1202226A 3065..3070


SHORT REPORT

Genomic structure of the gene for the SH2 and pleckstrin homology
domain-containing protein GRB10 and evaluation of its role in
Hirschsprung disease

Misha Angrist1, Stacey Bolk1, Kimberly Bentley1, Sudha Nallasamy1, Marc K Halushka1 and
Aravinda Chakravarti1,2

1Department of Genetics and 2Center for Human Genetics, Case Western Reserve University and University Hospitals of Cleveland,
Cleveland, Ohio, 44106-4955, USA

Hirschsprung disease (HSCR), or congenital aganglionic
megacolon, is the most frequent cause of congenital
bowel obstruction. Germline mutations in the RET
receptor tyrosine kinase have been shown to cause
HSCR. Mice that carry null alleles for RET or for its
ligand, glial cell line-derived neurotrophic factor
(GDNF), both exhibit complete intestinal aganglionosis
and renal defects. Recently, the Src homology 2 (SH2)
domain-containing protein Grb10 has been shown to
interact with RET in vitro and in vivo, early in
development. We have con®rmed the map location of
GRB10 on human chromosome 7, isolated human BACs
containing the gene, elucidated its genomic structure,
isolated a highly polymorphic microsatellite marker
adjacent to exon 14 and scanned the gene for mutations
in a large panel of HSCR patients. No evidence of
linkage was detected in HSCR kindreds and no
mutations were found in patients. These data suggest
that while GRB10 may be important for signal
transduction in developing embryos, it does not play an
obvious role in HSCR.

Keywords: Hirschsprung disease; GRB10; GRB-IR;
RET receptor tyrosine kinase; mutation detection;
genomic structure; mapping

Congenital aganglionic megacolon, commonly known
as Hirschsprung disease (HSCR), is associated with a
lack of intrinsic ganglion cells in the myenteric and
submucosal plexuses along variable lengths of the
gastrointestinal tract (Holschneider, 1982). Enteric
ganglion cells are derived primarily from the vagal
neural crest. Thus, HSCR, like other disorders whose
a�ected tissues are of neural crest origin, is best
characterized as a neurocristopathy, albeit one that
frequently co-occurs with additional phenotypes
a�ecting neural crest-derived tissues (Bolande, 1997;
Martucciello, 1997). HSCR is relatively common, with
an incidence of approximately 1 in 5000 live births
(Spouge and Baird, 1985).

Evidence that HSCR susceptibility has a large
genetic component has come from pedigrees segregat-
ing HSCR as an incompletely penetrant autosomal
dominant trait and formal segregation analysis

supporting the hypothesis of dominant inheritance in
at least 20% of cases, with the remainder of cases
explainable by recessive or multigenic inheritance
(Badner et al., 1990; Bodian and Carter, 1963).
Subsequently, genetic mapping of HSCR to the
pericentromeric region of chromosome 10 in a subset
of families (Angrist et al., 1993; Lyonnet et al., 1993,
and to chromosome 13q22 in a large Mennonite
kindred (Pu�enberger et al., 1994a) was reported.

In 1994, mutations in HSCR patients were described
in several genes, most notably in the RET receptor
tyrosine kinase (Edrey et al., 1994; Romeo et al., 1994)
and in the G-protein coupled endothelin-B receptor
(EDNRB; Pu�enberger et al., 1994a). Mutations have
since been reported in EDNRB's physiological ligand,
endothelin 3 (Hofstra et al., 1996; Kusafuka et al.,
1996, 1997. Mouse knockout phenotypes for these
genes, and for RET's ligand GDNF, support roles for
them in the development of the enteric nervous system
(Jing et al., 1996; Moore et al., 1996; Sanchez et al.,
1996; Schuchardt et al., 1994), as mice carrying null
alleles in each case exhibit intestinal aganglionosis.
However, given the high likelihood that mutations in
RET and the endothelins account for no more than
50% of all cases of HSCR (Attie et al., 1995;
Chakravarti, 1996; Seri et al., 1997), substantial
additional genetic factors remain to be discovered.

The importance of RET in the development of
neural crest derivatives, and HSCR in particular,
suggested that downstream components of the RET
signal transduction pathway might also predispose to
HSCR susceptibility. Recently, the SH2 domain-
containing protein Grb10 (Ooi et al., 1995) was found
to be a constituent of this pathway. Using mouse Ret
as bait in a yeast two-hybrid assay, Pandey et al. (1995)
isolated Grb10 as prey from an embryonic day 10.5
expression library. In vitro binding studies and co-
immunoprecipitation experiments con®rmed these
results. Among other prey, Grb10 was also obtained
in a Ret two-hybrid screen by another group (Durick
et al., 1996). Using the cytoplasmic domain of human
RET as bait, we conducted another two-hybrid screen
of a mouse embryonic day 11 cDNA library. Among
the 21 positives selected for signal strength in the
b-galactosidase assay was a mouse cDNA correspond-
ing to the SH2 and pleckstrin homology domains of
Grb10 (Angrist, 1996). Thus, in several independent
two-hybrid screens of early embryonic libraries, the
SH2 domain of Grb10 was found to interact with the
intracellular domain of Ret.

Correspondence: A Chakravarti
Received 16 April 1998; revised 18 June 1998; accepted 18 June 1998

Oncogene (1998) 17, 3065 ± 3070
ã 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00
http://www.stockton-press.co.uk/onc



GRB10 was also viewed by us as a candidate for
HSCR susceptibility for reasons other than its
association with RET. Its expression in the early
embryo is consistent with a role in enteric nervous
system development (Angrist, 1996; Okamoto and Ueta,
1967; Pandey et al., 1995; Webster, 1973). In addition to
an SH2 domain at its carboxy terminus, Grb10 also
contains a pleckstrin homology (PH) domain of *100
amino acids (Margolis, 1994; Ooi et al., 1995). PH
domains have been implicated in cellular signaling and
cytoskeletal organization, especially in those molecules
associated with cell membranes (Shaw, 1996).

The sequence that codes for the PH domain that is
present in the so-called hGrb-IRb/Grb10 and
hGRB10/IR-SV1 isoforms of GRB10 (Frantz et al.,
1997; O'Neill et al., 1996) is part of a larger region in
the coding sequence that bears striking identity to the
C. elegans cosmid F10E9.6 and the gene it contains,
mig-10. Mig-10 is critical for migration of several
groups of neurons. What these neurons have in
common is that, like presumptive enteric neurons in
the mammal, they all undergo long-range migrations
along the antero-posterior axis. Additionally, mutant
mig-10 animals all have shortened posterior excretory
canals. Manser and Wood (Manser et al., 1997;
Manser and Wood, 1990) have suggested that the
pleiotropic nature of this mutation may arise as the
result of a defect in a component of the basal lamina
that is critical for both canal outgrowth and neuronal
migration. Thus, given the failure of excretory cell
migration and the presence of non-cell autonomous
defects, pleiotropy, incomplete penetrance as well as
the potential involvement of the extracellular matrix, it
is clear that mig-10 mutant and HSCR phenotypes
share several salient genetic features.

In order to assess GRB10's role in HSCR and
further characterize the gene, we have con®rmed its
map location, isolated bacterial arti®cial chromosomes
(BACs) containing human genomic GRB10 sequence,
determined its intron-exon boundaries, and identi®ed a
highly polymorphic microsatellite in intron 13. We
have also screened a panel of HSCR patients and
families for linkage to chromosome 7p by genotyping
microsatellite polymorphisms and for mutations in
GRB10 by nucleotide sequencing. No evidence of
linkage was observed and no mutations were detected
in HSCR patients.

Initially, GRB10 was mapped to human chromo-
some 7 by somatic cell hybrid mapping. Using unique
primers from the GRB10-derived expressed sequence
tag (EST) c-1kf03 (GenBank accession no. Z43779), a
103 bp product was PCR-ampli®ed essentially as
described (Angrist et al., 1995a; 538 annealing
temperature). Primers used were: C1KF03.F:
5'-GAGAGGTGCTTGGAAGACCAT-3' and CIK-
F03.R: 5'-AGAACTCGTATTTTGCGTAAT-3'. Chro-
mosomal mapping was accomplished by PCR genotyp-
ing against the National Institute of General Medical
Sciences (NIGMS) human 6 rodent somatic cell
hybrid mapping panel 2 (Coriell Institute for Medical
Research, Camden, NJ, USA. Regional chromosomal
localization was performed using the Stanford G3
Radiation Hybrid Mapping Panel as DNA template, as
previously described (Angrist et al., 1995b). Both
somatic cell hybrid and radiation hybrid mapping
experiments yielded a single, unambiguous band. Data

were submitted to the Stanford RH Web Server (http://
www-shgc.stanford.edu/rhserver2/rhserver_form.html)
for map analysis. RH mapping re®ned the gene's
position to within 35.2 centiRays (&700 kb) of
D7S2467 and 52.2 centiRays (&1050 kb) of D7S2422
by radiation hybrid mapping. The best location is
depicted in Figure 1. Based on a recent integrated
physical and genetic map of human chromosome 7
(Bou�ard et al., 1997), the cytogenetic location of
GRB10 can be deduced to be 7p11.2 ± 7p12. This map
placement is in close agreement with other recently
reported ¯uorescence in situ hybridization and radia-
tion hybrid mapping data from two independent
groups (Dong et al., 1997; Jerome et al., 1997).

Genomic DNA containing GRB10 was obtained by
screening a BAC library with a fragment derived from
the cDNA clone obtained in an earlier two-hybrid
screen (Angrist, 1996) and that most closely resembled
the sequence of human GRB-IR (Liu and Roth, 1995).
A 1164 bp SpeI fragment (nucleotides 661 ± 1824)
derived from this clone was excised from a two-hybrid
system prey vector (pGAD10, a gift from Dr Stanley
Fields, University of Washington, Seattle, WA),
cleaned using the Wizard DNA Clean-up System
(Promega, Madison, WI, USA) and used to probe
the Research Genetics (Huntsville, AL) Bacterial
Arti®cial Chromosome (BAC) Library ®lters. Hybridi-
zations, isolation of BAC DNA and manual sequen-
cing were performed as described (Angrist et al. 1998).
The gene was found to contain at least 16 exons
(Figure 1, Table 1). Exon and approximate intron sizes
are listed in Table 1. The genomic boundaries of
human GRB10 are estimated to encompass at least
47 kb.

A potentially polymorphic dinucleotide repeat
([TG]25TAA[GA]2G[TG]6) was detected 123 bp up-
stream of the 5' end of exon 14. We ampli®ed this
sequence using radioactively end-labeled primer in 50
CEPH control individuals and 161 HSCR patients and
their families. Primers used were: GRBIRCAR.F1: 5'-
GTCTTGGT-GCTTGCCTGGTGTG-3' and GRBIR-
CAR.R1: 5'-GGCTGTCACGGAGGAGAAAAAG-3'.
Ampli®cation conditions were as described (Puffen-
berger et al., 1994b), except the annealing temperature
was 568C and the Mg2+ concentration was 0.75 mM.
Allele sizes and frequencies of the microsatellite
marker, designated GRB10-CAn, are listed in Table 2,
with a heterozygosity of 0.82 determined empirically in
CEPH control individuals. When this marker was
tested for linkage in HSCR families and sib pairs, no
evidence of linkage or increased allele sharing among
a�ected individuals was detected. In order to assess
allele sharing among a�ecteds, we utilized the
nonparametric linkage (NPL) test, as implemented in
the program GENEHUNTER (Kruglyak et al., 1996).
This analysis yielded NPL Z scores of 70.42 (P=0.69)
for nine large kindreds, 70.38 (P=0.64) for 30 sib
pairs, and 70.55 (P=0.71) for all families segregating
HSCR combined. Additionally, we obtained maximum
two-point parametric lod scores for HSCR versus
GRB10-CAn of 77.3 for nine large families, 75.3 for
30 sib pairs, and 712.6 for the combined family data.
Parametric analyses assumed a rare autosomal
dominant gene (P=0.001) and sex-dependent reduced
penetrance in males (58%) and females (29%), as
described in Angrist et al. (1993).

GRB10 structure and analysis in Hirschsprung disease
M Angrist et al

3066



After obtaining informed consent, a panel of 85+
biopsy-proven HSCR patients was screened for
mutations in GRB10. Patients were chosen without
regard to segment length or accompanying pheno-
types; the panel used here closely resembled that used

in our previous non-Mennonite HSCR studies
(Angrist 1995a, 1996) and represented the broad
spectrum of segment length and associated manifesta-
tions of neural crest disorders. Using the intron-exon
boundary information obtained from direct BAC

Figure 1 (Top) Genetic map of human chromosome 7p11.2-p12. Horizontal bar indicates most likely location of GRB10 based on
radiation hybrid mapping; marker locations are from Dib et al. (1996). (Bottom) Schematic representation of GRB10 exons. The
relative size of each exon is indicated by the size of its box; intronic sequence is not represented. Below the exons, the corresponding
protein domains are depicted. `Exon 7a' is presumed to exist 3' of exon 7 based on reports of another GRB10/GRB-IR/hGRB10g
isoform (Frantz et al., 1997; O'Neill et al., 1996). The top and bottom portions of the ®gure are not drawn to the same scale

Table 1 Human GRB10 genomic structure

Exon Size (bp) Position in
cDNA

a
(nt)

Intron size (kb) Splice acceptor Splice donor

1
2
3
4
5
6
7
7A

b

8
9
10
11
12
13
14
15

82
88
223
142
157
118
69
138
111
99
78
117
67
88
94
539

1 ± 82
83 ± 170

171 ± 393
394 ± 535
536 ± 692
693 ± 810
811 ± 879
880 ± 1017
880 ± 990
991 ± 1089
1090 ± 1167
1168 ± 1284
1285 ± 1351
1352 ± 1439
1440 ± 1533
1534 ± 2072

*2.9
*6.0
*5.7
*1.6
*2.2
*1.2
*3.5
53.3
*2.0
*6.7
*1.1
*1.1
*0.3
*9.0
*2.0
±

5' UTR
ttttttttagGACAAGGTG
ttctgtgcagAGGATGATG
tgccttccagGCGCCTTCA
tgtgttgcagGATGTTAAA
gcccctgcagAGAGAGGTG
attttcttagAATTTCTTC
tcttcaacagAATTTTCTG
gttgtcacagGAACCCAGA
cctcctgtagCCAAACAAA
tcctctccagTATGGAATG
tccttttcagGCCAGTGTC
tcttttgcagAAGCGAAGC
ccccccacagTGATTCACA
ccttccccagGCTTTTTCT
gtctctccagTGCGAGGAC

TACTACCAGgtatgggcga
ATCACCAGCgtaaggaatg
CGCTGTCAGgtaggtccag
GCAAAGCAGgtgagtgtgc
TAGGATTAGgtagggaacc
AATCCCATGgtgagtctta
CTTTTGCAGgtactgggcc
ACTTCAAAGgtgagcttta
TGCATAAAGgtacccacga
CTCCTCAAGgtatgtgaca
ACGCCAGTGgtaagtaaag
CCCTGGAGGgtaaggcccc
TAAGTACAGgtaaacaggg
CGTGGATGGgtaaggaacc
ATCTTACCTgtaagtattg
3' UTR

a
cDNA sequence from Liu and Roth (1995).

b
Position based on additional GRB10 splice variants reported by others (Frantz et al., 1997).

GRB10 structure and analysis in Hirschsprung disease
M Angrist et al

3067



sequencing, primers were designed to PCR amplify the
16 exons of human GRB10 (Table 3). PCR
ampli®cation and sequencing were carried out as
described (Angrist et al., 1998). Beyond the micro-
satellite marker, several sequence variants were found
in GRB10 in patients (Table 4). All were silent

changes, none of which are expected to disrupt
splicing or otherwise interfere with proper GRB10
function, although this cannot be demonstrated
conclusively in the absence of functional studies.
Interestingly, two of the three intronic variants
altered two closely positioned but non-consecutive
nucleotides (Table 4), suggesting that GRB10 might be
undergoing gene conversion. We consider this to be a
reasonable hypothesis, given that these sequence
changes reside outside canonical motifs necessary for
proper splicing (Senapathy et al., 1990) and that there
are at least three other extant genes in the genome
closely related to GRB10 (Daly et al., 1996; Margolis,
1994).

As reported here, GRB10 was found to span a
genomic region of *47 kb and to include at least 16
exons (Figure 3). These exons encompass all published
GRB10 splice variants and include both the unique N-
terminus of hGRB-IR and the full-length PH domain
of hGRB-IRb (also called GRB-IRPH and hGRB10/IR-
SV1; Frantz et al., 1997; Liu and Roth, 1995, 1998;
O'Neill et al., 1996) Each intron-exon boundary listed

Table 2 Allele sizes and frequencies for microsatellite marker
GRB10-CAn in 50 unrelated individuals

Allele Size (bp) Frequency

1
2
3
4
5
6
7
8
9
10
11

224
226
228
230
232
234
236
238
240
242
244

0.01
0.05
0.01
0.34
0.09
0.02
0.07
0.05
0.13
0.14
0.09

Table 3 Primers used for genomic ampli®cation of human GRB10 exons

Exon

Exon

size
(bp)

Product

size
(bp)

Annealing
temperature
(8F) Forward primer (5'®3')

Distance to
5' end of
exon (bp) Reverse primer (5'®3')

Distance
from 3' end
of exon (bp)

1
2
3
4
5
6
7
7A
8
9
10
11
12
13
14
15

82
88
223
142
157
118
69
138
111
99
78
117
67
88
94
539

213
159
321
298
242
248
172
242
213
231
198
188
164
278
316
514

58
55
56+DMSO
58
55
53
50
51
59
54
56
59
55
58+DMSO
53
59

GGCTTGGCTTCTCACAGTCTG
TTCTGCTGCGGTCCTGTTTTT
CCTGATCACCAAGATGTACA
GTGTAAGGCTGGGTCAT
TTTCTTGAAAGCCCGAAGTT
TACCATGAATTTCCCACCTGT
CTTTGQAGCTAACCTTTTACG
GCTGATAACATGTCTGCTTTA
TTGCCTTTGCTGTGCTTGAG
TTCTGACTCCCTGTGTGAACA
TGCTGTGGCGTTTGTCAC
CTGCGGCCTTTCCTTTTC
GTCTGCTGTCCTCGGTGCTAA
GGCCAGAGTGCACCACACAAAG
ACTTAAATGCCAAAGCACTGC
CGTTCTGTTCCCTGAGGTGGC

58 to ATG
5
63
36
40
32
74
47
27
29
55
2
19
113
147
37

ATCTATGGCTGGTGGCGACAT
CAAGCTAGAACTGGGAGTGT
GAGGCCTGGACCTACCTG
ACTTCCCGCCCTTCTTCC
AGGTGCCAATCCTGTTCTGA
TGATACTATGAAAACCCAAGT
CAAGAGGATTTCTATTCTGAA
ATTAGGGCTGGTGGTGGTAGC
AGTCTCCTGTGGGCTGCTGAG
CTGCCAGAATAGACATCAAGT
TGAAGCTGAAAAGGCACT
GGCTACCACCTTGAGGGT
GGGGTGCTGTTTGATTTTCTT
CCCCAGCACAATAAAACCTTAG
GAATGAAACCAGAAAACAAAC
CTCCGGTTCTTGTTCCTAAGC

62
24
0
84
12
56
94
12
33
61
29
33
36
35
33
288 to TGA

Table 4 Polymorphisms detected in human GRB10

GRB10
intron/ Nucleotide

Amino
acid

Enzyme
site

HSCR patients Controls

exon change change Domain change +/+ +/ ± ± / ± +/+ +/± ± / ± w2 (P)

intron 1 ATA?GTC 15 bp 3'
of exon 1

unique to
GRB-IR

TaiI 102 1 6 275 25 15 1.33
(0.249)

exon 3 CCG?CCA P105P unique to
GBR-IR

AvaI
StyI

38 26 60 16 53 38 0.045
(0.832)

exon 6 TAC?TAT Y249Y GM ± 75 8 0 74 8 0 0.857
(0.355)

exon 6 CCC?CCA P258P GM StyI 78 5 0 74 8 0 0.346
(0.556)

intron 6 G?A 3 bp 3'
of exon 6

GM HphI
PleI, HinfI

79 4 0 74 8 0 0.760
(0.383)

intron 11 GTGGG?
ATGGA

18 bp 5'
of exon 12

GM ± 72 6 0 80 3 0 0.552
(0.457)

GM=Grb and Mig (see text). `+' refers to previously published alleles in GRB10 cDNA (Frantz et al., 1997; Liu and Roth, 1995; O'Neill et al.,
1996), `±' refers to alleles not previously described

GRB10 structure and analysis in Hirschsprung disease
M Angrist et al

3068



is described in Table 1 and contains the GT-AG
consensus sequences for eukaryotic donor and acceptor
splice sites (Senapathy et al., 1990).

Several factors might explain why GRB10 was not
found to predispose to HSCR in our study. First,
although there is little doubt that Grb10 and Ret
interact in the mouse, this may not be the case in the
human. Also, it is not clear that this interaction is
essential in vivo; in the absence of targeted Grb10
mutations in mice, it is di�cult to gauge whether
Grb10 is truly unique in its function, or is merely a
redundant member of a large family of SH2 adapter
proteins. Closely-related family members Grb7 and
Grb14 also contain proline-rich, GM, PH and SH2
domains. Moreover, Ret appears to be one of several
signaling molecules that exhibits high a�nity for
Grb10. Both the insulin receptor (IR) and the type I
insulin-like growth factor receptor (IGF-IR) have been
shown to interact directly with Grb10 (reviewed in
(Morrione et al., 1997). Also, recent reports have
shown that human GRB10 isoforms are di�erentially
expressed in insulin target cells such as skeletal muscle,
liver and adipocytes (Dong et al., 1997). Thus, GRB10
may be more important in mediating insulin signaling
than in enteric neurogenesis. Lastly, although we were
able to screen all of the GRB10/GRB-IR exons in
patients and controls, we did not screen the introns or
the upstream and downstream untranslated sequences.
In addition, given that exon 7A was found to reside in
intron 7, it is not unlikely that additional exons may
exist within other introns. Indeed, there are now four
known isoforms in the human (Dong et al., 1997).
Nevertheless, linkage analysis of HSCR families using

a highly polymorphic microsatellite marker within the
gene suggests that GRB10 mutations cannot account
for a substantial fraction of familial HSCR.

To date, all reported HSCR susceptibility genes are
members of the RET signaling pathway (RET,
GDNF), the G protein-coupled receptor (GPCR)
endothelin-B pathway (EDNRB, EDN3), or the
SRY-like HMG-box family of transcription factors
(SOX10; Pingault et al., 1998; Southard-Smith et al.,
1998). We believe that the presence of both SH2 and
PH domains in the Grb10 family of proteins suggest a
possible model for uniting the RET and GPCR
pathways. There is now evidence that human
GRB10 is a common target for kinases existing in
both the MAP kinase and phosphatidylinositol 3-
kinase signal transduction pathways (Dong et al.,
1997). These data, as well as the importance of SH2
domains in RTK signaling and PH domains' possible
role in G protein-coupled receptor-mediated signaling,
suggest that GRB10 family members might serve as a
conduit for transducing extracellular neuroenteric
signals to the nucleus and perhaps, SOX10 or other
related transcription factors therein.

Acknowledgements
We are extremely grateful to the families who make all of
our studies possible, to Dr Akhilesh Pandey for reagents
and helpful discussion, and to Nydia Bringht-Twumasi for
expert technical assistance. This research was partially
supported by a grant from the National Institutes of Child
Health and Development, National Institutes of Health
(HD-28088).

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	Genomic structure of the gene for the SH2 and pleckstrin homology
domain-containing protein GRB10 and evaluation of its role in
Hirschsprung disease
	Acknowledgements
	References