Absence of mutations in NR2E1 and SNX3 in five patients with MMEP (microcephaly, microphthalmia, ectrodactyly, and prognathism) and related phenotypes

Abstract

A disruption of sorting nexin 3 (SNX3) on 6q21 was previously reported in a patient with MMEP (microcephaly, microphthalmia, ectrodactyly, and prognathism) and t(6;13)(q21;q12) but no SNX3 mutations were identified in another sporadic case of MMEP, suggesting involvement of another gene. In this work, SNX3 was sequenced in three patients not previously studied for this gene. In addition, we test the hypothesis that mutations in the neighbouring gene NR2E1 may underlie MMEP and related phenotypes.
Mutation screening was performed in five patients: the t(6;13)(q21;q12) MMEP patient, three additional patients with possible MMEP or a related phenotype, and one patient with oligodactyly, ulnar aplasia, and a t(6;7)(q21;q31.2) translocation. We used sequencing to exclude SNX3 coding mutations in three patients not previously studied for this gene. To test the hypothesis that mutations in NR2E1 may contribute to MMEP or related phenotypes, we sequenced the entire coding region, complete 5' and 3' untranslated regions, consensus splice-sites, and evolutionarily conserved regions including core and proximal promoter in all five patients. Two-hundred and fifty control subjects were genotyped for any candidate mutation.
We did not detect any synonymous nor nonsynonymous coding mutations of NR2E1 or SNX3. In one patient with possible MMEP, we identified a candidate regulatory mutation that has been reported previously in a patient with microcephaly but was not found in 250 control subjects examined here.
Our results do not support involvement of coding mutations in NR2E1 or SNX3 in MMEP or related phenotypes; however, we cannot exclude the possibility that regulatory NR2E1 or SNX3 mutations or deletions at this locus may underlie abnormal human cortical development in some patients.

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BioMed Central
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BMC Medical Genetics
Open Access
Research article
Absence of mutations in NR2E1 and SNX3 in five patients with
MMEP (microcephaly, microphthalmia, ectrodactyly, and
prognathism) and related phenotypes
Ravinesh A Kumar
1
, David B Everman
2
, Chad T Morgan
2
, Anne Slavotinek
3
,
Charles E Schwartz
2
and Elizabeth M Simpson*
1
Address:
1
Centre for Molecular Medicine and Therapeutics, Child & Family Research Institute, Department of Medical Genetics, University of
British Columbia, 950 West 28th Ave, Vancouver, V5Z 4H4, Canada,
2
Center for Molecular Studies, J.C. Self Research Institute, Greenwood Genetic
Center. One Gregor Mendel Circle, Greenwood, South Carolina, 29646, USA and
3
Department of Pediatrics, Division of Medical Genetics,
University of California, Box 0748, 533 Parnassus St., San Francisco, California, 94143-0748, USA
Email: Ravinesh A Kumar - rakumar@cmmt.ubc.ca; David B Everman - deverman@ggc.org; Chad T Morgan - morgan114@charter.net;
Anne Slavotinek - slavotia@peds.ucsf.edu; Charles E Schwartz - ceschwartz@ggc.org; Elizabeth M Simpson* - simpson@cmmt.ubc.ca
* Corresponding author
Abstract
Background: A disruption of sorting nexin 3 (SNX3) on 6q21 was previously reported in a patient
with MMEP (microcephaly, microphthalmia, ectrodactyly, and prognathism) and t(6;13)(q21;q12)
but no SNX3 mutations were identified in another sporadic case of MMEP, suggesting involvement
of another gene. In this work, SNX3 was sequenced in three patients not previously studied for this
gene. In addition, we test the hypothesis that mutations in the neighbouring gene NR2E1 may
underlie MMEP and related phenotypes.
Methods: Mutation screening was performed in five patients: the t(6;13)(q21;q12) MMEP patient,
three additional patients with possible MMEP or a related phenotype, and one patient with
oligodactyly, ulnar aplasia, and a t(6;7)(q21;q31.2) translocation. We used sequencing to exclude
SNX3 coding mutations in three patients not previously studied for this gene. To test the
hypothesis that mutations in NR2E1 may contribute to MMEP or related phenotypes, we sequenced
the entire coding region, complete 5' and 3' untranslated regions, consensus splice-sites, and
evolutionarily conserved regions including core and proximal promoter in all five patients. Two-
hundred and fifty control subjects were genotyped for any candidate mutation.
Results: We did not detect any synonymous nor nonsynonymous coding mutations of NR2E1 or
SNX3. In one patient with possible MMEP, we identified a candidate regulatory mutation that has
been reported previously in a patient with microcephaly but was not found in 250 control subjects
examined here.
Conclusion: Our results do not support involvement of coding mutations in NR2E1 or SNX3 in
MMEP or related phenotypes; however, we cannot exclude the possibility that regulatory NR2E1
or SNX3 mutations or deletions at this locus may underlie abnormal human cortical development
in some patients.
Published: 26 July 2007
BMC Medical Genetics 2007, 8:48 doi:10.1186/1471-2350-8-48
Received: 13 February 2007
Accepted: 26 July 2007
This article is available from: http://www.biomedcentral.com/1471-2350/8/48
© 2007 Kumar 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.

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Background
The MMEP phenotype and EEC syndrome represent syn-
dromic forms of split-hand/foot malformation (SHFM),
which occurs either as an isolated malformation or as a
feature of many other conditions with overlapping clini-
cal findings [1]. SHFM can be caused by chromosome
rearrangements or single gene mutations and is a geneti-
cally heterogeneous malformation with multiple gene loci
having been identified [1].
Sorting nexin 3 (SNX3) is ubiquitously expressed and
belongs to the sorting nexin family, which is involved in
intracellular protein trafficking [2]. Previously, SNX3 was
found to be disrupted in its 3rd intron by a de novo bal-
anced translocation t(6;13)(q21;q12) in a patient with
MMEP (microcephaly, microphthalmia, ectrodactyly, and
prognathism) and severe mental retardation [3]. Thus,
SNX3 was initially proposed as a reasonable candidate for
MMEP in this patient, who did not harbor any SNX3
mutations on the normal chromosome [3]. However,
mutations involving SNX3 were not identified in a spo-
radic case with possible MMEP and normal karyotype
[3,4]. Thus, mutations in a gene close to SNX3 may con-
tribute to MMEP and related phenotypes.
Nuclear receptor 2E1 (NR2E1; previously, TLX [MIM
603849]) is the closest gene to SNX3 (Figure 1) and there-
fore represents a strong positional candidate that may
contribute to the brain phenotype of MMEP. NR2E1 is
also a strong functional candidate, given that mice deleted
for Nr2e1 present with a complex MMEP-related pheno-
type that includes forebrain hypoplasia, eye abnormali-
ties, and cognitive impairment [5-7], which is consistent
with the brain and eye expression pattern of this gene
[8,9]. Further evidence is supported by the possibility that
the der(6) breakpoint in the t(6;13)(q21;q12) patient
could have created a position effect that altered the expres-
sion of NR2E1 resulting in the MMEP phenotype (Figure
1) [3]. Such a hypothesis, however, is difficult to test in
light of the predominantly brain-specific transcription of
NR2E1 [8,10] and lack of suitable patient material.
We propose that a gene(s) near the 6q21 translocation
may underlie MMEP and related phenotypes in some
patients. Here, we test the hypothesis that patients with
MMEP or a related phenotype may harbor mutations in
NR2E1 and/or SNX3. To test this hypothesis, we
sequenced the entire NR2E1 coding region, consensus
splice-site regions, complete 5' and 3' untranslated
regions and evolutionarily conserved elements including
core and proximal promoter in one patient with MMEP,
three patients with possible MMEP or related phenotypes,
and one patient with oligodactyly, ulnar aplasia, and a
t(6;7)(q21;q31.2) translocation [11]. We also sequenced
the complete coding regions of SNX3 in three of five
patients not previously examined for this gene.
Methods
Human subjects
Approval for this study was obtained from The University
of British Columbia and Child & Family Research Insti-
tute. The research followed Canada's Tri-Council State-
ment on 'Ethical Conduct for Research Involving
Humans'. Approval was also obtained through the Institu-
tional Review Board of Self Regional Healthcare (Green-
wood, SC). Consent from all patients was obtained for
research purposes. Patients were ascertained and exam-
ined from five centers: The University of the Witwater-
strand, Johannesburg; The University of Cape Town,
South Africa; Cedars-Sinai Medical Center, Los Angeles;
Universita Cattolica, Rome; and The University of Califor-
nia, San Francisco. Patients were referred to the Green-
wood Genetic Center for molecular research studies on
split-hand/foot malformation. Controls were ascertained
from the Greenwood Genetic Center.
All cases were sporadic and born to non-consanguineous
parents. Patient 1 is the original patient with MMEP and
t(6;13)(q21;q12) described previously by Viljoen and
Smart [12]. This is a 44-year-old Caucasian female with
severe mental retardation, congenital microphthalmia
causing complete blindness, central cleft lip and palate,
ectrodactyly with absence of toes 2–4 on both feet, finger-
like thumbs, and a broad, prominent jaw.
Patients 2–4 had normal blood chromosome analyses
and were included in this study on the basis of having
clinical features that significantly overlapped those of the
MMEP phenotype. Patient 2 was felt to have possible
MMEP versus an unusual variant of the ectrodactyly-ecto-
dermal dysplasia-clefting (EEC) syndrome and has not
Schematic drawing of 6q21 demonstrates relative positions of genes flanking SNX3Figure 1
Schematic drawing of 6q21 demonstrates relative
positions of genes flanking SNX3. Green bar indicates
location of breakpoint within intron 3 of SNX3 in patient
with t(6;13)(q21;q12) translocation [3]. Red arrows indicate
direction of transcription. Genetic locations of transcrip-
tional start and end sites are from RefSeq genomic assembly
NC_000006.10.
6q21
NR2E1 SNX3 LACE1
108,593,955
108,616,706
108,639,119
108,689,157
108,722,791
108,950,951
to centromere

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been described previously. She is a Hispanic female with
congenital microcephaly, bilateral microphthalmia, colo-
bomas of the right iris and retina, left chorioretinal colo-
boma, left lacrimal duct stenosis, unilateral cleft lip and
palate, dysplastic ears, soft tissue syndactyly of fingers 3–
4 on the right hand, ectrodactyly of the left foot with a hal-
lux and two digital rays, unilateral mixed versus sen-
sorineural hearing loss requiring a hearing aid, sparse
scalp hair and eyebrows, and narrow, deep-set nails. She
had a prolonged hospitalization after birth and required
gastrostomy tube placement. She had no evidence of a
TP73L (previously, TP63, P63) mutation, which is known
to be involved in split hand/foot malformation [13].
Patients 3 and 4 were felt to have an unusual variant of the
EEC syndrome. A brief summary of their clinical findings
was reported previously [13]. Patient 3 is a 10-year-old
Caucasian female with microcephaly, bilateral iris colo-
bomas, microphthalmia with significant vision impair-
ment, bilateral ectrodactyly of the hands and feet,
unilateral cleft lip, and patchy alopecia of the scalp hair.
She required gastrostomy feedings until age 9 due to
ongoing problems with feeding, failure to thrive, and
severe gastroesophageal reflux. A gastric emptying study
revealed delayed emptying with a non-functioning sec-
tion of the stomach and reverse peristalsis, which resolved
with gastric Botox therapy. She has experienced significant
dental problems due to ectodermal dysplasia. She attends
a regular school program with visual assistance. Patient 4
is an African-American female seen at 3 months of age
with congenital microcephaly, congenitally sealed eyelids
with small to absent globes, ectrodactyly of the hands and
feet, a notch in the upper lip resembling a mild midline
cleft, absent scalp hair, underdeveloped eyelashes and
eyebrows, underdeveloped nails, minor differences in ear
shape, unilateral hearing loss, pelvic kidney, anteriorly
placed anus, and tethered spinal cord. Both patients had
no evidence of a TP73L mutation [13].
Patient 5 was included in the study on the basis of having
a de novo chromosome translocation involving the 6q21
region (t(6;7)(q21;q31.2)) and congenital ulnar ray apla-
sia. His findings were described previously by Gurrieri et
al. [11]. He is a Caucasian male evaluated in the newborn
period with congenitally bowed radii, absent ulnae,
absence of fingers 3–5 on the right hand, syndactyly of
fingers 2–3 and absence of fingers 4–5 on the left hand,
and a cyst of the septum pellucidum. He did not have
microcephaly, ocular abnormalities, or other features of
the MMEP phenotype. We cannot exclude the possibility
that breakage at 7q31 may disrupt a gene(s) involved in
MMEP.
DNA amplification and sequencing
Sequencing was used to screen for SNX3 coding muta-
tions in all 4 exons as previously described [3]. We
sequenced genomic NR2E1 using 20 PCR amplicons that
covered the coding regions (1,146 bp), complete 5' and 3'
UTRs (1,973 bp), exon-flanking regions including con-
sensus splice-sites (1,719 bp), and evolutionarily con-
served regions including the core and proximal promoter
(1,528 bp). Polymerase chain reactions (PCR) and
sequencing were performed as previously described [14].
Sequences were visually inspected and scored blindly by
at least two individuals using either Consed [15] or
Sequencher (Gene Codes, Ann Arbor, MI). Every variant
identified was confirmed by repeating the PCR and
sequencing process. Confirmation of the g.21502T>C
change in patient 2 and genotyping of unaffected parents
and 250 controls without MMEP or related phenotypes
was performed by restriction enzyme digestion of a 505-
base pair PCR product using BsmBI. Primers and PCR con-
ditions were as previously reported for 3' UTRb [14]. The
alteration created an additional BsmBI site, producing
bands of 505, 278, and 227 base pairs in the heterozygous
state.
Results and discussion
Absence of SNX3 coding mutations in MMEP or related
phenotypes
To exclude the role of coding mutations in SNX3 in
MMEP or related phenotypes, we sequenced all four cod-
ing exons in all patients not previously studied for this
gene. No mutations were detected. The absence of coding
mutations in SNX3 supports the role of other loci, such as
NR2E1, in these disorders. We cannot, however, exclude
the role of regulatory SNX3 mutations given that pro-
moter and UTRs were not examined.
Absence of NR2E1 coding mutations in MMEP or related
phenotypes
To determine whether patients with MMEP or related phe-
notypes harbor NR2E1 mutations, we sequenced the com-
plete coding region, complete 5' and 3' UTR, consensus
splice-sites, and evolutionarily conserved regions includ-
ing the core and proximal promoter in all five patients.
We generated approximately 33.5 kb of sequence data. We
did not detect any synonymous nor nonsynonymous cod-
ing mutations.
We identified one candidate regulatory mutation in the 3'
UTR, g.21502T>C, in patient 2, that was not found in
dbSNP (Build 127) [16]. We confirmed this variant by
resequencing both strands of DNA. The variant was not
predicted to alter binding of neural transcription factors
[14]. We genotyped g.21502T>C in the unaffected parents
and identified the candidate regulatory mutation in the
mother but not the father (Figure 2). In addition, we did

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not find g.21502T>C in 500 chromosomes from approxi-
mately 250 controls without MMEP or related pheno-
types. Interestingly, we previously reported the
g.21502T>C candidate regulatory mutation (in addition
to two other mutations) in a patient with microcephaly
and the unaffected father but not in 344 control chromo-
somes nor in 188 ethnically-diverse chromosomes [14].
Thus, g.21502T>C has so far been identified only in fam-
ilies that present with microcephaly but not in 1032 con-
trol chromosomes from subjects that do not present with
microcephaly.
One possibility is that the g.21502 T>C is an innocuous
substitution that does not contribute to disease but rather
represents a rare variant in the general population. An
alternative interpretation is that heterozygous NR2E1
mutations may contribute to disease, which is supported
by studies in mice heterozygous for Nr2e1 deletions that
show premature cortical neurogenesis early in develop-
ment [17], thereby suggesting dosage sensitivity for
NR2E1. The presence of g.21502T>C in an unaffected par-
ent may be due to incomplete penetrance of this variant.
Alternatively, g.21502T>C may interact with another sus-
ceptibility variant arising de novo in the patient or inher-
ited from the non-g.21502T>C-transmitting parent. Such
a mechanism is supported by genetic studies in mice in
which double heterozygous mutations at Nr2e1 and Pax6
are shown to enhance cortical phenotypes [18]. It is
important to note that the clinical phenotypes of the
patients in this study may be attributable to different
genetic mechanisms affecting similar developmental
pathways.
Conclusion
MMEP and related phenotypes represent a spectrum of
heterogeneous conditions for which multiple loci may be
involved, including NR2E1 and SNX3 on Chromosome
6q21–22 [11,12]. The present study does not support
involvement of NR2E1 or SNX3 coding mutations in
MMEP or related phenotypes. However, we cannot
exclude the possibility that regulatory NR2E1 or SNX3
mutations, such as g.21502T>C of NR2E1, may underlie
abnormal human cortical development in some families.
In addition, we cannot exclude the possibility that dele-
tions at NR2E1 or SNX3 may underlie MMEP, given that
sequencing is unable to distinguish between homozygos-
ity across loci versus large deletions. The lack of obvious
mutations in NR2E1 and SNX3 contribute to the genetic
complexity underlying this heterogeneous syndrome. Fol-
low-up studies of other positional candidates such as
LACE1, would be a next logical undertaking.
Competing interests
The author(s) declare that they have no competing inter-
ests.
Authors' contributions
RAK conducted the NR2E1 molecular genetic studies, per-
formed data analysis, and wrote the manuscript. DBE, AS,
and CES collected families and provided DNA. CTM car-
ried out the SNX3 sequencing and contributed to NR2E1
genotyping. EMS initiated the study and finalized the
analyses as well as the paper. All authors read and
approved the final manuscript.
Acknowledgements
The authors acknowledge the patients and families who donated their time
and blood samples. We thank Dr. Denis L. Viljoen (University of the Wit-
waterstrand, Johannesburg), Dr. Ronald Smart (University of Cape Town,
South Africa), Dr. John M. Graham, Jr. (Cedars-Sinai Medical Center, Los
Angeles), and Dr. Fiorella Gurrieri (Universita Cattolica, Rome) for provid-
ing us with patient samples. We also thank Tonya Moss for assistance in
sequence analyses of SNX3. The authors are grateful to Brett S. Abrahams
and Tracey D. Weir (Centre for Molecular Medicine and Therapeutics,
Canada) for insight and helpful comments on the manuscript. This work
was supported in part by grants from Jack and Doris Brown Foundation and
British Columbia Institute for Children's & Women's Health (to RAK);
Shriners Hospitals for Children, Grant #8510 (to DBE); South Carolina
Department of Disabilities and Special Needs (SCDDSN) (to CES); and
Canadian Institutes for Health Research (CIHR), CIHR Research and
Development, and Canada Research Chair in Genetics and Behaviour (to
EMS).
Genotyping NR2E1 candidate regulatory mutation in patient 2 and family membersFigure 2
Genotyping NR2E1 candidate regulatory mutation in
patient 2 and family members. A) DNA sequencing
detects g.21502T>C in the 3' UTR of patient 2 but not in
unrelated controls. B) Restriction digest detects g.21502T>C
in the unaffected mother but not in the unaffected father nor
in an unrelated control.
G A T/C G A G A T G A
A
B
Patient 2
Mother
Father
Control
Ladder
Patient 2 Control

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