Identification of gene variants in 130 Han Chinese patients with hypospadias by targeted next-generation sequencing.

BACKGROUND: Hypospadias is a common congenital malformation of male external genitalia, which mainly manifests as an abnormal urethral opening on the ventral side of the penis. The etiology and clinical phenotype of hypospadias is highly heterogeneous, and its clinical diagnosis is challenging. Currently, over 70% of patients have an unknown etiology. Here, we performed a targeted analysis of gene mutations in 130 patients with hypospadias of unknown etiology to find the precise genetic cause. METHODS: We developed a targeted next-generation sequencing (NGS) panel, encompassing the exon coding regions of 105 genes involved in external genitalia and urogenital tract development and performed sequencing analysis on 130 children with hypospadias of unknown etiology. RESULTS: In total, 25 patients with hypospadias (19.2%) were found to have 20 mutations among the nine genes involved in external genitalia and urogenital tract development, including 16 reported and four novel mutation sites. Twenty-two patients (16.9%) had diagnostic variants. Multiple genetic mutations were identified in three of the 25 patients. Hypospadias combined with micropenis was the most common phenotype (68%) in 25 patients. CONCLUSIONS: Higher frequency mutations were identified in SRD5A2 (52%) and AR (24%) in our patient cohort. Middle or posterior hypospadias with micropenis may be significant indicators of genetic variations. Polygenic inheritance may be a rare genetic cause of hypospadias.

Management of failed hypospadias: choosing the right method and achieving optimal results.

OBJECTIVE: Because of the complexity of the abnormalities and limited options for reconstruction of failed hypospadias, creating a neourethra presents a challenge to surgeons. We reviewed our experiences with staged urethroplasty strategies to repair the penis of failed hypospadias. MATERIALS AND METHODS: We retrospectively reviewed 56 consecutives patients following multiple unsuccessful hypospadias repairs from 2010 to 2016. Patients were divided into the following two groups based on their penile conditions and urethroplasty procedures: staged buccal mucosa graft Bracka urethroplasty (group1) and two-stage urethroplasty with additional buccal mucosa graft augmentation of the dorsal urethral plate (group2). RESULT: Median follow-ups were 26.5 months (12-59 months) and 28.6 months (14-59 months) in the group 1 and group 2. After the second stage, three patients (11.1%) in group 1 and two patients (6.89%) in group 2 did not have a meatal opening at the top of the glans. Three patients (11.1%) in group 1 and 4 patients (13.79%) in group 2 had urethrocutaneous fistulas. One patient (3.70%) in group 1 and no patients in group 2 had meatal stenosis. Two patients (6.89%) in group 2 and no patients in group 1 had urethral strictures; all patients with strictures were cured using dilations, so follow-up surgeries were not required. No patients in either group had signs of diverticulum or residual chordee. Three patients (11.1%) in group 1 and 4 patients (13.79%) in group 2 needed reoperations. CONCLUSION: Failed hypospadias repairs were often due to the underestimation of the penile conditions at the prior surgery. The results indicated that two-staged strategies were preferred for treating complex situations during the intermediate period of our study. Staged buccal mucosa graft Bracka urethroplasty and two-stage urethroplasty with additional buccal mucosa graft augmentation of the dorsal urethral plate severed as reliable approaches in complex hypospadias cases and could improve the overall success rate.

Association of MAMLD1 single-nucleotide polymorphisms with hypospadias in Chinese Han population.

Hypospadias is one of the most common congenital malformations among children. Both gene mutations and environmental factors are thought to be involved in the development of hypospadias. The mastermind-like domain-containing 1 gene (MAMLD1, formerly CXorf6) is a new candidate gene and its mutation has been shown in some cases of hypospadias. Here, by direct sequencing of PCR products, we assessed and found mutations that occur in 220 sporadic cases of hypospadias. The mutations p.N589S (c.1766A>G) was found at a significantly higher rate among patients with hypospadias.

[Risk factors for the complications of primary hypospadias urethroplasty with the urethral plate reserved].

OBJECTIVE: To investigate the risk factors for the complications of urethroplasty in patients with primary hypospadias by postoperative follow-up observation. METHODS: We retrospectively analyzed 110 cases of primary hypospadias repair performed from November 2010 to October 2015, including 70 cases of tubularized incised plate (TIP) urethroplasty and 40 cases of inlay internal preputial graft (IIPG) urethroplasty, all with the urethral plate reserved. We followed up the patients for 15.6－36 months, (27.3 ± 0.52) mo for those with and (26.9 ± 0.22) mo for those without complications. The mean age of the two groups of patients was (7.5 ± 0.2) and (7.0 ± 0.5) yr, respectively. RESULTS: The follow-up data were collected from all the patients, 17 (15.5%) with and 93 (84.5%) without complications. The success rate of surgery was 84.5%. There were no statistically significant differences in the follow-up time and age between the two groups of patients (P >0.05). Single-factor analysis of variance showed significant differences between the complication and non-complication groups in the preoperative urethral opening (P <0.01), ventral penile curvature (P <0.01), and length of urethral defect (P = 0.04), while multiple linear regression analysis exhibited that only ventral curvature was associated with the postoperative complications of the patients (OR = 1.12, 95% CI: 1.06－1.19, P<0.01). CONCLUSIONS: We chose single-stage urethroplasty with the urethral plate reserved for the treatment of primary hypospadias and achieved satisfactory outcomes. Ventral penile curvature is an independent risk factor for the complications of primary hypospadias, and a higher degree of curvature is associated with a higher incidnece of complications.

The Effects of Guizhi Fuling Capsule Drug Serum on Uterine Leiomyoma Cells and Its Mechanism.

Aims. To observe the effects of Guizhi Fuling Capsule (GZFLC) drug serum on uterine leiomyoma cells and explore its mechanism. Main Methods. Sixty Sprague Dawley rats were randomly divided into two groups (normal saline lavage group and GZFLC lavage group), then, respectively, blank serum and GZFLC drug serum were collected, and finally human uterine leiomyoma cells were treated. Human leiomyoma tissues were collected from 20 patients who underwent uterine leiomyomas operations, and leiomyoma cells were primary cultured. The leiomyoma cells were treated by GZFLC drug serum in different concentrations (10%, 20%, and 30%) and variable treatment time (12 h, 24 h, 36 h, 48 h, and 72 h). Cell proliferation was observed using CCK8 assay. Flow cytometry and Annexin V/PI were used to assay the effects of GZFLC drug serum on cell apoptosis. Western blot analysis was used to assay the effects of GZFLC drug serum on TSC2, FOXO, and 14-3-3γ expression in uterine leiomyoma cells. Key Findings. In the concentrations of 10%～30%, GZFLC drug serum could inhibit proliferation of leiomyoma cells in dose-dependent manner; at the time of 36 h, cell inhibition rate was at the peak; GZFLC drug serum could induce apoptosis of leiomyoma also in a dose-dependent manner, and apoptosis rate quickly achieved maximum at 12 h time points, and then second apoptosis peak appeared at 36 h. Compared to nontreatment group, TSC2, FOXO, and 14-3-3γ expressions in drug serum group were significantly changed after 12 h treatment. Significance. GZFLC drug serum can efficiently inhibit the proliferation and induce apoptosis of leiomyoma cells, which is related to the 14-3-3γ pathway.

Expression of AQP3 protein in hAECs is regulated by Camp-PKA-CREB signalling pathway.

Previous studies by others and our group have demonstrated the expression of AQP3 protein in human chorioamniotic membranes. Here, we investigated whether cyclic adenosine monophosphate (cAMP) up-regulation of aquaporin 3 (AQP3) protein expression in human amniotic epithelial cells (hAECs) is mediated by protein kinase A (PKA) dependent or independent pathway. Cells were treated with various concentrations of Forskolin, SP-cAMP, H-89 at various time intervals or optimal concentration of Forskolin in combination with H-89 in the blocking experiments. Forskolin significantly increased cAMP levels and the expression of PKA, p-CREB and AQP3. SP-cAMP had similar effects. H-89 inhibited PKA, p-CREB and AQP3 protein expression, and attenuated the stimulatory effect of Forskolin. These results show that the AQP3 protein expression in hAECs was regulated by cAMP-PKA-CREB signal pathway. A relatively short biological half life of AQP3 renders its rapid responsiveness to regulation.

One-stage dorsal inlay oral mucosa graft urethroplasty for anterior urethral stricture.

BACKGROUND: Anterior urethral stricture remains a great challenge. We reported our clinical technique and results by using inlay dorsal buccal mucosal graft urethroplasty for repair of anterior urethral stricture. METHODS: From January 2005 to July 2008, 87 male patients (range from 9 to 72 years old) with anterior urethral stricture have been treated by one-stage dorsal inlay oral mucosal graft (OMG) urethroplasty. All patients gave written informed consent for their participation. All patients showed that urethral plate had been either scarred or removed previously. In our surgery, the urethra was dissected dorsally and scar of the urethral plate was removed. The remnant urethral plate was split at midline and a buccal mucosa patch was inserted between the two parts. Neourethra was tubularized and covered with dartos flap. The pre-operative assessments included clinical data, urine analysis, uroflowmetry, retrograde and voiding cystogram, urethral ultrasonography and endoscopy. Postoperatively, the flow rate and void residual volume were analyzed by uroflowmetry and sonography. Any further instrumentation to assist voiding was considered as failure. RESULTS: After 12 to 48 months (mean: 25.8 months), 78 patients (89.66%) have shown good results by the one-stage urethroplasty. However, 9 patients (10.3%) required further treatment due to recurrence, while 6 patients (6.9%) had fistula. CONCLUSIONS: This one-stage dorsal inlay approach using dorsal oral mucosal grafts is a reliable method to create a substitute urethral plate for tubularization. This approach represents a safe option for anterior urethral stricture patients especially with grossly scarred urethral plate.

[Single nucleotide polymorphisms of the DGKK gene and hypospadias in Chinese children].

OBJECTIVE: To investigate the role of single nucleotide polymorphisms of the gene of diacylglycerol kinase κ (DGKK) in hypospadias in Chinese children. METHODS: We performed direct sequencing on 2 hypospadias-related candidate single nucleotide polymorphisms of the DGKK gene (rs1934179 and rs7063116, never previously reported in the Chinese population) from 300 children with sporadic hypospadias and 200 healthy controls, and compared the results between the two groups. RESULTS: The mutation frequencies of rs1934179 and rs7063116 were 5.0% (15/300) and 5.67% (17/300) respectively in the hypospadias patients, significantly higher than 1.5% (3/200) and 2.0% (4/200) in the normal controls (P <0.05). The mutation frequencies of rs1934179 and rs7063116 in the cases of distal and middle hypospadias were also remarkably higher (6.5%, [13/200] and 7.5% [15/200], P <0.05), but those in the proximal cases (both 2.0% [2/100]) showed no statistically significant difference from the control (P >0.05). CONCLUSION: The polymorphisms of the DGKK gene may be associated with hypospadias, particularly distal and middle hypospadias, in Chinese children.

Botryoid Wilms' tumor: report of two cases.

BACKGROUND: Botryoid Wilms' tumor is a rare kind of Wilms' tumor. We report two cases of this tumor. METHODS: Case 1, a 2-year-old boy, was admitted with macrohematuria for 5 months. Case 2, a 19-month-old boy, was referred for a palpable abdominal mass. The two cases were checked by 64-row multi-slice spiral CT (MSCT) and scanned with the dynamic contrast enhancement. The masses were excised and pathologically confirmed. RESULTS: In case 1, the mass occurred in the renal pelvis and calyx bilaterally, with heterogeneous density and prominent calcification. By contrast enhanced CT scan, the mass was mildly enhanced. In case 2, the left renal pelvis and ureter were filled with the tumor. Unenhanced scan revealed that the mass was homogeneous and non-calcified. In contrast, the mass was slightly and heterogeneously enhanced. Macroscopically, the mass filled in the pelvicalyceal system and had a botryoid appearance. Microscopically, the typical features of Wilms' tumor with blastemal, epithelial, and stromal components were evident. CONCLUSION: Botryoid Wilms' tumor should be included in the differential diagnosis of tumors in the pelvicalyceal system no matter it is unilateral or bilateral.

[Correction of penile curvature by various tunica albuginea plasty].

OBJECTIVE: To explore various tunica albuginea plasty for penile curvature and their efficacies. METHODS: From July 2005 to July 2008, there were a total of 8 patients with a mean age of 34 ± 9 years old (range: 23 - 47) undergoing corrective surgery for various types of penile curvature. Among them, 4 patients (3 with ventral curvature, 1 with lateral curvature) were treated by 16-dot plication technique, 2 patients of lateral curvature with tightening ring in tunica albuginea corrected by V-Y plasty and 2 patients (one caused by Peyronie's disease while the other by tunica scar contracture) repaired by venous graft patch. The pre-and post-operative levels of sexual satisfaction were evaluated by the International Index of Erectile Function (IIEF-5) scoring system. RESULTS: All cases were followed up at 3, 6 & 9 months postoperatively. Their mean curvature at maximal erection was (58.2 ± 9.7)°. And a postoperative office assessment revealed a functional straight in all patients [mean degree (11 ± 4)°]. One patient (12.5%) reported penile shortening without dissatisfaction about it. No patients complained of anesthesia or other sensory abnormality on their penile skin. An improvement of IIEF-5 scores was noted (21 ± 3 vs 15 ± 5, P < 0.05) and all patients were satisfied with their outcomes. No complications, such as hematoma, infection or erectile dysfunction, occurred. CONCLUSION: To achieve a better penile morphology and functional outcomes with fewer complications, various arthroplasty should be adopted according to the cause, degree and direction of penile curvature.

Single stage dorsal inlay buccal mucosal graft with tubularized incised urethral plate technique for hypospadias reoperations.

AIM: To report the experience with single stage dorsal inlay buccal mucosal grafts using the Snodgrass technique for complex redo cases. METHODS: From May 2004 to December 2005, a total of 53 patients aged from 3 to 34 years old (average 11.62 +/- 7.18 years) with failed previous hypospadias surgery were included in the present study. Indications included urethral strictures and repair breakdown. The unhealthy urethra was unroofed from the meatus in the ventral midline, a buccal mucosal graft was inlayed between the incised urethral plate and fixed to the corpora cavernosa. The neourethra was tubularized, and covered with subcutaneous (dartos) tissue and penile skin. Glanuloplasty was also performed in all cases. Outcome analysis included clinical follow-up, and endoscopy in 2 selected cases. RESULTS: The buccal mucosal graft was 3.0-7.5 cm in length and 0.7-2.0 cm in width. All patients required glanuloplasty, with buccal mucosal grafts extended to the tip of the glans. After a follow-up of 14-30 months (mean 22.6 months), the total complication rate was 15.1%, with five cases of fistula and three cases of stricture. CONCLUSION: Inlaying dorsal buccal mucosal grafts applying the Snodgrass technique is a reliable method for creating a substitute urethral plate for tubularization. The recurrent rate of urethral stricture and fistula is at an acceptable level for redo cases. This approach represents an effective, simple and safe option for reoperations.

Bilateral intrarenal pelvis Wilms' tumor with fibroepithelial polyp.

Intrarenal pelvis Wilms' tumor is rare in children. A case of a 28-month-old boy with bilateral intrarenal pelvis Wilms' tumor associated with a fibroepithelial polyp is reported in this article. The tumor was evaluated by ultrasonography, computed tomography, and intravenous pyelography. The boy underwent bilateral renal pelviotomies. Now he is being treated and followed up by pediatric oncologists.