Xiao-Jun Li

Altered Profile of Seminal Plasma MicroRNAs in the Molecular Diagnosis of Male Infertility

BACKGROUND Although microRNAs (miRNAs) play essential roles in spermatogenesis, little is known about seminal plasma miRNAs in infertile men. We investigated the profile of seminal plasma miRNAs in infertile men to identify miRNAs that are altered in infertility; we then evaluated their diagnostic value. METHODS Seminal plasma samples were obtained from 289 infertile men and 168 age-matched fertile control individuals. The stability of the miRNAs was first assessed by time-course and freeze-thaw cycle analyses. The Solexa sequencing technology was used for an initial screen of the miRNAs in samples pooled from 45 patients with nonobstructive azoospermia, 58 patients with asthenozoospermia, and 100 fertile controls. A stem-loop quantitative reverse-transcription PCR (RT-qPCR) assay was conducted in the training and verification sets to confirm the concentrations of the altered miRNAs in 73 patients with nonobstructive azoospermia, 79 patients with asthenozoospermia, 34 patients with oligospermia, and 68 fertile controls. RESULTS The miRNAs in seminal plasma were stable. The Solexa sequencing analysis demonstrated 19 markedly altered miRNAs in the patient groups, compared with the control group. RT-qPCR analysis identified 7 miRNAs (miR-34c-5p, miR-122, miR-146b-5p, miR-181a, miR-374b, miR-509-5p, and miR-513a-5p) as markedly decreased in azoospermia but increased in asthenozoospermia. The area under the ROC curve for these miRNAs ranged from 0.733 to 0.921, markedly higher than for routine biochemical parameters (0.510-0.622). Moreover, the concentrations of some selected miRNAs were also increased in the semen sperm of the asthenozoospermia patients. CONCLUSIONS The measurement of miRNAs in seminal plasma provides a novel, noninvasive approach for diagnosing male infertility.

Molecular cytogenetic characterization of a new wheat-Thinopyrum intermedium partial amphiploid resistant to powdery mildew and stripe rust.

A partial amphiploid, TE253, derived from crosses between the common wheat (Triticum aestivum L.) cultivar Yannong 15 and Thinopyrum intermedium (Host) Barkworth & D.R. Dewey was characterized by cytogenetic observations, disease resistance tests and genomic in situ hybridization (GISH). Mitotic observations showed that most plants of TE253 had 56 chromosomes, but a few had 54 or 55 chromosomes. The chromosomes in most pollen mother cells of plants with 2n = 56 formed 28 bivalents. Univalents (0.89 per cell) and tetravalents (0.087 per cell) occasionally occurred at meiotic metaphase I, showing a high degree of cytogenetic stability. After inoculation with the powdery mildew and stripe rust pathogens, Yannong 15 was highly susceptible, whereas TE253 and Th. intermedium were immune to both diseases. This indicated that the resistance of TE253 to powdery mildew and stripe rust was derived from Th. intermedium. GISH analysis using St-genomic DNA from Pseudoroegneria strigosa (M. Bieb) Á. Löve as a probe and ABD-genomic DNA from Chinese Spring wheat as a blocker demonstrated that TE253 consisted of 2 St-genome chromosomes, 8 JS-genome chromosomes, 2 SAT J chromosomes and 2 J-St translocated chromosomes. Line TE253 is a new partial amphiploid with resistance to both powdery mildew and stripe rust and can be used as a source of resistance genes in wheat improvement.

Gonadotropin-releasing hormone positively regulates steroidogenesis via extracellular signal-regulated kinase in rat Leydig cells

Gonadotropin-releasing hormone (GnRH) is secreted from neurons within the hypothalamus and is necessary for reproductive function in all vertebrates. GnRH is also found in organs outside of the brain and plays an important role in Leydig cell steroidogenesis in the testis. However, the signalling pathways mediating this function remain largely unknown. In this study, we investigated whether components of the mitogen-activated protein kinase (MAPK) pathways are involved in GnRH agonist (GnRHa)-induced testis steroidogenesis in rat Leydig cells. Primary cultures of rat Leydig cells were established. The expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) and the production of testosterone in response to GnRHa were examined at different doses and for different durations by RT-PCR, Western blot analysis and radioimmunoassay (RIA). The effects of GnRHa on ERK1/2, JNK and p38 kinase activation were also investigated in the presence or absence of the MAPK inhibitor PD-98059 by Western blot analysis. GnRHa induced testosterone production and upregulated 3β-HSD expression at both the mRNA and protein levels; it also activated ERK1/2, but not JNK and p38 kinase. Although the maximum effects of GnRHa were observed at a concentration of 100 nmnol L⁻¹ after 24 h, activation of ERK1/2 by GnRHa reached peak at 5 min and it returned to the basal level within 60 min. PD-98059 completely blocked the activation of ERK1/2, the upregulation of 3β-HSD and testosterone production. Our data show that GnRH positively regulates steroidogenesis via ERK signalling in rat Leydig cells. ERK1/2 activation by GnRH may be responsible for the induction of 3β-HSD gene expression and enzyme production, which may ultimately modulate steroidogenesis in rat Leydig cells.

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.

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.

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.

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.