MAMLD1 Mutation and Phenotypes of Hypospadias

MAMLD1 Mutation and Phenotypes of HypospadiasThe relationship amongst clinical phenotypes and revolutions of MAMLD1 in children with hypospadiasYong-fen Lv, Lu-lu Cui, Pin Li*Department of Endocrinology, Shanghais Children Hospital, Shanghai Jiaotong UniversityAcknowledgementsThe work was financi whollyy support by the key project of Shanghai municipal health bureau (2011111), youth project of Shanghai municipal health bureaumajor uncover 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 in the midst of clinical phenotypes of hypospadias and variances of MAMLD1.Methods Seventy-deuce patients were diagnosed to be hypospadias in department of endocrinology and department of urinary surgery in our hospital. Among all the patients, 69 were with normal karyotype and enrolled as the flush toiletvas group. liter firm boys were employed as the controls. Peripheral Blood were collected for DNA extraction. For the examine group, PCR primer was designed and direct sequencing was performed for screening for MAMLD1 mutations in six cryptogram exons and the flanking region. Those mutated exons were examined for the control group.Results Thirty-five of all the 72 patients (48.6 %) were isolated hypospadias. The former(a) 37 cases (51.4%) were complicated by other genitourinary system malformations, including 12 cases with micropenis and/or underdeveloped testicles. Abnormal karyotype was identified 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 find 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 single nucleotide polymorphism except c.5AG (p.D2G), a newly discovered percentage point mutation. The relative absolute frequency of IVS4-364C/A was significantly opposite between patients and controls, and it was also significantly contrastive between patients with and without micropenis and/or underdeveloped testicles.Conclusion Chromosome abnormality is not the spark advance cause of other genitourinary system malformations complicated with hypospadias. Mutations of MAMLD1 maybe closely colligate 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.IntroductionHypospadias is one of the most common congenital genitourinary system malformations in males, with incidence 11%. As one of the Juvenile-types of testicular dys divisorsis syndrome,(1-3) the prevalence of hypospadias is obviously increasing in these years. From 1987 to 2001, the prevalence rate was doubly increased in China.(4) Hypospadias will asterisk to different degrees of genital malformation, and the clinical p henotypes vary when with other complications. For instance, besides the factorral signs of hypospadias, e.g., ectopic ureteral orifice, phallocampsis, redundant dorsal prepuce, etc, the patient may be also suffered from other malformations including penoscrotal transposition, cryptorchidism, hydrocele, oblique inguinal hernia, micropenis and underdeveloped testicles. Hypospadias is a complicated disease due to various causes. The causes of most cases atomic number 18 not able to be verified, specially for those mild cases. For these cases, environmental factors, endocrine factors and abnormal agent expression may be the leading 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) with molecular len gth 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 reproductive systems of patients are normally developed with mutations in Myotubularin MTM-1, maculation different degrees of malformations occur in cases with deletion of MTM1 gene.(8-11) The subsequent experiments indicated 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 indicate that MAMLD1 is the perfect candidate gene for the sphere of 46, XY DSD, especially for hypospadias.This work aimed to evaluate 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-two children with hypospadias admitted to Shanghai Childrens Hospital Affiliated to Jiaotong University from March 2011 to Decemb er 2012 were enrolled in this study. Definite diagnosis was ground on the clinical signs, and patients with adrenogenital syndrome were excluded through clinical examination.Clinical examinationClinical survey was performed including patients complain, present medical history, past medical history, in-person history, family history, birth history, mothers medical history in pregnancy, previous exposure to environmental pollution, etc. Physical examination was performed to measure the hight, weight, heart rate, daub pressure and the status of gonad development, etc. For adolescent, the development of secondary sexual characteristics was also assessed.Regular auxiliary examinations were performed including blood and urine routine test, biochemistry test, gonadal endocrine level, adrenal cortex function and abdominal ultrasound exam, etc.Karyotype analysis and detection of SRY geneLymphocytes were isolated from peripheral blood of patients, cultured and smeared on slides, and G-ban ds 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 Center for Biotechnology culture (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 Blood DNA Kit (TIANGEN Biotech (Beijing) Co., Ltd, China) and chastity 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 used for statistical evaluation. Chi-square test was used to compare the two groups, frequency of single nucleotide polymorphism between the two groups was analyzed using Fisher 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). Two 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 opening position was located at the middle segment of penis. Due to the limited number of mutation cases, the sample size should be increased to study the relationship between c.5AG (p.D2G) and phenotypes of hypospadias. Analyzed by Chi-squared test with Yates continuity correction, the frequency of IVS4-364C/A was significantly different between patients with and without micropenis and/or underdeveloped testicles (p=0.001).DiscussionChromosome abnormality and karyotype change is one of the causes of hypospadias. Till now, ten types of chromosome abnormalities were confirmed involving 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, womb and ovary were not found through the laparoscopic exploration. Therefore, they were diagnosed to be 46, XX male sex reversal 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) Except MAMLD1, no other candidate genes were found in the deletion regi on. These results indicate that MAMLD1 is the perfect candidate gene for the study of 46, XY DSD, especially for hypospadias.In the works 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 opening position located on scrotum and the interchangeable 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 subgenus 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 isolated hypospadias,(15) where 200 healthy participants were randomly 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 population between China and abroad, i.e., the studied populations of foreign works were mostly composed 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 exceedingly conserved, and analyzed via polyphe n, the mutation c.5AG (p.D2G) of MAMLD1 is predicted to be probably damaging with a score of 0.996 from thrumDiv and 0.993 from HunVar, which indicates that c.5AG (p.D2G) is highly related to hypospadias. SIFT predicts that the mutation can affect 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 hulky sample size and the information of the exact protein function are required to elucidate whether c.5AG (p.D2G) of MAMLD1 is the cause of isolated hypospadias and its role in human sexual differentiation.The meaty SNP detected in this work is the full mutation in introns close 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 openings 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 before the mature mRNA can be transported, thus do not exist in the mRNA sequence. However, there may be several 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 jump 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 crop mode, thus lead to the change of protein function. Next step of our study plan is to verify whether the mutation could affect mRNA shear mode through reverse transcription, amplificatio n 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 tumult during male sexual differentiation and masculinization. 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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. Research 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