doi:10.1086/302618


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� 1999 by The American Society of Human Genetics. All rights reserved.
0002-9297/1999/6505-0033$02.00

Am. J. Hum. Genet. 65:1469–1473, 1999

Association of RET Protooncogene Codon 45
Polymorphism with Hirschsprung Disease

To the Editor:
The RET protooncogene (MIM 164761) is expressed in
human tissues of neural crest origin and has been rec-

ognized as a susceptibility gene for several autosomal
inherited diseases, such as Hirschsprung disease (HSCR
[MIM 142623]) and multiple endocrine neoplasia type
2 syndromes (MEN 2 [MIM 171400]) comprising med-
ullary thyroid carcinoma (MTC [MIM 155240]) as an
obligatory feature (Eng et al. 1997). Of the patients with
HSCR, 10%–40% have been reported to harbor germ-
line mutations of the RET protooncogene, which are
primarily point mutations scattered throughout the ex-
tracellular domain and within the intracellular tyrosine
kinase domain of RET (Edery et al. 1994b; Romeo et
al. 1994; Angrist et al. 1995; Seri et al. 1997). MEN 2
syndrome germline mutations of the RET protoonco-
gene have been found to affect exons 10, 11, and 13–16
(Donis-Keller et al. 1993; Mulligan et al. 1993; Carlson
et al. 1994; Eng et al. 1994; Hofstra et al. 1994; Bolino
et al. 1995). Functional studies have demonstrated that
RET mutations that characterize the autosomal domi-
nant–inherited MEN 2 cause activation of the RET-sig-
naling pathway, often in a constitutive manner or by
altering the substrate specificity (Borrello et al. 1995;
Santoro et al. 1995; Ceccherini et al. 1997; Pasini et al.
1997; Chappuis-Flament et al. 1998). In contrast, RET
mutations found in HSCR presumably result in either
RET-protein truncation or functional inactivation of the
molecule. Although loss of one allele in some patients
with HSCR suggests haploinsufficiency (Martucciello et
al. 1992), the retention of one wild-type allele in patients
with HSCR who have an inactivating RET mutation
seems to explain the presumed autosomal dominant in-
heritance and implicates a dominant-negative action of
the mutated RET allele (Badner et al. 1990; Cosma et
al. 1998).

Furthermore, several polymorphisms in the coding re-
gion of the RET protooncogene have been described. A
panel of the most frequent polymorphisms has been re-
ported by Mulligan et al. (1993), Ceccherini et al.
(1994), and Sáez et al. (1998), comprising those in co-
dons 45, 125, 432, 691, 769, 836, and 904. In the study
by Ceccherini et al., the allele frequencies of these poly-
morphisms were evaluated in a normal control group.
These data were confirmed by a study by Gimm et al.
(1999), and similar allele frequencies of the codon 45
polymorphism have been described by Edery et al.
(1994a), who focused on a control population only. All
of the investigated polymorphisms are silent mutations,
except for the codon 691 polymorphism, which results
in a change in the amino acid residue, from glycine to
serine. Bugalho et al. (1994) investigated the frequency
of the codon 691 polymorphism in a small population
of clinically defined sporadic medullary thyroid carci-
nomas (MTC) and found no significant differences from
a normal control population. Elsewhere, Gimm et al.
(1999) have investigated all seven RET polymorphisms
in a population with sporadic MTC and have found an



1470 Letters to the Editor

Table 1

Allele Frequencies of Polymorphic Variants of RET in 62 Patients with Sporadic HSCR and
in 156 Control Individuals

EXON

NUCLEOTIDE
CHANGE

(CODON)a

RESTRICTION
SITE

CHANGED

ALLELE FREQUENCY INb

(%) STATISTIC

Controls
Patients

with HSCR x2 P

2 GCGrGCA (A45A) EagI 76.3 26.6 93.064 !.001
3 GTCrGTA (V125V) MboII 98.1 97.6 .108 .742
7 GCGrGCA (A432A) BsmI 72.4 74.2 .139 .709
11 GGTrAGT (G691S) BanI 79.8 89.5 5.811 .016
13 CTTrCTG (L769L) TaqI 76.3 57.3 15.556 !.001
14 AGCrAGT (S836S) AluI 96.4 c 100 4.575 .032
15 TCCrTCG (S904S) RsaI 80.1 88.7 4.540 .033

a The wild-type allele is underlined.
b Of the wild-type allele.
c Only 153 control individuals were tested.

overrepresentation of the rare codon 836 polymorphism,
compared with the frequency in normal controls. Inter-
estingly, in this study the rare germline codon 836–se-
quence variant seems to be associated with the presence
of a common somatic M918T mutation in the corre-
sponding tumor DNA of patients with sporadic MTC.

To reveal the potential impact that RET polymor-
phisms for etiology have for HSCR in particular, we
investigated the genotype distribution of polymorphisms
of codons 45, 125, 432, 691, 769, 836, and 904 of the
coding region of the RET protooncogene in patients
with HSCR but without a family history of the disease.
The population that we studied comprised 62 individ-
uals with sporadic HSCR who were from two different
areas of Germany, around the cities of Dresden (n =

) and Erlangen ( ). The male:female ratio of37 n = 25
these individuals was 3.8:1. For inclusion in the study,
histopathological criteria of HSCR were (a) increased
acetylcholinesterase histochemical staining in nerve fi-
bers, in suction biopsies of the rectal submucosa, and
(b) absence of neuronal ganglia, in operative histochem-
ical and histological evaluation of the aganglionic tract.
Patients with additional features or associated diseases
were excluded from the study. Anonymous healthy
blood donors from each region served as controls (n =

for Dresden; for Erlangen). Controls were117 n = 39
not matched for age or race, although all individuals
were white. There was, therefore, a slight potential for
population stratification in the patients with HSCR, rel-
ative to that in the controls. Genomic DNA was obtained
from leukocytes from peripheral venous blood samples
isolated by standard protocols. The seven investigated
exons were amplified from genomic DNA by use of
primers and reaction conditions described by Ceccherini
et al. (1994), for exons 2 (codon 45), 3 (codon 125), 11
(codon 691), and 14 (codon 836), and by Mulligan et
al. (1994), for exons 7 (codon 432) and 13 (codon 769).

To amplify exon 15 (codon 904), we generated a new
primer pair (sense, 5′CCCCCGGCCCAGGTCTCAC-3′;
antisense, 5′GCTCCACTAATCTTCGGTATCTTT-3′).
All analyzed polymorphisms generate or destroy a re-
striction site of an endonuclease—namely, EagI, MboII,
BsmI, BanI, TaqI, AluI, or RsaI (Ceccherini et al. 1994).
Genotypes were determined by digestion of the PCR
product and electrophoresis on a polyacrylamide gel. In
addition, these results from the patient population were
confirmed by DNA-sequencing analysis by use of the
Thermo Sequenase� Fluorescent Cycle Sequencing kit
(Amersham Pharmacia Biotech), according to the man-
ufacturer’s protocol. The sequencing primers were the
same as the PCR primers, with an additional Cy5TM
labeling, allowing sequence analysis on A.L.F. express
devices (Amersham Pharmacia Biotech). Statistical anal-
ysis was performed with the Pearson x2 test. Written
informed consent was obtained from all patients.

Our data revealed that allele frequencies of all poly-
morphisms in the control population were similar to
those reported by Ceccherini et al. (1994), Gimm et al.
(1999), and Edery et al. (1994a), suggesting that the
allele frequency is similar in the German, European, and
American populations tested, but the study does not in-
clude data of an ethnically diverse, nonwhite population.
The genotype distribution for each of the seven poly-
morphic loci did not deviate significantly from Hardy-
Weinberg equilibrium. Although the wild-type allele of
the codon 45 polymorphism was detected in 76.3% of
312 control chromosomes, the same allele was found in
26.6% of 124 HSCR chromosomes, an almost inverted
relationship (table 1) (for allele frequencies in patients
with HSCR vs. those in controls, , ).2x = 93.06 P ! .001
This highly significant difference between these allele fre-
quencies resulted from a strong overrepresentation of
the homozygous codon 45–polymorphism variant in the
population with HSCR (34 of 62 patients with HSCR,



Letters to the Editor 1471

vs. 9 of 156 controls). Ceccherini et al. (1994) found
the wild-type allele of the codon 45 polymorphism in
71% of 104 chromosomes, the same frequency as later
was reported, by Gimm et al. (1999), in an analysis of
96 chromosomes. Furthermore, we found this highly sig-
nificant association of the codon 45 polymorphism also
in the two independent populations with HSCR and in
controls from the regions around Erlangen and Dresden
(for the allele frequency in Dresden patients with HSCR
vs. that in Dresden controls, , ; for2x = 60.65 P ! .001
the allele frequency in Erlangen patients with HSCR vs.
that in Erlangen controls, , ).2x = 31.65 P ! .001

Within the population with HSCR, a tendency toward
overrepresentation of the codon 769 polymorphism,
similar to that of the codon 45 polymorphism, was
found, compared with the frequency in the controls (ta-
ble 1). In addition, we found the codon 769 polymor-
phism to be associated with HSCR in both populations,
compared with what was found in the controls (for the
allele frequency of Dresden patients with HSCR vs. that
in Dresden controls, , ; for the fre-2x = 9.26 P = .002
quency in Erlangen patients with HSCR vs. that in Er-
langen controls, , ).2x = 5.72 P ! .017

Although in codons 45 and 769 the polymorphic allele
was overrepresented in the population with HSCR, in
codons 691, 836, and 904 we found the wild-type allele
to be more frequent in the population with HSCR pop-
ulation than in the control group, although the difference
was not statistically significant (table 1).

In this study we have demonstrated that the codon
45–polymorphism allele frequency is overrepresented in
patients with sporadic HSCR compared with the normal
population, a finding that is highly significant statisti-
cally. In agreement with our findings, Puffenberger et al.
(1994) described a significant excess of this polymor-
phism (for allele frequencies, , ) on2x = 12.08 P ! .001
the HSCR haplotype that is transmitted to affected mem-
bers of Mennonite families with HSCR. However, the
predominant mutation identified in this kindred is a
founder homozygous W276C EDNRB (MIM 131244)
gene mutation, which is an interesting association in
itself and supports the polygenic, complex inheritance
of HSCR. In addition, one patient has been described
with both an EDNRB mutation and a RET mutation
that apparently result in aberrant RET RNA splicing
(Auricchio et al. 1999).

The mechanism by which the silent codon 45 poly-
morphism may act in HSCR genesis is unknown, but
speculations have been made regarding the possible
mechanisms. It has, for instance, been proposed that the
silent sequence variant could lead to aberrantly spliced
products, resulting in a protein with a 21-amino-acid
deletion in the extracellular domain, altering a part of
the extracellular signal-peptide sequence (Borrego et al.
1998).

In addition, it has been suggested that a seemingly
nonfunctional polymorphism may create an unstable
downstream sequence, which results in a functional so-
matic mutation (Gimm et al. 1999). Such a mechanism
has been observed in the APC (MIM 175100) gene in
Ashkenazim with familial colorectal cancer, in which ad-
ditional somatic mutations were more often found on
the allele carrying a conservative amino acid change
(I1307K) (Laken et al. 1997).

If no pathogenic effect can be associated with the co-
don 45 RET polymorphism, then the possibility has to
be considered that the base substitution is in linkage
disequilibrium with an unknown functional variant up-
stream or downstream. For example, an MspI RFLP of
the 3′ end of the human CYP1A1 (MIM 108330) gene
has been shown to be in linkage disequilibrium with an
adenine-to-guanine mutation at residue 462 in exon 7.
The latter mutation causes an amino acid substitution,
which results in increased enzymatic activity of CYP1A1
(Hayashi et al. 1991). Similarly, the silent codon 45 poly-
morphism may be either closely linked with a functional
genetic variant or be functional itself.

Nevertheless, the observed difference in the homo-
zygous genotype of the silent polymorphism—5.8% in
the normal population of 156 individuals versus 54.8%
in 62 analyzed patients with HSCR—suggests a strong
association with the HSCR phenotype.

GUIDO FITZE,1 MATTHIAS SCHREIBER,4

EBERHARD KUHLISCH,3 HANS K. SCHACKERT,2

AND DIETMAR ROESNER1

Departments of 1Pediatric Surgery and 2Surgical
Research and 3Institute of Medical Informatics and
Biometry, University of Technology Dresden, Dresden;
and 4Department of Pediatric Surgery, University of
Erlangen, Erlangen, Germany

Electronic-Database Information

Accession numbers and URL for data in this article are as
follows:

Online Mendelian Inheritance in Man (OMIM), http://www
.ncbi.nlm.nih.gov/Omim (for APC [MIM 175100], CYP1A1
[MIM 108330], EDNRB [MIM 131244], HSCR [MIM
142623], MEN 2 [MIM 171400], and MTC [MIM
155240], and RET [MIM 164761])

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Buys CHC, Cass DT, et al (1995) Mutation analysis of the
RET receptor tyrosine kinase in Hirschsprung disease. Hum
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Auricchio A, Griseri P, Carpentieri ML, Betsos N, Staiano A,
Tozzi A, Priolo M, et al (1999) Double heterozygosity for
a RET substitution interfering with splicing and an EDNRB



1472 Letters to the Editor

missense mutation in Hirschsprung disease. Am J Hum Ge-
net 64:1216–1221

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genetic study of Hirschsprung disease. Am J Hum Genet 46:
568–580

Bolino A, Schuffenecker I, Luo Y, Seri M, Silengo M, Tocco
T, Chabrier G, et al (1995) RET mutations in exons 13 and
14 of FMTC patients. Oncogene 10:2415–2419

Borrego S, Eng C, Sánchez B, Sáez ME, Navarro E, Antinolo
G (1998) Molecular analysis of the ret and GDNF genes
in a family with multiple endocrine neoplasia type 2A
and Hirschsprung disease. J Clin Endocrinol Metab 83:
3361–3364

Borrello MG, Smith DP, Pasini B, Bongarzone I, Greco A,
Lorenzo MJ, Arighi E, et al (1995) RET activation by
germline MEN2A and MEN2B mutations. Oncogene 11:
2419–2427

Bugalho MJM, Cote GJ, Khorana S, Schultz PN, Gagel RF
(1994) Identification of polymorphism in exon 11 of the
RET proto-oncogene. Hum Mol Genet 3:2263

Carlson KM, Bracamontes J, Jackson CE, Clark R, Lacroix
A, Wells SA, Goodfellow PJ (1994) Parent-of-origin effects
in multiple endocrine neoplasia type 2B. Am J Hum Genet
55:1076–1082

Ceccherini I, Hofstra RMW, Luo Y, Stulp RP, Barone V, Stel-
wagen T, Bocciardi R, et al (1994) DNA polymorphisms
and conditions for SSCP analysis of the 20 exons of the ret
proto-oncogene. Oncogene 9:3025–3029

Ceccherini I, Pasini B, Pacini F, Gullo M, Bongarzone I, Romei
C, Santamaria G, et al (1997) Somatic in frame deletions
not involving juxtamembranous cysteine residues strongly
activate the RET proto-oncogene. Oncogene 14:2609–2612

Chappuis-Flament S, Pasini A, DeVita G, Segouffin-Cariou C,
Fusco A, Attie T, Lenoir GM, et al (1998) Dual effect on
the RET receptor of MEN 2 mutations affecting specific
extracytoplasmic cysteins. Oncogene 17:2851–2861

Cosma MP, Cardone M, Carlomagno F, Colantuori V (1998)
Mutations in the extracellular domain cause RET loss of
function by a dominant negative mechanism. Mol Cell Biol
18:3321–3329

Donis-Keller H, Dou S, Chi D, Carlson KM, Toshima K, Lair-
more TC, Howe JR, et al (1993) Mutations in the RET
proto-oncogene are associated with MEN2A and FMTC.
Hum Mol Genet 2:851–856

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Genet 94:579–580

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Holder S, et al (1994b) Mutations of the RET proto-on-
cogene in Hirschsprung’s disease. Nature 367:378–379

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oncogene in the multiple endocrine neoplasia type 2 syn-
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MA, Gardner E, et al (1994) Point mutations within the
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Fournier L, Rudkin BB, et al (1997) Oncogenetic activation
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tat 9:243–249



Letters to the Editor 1473

Table 1

Numbers of Males and Females in Populations of Unrelated
Persistent Stutterers, and x2 Analysis of the Differences in
Gender Ratios between Groups

SEX

NO. OF PATIENTS IN POPULATION

HCRI
Alumni

NSP
Members

SFA
Records

AIS
Alumni Total

Males 810 285 131 826 2052
Females 156 112 52 212 532

NOTE.—Overall, familial cases versus general stuttering pop-
ulation ; df 1; . Familial cases versus cases as-2x = 43 P ! .00001
certained via therapy programs . . Familial2x = 63 P ! .000001
cases versus cases ascertained without respect to treatment 2x =

. .13 P ! .002

Address for correspondence and reprints: Dr. Guido Fitze, Department of
Pediatric Surgery, University of Technology Dresden, Fetscherstrasse 74, D-
01307 Dresden, Germany. E-mail: Guido.Fitze@mailbox.tu-dresden.de

� 1999 by The American Society of Human Genetics. All rights reserved.
0002-9297/1999/6505-0034$02.00

Am. J. Hum. Genet. 65:1473–1475, 1999

The Sex Ratio in Familial Persistent Stuttering

To the Editor:
Stuttering is a speech disorder characterized by invol-
untary syllable repetitions, syllable prolongations, or in-
terruptions, known as blocks, in the smooth flow of
speech (World Health Organization 1992; Bloodstein
1995). Stuttering typically arises in young children,
where it affects �15% of children in the age range of
4–6 years (Bloodstein 1995). Stuttering often resolves
spontaneously before adolescence, leading to a popu-
lation prevalence of 1%–2% among adults. Stuttering
beyond childhood is characterized by a significant bias
toward males, with males outnumbering females by a
ratio of 3:1–5:1 (Yairi et al. 1996).

Many studies support the view that inherited factors
contribute to stuttering (Howie 1981; Yairi et al. 1996;
Felsenfeld and Plomin 1997). As part of a linkage study
to identify predisposing loci for this disorder, we assem-
bled 1100 small-to-medium–sized unrelated families
with multiple cases of persistent stuttering, chosen to
represent the typical presentation of familial stuttering
in the adult population. In these families, we have ob-
served a male-to-female ratio among the affected indi-
viduals that is strikingly different from the generally ac-
cepted ratio in the overall adult stuttering population.

Family ascertainment was designed to obtain the most
diverse sample possible from the North American pop-
ulation. The NIH families were ascertained under NIH
IRB-approved protocol 97-DC-0087, through a broad
variety of appeals directed at stuttering interest groups,
stuttering support groups, professional speech and lan-
guage organizations, alumni of stuttering therapy pro-
grams—including intensive residential programs and
part-time, outpatient programs—and the general public.
The enrolled families included whites, African Ameri-
cans, Hispanics, and Asians, with no evidence for over-
or under-representation of any group compared to the
general population. Among the identifiable probands in
these families, 56% were male and 44% were female.
We exhaustively ascertained and evaluated family mem-
bers aged 18 years according to well-established diag-
nostic criteria for stuttering (Webster 1978; World

Health Organization 1992), using videotaped speech
samples and counting the number of stuttering-like dys-
fluencies, in both conversation and reading. In some
cases, audio tape recordings were substituted. The stan-
dardized reading passage was 500 words in length and
contained balanced numbers of each of the different clas-
ses of speech sounds. This tool has been used for 110
years and has well-established performance norms (R.
Webster, personal communication; copy available, on
request, from corresponding author). For individuals to
be classified as affected, a score of �4% dysfluent words
(representing the 25th percentile among individuals who
present themselves for stuttering therapy) was required
in the individual’s speech in both conversation and read-
ing. In some cases, videotaped speech samples were not
obtainable, and audio recordings of speech were sub-
stituted. By these criteria, 224 individuals were classified
as affected in our families. Affection status, as deter-
mined by professional speech evaluation, was generally
in agreement with self-reported affection status. The few
discrepancies showed no evidence of bias between males
and females. The affected individuals had an age range
of 10–86 years, with a mean age of 39.9 years. Among
these affected individuals, 137 are male and 87 are fe-
male, yielding a male-to-female ratio of 1.57.

To compare this ratio to the male-to-female ratio in
the general stuttering population, we examined four dif-
ferent populations of unrelated, persistent stutterers. We
chose four different groups of persistent stutterers, be-
cause each group was subject to individual ascertain-
ment biases. For example, therapy programs are gen-
erally believed to ascertain males preferentially, while
support groups are believed to attract more females, fre-
quently affected mothers of affected children. We sought
the largest available sources of such populations of stut-
terers and derived data from the clinical records of two
large therapy programs, the Hollins Communications
Research Institute (HCRI) and the American Institute
for Stuttering (AIS), plus data on two groups, ascertained


	Association of RET Protooncogene Codon 45 Polymorphism with Hirschsprung Disease
	References