A duplication upstream of SOX9 was not positively correlated with the SRY­negative 46,XX testicular disorder of sex development: A case report and literature review.

The 46,XX male disorder of sex development (DSD) is rarely observed in humans. Patients with DSD are all male with testicular tissue differentiation. The mechanism of sex determination and differentiation remains to be elucidated. In the present case report, an 46,XX inv (9) infertile male negative for the sex­determining region of the Y chromosome (SRY) gene was examined. This infertile male was systemically assessed by semen analysis, serum hormone testing and gonadal biopsy. Formalin­fixed and paraffin­embedded gonad tissues were assessed histochemically. The SRY gene was analyzed by fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR). The other 23 specific loci, including the azoospermia factor region on the Y chromosome and the sequence-targeted sites of the SRY­box 9 (SOX9) gene were analyzed by PCR. The genes RSPO1, DAX1, SOX3, ROCK, DMRT1, SPRY2 and FGF9 were also assessed using sequencing analysis. Affymetrix Cytogenetics Whole Genome 2.7 M Arrays were used for detecting the genomic DNA from the patient and the parents. The patient with the 46,XX inv (9) (p11q13) karyotype exhibited male primary, however, not secondary sexual characteristics. However, the patient's mother with the 46, XX inv (9) karyotype was unaffected. The testicular tissue dysplasia of the patient was confirmed by tissue biopsy and absence of the SRY gene, and the other 23 loci on the Y chromosome were confirmed by FISH and/or PCR. The RSPO1, DAX1, SOX3, ROCK, DMRT1, SPRY2 and FGF9 genes were sequenced and no mutations were detected. A duplication on the 3 M site in the upstream region of SOX9 was identified in the patient as well as in the mother. The patient with the 46,XX testicular DSD and SRY­negative status was found to be infertile. The duplication on the 3 M site in the upstream region of SOX9 was a polymorphism, which indicated that the change was not a cause of 46,XX male SDS. These clinical, molecular and cytogenetic findings suggested that other unidentified genetic or environmental factors are significant in the regulation of SDS.

46,XX testicular disorder of sexual development with SRY-negative caused by some unidentified mechanisms: a case report and review of the literature.

BACKGROUND: 46,XX testicular disorder of sex development is a rare genetic syndrome, characterized by a complete or partial mismatch between genetic sex and phenotypic sex, which results in infertility because of the absence of the azoospermia factor region in the long arm of Y chromosome. CASE PRESENTATION: We report a case of a 14-year-old male with microorchidism and mild bilateral gynecomastia who referred to our hospital because of abnormal gender characteristics. The patient was treated for congenital scrotal type hypospadias at the age of 4 years. Semen analysis indicated azoospermia by centrifugation of ejaculate. Levels of follicle-stimulating hormone and luteinizing hormone were elevated, while that of testosterone was low and those of estradiol and prolactin were normal. The results of gonadal biopsy showed hyalinization of the seminiferous tubules, but there was no evidence of spermatogenic cells. Karyotype analysis of the patient confirmed 46,XX karyotype and fluorescent in situ hybridization analysis of the sex-determining region Y (SRY) gene was negative. Molecular analysis revealed that the SRY gene and the AZFa, AZFb and AZFc regions were absent. No mutation was detected in the coding region and exon/intron boundaries of the RSPO1, DAX1, SOX9, SOX3, SOX10, ROCK1, and DMRT genes, and no copy number variation in the whole genome sequence was found. CONCLUSION: This study adds a new case of SRY-negative 46,XX testicular disorder of sex development and further verifies the view that the absence of major regions from the Y chromosome leads to an incomplete masculine phenotype, abnormal hormone levels and infertility. To date, the mechanisms for induction of testicular tissue in 46,XX SRY-negative patients remain unknown, although other genetic or environmental factors play a significant role in the regulation of sex determination and differentiation.

C/EBPα inhibits hepatocellular carcinoma by reducing Notch3/Hes1/p27 cascades.

BACKGROUND AND AIMS: CCAAT/enhancer binding protein α is one of the key transcription factors of the hepatocyte nuclear factors family, which plays a critical role in liver cell proliferation and differentiation. However, the role of CCAAT/enhancer binding protein α in hepatocarcinogenesis remains to be defined. METHODS: A recombinant adenovirus carrying the C/EBPα gene was constructed to determine its effect on hepatocarcinogenesis in vitro and in vivo. RESULTS: We demonstrated that overexpression of CCAAT/enhancer binding protein α inhibited the tumourigenicity of Huh7 cells, re-established the expression of certain liver-specific genes and induced G0/G1 arrest. Overexpression of CCAAT/enhancer binding protein α significantly suppressed the proliferation of primary hepatocarcinogenesis cells and tumour associated fibroblasts in vitro. Additionally, intratumoural injection of adenovirus carrying the C/EBPα reduced the growth of subcutaneous hepatocarcinogenesis xenografts in nude mice. Systemic administration of adenovirus carrying the C/EBPα resulted in the eradication of orthotopic liver hepatocarcinogenesis nodules in nude mice. Further, up-regulation of CCAAT/enhancer binding protein α reduced the expression of Notch3, thereby suppressing Hes1 transactivation activity and leading to decreased p27 expression. Overexpression of Hes1 partially abolished the anti-proliferation effect of CCAAT/enhancer binding protein α on Huh7 cells. CONCLUSION: These results suggested that the effect of CCAAT/enhancer binding protein α on hepatocarcinogenesis is partially through by reducing Notch3/Hes1/p27 cascades and CCAAT/enhancer binding protein α may possess a novel therapeutic potential for human hepatocarcinogenesis.

[Susceptibility to prostate cancer in Han Chinese: single nucleotide polymorphism analysis of 1 667 cases].

OBJECTIVE: Prostate cancer (PCa) has the highest incidence among male malignancies in Western industrialized countries and, as a most common malignant disease in urology, its incidence has been increasing in recent years in Chinese men. This study was to investigate the risk loci associated with PCa susceptibility in Han Chinese by analyzing single nucleotide polymorphisms (SNP). METHODS: We collected peripheral blood samples from 1 667 PCa patients and 1 525 healthy men, and detected 40 loci associated with PCa susceptibility by analyzing SNPs using Sequenom technology. RESULTS: Of the 40 known loci, 16 were confirmed to be significantly associated with PCa susceptibility (P < 0.05). The loci 1, 2 and 5 at 8q24, 10q11 and 22q13.2 also contributed to PCa susceptibility in different ethnic groups. CONCLUSION: PCa susceptibility is obviously associated with the risk loci rs1465618, rs721048, rs12621278, rs7679673, rs12653946, rs339331, rs1512268, rs10086908, rs16901979, rs1447295, rs10993994, rs10896449, rs902774, rs9600079, rs11649743 and rs5759167 in Chinese Han population.

Association between ubiquitin-specific protease USP26 polymorphism and male infertility in Chinese men.

BACKGROUND: Increased sperm ubiquitin was inversely associated with sperm count and motility. Ubiquitin-specific protease 26 (USP26), which is an X-linked gene, has been studied as a potential infertility gene. There are conflicting reports on whether variations in USP26 are associated with spermatogenesis. METHODS: In order to assess that USP26 polymorphisms contribute to male infertility, we screened 221 infertile men with azoospermia, oligozoospermia, asthenozoospermia, or oligoasthenozoospermia, and 101 control fertile men using DNA sequencing. RESULTS: There were six polymorphisms identified, including an unreported variation (508G>A, G170R). Only the allele frequency of 576G>A was significantly higher in fertile men than infertile patients (p<0.001), although this variant does not result in an amino acid change. The major haplotypes in fertile and infertile men were TGATC (76.2% vs 47.5% of the population, p<0.001) and TGGTC (14.9% vs 39.4%, p<0.001). The haplotype TGATC was under-transmitted, whereas the haplotype TGGTC was over-transmitted in infertile men with asthenozoospermia and oligoasthenozoospermia. CONCLUSIONS: Our results indicated the variation of USP26 was not directly associated with human sperm count but suggested it might be a potential role in sperm motility. The 576G>A synonymous single nucleotide polymorphism (SNP) might have a role in improving the sperm motility.

[Mutation of the USP26 gene in spermatogenesis dysfunction].

The ubiquitin specific protease 26 (USP26) gene is located at Xq26.2 and present as a single exon on the X chromosome encoding for a protein of 913 amino acids. It belongs to a large family of deubiquitinating enzymes, and is exclusively expressed in the testis. There are conflicting reports on whether mutations in USP26 are associated with male infertility. This article updates the researches on the USP26 gene, its complicated relationship with male spermatogenesis dysfunction, the role of its mutation in male infertility, its geographical or ethnic distribution, and its evolution.

A novel insertion mutation in the SEDL gene results in X-linked spondyloepiphyseal dysplasia tarda in a large Chinese pedigree.

BACKGROUND: Spondyloepiphyseal dysplasia tarda (SEDT) is an X-chromosome linked primary skeletal dysplasia characterized by a disproportionate short-trunked short stature, dysplasia of the large joints and flattened thoracic and lumber vertebral bodies. The objective of this study is to describe a large Chinese SEDT family with a milder phenotype and describe the molecular and clinical findings. METHODS: Eight affected males of the family were diagnosed with SEDT according to their clinical and radiological features. Direct DNA sequencing of the SEDL gene was performed. RT-PCR experiments on total RNA from blood lymphocytes were performed to confirm the defect on the SEDL gene. A short summary of all currently known SEDL gene mutations is presented. RESULTS: DNA sequencing revealed that all the affected males carried an insertion mutation (c.370-371insA) unreported previously, predicted to result in frameshifts and generate a premature stop codon (p.S124fsX127). The identical mutation was also observed in a 10-year old presymptomatic boy of the family. Eight female carriers had the typical sequencing chromatograms of heterozygotes. CONCLUSIONS: Identification of the novel insertion mutation (c.370-371insA) in this SEDT family enables carrier detection and presymptomatic/prenatal diagnosis, but also the detailed molecular and clinical features will be useful for extending the evidence for genetic and phenotypic heterogeneity in SEDT.

Rapid molecular prenatal diagnosis of spondyloepiphyseal dysplasia congenita by PCR-SSP assay.

Heterozygous mutations of COL2A1 gene are responsible for type II collagenopathies. The common skeletal phenotypes include achondrogenesis type II, hypochondrogenesis, Stickler dysplasia, Kniest dysplasia, late onset spondyloepiphyseal dysplasia, and spondyloepiphyseal dysplasia congenita (SEDC). Prevention of SEDC can be achieved by prenatal diagnosis. This study reports the first rapid molecular prenatal diagnosis of SEDC performed in China by polymerase chain reaction sequence-specific primer (PCR-SSP) analysis. The pregnant woman we previously reported with SEDC carried the G to A substitution at nucleotide 1510 in exon 23 of COL2A1 gene, which caused a change from glycine to serine at codon 504 (G504S). By the time the woman got pregnant again, she had terminated two pregnancies and still had no child. In the first pregnancy, the molecular mutation of the family was not yet identified, and therefore prenatal diagnosis was unable to be performed by DNA analysis. In the second pregnancy, G504S mutation was found from fetal DNA. At the time of her third pregnancy, the woman and her husband became extremely worried about the potential SEDC for the fetus. For this reason, a quick and reliable molecular prenatal diagnosis of SEDC was performed by a PCR-SSP on an amniocyte sample collected at the 14th week of pregnancy. No mutation of the fetal DNA was identified. The result was obtained within 24 h after the sample was collected. The technique could be applied in confirmatory diagnosis and prenatal diagnosis for the affected family.

[Observation of spermatogenic cells for infertile patients with Y-chromosomal microdeletion].

OBJECTIVE: To assess the spermatogenic function of the infertile patients with Y-chromosomal microdeletion. METHODS: Thirty-five 23-44 years old patients with microdeletions of Y chromosome were included in this study. Three semen analyses confirmed that 26 cases were non-obstructive azoospermia and 9 oligospermia with sperm count < 1 x 10(6)/ml. They were divided into 3 groups by the locus of deletion, 5 cases of AZFa + b + c deletion in group 1, 4 cases of AZFb + c and 3 cases of AZFb deletion in group 2, and 23 cases of AZFc deletion in group 3. Semen was collected and centrifuged, the supernatant removed and the centrifugate applied on the clean slides after dilution. Following Wright's-Giemsa staining, the slides were viewed under the microscope. Testis histopathological biopsy was performed for 6 of the cases. RESULTS: In group 1, no spermatogenic cells were observed but only Sertoli cells in 1 case, with a consistency between the result of spermatogenic cell test and that of testis biopsy. In group 2, spermatogenic cell tests revealed spermatocytes in 6 cases, 2 were proved by testis biopsy with sperm maturation arrest in the primary spermatocyte stage, and spermatogenic cells of all developmental stages were seen in 1 AZFb deletion patient with the same sperm maturation arrest as the above two. In group 3, only primary spermatocytes were detected by spermatogenic cell test in 5 oligospermia patients but no spermatogenic cells in the 15 azoospermia cases, and biopsy revealed 2 cases of Sertoli cell-only syndrome. CONCLUSION: The spermatogenic cell test can effectively assess the spermatogenic function of AZF deletion patients. Non-invasive and easily accepted by patients, it is highly recommendable for the evaluation of spermatogenesis of patients with Y-chromosomal microdeletion.

Molecular prenatal diagnosis in 2 pregnancies at risk for spondyloepiphyseal dysplasia congenita.

BACKGROUND: Spondyloepiphyseal dysplasia congenita (SEDC) is an autosomal dominant skeletal dysplasia characterized by short stature, abnormal epiphyses, and flattened vertebral bodies. Secondary prevention of SEDC can be achieved by prenatal diagnosis. Reports of antenatally-diagnosed SEDC fetuses have been very rare and molecular prenatal diagnosis even rarer. We previously reported a familial G504S mutation in the type II collagen (COL2A1) gene resulting in SEDC. In this study, molecular prenatal diagnosis was performed to 2 couples in this family with pregnancies at risk for SEDC. METHODS: Amniotic fluid was sampled by amniocentesis under ultrasound guidance at 19+3 and 18+6 weeks' gestation, respectively. Karyotype and molecular genetic analysis were performed on cultured amniotic fluid cells. Maternal cell contamination was excluded by short tandem repeat (STR) analysis. Direct DNA sequencing and DHPLC were conducted to detect the potential mutation in exon 23 of COL2A1 gene. Both women underwent serial sonograms because they insisted that the molecular diagnosis should be confirmed by another method, although they had been informed that mutation analysis is predictive of the disease. RESULTS: Karyotype of both fetuses was normal and molecular genetic analysis revealed that fetus 1 carried a G504S mutation in exon 23, while fetus 2 was normal. In case 1, femur length of the fetus was markedly below the 5th centile at 23 weeks' gestation, which confirms the accuracy of molecular diagnosis. A medical termination was carried out at 27+5 weeks' gestation and a male fetus with a relatively large head and short limbs was delivered. The fetal radiograph demonstrated a number of features, including generalised platyspondyly, absent ossification of the vertebral bodies in the cervical region and significant shortening of the long bones. The diagnosis of SEDC was thus confirmed clinically. Ultrasound monitoring of fetus 2 showed that its femur length was normal for gestational age at repeated scans, which was consistent with the molecular diagnosis. CONCLUSIONS: Molecular analysis allows early and accurate prenatal diagnosis for SEDC once mutation is known in a family. However, considering the poor genotype/phenotype correlation in many cases of SEDC, the combination of ultrasound as well as molecular genetic approach might be needed.