MAMLD1 Mutation and Phenotypes of Hypospadias

MAMLD1 Mutation and Phe nonypes of HypospadiasThe consanguinity amongst clinical pheno theatrical roles and variants of MAMLD1 in children with hypospadiasYong-fen Lv, Lu-lu Cui, declension Li*Department of Endocrinology, yarn-dyes Children Hospital, Shanghai Jiaotong UniversityAcknowledgementsThe work was financi eachy supported by the key project of Shanghai municipal health bureau (2011111), spring chicken project of Shanghai municipal health bureaumajor pop subprojects of Shanghai science and technology commission (12411952408), Yangtze river delta research project of Shanghai science and technology commission (13495810300).AbstractPurpose To verify the relationship between clinical phenotypes of hypospadias and mutations of MAMLD1.Methods Seventy- dickens perseverings were diagnosed to be hypospadias in plane section of endocrinology and department of urinary surgery in our hospital. Among either the patients, 69 were with normal karyotype and enrolled as the analyze group. Fifty healthy boys were employed as the controls. Peripheral parentage were collected for DNA extraction. For the studied group, PCR primer was in cardinaltional and direct sequencing was performed for blanket for MAMLD1 mutations in hexad coding exons and the flanking sphere. Those mutated exons were examined for the control group.Results Thirty-five of in all the 72 patients (48.6 %) were isolated hypospadias. The other 37 cases (51.4%) were entangled by other genitourinary form malformations, including 12 cases with microphallus and/or under certain testicles. Abnormal karyotype was determine in 3 patients, and all were karyotype as 46, XX (SRY+ in 1 case and SRY- in 2 cases). Six types of MAMLD1 mutations were detected in exon 2, 3, 5, 7 in studied group, including c.5AG (p.D2G), IVS4-364C/A, c.1910AGp.N637S), c.2208TC, c.2227 GA (p.E742K) and IVS8-144C/T. All were mavin nucleotide pleomorphism tho c.5AG (p.D2G), a newly discovered point mutation. The often ness of IVS4-364C/A was importantly distinct between patients and controls, and it was in like manner significantly different between patients with and without microphallus and/or develop testicles.Conclusion Chromosome brachydactylicity is not the track ca intake of other genitourinary system malformations complicated with hypospadias. Mutations of MAMLD1 whitethornbe fast related to hypospadias in Chinese. c.5AGp.D2Gis the newly discovered point mutation in this work. IVS4-364C/A is associated with underdeveloped testicles and/or micropenis in hypospadias patients. establishmentHypospadias is one of the nigh common congenital genitourinary system malformations in males, with incidence 11%. As one of the Juvenile-types of testicular dys elementsis syndrome,(1-3) the prevalence of hypospadias is obviously increasing in these years. From 1987 to 2001, the prevalence rate was doubly change magnitude in China.(4) Hypospadias will lead to different degrees of genital malformat ion, and the clinical phenotypes straggle when with other complications. For instance, besides the agentral signs of hypospadias, e.g., ectopic ureteral orifice, phallocampsis, redundant dorsal prepuce, etc, the patient may be also suffered from other malformations including penoscrotal trans rig, cryptorchidism, hydrocele, oblique inguinal hernia, micropenis and underdeveloped testicles. Hypospadias is a complicated disease due to various suffers. The causes of most cases are not able to be verified, especially for those mild cases. For these cases, environmental factors, endocrinal factors and abnormal gene expression may be the preeminent causes.(5)The sex differentiation of males is a continuous series of processes related on the balancing and interaction of various genes like SRY, WTl, ATF3, SF-1, etc. MAMLD1, which is previously called chromosome X open reading frame6 (Cxorf6), is the important candidate gene widely studied recently. This gene is located in Xq28,(6,7) wit h molecular length of 157898 bp and containing 8 exons, among which exon 2, 3, 4, 5, 6, 7 are coding exons. MAMLD1 is initially detected in patients with X-linked myotubular myopathy. The fruitful systems of patients are normally developed with mutations in Myotubularin MTM-1, while different degrees of malformations go along in cases with deletion of MTM1 gene.(8-11) The subsequent experiments taperd that, for patients with 46, XY disorder of sex development (DSD), except MAMLD1, no other candidate genes were found in the deletion region. These results request that MAMLD1 is the perfect candidate gene for the study of 46, XY DSD, especially for hypospadias.This work aimed to approximate the mutations of MAMLD1 and clinical phenotypes in children with hypospadias in China, and thus to illustrate the role of MAMLD1 mutation in hypospadias.MethodsPatientsSeventy- 2 children with hypospadias admitted to Shanghai Childrens Hospital Affiliated to Jiaotong University from March 2011 to declination 2012 were enrolled in this study. Definite diagnosis was based on the clinical signs, and patients with adrenogenital syndrome were excluded with clinical examination.Clinical examinationClinical survey was performed including patients complain, present medical history, past medical history, personal history, family history, birth history, inducts medical history in pregnancy, previous exposure to environmental pollution, etc. Physical examination was performed to measure the hight, weight, heart rate, blood pressing and the status of gonad development, etc. For adolescent, the development of warrantary sexual characteristics was also assessed.Regular auxiliary examinations were performed including blood and urine routine test, biochemistry test, gonadal ductless gland level, adrenal cortex function and abdominal ultrasound exam, etc.Karyotype analytic thinking and detection of SRY geneLymphocytes were isolated from peripheral blood of patients, cultured and s meared on slides, and G-bands were produced by treatment with trypsin. Thirty split-phases were selected for each case, and karyotype analysis was performed according to ISCN-1995. SRY gene detection was performed for all the patients.Screening for MAMLD1 mutationsThe gene sequence of MAMLDI was obtained from National sharpen for Biotechnology Information (NCBI), which was the same as obtained from Ensembl Genome Browser NC_000023.10 (NCBI) versus ENSG00000013619 (Ensembl release 70-January 2013). Primers were designed for the coding exon 2, 3, 4, 5, 6, 7 of MAMLD1. DNA extraction was performed using TIANamp line of merchandise DNA Kit (TIANGEN Biotech (Beijing) Co., Ltd, China) and purity test was done. Ploymerase chain reaction (PCR) was performed with use of LONGgene A300 PCR and Premix Ex Taq Version2.0 (TaKaRa D332A), GC buffer (TaKaRa DRR20GC1) and rTag (TaKaRa DR001BM), 35 cycles of denaturation at 94 for 30 seconds,extension at 72 for 60 seconds. Mutations were identified in the six coding exons and flanking regions of MAMLD1, and those mutated exons were examined for the control group.Statistical analysisThe SPSS 18 software was employ for statistical evaluation. Chi-square test was used to compare the two groups, frequency of single nucleotide polymorphism between the two groups was canvas using pekan Exact test, and differences were considered statistically significant when the p-value was G (p.D2G), IVS4-364C/A (rs1209024), c.1910AG (p.N637S), c.2208TC, c.2227 GA (p.E742K, rs5925166) and IVS8-144C/T (rs658748). ii types of mutations were detected in exon 5 and 7 in all healthy controls, including c.1910 AG (p.N637S, rs2073043) and c.2208TC. Among all the mutations, c.5AG (p.D2G) was a newly discovered point mutation, others were all single nucleotide polymorphism. The studied group compared to the control group, the frequency analyzed by Fisher Exact test, the P value for IVS4-364C/A, c.1910AG (p.N637S), c.2208TC, c.2227 GA (p.E742K) and IVS8 -144C/T were 0.002, 0.638, 0.362, 1 and 0.509 respectively. Therefore, the frequency of IVS8-144C/T was significantly different between the two groups, and the frequency of the other 4 SNPs were not significantly different between the two groups.Relationship between mutations of MAMLD1 and clinical phenotypes of hypospadiasOne case with c.5AG (p.D2G) was isolated hypospadias, the urethral go-ahead position was located at the middle segment of penis. imputable to the limited hail of mutation cases, the prove size should be change magnitude to study the relationship between c.5AG (p.D2G) and phenotypes of hypospadias. Analyzed by Chi-squared test with Yates doggedness correction, the frequency of IVS4-364C/A was significantly different between patients with and without micropenis and/or underdeveloped testicles (p=0.001).DiscussionChromosome abnormalcy and karyotype change is one of the causes of hypospadias. Till now, ten types of chromosome abnormalities were confirmed involvi ng chromosome 1, 4, 6, 8, 11, 13 19, 20, 21, X, Y, etc. In the studied 72 patients, abnormal karyotype was identified in 3 patients, and all were karyotype as 46, XX (SRY+ in 1 case and SRY- in 2 cases). For these three patients, uterus and ovary were not found through the laparoscopic exploration. Therefore, they were diagnosed to be 46, XX male sex shiner syndrome. Karyotype analysis is important for hypospadias patients with sex reversal syndrome in exploring candidate gene and pathogenesis, in clinical diagnosis as well as in making therapeutic plan. However, there are only 3 cases with karyotype abnormality in the 72 patients studied, which means chromosome abnormality is not the leading cause of hypospadias.MAMLD1 is initially detected in patients with X-linked myotubular myopathy. The reproductive systems of patients are normally developed with mutations in Myotubularin MTM-1, while different degrees of malformations occur in cases with deletion of MTM1 gene.(8-11) neglect MAMLD1, no other candidate genes were found in the deletion region. These results indicate that MAMLD1 is the perfect candidate gene for the study of 46, XY DSD, especially for hypospadias.In the full treatment of Fukami et al., three nonsense mutations were detected, i.e., p.E124X, p.Q197X and p.R653X, in 4 XY DSD cases, involving micropenis and hypospadias with urethral enterprisingness position located on scrotum and the joint at penis and scrotum.(12) Kalfa et al. have studied the mutations of MAMLD1 in hypospadias patients and discovered 3 mutations, including p.V432A, p.E109fsX121 and P.531ins3Q, and they proposed that 10 percent of all the severe hypospadias cases was caused by mutations of MAMLD1.(13) In the study of Chen et al., three mutations of MAMLD1 were discovered, i.e., p.Q529K, p.D686D and noncoding region c.2065+8at, in 99 Swedish with hypospadias.(14) However, this is not the case in China. Qian et al. have studied the mutations of MAMLD1 in 100 cases of isolate d hypospadias,(15) where 200 healthy participants were willy-nilly selected as control. In their work, two point mutations, c.1699CT and c.1985AG, were detected and all were SNPs, and statistical analysis revealed that MAMLD1 is not the candidate gene for isolated hypospadias in China. The different results may be due to the different inclusion criteria of studied people between China and abroad, i.e., the studied populations of foreign works were mostly make up by hypospadias cases complicated by other genitourinary system malformations, including gonadal dysgenesis, while mostly isolated hypospadias cases were selected in the works of Chinese.In this study, a new point mutation c.5AG was detected in exon 2 of one patient, and this mutation was not found in controls. The mutation makes the second amino acid position, originally the hydrophilic negatively charged aspartic acid, substituted by a neutrally charged glycine. For various species, the second amino acid position in exon 2 of MAMLD1 is highly conserved, and analyzed via polyphen, the mutation c.5AG (p.D2G) of MAMLD1 is predicted to be probably damaging with a score of 0.996 from bombilateDiv and 0.993 from HunVar, which indicates that c.5AG (p.D2G) is highly related to hypospadias. screen predicts that the mutation can impinge on protein function, since there is no protein diversity on the site (supplementary figure 20). The case with c.5AG was isolated hypospadias, and the urethral opening was located in the middle segment of penis. A big sample size and the information of the exact protein function are required to unclutter whether c.5AG (p.D2G) of MAMLD1 is the cause of isolated hypospadias and its role in human sexual differentiation.The significant SNP detected in this work is the full mutation in introns crocked to exon 3, i.e., IVS4-364C/A (rs1209024), in 12 patients, which is not found in controls. The 12 cases including 2 cases with anterior hypospadias, 9 cases middle urethral openi ngs and 1 case posterior urethral opening. Among all the 12 patients, 6 were complicated by 2 other malformations, 8 cases were complicated by micropenis and/or underdeveloped testicles. Generally, introns are non-coding sections of a gene, which are removed sooner the mature mRNA can be transported, thus do not exist in the mRNA sequence. However, there may be several(prenominal) mini genes in some introns, the so called genes-within-genes. The frequency of IVS4-364C/A was significantly different between patients and controls, and it was also significantly different between patients with and without micropenis and/or underdeveloped testicles. Therefore, two possibilities could be speculated, the first is that there may be coding sequence related to micropenis and/or underdeveloped testicles in the introns close to exon 3 of MAMLD1, the second is that the mutation could affect mRNA cut back mode, thus lead to the change of protein function. Next tone of voice of our study plan is to verify whether the mutation could affect mRNA shear mode through reverse transcription, amplification and sequencing of extracted mRNA.References1. Skakkebaek NE, Rajpert-De ME, Main KM. Testicular dysgenesis syndrome an increasingly common developmental disorder with environmental aspects. Hum Reprod 200116972-8.2. Sharpe RM. Pathways of endocrine disruption during male sexual differentiation and masculinization. Best Pract Res Clin Endocrinol Metab 20062091-110.3. Sharpe RM, Skakkebaek NE. Testicular dysgenesis syndrome mechanistic insights and potential new downstream effects. Fertil Steril 200889(2 Suppl)e33-8.4. Wu YQ, Dai L, Wang YP, Liang J, Zhu J, Wu DS. Secular Trends of Hypospadias in Chinese Perinatals. J Sichuan Univ (Med Sci Edi) 200536274-6.5. Liang WQ, Ji CY, Zhang JM, et al. The correlation between the type of hypospadias and external genital system malformations. Chin J Urol 201132126-9.6. Laporte J, Kioschis P, Hu LJ, et al. copy and char acterization of an al ternatively spliced gene in proximal Xq28 deleted in two patients with intersexual genitalia and myotubular myopathy. Genomics 19974145862.7. Laporte J, Guiraud-Chaumeil C, Vincent MC, et al. Mutations in the MTM1 gene implicated in X-linkedmyotubular myopathy. ENMC International Consortium on Myotubular Myopathy. European NeuroMuscular Center. Hum groyne genet 199761505-11.8. Bartsch O, Kress W, Wagner A, et al. The novelcontiguous gene syndrome of myotubular myopathy(MTM1), male hypogenitalism and deletion in Xq28 root of the first familial case. Cytogenet Cell Genet 199985310-4.9. Bates PA, Kelley LA, MacCallum RM, et al. Enhancement of protein mannikin by human intervention inapplying the automatic programs 3D-JIGSAW and3D-PSSM. Proteins 2001S5(Suppl 5)39-46.10. Biancalana V, Caron O, Gallati S, et al. Characterisation of mutations in 77 patients with X-linked myotubular myopathy, including a family with a very mild phenotype. Hum Genet 2003112135-42.11. Hu LJ, Laporte J, Kre ss W, et al. Deletions in Xq28 in two boys with myotubular myopathy and abnormal genital development define a new contiguous gene syndrome in a 430 kb region. Hum Mol Genet 19965139-43.12. Fukami M, Wada Y, Miyabayashi K, et al. CXorf6 is a causative gene for hypospadias. Nat Genet 2006381369-71.13. Kalfa N, Liu B, Klein O, et al. Mutations of CXorf6 are associated with arrange of severities of hypospadias. Eur J Endocrinol 2008159453-8.14. Chen Y, Thai HT, Lundin J, et al. mutational study of the MAMLD1-gene in hypospadias. Eur J Med Genet 201053122-6.15. Qian C, Lin HW, Sun P, et al. enquiry of MAMLD1 gene in Hypospadias. J Clin Pediatr Surg 201211106-11.Figure LegendsFigure 1. Mutations of MAMLD1, the number represents the exon serial number, the black area represents the coding region.Table 1Clinic phenotypes of 72 patients with hypospadiasTable 2. Patients complicated by other genitourinary system malformationsTable 3. Mutations of MAMLD1 gene screened in patients and controls