Deleted and normal chromosome 10 homologs from a patient with Hirschsprung disease isolated in two cell hybrids through enrichment by immunomagnetic selection

Abstract

A cytogenetically detectable deletion, del(10) (q11.2→q21.2), was observed in a patient with total colonic aganglionosis with small bowel involvement (TCSA), a variant of Hirschsprung disease (HSCR). A similar deletion is present in another TCSA patient (S.M. Huson, personal communication). To reveal cytogenetically undetectable deletions of chromosome 10 in further patients, we developed a strategy for mapping chromosome 10 DNA markers with respect to the observed deletions. To this end, the two chromosome 10 homologs (deleted and normal) were segregated in two distinct somatic cell hybrids obtained after fusion of the patient’s fibroblasts with a Chinese hamster ovary cell line (YH21). Hybrid cells containing chromosome 10 were selected for the expression of the gene coding for the β subunit of the fibronectin receptor (FNRB), which maps to lOpl 1.2, using a monoclonal antibody against FNRB. Hybrid 185.0 contains the deleted chromosome, whereas hybrid 179.Q contains the nondeleted one. Southern blot and PCR analysis of DNA from these two hybrids mapped the markers RBP3H4, RET, D10S15, D10S5, D10S22, and D10S88 inside the deletion and D10S170, CDC2, EGR2, and D10S19 outside the deletion. MEN2A and MEN2B have recently been mapped within the centromeric region closely linked to RBP3 and D10S15 (which are located inside the deletion) and cosegregate with HSCR in at least two different pedigrees. Since HSCR, MEN2A, and MEN2B represent defects of neural crest cell development, we hypothesize that they originate from mutations in different genes clustered in the centromeric region of 1 Oq.

Recurrent deletions and reciprocal duplications of 10q11.21q11.23 including CHAT and SLC18A3 are likely mediated by complex low-copy repeats

“…Comparison with previously reported individuals with deletions encompassing 10q11.2 [Bisgaard et al, 2007; Fewtrell et al, 1994; Fryns et al, 1991; Ghai et al, 2011; Holden and MacDonald, 1985; Kirchhoff et al, 2005; Lobo et al, 1992; Puliti et al, 1993; Shapiro et al, 1985; Zenger‐Hain et al, 1993] is uninformative because most of these deletions were identified by standard cytogenetic analysis and are substantially larger than the deletions reported here. Only two of the 15 deletions in our study for which parental samples were available were de novo.…”

“…Mutations in RET have been reported in patients with multiple endocrine neoplasia, type IIA (MEN2A; MIM# 171400), MEN, type IIB (MEN2B; MIM# 162300), Hirschsprung disease (HSCR; aganglionic megacolon; MIM# 142623), and medullary thyroid carcinoma (MTC; MIM# 155240). Interestingly, Puliti et al [1993] described a patient with a cytogenetically visible deletion 10q11.2q21.2 and a variant of HSCR.…”

Close linkage with the RET protooncogene and boundaries of deletion mutations in autosomal dominant Hirschsprung disease

“…The segregation in a somatic cell hybrid of the deleted chromosome 10 from our HSCR patient (corresponding to patient 1 of table 2) was previously described (6). Using the same approach, two additional hybrids retaining the deleted chromosome 10 respectively from patients 2 and 3 of table 2 were isolated.…”

“…These findings suggested the presence of a HSCR gene in the proximal long arm of chromosome 10. Based on this hypothesis, we segregated the deleted and normal chromosomes 10 from one of the patients(5) into 2 distinct somatic cell hybrids (6) and mapped a series of chromosome 10 markers with respect to the deletion. Linkage analysis in 15 HSCR pedigrees showing autosomal dominant inheritance indicated the interval between loci D10S208/D10S199 and D10S196/D10S220/D10S225, spanning 9.9 cM, as the most likely location of the gene (7).…”

Identification and Computational Analysis of Gene Regulatory Elements

“…For example, a recent study revealed a strong association between Hirschsprung disease risk and the presence of a regulatory mutation located in the first intron of the RET proto-oncogene (Emison et al 2005). RET was previously linked with Hirschsprung disease (Puliti et al 1993) and the development of multiple types of cancer (Donahue and Hines 2009; Pacini et al 2010). This particular mutation, however, has a 20-fold greater contribution to risk than do rare alleles, and the authors of the study showed that the acquired mutation is capable of significantly decreasing the level of RET expression.…”