A human model for multigenic inheritance: phenotypic expression in Hirschsprung disease requires both the RET gene and a new 9q31 locus

Abstract

Reduced penetrance in genetic disorders may be either dependent or independent of the genetic background of gene carriers. Hirschsprung disease (HSCR) demonstrates a complex pattern of inheritance with approximately 50% of familial cases being heterozygous for mutations in the receptor tyrosine kinase RET. Even when identified, the penetrance of RET mutations is only 50-70%, gender-dependent, and varies with the extent of aganglionosis. We searched for additional susceptibility genes which, in conjunction with RET, lead to phenotypic expression by studying 12 multiplex HSCR families. Haplotype analysis and extensive mutation screening demonstrated three types of families: six families harboring severe RET mutations (group I); and the six remaining families, five of which are RET-linked families with no sequence alterations and one RET-unlinked family (group II). Although the presence of RET mutations in group I families is sufficient to explain HSCR inheritance, a genome scan reveals a new susceptibility locus on 9q31 exclusively in group II families. As such, the gene at 9q31 is a modifier of HSCR penetrance. These observations imply that identification of new susceptibility factors in a complex disease may depend on classification of families by mutational type at known susceptibility genes.

Figure 1

HSCR families used in linkage analysis. Individuals with confirmed HSCR and clinical diagnosis of chronic severe constipation are indicated by filled and shaded symbols, respectively. Asterisks indicate individuals used in genotyping.

Figure 2

Linkage analysis of HSCR. (a) Chromosome 10. (b) Chromosome 9. The x axis indicates the genetic map of the indicated chromosome with intermarker distances in centimorgans (cM), and the y axis indicates the linkage score. (Solid line, NPL score; dashed line, parametric lod score. Although depicted on the same graph, NPL and lod scores have distinct properties and are not synonymous; thus, they should not be numerically compared against each other.)

Figure 3

Functional consequences of the S649S variant in RET splicing. (a) Normal and mutant splicing events of RET exon 11 and its resulting translation. The site of the G → A transition observed in HSCR family 7 does not alter the Serine649 residue but does create a sequence closely resembling the consensus splice acceptor site [(ct)₁₀ncagG]. (b) RT-PCR on RNA isolated from a lymphoblastoid cell line. One band of the expected size (383 bp) on normal splicing and a smaller band (314 bp) resulting from aberrant exon 11 splicing are amplified in affected carriers of the Serine variant (lanes 2–6). The unaffected grandmother (lane 1) who does not carry the S649S variant has only one RT-PCR product of the expected size (383 bp) on normal splicing. (c) BanI restriction digest of RT-PCR products. An expressed polymorphism in RET, which alters a BanI restriction site in exon 11, was used to analyze RT-PCR products. The wild-type band in lane 3 (labeled as 3+) and the mutant band in lane 3 (3−), as well as the band in lane 1 (1+), were gel-extracted. Two bands (239 bp and 144 bp) are observed after the digestion of lane 1, indicating homozygosity for the BanI site. Lane 3+ contains three bands: one undigested band (383 bp) and two bands resulting from the BanI digest (239 bp and 144 bp), indicating that the wild-type band observed in b has been amplified from both the wild-type and mutant alleles. The novel acceptor splice site is thus only partially used when present in an affected individual.

Figure 4

Nonparametric linkage (NPL) analysis of chromosome 9 markers in HSCR on subdividing families by RET mutation status. Dashed line, those with RET coding sequence mutations (families 1–6); solid line, those without RET coding sequence mutations (families 7–12).