Genetic Mutation Analysis Of Hypospadias

Hypospadias, one of the most common congenital abnormalities of the male extemal genitalia withelusive etiology, are caused by a defect in normal development of the urethral, foreskin and ventralaspect of the penis. The main symptoms are abnormally located urethral orifice, downward curvaturepenis and excess dorsal prepuce. The incidence of hypospadias is 1/300-1/125 and the incidence isincreasing in many counties.The formation of the male sex organ is fully androgen-dependent. At about the 8th week ofgestation, a sex determination cascade begins with SRY (sex-determining region Y) triggeringdevelopment of Sertoli cells. SOX9 (SRY box 9) respond to the stimulation, participate in the regulationof Sertoli cell-specific expression of anti-Mullerian hormone and the synthesis of testosterone (T) infetal testes. The WT1 (Wilms' tumor 1) also has a male-specific role from earlier to later sexdetermination. At the time course of early sex differentiation, maternal hCG binds to the LHR of Leydigcell of the fetus testis, and the Leydig cell were activated and secrets testosterone. Meanwhile, SRD5A2(steroid 5 alpha reductase type 2) catalyzes testosterone into DHT (dihydrotestosterone), a more potentform than dihydrotestosterone in binding the AR (androgen receptor). Therefore, for hypospadias, itseemed that a more reasonable explanation is an abnormality in androgen metabolism. Indeed, thedefects in SRY, SOX9, AR, SRD5A2 and WT1 might contribute to the abnormal male sex developmentincluding a subset of hypospadias. The modes of inheritance of hypospadias might vary. For example,the inheritances of AR defects, SRD5A2 defects are X-linked recessive, autosomal recessiverespectively. However, all these defects only account for no more than 30％of cases. Thus, the fact thatthe causes have not yet been identified for a large number of cases highlights the importance of seekingother possible genetic explanations for the congenital defect.Knowledge about molecular mechanisms of the normal formation of the male urethra, includingthe glandular urethra might elucidate the etiology of hypospadias. To a great extent, the development of male urethra is similar to those of palate and limbs. During the formations of these organs, tissuephenotypic transformations always occur. The classic mechanism of the phenotypic transformation is anepithelial-mesenchymal cell change or mesenchymal-to-epithelial transformation. The transformationinvolves in a signaling cascade composed of Hedgehog, TGFβsignaling pathway as well as HOXmolecules and so on. These molecules are always as mediators of morphogenetic cell-cell interactions.This signaling cascade is conserved throughout organisms from Drosophila to humans and in manytissues, including skeleton, limb, lung, gut and genitalia. Among these signaling molecules of thecascade, SHH, HOXA13, BMP4, BMP7 and FGFS, FGF10 etc. are major ones. Studies in mice havealready shown that defects or loss of functions of these molecules could result in an altered genitalphenotype including hypospadias, either alone or in a combination.As mentioned above, the development of the penile shaft skin and the preputial skin is alsofundamental stages in the formation of male urethra. Any disturbance in these stages may also causehypospadias. Although there is no direct evidence in the urogenital system, experimental evidence hassuggested that HOXB6 (former Hox2.2) and HOXA4 play an important role in skin development ofvarious tissues at week 10-17 of gestation, which is consistent with the time course of the developmentof the penile shaft skin and the preputial skin. In addition, mice and chicken models also show thatHOXB6 gene products are involved in controlling pattem formation in developing limbs.Based on the above knowledge, we hypothesized that mutations of SHH, HOXA13, BMP4, BMP7and FGF8, FGF10 might result in an altered genital phenotype including hypospadias. However, therehave already been evidences excluding HOXA13 and some of FGFs as candidate genes for isolatedhypospadias. In the present study, we attempt to investigate whether there are some nucleotidealternations in BMP4 and BMP7, two downstream molecules of the above signaling cascade, and inHOXA4, HOXB6 in hypospadias.Our results in the part of studies on BMP4, BMP7, HOXA4 and HOXB6 analysis are: a total of 13different heterozygous nucleotide sequence variations were found. They include three in BMP4, six inBMP7, two in HOXA4 and two in HOXB6 in the exons and introns of the four genes in 14 of 90patients. Of these variations, eight (c.619C＞G, c.668G＞A, c.751C＞T; c.907C＞T; c.385G＞T, c.869C＞G;c.124C＞A, c.367T＞C) are missense variations. The others, c.597G＞A, c.1465T＞A (3'UTR), c. 1567A＞G (3'UTR), c.612-33C＞T, c.1146+39C＞T are silent because c.597G＞A does not change aminoacid, while, c.1465T＞A, c.1567A＞G locate in the 3'UTR and c.612-33C＞T, c.1146+39C＞T are inintrons. All these variations found in the study were not found in 380 control chromosomes. Amino acidsequence alignments of the four human genes with respective vertebrate orthologs available from NCBIshow that p.H207, p.R223 and p.H251 in BMP4; p.Q199 and p.R303 in BMP7 are highly conservedacross all their respective vertebrate orthologs available from NCBI database, p.G129 and p.S290 inHOXA4, p.P42 and p.C123 in HOXB6 are partly conserved across all their respective vertebrateorthologs available from NCBI database. These facts indicate that so many nucleotide variations foundin the present study in the four genes shouldn't be random events. Furthermore, they merit furtherfunctional studies to make the associations clear between the variants and hypospadias. Of course, ourcurrent work is a start for seeking the causes for hypospadias from development-related pathways.In addition, due to the central roles of LHR in normal male sex differentiation, the defects in LHRcan affect normal sex development. It is reported that there are two kinds of mutations in LHR,inactivating mutations and activating mutations. Activating mutations are inherited in autosomaldominant mode. The hot spots are in TM6 and IL3. No mutation has yet been found in extracellulardomain, hinge domain, and TM4, TM7 of the LHR. Inactivating mutations might cause Leydig cellhypoplasia (LCH). Two types of LCH have been described. TypeⅠis the severe form of LCH identifiedin 46 XY individuals displaying a predominantly female phenotype, which is caused by mutations in theLHR that completely disrupt LH signaling. Milder forms of LCH (typeⅡ) are also caused by amutation in the LHR gene, but this defect disrupts LHR signaling less severely, and patients arecharacterized by hypospadias or micropenis. Inactivating mutations are inherited in autosomal recessivemode, which can influence the differentiation of male fetus sex organ, even cause infertility in man.Inactivating mutations may occur anywhere, and no hot spots have been found for the inactivatingmutations.In the present study, we have also screened the LHR gene, and further performed functional studiesof those mutants found by us.Our studies for LHR in the study identified that two patients carded the LHR mutation, patient 85 isheterozygous T108I. He is perineal hypospadias with cryptorchidism. Though confocal and cAMP studies show the slight defects in trafficking to cell surface membrane and signal transduction forp.T108I mutant, based on the mode of inheritance of inactivating mutation in LHR, we can not nowconclude the c.C323T (p.T108I) is the disease-causing mutation for patient 85. Further studies arerequired to make clear the associations between the heterozygous T108I and the phenotype of patient 85.Another patient, patient 96 carries a homozygous E188del, and was also perineal hypospadias with bifidscrotal and gynecomastia. Confocal and cAMP studies show the severe defects in trafficking to cellsurface membrane and signal transduction for p.E188del mutant. So we can conclude that thehomozygous E188del was the disease-causing mutation for the patient 96, and she was the typeⅠofLCH.