Differential contributions of rare and common, coding and noncoding Ret mutations to multifactorial Hirschsprung disease liability

Abstract

The major gene for Hirschsprung disease (HSCR) encodes the receptor tyrosine kinase RET. In a study of 690 European- and 192 Chinese-descent probands and their parents or controls, we demonstrate the ubiquity of a >4-fold susceptibility from a C-->T allele (rs2435357: p = 3.9 x 10(-43) in European ancestry; p = 1.1 x 10(-21) in Chinese samples) that probably arose once within the intronic RET enhancer MCS+9.7. With in vitro assays, we now show that the T variant disrupts a SOX10 binding site within MCS+9.7 that compromises RET transactivation. The T allele, with a control frequency of 20%-30%/47% and case frequency of 54%-62%/88% in European/Chinese-ancestry individuals, is involved in all forms of HSCR. It is marginally associated with proband gender (p = 0.13) and significantly so with length of aganglionosis (p = 7.6 x 10(-5)) and familiality (p = 6.2 x 10(-4)). The enhancer variant is more frequent in the common forms of male, short-segment, and simplex families whereas multiple, rare, coding mutations are the norm in the less common and more severe forms of female, long-segment, and multiplex families. The T variant also increases penetrance in patients with rare RET coding mutations. Thus, both rare and common mutations, individually and together, make contributions to the risk of HSCR. The distribution of RET variants in diverse HSCR patients suggests a "cellular-recessive" genetic model where both RET alleles' function is compromised. The RET allelic series, and its genotype-phenotype correlations, shows that success in variant identification in complex disorders may strongly depend on which patients are studied.

Copyright 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Genetic Associations between RET and HSCR in Two Populations Transmission disequilibrium test (TDT) of polymorphisms across the RET locus. -logP values for each of 14 SNPs, with purple triangles representing genotypes from individuals of European descent (Italy, France, Netherlands, Spain, and US) and green circles those of Chinese descent (trio and case-control p values were combined via Fisher's method), are shown on the y axis against the physical locations of SNPs with respect to RET exons (orange). The circled SNP rs2435357 segregates a functional hypomorphic enhancer mutation; rs3026785 is monomorphic in the Chinese.

Figure 2

SOX10 Transactivation and Binding to MCS+9.7 (A) Location and conservation of putative SOX10 transcription factor binding sites (TFBS). Human genomic sequence is aligned with the orthologous sequence interval in eight different mammals via multi-PIPmaker. The putative SOX10 TFBS (SOX10-BS1 and SOX10-BS2) are highlighted in orange. The HSCR-associated variant rs2435357 is boxed in black. (B) Reporter expression in HeLa cells of MCS+9.7 when cotransfected with SOX10 cDNA. Mutant (mut) and wild-type (WT) correspond to nucleotides T and C, respectively. Expression difference between mut and WT constructs is significant p < 0.01 by the two-tailed unpaired t test as indicated by the asterisk; error bars report standard errors of triplicate quantitative PCR reactions here and in (C)–(E). (C and D) Deletion of MCS+9.7 putative SOX10 binding sites reduces luciferase reporter expression in vitro. Expression in HeLa cells of altered MCS+9.7 luciferase reporter constructs when cotransfected with SOX10. MCS+9.7ΔBS1 and MCS+9.7ΔBS2 correspond to the deletions of the two putative SOX10 TFBS delineated in (A). Luciferase expression values for MCS+9.7ΔBS1 are shown in (C). Luciferase expression values for MCS+9.7ΔBS2 are shown in (D). (E) SOX10 physically associates with MCS+9.7 as demonstrated by chromatin immunoprecipitation. x axis is fold enrichment normalized to the no antibody control. Also included are a negative transfection control and a mock (no sample) control.

Figure 3

Proportion of Patients with Rare and Common Mutations Classified by Gender, Segment Length, and Familiality Frequencies of RET coding sequence (CDS) and enhancer mutation distribution in probands within HSCR subtypes. In each case, the percent of patients with no identified mutation, enhancer mutation only, CDS mutation only, and both enhancer and CDS mutations are shown (plus and minus signs indicate presence/absence) together with the respective sample size in each subgroup. A qualitative scale, demonstrating the direction of increase (decrease) in recurrence risk (incidence) of the categories from left to right, is shown.

Figure 4

Mendelian Errors Compatible with Gene Deletion Nine affected individuals with patterns of Mendelian inconsistencies suggestive of deletion are shown (each row represents a proband). RET exons (brown) and genotyped SNPs (blue) are shown across the top, in scale. For each proband, the longest RET region consistent with a deletion is represented: SNPs that are Mendelian inconsistent between parents and children are shown in solid color; SNPs that are Mendelian consistent but for which the child is homozygous are stippled in color. The entire genomic region is shown for illustrative purposes but only the SNPs were examined. Putative deletions occurring in the maternal and paternal germlines, or those with uncertain origin, are colored in purple, orange, and green, respectively. All deletions, but two, involve multiple exons.

Figure 5

Segregation of Both a Rare and Common Mutation in a HSCR Pedigree A family segregating S-HSCR, L-HSCR, and TCA with two RET mutations, a rare nonsense mutation (Y981X) and the common enhancer variant (rs2435357), is shown with genotypes of relevant individuals only. The Y981X mutation is inherited by all affected individuals from the paternal grandfather (I-I) and is present in two unaffected individuals (I-I and II-4). However, the severe forms of L-HSCR and TCA in the grandchildren (III-I and III-3) also harbor the common enhancer variant inherited instead from the individuals marrying into this family (II-I and II-5). This represents a case of allelic penetrance modification of a nonsense mutation.