MAMLD1 Mutation and Phenotypes of Hypospadias

MAMLD1 Mutation and Phenotypes of Hypospadias The relationship between clinical phenotypes and mutations of MAMLD1 in children with hypospadias Yong-fen Lv, Lu-lu Cui, Pin Li* Department of Endocrinology, Shanghais Children Hospital, Shanghai Jiaotong University Acknowledgements: The work was financially supported by the key project of Shanghai municipal health bureau (2011111), youth project of Shanghai municipal health bureauà ¯Ã‚ ¼Ã…’major issue subprojects of Shanghai science and technology commission (12411952408), Yangtze river delta research project of Shanghai science and technology commission (13495810300). Abstract Purpose: To verify the relationship between clinical phenotypes of hypospadias and mutations of MAMLD1. Methods: Seventy-two 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 studied group. Fifty healthy boys were employed as the controls. Peripheral Blood were collected for DNA extraction. For the studied group, PCR primer was designed and direct sequencing was performed for screening for MAMLD1 mutations in six coding 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 other 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 detected in exon 2, 3, 5, 7 in studied group, including c.5A>G (p.D2G), IVS4-364C/A, c.1910A>Gà ¯Ã‚ ¼Ã‹â€ p.N637S), c.2208T>C, c.2227 G>A (p.E742K) and IVS8-144C/T. All were single nucleotide polymorphism except c.5A>G (p.D2G), a newly discovered point mutation. 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. Conclusion: Chromosome abnormality is not the leading cause of other genitourinary system malformations complicated with hypospadias. Mutations of MAMLD1 maybe closely related to hypospadias in Chinese. c.5Aà ¯Ã‚ ¼Ã… ¾Gà ¯Ã‚ ¼Ã‹â€ p.D2Gà ¯Ã‚ ¼Ã¢â‚¬ °is the newly discovered point mutation in this work. IVS4-364C/A is associated with underdeveloped testicles and/or micropenis in hypospadias patients. Introduction Hypospadias is one of the most common congenital genitourinary system malformations in males, with incidence 1†°Ãƒ ¯Ã‚ ½Ã… ¾1%. As one of the Juvenile-types of testicular dysgenesis 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 lead to different degrees of genital malformation, and the clinical phenotypes vary when with other complications. For instance, besides the general 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 are not able to be verified, especially for those mild cases. For these cases, environmental factors, endocrine fa ctors and abnormal gene 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 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 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) 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 study of 46, XY DSD, especially for hypospa dias. 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. Methods Patients Seventy-two children with hypospadias admitted to Shanghai Childrens Hospital Affiliated to Jiaotong University from March 2011 to December 2012 were enrolled in this study. Definite diagnosis was based on the clinical signs, and patients with adrenogenital syndrome were excluded through clinical examination. Clinical examination Clinical survey was performed including patient’s complain, present medical history, past medical history, personal history, family history, birth history, mother’s medical history in pregnancy, previous exposure to environmental pollution, etc. Physical examination was performed to measure the hight, weight, heart rate, blood 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 hormone level, adrenal cortex function and abdominal ultrasound exam, etc. Karyotype analysis and detection of SRY gene Lymphocytes were isolated from peripheral blood of patients, cultured and smeared 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 mutations The gene sequence of MAMLDI was obtained from National Center 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 Blood 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 94à ¢Ã¢â‚¬Å¾Ã†â€™ for 30 seconds,extension at 72à ¢Ã¢â‚¬Å¾Ã†â€™ 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 analysis The 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.1910A>G (p.N637S), c.2208T>C, c.2227 G>A (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 A>G (p.N637S, rs2073043) and c.2208T>C. Among all the mutations, c.5A>G (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.1910A>G (p.N637S), c.2208T>C, c.2227 G>A (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 be tween 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 hypospadias One case with c.5A>G (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.5A>G (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). Discussion Chromosome 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, uterus 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 region. 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 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+8a>t, 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.1699C>T and c.1985A>G, were detected and all were SNPs, and statistical analysis revea led 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.5A>G 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.5A>G (p.D2G) of MAMLD1 is predicted to be probably damaging with a score of 0.996 from HumDiv and 0.993 from HunVar, which indicates that c.5A>G (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.5Aà ¯Ã‚ ¼Ã… ¾G 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 elucidate whether c.5A>G (p.D2G) of MAMLD1 is the cause of isolated hypospadias and its role in human sexual differentiation. The meaningful 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 first is that there may be coding sequence relat ed to micropenis and/or underdeveloped testicles in the introns close to exon 3 of MAMLD1, the second is that the mutation could affect mRNA shear 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, amplification and sequencing of extracted mRNA. References 1. Skakkebaek NE, Rajpert-De ME, Main KM. Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects. Hum Reprod 2001;16:972-8. 2. Sharpe RM. Pathways of endocrine disruption during male sexual differentiation and masculinization. Best Pract Res Clin Endocrinol Metab 2006;20:91-110. 3. Sharpe RM, Skakkebaek NE. Testicular dysgenesis syndrome: mechanistic insights and potential new downstream effects. Fertil Steril 2008;89(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) 2005;36:274-6. 5. Liang WQ, Ji CY, Zhang JM, et al. The correlation between the type of hypospadias and external genital system malformations. Chin J Urol 2011;32:126-9. 6. Laporte J, Kioschis P, Hu LJ, et al. Cloning and char acterization of an alternatively spliced gene in proximal Xq28 deleted in two patients with intersexual genitalia and myotubular myopathy. Genomics 1997;41:458–62. 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 Mol Genet 1997;6:1505-11. 8. Bartsch O, Kress W, Wagner A, et al. The novelcontiguous gene syndrome of myotubular myopathy(MTM1), male hypogenitalism and deletion in Xq28: report of the first familial case. Cytogenet Cell Genet 1999;85:310-4. 9. Bates PA, Kelley LA, MacCallum RM, et al. Enhancement of protein modeling by human intervention inapplying the automatic programs 3D-JIGSAW and3D-PSSM. Proteins 2001;S5(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 2003;112:135-42. 11. Hu LJ, Laporte J, Kress 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 1996;5:139-43. 12. Fukami M, Wada Y, Miyabayashi K, et al. CXorf6 is a causative gene for hypospadias. Nat Genet 2006;38:1369-71. 13. Kalfa N, Liu B, Klein O, et al. Mutations of CXorf6 are associated with arrange of severities of hypospadias. Eur J Endocrinol 2008;159:453-8. 14. Chen Y, Thai HT, Lundin J, et al. Mutational study of the MAMLD1-gene in hypospadias. Eur J Med Genet 2010;53:122-6. 15. Qian C, Lin HW, Sun P, et al. Research of MAMLD1 gene in Hypospadias. J Clin Pediatr Surg 2012;11:106-11. Figure Legends Figure 1. Mutations of MAMLD1, the number represents the exon serial number, the black area represents the coding region. Table 1à ¯Ã‚ ¼Ã… ½Clinic phenotypes of 72 patients with hypospadias Table 2. Patients complicated by other genitourinary system malformations Table 3. Mutations of MAMLD1 gene screened in patients and controls

Noli me tangere reflection Essay

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Karl Marx and The Russian Revolution Essay example

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Phrasal verbs are verbs that are made up of a main verb and followed by particle, usually prepositions. Most phrasal verbs are two or three words and can be quite challenging for English learners as they can be literal or figurative in meaning. In other words, sometimes it is easy to understand the meaning (such as get up), but in the case of figurative meanings can be quite confusing (such as pick up). Begin learning phrasal verbs with a limited list. The list below provides a good starting point for intermediate level English learners. Teachers can use this introducing phrasal verbs lesson plan to help students become more familiar with phrasal verbs and start building phrasal verb vocabulary. Finally, there are a wide variety of phrasal verb resources on the site to help you learn new phrasal verbs and test your understanding with quizzes. This ESL phrasal verb reference guide is intended for English learners. The guide contains some of the most important phrasal verbs used in everyday English. There are many, many more phrasal verbs, but I have chosen these verbs as a good starting point for English learners. Each phrasal verb is defined, has an example sentence for context, and states whether the definition is separable or inseparable, transitive or intransitive. For more information on how to use phrasal verbs, read the phrasal verb guide on this site. Important phrasal verbs in English starting with the letter A. Includes examples and whether the phrasal verb is separable / inseparable, transitive / intransitive. S Separable IS Inseparable T Transitive IT - Intransitive account for explain, be the reason for His lack of interest accounts for his poor grades. IS T act on take an action Tom acted on the information. IS T add to increase the size This chair will add to the furniture we already have. S T add up make sense Your guess adds up based on all the facts. IS IT agree with have the same opinion as someone I agree with Tom about the need for better schools. IS T allow something for provide time, money, or other resource for something You need to allow two hours for traffic. S T answer for something be responsible for something The director answers for the drop in sales last quarter. IS T argue something out discuss all the details to come to an agreement We argued our differences out and signed a contract. S T arrive at something agree upon something We arrived at a contract last week. IS T ask after somebody ask how someone is doing I asked after Kate last week and her mother told me she was doing well. IS T attend to something take care of something you need to do Peter attended to preparations for the party while his wife cooked the dinner. IS T average something out arrive at the average figure I average the contracts out and well make a profit of $250,000. S T