Hypospadias : Gene mapping and candidate gene studied

Abstract: Hypospadias is a common congenital malformation in boys, characterized by incomplete fusion of the urethral folds, abnormal opening of urethra and different degrees of curvature of the penis. In Sweden, the incidence of hypospadias is 1.14 per 300 male live-births according to the annual Swedish Malformation Registry. Hypospadias is considered to be a complex genetic disorder caused by the interplay between environmental factors and the additive effects of multiple genes. Several observations suggest that hypospadias are under genetic influence. To identify disease genes in complex disorders, two main strategies have been used. We have performed a whole genome screening for linkage in families with additional cases of hypospadias and also performed DNA sequencing of candidate genes for hypospadias To identify the chromosomal loci involved in the pathogenesis of hypospadias, a genome-wide linkage analysis in a three-generational family showing autosomal dominant inheritance of hypospadias was performed. Fifteen individuals, whereof seven affected, were genotyped within a total of 426 microsatellite markers and the genotyping results were analyzed using parametric and non-parametric linkage analyses. The genome-wide linkage analysis and subsequent fine mapping gave a maximum linkage in both parametric (LOD score 2.71) and non-parametric (NPL score 5.01) single-point analyses for marker D7S640. A susceptibility haplotype shared by all affected boys was identified by markers D7S2519 and D7S2442, respectively. This finding suggests a novel hypospadias locus (25 cM) at chromosome 7q32.2- q36.1 (Study I). In a previous genome-wide scan for familial hypospadias, we have identified suggestive linkage in nine chromosomal regions. To extend this analysis new families and additional markers were included. The fine mapping analysis displayed an increased LOD score on chromosome 8q24.1 and 10p15 in altogether 82 families. From the chromosome region 10p15, we sequenced the AKR1C3 and KLF6 genes that have possible roles during male urethra development. Sequencing analysis showed one mutation (c.697A>G, p.A215T) in the AKR1C3 gene and one mutation (c.496T>C, p.P166S) in the KLF6 gene. In addition, three polymorphisms (rs3763676, rs12529 and rs7741) in the 5´end of the AKR1C3gene showed a significant association with hypospadias. These findings indicate that the AKR1C3 and possibly also the KLF6 genes function as geneticrisk factors for hypospadias (Study II). Several different pathways are implicated in the male genital development; of those the androgen pathway iscrucially important. Therefore the androgen receptor (AR) and the 5 alpha reductase (SRD5A2) genes were sequenced in 38 isolated hypospadias cases. One mutation in the AR gene (p.Q798E) and another in the SRD5A2 gene (p.K199S) was detected. Interestingly, also a high frequency of the rare leucine allele in theV89L polymorphism of the SRD5A2 gene was found. The leucine allele has been shown to confer a decreased activity of enzyme, thus this allele may be a risk factor for hypospadias. This finding was confirmed in total 158 hypospadias cases compared to 96 controls (Study III). The MAMLD1 (CXorf6) gene is the first gene causing isolated hypospadias. The MAMLD1 gene was sequenced in DNA from 97 sporadic hypospadias cases to elucidate the role of this gene in hypospadias. One new mutation, p.Q529K, that is predicted to affect the splicing process, was found in a boy with severe hypospadias. The variants, p.V432A and p.531ins3Q, have been reported previously and are indicated in this study as polymorphisms. Additionally, a significant association between the p.N589S polymorphism (rs2073043) that may alter the predicted protein structure and hypospadias was detected (p-value <0.05). The combination of two rare alleles of p.N589S and p.P286S (rs41313406) is over represented among cases. It may be that these two polymorphisms are inherited together as a haplotype (T-G), thus increasing the risk for hypospadias. These findings suggest that in a few cases hypospadias is caused by mutations in the MAMLD1 gene, and that the polymorphism (p.N589S) is a genetic risk factor (Study IV).