Kaixiang Cheng

A splice site mutation combined with a novel missense mutation of LHCGR cause male pseudohermaphroditism.

Leydig cell hypoplasia (LCH) is a rare form of male pseudohermaphroditism caused by inactivating mutations in the luteinizing hormone receptor gene (LHCGR). The majority of LHCGR mutations are located in the coding sequence, resulting in impairment of either LH/CG binding or signal transduction. We report a Chinese family with two siblings (46, XY and 46, XX) carrying a missense mutation (c. 455 T>C, p. Ile152Thr) and a splice site mutation (c. 537‐3 C>A). Computational analysis of the missense mutation in the three‐dimensional structural model predicted it might influence the distribution of hydrogen bonds and intermolecular contacts between the hormone and receptor. Consistent with these findings, in vitro mutant analysis revealed a marked impairment of human chorionic gonadotropin binding and signal transduction. The splice‐acceptor mutation (c. 537‐3 C>A) resulted in abnormal splicing of LHCGR mRNA, skipping exon 7. This report expands the genotypic spectrum of LHCGR mutations, with relevant implications for the molecular analysis of this gene.

Phenotypic and molecular characteristics in eleven Chinese patients with 5α-reductase Type 2 deficiency

Steroid 5α‐reductase type 2 deficiency (5α‐RD2) is a male‐limited, autosomal recessive inherited disease. Affected 46, XY individuals usually present with ambiguous genitalia at birth. An early and precise diagnosis is of great value to the long‐term prognosis of the disease.

Sensibility following innervated free radial forearm flap for penile reconstruction.

Background: The free radial forearm flap has proven to be reliable for penile reconstruction. The purpose of this study was to determine whether neurotization of this flap improved sensation of the reconstructed penis. Methods: A long-term follow-up study of 45 patients undergoing penile reconstruction using free radial forearm flap was performed; 28 of the 45 patients received an innervated flap, and 17 received a noninnervated flap. A nerve repair between the dorsal nerve of the penis and the lateral antebrachial cutaneous nerve was performed for innervation. Sensory testing, including pain perception, temperature perception, vibratory threshold, and static two-point discrimination, was performed by one blinded examiner in a standardized pattern. Results: Mean follow-up time was 9.1 years. Mean patient age was 26.4 years (range, 18 to 48 years). Postoperative pain perception and vibratory threshold were similar between the two groups in the proximal part of the neophallus but were significantly better in the innervated flaps (p < 0.01) in the distal part. Noninnervated flaps displayed a pattern of increasing sensibility from the distal part toward the proximal part, whereas innervated flaps regained sensation throughout. The innervated group had a better result of temperature perception and static two-point discrimination in both the proximal and the distal parts. Conclusions: Innervation of free radial forearm flap provides improved sensation to the reconstructed penis. If it is possible to find the functional recipient nerve, innervation should always be conducted.

Long-term follow-up of sensation recovery of the penis reconstructed by Cheng's method.

Background: Chengs method, a new surgical method for penile reconstruction in which the original glans or the residual penile stump is transferred to the anterior portion of the newly reconstructed penile body with microsurgical techniques, was first introduced in 1997. The purpose of this article was to show the recovery of sensation of the reconstructed penis. Methods: A long-term follow-up of 27 patients undergoing penile reconstruction using Chengs method was performed. Nerve repair between the dorsal nerve of the penis, the lateral antebrachial cutaneous nerve, and the dorsal nerve of the penis was performed for innervation. Four measurements for tactile sensation assessment were performed. Results: The mean follow-up was 10.3 years and the mean age of the patients was 25.7 years. The sensation of the distal part of the reconstructed penis (glans or the residual penile stump) was similar to the preoperative one (p > 0.05). There was no significant difference in sensation between the unoperated forearm and the postoperative flap (proximal part of the neophallus), except in two-point discrimination, in which the neophallus was better (p < 0.01). In the reconstructed penis, the distal part had better two-point discrimination than the proximal part (p < 0.01). Compared with our other group of penises reconstructed by free radial forearm flaps, the distal part with Chengs method had superior pain perception (p < 0.01), vibratory threshold (p < 0.05), and two-point discrimination (p < 0.01). Conclusion: Chengs method of penile reconstruction provides good sensation to the reconstructed penis, especially in the neoglans, in transferring either the original glans or the residual penile stump.

Identifying a novel mutation of CYP17A1 gene from five Chinese 17α-hydroxylase/17, 20-lyase deficiency patients

Mutations of CYP17A1 gene could cause complete or partial, combined or isolated 17α-hydroxylase/17,20-lyase enzyme deficiencies (17OHD). We intended to investigate the CYP17A1 mutation in five unrelated patients and analyze its possible influence on phenotype of an atypical 17OHD patient presented with micropenis, hypertension and intermittent hypokalemia. Steroid hormones were assayed in these patients. A novel missense mutation (c.1169C>G, p. Thr390Arg) located in exon 7 was detected in one of the patients. Homozygous c. 985_987delinsAA, p. Tyr329fs mutation was found in two patients, while compound heterozygous mutations (c. 985_987delinsAA, p. Tyr329fs/c. 932-939 del, p. Val311fs and c. 287G>A, p. Arg96Gln/c. 985_987delinsAA, p. Tyr329fs) were found in two other patients, respectively. Then, steric model analysis of CYP17A1 showed that the novel mutation T390R changed the local structure as well as the electrostatic potential of the nearby beta sheet. Finally, site-directed mutagenesis and in vitro expression were used to analyze the activity of novel mutant CYP17A1. It indicated the T390R mutant retained part of enzyme activity, which was consistent to the clinical features. In conclusion, we identified a novel missense mutation of CYP17A1 gene from a patient with micropenis, hypertension and intermittent hypokalemia, which varied from other four patients. It also expanded our understanding of genotype-phenotype correlation of the disease.

Bone morphogenetic protein 7-transduced human dermal-derived fibroblast cells differentiate into osteoblasts and form bone in vivo

ABSTRACT Background: Human dermal-derived fibroblast cells (hDDFCs) are multipotent. Bone morphogenetic proteins (BMPs) are a group of cytokines that promote different developmental processes, including the formation of bone. BMPs can promote hDDFC osteogenesis, but the role of BMP7 in hDDFC osteogenesis in vitro and bone formation in vivo has not been investigated in depth. Materials and Methods: hDDFCs were stably transfected with a human BMP7 recombinant adenovirus and osteogenic differentiation was examined by alkaline phosphatase staining and calcium accumulation. In addition, we measured the expression of osteoblast-related genes. To examine osteogenesis in vivo, we injected C57BL/6 nude mice with adenovirus-transfected hDDFCs in a calcium alginate hydrogel and examined bone formation using soft X-ray, histological, and immunohistochemical analyses. Results: Our findings showed that adenovirus-mediated BMP7 expression promoted osteogenic differentiation of hDDFCs and enhanced expression of osteoblast-related genes in vitro. Cells infected with BMP7 adenoviruses showed enhanced bone formation and osteoblast-related gene expression in vivo after the injection of hDDFC–hydrogel mixture. Conclusions: Taken together, our data indicate that BMP7 significantly promotes hDDFC osteogenesis, and confirm that infecting hDDFCs with BMP7-expressing adenoviruses is a useful tool for bone tissue engineering.

A New Surgical Procedure for Penile Reconstruction by Combined Free Radial Forearm Flap and Dorsalis Pedis Flap

OBJECTIVE To introduce a new surgical procedure for penile reconstruction, emphasizing both the aesthetic appearance and the function by combined free radial forearm flap and dorsalis pedis flap. PATIENTS AND METHODS In this procedure, the penis was subdivided into 2 anatomic subunits: the penile shaft and the glans penis. A sequential innervated radial forearm free flap was combined with a dorsalis pedis free flap to reconstruct the penile shaft and the glans, separately. Cartilage prosthesis was implanted at the same time. Since May 2011, 14 biologically male patients with total penile losses by various reasons were treated with this procedure. Patient satisfaction was evaluated by questionnaire, and sensory testing was performed. RESULTS The ages of the patients ranged between 21 and 53 years (mean, 35.2 years). The average follow-up period was 38.1 months (range, 25.5-56 months). Twenty-five flaps in 11 patients were 100% viable. One dorsalis pedis flap in a patient underwent partial necrosis. There were no cases of urethral fistula or urethral stenosis, but 1 case of prosthesis infection and 1 case of abdominal hernia were recorded. The sensation of the neophallus recovered 3-6 months after surgery, and the patient satisfaction rate was quite high. CONCLUSION The new surgical procedure of combined free radial forearm flap and dorsalis pedis flap for penile reconstruction achieves both satisfactory aesthetic and functional results. We recommend this procedure as an alternative ideal method for total penile reconstruction.

Bone morphogenetic protein 7 enhances the osteogenic differentiation of human dermal-derived CD105+ fibroblast cells through the Smad and MAPK pathways

The skin, as the largest organ of the human body, is an important source of stromal stem cells with multipotent differentiation potential. CD105+ mesenchymal stem cells exhibit a higher level of stemness than CD105− cells. In the present study, human dermal-derived CD105+ fibroblast cells (CD105+ hDDFCs) were isolated from human foreskin specimens using immunomagnetic isolation methods to examine the role of bone morphogenetic protein (BMP)-7 in osteogenic differentiation. Adenovirus-mediated recombinant BMP7 expression enhanced osteogenesis-associated gene expression, calcium deposition, and alkaline phosphatase activity. Investigation of the underlying mechanisms showed that BMP7 activated small mothers against decapentaplegic (Smad) and p38/mitogen-activated protein kinase signaling in CD105+ hDDFCs. The small interfering RNA-mediated knockdown of Smad4 or inhibition of p38 attenuated the BMP7-induced enhancement of osteogenic differentiation. In an in vivo ectopic bone formation model, the adenovirus-mediated overexpression of BMP7 enhanced bone formation from CD105+ hDDFCs. Taken together, these data indicated that adenoviral BMP7 gene transfer in CD105+ hDDFCs may be developed as an effective tool for bone tissue engineering.

Next-generation sequencing reveals genetic landscape in 46, XY disorders of sexual development patients with variable phenotypes

Disorders of sexual development (DSD) are rare congenital conditions in which chromosomal, gonadal, or anatomical sex is atypical. Currently, less than 20% of patients receive an accurate genetic diagnosis. Targeted next-generation sequencing, consisting of 33 candidate genes and 47 genes involved in sexual differentiation and development, was performed on 70 46, XY DSD patients. Functional assays were performed to evaluate the expression and transcriptional activity of one reported and nine novel mutations of NR5A1. In total, 113 mutations, including 86 novel and 27 reported sites in 40 genes, were identified in 52 patients. Among them, 37 mutations from 19 genes were first identified in 46, XY DSD patients, including EGF, LHX9, and CST9. Nine patients displayed biallelic mutations, 12 had mutations in sex chromosome genes and 14 had monoallelic mutations in NR5A1, BMP4, and WT1. Higher frequency mutations were identified in AR, SRD5A2, and NR5A1. Six missense, one frameshift, and one three-nucleotide deletion mutations of NR5A1 were shown to impair the transactivation ability with an altered nuclear aggregation of p.T29K and p.N44del variants. Multiple genetic mutations were identified in 33 of the 70 patients. The targeted sequencing panel provides an efficient method for the etiological diagnosis of 46, XY DSD patients and expands the candidate genes and inherited patterns.