Ying-Xia Cui

Long-Term Effects of Phytoestrogen Daidzein on Penile Cavernosal Structures in Adult Rats

OBJECTIVES Daidzein is a soy isoflavone with estrogenic activity present in plant-based food items and health foods and used as an alternative therapy for cancer and cardiovascular diseases. The present study was designed to investigate the effects of daidzein on the cavernosal components, including smooth muscle cells, collagen fibers, and elastic fibers, that are the key structures fundamental for erection. METHODS A total of 30 adult male Sprague-Dawley rats were equally divided into a normal control group, three experimental groups, and one positive control group. The three experimental groups were given daidzein at a dose of 2, 20, and 100 mg/kg body weight daily, and the positive control group received 0.1 mg diethylstilbestrol per animal daily for 90 days. The collagen fibers and elastic fibers in the corpora cavernosa were measured using histochemical or immunohistochemical techniques, and their relative contents were evaluated quantitatively or semiquantitatively. RESULTS The relative content of collagen fibers in the corpus cavernosa in rats treated with low-dose daidzein (2 mg/kg) was not significantly different from that of controls, as was the case for the smooth muscle and elastic fiber content (all P >0.05). However, the relative content of the collagen fibers was significantly increased in rats treated with a medium dose (20 mg/kg) and a high dose (100 mg/kg) of daidzein. The smooth muscle cell and elastic fiber content was reduced significantly compared with that of the controls (all P <0.01). Similar alterations were observed in the diethylstilbestrol-treated rats. CONCLUSIONS These results suggest that daidzein, if ingested in a relatively large amount, could induce histopathologic alterations in the penile cavernosal structures characterized by an increase in the collagen content and a reduction in smooth muscle cell and elastic fiber content, which might be suggestive of erectile dysfunction.

Clinical, molecular and cytogenetic analysis of 46, XX testicular disorder of sex development with SRY-positive

BackgroundTo review the possible mechanisms proposed to explain the etiology of 46, XX sex reversal by investigating the clinical characteristics and their relationships with chromosomal karyotype and the SRY(sex-determining region Y)gene.MethodsFive untreated 46, XX patients with SRY-positive were referred for infertility. Clinical data were collected, and Karyotype analysis of G-banding in lymphocytes and Fluorescence in situ hybridization (FISH) were performed. Genomic DNA from peripheral blood of the patients using QIAamp DNA Blood Kits was extracted. The three discrete regions, AZFa, AZFb and AZFc, located on the long arm of the Y chromosome, were performed by multiplex PCRs(Polymerase Chain Reaction) amplification. The set of PCR primers for the diagnosis of microdeletion of the AZFa, AZFb and AZFc region included: sY84, sY86, sY127, sY134, sY254, sY255, SRY and ZFX/ZFY.ResultsOur five patients had a lower body height. Physical examination revealed that their testes were small in volume, soft in texture and normal penis. Semen analyses showed azoospermia. All patients had a higher follicle-stimulating hormone(FSH), Luteinizing Hormone(LH) level, lower free testosterone, testosterone level and normal Estradiol, Prolactin level. Karyotype analysis of all patients confirmed 46, XX karyotype, and FISH analysis showed that SRY gene were positive and translocated to Xp. Molecular analysis revealed that the SRY gene were present, and the AZFa, AZFb and AZFc region were absent.ConclusionsThis study adds cases on the five new 46, XX male individuals with SRY-positive and further verifies the view that the presence of SRY gene and the absence of major regions in Y chromosome should lead to the expectance of a completely masculinised phenotype, abnormal hormone levels and infertility.

A novel splice site mutation in the dentin sialophosphoprotein gene in a Chinese family with dentinogenesis imperfecta type II.

Twenty-four individuals were investigated that spanned six generations in a Chinese family affected with an apparently autosomal dominant form of dentinogenesis imperfecta type II (DGI-II, OMIM #125490). All affected individuals presented with typical, clinical and radiographic features of DGI-II, but without bilateral progressive high-frequency sensorineural hearing loss. To investigate the mutated molecule, a positional candidate approach was used to determine the mutated gene in this family. Genomic DNA was obtained from 24 affected individuals, 18 unaffected relatives of the family and 50 controls. Haplotype analysis was performed using leukocyte DNA for 6 short tandem repeat (STR) markers present in chromosome 4 (D4S1534, GATA62A11, DSPP, DMP1, SPP1 and D4S1563). In the critical region between D4S1534 and DMP1, the dentin sialophosphoprotein (DSPP) gene (OMIM *125485) was considered as the strongest candidate gene. The first four exons and exon/intron boundaries of the gene were analyzed using DNA from 24 affected individuals and 18 unaffected relatives of the same family. DNA sequencing revealed a heterozygous deletion mutation in intron 2 (at positions -3 to -25), which resulted in a frameshift mutation, that changed the acceptor site sequence from CAG to AAG (IVS2-3C-->A) and may also have disrupted the branch point consensus sequence in intron 2. The mutation was found in the 24 affected individuals, but not in the 18 unaffected relatives and 50 controls. The deletion was identified by allele-specific sequencing and denaturing high-performance liquid chromatography (DHPLC) analysis. We conclude that the heterozygous deletion mutation contributed to the pathogenesis of DGI-II.

A rare Y chromosome constitutional rearrangement: a partial AZFb deletion and duplication within chromosome Yp in an infertile man with severe oligoasthenoteratozoospermia

We report a case of an infertile man with severe oligoasthenoteratozoospermia with a partial azoospermia factor b (AZFb) deletion and duplication region within chromosome Yp11.2. The hormonal profile was normal for serum concentrations of follicle-stimulating hormone, luteinizing hormone, testosterone and oestradiol. The patient, who showed a 46,XY karyotype, had an approximate 2.4 Mb inherited duplication region in Yp11.2 and a de novo partial AZFb deletion, which spanned 5.25 Mb including eight protein coding genes and four non-coding transcripts, but did not remove the RBMY gene family. Both proximal and distal breakpoints of the deletion were outside any palindromic region or inverted repeat sequence and intra-chromosomal non-allelic homologous recombination could not have been the deletion mechanism. The partial AZFb deletion in our case diminished sperm production, but did not completely extinguish spermatogenesis. Considering severe oligozoospermia, spermatozoa in the patients ejaculate were used for intracytoplasmic sperm injection, resulting in two twin pregnancies.

Meiotic Chromosome Behavior in a Human Male t(8;15) Carrier

Reciprocal translocation is one of the most common structural chromosomal rearrangements in human beings; it is widely recognized to be associated with male infertility. This association is mainly based on the abnormal chromosome behavior of the translocated chromosomes and sex chromosomes during meiosis prophase I in reciprocal translocation carriers. However, the underlying mechanisms are not completely known. Here we report a reciprocal translocation carrier of t(8;15), who is oligozoospermic due to apoptosis of primary spermatocytes and to premature germ cell desquamation from seminiferous tubules. Further analysis showed abnormal synapsis and recombination frequency in this patient, indicating a connection between chromosome behavior and apoptosis of primary spermatocytes. We also compared these observations with recently reported findings on spermatogenesis defects in reciprocal translocation carriers, and discuss the possible mechanisms underlying both common and unique phenotypes of reciprocal translocations involving different chromosomes with the aim of further understanding the regulation of human spermatogenesis.

Novel Mutations of ABCB6 Associated with Autosomal Dominant Dyschromatosis Universalis Hereditaria

Objective Dyschromatosis universalis hereditaria (DUH) is a rare heterogeneous pigmentary genodermatosis, which was first described in 1933. The genetic cause has recently been discovered by the discovery of mutations in ABCB6. Here we investigated a Chinese family with typical features of autosomal dominant DUH and 3 unrelated patients with sporadic DUH. Methods Skin tissues were obtained from the proband, of this family and the 3 sporadic patients. Histopathological examination and immunohistochemical analysis of ABCB6 were performed. Peripheral blood DNA samples were obtained from 21 affected, 14 unaffected, 11 spouses in the family and the 3 sporadic patients. A genome-wide linkage scan for the family was carried out to localize the causative gene. Exome sequencing was performed from 3 affected and 1 unaffected in the family. Sanger sequencing of ABCB6 was further used to identify the causative gene for all samples obtained from available family members, the 3 sporadic patients and a panel of 455 ethnically-matched normal Chinese individuals. Results Histopathological analysis showed melanocytes in normal control’s skin tissue and the hyperpigmented area contained more melanized, mature melanosomes than those within the hypopigmented areas. Empty immature melanosomes were found in the hypopigmented melanocytes. Parametric multipoint linkage analysis produced a HLOD score of 4.68, with markers on chromosome 2q35-q37.2. A missense mutation (c.1663 C>A, p.Gln555Lys) in ABCB6 was identified in this family by exome and Sanger sequencing. The mutation perfectly cosegregated with the skin phenotype. An additional mutation (g.776 delC, c.459 delC) in ABCB6 was found in an unrelated sporadic patient. No mutation in ABCB6 was discovered in the other two sporadic patients. Neither of the two mutations was present in the 455 controls. Melanocytes showed positive immunoreactivity to ABCB6. Conclusion Our data add new variants to the repertoire of ABCB6 mutations with DUH.

A Novel p. Gly630Ser Mutation of COL2A1 in a Chinese Family with Presentations of Legg–Calvé–Perthes Disease or Avascular Necrosis of the Femoral Head

Objective Mutations in the type II collagen gene are associated with certain human disorders, collectively termed type II collagenopathies. They include Legg–Calvé–Perthes disease (LCPD) and avascular necrosis of the femoral head (ANFH). These two diseases are skeletal dysplasias, inherited in an autosomal dominant fashion, characterized by groin pain, dislocation of the hip and diminished joint mobility. Coxa vara and elevation of the greater trochanter of the femur comprise the typical phenotype of LCPD, but do not occur in ANFH. Lack of synthesis of type II collagen and structural defects are responsible for the major clinical outcomes, because collagen is the essential matrix protein of all connective tissues. Type II collagen, encoded by the COL2A1 gene, contains N- and C- terminal regions that are cleaved after secretion into the extracellular matrix, and the core area is composed of a triple helical (Gly–X–Y) domain. If the Gly in this specific region is replaced by other amino acids, the structure of type II collagen will be destroyed. Method Forty-five members of a four-generation family were recruited and investigated. Diagnosis was made by independent orthopedic surgeons and radiologists. A mutation of the COL2A1 gene was detected. Result In our research, we identify a heterozygous mutation (c.1888 G>A, p. Gly630Ser) in exon 29 of COL2A1 in the Gly–X–Y domain, in a Chinese family affected by LCPD and ANFH. Our findings provide significant clues to the phenotype–genotype relationships in these syndromes and may be helpful in clinical diagnosis. Furthermore, these results should assist further studies of the mechanisms underlying collagen diseases. Conclusion Our data add new variants to the repertoire of COL2A1 mutation resulting in related collagenopathies.

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.

A girl with distinctive features of borderline high blood pressure, short stature, characteristic brachydactyly, and 11.47 Mb deletion in 12p11.21–12p12.2 by oligonucleotide array CGH

Interstitial deletions of the short arm of chromosome 12 are rare. Since Tenconi et al. [1975] described a male baby with a de novo interstitial deletion of 12p, a total of 10 additional patients have been reported [Orye and Craen, 1975; Magenis et al., 1981; Boilly-Dartigalongue et al., 1985; Fryns et al., 1990; Nagai et al., 1995; Gl€aser et al., 2003; Stumm et al., 2007]. Interstitial deletion of 12p could be divided into four groups according to the region of deletion: (1) deletions including the p1! p11; (2) deletions extending more distally (p13! p11); (3) deletions in band 12p13, and (4) deletions encompassing the terminal of 12p [Gl€aser et al., 2003]. Common phenotypic findings constituting the interstitial deletions of 12p include mental retardation, developmental delay, craniofacial anomalies, microcephaly, brachydactyly, and clinodactyly. Only one patient with borderline high blood pressure, short stature, brachydactyly, and a deletion in 12p11.21! 12.2 region was reported by Nagai et al. [1995]. We encountered a 13-year-old girl with moderate mental retardation, short stature, and characteristic brachydactyly. She was born at term of gestation with normal birth weight of 3,300 g and short stature of 46 cm ( 2.4 SD). The girl spoke at the age of 2 years and walked without assistance at the age of 3 years. Clinical observation showed a small slender girl with height 126 cm ( 4.4 SD), weight 25 kg ( 2.66 SD), borderline high blood pressure (120/80 mmHg), round face, strabimus and chaotic tooth arrangement (Fig. 1). The fifth toe overlapped the fourth one and both of them showed brachydactyly. Serum hormone levels of GH, TSH, T3, and T4 were normal for her age. Cerebral CT scan and MRI were normal. Roentgenograms of hands and feet disclosed the bilateral brachydactyly, with shortened metacarpals of digits 3–5, middle phalanges of digit 5, and metatarsals of digits 4 and 5 (Fig. 2). Cone-shaped epiphyses were not evident. No obvious skeletal abnormalities were found in vertebrae, pelvis, and other tube bones. Now she is enrolled in a special school for children with mental retardation. She can count numbers to 10. Chromosome analysis of G-banding at 400 band showed a possible deletion of 12p11.2 in all metaphases analyzed (Fig. 3A). Family history was negative. Chromosome analysis of both parents showed normal karyotypes, indicating a de novo deletion. This research was approved by Ethics Committee of the hospital, and the written informed consent was obtained from the patient and the parents. Multicolor fluorescent in situ hybridization (M-FISH) analysis using the Spectra Vysion WCP Probe (Vysis, Downers Grove, IL) was performed on metaphases. A reciprocal translocation was excluded. To refine the extent of the deletion on the molecular cytogenetic level, analysis of using a genomic-wide high-density oligo array (OaCGH44K) with 44,000 probes covering more than 30,000 mapped genes was carried out according to Agilent manufacturer’s procedures and statistical algorithms [Fan et al., 2007; www.agilent. com.chem/gocgh]. An 11.47 Mb interstitial deletion at genomic position 20, 724, 852 bp! 32, 201, 544 bp in

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.