Molecular Studies Of The MAMLD1,SF-1 Gene In Hypospadias

Characterized by incomplete fusion of the urethral folds resulting in an abnormal opening of urethra and different degrees of curvature of the penis, hypospadias is one of the most prevalent urogenital malformations in boys. The estimations of frequency are placed between 0.3%and 0.8%of male births in Asian, Europe and North America, and the international trends of the incidence is increasing.Hypospadias is considered as a complex disorder, the etiology is just beginning to be elucidated. Environmental and endocrinal agents were discovered involved in, more investigations demonstrating strong genetic background. Earlier molecular analyses have revealed mutations of gene responsible for the enzyme activity of 5-alpha-reductase, leading to disturbance of the production of dihydrotestosterone which is necessary for development of the malegenito-urinary tract, are contribute to the etiology of hypospadias. So far, lots of genes such as AR (androgen receptor),5 a-reductases, LH-receptor, ATF3, FGF8/10 were unveiled to be candidate gene for hypospadias.The normal development of external genitalia in male occurs during the 8th to 16th gestational week under the influence of androgens that secreted by the fetal testes. The genital tubercle elongates to form the shaft and glands of the penis. Since hypospadias are caused by arrested development of the urethra during the period of embryological sexual differentiation, the severity of hypospadias can be seen as a continuum. The earlier the fusion process is interrupted, the more severe or complex it will be.The formation of male genitalia needs the action of a specific androgen: testosterone (T). Placenta human chorionic gonadotrophin (hCG), stimulate Leydig cells in fetal testis to produce testosterone, In Leydig cells,17beta-hydroxysteroid dehydrogenase (HSD17B3) catalyzes the conversion of androsternedione to testosterone. Testosterone is converted to the more active dihydrotesterone (DHT) by the enzyme steroid 5a-reductase (SRD5A2). Both hormones (T and DHT) bind to the androgen receptor (AR) in the genital target tissue. T induces the differentiation of Wolffian duct into epididymis, and so on. DHT bound to the same androgen receptor, modulates the differentiation of the penis and scrotum.Thus, any factor that could affect the elongation of the urogenital tubercle, or interfere with the fusion of urogenital folds, could be a susceptibility of hypospadias. Mutations in the crucial genes in the androgen pathway, the androgen receptor and the 5-alpha-reductase genes, could cause arrested development of the urethra during the period of embryological sexual differentiation could account for a subset of hypospadias.Deletions at the Xq28 locus were first found in patients with myotubular myopathy:genital development is normal in patients with intragenic MTM1 mutations, and invariably abnormal in patients with microdeletions involving MTM1. Subsequent studies have shown loss of Mastermind-like domain containing 1 (MAMLD1) gene MAMLD1 in all patients with myotubular myopathy and 46, XY DSD, and no other candidate gene for 46, XY DSD has been identified within the commonly deleted region. These findings imply that MAMLD1 is an excellent candidate gene for 46, XY DSD, especially hypospadias.Further studies of MAMLD1 have shown that the gene is expressed in fetal Sertoli and Leydig cells around the critical period for sex development and that transient knockdown of MAMLD1 mRNA expression result in significantly reduced testosterone production in mouse Leydig tumor cells. MAMLD1 is further co-expressed with steroidogenic factor (SF-1), which regulates the transcription of genes involved in sex development, and a SF-1 target site is found within the MAMLD1 gene.SF-1 expression is consistently maintained in the somatic cells of the early testis after testis determination, where it may play a crucial role together with SRY in supporting SOX9 expression. In Sertoli cells, SF-1 activates expression of Mullerian inhibiting substance (MIS, anti-Mullerian hormone (AMH) from around 7 weeks gestation, which leads to the regression of Mullerian structures in the developing male fetus. In Leydig cells, SF-1 activates the expression of steroidogenic enzyme systems from 8 weeks gestation, which results in androgenization of the external genitalia.SF-1 transcriptionally regulates a vast array of genes involved in adrenal and gonadal development, sex differentiation, steroidgenesis and reproduction by the hypothalamic-pituitary-steroidogenic axis. So relatively small changes in SF-1 activity may cause'cascade reaction'of endocrine systems and clinically effects in the critical period.However, much less is known about the regulation of SF-1 expression itself. Despite reports of SF-1 promoter or enhancer regulation by factors such as WT1, many of the key mechanisms influencing SF-1 expression remain to be elucidated. Further research in this area could also have important implications for understanding the role of SF-1 in human disease.The aim of this thesis is to study candidate genes in hypospadias and composed by two parts:the first segment was focused on the MAMLD1 gene in sporadic cases by associated studies and function prediction; the second part is to study the SF-1 gene on hypospadias by sequencing and Genotyping. Part One:Study the mechanism of MAMLD1 gene in sporadic hypospadiasObjective:We have analyzed the occurrence of sex chromosome aberrations in hypospadias, further we have screened the MAMLD1 gene for mutations in a large number of sporadic hypospadias cases to further elucidate the mutation spectrum in the pathogenesis of hypospadias.Material:500 boys surgically treated for different severities of hypospadias in Sweden were recruited for the study. The control group consists of healthy voluntary anonymous blood donors at the Karolinska University Hospital.Methods:(1) DNA was extracted from peripheral blood or penile skin tissue.(2) Exon-flanking primers were designed by Primer 3 program.(3) After ExoSap-IT enzyme treatment, PCR fragments were sequenced on both directions using BigDye(?) Terminator v3.1 kit and analyzed in ABI Prism 3730 Sequencer. Sequence analysis was preformed with the program SeqScape v2.5.(4) Genotyping and statistical analyses:Three SNPs in the MAMLD1 gene (rs41313406, rs61740566, rs2073043) were selected for association study.(5) Splice site prediction:NetGene 2.0 NNSplice and HSF 2.4. HomoloGene: ClustalW2. Three-dimensional structure protein structure:Protein Homology/analogy Recognition Engine (Phyre).Results:(1) Screening for sex chromosomal aneuploidyPatients with sex chromosomal aneuploidy or mosaicism were removed from further analysis. (2) Direct sequencing 99 cases and 95 male controls.The p.V432A, p.Q529K, c.2065+8a>t and p.D686D variants were separately detected in one case each and none of the controls. The p.P286S and p.N589S variants were each detected in 11 cases and not in any of the controls while the CAG repeat polymorphism was detected in three cases and one control. The nonsynonymous mutation p.Q529K as well as the synonymous p.D686D and the non-coding c.2065+8a>t mutations are new mutations.(3) In 370 patients and 418 male controls, the p.P589S showed a weak association with hypospadias (p<0.05) while the p.P286S didn't make it.The [p.286S, p.589S] haplotype was slightly over represented in controls while the p-value is around the border of significance (p=0.05).No significance in the p.V432A (rs61740566).(4) Homology analysis showed that p.P286S and p.Q529K are located in regions of proline (P) and glutamine (Q) repeats respectively. The p.531ins3Q is located in the same stretch of glutamine (Q) residues as the p.Q529K mutation and structure prediction suggests it can change the protein structure.Conclusion:our findings suggest that a few hypospadias cases are caused by mutations in the MAMLD1 gene and that the haplotype [p.286S, p.589S] possibly is a risk factor for hypospadias. Part Two:The association between steroidogenic factor-1 and hypospadiasObjective:Steroidogenic factor-1 (SF-1, NR5A1, Ad4BP) plays key role in the hypothalamic-pituitary-steroidogenic organ axis. More and more mutations of SF-1 were discovered in 46 XY DSD (disorders of sex development).We tried to identify its association with hypospadias.Material and methods:95 patients with different degrees of severities of sporadic hypospadias (recruited by the Swedish Malformation Registry) were selected for direct sequencing of human SF-1 gene in coding exons and flanking region.380 sporadic and familial cases,760 healthy voluntary blood samples were chosen for genotyping:SF-1 polymorphism SNP (rs1110061):G>C.Results:We revealed 2 SNPs in 95 sporadic patients by direct sequencing:p.P125P (rs1110062) and p.G146A (rs1110061):3 cases are [p.P125P, p.G146A] haplotype, all of them are severe hypospadias; 2 cases are homozygous of p.G146A, and 3 more patients are simply heterozygous of p.G146A.The polymorphism rs1110061 was chosen for Taqman analysis:30 of 368 patients (8.15%) had G/C genotype (heterozygous,21 cases) or C/C (homozygous,9 cases). In the control group,21 of 753 contained allele C (heterozygous, no homozygous be discovered), a frequency of 2.76%:making significant difference (p<0.0001) between the cases and controls on C allele frequency.Conclusion:The allele C in Gly146Ala polymorphism of SF-1 increased significantly in hypospadias, the compound heterozygous carrying the p.G146A polymorphism in combination with other mutated alleles needed to further identified. SF-1 should be proposed as a susceptibility factor for hypospadias.