Objective Usher syndrome(USH) is an autosomal recessive disorder characterized by hearing impairment and vision dysfunction due to retinitis pigmentosa. Phenotypic and genetic heterogeneities of this disease make it i...
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Objective Usher syndrome(USH) is an autosomal recessive disorder characterized by hearing impairment and vision dysfunction due to retinitis pigmentosa. Phenotypic and genetic heterogeneities of this disease make it impractical to obtain a genetic diagnosis by conventional Sanger sequencing. Method This study explored an approach for detecting disease-causing genetic mutations in candidate genes in 25 cases from unrelated USH families based on targeted next-generation sequencing(NGS) technology. Through systematic data analysis using an established bioinformatics pipeline, all variants that passed filter criteria were validated by Sanger sequencing and co-segregation analysis. Result 44 pathogenic mutations in the USH disease genes were identified in the 25 USH families. We identified 7 novel mutations in CDH23 gene, 9 novel mutations in GPR98 gene, 2 novel mutations in MYO7 A gene, one mutations in PCDH15 gene, one mutations in USH1 C gene, and 12 novel mutations in USH2 A gene. All novel variations segregated with the disease phenotypes in their respective families and were absent from ethnically matched control individuals. Conclusion Targeted exome sequencing precisely and rapidly identified the genetic defects in 25 Chinese USH families and this technique can be applied as a routine examination for these disorders with significant clinical and genetic heterogeneity.
Objective Pseudoachondroplasia（PSACH） is an autosomal dominant osteochondrodysplasia ch aracterized by short?limb short stature, brachydactyly and early?onset osteoarthropathy. As so fa r, most cases of PSACH were c...
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Objective Pseudoachondroplasia（PSACH） is an autosomal dominant osteochondrodysplasia ch aracterized by short?limb short stature, brachydactyly and early?onset osteoarthropathy. As so fa r, most cases of PSACH were caused exclusively by mutations in the gene for cartilage oligomeri c matrix protein（COMP）. In this study,we report a family with the typical PSACH. The pedigree analysis revealed that the proband and her daughter affected by severe short stature within a spa n of two generations and the disorder exhibited transmission form of the autosomal dominant. Th e aim of this study is to identify the causative mutation in the family. Method Genomic DNA was extracted from peripheral blood by standard phenol/chloroform meth od. Polymerase chain reaction（PCR） was adopted to amplify the all the 19 exons and the exon/intron boundaries of COMP gene. Then the PCR fragments were screened by DNA Sanger sequen cing to find the causative mutation. Result A heterozygous deletion mutation ***1371385 GGACTCAGACCACGA in the exon 13 was identified in the proband, which resulted in a five-amino-acid deletion（Glu457 Aspdel DSDHD） in the sixth calmodulin-like repeat of the COMP protein. Then this mutation was confirmed to cosegregate with PSACH manifestations of the affected in the family, but not detected in the unaffec teds including 3 familial members and 50 population controls. Conclusion The mutation of ***1371385 GGACTCAGACCACGA is a novel mutation respons ible for severe familial PSACH（not reported in HGMD）. Our result expanded the mutation spectru m of the COMP gene, and indicated that molecular analysis will be the most promising method for the clinical diagnosis of PSACH in future.
Objective Spinal muscular atrophy(SMA) is an autosomal recessive disease caused by mutation s in the survival motor neuron1 gene(SMN1). Global carrier frequency is around 1 in 50 and carri er detection is crucial to d...
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Objective Spinal muscular atrophy(SMA) is an autosomal recessive disease caused by mutation s in the survival motor neuron1 gene(SMN1). Global carrier frequency is around 1 in 50 and carri er detection is crucial to define couples at risk to have SMA offspring. Most SMA carriers have on e SMN1 copy and are currently detected using quantitative methods. A few, however, have two SMN1 gen es in cis(2/0 carriers), complicating carrier diagnosis in SMA. Method We detected 2/0 carriers from a cohort of 1328 individuals, including SMA parents, SMA relatives, and unrelated individuals of the general population, by multiplex ligationdependent prob e amplification(MLPA) methodology and haplotype analysis. Result In two couples who had an SMA child, both the parents had two SMN1 copies. Conclusion Our results emphasize the importance of performing a detailed carrier study in SMA parents with two SMN1 copies. Expanding the analysis to other key family members might confir m potential 2/0 carriers. When a partner of a known carrier presents two SMN1 copies, the study of both parents will provide a more accurate diagnosis, thus optimizing genetic counseling.
Objective Objective: To establish an effective, feasible, economic, rapid and pollution-free practic al assay for genetic diagnosis of glucose-6-phosphate dehydrogenase(glucose-6-phosphate dehydrogenase, G6 PD) defici...
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Objective Objective: To establish an effective, feasible, economic, rapid and pollution-free practic al assay for genetic diagnosis of glucose-6-phosphate dehydrogenase(glucose-6-phosphate dehydrogenase, G6 PD) deficiency. Method Methods: A multiple fluorescence PCR system based Taqman MGB probes was perform ed to amplify the target fragments of G6 PD gene, and detect the domestic main 10 mutation sites kinds(1376 G>T, 1388 G>A, 95 A>G, 871 G>A, 392 G>T, 1024 C>T, 1004 C>A, 517 C>T, 592 C>T, 487 G>A). At the same time, the beta actin gene monitors the whole PCR system. Repeatability w as tested in 50 samples with known genotype. A double-blind trial for 346 unknown samples was performed by using multiplex fluorescence PC R assay and DNA sequencing simultaneously. Result Results: The established multiple fluorescence PCR assay based Taqman MGB probes w as able to detect the most common 10 mutations of G6 PD gene simultaneously in 1 hour, and bot h specificity and accuracy reached 100%. Among the 346 unknown samples, 57 mutational cases was detected. All the genotyping results of multiple fluorescence PCR assay based Taqman MG B probes were in complete concordance with direct DNA sequencing, and both specificity and ac curacy reached 100%. Conclusion Conclusion: The established multiple fluorescence PCR assay based Taqman MGB probes, detecting samples in 1 hour without open tubes after PCR, is a rapid, effective and feasibl e method of G6 PD deficiency gene diagnosis.
Objective Inherited cardiomyopathy(IC) is the most common genetic heterogeneity and clinical h eterogeneity cardiac disease. It also causes a significant proportion of sudden cardiac deaths and heart failure in young....
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Objective Inherited cardiomyopathy(IC) is the most common genetic heterogeneity and clinical h eterogeneity cardiac disease. It also causes a significant proportion of sudden cardiac deaths and heart failure in young. The purpose of this study was to investigate cases of primary cardiomyop athy for potential disease-causing mutations in 64 genes reported to be associated with IC. Method A total of 110 independent cases and families diagnosed with primary cardiomyopathy in cluding hypertrophic cardiomyopathy(n=34), dilated cardiomyopathy(n=22), restrictive cardiomy opathy(n=13), arrhythmogenic right ventricular cardiomyopathy(n=7), left ventricular non-compa ction(n=9), and phenotype overlap or undefined(n=25) were collected after informed consent. A custom designed Ampli Seq panel, including 64 genes, were screened by Ion Torrent PGM Seque ncing platform. The most likely disease-causing variants were validated by Sanger sequencing. Result As a result, 62 variants in 61 patients(55.5%) had been found with likely functional effects based on conservation, computational prediction, allele-frequency and supportive literature. In th ese variants, 22 are reported to be damaging mutations in the Human Gene Mutation Database, 38 are novel and predicted to be potential or possible pathogenic in silico. The distribution of mut ations among the cardiomyopathy genes were not equal in different type of cardiomyopathy. TTN truncating mutations is not so prevalent in our DCM cases(13%). Conclusion This study provides a comprehensive characterization of the genetic aberrations in C hinese primary cardiomyopathy patients and also highlights the diagnostic value of NGS analysis in IC.
Objective Marfan syndrome(MFS) is an autosomal dominant inherited connective tissue disorde r. The phenotypes are variable from person to person, but mainly affect multiple systems includin g skeletal, cardiovascular,...
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Objective Marfan syndrome(MFS) is an autosomal dominant inherited connective tissue disorde r. The phenotypes are variable from person to person, but mainly affect multiple systems includin g skeletal, cardiovascular, ocular, pulmonary, dura, skin and integument. The Ghent criteria is the internationally accepted criteria for MFS diagnosis. The two patients in this study are both diagno sed with MFS. Thus we performed the genetic testing of FBN1 which is the main disease causing gene of MFS to make a definite diagnosis. Method We collected the peripheral blood samples of patients and their family members, trying to find the causal FBN1 mutation using different methods including high-throughput sequencing, PC R and Sanger sequencing. Bioinformatics websites are essential for our follow up data analysis. Result We identified two novel FBN1 gene splicing mutations in our patients. Both of them are he terozygous splicing mutations. These mutation sites have been reported in the Human Gene Mut ation Database as Marfan syndrome disease causing mutations, but the base changing are differ ent. Based on this results, RNA of one patient was extracted and Sanger sequencing of the RT-P CR product confirm the splicing change. The RT-PCR of the other patient is not finished yet. Conclusion The two novel FBN1 gene splicing mutations of Marfan syndrome are first reported i n our study. However, the pathogenicity of these mutations still need further confirmation. Becaus e of the phenotypic similarity of the two patients, we hypothesise that there is a correlation betwe en the phenotype and mutation type. Our study has confirm the clinical diagnosis, and enlarge th e spectrum of FBN1 mutation. It will be helpful for prenatal diagnosis and genetic counseling.
Objective Congenital heart disease is the most common birth defects in the world. Cardiac transc ription factors play critical roles in the heart development. Abnormalities in those transcriptional fa ctors are the ma...
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Objective Congenital heart disease is the most common birth defects in the world. Cardiac transc ription factors play critical roles in the heart development. Abnormalities in those transcriptional fa ctors are the major contributors to the pathogenesis of CHD. Especially, genetic defects in those genes commonly result in CHD but largely remain unknown. Method In this study, twenty six candidate genes for CHD was screened for mutation analysis in 103 patients with TOF using next-generation sequencing *** Results showed novel pathogenic mutations concentrated in GATA family members, GAT A5(c.943 T>A p.S315 T and c.274 G>T p.A92 S) and GATA6(c.331 G>A p.D111 N and c.972 C>G p.H324 Q), their partner FOG2(c.3442 G>A p.E1148 K and c.3014 A>G p.E1005 G) and TBX5(c.G259 T, p.V87 L). Overexpression of the mutant of FOG2(***1148 Lys) in zebrafish leaded to ca rdiac abnormalities. Moreover, the mutant impaired the interaction with GATA4 in vitro. It proved t he mutation in FOG2 is pathogenic. Conclusion Therefore, our results suggested defects in the network involved in GATA family me mbers and their partners are the high risk for CHD.
Objective The objective of this study is to report the feasibility of cell-free DNA screening(cf DNA screening) as an indicator of parental balanced chromosome translocation. Method From February 2015 to September 201...
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Objective The objective of this study is to report the feasibility of cell-free DNA screening(cf DNA screening) as an indicator of parental balanced chromosome translocation. Method From February 2015 to September 2015, cf DNA screening was offered to 4992 pregnant women. Positive cf DNA screening results for aneuploidies were confirmed by karyotyping, while 4796/4932 negative results were interviewed after delivery. To validate the subchromosomal copy number variations(CNVs), we collected fetal amniotic fluids and tested the samples by chromosome microarray analysis(CMA) and karyotyping. Result In the 4992 cases, cf DNA screening was positive for common trisomies in 33 cases(25 trisomy 21, seven trisomy 18 and one trisomy 13). In addition, 22 cases were positive for sexchromosomal abnormalities, five cases were positive for other deletion/duplication in which two were identified as terminal duplication and deletion on different chromosomes. The CMA results or karyotyping confirmed the cf DNA screening findings and the origin of CNVs were validated afterward by karyotyping or fluorescence in situ hybridization(FISH) using parental blood samples. Conclusion Cf DNA screening may help identify deletions and duplications in fetus, which in some cases may indicate risk of a parent being a balanced rearrangement carrier, and that the diagnostic follow-up testing is necessary.
Objective Tissue factor pathway inhibitor(TFPI) is a major physiological inhibitor of TF-initiated c oagulation, but its precise role and signal pathway in vascular smooth muscle cells(SMCs) during the development of ...
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Objective Tissue factor pathway inhibitor(TFPI) is a major physiological inhibitor of TF-initiated c oagulation, but its precise role and signal pathway in vascular smooth muscle cells(SMCs) during the development of atherosclerosis are not clear. Method We constructed short hairpin RNA targeting TFPI gene(TFPI-sh RNA) in SMCs and the TFPI conditional knockout in SMCs of mouse. TFPI knockdown promoted migration of SMCs through a wound healing assay and a modified Boyden`s chamber method. A mouse model of special TFPI knockdown in SMCs was generated, and the mice were fed a high fat diet for 18 weeks and they were then euthanized. Aortas were stained wi th Sudan IV and more atherosclerotic lesions were found in the TFPI conditional knockout mice th an the control mice, but there were not different among the TFPIfl/fl Sma-Cre, TFPIfl/fl and TFPI+/+ mice. Result The result emphasized that the TFPI deficiency promoted the development and not the oc currence of atherosclerosis. In vitro and in vivo experiments identified that TFPI knockout in SMC s enhanced the phosphorylation of P38/HSP27, and it was more important that genes regulated by the p38 MAPK signaling pathway, such as IL-1β, IL-6, IL-8 and CXCL-10, further confirmed th e tendency. Conclusion TFPI deficiency in SMCs promoted the migration of SMCs and accelerated atheroscl erotic development through the phosphorylation of P38/HSP27.
Objective Conventional karyotyping has been a routine method to identify chromosome abnormalities in products of conception(POC). However, this manner is being transformed by single nucleotide polymorphism(SNP) array,...
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Objective Conventional karyotyping has been a routine method to identify chromosome abnormalities in products of conception(POC). However, this manner is being transformed by single nucleotide polymorphism(SNP) array, which has advantages over karyotyping including higher resolution, dispensing with cell culture and so on. This study was to evaluate the advantage of high-resolution SNP array in identifying genetic aberrations in POC. Method 155 POC specimens including 139 from first-trimester miscarriage and 16 from secondtrimester miscarriage were collected consecutively. SNP array was performed on these samples in parallel with G-banded *** SNP array obtained test success rate of 98.1%(152/155) that was higher than that in karyotyping(133/155, 85.8%). It yielded a 63.8%(97/152) abnormality rate, and the frequency of various chromosome abnormalities was agreement with other previous studies. Results between array and karyotyping demonstrated 94.0%(125/133) concordance. SNP array obtained additional aberrations in 3.8%(5/133) cases unidentified by karyotyping, which included 3 cases with whole-genome uniparental disomy(UPD), 1 case with pathogenic copy number variation(CNV) and 1 case with del(4)(q35.1 q35.2) and dup(12)(q24.31 q24.33). However, chromosome translocations presented in 2 cases and tetraploidy presented in 1 case were detected by karyotyping instead of array. Additionally, 2 out of 3 cases with mosaic trisomy were revealed by array but recognized as pure trisomy by karyotyping. Conclusion This study demonstrated that SNP array had certain advantages over G-banded karyotyping, including higher success rate, additional detection of CNVs and UPD, and improved sensitivity to mosaicism, so it would be an alternative method to karyotyping in clinical genetic practice.
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