Biallelic mutations in DCDC2 cause neonatal sclerosing cholangitis in a Chinese family.

BACKGROUND: Neonatal sclerosing cholangitis (NSC) is a severe cholestatic liver disease, which often develops into end-stage liver disease in childhood and requires liver transplantation. Mutations in CLDN1 and DCDC2 are confirmed to be the main pathogenic mechanism of NSC. METHODS: Whole exon sequencing (WES) was performed to find the possible disease-causing mutations of this family. The mutation was confirmed by Sanger sequencing, and large fragment copy number variation was confirmed by qPCR. RESULTS: We found novel biallelic mutations c.[705-2A>G];[923_1023del] in the DCDC2 gene of the proband. The proband's father had the heterozygous mutation c.705-2A>G, and his mother had a heterozygous c.923_1023del. The proband's younger brother, who had similar clinical manifestations, was found the same biallelic mutations with the proband. CONCLUSION: Novel biallelic mutations were identified in DCDC2 of this Chinese family, according to the American College of Medical Genetics and Genomics (ACMG) guidelines for interpretation of sequence variants, both mutations were classified as pathogenic, which might be the cause of NSC in this 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.

Hybrid minigene splicing assay verifies the pathogenicity of a novel splice site variant in the COL1A1 gene of a chinese patient with osteogenesis imperfecta type I.

BACKGROUND: Osteogenesis imperfecta (OI) is a rare genetic bone disease associated with brittle bones and fractures. Among all known types, OI type I is the most common type and characterized by increased bone fragility, low bone mass, distinctly blue-gray sclera, and susceptibility to conductive hearing loss beginning in adolescence. Mutations in genes encoding type I collagen (COL1A1 and COL1A2) contribute to the main pathogenic mechanism of OI. METHODS: Subtle mutation of the COL1A1 gene in the proband was detected by targeted next-generation sequencing (NGS) and confirmed by Sanger sequencing. We then assessed the effect of the mutation on the splicing of the COL1A1 gene by bioinformatics prediction and hybrid minigene splicing assay (HMSA). RESULTS: A novel splice site mutation c.1821+1 G>C was discovered in the proband by NGS and further confirmed by Sanger sequencing, which was also simultaneously identified from the proband's mother and elder sister. Bioinformatics predicted that this mutation would result in a disappearance of the 5' donor splice site in intron 26, thereby leading to abnormal splicing and generation of premature stop codon. The follow-up experimental data generated by HMSA was consistent with this prediction. CONCLUSION: Our study identified a novel splice site mutation that caused OI type I in the proband by abnormal splicing and demonstrated that combined applications of NGS, bioinformatics and HMSA are comprehensive and effective methods for diagnosis and aberrant splicing study of OI.

Whole-exome sequencing identifies compound heterozygous mutations in ARSA of two siblings presented with atypical onset of metachromatic leukodystrophy from a Chinese pedigree.

BACKGROUND: Metachromatic leukodystrophy (MLD) is a rare inherited lysosomal storage disorder caused mainly by variants in arylsulfatase A (ARSA) gene. MLD can be divided into three major clinical forms according to the age of onset: late infantile, juvenile, and adult. We report two siblings of late infantile MLD presenting with cerebellar ataxia as the only first clinical symptom. METHODS: Because of the unspecific neurological manifestation, whole-exome sequencing (WES) was performed to find disease-causing mutations for molecular diagnosis. Then successive MRI and ARSA activity determination were performed to further confirm the diagnosis. Moreover, the prenatal diagnosis was carried out on the basis of molecular diagnosis. RESULTS: The siblings exhibited compound heterozygous variants {[c.302G>T]+[c.1344dupC]} in the ARSA gene, and both of the variants have been reported as disease-causing mutations previously. The results of MRI and low ARSA activity confirmed the diagnosis of MLD. Prenatal diagnosis showed that the fetus was a heterozygous carrier. CONCLUSIONS: It is recommended that WES be considered as a first line diagnostic procedure to discover potential disease-causing genetic variants in affected individuals with hereditary traits but without definite clinical diagnosis. However, the final diagnosis should be confirmed by comprehensive evaluations including biochemical, enzymatic or imaging investigations.

Improvement and Evaluation of a Staining Method for Measuring Sperm Lactate Dehydrogenase C4 Activity.

BACKGROUND: This study sought to improve and evaluate a 2-hydroxyvaleric acid based staining method for detection of lactate dehydrogenase C4 (LDH-C4) activity in human spermatozoa. METHODS: A staining method for measuring sperm LDH-C4 activity with the substrate 2-hydroxyvaleric acid was improved. Expression level of LDH-C4 was assessed by Western blotting. The diagnostic performance was evaluated by plotting the receiver operating characteristic (ROC) curve. RESULTS: The positive products were black purple lumps concentrated in the neck segment of spermatozoa. Expression level of LDH-C4 was significantly reduced in the low activity infertile cases as compared to the matched contrasts. Decreased LDH-C4 level was significantly correlated with the declined enzyme activity (r = 0.702, p = 0.000). The ROC curve allowed for the discrimination between low and normal LDH-C4 activity cases with a sensitivity of 0.912 and specificity of 0.895, corresponding to an area under curve (AUC) of 0.941. CONCLUSIONS: The improved method hallmarks a promising accuracy in evaluating sperm LDH-C4 activity. Down-regulated LDH-C4 level is a culprit for the decreased LDH-C4 activity in spermatozoa.

The Value of Preoperative Neutrophil to Lymphocyte Ratio in Indicating Lymph Node Metastasis in Patients with Resectable T2 Stage Gastric Adenocarcinoma.

BACKGROUND: Lymph node metastasis (LNM) is closely associated with poor prognosis in patients with resectable T2 stage gastric adenocarcinoma (RT2-GA). Preoperative blood neutrophil to lymphocyte ratio (NLR) has been identified to be a very valuable predictor for prognosis in patients with diverse cancers. The aim of this investigation was to assess the relationship between NLR and LNM in RT2-GA. METHODS: This retrospective study reviewed 230 patients who underwent surgery for removal of primary T2-GA from August 2002 to December 2013 in a single hospital. Preoperative routine blood test data were collected and the relationship between NLR and LNM in RT2-GA was evaluated by X2 test and multivariate logistic regression analysis. RESULTS: The median value of NLR was 2.18 among 230 patients. Based on the median NLR value, the patients were categorized into two groups: low NLR group (NLR ≤ 2.18) and high NLR group (NLR > 2.18). χ2 test results exhibited that the preoperative NLR was significantly associated with the numbers of metastatic lymph nodes (≤ 6 and > 6) (p = 0.003) and status of lymph node involvement (N0, N1, and N2 stage) (p = 0.032). Multivariate analyses further confirmed that NLR > 2.18 was significantly associated with increased risk of appearing more numbers of metastatic lymph node or higher N stage which exhibited a 4.15- or 7.09-fold elevated risk compared to that of NLR ≤ 2.18. CONCLUSIONS: The preoperative NLR is closely associated with LNM in patients with RT2-GA, which may be used as a predictor indicating more serious LNM in this type of cancer.

Hybrid minigene splicing assay verified the pathogenicity of a novel splice site variant in the dystrophin gene of a Chinese patient with typical Duchenne muscular dystrophy phenotype.

BACKGROUND: Duchenne muscular dystrophy (DMD) is typically caused by disrupting the reading frame of the dystrophin gene: approximately 70%-80% of mutational events are represented by deletions or duplications of one or more exons in the dystrophin gene, and the remaining cases by subtle mutations, including point mutations, small indels, small inversions, and complex small rearrangements. The dystrophin gene is the largest known gene with one of the highest known rates of new mutations. METHODS: Deletions and duplications were detected in the DMD gene of the proband by using multiple ligation-dependent probe amplification (MLPA). Targeted next-generation sequencing (NGS) was used in the subtle mutation detection, followed by Sanger sequencing confirmation. The effect of the mutation on the splicing of the DMD gene was assessed by bioinformatics prediction and hybrid minigene splicing assay (HMSA). RESULTS: Neither duplication nor deletion was found in the DMD gene of the proband. While a novel splice site mutation c.6762+1G>C was identified in the proband by NGS and Sanger sequencing, and his mother was heterozygous at the same site. Bioinformatics predicted that the 5' donor splice site of intron 46 disappeared because of the mutation, which would lead to aberrant splicing and introduce premature stop codon. The HMSA results were in agreement with the prediction. CONCLUSIONS: The novel splice site mutation caused DMD in the proband by aberrant splicing. We suggested that combined applications of MLPA, NGS, HMSA and bioinformatics are comprehensive and effective methods for diagnosis and aberrant splicing study of DMD.

Diagnostic Accuracies of the TUNEL, SCD, and Comet Based Sperm DNA Fragmentation Assays for Male Infertility: a Meta-analysis Study.

BACKGROUND: Recent studies have provided new insights into the diagnostic value of sperm DNA fragmentation (SDF) for male factor sterility. This study aimed to systematically evaluate the diagnostic accuracy of the SDF test for male infertility. METHODS: Eligible studies were retrieved by searching electronic databases. The quality of the studies was assessed on the basis of quality assessment for studies of diagnostic accuracy (QUADAS) criteria tool. The bivariate metaanalysis model was employed to summarize the diagnostic indices and plot the summary receiver operator characteristic (SROC) curve by using Meta-disc 1.4 software. Influence analysis, meta-regression, and publication bias assay were all conducted through Stata 12.0 software. RESULTS: Our bivariate random effect meta-analysis yielded an AUC (area under curve) value of 0.9211 with a sensitivity (95% confidence interval) of 0.80 (0.78 - 0.82) and specificity of 0.83 (0.80 - 0.86) for the use of the SDF test in differentiating infertile males from normal fertile controls. Moreover, our subgroup analysis suggested that SDF analysis with a single TUNEL test resulted in an AUC value of 0.9506, with a pooled sensitivity of 0.77 (0.74 - 0.80) and specificity of 0.91 (0.87 - 0.94), while SCD and Comet assays displayed a combined sensitivity of 0.77 (0.67 - 0.81) or 0.91 (0.88 - 0.94), and specificity of 0.84 (0.75 - 0.91) or 0.63 (0.54 - 0.70), accompanied by an AUC value of 0.8408 or 0.9473. CONCLUSIONS: The SDF assay confers a relatively high diagnostic accuracy for infertility detection, among which the TUNEL based methodology seems to achieve higher accuracy than the SCD and Comet assays.

Alternatively spliced products lacking exon 12 dominate the expression of fragile X mental retardation 1 gene in human tissues.

Fragile X mental retardation 1 gene (FMR1) expression is associated with fragile X syndrome (FXS) and exhibits several splicing products. However, the proportion of spliced isoforms that are expressed in different tissues remains unclear. In the present study, long-chain reverse transcription-polymerase chain reaction with a T cloning-sequencing method was conducted in order to analyze the entire coding region of the FMR1 gene in human tissues. In particular, FXS-associated tissues were analyzed, including the brain and testis. Twenty alternatively spliced isoforms were observed among 271 recombinants, including six novel ones. The isoform that consisted of the entire FMR1 coding region (ISO1) accounted for a small proportion of all isoforms. Isoforms lacking exon 12 were the most abundant. In particular, spliced isoforms ISO7 and ISO17 were the most abundant. However, their relative abundance varied between the peripheral blood cells, and the testis and brain tissues. Bioinformatic analyses suggested that exon 12 may be the sole exon undergoing positive selection. The results of the present study suggested that the mechanisms underlying alternative splicing (AS) of the FMR1 gene may be more complex. Furthermore, the functions of alternatively spliced products lacking exon 12 require further investigation. The results of the present study provide novel insights into the association between AS and the structure and function of the FMR1 gene.

[A novel mutation of NTRK1 gene in a family with congenital insensitivity to pain with anhidrosis].

OBJECTIVE: To screen for mutations in the neurotrophic tyrosine kinase receptor type 1 (NTRK1) gene in a Chinese family affected with congenital insensitivity to pain with anhidrosis (CIPA). METHODS: With informed consent obtained, peripheral blood samples were obtained from the patient and his family members. Seventeen coding exons and intron-exon boundaries of the NTRK1 gene were amplified with PCR and analyzed by direct sequencing. RESULTS: A novel mutation c.2086_2087insC (p.Arg696 fsx) was identified in exon 16 of the NTRK1 gene in the proband. This insertion has caused open reading frame shifting and a premature termination has occurred just one codon downstream. Truncation of 72 amino acids at the C terminus has wiped out part of the tyrosine kinase domain (TKD) of the protein. Both of the proband's parents and two grandmothers have carried the c.2086_2087insC (p.Arg696 fsx) mutation. No mutation was found in the NTRK1 gene of other siblings. CONCLUSION: Mutation analysis of the NTRK1 gene has been carried out in a Chinese family affected with CIPA, and a novel NTRK1 gene mutation was identified.

[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.

[Analysis of small supernumerary marker chromosome 15q11 in four infertile males].

OBJECTIVE: To delineate the origins of small supernumerary marker chromosomes (sSMCs) identified in 4 infertile males. METHODS: The sSMCs were analyzed with combined G-banding, N-banding, multiplex ligation-dependent probe amplification (MLPA), fluorescence in situ hybridization (FISH) and single nucleotide polymorphisms array (SNP-array) techniques. RESULTS: G-banding analysis has suggested a 46,X,-Y,+mar karyotype in all of the 4 cases. N-banding revealed that all of the sSMCs have possessed two satellites located on both sides. By MLPA, 1 patient showed copy number gains for 15q11.2 region. SNP-array analysis suggested that all had duplication for 15q11.1-q11.2 region, spanning 3.06 Mb, 0.9118 Mb, 1.728 Mb and 0.287 Mb, respectively. By FISH analysis, all of the sSMCs showed two hybridization signals, indicating that they were dicentric chromosomes. CONCLUSION: In all of the four cases, the marker chromosomes have derived from chromosome 15 and were bisatellited and dicentric, which gave rise to a karyotype of 47,XY,+ish,inv dup(15)(q11)(D15Z4++). sSMC 15q11 therefore may be a major cause for male infertility.

[Delineation of three structural Y chromosome aberrations combined molecular techniques].

OBJECTIVE: To delineate the structure of Y chromosome aberrations and recombinant mechanisms for three patients. METHODS: Karyotype analysis, multiplex ligation dependent probe amplification (MLPA), fluorescence in situ hybridization (FISH), Y chromosome sequence tagged sites (STS) analysis, human whole genome-wide SNP array were used. RESULTS: The karyotypes of the three patients were 46, X, +mar. As suggested by MLPA analysis, case 1 has increased copy numbers of SRY, ZFY and UTY genes, case 2 had increased copies of SRY and ZFY genes, and deletion of UTY gene, and case 3 had decreased copies for subtelomeric regions of X/Yp and X/Yq. By STSs analysis, case 1 has retained SRY, sY84 and sY86 in the AZFa region, sY1227 in the AZFb region, whilst lost sY1228 in the AZFb region and other STSs in the AZFc region. Its breakpoint was thereby mapped between sY1227 and sY1228. Case 2 has retained SRY and sY1200 in the centromeric region, whilst has deletion of other STSs. Case 3 has retained SRY and STSs in the AZF regions. By SNP array, case 1 had duplicated Yp11.31-p11.2 and deletion of Yq11.22-q11.23 (approximately 5.18 Mb). Case 2 had duplicated Yp11.31-p11.2 and deletion of Yq11.21-q11.23 (approximately 14.644 Mb). Case 3 had single copy number deletion of p22.33 and q28 in the subtelomeric region of X/Yp and X/Yq. By FISH, cases 1 and 2 showed two signals for SRY and DYZ3 but no signal for DYZ1 on their marker chromosomes. Combining above results, the karyotypes of cases 1, 2 and 3 were determined as 46, X, idic(Y) (q11.23), 46, X, idic(Y) (q10) and 46, X, r(Y) (p11q12), respectively. CONCLUSION: Y chromosome aberrations are variable. Combined use of MLPA, STSs, FISH and SNP array is effective for revealing the breakpoints and recombinant mechanisms.

A rapid and sensitive protocol for prenatal molecular diagnosis of X-linked adrenoleukodystrophy.

BACKGROUND: X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative genetic disease characterized by progressive demylination of the brain, adrenal insufficiency and elevated VLCFA level. ABCD1gene is the disease gene and more than 500 unique mutations in the ABCD1gene have been recorded in the database, approximately 60% of which are noncurrent ones. Although great progress has been made in the treatment of X-ALD, prenatal diagnosis is still badly needed by X-ALD-stricken families. METHODS: Twelve high-risk fetuses entered this study. Amniotic fluid (AF) was divided into two parts, with one part being used directly to isolate genomic DNA and debris from the other part for amniotic fluid cells (AFC) culturing. STR profiling was performed to evaluate maternal contamination of AFC genomic DNA. Two different molecular approaches, be they any two of direct sequencing, PCR-RFLP, ARMS, dot hybridization and DHPLC, were applied to determine whether the mutation identified in the index patient was found in the fetus. RESULTS: The genotypes of all 12 fetuses were determined, among which 2 were diagnosed as ALD males, 5 unaffected males, 1 heterozygote, and 4 normal unaffected females. A total of 9 families sent samples of umbilical blood at the time of delivery, and results of molecular checking of these samples agreed with those of prenatal diagnosis. Up until now, no ALD-related abnormalities were reported postnatally. CONCLUSION: An in-house protocol for the prenatal molecular diagnosis of X-ALD was established, and this protocol would provide accurate and rapid prenatal genetic service to X-ALD-stricken families.

Evaluation of an in-house protocol for prenatal molecular diagnosis of SMA in Chinese.

BACKGROUND: Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder characterized by degeneration of the anterior horn of the spinal cord, leading to symmetric muscle weakness and atrophy. About 95% of SMA patients have homozygous loss of SMN1 which can be detected by conventional PCR-RFLP testing. However, the method cannot distinguish heterozygous healthy carriers. A quantitative method named multiple ligation-dependent probe amplification (MLPA) was introduced in our protocol for prenatal molecular diagnosis of SMA in our laboratory. METHODS: DNA was extracted from amniotic fluid cells of 13 fetuses from 11 Chinese SMA families. STR profiling was carried out to evaluate the contamination of amniotic DNA by maternal genomic DNA. Three methods, PCR-RFLP, allele-specific PCR and MLPA, were used to analyze SMN1 exon 7 of amniotic DNA. RESULTS: There was no contamination of amniotic DNA by maternal genomic DNA. In conventional PCR-RFLP, allele-specific PCR, and MLPA, homozygous loss of SMN1 was observed in 4 fetuses. Among the remaining 9 fetuses, 6 with 1 copy of SMN1 and 3 with 2 copies of SMN1 were showed by MLPA. CONCLUSION: This in-house protocol was reliable and efficient for prenatal molecular diagnosis of SMA.

Increased expression of hLRH-1 in human gastric cancer and its implication in tumorigenesis.

Altered signaling pathways or deregulated transcription factors represent an important category of molecular events leading to aberrant gene regulation in gastric cancer, among which the role of WNT/beta-catenin pathway remains unclear. LRH-1 is a critical transcription factor in controlling cell proliferation via crosstalk with the beta-catenin signaling pathway. In order to gain a knowledge of the expression of hLRH-1v1 and hLRH-1 in gastric cancer, a Q-PCR analysis was carried out. Our results showed that in about 50 and 47.6% of 42 tested patients with gastric cancer, the mRNA expression of hLRH-1v1 and hLRH-1 was significantly upregulated, as compared with self-paired normal control, respectively. Besides, overexpression of hLRH-1 was shown to promote the proliferation of gastric adenocarcinoma cell SGC-7901 via induction of cyclin E1. Taken together, our present study demonstrated for the first time the increased expression of hLRH-1v1 and hLRH-1 in human gastric cancer, an alteration which may implicate in tumorigenesis.

[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.

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.

[Expression and characterization of subunit C of mouse lactate dehydrogenase in Escherichia coli].

OBJECTIVES: To construct a prokaryotic recombinant vector for mouse lactate dehydrogenase-C and to detect its expression in BL21. METHODS: The coding sequence of mouse lactate dehydrogenase subunit C was amplified from mouse testis RNA with specific primers, and cloned into pGEX-2T after the restriction digestion with BamH I and EcoR I. GST fusion protein was expressed after induction with IPTG. RESULTS: Sequencing and restriction digestion of the recombinant plasmid revealed the existence of coding sequence for mouse lactate dehydrogenase subunit C. A protein band of about 60,000 could be induced by IPTG in the recombinant plasmid. CONCLUSIONS: The coding sequence of mouse lactate dehydrogenase subunit C was introduced into the pGEX-2T plasmid and a GST-fused protein could be induced at a high level.