unused samples to provide a
long-term resource for future studies;
7. Maintaining a database of samples with tracking
information and the ability to link with demographic
and clinical data; and
8. Analyzing the processed samples for vitamin (serum
or red cell folate, B12) status.
The Research Unit is also responsible for other unlisted
duties that may be mentioned in the Terms of Award.
The U.S. unit provides oversight, assistance in writing,
budget management, and advice on study design.
The Principal Investigator is responsible for the
following:
1. Providing the study protocol and other documents a s
listed in the Terms of Award;
2. Submitting to NIDCR and the Data Center
documentation of current approval and the
comments of the IRBs of local research unit and the
University of Iowa;
3. Informing NIDCR Program Official and Project
Officer and the Data Center of all major changes in
the status of the ongoing protocol, informed consent,
or IRB approval within specific timeframes as
indicated in the Terms of Award;
4. Submitting annual progress reports to NIDCR for
evaluating the Research Unit Performance; and,
5. Reporting serious adverse events to the Data Center,
and NIDCR Program Official and Project Officer and
any additional assigned contacts, within specific
timeframes (see Section 7.9.6).
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A chart of the lines of aut hority of the staff of the
study is provided in Addit ional file 1.
Duties of the data coordination center
Responsibilities of the Data Center (RTI) include the
following:
Provide advice on study design, data collection, data
analysis, and publication development.
Prepare operations manuals and data collection forms.
Compile monthly and quarterly subject enrollment
reports, meeting summaries, progress reports, and
other reports as needed.
Design and manage study databases; Assure
maintenance of high quality databases, supervise all
data collection procedures, and arrange for the most
efficient transfer of study data where indicated.
Ensure full compliance with NIH regulat ory
requirement s, including informed consent, reporting of
adverse events, human subject safety and welfare
provisions, and the requirements of international
collaboration.
Provide training to all Research Unit site personnel as
needed on data management and analysis, and quality
control and quality assurance.
In coordination with the NIH cosponsors, provide
periodic on-site monitoring to the Research Units for
those studies being performed at that site.
Duties of NIDCR
The NID CR Program Official and Proje ct Officer serve
as the principal representatives of NIH, and will pro-
vide overall programmatic oversight, coordination, and
assistance.
Specific responsibilities include:
 Oversee site participation and performance with the
support of the Data Center.
 Participate in study design, data analysis,
interpretation, and publication of study results.
Human subjects
Description of participants
Potential study participants include all women attending
or registered in the participating craniofacial clinics, for
their own craniofacial treatment (when they have NSCL/P)
or the treatment of their child with NSCL/P, who meet the
inclusion criteria as described in 3.2.1 and who do not
meet exclusion criteria as described in 3.2.2.
Recruitment
Subjects include women with NSCL/P or mothers of
children with NSCL/P who are currently receiving or
have received in the past care at the participating clinics,
are eligible, and prov ide consent to participate in the
study. Women who are married to men with NSCL/P
and at the same time do not fit in the above two groups
are not included as a group because of concern about
the staff’s ability to convince the women to take folic
acid in this situation. Therefore, it is exp ected that com-
pliance with taking folic acid will be achieved best in
women who are included in the above two groups.
Informed consent
Informed consent will be obtained from all subjects. The
benefits and possible adverse effects of the intervention
will be explained to subjects. Subjects will be encouraged
to seek clarification regarding the intervention, and
efforts will be made to ensure that the information wa s
comprehended. The Clinical Coordinators will read the
informed consents to all subjects, and confirmation will
be obtained via a signature for literate subjects, and via
thumb impression in the presence of a witness for
illiterate subjects. Potential subjects will be assured that
refusal to participate in the study will in no way affect
further treatment or care. A copy of the consent form
being used for this stud y is provided in Additional file 1.
Incentives and other benefits
See section 3.8.
Cultural issues
This is both a high-risk and a motivated population.
There is a risk that during the course of the study fa-
milies will be found with specific causes of clefts that
will change their recurrence risk from what was pre-
viously known and that might have relevance for other
family members not in the study. This can be dealt with
through the usual process of genetic counseling available
through the study clinics.
Reporting to local IRBs
Each year an annual report of activities undertaken will
be prepared. These reports will also include any modifi-
cation in the original protocol. Reports will be sent to all
entities involved in the project, including the local IRBs.
Publications and presentations
Review process
Prior to the ir submission or application for presentation,
all manuscripts, posters, or oral presentations, or other
reports of the outcomes of this research effort will be
approved by a majority of the senior study staff includ-
ing (1) the Principal Investigator, (2) the Co-Principal
Investigators, (3) the NIDCR Project Officer, and (4) a
Senior research officer from the Data Center.
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Authorship
The authorship of manuscripts, poster or oral presenta-
tions , or other reports of the results of this study will be
guided by the criteria for authorship formulated by the
International Committee of Medical Journal Editors and
published in its Uniform Requirements for Manuscripts
Submitted to Biomedical Journals (updated October,
2001; available at: http://www.icmje.org). According to
these criteria, each author should have participated suffi-
ciently in the work to take public responsibility for the
content. Authorship credit should be based only on sub-
stantial contributions to (a) conception and design or
analysis and interpretation of data; and, to (b) drafting
the article or revising it critically for important intellec-
tual content; and, on (c) final approval of the version to
be published. Conditions (a), (b), and (c) must all be
met.
As a multicenter study, up to twelve authors, as per-
mitted by journals such as the New England Journal of
Medicine and the American Journal of Obstetrics and
Gynecology, may be identified for a given manuscript,
but only those authors fulfilling the criteria above are
eligible. As a general, but not absolute, rule, at least one
individual from the NIDCR, one individual from the
Data Center, the Principal Investigator and the Co-
Principal Investig ators will be authors for all publications
that result from this research.
Discussion
The costs related to oral clefts are high, and there is
converging research evidence of long term psychological
and socio-economic effects [125]. Since it is estimated
that about 1000 cases of CL/P born each year in the US
to women with CL/P or mothers of a child with CL/P,
demonstration that a folic acid dose of 4 mg per day
results in a 50% reduction could prevent CL/P from oc-
curring in 500 births in the US per year. Application of
the strategy in all developed countries would eliminate
CL/P in 1,500 children and if applied worldw ide roughly
10,000 births a year would be free of CL/P. In the US,
the economic costs of oral clefts (both direct and indi-
rect) have been estimated to be about $200,000 per case
in present dollars [126], while the cost of folic acid is
pennies per day. This study provides an opportunity for
huge savings in not only money but the overall quality
of life. If supplementation with high dosage folic acid
proved effective, it is estimated that supplementation of
all at-risk women who would give birth in a particular
year in the United States would cost $132,000, compared
to avoided costs of about $100 million dollars.
This study will also provide an infrastructure to assess
the effectiveness of the proposed folic acid intervention
in subgroups of women of different genetic characteris-
tics and risks, particularly in folate metabolism genes.
The proposed study will estimate the average treatment
effect of 4 mg folic acid supplementation compared to
the 0.4 dose, with the gene interaction studies to further
check for potential mediation of the effect by genotype.
This may help establish more specific clinical guidelines
for oral cleft prevention so that the intervention can be
better tailored for at-risk women. In this proposed cli-
nical trial, we intend to collect and store DNA samples
for each participating subject, but we will not request a
specific budget in this grant for DNA analysis. We will
be able to conduct DNA analysis and implement the
gene-interaction studies under the auspices of other on-
going research support.
Additional file
Additional file 1: Appendix.
Abbreviations
ANVISA: Agência Nacional de Vigilância Sanitária; CI: Confidence interval;
CLO: Cleft lip only; CLP: Cleft lip with cleft palate; CL/P: Cleft lip with or
without cleft palate; CONEP: National Committee of Research Ethics;
CPO: Cleft palate only; DSMB: Data Safety and Monitoring Board;
GEE: Generalized Estimating Equations; FA: Folic Acid; NS: Non-syndromic;
HRAC: Hospital de Reabilitação de Anomalias Craniofaciais; HCPA: Hospital de
Clínicas de Porto Alegre; IMIP: Instituto Materno Infantil Prof. Fernando
Figueira; LMP: Last Menstrual Period; MOO: Manual of Operations;
NIDCR: National Institute of Dental and Craniofacial Research;
NICHD: National Institute of Child Health and Human Development; NSCL/
P: Non-syndromic cleft lip with or without cleft palate; NTDs: Neural Tube
Defects; OSID: Obras Sociais Irmã Dulce; RTI: RTI International.
Competing interests
None of the authors has any competing interests in this work.
Authors’ contributions
JCM and DMF conceived and designed the study. GW co-designed the
study, developed study measures and procedures, and contributed to
designing the statistical analyse s. ARC, TF, and CP and FQ and CVG
contributed to designing the study procedures and data questionnaires. NG
contributed to designing and writing the study procedures. THartwell and
HC contributed to designing the statistical analyses. Mr. SL contributed to
designing and writing the data management procedures. LJ and Atkinson
contributed to designing the study. All authors read and approved the final
manuscript.
Acknowledgements
TThe authors would like to thank all the staff who have contributed to this
study especially Pricila Copedê Frascareli Freitas, Priscila Padilha Moura,
Cinthia Carolina Dalastti, Juliana Mercado Santos, Renata Belmonte Ramalho,
Daniela Vera Cruz dos Santos, Kenzo Martins Matuzawa, Mariza Branco,
Lorena Silva de Argolo, Lorene Lins, Eduardo Varella, Daniela Bezerra de
Melo, Stela Brandao, Amanda Oliveira, Cirlei Ribeiro dos Santos, Denise de
Souza, Rita Tonochi, Miriam Neis, Ana Paula Vanz, Fabiane Dresch, and Liliane
Todeschini de Souza. We also thank the following co-investigators: José
Alberto de Souza Freitas, Josiane Souza, Alain Viegas, and Lucildo Drebes
and collaborators: João Henrique Nogueira Pinto, Regina Célia Bortoleto
Amantini, Zeus Moreira, and Lauro Consentino Filho, for their contributions.
Funding
This work has been funded by NIH/NICHD grant U01HD040561 awarded as
part of the Global Network for Women's and Children's Health Research and
by NIH/NIDCR grant U01 DE017958. The content is solely the responsibility
of the authors and does not necessarily represent the official views of the
National Institutes of Health.
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Ethics
The study was approved by the University of Iowa IRB, the National
Committee of Research Ethics (CONEP) in Brazil, and the ethics committees
of all the study sites in Brazil.
Data Sharing
The investigators will share the study data with other investigators with
approved IRB protocols to access and analyze the data. We will also share
the study data collection forms and manual of operations with other
investigators who would benefit from this experience.
Author details
1
University of Iowa, Iowa City, IA, USA.
2
Genetic Counseling Service –
Bioscience Institute, São Paulo State University, Botucatu, Sao Paulo, Brazil.
3
Hospital de Reabilitação de Anomalias Craniofaciais, Bauru, Sao Paulo, Brazil.
4
Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil.
5
Hospital Santo Antônio- Centrinho: Obras Sociais Irmã Dulce, Salvador,
Bahia, Brazil.
6
Instituto Materno Infantil Prof. Fernando Figueira, Recife,
Pernambuco, Brazil.
7
RTI International, Durham, NC, USA.
8
Eunice Kennedy
Shriver National Institute of Child Health and Human Development,
Bethesda, MD, USA.
Received: 5 October 2012 Accepted: 13 November 2012
Published: 26 November 2012
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doi:10.1186/1471-2431-12-184
Cite this article as: Wehby et al.: Oral cleft prevention program (OCPP).
BMC Pediatrics 2012 12:84.
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