Identification of novel mutations and risk assessment of Han Chinese patients with autosomal dominant polycystic kidney disease.

AIM: Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disease in humans and is caused by mutations in the PKD1 or PKD2 gene. ADPKD is heterogeneous with regard to locus and allele heterogeneity and phenotypic variability. METHODS: Using targeted capture associated with next generation sequencing (NGS), we performed a mutational analysis of Han Chinese patients with ADPKD from 62 unrelated families. Multivariate Cox proportional hazard modelling of their different clinical characteristics and mutation classes was performed. RESULTS: The detection rate for a PKD1 and PKD2 mutation in the Chinese ADPKD patients was 95.2% (59/62). We identified pathogenic mutations in 64.4% (38/59) of patients, including 32PKD1 mutations (15 nonsense mutations, 15 frameshift mutation, one splice mutation, and one large deletion) and six PKD2 mutations (three nonsense mutations and three frameshift mutations). Of the pathogenic variants we identified, 50% (19/38) were novel variants and 50% (19/38) were known variants. Patients with PKD2 mutations had milder and indistinguishable phenotypes. Significant phenotypic differences were observed among the various types of PKD1 mutations. CONCLUSION: Our results show that targeted capture associated with next-generation sequencing is an effective strategy for genetically testing ADPKD patients. This mutation analysis of ADPKD in Han Chinese extends our understanding of the genetic diversity of different ethnic groups, enriches the mutation database, and contributes to the genetic counselling of ADPKD patients.

Molecular and biological characterization of gamma-interferon-inducible lysosomal thiol reductase in silver carp (Hypophthalmichthys molitrix).

Gamma-interferon-inducible lysosomal thiol reductase (GILT) plays an important role in the processing of major histocompatibility complex (MHC) class II-restricted antigens by catalyzing disulfide bonds reduction. Herein, a GILT homolog (ScGILT) was identified from silver carp. Its open reading frame covers 771 base pairs, encoding a protein of 256 amino acids that possesses GILT signature sequence CQHGX2ECX2NX4C, active-site CXXC motif, and two potential N-linked glycosylation sites. The predicted tertiary structures of ScGILT and other GILTs were quite similar in shape and positional arrangement of the key motifs. ScGILT mRNA was constitutively expressed in all detected tissues, with high-level expression in fish immune organs, spleen and head kidney. After stimulation with lipopolysaccharide, the expression of ScGILT mRNA significantly increased in spleen and head kidney cells, and ScGILT protein translocated to late endosomes and lysosomes in HeLa cells. Recombinant ScGILT fused with a His6 tag was expressed and purified, and could reduce the interchain disulfide bonds of IgG at pH 4.5. These results suggested that ScGILT was capable of catalyzing disulfide bonds reduction, and then might play an important role in the processing of MHC class II-restricted antigens in silver carp.

The P20R mutation of αB-crystallin diminishes its anti-apoptotic activity in human lens epithelial cells.

αB-crystallin acts as an anti-apoptosis protein in human lens epithelial (HLE) cells. We recently identified a missense mutation in αB-crystallin that changes proline 20 to an arginine (P20R) in a Chinese family with autosomal dominant congenital posterior polar cataract. The impact of the P20R mutation on the anti-apoptosis function remains unclear. To explore the anti-apoptotic activity of αB-crystallin wild type (αB-wt) and its P20R mutant under oxidative stress, HLE cells were transfected with αB-wt and αB-P20R constructs and expression was measured by western blotting. Flow cytometry and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP digoxigenin nick end-labelling (TUNEL) staining were performed to investigate apoptosis. We found that αB-wt performed a dominant role in inhibiting stress-induced apoptosis, but this function was impeded in cells expressing αB-P20R. The P20R mutant of αB-crystallin exhibits diminished anti-apoptotic activity compared with the native protein.

The susceptibility of FSHB -211G > T and FSHR G-29A, 919A > G, 2039A > G polymorphisms to men infertility: an association study and meta-analysis.

BACKGROUND: Male infertility is a complex disorder caused by genetic, developmental, endocrine, or environmental factors as well as unknown etiology. Polymorphisms in the follicle stimulating hormone beta subunit (FSHB) (rs10835638, c.-211G > T) and follicle stimulating hormone receptor (FSHR) (rs1394205, c.-29G > A; rs6165, c.919A > G; rs6166, c.2039 A > G) genes might disturb normal spermatogenesis and affect male reproductive ability. METHODS: To further ascertain the aforementioned effects, we conducted a case-control study of 255 infertile men and 340 fertile controls from South China using the Mass ARRAY method, which was analyzed by the t-tests and logistic regression analysis using SPSS for Windows 14.0. In addition, a meta-analysis was performed by combining our results with previous reports using STATA 12.0. RESULTS: In the FSHB or FSHR gene single nucleotide polymorphism (SNP) evaluation, no statistically-significant difference was found in the frequency of allelic variants or in genotype distribution between cases and controls. However, a significant association for the comparison of GAA (P: 0.022, OR: 0.63, 95%CI: 0.43-0.94) was seen between the oligozoospermia and controls in haplotype analysis of rs1394205/rs6165/rs6166. In the meta-analysis, rs6165G allele and rs6166 GG genotype were associated with increased risk of the male infertility. CONCLUSIONS: This study suggested that FSHR GAA haplotype would exert protective effects against male sterility, which indicated that the combination of three SNP genotypes of FSHR was predicted to have a much stronger impact than either one alone. Then in the meta-analysis, a significant association was seen between FSHR rs6165, rs6166 polymorphisms and male infertility. In terms of male infertility with multifactorial etiology, further studies with larger sample sizes and different ethnic backgrounds or other risk factors are warranted to clarify the potential role of FSHB and FSHR polymorphisms in the pathogenesis of male infertility.

A novel compound heterozygous mutation of the SMARCAL1 gene leading to mild Schimke immune-osseous dysplasia: a case report.

BACKGROUND: Schimke immune-osseous dysplasia (SIOD, OMIM 242900) is characterized by spondyloepiphyseal dysplasia, T-cell deficiency, renal dysfunction and special facial features. SMARCAL1 gene mutations are determined in approximately 50% of patients diagnosed with SIOD. CASE PRESENTATION: The case presented here is that of a 6-year-old boy who was born at 33 weeks to healthy, non-consanguineous Chinese parents. He presented with short stature (95 cm; <3rd percentile) and proteinuria. Initially suspected of having IgM nephropathy, the patient was finally diagnosed with mild Schimke immune-osseous dysplasia. One novel mutation (p.R817H) and one well-known mutation (p.R645C) was identified in the SMARCAL1 gene. CONCLUSION: This report describes a clinical and genetic diagnostic model of mild SIOD. It also highlights the importance of molecular testing or clinical diagnosis and the guidance it provides in disease prognosis.

[Single nucleotide polymorphism of the TP53 gene is not correlated with male infertility].

OBJECTIVE: To investigate the correlation of the single nucleotide polymorphism (SNP) rs1042522 of the tumor protein p53 (TP53) gene with the risk of male infertility. METHODS: This casecontrol study included 380 male patients with idiopathic infertility and 398 normal fertile men as controls from the Nanjing area. We genotyped the SNP rs1042522 of the TP53 gene by Sequence Mass Array and analyzed the correlation of the SNP with male infertility using the logistic regression model. RESULTS: Compared with the normal controls, the patients with idiopathic infertility showed significantly decreased sperm concentration (ï¼»77.34±49.24ï¼½ vs ï¼»13.13±24.96ï¼½ ×106/ml), percentage of progressively motile sperm (ï¼»42.55±9.57ï¼½ vs ï¼»10.38±5.57ï¼½%), serum testosterone level (ï¼»14.07±5.36ï¼½ vs ï¼»11.89±4.50ï¼½ nmol/L), and folliclestimulating hormone level (ï¼»16.80±18.20ï¼½ vs ï¼»4.55±7.17ï¼½ U/L) (P < 0.05) but no statistically significant differences in other parameters. No correlation was observed between the SNP frequencies and male infertility and similar results were found in the subgroups of the cases. CONCLUSIONS: SNP rs1042522 of the TP53 gene is not significantly correlated with the risk of male infertility.

[Nucleotide polymorphism rs4880 of the SOD2 gene and the risk of male infertility].

OBJECTIVE: To investigate the correlation of the single nucleotide polymorphism (SNP) rs4880 of the superoxide dismutase 2 (SOD2) gene with the risk of male infertility. METHODS: This casecontrol study included 519 male patients with idiopathic infertility (aged 19－40 ï¼»28.93±4.93ï¼½ years) in the case group and 338 fertile men (aged 19－40 ï¼»28.40±4.25ï¼½ years) in the control group. We collected the clinical data, genotyped the SNP rs4880 of the SOD2 gene by Sequenom Mass Array, and analyzed the association of different genotypes with male infertility using the logistic regression model. RESULTS: Statically significant differences were observed between the case and control groups in the level of folliclestimulating hormone (FSH) (ï¼»4.72±2.51ï¼½ vs ï¼»15.65±17.24ï¼½ U/L, P< 0.01), the percentage of progressively mobile sperm (ï¼»9.12±13.5ï¼½ vs ï¼»41.95±9.03ï¼½%, P< 0.01), and sperm concentration (ï¼»12.95±24.38ï¼½ vs ï¼»72.88±45.60ï¼½ ×106/ml, P< 0.01), but not in other parameters. No correlation was found between male infertility and the heterozygous genotype TC (OR = 0.90, 95% CI: 0.65－1.25, P = 0.516) or the homozygous genotype CC (OR=1.49, 95% CI: 0.38－5.81, P = 0.566) as compared with the wild genotype TT, and similar results were obtained in the analysis of the subgroups. CONCLUSIONS: The SNP rs4880 of the SOD2 gene was not correlated with male infertility, which, however, is to be supported by further studies with larger samples from more areas.

[CYP1A1 rs4646422 gene polymorphisms not correlated with male infertility in Chinese Han population].

OBJECTIVE: To determine the correlation of the CYP1A1 (rs4646422) gene polymorphisms with male infertility in the Chinese Han population. METHODS: Using the Mass ARRAY iPLEX GOLD technique, we conducted a case-control study on theCYPlA1 (rs4646422) gene polymorphisms in 636 infertile males aged 21-49 years (case group) and 442 normal healthy men aged 23-47 years (control group) of the Chinese Han population. We analyzed the genotypes and allele frequencies in the two groups ofsubjects with the SPSS 20.0 software. RESULTS: Compared with the wild homozygous genotype GG, the heterozygous genotype AG (OR = 1.06, 95% CI 0.81-1.38) and homozygous genotype AA (OR = 1.11, 95% CI 0.56-2.21) showed no correlation with male infertility, nor did the mutant allele A (OR = 1.06, 95% CI 0.85-1.32) in comparison with the wild allele G. CONCLUSION: The CYP1A1 (rs4646422) gene polymorphisms might not be correlated with male infertility in the Chinese Han population.

[Single nucleotide polymorphisms of the N-acetyltransferase 2 gene not correlated with male infertility].

Objective: To investigate the correlation of the single nucleotide polymorphisms（SNPs） rs1799930 and rs1799931 of the N-acetyltransferase 2 gene（ NAT2） with the risk of male infertility in Nanjing area. Methods: We made a case-control study of 636 cases of male idiopathic infertility and 442 normal fertile men as controls. We genotyped the two SNPs by Sequenom Mass Array, analyzed the correlation of different genotypes with male infertility using the logistic regression model, and determined the association of the linkage effect of the two SNPs with male infertility by haplotype analysis. Results: Statistically significant differences were found between the case and control groups in sperm concentration（[32. 32 ± 45. 49] vs [72. 77 ± 45. 21] × 106/ ml, P < 0. 01）,the percentage of progressively motile sperm（[15. 29 ± 5. 06] vs [42. 02 ± 9. 04]%,P < 0. 01）,and the level of follicle-stimulating hormone（[14. 69 ± 12. 37] vs [4. 72 ± 2. 51] U / L,P < 0. 01), but not in other parameters. No correlation was observed between the frequencies of the two SNPs or alleles in different models and male infertility. Haplotype analysis suggested a linkage effect within rs1799930 and rs1799931（D' = 0. 998,r2= 0. 05） but no evident correlation between male infertility and genotype combination. Conclusion: The SNPs rs1799930 and rs1799931 of the NAT2 gene were not found to be correlated with the risk of idiopathic infertility in men.

Application of next­generation sequencing for molecular diagnosis in a large family with osteogenesis imperfecta type I.

Increased bone fragility and low bone mass are common features of osteogenesis imperfecta (OI), which is associated with connective tissue. Its type is distinguished by clinical phenotypes and molecular genetics. Although fifteen types (I­XV) of OI have been identified at present, the majority of patients are diagnosed as OI type I­IV. Type I collagen is responsible for OI type I­IV, consists of α1 (I) and α2 (I) chains and is encoded by COL1A1 and COL1A2. To identify the pathogenic gene of a large Chinese family with OI type I and explain genetic heterogeneity of the patients, next­generation sequencing (NGS) was conducted in a female with OI type I and her affected niece and daughter to search for the mutation. Subsequently, it was confirmed in other family members by Sanger sequencing. Analysis of COL1A1 gene identified a splicing mutation (c.471+1G>A, also termed IVS5+1G>A) that converted the 5' end of intron 5 from GT to AT. The current study aimed to investigate why there are different phenotypes with the same mutation observed within the same OI pedigree, and the results suggested that there may be environmental factors involved. The present study provided genetic counseling and prenatal diagnosis for the family members, however additionally provided insight into the phenotype­genotype association in OI.

Polymorphisms of protamine genes contribute to male infertility susceptibility in the Chinese Han population.

Protamine (PRM) plays important roles in the packaging of DNA within the sperm nucleus. To investigate the role of PRM1/2 and transition protein 1 (TNP1) polymorphisms in male infertility, 636 infertile men and 442 healthy individuals were recruited into this case-controlled study of the Chinese Han population, using MassARRAY technology to analyze genotypes. Our analysis showed that there were no significant differences between controls and infertile cases among the five single nucleotide polymorphisms identified in PRM1, PRM2 and TNP1 [rs737008 (G/A), rs2301365 (C/A), rs2070923 (C/A), rs1646022 (C/G) and rs62180545 (A/G)]. However, we found that the PRM1 and PRM2 haplotypes GCTGC, TCGCA and TCGCC exhibited significant protective effects against male infertility compared to fertile men, while TCGGA, GCTCC and TCGGC represented significant risk factors for spermatogenesis. Our data showed that rs737008 and rs2301365 in PRM1, and rs1646022 in PRM2, were significantly associated with male infertility and that gene-gene interaction played a role in male infertility. A linkage disequilibrium plot for the five SNPs showed that rs737008 was strongly linked with both rs2301365 and rs2070923. These findings are likely to help improve our understanding of the etiology of male infertility. Further studies should include a larger number of genes and SNPs, particularly growing critical genes; such studies will help us to unravel the effect of individual genetic factors upon male infertility.

A novel missense mutation of ADAR1 gene in a Chinese family leading to dyschromatosis symmetrica hereditaria and literature review.

Dyschromatosis symmetrica hereditaria (DSH) is a rare autosomal dominant pigmentary genodermatosis, which is characterized by a mixture of hyperpigmented and hypopigmented macules on the dorsal of the hands and feet, and on the face presented like freckle. Identification of RNA-specific adenosine deaminase 1 (ADAR1) gene results in DSH. This study was mainly to explore the pathogenic mutation of ADAR1 gene and provide genetics counselling and prenatal diagnostic testing for childbearing individuals.Mutational analysis of ADAR1 gene was performed by polymerase chain reaction (PCR) and electrophoretic separation of PCR products by 1.5% agarose gel electrophoresis. The coding exons and intron/exon flanking regions followed by bidirectional sequencing was performed on all participants. In this study, we found that a 28 year-old male patient harbouring a deleterious substitution of Leu1052Pro in the ADAR1 gene in a typical DSH family. His mother suffered from the DSH also owns the same mutation. This mutation, however, is not identified in the unaffected members in this family and those 200 normal controls. In summary, this new mutation Leu1052Pro reported here is pathogenic and detrimental for DSH. Our finding not only enriches mutation database and contributes to dissecting further the correlation between mutation position and phenotypical features of DSH, but also provides genetics counselling and prenatal diagnostic testing for childbearing couple.

Predictive value of GGN and CAG repeat polymorphisms of androgen receptors in testicular cancer: a meta-analysis.

The risk of testicular cancer (TC) is markedly increased in subjects with androgen insensitivity, and previous studies have proposed that GGN and CAG repeats in androgen receptors (AR) could be related to the risk of TC. To evaluate the association between the length of GGN and CAG repeats in AR and TC, a meta-analysis involving 3255 TC cases and 2804 controls was performed. The results suggested that long GGN repeats are associated with an increased risk of TC compared with those < 23 [odds ratio (OR) = 1.22, 95% confidence interval (CI) = 1.05-1.41]; similarly, a subgroup analysis revealed that this association occurred in studies with case sizes > 200, and in the mid-latitude, and seminoma subgroups. The subgroup analysis based on populations, high-latitude, and seminomas/non-seminomas suggested that AR CAG repeat polymorphisms with > 25 and < 21 + > 25 repeats might confer a protective effect to the patients with TC (in the high-latitude subgroup analysis, for > 25 vs. 21-25: OR = 0.54, 95% CI = 0.41-0.70). In contrast, an increased risk of TC was observed for AR CAG repeat polymorphisms with > 25 and < 21 + > 25 repeats in the mid-latitude subgroup (for > 25 vs. 21-25: OR = 1.65, 95% CI = 1.09-2.50). In addition, no associations between the remaining subgroups and male infertility were observed. In short, this meta-analysis suggested that AR GGN and CAG repeat polymorphisms may be involved in the etiology of TC.