Download Highly Recurrent RET Mutations and Novel Mutations in

Clinical Chemistry 50:1
93–100 (2004)
Molecular Diagnostics
and Genetics
Highly Recurrent RET Mutations and Novel
Mutations in Genes of the Receptor Tyrosine
Kinase and Endothelin Receptor B Pathways
in Chinese Patients with Sporadic
Hirschsprung Disease
Mercè Garcia-Barceló,1,2 Mai-Har Sham,2 Wing-Shan Lee,1 Vincent Chi-Hang Lui,1
Benedict Ling-Sze Chen,1 Kenneth Kak-Yuen Wong,1 Joyce Suet-Wan Wong,1 and
Paul Kwong-Hang Tam1,3*
Background: Hirschsprung disease (HSCR) is a congenital disorder characterized by an absence of ganglion
cells in the nerve plexuses of the lower digestive tract.
HSCR has a complex pattern of inheritance and is
sometimes associated with mutations in genes of the
receptor tyrosine kinase (RET) and endothelin receptor
B (EDNRB) signaling pathways, which are crucial for
development of the enteric nervous system.
Methods: Using PCR amplification and direct sequencing, we screened for mutations and polymorphisms in
the coding regions and intron/exon boundaries of the
RET, GDNF, EDNRB, and EDN3 genes of 84 HSCR
patients and 96 ethnically matched controls.
Results: We identified 10 novel and 2 previously described mutations in RET, and 4 and 2 novel mutations
in EDNRB and in EDN3, respectively. Potential diseasecausing mutations were detected in 24% of the patients.
The overall mutation rate was 41% in females and 19%
in males (P ⴝ 0.06). RET mutations occurred in 19% of
the patients. R114H in RET was the most prevalent
mutation, representing 7% of the patients or 37% of the
patients with RET mutations. To date, such a high
frequency of a single mutation has never been reported
in unrelated HSCR patients. Mutations in EDNRB,
EDN3, and GDNF were found in four, two, and none of
the patients, respectively. Two patients with mutations
in genes of the EDNRB pathway also harbored a mutation in RET. Three novel and three reported polymorphisms were found in EDNRB, EDN3, and GDNF.
Conclusion: This study identifies additional HSCR disease-causing mutations, some peculiar to the Chinese
population, and represents the first comprehensive genetic analysis of sporadic HSCR disease in Chinese.
© 2004 American Association for Clinical Chemistry
Hirschsprung disease (HSCR)4 is a developmental disorder characterized by the absence of ganglion cells in the
nerve plexuses of the lower digestive tract. The HSCR
phenotype is variable and can be classified into two
groups: short-segment aganglionosis (SSA; 80% of cases),
which includes patients with aganglionosis as far as the
rectosigmoid junction; and long-segment aganglionosis
(LSA; 20% of cases), which includes patients with aganglionosis beyond the rectosigmoid junction (1 ). Aganglionosis is attributable to a disorder of the enteric nervous
system (ENS) in which ganglion cell precursors fail to
populate the lower gastrointestinal tract during embryonic development. The condition presents in the neonatal
period as a failure to pass meconium, chronic severe
1
Division of Paediatric Surgery, Department of Surgery, University of
Hong Kong Medical Center, Queen Mary Hospital, Hong Kong SAR, China.
2
Department of Biochemistry and 3 Genome Research Centre, The University of Hong Kong, Hong Kong SAR, China.
*Author for correspondence: Fax 852-2817-3155; e-mail paultam@hkucc.
hku.hk.
Received May 15, 2003; accepted October 6, 2003.
Previously published online at DOI: 10.1373/clinchem.2003.022061
4
Nonstandard abbreviations: HSCR, Hirschsprung disease; SSA, shortsegment aganglionosis; LSA, long-segment aganglionosis; ENS, enteric nervous system; RET, receptor tyrosine kinase; EDNRB, endothelin receptor B;
GDNF, glial cell-line-derived neurotrophic factor; EDN3, endothelin 3; TCA,
total colonic aganglionosis; CCHS, congenital central hypoventilation syndrome; CD, cadherin domain; TK, tyrosine kinase; and SNP, single-nucleotide
polymorphism.
93
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Garcia-Barceló et al.: Genetic Analysis of Chinese Patients with HSCR
constipation, colonic distention, secondary electrolyte disturbances, and sometimes, enterocolitis and bowel perforation (1 ). The estimated population incidence is 1 in 5000
live births, although this is a representative value. The
highest incidence is in Asian populations (2.8 per 10 000
live births), and the lowest is in Hispanics (1 per 10 000
live births) (1 ). The M:F ratio is ⬃4:1 for SSA-HSCR
patients and ⬃1:1 for LSA-HSCR patients (1 ). Approximately 20% of HSCR cases are familial. The recurrence
risk for siblings of SSA-HSCR probands varies from 1.5%
to 3.3%, whereas the risk for siblings of LSA-HSCR
probands varies from 3% to 18% (1 ). HSCR is frequently
associated with chromosomal abnormalities, with other
neurodevelopmental disorders, such as Waardenburg
syndrome type 4, and with a variety of additional isolated
anomalies and syndromes (1, 2 ).
HSCR has a complex genetic etiology, with many
studies indicating the receptor tyrosine kinase gene (RET)
as the major susceptibility gene for HSCR (1–14 ). Mutations in the RET gene account for up to 50% of familial
cases and 7%–35% of sporadic cases (5–14 ). Other HSCR
genes identified to date mainly code for protein members
of the RET and endothelin receptor B (EDNRB) signaling
pathways, which are interrelated and involved in the
development of enteric ganglia from specific lineage of
neural crest cells (15–27 ). Mutations in these genes account for a small proportion of HSCR patients (7%) and
have reduced penetrance and various effects on the length
of the aganglionosis (1, 2 ), suggesting that modifier genes
influence the penetrance and severity of the phenotype
(28, 29 ). Receptor–ligand relationships underlie the physiologic basis for some types of complex inheritance diseases, of which HSCR is one.
We analyzed germline mutations in genes encoding
protein members of the RET and EDNRB signaling pathways to investigate how mutations in those genes correlate with the manifestation of the disease in Chinese
HSCR patients. In addition to the RET gene, we investigated the gene coding for the glial cell-line-derived neurotrophic factor (RET ligand; GDNF), the EDNRB gene,
and the gene coding for its ligand, endothelin 3 (EDN3).
To our knowledge, this is the largest sample of HSCR
patients concomitantly studied for these genes and the
first comprehensive genetic analysis of HSCR in the
Chinese population.
Materials and Methods
patients and control samples
Eighty-four ethnic Chinese patients diagnosed with sporadic HSCR were included in this study. Diagnosis, based
on histologic examination of either biopsy or surgical
resection material for absence of enteric plexuses, was
made at Queen Mary Hospital, Hong Kong SAR, between
January 1984 and January 2003. Nine patients were affected with total colonic aganglionosis (TCA), 8 with LSA,
and 67 with SSA. Sixteen patients presented with the
following associated anomalies: Down syndrome (SSA;
n ⫽ 5; of whom 2 had severe conductive hearing loss);
Waardenburg–Shah syndrome with severe sensorineural
hearing loss (TCA; n ⫽ 1); renal agenesis (SSA; n ⫽ 1);
parathyroid adenoma (SSA; n ⫽ 1); parathyroid nodules
(TCA; n ⫽ 1); desmoid tumor (SSA; n ⫽ 1); congenital
central hypoventilation syndrome (CCHS; Ondine’s
curse; SSA; n ⫽ 1); slight mental retardation (SSA; n ⫽ 1);
Meckel diverticulum (SSA; n ⫽ 1); and sensorineural
hearing loss (n ⫽ 3; 1 SSA and 2 LSA).
Healthy controls (96 individuals) were unselected, unrelated, ethnic Chinese individuals. Patients and controls
assented to molecular analysis. The study was approved
by the Institutional Review Board of the University of
Hong Kong.
dna sequence analyses
DNA was extracted from peripheral blood by use of a
QIAamp Blood Kit (Qiagen). Unaffected parents were
also screened when available. Using PCR and direct
sequencing, we screened all exons of the RET, EDNRB,
EDN3, and GDNF genes, including intron/exon boundaries, for mutations and polymorphisms. The primers and
PCR conditions for amplification of the RET and EDN3
genes have been described previously (6, 12 ). For amplification of RET exon 21 and the EDNRB and GDNF genes,
we generated new pairs of primers (Table 1). PCR products were sequenced using an ABI PRISM® Big DyeTM
Terminator, Ver. 2.0, Cycle sequencing assay (Applied
Biosystems) and an ABI 3100 automated sequencer (Applied Biosystems). For those samples in which a DNA
sequence variation was observed, PCR amplification from
genomic DNA and sequencing using both forward and
reverse primers were repeated.
Results and Discussion
The RET and the EDNRB signaling pathways are critical
for the normal development of the ENS (18, 20, 21 ). We
analyzed the coding regions of the RET, GDNF, EDNRB,
and EDN3 genes in 84 Chinese patients with sporadic
HSCR. Twenty patients had at least one mutation in the
genes investigated, representing 24% of the total number
of HSCR patients studied. In total, we identified 16 novel
(10 in RET, 4 in EDNRB, and 2 in EDN3) and 2 previously
described mutations. Mutations in RET were found in
19% (16 of 84) of the patients, whereas mutations in
EDNRB, EDN3, and GDNF were found in 4, 2, and none
of the 84 patients, respectively. In total, eight mutations
could be confirmed to be de novo. The mutations identified and their distributions in the patients are summarized in Table 2. Of 16 patients with associated anomalies,
only 4 had mutations in the genes investigated. The
distribution of the patients according to gender, mutation
status, and length of aganglionosis is depicted in Table 3.
On the basis of previous reports (29 ), we defined a
sequence alteration as a mutation if it failed to occur in 96
ethnically matched controls. Otherwise it was termed a
polymorphism.
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Clinical Chemistry 50, No. 1, 2004
Table 1. Primers and PCR conditions for amplification of exon 21 of the RET gene and intron/exon boundaries of the
EDNRB and GDNF genes.
RET-21Fa
RET-21R
EDNRB-1Fa
EDNRB-1R
EDNRB-2Fb
EDNRB-3Ra
EDNRB-4F
EDNRB-4Ra
EDNRB-5Fa
EDNRB-5R
EDNRB-6Fa
EDNRB-6R
EDNRB-7Fa
EDNRB-7R
GDNF-1Fa
GDNF-1R
GDNF-2Fa
GDNF-2R
a
b
Primers
Product size, bp
5⬘-AAAGGGAGTTTTGCCAAGGCC-3⬘
5⬘-TTTAAGTCTGAAGAGCAGGC-3⬘
5⬘-ATTAGCGTTTGCAGCGACTT-3⬘
5⬘-CTCAAGCCCACCATGATTTC-3⬘
5⬘-GTGATACAATTCAGAGGGCATC-3⬘
5⬘-GGGAACAGGGGAAAAATAGC-3⬘
5⬘-TAATCATTCCCTGATGAATTTTT-3⬘
5⬘-AAATTCAACCACGAGTTATCAAA-3⬘
5⬘-TGCTATGAGTAAAATGAGCCATC-3⬘
5⬘-TCGATGGAAACACTTCTGAGT-3⬘
5⬘-GCACAGAAGCTACAATGACTACA-3⬘
5⬘-AGCAGTTTTGAAAGCTTATATTTGA-3⬘
5⬘-AAGAGTTGGGAAAGGTGACTGA-3⬘
5⬘-TGTTTTAATGACTTCGGTCCAA-3⬘
5⬘-CAGGCTTAACGTGCATTCTGC-3⬘
5⬘-GCTGGCTTGGGGTACGTGC-3⬘
5⬘-GGTCCTATAGCTTAATCGGCTG-3⬘
5⬘-TCTTTGCACTGTAGCAGGAATGC-3⬘
157
1.5 mM MgCl2; Tm ⫽ 60 oC
Conditions
645
1.5 mM MgCl2; Tm ⫽ 56 oC
590
1.5 mM MgCl2; Tm ⫽ 56 oC
287
2 mM MgCl2; Tm ⫽ 56 oC
250
1.5 mM MgCl2; Tm ⫽ 56 oC
250
2 mM MgCl2; Tm ⫽ 56 oC
252
2 mM MgCl2; Tm ⫽ 56 oC
303
1.5 mM MgCl2 ⫹50 mL/L dimethylsulfoxide; Tm ⫽ 60 oC
649
1.5 mM MgCl2; Tm ⫽ 56 oC
Sequencing primer.
Exons 2 and 3 in the same amplicon.
sequence alterations in ret and gdnf
In the RET gene we identified 10 novel as well as 2
previously described mutations (Fig. 1). The new mutations consisted of two point deletions, two small deletions, and six nucleotide substitutions [5⬘UT ⫺37G⬎C,
c360 C⬎T (T120T), c434 T⬎G (V145G), c2081 G⬎A
(R694Q), c2862 G⬎A (G954G), and c2881 T⬎C (F961L)].
Seven of 12 (58%) of the RET mutations were de novo,
which reaffirmed the pathogenic role of RET in sporadic
HSCR.
We predict that the four novel deletions found in this
study are all probably disease-causing mutations. Both
point deletions, c1449delC (Y483X) in the cadherin domain (CD) and c1908delG (V636fsX1) in the cysteine
domain, lead to stop codons. In these cases, the mRNA
transcripts are likely to be degraded by nonsense-mediated mRNA decay surveillance mechanisms (30 ) and, if
translated, would lead to an altered or truncated RET
protein. The 10-nucleotide deletion c1685_1690 ⫹ 4del
abolishes the splice site between exon and intron 8,
probably leading to an abnormally spliced mRNA. The
36-nucleotide deletion IVS11 ⫹ 15 (15_36del), starting at
nucleotide ⫹15 of intron 11 and spanning to nucleotide
⫹36, probably affects intronic cryptic regulatory sites.
We found two missense mutations in the extracellular
domain of the RET protein, the novel V145G and R114H.
Generally, HSCR missense mutations located in the extracellular domain would affect the folding of the RET
protein, impairing its maturation and preventing it from
reaching the cell surface (31 ). V145 is a conserved residue
of the prototypical strand of the cadherin fold in CD1 (Fig.
1) and part of a hydrophobic core crucial to the structure
of the protein (32 ). R114H was the most prevalent mutation in our series (observed in six patients). R114H was
described recently (33 ) in one Japanese patient with
isolated CCHS, a condition that has an incidence of 1.9%
in HSCR (34 ). In this study, none of the HSCR patients
with R114H presented with CCHS features. The only
HSCR patient with CCHS (patient 97) had a G⬎C change
at nucleotide ⫺37 of the 5⬘-untranslated region (5⬘UT
⫺37G⬎C). Because R114H has been found only in Asian
patients, it would seem that R114H is a founder mutation
contributing to HSCR in Asia.
The other novel missense mutations that we found
(R694Q and F961L) are located in the intracellular domain. R694Q is in a transition zone between the transmembrane domain and the tyrosine kinase (TK) domain,
whereas F961L is in the TK domain. HSCR mutations in
the TK domain usually cause impairment of the kinase
activity (31 ). Even mutations affecting the nonconserved
amino acids of the TK domain may partially impair the
kinase activity (35 ).
We observed the R982C change in three patients (patients 9, 55, and 92) but not in the 96 controls. R982C has
been debated widely (36, 37 ); it was initially described as
a mutation but later was observed in control chromosomes (11, 38 ). Interestingly, patient 55, who inherited
R982C from her mother, also had two de novo silent
mutations, T120T and G945G, both novel. The fact that
both mutations are de novo, both are in the same patient,
and neither was found in the controls could indicate that
they contribute to the disease, either independently, combined, or having a joint effect with R982C. Other silent
mutations are known to alter mRNA processing, and in
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Garcia-Barceló et al.: Genetic Analysis of Chinese Patients with HSCR
Table 2. Distribution of the mutations found in the 84 sporadic HSCR patients analyzed.a
Patients
Phenotype/Gender
22b
66
83
99A
13 patients
1b
6
17
19b
25
26
32
36
42
55
59
61
72
74
93
97b
51 patients
LSA/F
TCA/F
TCA/F
TCA/M
7 LSA and 6 TCA
SSA/M
SSA/M
SSA/M
SSA/F
SSA/M
SSA/M
SSA/M
SSA/M
SSA/F
SSA/F
SSA/M
SSA/M
SSA/M
SSA/M
SSA/F
SSA/M
SSA
RET
EDNRB
R114Hc
V145Gd (new)
c1685_1690 ⫹ 4deld (new)
V636fsX1d (new)
IVS11 ⫹ 15(15_36del)f (new)
R114Hg
M1064Tg
R114He
F961Ld (new)
Y483Xd (new)
R694Qe (new)
R114He
EDN3
GDNF
E48De (new)
N426Nc (new)
p.P383_L386delinsPf (new)
d
d
T120T (new)/G954G (new)
D241Dc (new)
IVS4 ⫺14T⬎Ce (new)
5⬘UT ⫺19C⬎Ad (new)
g
R114H
R114Hf
5⬘UT ⫺37G⬎Ce (new)
a
All mutations found were in heterozygous state. Blanks indicate that no mutations were found; (new) indicates a mutation never before described.
HSCR-associated anomalies: patient 1, slight mental retardation; patient 19, Down syndrome and severe bilateral conductive hearing loss; patient 22,
sensorineural hearing loss; patient 97, Ondine’s curse.
c
Inherited from unaffected mother.
d
De novo mutation.
e
Inherited from unaffected father.
f
Parental DNA not available.
g
Paternal DNA not available and maternal DNA negative for the mutation.
b
the RET gene in particular, the silent mutation I647I,
interferes with normal transcription, leading to decreased
protein concentrations (39 ).
The M1064T mutation in exon 20 was found in a male
affected with SSA. DNA from the father was not available,
and analysis of maternal DNA was negative for the
mutation (see Table 2). The M1064T mutation was first
described in a HSCR patient with SSA who had inherited
it from an unaffected parent. Because this mutation is
Table 3. Classification of the 84 sporadic HSCR patients
according to gender, mutation status, and length of
the aganglionosis.
Total (n ⫽ 84; M:F ⫽ 3.94)
Males (n ⫽ 67)
Females (n ⫽ 17)
SSA (n ⫽ 67; M:F ⫽ 5.09)
Males (n ⫽ 56)
Females (n ⫽ 11)
LSA (n ⫽ 17; M:F ⫽ 1.83)
Males (n ⫽ 11)
Females (n ⫽ 6)
Patients with mutation/s
in the HSCR genes
investigated, n (%)
Patients with
mutations
in RET, n (%)
20/84 (24)
13/67 (19)
7/17 (41)
16/67 (24)
12/56 (21)
4/11 (36)
4/17 (24)
1/11 (9.1)
3/6 (50)
16/84 (19)
10/67 (15)
6/17 (35)
12/67 (18)
9/56 (16)
3/56 (5.4)
4/17 (24)
1/11 (9.1)
3/6 (50)
specific to the isoform RET51, it was suggested that this
isoform was essential for normal enteric development (7 ).
Functional analyses of M1064 failed to demonstrate significant changes in the activity of RET (31 ), and experiments in transgenic mice indicated that RET51 is not
required for the development of the ENS (40 ). However,
based on the demonstration of the formation of homoand heterodimers between RET9 and RET51, it has recently been suggested that there may be a critical role for
the RET51:RET9 ratio in addition to that of the absolute
amounts of the two isoforms (41 ).
A detailed analysis of the RET single-nucleotide polymorphisms (SNPs) is reported elsewhere (42 ). In brief,
using case– control statistics and the Transmission Disequilibrium Test, we found that the variant alleles of both
A45A (c135G⬎A in exon 2) and L769L (c2307T⬎G in exon
14) were overrepresented in the patient population. Conversely, the variant allele of A432A (c1296G⬎A in exon 7),
was significantly underrepresented. The frequencies of
these associated alleles were significantly higher than
those reported in other populations, including those of the
controls, which may explain the higher incidence of HSCR
in Asians. As for RET haplotypes comprising the diseaseassociated SNPs, we found that 66% of the Chinese HSCR
patients were represented by one main haplotype: allele A
Clinical Chemistry 50, No. 1, 2004
97
Fig. 1. Schematic of the RET gene and HSCR
mutations identified in this study.
Exons of the RET gene are represented by numbered boxes. Introns are represented by solid bars.
Arrows represent the relative locations of the mutations in the RET gene. Correspondence of the RET
coding regions with the RET protein is also represented. The numbering of the amino acid residues
corresponds to the unprocessed protein, starting at
the initiator methionine residue. Numbers in parentheses indicate residues comprising each domain
of the RET protein. CD, cadherin domain; CYS,
cysteine-rich domain; TMD, transmembrane domain.
of A45A (c135G⬎A), allele G of A432A (c1296G⬎A), and
allele G of L769L (c2307T⬎G; A-G-G, P ⫽ 0.000002). This
association was independent of the RET mutational status
of the patients. Thus, this haplotype is either functional
itself, conferring susceptibility to HSCR and/or acting as
a modifier and contributing to the variable expression of
the phenotype, or represents linkage disequilibrium with
a susceptibility locus located in noncoding regions of RET.
This would explain the paucity of RET mutations and
their low penetrance and variability in the presentation of
the HSCR phenotype.
The only sequence alteration found in the GDNF gene
was the transition c278G⬎A in exon 2, which causes a
nonsynonymous change in the protein (R93Q). This
change was observed in one control and one SSA patient,
who was also affected with parathyroid adenoma. No
other mutations were found in this patient. Interestingly,
a nucleotide substitution, also affecting codon 93 (R93W),
was identified as a GDNF familial mutation leading to
HSCR in conjunction with a RET mutation (23 ). To date,
GDNF mutations have been found in five patients and are
a rare cause of HSCR. Four of those patients had additional contributory factors, such as mutations in RET
(23–26 ).
sequence alterations in ednrb and edn3
We found four novel mutations [p.P383_L386delinsP,
D241D (c723T⬎C), N426N (c1278T⬎C), and IVS4
⫺14T⬎C] in the EDNRB gene. The p.P383_L386delinsP
mutation encodes a 9-bp deletion (nucleotides c1149 –
1157) in exon 6, affecting the codons for proline-383,
isoleucine-384, alanine-385, and lysine-386 and substituting them for a proline. The result would be a shorter
protein lacking highly conserved residues of the seventh
transmembrane domain of the receptor. A missense mutation affecting the same codon (P383L) is known to be
responsible for the HSCR phenotype (17 ). Functional
analysis of P383L demonstrated that ligand-binding sites
were reduced and that signal transduction was impaired
(43 ). Thus, p.P383_L386delinsP could indeed lead to
HSCR. Interestingly, N426N was found in a patient who
also had a mutation in RET. Although D241D (exon 3),
N426N (exon 7), and IVS4 ⫺14T⬎C (intron 4) were not
found in the controls, their contribution to disease should
be taken with caution.
Three polymorphisms [5⬘UT ⫺26G⬎A, V185M
(c553G⬎A), and L277L (c831G⬎A)] in the 5⬘-untranslated
regions of exons 2 and 4, respectively, were also detected.
The nonsynonymous change V185M is new and was
found in two mutation-free SSA patients (maternal inheritance) and in one control. 5⬘UT ⫺26G⬎A, initially reported as a mutation (14, 17 ), has recently been reported
as a SNP (rs2070591) after a comprehensive study of SNPs
in a Japanese population (44 ). In our study, 5⬘UT
⫺26G⬎A was found in one control and two SSA patients,
one of whom (patient 61) also harbored IVS4 ⫺14T⬎C
(both inherited from the father). L277L [(c831G⬎A; dbSNP (rs5351)] was found in both the heterozygous and
homozygous states.
We found two novel mutations in the EDN3 gene: a
missense mutation (E48D) in exon 2 resulting from a G⬎C
transversion; and a de novo C⬎A transversion at nucleotide ⫺19 from the start codon (5⬘UT ⫺19C⬎A), which
could be affecting as yet unexplored regulatory regions.
E48D was found in patient 83, who also harbored a
deletion in RET and was affected with TCA.
Interestingly, we identified the G⬎A transition leading
to the A17T nonsynonymous change in 7% of the patients
(none syndromic) and in 5% of the controls. Remarkably,
one of the controls was homozygous for A17T. A17T was
previously identified as a mutation in a nonsyndromic
sporadic HSCR patient (22 ). To our knowledge, this is the
first time that A17T has been detected in a healthy
individual. A new polymorphism, (T insertion at nucleotide ⫺35 of exon 5; IVS4 ⫺35insT), was found in 9 patients
and 11 controls.
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Garcia-Barceló et al.: Genetic Analysis of Chinese Patients with HSCR
Mutations in EDNRB and EDN3 were scarce and were
mainly inherited from unaffected parents. It is worthwhile noting that at least two EDNRB and EDN3 mutations previously described in isolated HSCR (14, 17, 22 )
have recently been observed in healthy individuals of
different ethnic origins [(44 ) and this study]. Therefore,
either those “mutations” do not contribute to HSCR or if
they do, they require the contribution of modifiers, which
could be population- or/and individual-specific.
gender bias in the incidence of the disease
For the whole cohort, the sex ratio observed was 3.94 (67
males and 17 females). The ratio was increased in SSAHSCR (5.09) but decreased in LSA-HSCR patients (1.83).
These values are consistent with a decrease in gender bias
with an increase in the length of aganglionosis (1 ). Gender
bias indicates the presence of additional gender-specific
susceptibility/modifying locus (loci) (21 ). If the main
HSCR genes harbor more severe mutations (more frequent in LSA/TCA), the effect of gender-specific modifiers on the phenotype would be diminished, explaining
the diminished gender bias in sporadic LSA-HSCR.
female patients have a higher mutation rate
than male counterparts
Remarkably, mutations were observed in 41% (7 of 17) of
the female patients and only in 19% (13 of 67) of the male
patients (Table 3). Although the number of female patients is too small for significant statistical analysis, to our
knowledge, this observation has never been reported.
genotype–phenotype correlation
Lack of genotype–phenotype correlation is a feature common to all mutations in HSCR genes. In our series, this is
exemplified by R114H of the RET gene. Of six patients
with R114H, five were affected with SSA and one with
LSA. Analysis of parental DNA showed that this mutation could be inherited from either the unaffected mother
or father. Remarkably, R114H is highly recurrent in our
series, representing 7% (6 of 84) of the patients or 30% (6
of 20) of the patients with mutations in any of the genes,
or 37% (6 of 16) of the patients with mutations in RET. To
date, no such frequency has been reported for any of the
RET mutations.
Of 17 LSA/TCA patients, 4 (24%) had potentially
HSCR-causing mutations in RET (including patient 83,
who also had E48D in EDN3). No mutations were found
in the other genes.
Of 67 SSA patients, 16 (24%) had mutations either in
RET (18%; 12 of 67) or in the other genes (Table 3). The
percentage of LSA/TCA patients harboring mutations
was quite similar to that of SSA patients with mutations,
probably because EDNRB mutations are more frequently
associated with SSA (1 ). RET mutations alone were
slightly more frequent in LSA/TCA patients (see Table 3).
More striking differences in mutation rates between LSA-
and SSA-HSCR patients have been documented in other
series of individuals (9 ).
The most important issue, however, is that 76% of the
patients had no mutation(s) in the genes investigated. It is
worth mentioning patient 38, who also had aganglionosis
in the small bowel. For this patient, the possibility of a
hemizygous deletion of RET cannot be ruled out because
analysis of RET polymorphisms showed a homozygous
state for each site. For the rest of the patients with no
mutations in RET, heterozygosity was observed for several SNPs distributed throughout the gene.
The overall paucity of mutations in HSCR patients
indicates that mutations exist in other as yet undiscovered
genes and/or in noncoding regions of the HSCR genes
identified. In addition, the fact that mutations leading to
disease are inherited from unaffected parents indicates
that the HSCR phenotype results from interaction of
several genes and/or modifiers, as has been demonstrated with genes of the RET and EDNRB pathways
(20, 21, 28, 29 ). In our study, only two patients had mutations in both RET and genes of the EDNRB pathway.
Interestingly, in those two patients (patients 25 and 83),
RET mutations were de novo, and the mutations in the
genes of the EDNRB pathway were inherited from unaffected parents. Whether E48D in EDN3 and N426N in
EDNRB, contribute to the phenotype in combination with
the mutations in RET is not known, but this certainly adds
force to the interaction between pathways (18 –21 ). Mutations in GDNF and EDNRB have been found previously in
the context of mutations in the RET locus (23, 38, 39 ).
Even specific EDNRB mutations are known to require
specific RET polymorphisms for transmission (20, 28 ).
Little is known about the signaling events governing
the fate of the enteric neurons during development. Each
of the genes coding for protein components of the RET
and EDNRB signaling pathways is to be considered a
HSCR candidate gene, contributing to HSCR on its own or
in combination. In patients without mutations, even polymorphisms in two or more loci of the RET and EDNRB
pathway genes could produce a simultaneous decrease in
protein dosage (39, 41 ). We have recently demonstrated
that PHOX2B (45 ), a gene encoding a transcriptional
activator necessary for the expression of RET, is also
associated with HSCR. Although the molecular mechanism underlying this association remains unclear, our
finding indicated the contribution of PHOX2B in the
enteric neuronal development. Recently, mutations in
PHOX2B have been found in patients with isolated CCHS
as well as in patients with CCHS and HSCR (46 ).
In oligogenic diseases such as HSCR, with a complex
mode of inheritance, functional analyses of mutations
have their limitations. A better understanding of the
molecular mechanisms of oligogenicity through the study
of the interactions between a discrete number of loci could
help to make genotype-based phenotypic predictions.
Clinical Chemistry 50, No. 1, 2004
We thank everyone who participated in the study. This
work was supported by research grants from the Hong
Kong Research Grants Council (HKU 7358/00M).
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