RESEARCH ARTICLE OFFICIAL JOURNAL www.hgvs.org Allele-Specific Expression at the RET Locus in Blood and Gut Tissue of Individuals Carrying Risk Alleles for Hirschsprung Disease Ivana Matera, 1,2 Marco Musso, 1 † Paola Griseri, 1 Marta Rusmini, 1,2 Marco Di Duca, 3 Man-ting So, 4 Domenico Mavilio, 3,5 Xiaoping Miao, 6 Paul HK Tam, 4 Roberto Ravazzolo, 1,7 Isabella Ceccherini, 1 ∗ and Merce Garcia-Barcelo 4 ∗ 1 UOC Genetica Medica, Istituto Giannina Gaslini, Genova, Italy; 2 Unit of Clinical and Experimental Immunology, Humanitas Clinical and Research Center, Milan, Italy; 3 Laboratorio di Fisiopatologia dell’ Uremia, Istituto Giannina Gaslini, Genova, Italy; 4 Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China; 5 Department of Medical Biotechnologies and Translational Medicine, University of Milan, Milan, Italy; 6 Department of Epidemiology and Biostatistics, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 7 DINOGMI, Universit ` a degli Studi di Genova, Genova, Italy Communicated by Stylianos Antonarakis Received 29 October 2012; accepted revised manuscript 6 February 2013. Published online 25 February 2013 in Wiley Online Library (www.wiley.com/humanmutation). DOI: 10.1002/humu.22302 ABSTRACT: RET common variants are associated with Hirschsprung disease (HSCR; colon aganglionosis), a con- genital defect of the enteric nervous system. We an- alyzed a well-known HSCR-associated RET haplotype that encompasses linked alleles in coding and noncod- ing/regulatory sequences. This risk haplotype correlates with reduced level of RET expression when compared with the wild-type counterpart. As allele-specific expres- sion (ASE) contributes to phenotypic variability in health and disease, we investigated whether RET ASE could con- tribute to the overall reduction of RET mRNA detected in carriers. We tested heterozygous neuroblastoma cell lines, ganglionic gut tissues (18 HSCR and 14 non-HSCR indi- viduals) and peripheral blood mononuclear cells (PBMCs; 16 HSCR and 14 non-HSCR individuals). Analysis of the data generated by SNaPshot and Pyrosequencing re- vealed that the RET risk haplotype is significantly more expressed in gut than in PBMCs (P = 0.0045). No ASE difference was detected between patients and controls, ir- respective of the sample type. Comparison of total RET expression levels between gut samples with and without ASE, correlated reduced RET expression with preferen- tial transcription from the RET risk haplotype. Nonran- dom RET ASE occurs in ganglionic gut regardless of the Additional Supporting Information may be found in the online version of this article. † Present address: Centro della Microcitemia e delle Anemia Congenite-Ematologia, Ospedale Galliera, Genova, Italy. ∗ Correspondence to: Merce Garcia-Barcelo, Department of Surgery; 21 Sas- soon Road, Pokfulam, Hong Kong, SAR, China. E-mail: mmgarcia@hku.hk or Isabella Ceccherini, UOC Genetica Medica, Istituto Giannina Gaslini, L.go G. Gaslini, 5–16148 Genova, Italy. E-mail: isa.c@unige.it Contract grant sponsors: Istituto Superiore di Sanit ` a; Italian Ministry of Health (Bando Giovani Ricercatori GR-2008–1135082); Hong Kong Research Grants Council HKU (778610M); The University of Hong Kong Seed Funding Programme (200611159028); The University of Hong Kong Genomics Strategic Research Theme. disease status. RET ASE should not be excluded as a dis- ease mechanism acting during development. Hum Mutat 34:754–762, 2013. C  2013 Wiley Periodicals, Inc. KEY WORDS: Hirschprung disease; RET; regulatory poly- morphisms; allele-specific expression Introduction Hirschsprung disease (HSCR; MIM #142623) is a complex ge- netic disorder affecting one in 5,000 live births, characterized by the absence of enteric ganglia in variable lengths of the distal gastroin- testinal tract. Patients are classified according to the length of the aganglionic segment into short (S-HSCR) or long (L-HSCR) seg- ment aganglionosis and into total colonic aganglionosis when the whole colon is affected. The RET proto-oncogene (MIM #164761) is the major gene involved in HSCR, with loss of function mutations found in approximately 50% of familial and 10%–15% of sporadic cases. On the other hand, positional cloning and candidate gene analyses have identified additional genes (GDNF, NRTN, SOX10, EDNRB, EDN3, ECE1, ZFHX1B, PHOX2B, TCF4, and KIAA1279) with mutations in a total of 5% of mostly syndromic patients [Amiel et al., 2008]. In addition to coding sequence mutations in RET or other genes, common SNPs throughout RET are also associated with predisposition to HSCR [Borrego et al., 1999; 2003; Burzynski et al., 2005; Fitze et al., 1999; Garcia-Barcelo et al., 2003; Lantieri et al., 2006]. One of most common HSCR-associated RET haplo- types comprises two SNP alleles (rs10900296G/ A; rs10900297A/ C; associated allele underlined throughout the text) that map at –5 and –1 base pairs, respectively, from the RET transcription start site, and, a coding sequence SNP allele in exon 2 (rs1800858; c135G/ A; A45A) [Fitze et al., 2003; Garcia-Barcelo et al., 2005; Griseri et al., 2005]. These two RET promoter SNPs have been widely investigated as they are likely to affect RET transcription. However, contradic- tory results were reported when the ability to drive the expression of a reporter gene was compared between the different SNP alleles combinations, namely A- C, A-A, G- C and G-A [Fitze et al., 2003; Garcia-Barcelo et al., 2005; Griseri et al., 2005]. Lying on the same C  2013 WILEY PERIODICALS, INC.