SHORT REPORT Allele dosage-dependent penetrance of RET proto-oncogene in an Israeli-Arab inbred family segregating Hirschsprung disease Lina Basel-Vanagaite*,1, Anna Pelet2, Zvi Steiner3, Arnold Munnich2, Yoram Rozenbach4, Mordechai Shohat1 and Stanislas Lyonnet2 1Department of Medical Genetics, Rabin Medical Center, Beilinson Campus, Petah Tikva, Israel; 2Université Paris- Descartes, Faculté de Médecine, Hôpital Necker-Enfant Malades, Paris, France; 3Division of Pediatric Surgery, Hillel- Yaffe Medical Center, Hadera, Affiliated with the Rappaport Faculty of Medicine, The Technion, Haifa, Israel; 4Division of Gastroenterology and Nutrition, Schneider Children’s Medical Center of Israel, Petah Tikva, Israel Hirschsprung disease (HSCR) is characterised by intestinal obstruction resulting from an absence of ganglion cells in the intestinal tract. The mutations in the major gene, RET, associated with isolated HSCR, are dominant loss-of-function mutations with incomplete penetrance and variable expressivity. We have ascertained a large inbred Israeli-Arab family segregating HSCR. Sequencing of the RET gene showed a splicing mutation, IVS6 þ 5G�4A, in the homozygous state in all the females with severe forms of HSCR and in the heterozygous state in the male patient with short-segment HSCR. The recently described hypomorphic-RET predisposing allele, rs2435357, was transmitted in the heterozygous state to the male patient, but was not transmitted to the three affected females. Although the heterozygous IVS6 þ 5G�4A is of low-penetrance for short-segment HSCR disease, the homozygous state is fully penetrant for total aganglionosis or long-segment HSCR. As in other inbred populations segregating a weakly penetrant RET allele (Mennonite), our findings support the hypothesis that the penetrance of RET gene mutations for the HSCR phenotype depends on: (i) the nature of the mutation, (ii) the allele dosage and (iii) modifier-loci. European Journal of Human Genetics (2007) 15, 242–245. doi:10.1038/sj.ejhg.5201733; published online 8 November 2006 Keywords: Hirschsprung; RET mutation; penetrance Introduction Hirschsprung disease (HSCR) is characterised by intestinal obstruction resulting from the absence of ganglion cells in a variable portion of the intestinal tract. The incidence of HSCR is 1:5000 live-births in Caucasian populations.1 Symptoms range from abdominal distension and failure to pass stools in neonates, to chronic constipation and enterocolitis in childhood. In 80% of individuals, agan- glionosis is restricted to the rectosigmoid colon (short- segment disease, S-HSCR), but in B15% the aganglionosis extends proximal to the sigmoid colon (long-segment disease, L-HSCR). In B5% of individuals, aganglionosis affects the entire large intestine (total colonic aganglio- nosis, TCA). Total intestinal aganglionosis (TIA) extending from the duodenum to the rectum is the rarest form and is usually fatal. The RET gene, located on chromosome 10q11.21, is the major gene in nonsyndromic HSCR.2,3 The vast majority of families with HSCR show linkage to the RET locus.4 Heterozygous mutations within the RET gene coding sequence are identified only in 50% of (linked) families and 10–20% of sporadic cases5 – 9 and are char- acterised by incomplete sex biased penetrance and a Received 27 July 2006; revised 26 September 2006; accepted 4 October 2006; published online 8 November 2006 *Correspondence: Dr L Basel-Vanagaite, Department of Medical Genetics, Rabin Medical Center, Beilinson Campus, Petah Tikva 49100, Israel. Tel: þ 972 3 937 7659; Fax: þ 972 3 937 7660; E-mail: basel@post.tau.ac.il European Journal of Human Genetics (2007) 15, 242–245 & 2007 Nature Publishing Group All rights reserved 1018-4813/07 $30.00 www.nature.com/ejhg variable extension of aganglionosis.10,11 The estimated penetrance is 72% in males and 51% in females.5 Recently a major role of noncoding variations in intron 1 of RET (hypomorphic alleles) has been demonstrated by several studies.9,12 – 15 In this report, we describe an inbred Israeli-Arab family with HSCR where a splicing mutation segregates in affected family members either in the homozygous state resulting in TCA or L-HSCR (females) or in the heterozygous state resulting in S-HSCR (male). Patients The patients are all members of a large consanguineous Israeli-Arab family. In one branch of the family three female siblings have TCA (individuals III-5 (deceased), III-6 and III-9, Figure 1) and one female has L-HSCR (individual III-11, Figure 1), and in the second branch of the family one male has S-HSCR (individual III-2, Figure 1). Patients III-4, III-8 and III-11 also have congenital autosomal recessive ichthyosis, which is unrelated to the HSCR. The clinical features of the patients are summarized in Table 1. The research study was reviewed and approved by the Ethics Committee (CCPPRB approval 95-05-03, AP-HP, Paris). Methods DNA was isolated by standard methods. Linkage analysis to the RET gene locus was performed using polymorphic genetic markers D10S141, RET INT-5 and StCl2, followed by sequencing of the SNP rs2435357 (IVS1-C4T). Mutation screening of the coding sequence of the RET gene was performed with primers designed for exons and the flanking splice sites. PCR products were directly sequenced in both directions on an ABI PRISM 3100 DNA sequencer (Perkin Elmer-Applied Biosystems) using the Big Dye Terminator method according to the manufacturer’s instructions. Results Linkage to the RET gene locus was established. The haplotype 165-C-184-144 segregated with the HSCR pheno- type (Figure 1). A splice-mutation, IVS6 þ 5G�4A, was identified; this was not found in 120 control chromo- somes. After the complete sequencing of the RET gene, no other sequence changes were found. The mutation IVS6 þ 5G�4A involves a known canonical splice site, where the intronic nucleotide þ 5 is known to participate in the donor splice site processes. When the IVS6 þ 5G�4A mutation was tested with a programme (http://www.fruitfly.org/seq_tools/splice.html), which is designed to predict the efficiency of splice sites, the score of the wild donor site sequence decreased from one to 0.54. All the patients with TCA and the patient with L-HSCR were homozygous for the mutation (Figure 1). The same mutation in heterozygous state was found in the boy with Figure 1 The haplotypes and mutation analysis in the affected and unaffected family members in the families with HSCR. The order of the genetic markers analysed is shown in the upper left-hand corner. The arrow indicates the proband. Penetrance of RET depends on allele dosage L Basel-Vanagaite et al 243 European Journal of Human Genetics S-HSCR and in seven healthy family members (Figure 1). Individuals II-1, II-4 and III-2 carried the hypomorphic allele T (SNP rs2435357) in trans. The rs2435357 allele was not present in any of the females homozygous for the IVS6 þ 5G�4A mutation. Discussion Heterozygous mutations of the RET proto-oncogene occur in families with TCA, L- or S-HSCR. 7,16 – 18 Homozygous RET gene mutations causing HSCR are extremely rare. Only one patient with TIA and three with TCA owing to homozygous RET mutations have been reported in the literature (Table 2). In the family described by Geneste et al,19 as in our patients, TCA was caused by a homozygous RET gene mutation, whereas in contrast to our family, L-HSCR in another set of their patients was caused by the same mutation in the heterozygous state. In this study, we illustrate the effect of RET gene dosage on the penetrance and expressivity of the HSCR pheno- type. Although the IVS6 þ 5G�4A mutation in hetero- zygous state is of low penetrance for S-HSCR disease (less than 12.5% if the obligate untyped carriers in generation I are included), there is full penetrance (100%) in the homozygous state. Homozygous patients show little varia- bility of expression (TCA with small bowel involvement and L-HSCR). In a study describing homozygous mutation inheritance in the EDNRB in a large inbred Mennonite kindred with HSCR, most of the affected individuals were homozygous for the mutated allele, although some heterozygotes were also described.20 Homozygotes and heterozygotes for the EDNRB mutation W276C had a 74 and 21% risk, respec- tively, of developing HSCR. The EDNRB-mutation showed incomplete penetrance, as some unaffected individuals from this family were also found to be homozygous. In addition, some affected individuals did not carry the mutation, suggesting the presence of additional suscept- ibility loci contributing to HSCR inheritance.21 Even in an isolated population, such as the Mennonites, HSCR is a multigenically inherited disease involving interaction between the hypomorphic-EDNRB allele and one or more RET HSCR-susceptibility variants.22 In our study, all the family members who are homozygous for the RET gene mutation have severe forms of HSCR. None of them carries the hypomorphic allele rs2435357, which we chose because of homology and evolutionary conservation between rodents and primates and because in vitro studies have highlighted an enhancer role for this region.15,23 Two males, II-1 and III-2, are heterozygotes for the splice mutation and the hypomorphic allele; however, individual Table 1 Clinical characteristics of the patients Patient Age Age at onset of the disease Clinical presentation Age at operation Extension of aganglionosis III-2 5 years 5 mo Constipation 6 mo S-HSCR III-5 Died at the age of 21 2 years from liver failure 2 days Abdominal distension, vomiting 15 mo TCA III-6 10 years 2 days Abdominal distension 14 mo TCA+6 cm of terminal ileum III-9 4 years 2 days Abdominal distension, bilious vomiting 7 mo TCA+10 cm of terminal ileum III-11 1 year 1 day Abdominal distension, vomiting 9 mo L-HSCR (2/3 of colon up to hepatic flexure) mo ¼ months. Table 2 Reported patients with homozygous RET mutations and their heterozygous siblings Mutation Genotype Gender Extension of aganglionosis Reference R313Q M/M ? TCA with small bowel involvement Seri et al7 A969T M/M or M/� Male TIA Inoue et al17; Shimotake et al18 L1061P M/M Female TCA Geneste et al19 M/M Female TCA M/wt Male L-HSCR IVS6+5G�4A M/M Female TCA with small bowel involvement This study M/M Female TCA with small bowel involvement M/M Female L-HSCR M/wt Male S-HSCR M ¼ mutated allele, wt ¼ wild-type allele, � ¼ deletion of entire RET exon(s). Penetrance of RET depends on allele dosage L Basel-Vanagaite et al 244 European Journal of Human Genetics III-2 is affected (S-HSCR), whereas his haplo-identical father, II-1, is unaffected. Additional genetic changes are thought to be responsible for the variable expressivity of the disease in the homozygous and heterozygous patients described in this study. As suggested in other inbred populations segregating a weakly penetrant RET predisposing allele, our findings support the hypothesis that the penetrance of RET gene mutations for the HSCR phenotype depend on: (i) the nature of the mutation, (ii) the allele dosage and (iii) the modifier-loci. The results of this study emphasise the importance of ascertaining the molecular basis of HSCR in families with more than one affected individual, especially if they originate from a small-inbred population. The detection of a RET gene mutation allows the families to be offered genetic counselling and enables early disease detection in the homozygous individuals. Acknowledgements We are grateful to the families who participated in this study. We thank Dr Gabrielle Halpern for her help with editing the manuscript and Irit Lis for preparing the figures. References 1 Bodian M, Carter C: A family study of Hirschsprung disease. 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Am J Med Genet 2000; 93: 278–284. 18 Shimotake T, Go S, Inoue K, Tomiyama H, Iwai N: A homozygous missense mutation in the tyrosine E kinase domain of the RET proto-oncogene in an infant with total intestinal aganglionosis. Am J Gastroenterol 2001; 96: 1286–1291. 19 Geneste O, Bidaud C, De Vita G et al: Two distinct mutations of the RET receptor causing Hirschsprung’s disease impair the binding of signalling effectors to a multifunctional docking site. Hum Mol Genet 1999; 8: 1989–1999. 20 Puffenberger EG, Kauffman ER, Bolk S et al: Identity-by-descent and association mapping of a recessive gene for Hirschsprung disease on human chromosome 13q22. Hum Mol Genet 1994; 3: 1217–1225. 21 Puffenberger EG, Hosoda K, Washington SS et al: A missense mutation of the endothelin-B receptor gene in multigenic Hirschsprung’s disease. Cell 1994; 79: 1257–1266. 22 Carrasquillo MM, McCallion AS, Puffenberger EG, Kashuk CS, Nouri N, Chakravarti A: Genome-wide association study and mouse model identify interaction between RET and EDNRB pathways in Hirschsprung disease. Nat Genet 2002; 32: 237–244. 23 McCallion AS, Stames E, Conlon RA, Chakravarti A: Phenotype variation in two-locus mouse models of Hirschsprung disease: tissue-specific interaction between Ret and Ednrb. Proc Natl Acad Sci USA 2003; 100: 1826–1831. Penetrance of RET depends on allele dosage L Basel-Vanagaite et al 245 European Journal of Human Genetics Allele dosage-dependent penetrance of RET proto-oncogene in an Israeli-Arab inbred family segregating Hirschsprung disease Introduction Patients Methods Results Discussion Acknowledgements References