doi:10.1086/302329


1216 Letters to the Editor

Table 1

RET and EDNRB Mutations in 50 Male Patients with Isolated HSCR

Patient Number Genea Exon
Nucleotide

Change
Amino Acid

Change
Type of

Case
Length of

Aganglionosis

HSCR20 EDNRB 4 G1151A S305N Sporadic Short
HSCR18 EDNRB 6 del1369A N378I Familial Short
HSCR46 RET 10 C1876A Q626K Sporadic Ultrashort
HSCR20 RET 11 C1941T I647I Sporadic Short
HSCR25 RET 11 G1947A S649S Sporadic Short
HSCR40 RET 14 G2438A R813Q Sporadic Short
HSCR01 RET 14 G2607�5A Sporadic Long

a For the RET, EDNRB, and EDN3 genes, primer sequences and PCR-SSCP conditions have
been described elsewhere (Ceccherini et al. 1994; Auricchio et al. 1996). To screen the GDNF
gene we used both SSCP (for exon 1) and denaturing gradient gel electrophoresis (DGGE) (for
exon 2) with the following primer sets: 1F/R (5′-AGGCTTAACGTGCATTCTG-3′ and 5′-
GGGAACGGTTCTTACAGT-3′), 2AF/R (5′-30-bp GC clamp-GATCATTTTTGTCTCATG-
TGCCA-3′ and 5′-TCCTCTAATTCTCTGGGT-3′), 2BF/R (5′-GAGCGGAATCGGCAGGCTG-3′

and 5′-30-bp GC clamp-CAAGAGCCGCTGCAGTACCT-3′) and 2CF/R (5′-CTTGGGT-
CTGGGCTATGAA-3′ and 5′-30-bp GC clamp-GCAATACACAGCAGTCTCTG-3′). PCR anneal-
ing temperatures were 57�C–63�C. SSCP was done as reported elsewhere (Ceccherini et al. 1994),
whereas a 20%–70% denaturing gradient in 1#TEA buffer at 160 V constant for 3.5–4.5 h was
applied for DGGE analysis.

Am. J. Hum. Genet. 64:1216–1221, 1999

Double Heterozygosity for a RET Substitution
Interfering with Splicing and an EDNRB Missense
Mutation in Hirschsprung Disease

To the Editor:
Hirschsprung disease (HSCR [MIM 142623]) is a de-
velopmental disorder resulting from the arrest of the
craniocaudal migration of enteric neurons from the neu-
ral crest along gastrointestinal segments of variable
length (Behrman 1992). The involvement of the Ret
proto-oncogene and the endothelin-B receptor-mediated
signaling pathways in the migration and differentiation
of enteric ganglion cells has been demonstrated in both
humans and mice.

Heterozygous, incompletely penetrant point muta-
tions and deletions of the RET proto-oncogene have
been described in sporadic and familial cases of HSCR
(Edery et al. 1994; Romeo et al. 1994; Angrist et al.
1995). In addition, homozygous RET-targeted disrup-
tion in mice results in megacolon with renal abnormal-
ities (Schuchardt et al. 1994). This phenotype is remi-
niscent of the knockout for GDNF, encoding for the glial
cell-line–derived neurotrophic growth factor (Moore et
al. 1996; Pichel et al. 1996; Sanchez et al. 1996), a pro-
tein that has been demonstrated to bind specifically and
to activate Ret with a glycophosphatidylinositol (GPI)-
anchored protein, the GDNF receptor-a (GFRA1) (Jing
et al. 1996; Treanor et al. 1996). Although no nucleotide
changes have been found at the GFRA1 locus (Angrist
et al. 1998; Myers et al. 1998), rare heterozygous mu-
tations of the GDNF gene, in some cases in combination

with RET mutations, have been detected in a few pa-
tients (Angrist et al. 1996; Ivanchuk et al. 1996; Salomon
et al. 1996). Similarly, a missense mutation in the neur-
turin (NTN) gene, a component of a second RET bind-
ing complex including the NTN receptor (also known
as GDNFR-a2, GDNFR-b, or GFRA2), has been found
in an HSCR family cosegregating with a RET missense
mutation (Doray et al. 1998). Rarely, megacolon pres-
ents in association with pigmentary anomalies in the
Shah-Waardenburg syndrome, a human autosomal re-
cessive disease similar to the mouse piebald lethal and
lethal spotting spontaneous mutants. The study of these
models of syndromic megacolon has led to the identi-
fication of homozygous mutations in the endothelin-B
receptor gene (EDNRB) and the endothelin 3 (EDN3)
gene in both humans and mice (Baynash et al. 1994;
Hosoda et al. 1994; Puffenberger et al. 1994; Attié et
al. 1995). In particular, in a large Mennonite pedigree,
a founder homozygous W276C EDNRB mutation has
been found in association with a specific RET haplotype,
thus suggesting a possible genetic interaction between
the two genes in the Mennonites affected with the Shah-
Waardenburg syndrome (Puffenberger et al. 1994).
EDNRB and EDN3, respectively, encode the endothelin-
B receptor, a G-protein–coupled receptor with seven TM
domains, and its ligand endothelin-3. We and others
have demonstrated heterozygous incompletely penetrant
mutations in the EDNRB gene in individuals with iso-
lated megacolon (Amiel et al. 1996; Auricchio et al.
1996; Kusafuka et al. 1996), although mutations at the
EDN3 locus are rarely found in HSCR (Bidaud et al.
1997). Recently, mutations of the transcriptional factor
SOX10 (Pingault et al. 1998) and of the endothelin-



Letters to the Editor 1217

Figure 1 SSCP analysis of the RET exon 11 transcript from
affected individuals carrying the I647I silent mutation. PCR products
corresponding to this exon from father, mother, and patient 1 genomic
DNA are represented in lanes 1, 2, and 3, whereas lanes 4, 5, and 6
show the PCR products obtained from the cDNA of patients 1 and 2
and a control individual, respectively. Note that the shifted upper band
corresponding to the mutated allele present in lanes 2 and 3 is absent
in the cDNAs of patients 1 and 2 (lanes 4 and 5), which show only
the normal allele present in the cDNA from a control individual (lane
6).

Figure 2 ARMS analysis of the RET exon 11 from patient 2
genomic DNA and cDNA. PCR products were obtained by use of
oligonucleotide primers specific for either the normal (lanes 1 and 3)
or the I647I (lanes 2 and 4) RET alleles. A band of the expected size
is amplified from patient genomic DNA with both sets of primers (lanes
1 and 2), whereas at the cDNA level the normal (lane 3), but not the
mutated, allele (lane 4) is present.

converting enzyme 1 (ECE1) (Hofstra et al. 1998) have
been reported in HSCR patients with pigmentary and
cardiac defects.

RET, GDNF, EDNRB, and EDN3 are mutated in a
variable proportion of individuals affected with isolated
HSCR, accounting for 30%–50% of all cases (Edery et
al. 1997; Eng and Mulligan 1997; Hofstra et al. 1997).
In addition, these mutations alone do not explain the
variable expressivity and the incomplete penetrance of
the disease for which, according to a complex model of
inheritance, the combined presence of mutations in more
than one of the known or still unknown HSCR suscep-
tibility genes can be inferred.

To better understand the contribution of RET, GDNF,
EDNRB, and EDN3 to the pathogenesis of HSCR, we
collected samples from 50 patients and analyzed them
for mutations at these loci. Forty-seven of these patients
represented unrelated sporadic cases, and three belonged
to two different families in which a polygenic or recessive
mode of inheritance could be inferred on the basis of
the pedigree. They were affected with isolated mega-
colon, including long, short (“classic”), and ultrashort
forms. None of them was born of consanguineous par-
ents, and all were of Italian origin.

Table 1 shows two heterozygous incompletely pene-

trant EDNRB mutations (S305N and N378I) we re-
cently described in two patients with isolated short-seg-
ment HSCR disease, inherited from healthy parents
(Auricchio et al. 1996). These two mutations were iden-
tified during a first screening of 20 patients and were
absent in 100 control chromosomes. We then analyzed
samples from 30 additional patients with isolated HSCR
and could not detect any novel mutations at the EDNRB
locus. The mutation screening of the 21 exons of the
RET proto-oncogene was done on all 50 patients. As
shown in table 1, two missense mutations in exons 10
and 14 (C1876A and G2438A, respectively) and a nu-
cleotide change affecting the 5′ end of intron 14
(G2607�5A) were identified in three different patients.
In particular, the Q626K missense mutation affects the
last codon of exon 10, namely the cys-rich region located
in the juxtamembranous extracellular domain, suggest-
ing either an effect on the folding of the ligand-binding
domain or an interference with RET dimerization, a cru-
cial step preceding its activation. Mutation R813Q, af-
fecting exon 14, is likely to alter the RET tyrosine kinase
activity, whereas the GrA transition at the 5′ end of
intron 14 might interfere with correct processing of the
primary transcript. In addition, we found two synony-
mous nucleotide changes in RET exon 11, leading to
neutral substitutions I647I and S649S in patients 20 and
25, respectively (table 1). These were absent in 150 con-
trol individuals. We could not detect any GDNF or
EDN3 mutations in our patients, thus confirming that
they play a minor role in HSCR pathogenesis.



1218 Letters to the Editor

Figure 3 In vitro analysis of the RET exons 10 and 11 splicing products from both the normal and the I647I mutated alleles (see text
for details). a, Schematic representation of the RET constructs used for the in vitro splicing analysis. b, RT-PCR amplification using primers
designed in exons 10 and 11, as indicated in a, from total RNA of COS-7 cells transiently transfected with the RET wild-type (lane 1) and the
I647I (lane 2) constructs. Note the abnormal products present in lane 2. c, Sequence analysis of the 255-bp products from lanes 1 and 2 shown
in b corresponded to the normal and mutated RET alleles, respectively, an observation consistent with the use of canonical “gt” and “ag”
donor and acceptor splice sites (underlined in the partial intronic sequence given in lowercase letters). Conversely, sequence analysis of the 148-
bp and 120-bp products revealed two exon 11 deleted forms resulting from the use of cryptic acceptor “ag” sites activated to generate the two
abnormal products (underlined in the sequence of the 5′ portion of exon 11 given in capital letters). The two alternative nucleotides involved
in the neutral substitution of codon 647 (T in the normal allele and C in the variant allele) are shown in boldface type.

Interestingly, patient 20 (from now on, patient 1) car-
ries both a RET synonymous nucleotide change,
C1941T (I647I), inherited from the unaffected mother,
and an EDNRB missense mutation, S305N, transmitted
by the healthy father. This latter mutation results in the
substitution of a serine, located in the third intracellular
loop, with an asparagine (Auricchio et al. 1996). This
serine, highly conserved between different species, has
been shown to be a site of in vivo phosphorylation (Roos
et al. 1998), suggesting that it plays an important role
in the receptor regulation and that its absence can result
in the protein loss of function. Intraexonic silent mu-
tations can alter correct mRNA processing, thus result-
ing in either altered mRNA levels or truncated proteins
(Steingrimsdottir et al. 1992; De Meirleir et al. 1994; Li
et al. 1995; Richard and Beckmann 1995; Jin et al. 1996;
Llewellyn et al. 1996; Ploos van Amstel et al. 1996; Liu
et al. 1997). To test the hypothesis that the RET I647I
variant could play a causative role in the development
of the disease phenotype in patient 1, we analyzed, both
in vitro and in vivo, the RET mRNA expression, using

cDNA and genomic DNA from patient 1 and from an
additional proband (patient 2) with the same I647I
change, described elsewhere (Ceccherini et al. 1994).

As an in vivo approach to study the RET transcrip-
tion, we performed reverse transcription (RT) PCR with
total RNA from lymphoblastoid cell lines of these two
patients, by means of two different techniques—the
SSCP (Orita et al. 1989) and the amplification-refractory
mutation system (ARMS) (Newton et al. 1989) analysis.
Results obtained from SSCP analysis of the RET exon
11 are shown in figure 1. The shifted upper band cor-
responding to the mutated allele present in the genomic
DNA of patient 1 and his healthy mother (lanes 2 and
3) is absent in the cDNAs of patients 1 and 2 (lanes 4
and 5). They depict only the normal allele present in the
cDNA from a control individual (lane 6). Similar results
have been obtained from ARMS analysis of the RET
exon 11 from patient 2 genomic DNA and cDNA (fig.
2). In particular, using oligonucleotide primers specific
for either the normal (lanes 1 and 3) or the I647I (lanes
2 and 4) RET alleles, we were able to amplify a band



Letters to the Editor 1219

of the expected size from patient genomic DNA with
both sets of primers (lanes 1 and 2), although at the
cDNA level the normal (lane 3), but not the mutated,
allele (lane 4) was present. In addition, direct sequence
analysis of the PCR product, as well as BsmI enzymatic
restriction of an exon 7 RET polymorphism, the phase
of which is known with respect to the I647I mutation,
confirmed the absence of the mutated allele in the cDNA
of the affected individuals (data not shown).

To elucidate whether the I647I silent mutation could
interfere with splicing, thus resulting in unstable prod-
ucts undetectable in vivo in lymphoblast cDNAs, we
cloned the 3-kb genomic region encompassing RET ex-
ons 10, 11, and 12, from both the normal and the I647I
mutated alleles, into the pSPL3 eukaryotic vector
(Church et al. 1994) (fig. 3a). RT-PCR amplification
from total RNA of SV40-transformed African green
monkey kidney cells (COS-7) transiently transfected
with the RET wild-type construct (fig. 3b) shows the
expected fragment of 255 bp (lane 1). Two additional
products of 148 bp and 120 bp were amplified when
the I647I construct was transfected (lane 2). As shown
in figure 3c, sequence analysis of the 255-bp products
from the above lanes 1 and 2 corresponded to the normal
and mutated RET alleles, respectively, an observation
consistent with the use of canonical “gt” and “ag” donor
and acceptor splice sites (underlined in the partial in-
tronic sequence given in lowercase letters). Conversely,
sequence analysis of the 148-bp and 120-bp products
revealed two exon 11 deleted forms. Both products re-
sulted from the fusion of the 3′ end of exon 10 to two
different sites located 107 bp and 135 bp downstream
of the exon 11 starting nucleotide in the largest and the
smallest abnormal products, respectively. If translated,
these mRNAs would result in two proteins: the one cor-
responding to the largest transcript truncated 758 bp
downstream of the newly activated splice site, and the
smallest mRNA resulting in an in-frame interstitial loss
of 45 amino acids, including the transmembrane do-
main. Nevertheless, we postulate that this abnormal pro-
cessing of the I647I allele may reduce the mutant RNA
to a level that escapes detection by the commonly used
in vivo RT-PCR approach. Thus, the presence in our
patient of an inactive form of the endothelin-B receptor
and of reduced levels of the Ret protein could impair
the normal enteric neuronal migration.

In conclusion, we confirm that in isolated HSCR the
major susceptibility locus is the RET proto-oncogene,
with EDNRB accounting for a minority of cases. More
relevantly, we demonstrate in two different patients,
both in vivo and in vitro, that the same silent RET mu-
tation can interfere with correct transcription, thus pos-
sibly leading to a reduced level of the Ret protein. Finally,
the coexistence, reported for the first time, in the same
patient of two functionally significant EDNRB and RET

mutations suggests a direct genetic interaction between
these two distinct transmembrane receptors in polygenic
HSCR disease.

Acknowledgments

The financial support of Telethon–Italy (grant E791) is grate-
fully acknowledged. This work was also funded by the Italian
Telethon Foundation, the Italian Ministry of Health, and the
European Community (contract MH4-CT97-2107).

ALBERTO AURICCHIO,1,∗ PAOLA GRISERI,2

MARIA LUISA CARPENTIERI,3 NICOLA BETSOS,2

ANNAMARIA STAIANO,3 ARTURO TOZZI,3

MANUELA PRIOLO,2 HELEN THOMPSON,2

RENATA BOCCIARDI,2 GIOVANNI ROMEO,4,5

ANDREA BALLABIO,1 AND ISABELLA CECCHERINI2
1Telethon Institute of Genetics and Medicine, Milan,
2Laboratorio di Genetica Molecolare, Istituto
Giannina Gaslini, Genova-Quarto, 3Dipartimento di
Pediatria, Università �Federico II,� Naples, and
4Dipartimento di Oncologia Biologia e Genetica,
Università degli Studi di Genova, Genova, Italy; and
5Genetic Cancer Susceptibility Unit, Lyon, France

Electronic-Database Information

Accession number and URL for data in this study are as
follows:

Online Mendelian Inheritance in Man (OMIM), http://
www.ncbi.nlm.nih.gov/Omim (for HSCR [142623])

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Address for correspondence and reprints: Dr. Isabella Ceccherini, Laboratorio
di Genetica Molecolare, Istituto Giannina Gaslini, Largo G. Gaslini, 5, 16148
Genova, Italy. E-mail: isa.c@unige.it

� 1999 by The American Society of Human Genetics. All rights reserved.
0002-9297/99/6404-0035$02.00

Am. J. Hum. Genet. 64:1221–1225, 1999

A Mutation (2314delG) in the Usher Syndrome Type
IIA Gene: High Prevalence and Phenotypic Variation

To the Editor:
Usher syndrome (USH), an autosomal recessive condi-
tion, is characterized by hearing impairment associated
with retinitis pigmentosa (RP). It is a clinically and ge-
netically heterogeneous condition. Three clinical forms
of USH have been described, and eight loci have been
mapped (Hereditary Hearing Loss home page). USH
type I (USH1) is the most severe form, manifested by
profound congenital deafness, constant vestibular dys-
function, and prepubertal onset of RP. USH type II
(USH2) is characterized by congenital moderate-to-se-
vere hearing impairment, normal vestibular responses,
and RP (Smith et al. 1994). USH type III (USH3) is
characterized by progressive hearing loss, variable ves-
tibular problems, and RP (Pakarinen et al. 1995). The
USH1B gene has been identified (Weil et al. 1995), and
a variety of mutations leading to USH1 have been cat-
alogued in the MYO7A gene (Liu et al. 1998). USH2 is

the most common of the three types of USH, accounting
for more than half of all cases (Hopes et al. 1997; Ro-
senberg et al. 1997).

Recently, Eudy et al. (1998) reported the identification
of the USH type IIA gene (USH2A; MIM 276901).
USH2A encodes a putative extracellular matrix protein
of 1,551 amino acids with laminin epidermal growth
factor and fibronectin type II motifs. Expression of the
USH2A gene has been detected by reverse transcriptase
PCR (RT-PCR) from fetal human cochlea, eye, and adult
human retina (Eudy et al. 1998). Three different mu-
tations were identified in patients with USH2A, the most
frequent of which is the 2314delG mutation. Of 96 pro-
bands tested, 21 carried the 2314delG mutation.

In the present study, we have undertaken mutational
analysis of the USH2A gene in 23 families with USH
(both USH2 and atypical USH), from the United King-
dom and China. We found that the majority of families
with USH2 carry the 2314delG mutation. Surprisingly,
we determined that the 2314delG mutation in the
USH2A gene can also lead to atypical USH.

Of 23 families with USH analyzed in this study, 15
have been described elsewhere (Hopes et al. 1997). All
available affected subjects were examined by one of the
authors. Full clinical histories were obtained, with em-
phasis on any potential causes of hearing impairment.
The audiovestibular and ophthalmic evaluations were
done on the basis of recommendations by the Usher
Syndrome Consortium (Smith et al. 1994). The details
of the clinical evaluations can be found in Hopes et al.
(1997). The cases studied here are classified clinically
into the following two groups: (1) USH2, consisting of
congenital sloping moderate/severe hearing impairment,
normal vestibular function, and RP (Smith et al. 1994);
and (2) atypical USH, consisting of bilateral sensorineu-
ral progressive hearing loss without other obvious fac-
tors as the cause of the progression of the hearing im-
pairment, along with variable vestibular dysfunction and
RP.

Of 23 families studied, 13 were given a diagnosis of
USH2 and 10 were given a diagnosis of atypical USH.
Eighteen families living in the United Kingdom and five
in China were included in our analyses. Twenty-three
probands with USH were screened for the 2314delG
mutation by combined SSCP/heteroduplex analysis.
Twelve families (52%) carried the 2314delG mutation—
8 (62%) of 13 patients with typical USH2 and 4 (40%)
of 10 patients with atypical USH (table 1). Of 12 families
with this mutation, 7 were identified with only one af-
fected member (sporadic cases). Of the remaining five
families with more than one affected individual, two
families (USH.05 and USH.10) with two affected sibs
were tested for cosegregation of the disease with the
USH2A locus (Eudy et al. 1998). The segregation anal-
ysis was consistent with linkage to USH2A (data not


	Double Heterozygosity for a RET Substitution Interfering with Splicing and an EDNRB Missense Mutation in Hirschsprung Disease
	Acknowledgments
	References