Genetic mutation analysis of 22 patients with congenital absence of vas deferens: a single-center study†.

Congenital absence of the vas deferens (CAVD), a congenital malformation of the male reproductive system, causes obstructive azoospermia and male infertility. Currently, the cystic fibrosis transmembrane conductance regulator (CFTR) has been recognized as the main pathogenic gene in CAVD, with some other genes, such as adhesion G-protein-coupled receptor G2 (ADGRG2), solute carrier family 9 isoform 3 (SLC9A3), sodium channel epithelial 1 subunit beta (SCNN1B), and carbonic anhydrase 12 (CA12), being candidate genes in the pathogenesis of CAVD. However, the frequency and spectrum of these mutations, as well as the pathogenic mechanisms of CAVD, have not been fully investigated. Here, we sequenced all genes with potentially pathogenic mutations using next-generation sequencing and verified all identified variants by Sanger sequencing. Further bioinformatic analysis was performed to predict the pathogenicity of mutations. We described the distribution of the p.V470M, poly-T, and TG-repeat CFTR polymorphisms and identified novel missense mutations in the CFTR and SLC9A3 genes, respectively. Taken together, we identified mutations in the CFTR, ADGRG2, SLC9A3, SCNN1B, and CA12 genes in 22 patients with CAVD, thus broadening the genetic spectrum of Chinese patients with CAVD.

Pathogenesis of acephalic spermatozoa syndrome caused by SUN5 variant.

Acephalic spermatozoa syndrome (ASS) is a rare teratozoospermia that leads to male infertility. Previous work suggested a genetic origin. Variants of Sad1 and UNC84 domain containing 5 (SUN5) are the main genetic cause of ASS; however, its pathogenesis remains unclear. Here, we performed whole-exome sequencing in 10 unrelated ASS and identified 2 homozygous variants, c.381delA[p.V128Sfs7*] and c.675C>A[p.Y225X], and 1 compound variant, c.88 C > T[p.R30X] and c.381 delA [p.V128Sfs7*], in SUN5 in 4 patients. The c.381delA variant had been identified as pathogenic in previous reports, while c.675C>A and c.88 C > T were two novel variants which could lead to a premature termination codon (PTC) and resulted in loss of SUN5, and may also be pathogenic. SUN5 mRNA and protein were present at very low levels in ASS patients with SUN5 nonsense mutation. Furthermore, the distribution of outer dense fiber protein 1 (ODF1) and Nesprin3 was altered in sperm of ASS patients with SUN5 variants. The co-immunoprecipitation analysis indicated that SUN5 and ODF1, SUN5 and Nesprin3, and ODF1 and Nesprin3 interacted with each other in transfected HEK293T cells. Thus, we propose that SUN5, Nesprin3, and ODF1 may form a 'triplet' structure through interactions at neck of sperm. When gene variants resulted in a loss of SUN5, the 'triplet' structure disappears and then the head-tail junction becomes fragile, leading to the occurrence of ASS.

Further identification of a 140bp sequence from amid intron 9 of human FMR1 gene as a new exon.

BACKGROUND: The disease gene of fragile X syndrome, FMR1 gene, encodes fragile X mental retardation protein (FMRP). The alternative splicing (AS) of FMR1 can affect the structure and function of FMRP. However, the biological functions of alternatively spliced isoforms remain elusive. In a previous study, we identified a new 140bp exon from the intron 9 of human FMR1 gene. In this study, we further examined the biological functions of this new exon and its underlying signaling pathways. RESULTS: qRT-PCR results showed that this novel exon is commonly expressed in the peripheral blood of normal individuals. Comparative genomics showed that sequences paralogous to the 140 bp sequence only exist in the genomes of primates. To explore the biological functions of the new transcript, we constructed recombinant eukaryotic expression vectors and lentiviral overexpression vectors. Results showed that the spliced transcript encoded a truncated protein which was expressed mainly in the cell nucleus. Additionally, several genes, including the BEX1 gene involved in mGluR-LTP or mGluR-LTD signaling pathways were significantly influenced when the truncated FMRP was overexpressed. CONCLUSIONS: our work identified a new exon from amid intron 9 of human FMR1 gene with wide expression in normal healthy individuals, which emphasizes the notion that the AS of FMR1 gene is complex and may in a large part account for the multiple functions of FMRP.

Aggregation of Ribosomal Protein S6 at Nucleolus Is Cell Cycle-Controlled and Its Function in Pre-rRNA Processing Is Phosphorylation Dependent.

Ribosomal protein S6 (rpS6) has long been regarded as one of the primary r-proteins that functions in the early stage of 40S subunit assembly, but its actual role is still obscure. The correct forming of 18S rRNA is a key step in the nuclear synthesis of 40S subunit. In this study, we demonstrate that rpS6 participates in the processing of 30S pre-rRNA to 18S rRNA only when its C-terminal five serines are phosphorylated, however, the process of entering the nucleus and then targeting the nucleolus does not dependent its phosphorylation. Remarkably, we also find that the aggregation of rpS6 at the nucleolus correlates to the phasing of cell cycle, beginning to concentrate in the nucleolus at later S phase and disaggregate at M phase. J. Cell. Biochem. 117: 1649-1657, 2016. © 2015 Wiley Periodicals, Inc.

[Advances in the studies of spermatogonial stem cells in primate mammals].

Spermatogonial stem cells(SSCs) are a type of spermatogonial cells that play an important role in the spermatogenesis of males. SSCs not only possess the properties of stem cells but also differentiate into sperm. They are the unique adult stem cells that transmit genetic information to subsequent generations. Therefore,SSCs are regarded as an ideal alternative source of pluripotent stem cells according with moral and ethical issues,legality,and safety. Long-term in vitro culture systems and identification of SSCs have paved the ground for the studies of their transplantation and pluripotency. Early relevant studies mainly focused on non-primate mammals. Recently,researches on SSCs have made great progress in primate mammals,especially in humans. This review focuses on the characterization,isolation,purification,cultivation,identification,and biological markers of SSCs,with a discussion on their unlimited pluripotency and application,and meanwhile provides an insight into the application potential of SSCs in the treatment of male infertility and human regenerative medicine.

[Molecular diagnosis and prenatal diagnosis in a hereditary multiple osteochondromas family].

OBJECTIVE: To identify the mutation in the disease gene and provide prenatal diagnosis for a hereditary multiple osteochondromas (HMO) family. METHODS: The exons of EXT1 gene in the proband with HMO and his family members were amplified by PCR. The products were analyzed by direct sequencing. Prenatal genetic diagnosis was performed by amniocentesis sampling after genotyping the proband. RESULTS: In the family, the affected proband was heterozygous of the mutation of 1476_1477delTC in the EXT1 gene, and the proband's father carried the same mutation in part of his somatic cells. No mutation was found in the EXT1 gene of the proband's mother and other 11 siblings of his father. CONCLUSION: METHODS for molecular diagnosis and prenatal diagnosis of HMO were established and applied to a family of HMO.

[Progress in molecular diagnosis of fragile X syndrome].

Fragile X mental retardation 1 is the gene underlying fragile X syndrome (FXS). Its product, fragile X mental retardation protein, is closely involved with development of brain and neurons. PCR and Southern blotting have been the major methods for laboratory diagnosis of FXS. In this article, the progress in the molecular diagnosis of FXS is reviewed.

[Identification of a mosaic mutation of NF1 gene in a sporadic case of neurofibromatosis type 1].

OBJECTIVE: To detect NF1 gene mutation in a patient with neurofibromatosis type 1. METHODS: Five fragments encompassing the entire coding sequence of the NF1 gene were amplified with reverse transcription PCR. PCR products were directly sequenced. Suspected mutations were verified by sequencing of DNA amplified by PCR using genomic DNA as template. Corresponding exon of family members was also sequenced. Furthermore, the PCR products were inserted into a pGEM-T cloning vector to quantify cells carrying the mutation in different samples derived from the three embryonic layers. RESULTS: The proband's clinical manifestation was consistent with neurofibromatosis type 1. Sequence analysis has identified a novel heterozygous mutation c.7911 C to T (p.Q2510X) in exon 51 of the NF1 gene in the proband. The same mutation was also detected in peripheral blood cells, uroepithelial cells and oral mucosal cells of the proband, though the signals of uroepithelial cells were significantly weaker. By T cloning-sequencing, recombinants carrying the NF1 gene mutation respectively accounted for 42%, 36% and 12% of all peripheral blood cells, oral mucosal cells and uroepithelial cells . CONCLUSION: It is likely that a mutation of NF1 gene has occurred in early embryogenesis of the proband, which in turn has led to generalized mosaicism of neurofibromatosis type 1.

Two novel multiple mutations in chinese patients with adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder caused by mutations in the ABCD1 gene. Up to now, more than 1 050 mutations have been reported in the ABCD1 gene, of which only 10 are multiple mutations in one allele of the gene. In this study, we report 2 novel multiple mutations in 2 patients with X-ALD from 2 unrelated Chinese families. Total RNA and genomic DNA were isolated from peripheral blood of the 2 patients, and the ABCD1 gene was analyzed by direct sequencing and denaturing high-performance liquid chromatography. We detected [p.Ser108X+p.Arg259Trp] in patient 1, [p.Lys217Glu+p.Val489Val] in patient 2 in one allele of the ABCD1 gene. Both novel multiple mutations have not previously been reported and this is the first report of multiple mutations identified in Chinese patients with X-ALD.

[A novel missense mutation resulting in X-linked adrenoleukodystrophy in female heterozygotes of a Chinese family].

OBJECTIVE: To identify ABCD1 gene mutation in a Chinese family with three heterozygous female patients. METHODS: Four fragments covering the entire coding sequence of the ABCD1 gene from one of the female patients were amplified by reverse transcription-PCR. The PCR products were directly sequenced. The result of sequencing was confirmed by restriction enzyme digestion of PCR products from genomic DNA. Human ABCD1 gene and ALD protein were aligned with those of rat, monkey, mouse and cattle by Clustal X 1.83. Softwares of Motif Scan, TMpred and ESYpred3D were used to predict the effect of the mutation on the structure of the ALD protein. RESULTS: A novel missense mutation, CAC to CGC, was found at codon 283 of the ABCD1 gene from the patient, resulting in the replacement of histidine by arginine. This mutation abolished an Msl I site in the gene. Her son was free from this mutation. The mutated amino acid residue (283H) was highly conservative in evolution, and the mutation caused a dramatic change in the structure of the ALD protein. CONCLUSION: Three female patients heterozygous for ABCD1 gene mutation were first reported in China, and a novel mutation, p.H283R, was identified in this X-ALD family.

Splicing of exon 9a in FMR1 transcripts results in a truncated FMRP with altered subcellular distribution.

FMRP is an RNA-binding protein, loss of which causes fragile X syndrome (FXS). FMRP has several isoforms resulted from alternative splicing (AS) of fragile X mental retardation 1 (FMR1) gene, but their biological functions are still poorly understood. In the analysis of alternatively spliced FMR1 transcripts in the blood cells from a patient with FXS-like phenotypes (normal CGG repeats and no mutation in coding sequence of FMR1), we identified three novel FMR1 transcripts that include a previously unidentified microexon (46 bp), terming the exon 9a. This microexon exists widely in unaffected individuals, inclusion of which introduces an in-frame termination codon. To address whether these exon 9a-containing transcripts could produce protein by evading nonsense-mediated decay (NMD), Western blot was used to analysis blood cell lysate from unaffected individuals and a 34 kDa protein that consistent in size with the molecular weight of the predicted truncated protein produced from mRNA with this microexon was found. Meanwhile, treatment of peripheral blood mononuclear cells with an inhibitor of NMD (Cycloheximide) did not result in significant increase in exon 9a-containing transcripts. Using confocal immunofluorescence, we found the truncated protein displayed both nuclear and cytoplasmic localization in HEK293T and HeLa cells due to lacking C-terminal domains including KH2, NES, and RGG, while the full-length FMRP protein mainly localized in the cytoplasm. Therefore, we hypothesize that the inclusion of this microexon to generate exon 9a-containing transcripts may regulate the normal functionality of FMRP, and the dysregulation of normal FMRP due to increased exon 9a-containing alternatively spliced transcripts in that patient may be associated with the manifestation of FXS phenotype.

[Molecular diagnosis of a Chinese pedigree with osteogenesis imperfecta type I].

OBJECTIVE: To perform molecular diagnosis for a Chinese pedigree with osteogenesis imperfecta type I. METHODS: Thirty pairs of primers were designed to amplify all the 52 exons, exon boundaries and promoter region of the COL1A1 gene from genomic DNA of peripheral blood cells of the family members. The PCR products were purified and directly sequenced. To check the mutation in normal controls, the genomic DNA from peripheral blood cells of the index patient, his mother and 60 normal controls were analyzed by amplification refractory mutation system. RESULTS: A missense mutation of GAT>CAT was identified at codon 1441 of the COL1A1 gene from the family, which resulted in the replacement of aspartic acid by histidine (D1441H). This mutation was not found in a group of 60 normal controls. CONCLUSION: The method for molecular diagnosis of osteogenesis imperfecta was established and a novel COL1A1 gene mutation, D1441H, was identified in the Chinese pedigree with osteogenesis imperfecta type I.

[Mutation analysis of SMN gene in a patient and his family with spinal muscular atrophy].

OBJECTIVE: To perform mutation analysis and describe the genotype of the SMN gene in a patient with spinal muscular atrophy (SMA) and his family. METHODS: Deletion analysis of the SMN1 exon 7 by conventional PCR-restriction fragment length polymorphism (RFLP) and allele-specific PCR, and gene dosage of SMN1 and SMN2 by multiplex ligation-dependent probe amplification (MLPA) were performed for the patient and his parents; reverse transcriptase (RT)-PCR and sequencing were performed for the patient. To determine whether the SMN variant was exclusive to transcripts derived from SMN1, the RT-PCR product of the patient was subcloned and multiple clones were sequenced directly; PCR of SMN exon 5 from the genomic DNA of the parents and direct sequencing were performed to confirm the mutation. RESULTS: In SMN1 exon 7 deletion analysis, no homozygous deletion of the SMN1 was observed in the family; the gene dosage analysis by MLPA showed that the patient had 1 copy of SMN1 and 1 copy of SMN2 his father had 2 copies of SMN1 and 2 copies of SMN2, and his mother had 1 copy of SMN1 and no SMN2. A previously unreported missense mutation of S230L was identified from the patient and this mutation was also found in his father. CONCLUSION: A novel missense mutation of S230L was identified in the SMA family and the genotype of the family members were investigated.

[Molecular diagnosis of spinal muscular atrophy by multiplex ligation-dependent probe amplification].

OBJECTIVE: To investigate the effect of multiplex ligation-dependent probe amplification (MLPA) in molecular diagnosis of spinal muscular atrophy (SMA). METHODS: Peripheral blood samples were collected from 13 SMA patients, 31 parents of SMA patients, 50 healthy individuals without family history of SMA, and 10 specimens of amniotic fluid from these families were collected too. Genomic DNA was analyzed by MLPA, conventional PCR-RFLP, and allele-specific PCR. RESULTS: In complete agreement with the results of conventional PCR-RFLP and allele-specific PCR, MLPA analysis showed that all of the 13 patients had homozygous deletion of the survival of motor neuron 1 (SMN1) gene, and there was significant difference between the SMA severity (type I to type III) and SMN2 copy number (P < 0.05). Of the 31 parents 29 (93.5%) had 1 copy of SMN1, 2 (6.5%) had 2 copies of SMN1. Of the 50 healthy individuals, 1 (2.0%) had 1 copy of SMN1, 48 (96.0%) had 2 copies of SMN1, and 1 (2.0%) had 3 copies. The SMN1 copy number of the parents was significantly higher than that of the healthy individuals (P < 0.01). Two of the 10 fetuses had homozygous deletion of SMN1. CONCLUSION: The MLPA technique has proved to be an accurate and reliable tool for the molecular diagnosis of SMA, both in patients and in healthy carriers.

[Prenatal molecular diagnosis of four fetuses at high risk for X-linked adrenoleukodystrophy].

OBJECTIVE: To investigate methods for prenatal molecular diagnosis of fetuses at high risk for X-linked adrenoleukodystrophy (X-ALD). METHODS: The amniotic fluid was obtained and genomic DNA was isolated from amniotic fluid cells. Maternal contamination was evaluated by paternity test. PCR-RFLP, sequencing and denaturing high performance liquid chromatography (DHPLC) were used to detect the ABCD1 gene of fetal genome. RESULTS: In the pedigree 1, the PCR product (799 bp) of the fetus 1 and her father (normal control) could be digested with BcnI. No P560L mutation, which was present in the index patient, was detected in the ABCD1 gene from the genomic DNA of the fetus 1 using direct sequencing. In the pedigree 2, the PCR product (232 bp) of the fetus 2 and her father could not be digested with MaeI and no Q177X mutation, which was present in the propositus, was detected in the ABCD1 gene from the genomic DNA of the fetus 2 using direct sequencing. In the pedigree 3, the PCR product (271 bp) was digested with AciI, the pattern of the fetus 3 and the propositus being the same, and the R617C mutation was found in the ABCD1 gene from the genomic DNA of the fetus 3 using direct sequencing. In the pedigree 4, the PCR product (269 bp) was analyzed with the DHPLC, and the pattern of elution peaks of the fetus 4 and her father was similar, but different from that of the propositus. No K276E mutation was detectable in the ABCD1 gene from the genomic DNA of the fetus 4 by using direct sequencing. Judging from the sex of the fetuses, fetuses 1 and 2 were normal homozygotes, fetus 3 was an ALD hemizygote, and fetus 4 was a normal hemizygote. CONCLUSION: A new protocol for X-ALD prenatal molecular diagnosis is proposed, which would ensure the accuracy of prenatal diagnosis.

[Genetic polymorphism of sperm-specifically expressed genes and male infertility].

The expression of sperm-specific genes is necessary in spermiogenesis, and the genetic polymorphism of these genes may impair spermatogenesis, leading to male infertility. Mamy investigations have been conducted on the polymorphism of some candidate genes that are specifically expressed in spermiogenesis, the relation of genetic polymorphism with male infertility and its possible mechanism. Further attempts have been made at the explanation of the causes of infertility from the genetic angle.

[Prenatal diagnosis of 5 fetuses with high risk of developing spinal muscular atrophy].

OBJECTIVE: To perform prenatal diagnosis for 5 pregnant women who had given birth to children with spinal muscular atrophy (SMA). METHODS: Thirty to forty mililiters of amniotic fluid was obtained by amniocentesis under ultrasonic monitoring. DNA was extracted directly from sediment of amniotic fluid. Short tandem repeat (STR) profiling was carried out to evaluate the contamination of amniotic DNA by maternal genomic DNA. Two methods, PCR-restriction fragment length polymorphism (PCR-RFLP) and allele-specific PCR, were used to analyze exon 7 of SMN gene from amniotic DNA. RESULTS: Comparing the 16 STR sites of each fetus with those of his/her parents, there was no or little contamination of amniotic DNA by maternal genomic DNA. In conventional PCR-RFLP, part of the PCR product (189 bp) from amniotic DNA of fetus A, C, or D remained intact after digestion with Dra I, while the PCR product from amniotic DNA of fetus B or E was completely digested by Dra I. In allele-specific PCR, exon 7 of both SMN1 and SMN2 gene could be seen when amniotic DNA of fetuses A, C, or D was analyzed, while only exon 7 of SMN2 could be seen when amniotic DNA of fetuses B or E was analyzed. CONCLUSION: Homozygous deletion of SMN1 is not detected in fetuses A, C, and D, predicting that the risk of developing SMA after birth would be extremely low. Homozygous deletion of SMN1 was present in fetuses B and E suggesting high risk of developing SMA after birth.

Experimental study on the suppression of human nuclear receptor hLRH-1 via a vector-based RNA interference.

To explore the inhibitions of human nuclear receptor hLRH-1 via RNA interference, siRNAs expressing vectors pShLRH-1.1 and pShLRH-1.2, and targeting hLRH-1 were designed and constructed. The recombinants were introduced into hepatocellular carcinoma cells, BEL-7402, mediated by lipofectamin. RT-PCR was carried out to examine the inhibition ratio of hLRH-1 expression. The same method was also applied to analyze the expression of farnesyl pyrophosphate synthetase (FPPS) gene. Our results demonstrated that after transient transfection, both pShLRH-1.1 and pShLRH-1.2 could trigger the efficient inhibition of hLRH-1 in cultured cells, BEL-7402. The inhibition ratios were up to 80%. By comparing with non-transfection and vector-transfection control, the expression of FPPS in cells with inhibition of hLRH-1 was up-regulated significantly. Thus, the inhibition of expression of hLRH-1 in cultured cells was achieved via RNA interference in this study. Our results also suggested that hLRH-1 acts as a negative regulator in FPPS expression.

Microarray analysis of gene-expression profile in hepatocellular carcinoma cell, BEL-7402, with stable suppression of hLRH-1 via a DNA vector-based RNA interference.

To establish a cell line with a permanent suppression of hLRH-1 in this study, a stable RNAi vector (pSineohLRH-1) targeting hLRH-1 was constructed and introduced into hepatocellular carcinoma cell, BEL-7402. By semiquantitative RT-PCR analysis, the expression of hLRH-1 in BEL-7402 cells carrying pSineohLRH-1 was shown to be significantly suppressed by up to approximately 60%. In addition, microarray analysis was carried out to assess the extent of altered gene expression in BEL-7402 cells with stable knockdown of hLRH-1. Direct comparison of gene-expression profiles of more than 18,000 genes showed that 405 of the expressed genes in hLRH-1-knockdown cells differed dramatically in expression levels from those in controls, which suggested the even extensive biological functions of hLRH-1. Interestingly, among those differentially expressed genes, some are cancer-associated such as Gadd45beta and PTEN, and their expressions were further validated. Although the identification of the exact relationship between these genes and hLRH-1 awaits intensive investigation, the findings of this study provide new insights into the mechanism by which hLRH-1 is involved in tumorigenesis.