The expanding phenotype of POMT1 mutations: From Walker-Warburg syndrome to congenital muscular dystrophy, microcephaly, and mental retardation

Abstract

The importance of O-glycosylation of alpha-dystroglycan (alpha-DG) is evident from the identification of POMT1 mutations in Walker-Warburg syndrome (WWS). Approximately one-fifth of the WWS patients show mutations in POMT1, which result in complete loss of protein mannosyltransferase activity. WWS patients are characterized by congenital muscular dystrophy (CMD) with severe brain and eye abnormalities. This suggests a crucial role for alpha-DG during development of these organs and tissues. Here we report new POMT1 mutations and polymorphisms in WWS patients. In addition, we report different compound heterozygous POMT1 mutations in four unrelated families that result in a less severe phenotype than WWS, characterized by CMD with calf hypertrophy, microcephaly, and mental retardation. Compared to WWS patients, these patients have milder structural brain abnormalities, and eye abnormalities were absent, except for myopia in some cases. In these patients we postulate that one or both transcripts for POMT1 confer residual protein O-mannosyltransferase activity. Our data suggest the existence of a disease spectrum of CMD including brain and eye abnormalities resulting from POMT1 mutations.

Full text

HUMAN MUTATION 27(5), 453^459,2006
RESEARCH ARTICLE
The Expanding Phenotype of
POMT1
Mutations:
From Walker-Warburg Syndrome to Congenital
Muscular Dystrophy, Microcephaly,
and Mental Retardation
Jeroen van Reeuwijk,
1y
Svetlana Maugenre,
2y
Christa van den Elzen,
1
Aad Verrips,
3
Enrico Bertini,
4
Francesco Muntoni,
5
Luciano Merlini,
6
Hans Scheffer,
1
Han G. Brunner,
1
Pascale Guicheney,
2
and Hans van Bokhoven
1

1
Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands;
2
Inserm U582, Institut de
Myologie, IFR 14, Groupe Hospitalier Pitie
´-Salpe
ˆtrie
`re, Universite
´Pierre et Marie Curie (UPMC), Paris, France;
3
Department of Pediatric
Neurology, University Medical Center, Utrecht, The Netherlands;
4
Unit of Molecular Medicine, Department of Laboratory Medicine, Bambino
Gesu’ Children’s Research Hospital, Rome, Italy;
5
Dubowitz Neuromuscular Centre, Imperial College, Hammersmith Hospital Campus, London,
United Kingdom;
6
Dipartimento di Medicina Sperimentale e Diagnostica, Sezione di Genetica Medica, Muscle Unit, Universita
`di Ferrara, Italy
Communicated by Jean-Louis Mandel
The importance of O-glycosylation of alpha-dystroglycan (a-DG) is evident from the identification of POMT1
mutations in Walker-Warburg syndrome (WWS). Approximately one-fifth of the WWS patients show mutations
in POMT1, which result in complete loss of protein mannosyltransferase activity. WWS patients are
characterized by congenital muscular dystrophy (CMD) with severe brain and eye abnormalities. This suggests
a crucial role for a-DG during development of these organs and tissues. Here we report new POMT1 mutations
and polymorphisms in WWS patients. In addition, we report different compound heterozygous POMT1
mutations in four unrelated families that result in a less severe phenotype than WWS, characterized by CMD with
calf hypertrophy, microcephaly, and mental retardation. Compared to WWS patients, these patients have milder
structural brain abnormalities, and eye abnormalities were absent, except for myopia in some cases. In these
patients we postulate that one or both transcripts for POMT1 confer residual protein O-mannosyltransferase
activity. Our data suggest the existence of a disease spectrum of CMD including brain and eye abnormalities
resulting from POMT1 mutations. Hum Mutat 27(5), 453–459, 2006. rr 2006 Wiley-Liss, Inc.
KEY WORDS: POMT1; Walker-Warburg syndrome; muscle-eye-brain; genotype–phenotype; dystroglycan
INTRODUCTION
Extensive studies on yeast protein mannosyltransferases (Pmt)
showed that proper O-mannosylation of proteins is required for
cell wall rigidity and cell integrity [Gentzsch and Tanner, 1996].
Human protein O-mannosyltransferase 1 (POMT1; MIM]
607423) is closely related to members of the yeast Pmt4 subfamily.
POMT1 O-mannosyltransferase activity appears to be necessary
for the first step in O-glycosylation of alpha-dystroglycan (a-DG),
which is transfer of a mannosyl residue from Dol-P-Man to Ser/Thr
residues [Willer et al., 2003; Manya et al., 2003]. Homozygous
POMT1 mutations account for Walker-Warburg syndrome
(WWS; ]MIM 236670) in nearly one-fifth of patients, and such
patients typically have hypoglycosylation of a-DG in affected
muscle tissue [Beltra
´n-Valero de Bernabe
´et al., 2002]. WWS
patients rarely live beyond the first year of life because of
multiorgan involvement. Patients have congenital muscular
dystrophy (CMD), eye abnormalities such as cataract, micro-
phthalmia, buphthalmia, and Peters’ anomaly, and severe struc-
tural brain defects. Brain defects include hydrocephalus, lissence-
phaly, agenesis of the corpus callosum, fusion of the hemispheres,
cerebellar hypoplasia, and neuronal overmigration, which causes
Published online 30 March 2006 in Wiley InterScience (www.
interscience.wiley.com).
DOI 10.1002/ hu mu .20313
The Supplementary Material referred to in this article can be
accessed at http://www.interscience.wiley.com/jpages/1059-7794/
suppmat.
Received 23 Au gu st 2005; acce pted rev ise d man u script 10 Jan uary
2006.
Grant sponsor: Prinses Beatrix Fonds; Muscular Dystrophy Cam-
paign; The Netherlan ds Genomics Initiative (Horizon Programme);
Fondazione Carisbo, Italy; Institut National de la Sante
Łet de la
Recherche Me
Łdicale (INSERM); Association Franc-aise contre les
Myopathies (AFM); GIS-Institut des Maladies Rares; Grant sponsor:
Stichting Spieren voor Spieren; Grant number: MAR02-226; Grant
sponsor: Dutch Foundation for Scienti¢c Research; Grant n umber:
NWO 903-42-190; Grant sponsor: Hersenstichting Nederland;
Grant number: 11F503.21.

Correspondence to: Dr. H. van Bokhoven, Department of Human
Genetics 417, Radboud University Nijmegen Medical Centre, Box
9101, 6500 HB Ni jmegen,T he Neth erlan ds.
E-mail: H.vanBokhoven@antrg.umcn.nl
y
Both a uthors con tribu ted equal ly to thi s work.
rr 2006 WILEY-LISS, INC.

a cobblestone cortex [Dobyns et al., 1989; Cormand et al., 2001;
Beltra
´n-Valero de Bernabe
´et al., 2002; van Reeuwijk et al.,
2005a]. We have shown that mutations in POMT2 (MIM]
607439), FCMD (MIM]607440), and FKRP (MIM]606596) also
give rise to WWS, in a small percentage of the patients [van
Reeuwijk et al., 2005b; Beltra
´n-Valero de Bernabe
´et al., 2003,
2004]. Muscle biopsies from these patients showed similar
hypoglycosylation of a-DG suggesting a common defective path-
way. Other CMDs with brain involvement, like muscle-eye-brain
disease (MEB; MIM]253280), and Fukuyama congenital
muscular dystrophy (FCMD; MIM]253800) reflect other genes
involved in this glycosylation pathway and a genotype–phenotype
correlation for those genes has been established or is likely to exist
[Kondo-Iida et al., 1999; Taniguchi et al., 2003; Diesen et al.,
2004; van Reeuwijk et al., 2005a]. POMT1 mutations in WWS
patients cause premature stop codons or create amino acid
substitutions and deletions in conserved domains. This suggests
that these mutations may reflect null alleles [Beltra
´n-Valero de
Bernabe
´et al., 2002; Kim et al., 2004]. In vitro experiments have
demonstrated the disruptive effect of these mutations on the
O-mannosyltransferase activity of POMT1 [Akasaka-Manya et al.,
2004]. In this study we report the results of POMT1 mutation
analysis in 28 WWS patients. In eight WWS cases we identified
seven novel mutations and one previously described mutation. In
addition, mutation analysis was performed in patients with a less
severe phenotype, consisting of CMD with calf hypertrophy,
microcephaly, and severe mental retardation. Causative compound
heterozygous mutations were identified in four unrelated families.
One family is of Dutch origin. Three families are of Italian origin,
including two families that were described earlier [Villanova
et al., 2000] and were subsequently labeled ‘‘Italian MEB’’
[Muntoni et al., 2003], although eye abnormalities (severe
myopia) were present in only a single patient. In this work, we
refer to these four families as congenital muscular dystrophy plus
mental retardation (CMD/MR).
PATIENTS, MATERIALS, AND METHODS
Patients
The clinical features as well as brain MRIs for Patients 1 and 2
(first cousins) and Patient 3 from Italian unrelated families are
strikingly similar and were described by Villanova et al. [2000]. In
short, these patients have severe mental retardation, microcephaly,
hypertrophy of the quadriceps and calf muscle, and structural brain
abnormalities (Fig. 1), but no eye abnormalities. Patients 1 and 2
never developed the ability to walk and are presently 18 and 17
years old. Patient 3 died at the age of 3.5 years from
bronchopneumonia due to swallowing problems and aspiration.
Patient 4 is a 16-year-old girl of Italian descent born from
nonconsanguineous parents. She presented at birth, following a
normal pregnancy and delivery at term, with hypotonia and
microcephaly. Her developmental milestones were characterized
by gross delay, with acquisition of the sitting posture at 16 months
and inability to walk unsupported. Bilateral hip dislocation was
noticed at 2 months and severe myopia at 9 months. The patient
had severe mental retardation, was unable to speak, and displayed
stereotypic hand-regard movements. Her serum creatine kinase
(CK) was markedly elevated at 5,000 U/L (normal value is o200
U/L). Moderate hypertrophy of the thigh muscles was present. A
brain MRI performed at the age of 2 years showed megacisterna
magna and cerebellar hypoplasia and dilatation of the IV ventricle.
The white matter signal was normal and no gross supratentorial
FIGURE 1. Cerebral MRIs of Patients 3 and 5, compared with a typical WWS patient. A: Sagittal £uid attenuation inversion recovery
(FLAIR) MRI (repetitiontime [ TR] 5512 msec/echo time [TE] 515 msec) showing hypoplasia of the corpus callosum, brainstem and
cerebellum. B:Transverse relaxation time (T 2) MRI (TR 53,475 msec/TE 5150 msec) shows frontal atrophy and abnormal thicken-
ing of the frontal cortex (cortical dysplasia) in the typical WWS patient and in Patient 5, but normal cortex in Patient 3. Arrows in-
dicate thefollowing abnormalities:1, occipital encephalocele; 2, cerebellar hypoplasia; 3, hypoplasia of the brainstem; 4, hypoplasia
of the corpus callosum; 5, severe (supratentorial) hydrocephalus; 6, abnormal thickening of the frontal cortex (cortical dysplasia);
and 7, no cortical abnormalities.
454 HUM AN MUTATION 27(5) , 453^ 459, 2006
Human Mutation DOI 10.1002/humu

cortical dysplasia was visible. At the age of 13 years her head
circumference was 46 cm (below 2.5 standard deviations [SD]);
her sitting posture was characterized by mild scoliosis, and flexion
contractures of her hips and knees, and equinovarus posture of
her feet. A muscle biopsy performed at the age of 5 years was
dystrophic with intact laminin a2 staining. There was no further
muscle tissue available to study a-dystroglycan expression.
Patient 5 is a Dutch boy from nonconsanguineous parents, who
recently died at the age of 4 years. At birth he was hypotonic and
contractures were absent. At the age of 4 months, severe visual
impairment was suspected. Serum CK level was 6,125 U/L.
Ultrasound imaging showed atrophy and increased echodensity of
muscles. A muscle biopsy was refused.
At the age of 6 months, after the cerebral MRI was done (Fig. 1)
a diagnosis of MEB was made. At the age of 4 years he was
microcephalic with a head circumference of 45 cm (below 2.5
SD). Binocular visual acuity (Teller Acuity Cards) was 0.25. There
was myopia, astigmatism, and bilateral optic nerve hypoplasia.
Ocular pressure was normal, cataract was absent. He had a
myopathic face with open mouth. Supine position was frog-like
with generalized muscle atrophy, paucity of spontaneous move-
ments, and severe hypotonia with absence of head balance.
Flexion contractures of both knees and Achilles tendons were
present. Sitting without support was not possible.
Patients 6 to 13 are typical WWS patients. Table 1 gives an
overview of the clinical features of the CMD/MR patients
(Patients 1 to 5) compared to WWS and MEB patients.
Informed consent was obtained from all analyzed subjects.
LinkageAnalysis
DNA was extracted from blood lymphocytes using standard
procedures. A genome-wide screening was performed in one family
(Patients 1 and 2) using a 400–microsatellite marker set (Applied
Biosystems; www.appliedbiosystems.com) at the French National
Centre of Genotyping (CNG; www.cng.fr). Linkage to the WWS
locus was assessed by genotyping subjects for microsatellite
markers flanking POMT1, D9S1863, D9S179, and the intragenic
marker, D9S64, which is located in intron 2 of the POMT1 gene
[Beltra
´n-Valero de Bernabe
´et al., 2002]. PCR was carried out with
40 ng of DNA. We used the touchdown PCR method, with
annealing temperature decreasing from 65 to 551C in the first
10 cycles, and fixed at 551C in the final 20 cycles. All PCRs
were done using 0.5 U Platinium Taq polymerase (Invitrogen;
www.invitrogen.com) for amplification in a final volume of 15 ml.
PCR products were amplified using forward primers labeled
at their 50end with 6-Fam or Hex fluorochromes, and migrated
on an ABI 377 automated sequencer. Data were analyzed by the
Genscan (version 3.1) Genotyper 2.5 software (Applied Biosys-
tems) and haplotypes were constructed.
Mutation Analysis
Primer sequences, conditions for PCR amplification, and DNA
sequencing of 19 coding exons of POMT1 (NM_007171.1) in all
families was performed as described previously [Beltra
´n-Valero de
Bernabe
´et al., 2002]. In the case of missense mutations, we
determined their causative involvement by checking the Mende-
lian segregation in family members, and by excluding the presence
of these mutations in at least 100 control chromosomes.
Amin o Acid Conserva tion
In order to determine amino acid conservation of POMT1 in
multiple species we made a multiple alignment of human POMT1
orthologous protein sequences using MUSCLE [Edgar, 2004].
Proteins with the following GenBank accession numbers were
included in the alignment: NP_009102.1 (Human); AAS76201.1
(Mouse); NP_445858.1 (Rat); NP_001025856.1 (Chicken);
CAF89480.1 (Fish); AAH75534.1 (Frog); XP_318526.2 (Mosqui-
to); NP_524025.2 (Fly); and NP_596807.1, EAK86491.1,
XP_460584.1, EAK95207.1, XP_451701.1, AAS50686.1,
NP_012677.1, XP_449354.1, XP_503607.1, EAA64589.1,
TABLE 1. Clinical Features of Patients1 ^5 (CMD/MR), Compared toW WS and MEB Patients
Feature WWS
a
MEB
a
Patient1 Patient 2 Patient 3 Patient 4 Patient 5
Brain
Cortical abnormalities Y111 Y11 Y1Y1NNY11
Encephalocele Y N N N N N N
Hydrocephalus Y111 Y11 Y1Y1NNY1
White matter abnormalities Y111 Y1Y1Y1Y1NY1
Fusion of hemispheres Y11 NN N Y1NN
Septum hypoplasia Y111 Y11 NNNNY1
Corpus callosum hypoplasia Y111 Y1NNNNY1
Cerebellar cortex hypoplasia Y111 Y11 Y1Y11 YYY1
Cerebellar vermis hypoplasia Y111 Y11 Y1Y1Y1YY1
Speech (words) N N Y1Y1Y1NN
Mental retardation Y111 Y111 Y11 Y11 Y11 Y11 Y11
Microcephaly N N 3SD 3SD 3SD 2.5 SD 2.5 SD
Eye
Microphthalmia Y11 YNNNNN
Myopia Y Y N N Y11 YY
Retinal dysplasia Y11 Y1NNNNN
Muscle
Serum CK (normal value:200 U/L) 45x 42x 20x 40x 410 x 410 x 30 x
Calf hypertrophy N N Y Y Y N (thigh) Y
Macroglossia N N Y Y N ND ND
Other
Able to walk N N N N N N N
Age (years) o3o30 18 17 3.5, y16 4.5, y
a
Clinicalfeatures of WWS and MEB patients as previouslyreported [Cormandet al., 2001; Beltra
¤n-Valero de Bernabe
Łet al. , 2002; Ta nig uchi et al. , 2003] .
Y, observed; N, not observed; 1, mild; 11, moderate; 111, severe; ND, no data;CK, creatinekinase; y, deceased; SD, standard deviation.
HUM AN M UTATION 27(5) , 453 ^459, 2006 455
Human Mutation DOI 10.1002/humu

XP_332024.1, AAP05785.1, and EAA67633.1 (Fungi). A Clustal
color scheme with a 10% conservation threshold was used to
indicate conserved residues in the alignment [Clamp et al., 2004].
RESULTS
LinkageAnalysis
To unravel the genetic basis of two Italian families in which
three affected individuals were diagnosed with CMD associated
with severe mental retardation, structural brain abnormalities, and
muscle hypertrophy, we undertook genome-wide linkage mapping
on the most informative family with two patients. Although these
patients are first cousins, born to nonconsanguineous parents,
we hypothesized that a founder mutation may be shared by this
and other previously reported Italian families in which linkage
to LAMA2 (MIM]156225), POMGnT1 (MIM]606822), and
FCMD was excluded [Villanova et al., 2000]. After analyzing
several potential loci, no common homozygous markers were found
for Patients 1 to 3 and additional patients originating from Italy
with similar phenotypes. We then analyzed the genome-wide
linkage data based on the hypothesis that Patients 1 and 2 may
carry the same compound heterozygous mutations. This allowed
the identification of POMT1 as a candidate, since the two affected
individuals shared identical haplotypes for intragenic and POMT1
flanking markers. Patient 3 appeared to have different haplotypes
at the POMT1 locus (Supplementary Fig. S1) (available online at
http://www.interscience.wiley.com/jpages/1059-7794/suppmat).
Mutation Analysis
POMT1 mutation analysis was performed in two related
(Patients 1 and 2) and 39 unrelated sporadic cases. Twenty-eight
cases were diagnosed as WWS, seven cases as intermediate
MEB/WWS, and seven cases as CMD including calf hypertrophy,
microcephaly, and mental retardation. This last group includes the
two Italian families described above (Supplementary Fig. S1) and
four other families in which no linkage analysis was performed.
Four out of these six families, including the two Italian families,
revealed causative compound heterozygous mutations for POMT1
(Fig. 2). In addition, we detected causative POMT1 mutations
in 7 out of 28 WWS cases. None of the missense mutations
was detected in more than 100 chromosomes from control
subjects. We found no causative mutations in seven cases of
an intermediate MEB/WWS phenotype. All causative POMT1
mutations identified in this study are listed in Table 2. A schematic
overview of all causative POMT1 mutations known to date is
given in Figure 2. In addition to causative POMT1 mutations we
identified three rare exonic polymorphisms and one intronic
polymorphism that have not been described before. An overview
of all exonic and rare intronic polymorphisms identified in affected
individuals is given in Table 3.
Genotype^Phenotype Correlation
for POMT1 Mutations
The expanding phenotype of POMT1 mutations is suggestive
for a genotype–phenotype correlation resulting from mutations
that affect transcript or protein in different degrees of severity.
We studied whether substituted or deleted amino acids were
conserved across multiple species and looked further to see if these
amino acids were located in an important domain or 2D structure
of the protein. In order to determine the conservation of amino
acids we made a multiple alignment of POMT1 orthologous
protein sequences from different species (Supplementary Fig. S2).
The amino acid substitution of highly conserved amino acids
(similar residue throughout the alignment) is underlined in
Figure 2. It is remarkable that most mutations in WWS patients
that result in amino acid substitution or deletion are located in
the highly conserved PMT and MIR domains, although not all
affected amino acids are highly conserved. The only exception
is the S537R mutation reported by Currier et al. [2005]. How-
ever, the causation of this S537R change is not proven, because
this was the only heterozygous change identified in the POMT1
gene of a WWS patient. In addition, amino acid S537 is poorly
conserved (Supplementary Fig. S2). In three patients affected with
a milder CMD/MR phenotype (Patients 1, 2, and 4), we found an
amino acid substitution (c.193G4A, p.G65R) and deletion
(c.418_420delATG, p.M140del) in the PMT domain, but these
changes did not involve highly conserved amino acids.
FIGURE 2. Schematic representation of the POMT1 protein including all mutations known to date.The highly conserved protein man-
nosyltransferase (PMT) domain is given in red. Another conserved region is indicated in blue where the mannosyl-IP3R-RyR (MIR)
motifs are located. Both conserved elements are thought to be involved in the recognition and/or binding of protein substrates, and/
or catalysis.Transmembranous, endoplasmic reticulum (ER), and cytoplasmic parts of the protein are indicated in black, gray, and
light gray, respectively. Mutations resultinginW WS are given below the schematic representation of the POMT1 protein while muta-
tions resulting in the milder phenotype are given on top, except for the mutation p.A200P between the asterisks that results in
LGMD2K [Balci et al., 2005]. Connecting lines between mutations indicate compound heterozygosity and homozygous mutation
are in bold. Novel mutations are indicated in red. Changes in highly conserved (human-yeast) amino acids are underlined.
456 HUM AN MUTATION 27(5) , 453^ 459, 2006
Human Mutation DOI 10.1002/humu

DISCUSSION
Here we describe seven novel POMT1 mutations, in seven
WWS patients that show typical WWS symptoms as described
earlier [Dobyns et al., 1989; Cormand et al., 2001]. In total we
identified POMT1 mutations in approximately one-fifth of the
WWS patients in our cohort, including the patients for which the
POMT1 locus was excluded by haplotype analysis. This is
consistent with our previous study, but different from a recent
report in which it was shown that the incidence of POMT1
mutations in WWS patients can be as low as 7% [Currier et al.,
2005].
The POMT1 mutations in WWS patients that are known to
date are predicted to be highly disruptive toward the mannosyl-
transferase activity of the protein, because in nearly all cases they
predict truncated proteins or amino acid substitution, or deletion,
at critical positions. Indeed, almost complete loss of POMT1
activity has been established for some of the WWS missense
TABLE 2. POMT1 SequenceVariants and Amino Acid Changes

Patients ( Origin) Diagnosis Nucleotide variant Amino acid Amino acidconservation
b
Domain 2D structure
1 and 2 (Italy) CMD/MR c.193G4Ap.G65R 11 PMT Coil
c.1746G4Cp.W582C 1111 No Coil
3 (Italy) CMD/MR c.1540C4Tp.R514X N.A. 3
0MIR Coil
c.1770G4Cp.Q590H 1111 No Strand
4 (Italy) CMD/MR c.418___420delATG p.M140del 1PMT Helix
c.2167dupG
a
p.D723 fs
a
N.A. No Coil
5 (Netherlands) CMD/MR c.1149-2A4G (splice site) N.A. MIR Coil
c.2174C4G p.S725X N.A. No Coil
6 (Netherlands) WWS c.427G4Ap.E143K 11 PMT Helix
c.1104delC p.F369fs N.A. MIR Coil
7 ( Pakistan) WWS Hom. c.313C4T p.R105C 1111 PMT Helix
8 and 9 (Lebanon) WWS Hom. c.314G4A p.R105H 1111 PMT Helix
10 ( I n d i a ) W W S c .6 20 G 4Tp.G207V 1111 PMT Helix
c.1332delG p.W444X N.A. MIR Strand
11 (Qatar) WWS Hom. c.1456delT p.W486fs N.A. MIR Coil
12 ( Ireland) WWS Hom c.2167dupG
a
p.D723 fs
a
N.A. No Coil
13 ( Turkey) WWS Hom. c.907 C4T
a
p.Q 303 X
a
N.A. No Helix

POMT1mutation numbering based on NM_007171.1; 11 is A of ATG start codon.
a
The POMT1 mutations p.Q303X and p.D723fs have previously been identi¢ed (p.D723fs as p.G722fs in compound heterozygosity with p.V428D)
inWWS patients [Beltra
¤n-Valero de Bernabe
Łet al., 2002].
b
Amino acid conservation of human POMT1 (SupplementaryFigure S2) in mammals (1), birds, ¢sh, frogs (11), insects (111), yeast (1111).
WWS,Walker-Warburg Syndrome; MEB, muscle-eye-brain disease; Hom, homozygous; N.A., not applicable.
PMT, protein mannosyltransferasedomain; MIR, mannosyltransferase, IP3R and RyR domain.
TABLE 3. POMT1Polymorphisms

Nucleotide variant Amino acid Amino acid conservation
b
Domain 2D structure Reference
a
c.718T 4Gp.C240GPMT Helix rs4997217
c.724 A4G p.M247V PMT Heli x rs4995933
c.930C4Tp.R251WPM T C oil rs 3887873
c.931G4Ap.R251QPMT Coil rs2296949
c.855G4C(1%) p.L285F 11 PMT Helix This study
c.942C4Tp.T314T111 No Coil rs10901065
c.979G 4Ap.V327I1MIR Strand rs4740164
c .1113 T 4Cp.D371D1111 MIR Coil rs 3739494
c.1299C4A p.D433E 111 MIR Coil rs11243406
c.1565G4A(1%) p. R 522K No Coil This study
c.1758G4A p.R586R 1No Coil Currier et al. [2005]
c.1922C4Tp.A641V1No Helix rs12115566
c.2097 C4T(1%) p. S 69 9 S 1No Helix This study
c.1148116 G 4A Intronic N.A. N.A. N.A. Balci et al. [2005]
c.1241198___99delCT Intronic N.A. N.A. N.A. Balci et al. [2005]
c.1764148G4C Intronic N.A. N.A. N.A. Balci et al. [2005]
c.17641107 A 4C Intronic N.A. N.A. N.A. Balci et al. [2005]
c.1765- 6___7CC4AA (3%) Intronic N.A. N.A. N.A. This study
c.2069113 T 4C Intronic N.A. N.A. N.A. Balci et al. [2005]

POMT1mutation numbering based on NM___007171.1; 11is A of ATG start codon.
a
Referenced in NCBI SNP Cluster Report. New polymorphisms in POMT1 are shown in bold with their frequency in between brackets.The changes
p.L285F and p.R522 K were designated as rare polymorphisms as they were identi¢ed only in heterozygosity in consanguineous patients. In addition
these are nonconserved residues, which are substituted by a residue that is also present at corresponding positions of orthologous POMT1
protein sequences.
b
Amino acid conservation of human POMT1 (Supplementary Figure S3) in mammals (1), birds, ¢sh, frogs (11), insects (111), yeast (1111),
no conservation ().
N.A., not applicable.
PMT, protein mannosyltransferasedomain; MIR, mannosyltransferase, IP3R and RyR domain.
HUMAN MUTATION 27(5),453^ 459, 2006 457
Human Mutation DOI 10.1002/humu

mutations [Akasaka-Manya et al., 2004]. We therefore postulated
that mild mutations in POMT1 give rise to a milder phenotype
than WWS. Haplotype analysis in two unrelated Italian families
with three CMD/MR patients (Patients 1, 2, and 3) identified
POMT1 as a candidate gene. Mutation analysis by direct
sequencing of POMT1 in these patients with a less severe pheno-
type revealed compound heterozygous mutations (c.193G4A,
p.G65R; c.1746G4C, p.W582C and c.1540C4T, p.R514X;
c.1770G4C, p.Q590H). Direct sequencing of POMT1 in two
more patients, from Italian (Patient 4) and Dutch (Patient 5)
origin, with similar clinical features also revealed compound
heterozygous mutations (c.418_420delATG, p.M140del; c.2167
dupG, p.D723fs and c.1149–2A4G; c.2174C4G, p.S725X,
respectively). If our hypothesis is correct then one or both
mutations in these CMD/MR patients should be relatively mild,
i.e., leading to a mutant POMT1 protein with residual activity.
Indeed, the amino acid substitutions and deletions identified in
these patients either affect nonconserved residues (p.G65R and
p.M140del) or affect amino acids that are located outside the
recognizable protein motifs (p.W582C and Q590H). One of the
CMD/MR patients (Patient 5) is compound heterozygous for a
splice-site mutation (c.1149–2A4G) and a nonsense mutation
(c.2174C4G) in the last exon. Although these appear to be
severe mutations, this is not necessarily the case. For example,
there are several alternative splice sites close to position c.1149,
which would give rise to transcripts in which the reading frame is
maintained. Also, the S725X nonsense mutation is the most
C-terminal truncation known to date and may yield a protein with
residual enzymatic activity. Additional experiments are required to
determine the actual effects of the POMT1 mutations, such as
measurement of protein O-mannosyltransferase activity in patient
cell lines or in transfected cell systems [Akasaka-Manya et al.,
2004]. Unfortunately, no cell lines are available for the patients
described here.
The existence of POMT1 mutations that result in a rela-
tively mild phenotype is suggestive for a genotype–phenotype
correlation for POMT1 mutations as pointed out previously for
other causative genes (i.e., FCMD) for WWS and other
congenital muscular dystrophies with brain involvement [Kondo-
Iida et al., 1999; Taniguchi et al., 2003; Diesen et al., 2004; van
Reeuwijk et al., 2005a]. Mild POMT1 mutations may result in
even milder disorders of O-glycosylation, as recently reported
for a homozygous founder missense mutation (c.598G4C,
p.A200P) in five Turkish patients with autosomal recessive limb
girdle muscular dystrophy (LGMD2K; #MIM 609308) and mild
mental retardation [Balci et al., 2005].
Two recent publications demonstrate the simultaneous require-
ment of POMT1 as well as POMT2 to obtain protein mannosyl-
transferase activity in human and in Drosophila [Manya et al.,
2004; Ichimiya et al., 2004]. We recently reported POMT2
mutations in three WWS patients [van Reeuwijk et al., 2005b].
This is suggests that mild mutations in POMT2 may also give rise
to a mild phenotype.
ACKNOWLEDGMENTS
We thank the families for contributing material for this study.
We also thank the following persons for DNA sample collection;
M. Janssen, Dr. M. Brockington, Dr. W.J. Kleier, Dr. L. Brueton,
Dr. M. Wilson, Dr. F. Collins, Dr. D. Chitayat, and Dr. M.
Sheridan. We thank Dr. D. Beltra
´n-Valero de Bernabe
´for his
previous contribution to the research described here.
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