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Non-syndromic cleft lip and/or cleft palate: Epidemiology and risk factors in
Lubumbashi (DR Congo), a case-control study
Sébastien Mbuyi-musanzayi, PhD, Tony J. Kayembe, MD, Marc K. Kashal, MD,
Prosper T. Lukusa, PhD, Prosper M. Kalenga, PhD, François K. Tshilombo, PhD,
Koenraad Devriendt, PhD, Hervé Reychler, MD
PII: S1010-5182(18)30210-5
DOI: 10.1016/j.jcms.2018.05.006
Reference: YJCMS 2976
To appear in: Journal of Cranio-Maxillo-Facial Surgery
Received Date: 7 May 2017
Revised Date: 10 April 2018
Accepted Date: 2 May 2018
Please cite this article as: Mbuyi-musanzayi S, Kayembe TJ, Kashal MK, Lukusa PT, Kalenga PM,
Tshilombo FK, Devriendt K, Reychler H, Non-syndromic cleft lip and/or cleft palate: Epidemiology and
risk factors in Lubumbashi (DR Congo), a case-control study, Journal of Cranio-Maxillofacial Surgery
(2018), doi: 10.1016/j.jcms.2018.05.006.
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Non-syndromic cleft lip and/or cleft palate: Epidemiology and risk factors in
Lubumbashi (DR Congo), a case-control study
Sébastien Mbuyi-musanzayi, PhD; Tony J. Kayembe, MD; Marc K. Kashal, MD; Prosper
T. Lukusa, PhD; Prosper M. Kalenga, PhD; François K. Tshilombo, PhD; Koenraad
Devriendt, PhD; Hervé Reychler,
MD
Department of Surgery, Cliniques Universitaires de Lubumbashi, DRC (Head : Pr
Tshilombo François, PhD)
Corresponding author: Pr ém. Hervé Reychler, MD, DMD
Department of Oral and Maxillofacial Surgery
Catholic University of Louvain
Cliniques universitaires saint-Luc
Avenue Hippocrate, 10
B- 1200 Brussels, Belgium
+3227645702
herve.reychler@uclouvain.be
Grants: Sébastien Mbuyi-musanzayi was supported by a scholarship from ARES
(Académie de Recherche et d’Enseignement Supérieur) and received scholarships from
the IRO, KU Leuven in 2011 and 2014.
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Summary
Purpose: To determine the incidence and risk factors of occurrence of non-
syndromic cleft lip and/or cleft palate (NSCLP) in Lubumbashi.
Method: A case-control study was conducted in the health district of Lubumbashi
from February 2012 to December 2015. An exhaustive sampling, collecting all
newborns with cleft lip and/or cleft palate (CL±P) in maternity wards was
conducted. From a total of 172 cases, 162 non-syndromic cases were recruited. For
each case, one clinically normal newborn control was selected.
Results: NSCLP had an incidence of 1/1258 live births (0.8/1000). We found
significant associations with a family history of cleft lip and palate (CLP) (x²
family
history
=11.5, p=0.0007), maternal alcohol intake (OR=19.3, 95% CI: 1.9- 197.1),
paternal alcohol during the periconceptional period and the first trimester of
pregnancy (OR=18.7, 95% CI: 3.9-89.2), maternal educational level lower than
high school (OR=9.5, 95% CI: 2.0- 44.7), clay (Pemba) consumption during
pregnancy (OR=38.3, 95% CI: 9.3-157.0), the use of insecticides in the evening
(OR=130.3, 95% CI: 13.2-1286.9), indoor cooking with charcoal (Makala)
(OR=6.5, 95% CI: 1.22-34.5), and regular consumption of Kapolowe fish,
supposedly contaminated with heavy metals (OR=29.5, 95% CI: 7.4-116.7).
Conclusion: Several environmental risk factors highly prevalent in Central Africa
for facial clefting were found.
Keywords: cleft lip and/or cleft palate, incidence, risk factors, Lubumbashi (DR
Congo)
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Introduction
Cleft lip and/or cleft palate (CL±P) is the most common congenital malformation
of the head and neck region, with an estimated prevalence of approximately one in
700 live births. However, a significant geographical and ethnic variation is
observed (Panamonta et al., 2015). Asian and Native Americans have the highest
prevalence, about one in 500 live births. In Europeans, the incidence is one in 1000
births and the lowest prevalence is observed in Africans, about one in 2500. This
difference may be due to difficulties in performing epidemiological studies in
Africa. A systematic review of epidemiological studies on clefting in Africa
confirmed the lack of reliable epidemiological data, and pointed towards the need
for systematic studies based on a birth defects surveillance system in
geographically defined regions (Butali and Mossey, 2009).
Non-syndromic CL±P (NSCLP) is considered to be a multifactorial disorder caused
by the interaction of genetic and environmental factors. The observed
geographic/ethnic differences in prevalence might therefore also reflect variation in
genetic and environment risk factors (Dixon et al., 2011). A recent large-scale study
replicated several known genetic risk factors in different African populations
(Gowans et al., 2016).
From a preventive point of view, the identification of environmental risk factors is a
priority (Burg et al., 2016). Several risk factors have been identified, of which
active and passive smoking are the most consistent (Dixon et al., 2011, Little et al.,
2004, Sabbagh et al., 2015). Alcohol exposure and nutritional factors such as low
folate consumption have also been suggested as risk factors (reviewed by Dixon et
al., 2011). Population-specific studies are crucial, to account for differences in
environmental exposure in the African population. In addition, there exist gene-
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environment interactions, e.g. between variants in GSST1, NOS3 or IRF6 and
maternal smoking (Dixon et al., 2011). Given the population differences in
incidence of genetic variants, gene-environment interactions may also differ. The
situation in sub-Saharan Africa is especially challenging, since in this region,
environmental factors cause the highest number of deaths (per capita) worldwide
(Prüss-Üstün et al., 2006). This is traditionally explained by infectious diseases, but
exposure to modern environmental health hazards (such as natural resource mining
and processing) poses an increasing burden (Nweke and Sanders, 2009).
In this study, we address critical questions regarding the epidemiology of non-
syndromic cleft lip and/or palate (NSCLP) and local teratogenic risk factors, by
means of a systematic ascertainment of cases using a birth registry, in a well-
defined region in Lubumbashi, Central Africa.
MATERIAL AND METHODS
Study location
We conducted an analytical case-control study in the city of Lubumbashi, from
February 2012 to December 2015 in 126 hospitals with a maternity ward. The
health district of Lubumbashi has 11 health zones. Each zone is constituted by a
Reference General Hospital and many health centers and private clinics. The study
was conducted by the team of researchers from the departments of Surgery and of
Toxicology of the University of Lubumbashi, in collaboration with the provincial
Ministry of health of the former province of Katanga and the health district of
Lubumbashi. In Lubumbashi, all births must be reported to the Provincial Health
Department within 5 days after birth. Community workers independently control
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whether pregnant women in health zone did report the birth of their child, thus
rendering the official health statistics a reliable source of the total number of births.
Selection of subjects
An exhaustive sampling was done, recruiting all newborns with CL±P in the
maternity hospitals. Health care professionals in the health district of Lubumbashi
were trained in the detection of birth defects in newborn and the identification of
CL±P, as part of an ongoing epidemiological study to register all birth defects. A
telephonic system was established to inform the research team that reached the
clinic within 12 hours after delivery. Included cases were newborns with cleft lip
and/or palate and whose mothers stayed in Lubumbashi during the periconceptional
period and the first trimester of pregnancy. Controls were newborns
morphologically normal at birth, born in the same maternity ward as the case and
whose mother lived in the same neighborhood as the mother of the case.
All newborn cases and controls were physically examined by the research team,
and a questionnaire was completed after obtaining the informed consent of mothers
and/or fathers. The questionnaire focused on the periconceptional period and the
first trimester of pregnancy to determine the possible contribution of known and
suspected risk factors for CL±P. It was completed during the hospitalization of the
mother. The questionnaire was simple and detailed. It was read by the researcher in
the language that the mother and/or her husband understood the best. Details were
discussed for more clarification as needed. Cases with CL±P were followed once a
month in the pre- and postoperative period. Controls were followed every 3 months
until the age of 1 year, to exclude any malformation not apparent at birth. No case
was lost to follow-up. One control was diagnosed with osteogenesis imperfecta at
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age of 3 months and was replaced by another control newborn. After birth, we have
organized a follow-up system of all controls one time for all 3 months. In
Lubumbashi, we have only one center for the management of patients with cleft lip
and palate. This facilitated follow-up of all cases. All non-syndromic cases were
followed up to 1 year after birth, and according to their surgery schedule, and no
cases were lost. However, 5 syndromic cases died before the fifth month after birth.
Controls were initially followed up according to their vaccination schedule and then
recalled every 3 months. For those who did not respond to the appointment, a call-
back was made. All controls were reviewed until the age of 1 year.
This protocol and the informed consent were approved by the medical ethics
committee of the University of Lubumbashi (CEM-UNILU:
UNILU/CEM/015/2012).
Definition of variables studied
Sociodemographic parameters included gestational age at the first prenatal visit,
maternal and paternal ages at childbirth, ethnic group, address of the parents,
maternal education level (categorized as illiterate, elementary school, high school,
university level) and consanguinity.
Maternal medical history included number of miscarriages, presence of birth
defects in other children, occurrence of urogenital infections and malaria during the
pregnancy and their mode of treatment
(traditional or modern medicine),
medication taken during pregnancy (time, type and dosage). Additional
periconceptional and first trimester parameters included maternal clay (Pemba)
consumption, folic acid intake, active or passive smoking, alcohol intake, the use of
traditional medicine, the use of skin lightening creams, the use insecticides at night,
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in house cooking using charcoal (Makala), regular contact with bitumen, and
contact with mining sites. The regular consumption of food rich in folates (i.e.
vegetables and fruits) was evaluated, regular consumption meaning at least five
times a week. For the father, we recorded alcohol intake before conception and
during the first trimester, active smoking, and contact with minerals (included
working in a mining company, transporting minerals, working in artisanal mineral
processing or stocking minerals at home). The clinical parameters of the newborn
recorded were sex, anthropometric parameters (birth weight, height, and head
circumference, additional malformations and dysmorphism).
The diagnosis of CL±P was made clinically and classified as syndromic when at
least one additional major malformation and/or dysmorphism was present. CL±P
was typed anatomically in cleft lip (CL), cleft lip and alveolar cleft (CLA), cleft
hard and/or soft palate (CP), cleft lip and palate (CLP), cleft uvula or bifid uvula.
No newborn cases of submucosal cleft were identified, probably because this
remains subclinical at that age. Furthermore, cases were classified as unilateral (left
or right), bilateral or, for the palate, median cleft.
Statistical analysis
Parameters were encoded by epi-info 7 (Centers for Disease Control and Prevention
[CDC], Atlanta, GA, USA), and analyzed using IBM SPSS Statistics 24.0 release
for Windows (IBM Corp., Armonk, NY, USA). We described samples by usual
descriptive statistics. Most of our variables were dichotomized encoded. The
comparison of quantitative variables using their mean or median was made by
analysis of variance or the Kruskal-Wallis test. The risk of infants born with CLP
was calculated by odds ratios with a 95% confidence interval. To estimate the
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power of each risk factor in the occurrence of CLP, we performed a multivariate
logistic regression analysis. The probability of less than 0.05 was the significance
level, and the probability of exclusion parameter was 0.10. ROC curve has allowed
us to establish the predictive variables for occurrence of CLP in Lubumbashi.
RESULTS
Incidence and distribution of types of clefting
From February 2012 to December 2015, we detected 172 cases of CL±P among
203,836 live births in the city of Lubumbashi. Of these, 162 had NSCLP (94.2%)
and 10 were syndromic (5.8%) (Figure 1). Thus, orofacial clefts had an incidence of
1/1185 live births (0.84/1000), with 1/1258 live births (0.8/1000) for NSCLP.
CL±P cases originated from different ethnicities, including Luba from former
province of Katanga (33.3%), Luba from Kasai (21.6%), Bemba (9.9%), Swahili
(8%), and Hemba (8%).
Syndromic cases were diagnosed clinically as trisomy 13 (2 cases), ectrodactyly-
ectodermal dystrophy cleft syndrome (2 cases), Van der Woude syndrome (1 case),
and possible diagnosis of frontonasal dysplasia (2 cases), and one of
mandibulofacial dysostosis with microcephaly, blepharo-cheilo-dontic syndrome
and Peter’s plus syndrome (one case each). There were excluded from further
studies of risk factors.
Of the NSCLP cases, CL±P was observed in 88.3% of cases. Complete CL±P was
the most frequent anomaly (92.6%), and incomplete CL (with or without alveolar
ridge), meaning microform cleft appearing as a little defect in the red part of the lip
or looking like a scar from the lip up to the nostril, was observed in 7.4%. Bilateral
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clefts and median cleft palate had a low frequency, respectively 11.1% and 9.3% of
cases. Unilateral clefts accounted for 79.6% of cases, with the left side involved in
67.4% and right side in 32.6%.
Sex distribution
Overall, clefting was slightly more common in males (52%) compared to females
(48%), with a male/female sex ratio of 1.08, but we observed differences depending
on the type of clefting. CLA and CLP had a sex ratio of 1.4:1 and 1.4:1, whereas
CL and CP had a slight female predominance with a sex ratio of respectively 1:1.2
and 1:1.1. Bilateral clefts were more common in males (55.6% of cases compared
to 44.4% in females). Unilateral clefts were more commonly on the left side in
males (41 out of 66) and female (46 out of 63).
Sociodemographic, parental, family, pregnancy and newborn characteristics
Table 1 provides an overview of the parental, family, pregnancy and newborn
characteristics. Risk factors for NSCLP in this population include advanced
maternal age between 25 and 35 years (OR=1.9, 95% CI: 1.2-3.1) and older than 35
years (OR=3.5, 95% CI: 1.7-7.2), paternal age after 35 years of age (OR=5.1, 95%
CI: 3.1-8.3), multiparity (OR=2.1, 95% CI: 1.3-3.4). History of miscarriage
(OR=1.6, 95% CI: 0.9-2.9) and consanguinity (OR=2.5, 95% CI: 0.5-13.3) (Table
2) were not significant risk factors.
The mean gestational age at birth was 38.8±1.4 weeks in the included cases, which
is shorter compared to 39.2±1.2 weeks in controls (OR=1.2, 95% CI: 0.6-2.5). The
mean birth weight was higher in the cases (3260.0±505.2 g) compared to controls
(3073.6±355.5 g) (OR=3.6, 95% CI: 1.3-10.0). In 7.6% of CL±P cases, family
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history was positive for clefting, of which 2 were syndromic (Van der Woude and
suspected mandibulofacial dysostosis with microcephaly). A positive family history
of CL±P was significantly associated with NSCLP (X
2
family history
=11.5, p=0.0007).
In all, 56.8% of CL±P cases were born from pregnant mothers who were
housewives. A total of 4.3% of mothers were working in mining companies. In the
control group, 68.6% of mothers were housewives, and 1.2% was working in
mining companies.
Being illiterate (X
2
=18.9, p=0.0000) or following primary school only (OR=3.6;
95% CI: 1.8-7.4) were associated significantly with the occurrence of CL±P in the
offspring.
Environmental risk factors during the first trimester of pregnancy
Table 2 shows the results of the analysis of known and suspected environmental
risk factors for NSCLP. Paternal smoking (OR=8.2, 95% CI: 4.7-14.2), paternal
alcohol intake (OR=16.9, 95% CI: 9.4-30.2) and maternal alcohol intake (OR=28.8,
95% CI: 8.8-94.3) during the periconceptional period and the first trimester of
pregnancy were significantly associated with the occurrence of orofacial clefts in
offspring. We reported statistically significant associations between clay
consumption (OR=37.1, 95% CI: 19.5-70.4), Kapolowe fish consumption regularly
(OR=13.8, 95% CI: 7.8-24.4), contact with minerals (OR=18.7, 95% CI: 8.8-94.3),
use of insecticide (OR=67.3, 95% CI: 16.1-277.8), contact with bitumen (OR=10.9,
95% CI: 4.5-26.5), and cooking with charcoal indoors (OR=6.1, 95% CI: 3.1-12.3)
and the occurrence of orofacial clefts. Using lightning cream (OR=1.3, 95% CI:
0.8-2.1) was not associated with the occurrence of orofacial clefts.
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Multiple logistic regression analysis
Multiple logistic regression analysis (Tables 3 and 4) revealed the following risk
factors during the first trimester of pregnancy: maternal alcohol consumption (OR=
19.3, 95% CI: 1.9-197.1) paternal alcohol consumption during the periconceptional
period (OR=18.7, 95% CI: 3.9-89.2), maternal educational level less to high school
graduation (OR=9.5, 95% CI: 2.0-44.7), clay (Pemba) consumption (OR=38.3,
95% CI : 9.3-157.0), the use of insecticides in the evening (OR=130.3, 95% CI:
13.2-1286.9), cooking with charcoal (Makala) indoor (OR=6.5, 95%CI: 1.2-34.5),
and eating Kapolowe fish (OR=29.5, 95% CI: 7.4-116.7).
With the area under the receiver operating characteristic (ROC) curve of 0.98,
variables used in the multiple logistic regressions are discriminatory and are
associated with facial clefting. The predictive power of the model is very
significant (Figure 2).
Taken together, the different risk factors identified in this study after multiple
logistic regressions explain about 39% of the total risk (Table 4).
DISCUSSION
In this study, we provide, for the first time, reliable epidemiological data on facial
clefting from a Central African region, Lubumbashi, and identify environmental
risk factors relevant for this area. Data regarding the epidemiology and causes of
CLP in the Central Africa are scarce. In particular, in the DRC, only one single
epidemiological study exists, reporting a prevalence of 1 in 2250 live births for
CLP, and 1 in 50,000 for isolated CP between 1977 and 1979 (Ogle, 1993).
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Our study in Lubumbashi detected orofacial clefts in 0.84/1000 live births, and
NSCLP had an incidence of 0.8/1000 live births. This is higher than previously
observed in Kinshasa in the DR Congo (0.4/1000 for CLP and 0.02/1000 for CP)
(Ogle, 1993), and also higher than other African studies that have reported an
incidence ranging between 0.3/1000 and 0.67/1000 (1/2500) (Simpkiss and Lowe,
1961, Robinson and Shepherd, 1970, Kromberg and Jenkins, 1982, Msamati et al.,
2000). However, the incidence reported in Sudan was 0.9/1000 live births
(Suleiman et al., 2005). This difference is most likely due to missed cases in other
studies. Indeed, the strength of our study is that it took place in the framework of a
registry of birth defects in a well-defined region, maximizing the identification of
cases. Other studies from sub-Saharan Africa are limited by incomplete
ascertainment, because recruitment is typically in a limited number of all hospitals
serving a region or because children were examined several months after birth, e.g.
when recruited through humanitarian surgical mass campaigns. An illustrative
example is the study conducted in Tanzania, which reported a mean age of
consultation of 1 year (Manyama et al., 2011).
Facial clefts can be classified according to different criteria, i.e. isolated versus
syndromic, cleft lip versus cleft palate, severity and sidedness. Overall, we did not
find major differences in these characteristics compared to those in other countries.
For instance, the male predominance for CL/P and female predominance for CP is
well known and was also observed in our study.
Parental socioeconomic including advanced parental age, educational level and
obstetric history (including high parity) were identified as risk factors for NSCLP,
but in the multivariate analysis they were not retained as significant risk factors
except for low maternal educational level (less than secondary school graduation).
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In the literature, conflicting results exist for each of these variables (Gonzalez et al.,
2008). A positive family history of CL±P was found in 8% of cases in our series,
and this represents a significant risk factor. Family history of CL±P is a known risk
factor, and recurrence risks in genetic counseling are based on severity of the
clefting, distance of the relationship, number of affected relatives and sex of the
index. Consanguinity is also considered as a risk factor for multifactorial disorders,
but in this region, consanguinity is exceptional (only 3.1%), and was not associated
with an elevated risk for CL±P.
Children with NSCLP were born at term but on average 1 week earlier compared to
controls. However, this was associated with an increased average weight in
newborns with CL±P, a finding that we cannot explain. Also, this is not supported
by other studies (Kianifar et al., 2015).
Besides studying epidemiological aspects, our study aimed to identify
environmental risk factors. In the literature, several such factors are known.
However, there is a special interest in conducting such studies in Central Africa,
given the different genetic background and unique local environmental factors.
We found that maternal genitourinary infections during the first trimester of
pregnancy were associated with the occurrence of CL±P. This has been reported
before in some studies (Krapels et al., 2006, Natsume et al., 2000) but not in others
(Murray, 2002, Reefhuis et al., 2002). Along the same line, we observed a
significant association between fever untreated or poorly treated in the first
trimester and NSCLP. This is similar to the previously reported increased risk of
CL±P when the pregnant mother had a febrile disease or influenza in the first
trimester of pregnancy (Natsume et al., 2000, Krapels et al., 2006). Most of the
pregnant women in our study did not receive medical care during the first trimester
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of pregnancy and were self-treated by traditional medications. These drugs may
have an active ingredient, but the doses are often unknown.
We confirmed the well-known association between paternal smoking and CL±P
(Zhang et al., 2011, Molina-Solana et al., 2013, Hoyt et al., 2016). Maternal
smoking was exceptional in our study, and therefore this could not be reliably
evaluated as a risk factor. We also observed that the consumption of alcohol in
periconceptional period and the first trimester of pregnancy by the mother was a
significant risk factor for CL±P, as was reported before by some studies, but not by
others (Boyles et al., 2009, Boyles et al., 2010, Mossey et al., 2009, Wilcox et al.,
2007, DeRoo et al., 2016).
In the literature, data on folate deficiency are inconsistent (Dixon et al., 2011). In
our study, only 3.1% of mothers of a child with CL±P took folic acid (0.4 mg/day)
during the first trimester of pregnancy, compared to 9.5% in control mothers, which
was not significantly different. Also the regular consumption of fresh vegetables
and fruits, an alternative source of folic acid, was not significantly different in cases
and controls.
One particular environmental factor in Lubumbashi is the use of charcoal (Makala)
for cooking by almost all households. In-house cooking on charcoal, typically
without ventilation, was found to be a significant risk factor for CL±P. Burning
coal releases volatile molecules and several studies have demonstrated an
association between air pollution by CO, NO
x
, O
3
, PM
10
, SO
2
and the occurrence of
birth defects (Gilboa et al., 2005, Hansen et al., 2009, Hwang and Jaakkola, 2008,
Marshall et al., 2010, Teng et al., 2016, Qian et al., 2016). More specifically, CP
was reported to be associated with exposure to carbon monoxide (CO) and
particulate matter in the periconceptional period and in the first trimester (Zhu et
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al., 2015). Exposure to sulfur dioxide was associated with the occurrence of CLP
(Zhu et al., 2015, Rao et al., 2016). A study conducted in China found a significant
association between poor ventilation during heating of the house and the occurrence
of CLP (Wang et al., 2009). It is likely that in-house cooking on charcoal in a
poorly ventilated room is associated with elevated levels of potentially teratogenic
compounds. This result should encourage pregnant women to preferably cook
outside the house or to use adequate ventilation.
In an environment infested with malaria-carrying mosquitoes, insecticide use is
highly recommended. However, regular use of insecticides in the evening during
the first trimester of pregnancy was a significant risk factor for CL±P, with an OR
of 67.3. This result is in line with the data from other studies (Xu et al., 2015,
Mirilas et al., 2011). Pyrethrins are an important component of domestic
insecticides. A recent experimental study demonstrated that pyrethrin in cultured
lymphocytes increased the occurrence of sister chromatid exchange, and affected
mitotic and proliferative indices (Azab et al., 2016).
Economic activities in developing countries are associated with an increased
exposure to modern environmental health hazards (Nweke and Sanders, 2009).
Exposure to bitumen during the first trimester of pregnancy was found to be a
significant risk factor for CL±P. Bitumen is petroleum derivative that is widely
present in the Lubumbashi environment as an important compound for roads. It is a
complex product composed primarily of hydrocarbons, and its derivatives and may
contain various additives such as naphtha. At elevated temperature, bitumen emits
hydrocarbon molecules and complex compounds including polycyclic aromatic
hydrocarbons (PAH) such as benzopyrene. The thermal decomposition of naphtha
gives rise to toxic gases and vapors, including carbon oxides (Lauby-Secretan et al.,
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2011, Olajire et al., 2007). Our results were similar to the literature demonstrating
the association between organic solvents and the occurrence of CL±P (Hao et al.,
2015). An experimental study on inhalation of High Flash Aromatic Naphtha at
near-lethal level in pregnant mice revealed an increased incidence of cleft palate
(McKee et al., 1990).
Lubumbashi is at the heart of a mining region, especially of heavy metals. A recent
study in Katanga, of which Lubumbashi is the capital, showed that populations in
or near mining areas had high concentrations of heavy metals in their urine,
regardless of age (Banza et al., 2009). Metals that showed statistically significant
differences in concentration in this study were lead, copper, cobalt, cadmium and
uranium. Exposure in pregnant mothers was estimated by the number of visits to a
mining site during the first trimester. This was found to be a significant risk factor
for CL±P. Another possible source of heavy metal exposure is the consumption of
food polluted by heavy metals. The Kapolowe River serves the mining areas with
high concentrations of heavy metals and a major source of fresh fish called
Kapolowe in Lubumbashi. We observed a significant association between regular
consumption of fish Kapolowe in the first trimester of pregnancy and the
occurrence of CL±P. We hypothesize that this fish may contain high concentration
of heavy metals, and thus constitute a risk for CL±P. The literature reports a
significant association between heavy metals and occurrence of CLP (Hao et al.,
2015). Heavy metals can cross the placenta. They can replace essential minerals,
produce free radicals, neutralize the amino acids essential for detoxification, and
have been shown to decrease cell proliferation of the embryonic neural tube and to
damage nerve cells (Morello-Frosch et al., 2016, Vandenbroucke et al., 2015, Chow
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et al., 2008, Papaconstantinou et al., 2003, Su and Okita, 1976, Webster and
Messerle, 1980).
The consumption of clay during the first trimester of pregnancy, a local habit, was
found to be a risk factor for CL±P. The clay consumed often contains heavy metals.
With an average amount of 20 g clay consumed daily by pregnant women this
exposes the embryo to toxic concentrations of heavy metals (Lar et al., 2015,
Odongo et al., 2016). In addition to this, the clay consumed by pregnant women
also contains helminths eggs, including Ascaris lumbicoïdes, Tricuris trichiura,
Taenia spp, Necator americanus and Ankylostoma duodenal (Odongo et al., 2016).
These heavy metals or parasite eggs can interfere with micronutrient absorption by
the digestive tract, necessary for embryonic development (McKinney et al., 2013).
The main limitation of our study is the lack of critical discussion of several
exceptionally strong associations with environmental factors that are specific to
sub-Saharan Africans and were not reported before in the literature, and the lack of
biological measures of exposure. An other limitation of our study is the small size
of our sample, which gave fairly wide intervals of confidence.
CONCLUSION
We report that the incidence of NSCLP in Lubumbashi is higher than previously
reported for sub-Saharan Africans, most likely due to a better ascertainment of
cases in our study. This highlights the value of systematic registries of birth defects,
as was suggested before. Moreover, we identified novel risk factors for clefting
characteristic for this region, including exposure to heavy metals from mining
activities, the use of insecticides and clay consumption during the first trimester of
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pregnancy. These results, when confirmed in follow-up studies, will form the basis
of guidelines for the prevention of facial clefting in this part of the world.
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Conflict of interest
None of the authors has a conflict of interest to disclose in relation to this work.
Acknowledgements
We thank patients’ families for their cooperation and the village of Holsbeek
(Belgium) for supporting our patients in 2014.
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Table 1. Parental, family, pregnancy and newborn characteristics.
Cases (n) Controls (n) OR 95% CI ꭓ² p
Maternal age (years)
<25 42 70 1
25-35 86 76 1.9 1.2 - 3.1 5.9 0.0076
>35 34 16 3.5 1.7 - 7.2 11.7 0.0003
Mean age 29.3±7.2 26.2±6.0
Paternal age (years)
<25 7 9 1.5 0.5 - 4.3 0.3 0.29
25-35 59 116 1
>35 96 37 5.1 3.1 - 8.3 43.2 <0.0001
Mean age 36.6±7.9 32.0±6.3
Gestational age
(weeks)
<38 21 17 1.2 0.6 - 2.5 0.3 0.3
38-42 141 145 1
Mean
Gestational age 38.8±1.4 39.2±1.2
Parity
Primiparity 38 63 1
Multiparity 124 99 2.1 1.3-3.4 8.3 0.0019
Average
parity 3.6±2.3 2.9±2.2
Miscarriage
Yes 36 24 1.6 0.9 - 2.9 2.5 0.0600
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No 126 138 1
Family history
Yes 13 0 11.5 <0.0001
No 149 162
Consanguinity
Yes 5 2 2.5 0.5 - 13.3 0.6 0.2200
No 157 160 1
Maternal educational
level
Illiterate 18 0 18.9 <0.0001
Primary school 34 12 3.6 1.8 - 7.4 12.7 0.0002
Secondary
school 100 128 1
University 10 22 0.6 0.3 - 1.3 1.3 0.122
Birthweight
<2500 g 4 8 0.5 0.2-1.8 0.6 0.2298
2500-4000 g 141 149 1
>4000 g 17 5 3.6 1.3-10.0 5.6 0.0079
Mean birth weight
(g) 3262.0±502.2 3073.6±355.5
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Table 2. Environmental risk factors in the first trimester of pregnancy
Case Controls
OR 95% CI p
n N Significance
Folic acid intake
Yes 5 14 0.3 0.1-1.0 0.0585 NS
No 157 148 1
Vegetables
consumption
Regularly 147 152 0.6 0.3 – 1.5 0.2020 NS
Irregularly 15 10 1
Contact with
minerals
Yes 52 4 18.7 8.8-94.3 <0.0001 SS
No 110 158 1
Maternal alcohol
Yes 57 3 28.8 8.8-94.3 <0.0001 SS
No 105 159 1
Paternal alcohol
Yes 143 50 16.9 9.4-30.2 <0.0001 SS
No 19 112 1
Clay (Pemba)
consumption
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Yes 130 16 37.1 19.5-
70.4 <0.0001 SS
No 32 146 1
Lightening cream
Yes 57 48 1.3 0.8-2.1 0.2853 NS
No 105 114 1
Bitumen
Yes 48 6 10.9 4.5-26.5 <0.0001 SS
No 114 156 1
Charcoal
(Makala) indoor
Yes 50 11 6.1 3.1-12.3 <0.0001 SS
No 112 151 1
Genitourinary
infection
Yes 60 18 4.7 2.6-8.4 <0.0001 SS
No 102 144 1
Insecticide
Yes 74 2 67.3 16.1-
277.8 <0.0001 SS
No 88 160 1
Traditional
medicine
Yes 10 2 5.3 1.1-24.4 0.0395 SS
No 152 160 1
Malaria
Yes 48 28 2.0 1.2-3.4 0.0127 SS
No 114 134 1
Maternal
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smoking
Yes 3 2 1.5 0.3-9.2 0.5000 NS
No 159 160 1
Paternal smoking
Yes 89 21 8.2 4.7-14.2 <0.0001 SS
No 73 141 1
Kapolowe fish
Yes 107 20 13.8 7.8 -
24.4 <0.0001 SS
No 55 142 1
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Table 3. Multiple logistic regression analysis of risk factors for NSCLP
Wald df Sig. OR 95% CI
Contact with
minerals 1.165 1 0.2805 3.622 0.349-37.486
Maternal alcohol 6.235 1 0.0125 19.301 1.890-197.095
Paternal alcohol 13.560 1 0.0002 18.748 3.939-89.229
Clay consumption 25.606 1 <0.0001 38.269 9.328-157.010
Bitumen 0.791 1 0.3737 0.403 0.054-2.988
Charcoal indoor 4.845 1 0.0277 6.536 1.229-34.48
Insecticides 17.370 1 <0.0001 130.306 13.194-1286.948
Genitourinary
infection 0.086 1 0.7698 1.231 0.306-4.953
Paternal smoking 0.307 1 0.5795 1.460 0.383-5.573
Kapolowe
consumption 23.212 1 <0.0001 29.470 7.441-116.716
Paternal age 1.128 1 0.2883 1.864 0.590-5.885
Maternal age 0.298 1 0.5853 0.744 0.256-2.156
Educational level 8.086 1 0.0045 9.480 2.012-44.676
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Table 4. Contribution of risk factors in the occurrence of NSCLP in Lubumbashi
Variables or risk factors Final X
2
X
2
without
one
identifie
d risk
factor
Contributio
n (%)
Clay consumption (Pemba) 358.79 321.18 10.48%
Kapolowe fish consumption 358.79 326.74 8.93%
Insecticide use 358.79 331.03 7.74%
Paternal alcohol consumption 358.79 341.19 4.91%
Maternal educational level 358.79 349.82 2.50%
Maternal alcohol consumption 358.79 351.41 2.06%
In-house cooking on charcoal
(Makala) 358.79 353.76 1.40%
Regular contact with minerals 358.79 357.59 0.33%
Paternal age 358.79 357.63 0.32%
Regular contact with bitumen 358.79 358.01 0.22%
Paternal smoking 358.79 358.48 0.09%
Maternal age 358.79 358.49 0.08%
First trimester urogenital infection 358.79 358.78 0.00%
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Figure 1. Distribution of different CL/P according to types, sex ratio, and familial
occurrence.
Figure 2. Receiver operating characteristic curve of risk factors in the multiple
logistic regression analysis.
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Figure 1. Distribution of different CL/P according to types, sex ratio, and familial
occurrence
1.1
:1
1
:1.1
3
:1
1
:1
8 (
5.6
%)
2
(10.5
%)
1
(
14.3%)
2 (
66.7
%)
7 (100%)
3 (100%)
1
7 (89.5%)
133
(93.0%
)
Sex ratio
(M/F)
Familial occurrence
Complete
CLP
2
(10.5
%)
10
(7.0
%)
0 (0%)
0 (0%)
Incomplete
CLP
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Figure 2. Roc curve of risk factors in the multiple logistic regressions
