Salud Borrego

Specific polymorphisms in the RET proto-oncogene are over-represented in patients with Hirschsprung disease and may represent loci modifying phenotypic expression

Hirschsprung disease (HSCR) is a common genetic disorder presenting with functional intestinal obstruction secondary to enteric aganglionosis. HSCR can be familial or sporadic. Although five putative susceptibility genes have been identified, only germline mutations in the RET proto-oncogene account for a significant minority (up to 50%) of familial HSCR; 3% of sporadic HSCR in a population based series carry RETmutations. From 1998 to February 1999, we prospectively ascertained 64 cases of sporadic HSCR from the western Andalusia region. To determine if polymorphic sequence variants within RETcould act as low penetrance predisposing alleles, we examined allelic frequencies at seven polymorphic loci in this population based series. Whether allele frequencies differed from those in the control population were determined by either chi-squared analysis or Fisher’s exact test. For two sequence variants, A45A (c 135G→A) (exon 2) and L769L (c 2307T→G) (exon 13), the rarer polymorphic allele was over-represented among HSCR cases versus controls (p<0.0006). In contrast, two other polymorphisms, G691S (c 2071C→A) (exon 11) and S904S (c 2712C→G) (exon 15), were under-represented in the HSCR patients compared to controls (p=0.02). Polymorphisms in theRET proto-oncogene appear to predispose to HSCR in a complex, low penetrance fashion and may also modify phenotypic expression.

EYS, encoding an ortholog of Drosophila spacemaker, is mutated in autosomal recessive retinitis pigmentosa.

Using a positional cloning approach supported by comparative genomics, we have identified a previously unreported gene, EYS, at the RP25 locus on chromosome 6q12 commonly mutated in autosomal recessive retinitis pigmentosa. Spanning over 2 Mb, this is the largest eye-specific gene identified so far. EYS is independently disrupted in four other mammalian lineages, including that of rodents, but is well conserved from Drosophila to man and is likely to have a role in the modeling of retinal architecture.

A Founding Locus within the RET Proto-Oncogene May Account for a Large Proportion of Apparently Sporadic Hirschsprung Disease and a Subset of Cases of Sporadic Medullary Thyroid Carcinoma

Hirschsprung disease (HSCR) is a common congenital disorder characterized by aganglionosis of the gut. The seemingly unrelated multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominant disorder characterized by medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism. Yet, germline mutations in the RET proto-oncogene are associated with both MEN 2 and HSCR. In the former, gain-of-function mutations in a limited set of codons is found, whereas, in the latter, loss-of-function mutations are found. However, germline RET mutation is associated with only 3% of a population-based series of isolated HSCR, and little is known about susceptibility to sporadic MTC. We have found previously that specific haplotypes comprising RET coding single-nucleotide polymorphisms (SNPs) comprising exon 2 SNP A45A were strongly associated with HSCR, whereas haplotypes associated with exon 14 SNP S836S were associated with MTC. In this study, we describe three novel intron 1 SNPs, and, together with the coding SNP haplotypes, the data suggest the presence of distinct ancestral haplotypes for HSCR and sporadic MTC in linkage disequilibrium with a putative founding susceptibility locus/loci. The data are consistent with the presence of a very ancient, low-penetrance founder locus approximately 20-30 kb upstream of SNP A45A, but the failure of the SNPs to span the locus presents challenges in modeling mode of transmission or ancestry. We postulate that this founding locus is germane to both isolated HSCR and MTC but also that different mutations in this locus would predispose to one or the other.

RET genotypes comprising specific haplotypes of polymorphic variants predispose to isolated Hirschsprung disease

BACKGROUND Hirschsprung disease (HSCR), which may be sporadic or familial, occurs in 1:5000 live births and presents with functional intestinal obstruction secondary to aganglionosis of the hindgut. Germline mutations of theRET proto-oncogene are believed to account for up to 50% of familial cases and up to 30% of isolated cases in most series. However, these series are highly selected for the most obvious and severe cases and large familial aggregations. Population based studies indicate that germline RETmutations account for no more than 3% of isolated HSCR cases. Recently, we and others have noted that specific polymorphic sequence variants, notably A45A (exon 2), are over-represented in isolated HSCR. PURPOSE In order to determine if it is the variant per se, a combination thereof, or another locus in linkage disequilibrium which predisposes to HSCR, we looked for association of RET haplotype(s) and disease in HSCR cases compared to region matched controls. METHODS Seven loci acrossRET were typed and haplotypes formed for HSCR cases, their unaffected parents, and region matched controls. Haplotype and genotype frequencies and distributions were compared among these groups using the transmission disequilibrium test and standard case-control statistic. RESULTS Twelve unique haplotypes, labelled A-L, were obtained. The distributions of haplotypes between cases and controls (χ11 2 =81.4, p<<0.0001) and between cases and non-transmitted parental haplotypes were significantly different (χ2 11=53.1, p<0.0001). Genotypes comprising pairs of haplotypes were formed for cases and controls. There were 38 different genotypes among cases and controls combined. Inspection of the genotypes in these two groups showed that the genotype distribution between cases and controls was distinct (χ37 2=93.8, p<<0.0001). For example, BB, BC, BD, and CD, all of which contain at least one allele with the polymorphic A45A, are prominently represented among HSCR cases, together accounting for >35% of the case genotypes, yet these four genotypes were not represented among the population matched normal controls. Conversely, AA, AG, DD, GG, and GJ, none of which contains A45A, are commonly represented in the controls, together accounting for 43% of the control genotypes, and yet they are never seen among the HSCR cases. CONCLUSIONS Our data suggest that genotypes comprising specific pairs of REThaplotypes are associated with predisposition to HSCR either in a simple autosomal recessive manner or in an additive, dose dependent fashion.

Germline sequence variant S836S in the RET proto‐oncogene is associated with low level predisposition to sporadic medullary thyroid carcinoma in the Spanish population

OBJECTIVE The molecular basis of sporadic medullary thyroid carcinoma (MTC) remains elusive. While germline gain‐of‐function mutations in the RET proto‐oncogene cause hereditary MTC, somatic activating RET mutations and loss of heterozygosity of markers in various chromosomal regions representing deletions of tumour suppressor genes, have been described in a variable number of sporadic MTC. A previous report suggested that the presence of a germline variant at RET codon 836 (S836S) was associated with the development of sporadic MTC and, furthermore, that the presence of S836S was highly correlated with somatic RET M918T mutation in the MTC. Thus, we sought to determine if the S836S variant would be associated with sporadic MTC from a completely different population base, that of Andalucia.

A major locus for autosomal recessive retinitis pigmentosa on 6q, determined by homozygosity mapping of chromosomal regions that contain gamma-aminobutyric acid-receptor clusters

Retinitis pigmentosa (RP) is the most common inherited retinal dystrophy, with extensive allelic and nonallelic genetic heterogeneity. Autosomal recessive RP (arRP) is the most common form of RP worldwide, with at least nine loci known and accountable for approximately 10%-15% of all cases. Gamma-aminobutyric acid (GABA) is the major inhibitory transmitter in the CNS. Different GABA receptors are expressed in all retinal layers, and inhibition mediated by GABA receptors in the human retina could be related to RP. We have selected chromosomal regions containing genes that encode the different subunits of the GABA receptors, for homozygosity mapping in inbred families affected by arRP. We identify a new locus for arRP, on chromosome 6, between markers D6S257 and D6S1644. Our data suggest that 10%-20% of Spanish families affected by typical arRP could have linkage to this new locus. This region contains subunits GABRR1 and GABRR2 of the GABA-C receptor, which is the effector of lateral inhibition at the retina.

Mutations in USH2A in Spanish patients with autosomal recessive retinitis pigmentosa: high prevalence and phenotypic variation

Retinitis pigmentosa (RP), which occurs in about one in 3000–7000 people in Spain, is inherited in an autosomal dominant manner in 12% of cases, in an autosomal recessive way in 39%, and in an X linked manner in 4% of cases. This leaves 41% of RP cases with a simplex form and 4% in which the transmission pattern is unclear.1 Direct analyses of rhodopsin, the alpha and gamma subunits of rod cGMP-phosphodiesterase, periferin/RDS, rod outer segment membrane protein, recoverin, guanilate cyclase activating protein, S antigen, interstitial retinol binding protein, and NRL have failed to detect any disease causing mutation in non-syndromic ARRP Spanish families. Mutations in the beta subunit of the rod cGMP-phosphodiesterase gene,2–5 in the ATP binding cassette receptor gene,6 and in the TULP1 gene7 account for a small percentage of Spanish ARRP families. These data indicate that genes other than these may be involved in the remaining families, emphasising the genetic heterogeneity of the disease and reinforcing the hypothesis that in ARRP a number of genes rather than one major gene will account individually for a small number of cases. The recent report that a missense mutation in the USH2A gene (C759F) is present in 4.5% of patients with non-syndromic ARRP8 prompted us to analyse the involvement of this mutation in a large set of Spanish ARRP families. A complete mutational analysis of the coding region of the USH2A gene was performed in all cases in which the C759F allele was found. Additional mutations were identified in the USH2A gene in non-syndromic ARRP patients. Interestingly, two C759F homozygotes belonging to a consanguineous ARRP family had no RP symptoms and no hearing impairment. A group of 196 unrelated ARRP patients plus four cases of retinitis punctata albescens were studied. The patients were diagnosed …

Mutation spectrum of EYS in Spanish patients with autosomal recessive retinitis pigmentosa

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal dystrophies characterised ultimately by the loss of photoreceptor cells. We have recently identified a new gene (EYS) encoding an ortholog of Drosophila spacemaker (spam) as a commonly mutated gene in autosomal recessive RP. In the present study, we report the identification of 73 sequence variations in EYS, of which 28 are novel. Of these, 42.9% (12/28) are very likely pathogenic, 17.9% (5/28) are possibly pathogenic, whereas 39.3% (11/28) are SNPs. In addition, we have detected 3 pathogenic changes previously reported in other populations. We are also presenting the characterisation of EYS homologues in different species, and a detailed analysis of the EYS domains, with the identification of an interesting novel feature: a putative coiled‐coil domain. Majority of the mutations in the arRP patients have been found within the domain structures of EYS. The minimum observed prevalence of distinct EYS mutations in our group of patients is of 15.9% (15/94), confirming a major involvement of EYS in the pathogenesis of arRP in the Spanish population. Along with the detection of three recurrent mutations in Caucasian population, our hypothesis of EYS being the first prevalent gene in arRP has been reinforced in the present study.

Identification of novel mutations in the ortholog of Drosophila eyes shut gene (EYS) causing autosomal recessive retinitis pigmentosa.

PURPOSE Recently, a novel gene was cloned for autosomal recessive retinitis pigmentosa (arRP), EYS, on 6q12. This study was conducted to determine the spectrum and frequency of EYS mutations in 195 unrelated patients with autosomal recessive and autosomal dominant RP (adRP). METHODS All cases had a complete ophthalmic examination, and the clinical diagnosis of RP was based on visual acuity, fundus photography, and electroretinography findings. The DNA extracted from all participants was subjected to molecular genetic analysis entailing amplification of the coding regions and exon-intron boundaries of EYS by polymerase chain reaction, followed by direct sequencing. Bioinformatics analysis was undertaken to study the effect of the identified mutations on protein structure and function. RESULTS Eleven novel missense, nonsense, and splice site mutations were identified within EYS in 10 unrelated arRP patients, with probable allele frequency of 11%. However, no mutations were observed in the adRP panel. In addition, 53 single-nucleotide polymorphisms (SNPs) were found, of which 12 were previously unreported. Bioinformatics analyses revealed that all mutations were highly conserved across other species and/or involved important domains on protein structure. Intrafamilial phenotypic variability was also observed in a family with double heterozygous mutations. CONCLUSIONS This is the first report of molecular genetic analysis of EYS in a cohort of unrelated British and Chinese patients with RP. The results further the initial hypothesis that EYS is a major causative gene for recessive RP and emphasize the role of different types of mutations in disrupting the function of EYS.

Interaction between a chromosome 10 RET enhancer and chromosome 21 in the Down syndrome–Hirschsprung disease association†

Individuals with Down syndrome (DS) display a 40‐fold greater risk of Hirschsprung disease (HSCR) than the general population of newborns implicating chromosome 21 in HSCR etiology. Here we demonstrate that the RET enhancer polymorphism RET+9.7 (rs2435357:C>T) at chromosome 10q11.2 is associated with HSCR in DS individuals both by transmission disequilibrium (P=0.0015) and case–control (P=0.0115) analysis of matched cases. Interestingly, the RET+9.7 T allele frequency is significantly different between individuals with DS alone (0.26±0.04), HSCR alone (0.61±0.04), and those with HSCR and DS (0.41±0.04), demonstrating an association and interaction between RET and chromosome 21 gene dosage. This is the first report of a genetic interaction between a common functional variant (rs2435357) and a not infrequent copy number error (chromosome 21 dosage) in two human developmental disorders. Hum Mutat 30:1–5, 2009.