Application Value Of Whole Exome Sequencing And Scrotal Mediastinal Flap In The Treatment Of Hypospadias

Objective:Hypospadias is a common congenital malformation of the male external genitalia.Numerous studies have shown that its etiology is highly heterogeneous,including genetic variation,endocrine abnormalities,and environmental disruptors,which is a co-product of multifactorial influences.Therefore,the clinical etiological diagnosis of hypospadias is extremely challenging.At present,the population base of hypospadias patients in China is huge,but the research on its genetic factors is relatively lacking.In this study,whole exome sequencing（WES）technology was used to genetically detect patients with hypospadias.On the one hand,the genetic pathogenicity of known variants can be verified,and on the other hand,new variants may be discovered and continuously enrich the gene variation database,which will provide a basis for future research on the function of pathogenic genes to clarify the pathogenesis of hypospadias molecules.Method:In this study,blood samples were collected from 42 patients with severe hypospadias with karyotype 46,XY from Nanhai District Maternal and Child Health Hospital of Foshan City.According to the GeneCards and Pubmed databases,163 candidate genes closely related to reproductive system development were selected.Whole exome sequencing technology was used to detect its genes,focusing on the analysis of 163 candidate genes.The results were compared with the human reference genome,and bioinformatics analysis was performed to screen out the potentially pathogenic genetic variants associated with the phenotype of the prospector.All clinically significant variants were sourced by Sanger validation of parent samples.Protein function damage prediction analysis is carried out through SIFT,PolyPhen2,ClinPred and other authoritative software.Finally,a comprehensive assessment of the pathogenicity grade of the variant was performed according to the ACMG guidelines.Results:1.In this study,a total of 27 gene variants were found in 20 patients with severe hypospadias,including:8 cases of SRD5A2 gene,4 cases of AR gene,3 cases of CYP17A1 gene,and 1 case each of WT1 gene,ANOS1 gene,NR5A1 gene,FGFR1 gene and DHX37 gene.2.Thirteen mutation sites of the SRD5A2 gene were found:7 cases of c.680G>A（p.Arg227Gln）,2 cases of c.607G>A（p.Gly203Ser）,2 cases of c.16C>T（p.Gln6Ter）,1 cases of c.408C>A（p.Tyr136Ter）,1 cases of c.142C>T（p.Pro48Leu）.Four different mutation sites were found in the four AR genes:c.302G>A（p.Arg101His）,c.2667C>T（p.Ser889=）,c.2593G>T（p.Asp865Tyr）and c.1705G>T（p.Gly569Trp）.Five different mutations were found in three CYP17A1 genes:c.316 T>C（p.Ser106Pro）、c.1263G>A（p.Ala421A1a）、c.1459₁467del GACTCTTTC（p.D487_F489del）、c.1298 T>C（p.Leu433Ser）and c.970-47C>T（Intron 5/7）.There was one mutation each in c.1373G>A（p.Arg458Gln）of WT1 gene,c.1369C>T（p.Arg457Ter）of ANOS1 gene,c.937C>T（p.Arg313Cys）of NR5A1 gene,c.995C>T（p.Ser332Phe）of FGFR1 gene and c.923G>A（p.Arg308Gln）of DHX37 gene.3.Six novel missense mutations were described for the first time in this study,including c.302G>A,c.2593G>T,c.1705G>T in the AR gene,c.1298 T>C in the CYP17A1 gene,c.995C>T in the FGFR1 gene,and c.923G>A in the DHX37 gene.In addition,a new intron mutation c.970-47C>T,Intron5/7 was found in the CYP17A1 gene.The remaining mutation sites have been reported in the Clinvar,HGMD,or Pubmed databases.4.This study sequenced the genes of some family members with a family of hereditary hypospadias.The results showed that 4 family members had AR gene common site mutations:p.Ser889=（c.2667C>T）.Conclusion:1.Genetic testing is helpful for the early diagnosis of hereditary hypospadias and provides scientific basis for individualized treatment of patients.2.Severe hypospadias is the most common clinical phenotype of genetic variation.Therefore,it is necessary to focus clinically on the presence of genetic variants in such patients.3.Hypospadias has a pronounced familial cluster.WES can provide patients with genetic counseling.4.SRD5A2 gene and AR gene are still the two most frequently detected pathogenic genes.In addition,we found rare variants in the FGFR1 gene and the DHX37 gene.Thus,the database of hypospadias gene variants was enriched.Objective:The only effective cure for hypospadias is surgical repair.Various literature reports that there are more than 300 surgical methods,each with its own advantages and disadvantages.However,there is currently no perfect procedure for the repair of all types of hypospadias.Due to complex factors such as large individual differences in hypospadias phenotypes,uneven technical levels of operators,and poor reproducibility of surgical methods,the incidence of postoperative complications is extremely high.Therefore,hypospadias surgery is very difficult to treat.To assess the surgical effects of patients with moderate to severe hypospadias who undergo surgery for scrotal mediastinal rectangular flap urethroplasty.By exploring the safety and efficacy of this procedure,we can determine whether this procedure is suitable for primary hospitals.Method:This study retrospectively analyzed the surgical effects of 88 patients with moderate to severe hypospadias who underwent the first phase of "scrotal mediastinal rectangular flap urethroplasty" in Foshan Nanhai District Maternal and Child Health Hospital from September 2021 to September 2022.Regular follow-up visits are conducted to eligible patients by WeChat,telephone,face-to-face interviews,etc.We keep detailed records of patients’ personal information,surgical conditions,complications,and Pediatric Penile Perception Score Scale（PPPS）scores.In this study,version25 of the SPSS program was used to perform simple descriptive statistics on survey data.The safety and efficacy of the procedure were evaluated by analyzing the success rate,postoperative complications,postoperative appearance and functional satisfaction.Results:1.Basic characteristics of patients:（1）.Classification:25 cases（28.4%）of penile radicular hypospadias,47 cases（53.4%）of scrotal hypospadias,16 cases（18.2%）of perineal hypospadias.（2）.Penile scrotal transposition:5 cases（5.7%）had no transposition,20 cases（22.7%）were mild,29 cases（33.0%）were moderate,and 34 cases（38.6%）were severe.（3）.Scrotal bifissure:16 cases（18.2%）had no fissure,27 cases（30.7%）were mild,24 cases（27.3%）were moderate,and 21 cases（23.9%）were severe.（4）.Penile curvature:1 case（1.1%）was mild,24 cases（27.3%）were moderate,and 63 cases（71.6%）were severe.（5）.The average length of the penis is 2.16cm（±0.42,range:1.50～3.00）.The average diameter of the glans penis is 1.10cm（±0.20,range:0.80～1.50）.The average length of the urethra is 3.61cm（±0.86,range:2～7）.（6）.The average operation time was 103min（± 24.62,range 63～250）.The average intraoperative blood loss was 37.44ml（±32.39,range 15～280）.（7）.Others:4 cases（4.5%）required dorsal penis folding suture to straighten downward bend.In 7 cases（8.0%）,the neourethra needed to be reinforced with testicular sheath covering.18 cases（20.5%）required drainage with indwelling scrotal film.48 cases（54.5%）required preoperative treatment with topical corticosteroids（methyltestosterone ointment/dihydrotestosterone ointment）.2.Postoperative success rate and complications:69 cases（78.4%）were cured in the first stage,and there were no serious complications after surgery and no special treatment was required.Nineteen（21.6%）had one or more complications of varying degrees after surgery,requiring further intervention.Including:10 cases（11.4%）had bleeding and hematoma,7 cases（7.9%）had urinary fistula,4 cases of urethral stricture（4.5%）,2 cases（2.3%）had urinary tract infection,1 case（1.1%）each had urethral diverticulum,urethral dehiscence,and surgical side injury,and there was no penis residual downward bend（>30°）or torsion（>90°）3.Satisfaction Survey:66 cases（75%）were very satisfied with 11～15 points,16 cases（18.2%）were dissatisfied with 6～10 points,and 6 cases（6.8%）were very dissatisfied with 0～5 points.The dissatisfactory conditions included slightly thin urine line in 11 cases（12.5%）,bifurcation in 5 cases（5.7%）,skewed urinary line in 5 cases（5.7%）,low position of external urethral ostia in 12 cases（13.6%）,poor penile exposure in 2 cases（2.3%）,and obvious hyperplasia of scars in 3 cases（3.4%）.Conclusion:In this study,the first phase of the operation "scrotal mediastinal rectangular flap urethroplasty" treated moderate and severe hypospadias,and achieved good function and satisfactory appearance results in short-term follow-up.It also has a low complication rate.Surgical techniques are easy to master.Therefore,it can be used as a safe and effective surgical method for the treatment of moderate and severe hypospadias in primary hospitals.