Novel Mutations of PRSS1 Gene in the Patients with Pancreatic Cancer among Han Population

Purpose: The aim of this study was to elaborate some novel mutations of PRSS1 gene in the patients with pancreatic cancer. Patients and Methods: There were 156 patients with pancreatic cancer and 220 unrelated individuals were studied as controls. The mutations of PRSS1 gene were analyzed by direct sequencing. K-ras Mutation Detection Kit was used to find the general k-ras gene disorder in the pancreatic cancer tissue. Then collected and analyzed the clinical data at the same time. Results: There were two patients carried novel mutations which is IVS 3 +157 G>C of PRSS1 gene from peripheral blood specimens and pancreatic cancer tissue. What’s more, we were surprised to find a novel complicated mutation of exon 3 in PRSS1 gene (c.409 A>G and c.416 C>T) in another young patient. The c complicated mutation made No.135 and No.137 amino acid transfer from Thr to Ala and Thr to Met respectively. All of the mutations weren’t found in the normal controls and no mutations of k-ras gene detected in the three patients. Conclusion: These observations imdicate that mutations of PRSS1 gene may be an important factor of pancreatic cancer.     

A novel mitochondrial tRNA gene mutation in a chinese family with dilated cardiomyopathy and sensorineural deafness

Objective： To determine whether a mutation of mitochondrial DNA induces familial dilated cardiomyopathy in Chinese families with cardiomyopathy, and analyzed the correlation between the genotype and phenotype. Methods： Affected members in three Chinese families of the familial dilated cardiomyopathy underwent clinical evaluation and DNA analysis. Polymerase chain reaction and direct DNA sequencing were used to screen for mitochondrial DNA mutation. The type of mtDNA variations and clinical situation were analysed on the patients with mitochondrial DNA mutation. Results： The mitochondrial A3434G mutation was identified in one of the three families,the 3434 th nucleotide A was replaced by G, which led to change of amino acid. No mutations were identified in the clinically unaffected members of the family and all members of the other two families. Conclusion： This study indicates that the mitochondrial A3434G mutation maybe related with familial dilated cardiomyopathy and deafness.     

Heteroplasmy Level of the Mitochondrial tRNA^Leu（UUR） A3243G Mutation in a Chinese Family Is Positively Associated with Earlier Age-of-onset and Increasing Severity of Diabetes

Objective To investigate the mutations of mitochondrial genome in a pedigree with suspected maternally inherited diabetes and deafness and to explore the correlations between the mutations and clinical features. Methods Genomic DNA was isolated from blood leucocytes of each member of the pedigree. The mitochondrial genome was amplified with 24-pair primers that could cover the entire mitochondrial DNA. Direct sequencing of PCR products was used to identify any mitochondrial DNA mutations. Results Family members on the maternal side all harbored the tRNA^Lcu（UUR） A3243G mutation. The paternal side family members did not have the mutation. The age-of-onset of diabetes of the 4 maternal side family members was 15, 41, 44, and 65 years old, and their corresponding heteroplasmy level of the mutation was 34.5%, 14.9%, 14.6%, and 5.9%, respectively. The age-of-onset of diabetes and heteroplasmy level of A3243G mutation were negatively correlated with a correlation coefficient of -0.980（P=0.02）. Meanwhile, patient with high heteroplasmy level of A3243G mutation had relatively low severity of disease. Moreover, 6 reported polymorphisms and 2 new variants were found. Conclusions The main cause of diabetes in this pedigree is the tRNA^Lcu（UUR） A3243G mutation. However, other gene variants may contribute to its pathogenicity. The heteroplasmy level of the tRNA^Lcu（UUR） A3243G mutation is positively associated with earlier age-of-onset and increasing severity of diabetes.     

Clinical and laboratory survey of 65 Chinese patients with Leigh syndrome

Background Leigh syndrome is an inherited neurodegenerative disease that emerges in infancy and childhood and presents with a clinically heterogeneous variety of neuromuscular and non-neuromuscular disorders. It can result from the inheritance of mutations in either nuclear or mitochondrial DNA. In the current study, we performed a retrospective study in 65 patients in order to investigate the clinical and genetic characteristics of Leigh syndrome in Chinese patients. Methods Sixty-five unrelated cases （35 men and 30 women） who were hospitalized in the past 12 years were reviewed. Diagnosis was based on both the clinical presentation and the characteristic neuropathologic findings of bilateral symmetric necrotizing lesions in the basal ganglia and brain stem as detected using cranial computed tomography （CT） scan or magnetic resonance imaging （MRI）. The differential diagnosis of organic acidurias and fatty acid IS-oxidation defects were performed. Specific point mutations and deletions in mitochondrial DNA （T8993G, T8993C, T9176C, A8344G, A3243G） were screened by PCR-restriction analysis and Southern blot. The SURF1 gene was sequenced. Skeletal muscle biopsies were performed in 17 （26.2%） of the patients. The diagnosis was confirmed by autopsy in 6 （9.2%） patients. Results The patients had various forms of metabolic encephalomyopathy. Filly-nine （90.8%） of the patients had the typical neuroradiological features of Leigh syndrome, including symmetrical necrotizing lesions scattered within the basal ganglia, thalamus and brain stem. Twenty （30.8%） patients were confirmed by genetic, biochemical analysis and autopsy. Specific point mutations in mitochondrial DNA were found in 5 cases （7.7%）. Of these, the A8344G mutation was detected in 2 patients. The T8993G T8993C, and A3243G point mutations were identified in 3 other patients, respectively. SURF1 mutations associated with cytochrome c oxidase deficiency were identified in 8 （12.3%） families by DNA sequencing. A G604C mutation was identified in 6 （9.2%） patients. The genotypes of 52 patients remained unknown. Conclusions Leigh syndrome presents as a diverse array of clinical features and can result from specific mutations in nuclear or mitochondrial DNA. In this study, SURF1 mutations associated with cytochrome c oxidase deficiency were identified in 8 （12.3%） out of 65 patients with Leigh syndrome. It indicates that SURF1 mutations might be a common cause of Leigh syndrome in China. The etiology of Leigh syndrome in Chinese patients represents a persistent challenge to clinicians.     

Mutations of connexin43 in fetuses with congenital heart malformations

Background Gap junction channels formed by connexin43 (Cx43) protein are important in cardiac morphogenesis, and Cx43 gene is thought to be associated with congenital heart malformation (CHM). This study was undertaken to detect the mutations of Cx43 in fetuses with CHM.Methods Cx43 extron DNA was amplified by PCR from 16 fetuses with a variety of CHM. The PCR products were analyzed by SSCP and DNA sequencing. Thirty children who had no CHM were selected as controls.Results Eight homozygous mutations of Cx43 were observed in a fetus with double outlet right ventricule (DORV) , fiveof the 8 mutations were missense mutations including Arg239Trp, Ser251Thr, Ala253Pro,Pro283Leu and Thr290Asn, and the remaining 3 were silent polymorphisms including Gly252Gly,Pro256Pro and Thr275Thr.No mutations were found in other fetuses and the control group.Conclusions Mutations of Cx43 may be associated with congenital conotruncal anomalies. PCR-SSCP is an effective method for screening the mutations of Cx43.     

MUTATIONS IDENTIFIED IN EXON 7 OF PHENYLALANINE HYDROXYLASE GENE IN CHINESE

Exon 7 of the phenylalanine hydroxylase (PAH) gene was analyzed in 45 children affected with classic phenylketonuria (PKU) from northern China by using PCR-single strand conformation polymorphism (PCR-SSCP)technique and DNA direct sequencing. Six missense mutatiorm(i.e. R243Q,R241H,G247V,L249H,F2541 and G257V)and one silent mutation (V245v) were identified. The latter three mlssense mutations were demonstrated as novel mutations in comparison with the PAH mutation database. One missense mutation (R241H) was first documented in Chinese. Our results showed population and region differences in the PAH mutation distribution,and suggest that there is more than one founding population for PKU in China. The finding of novel mutations will enhence the molecular diagnosis of PKU.     

Frequency of mitochondrial 12S rRNA gene A1555G and 961 insC mutations among children with sensorineural deafness in China

Objective： To investigate the frequency of mitochondrial 12S rRNA gene A1555G and 961 insC mutations among Chinese with sensorineural deafness. Methods： Blood samples from 78 sporadic cases with sensorineural deafness were obtained and DNA was extracted from the leukocytes, then the mitochondrial DNA target fragments were amplified by polymerase chain reaction（PCR）. The 1555G mutations were detected by BsmA 1 restriction endonuclease digestion, every fragment was analyzed by sequencing; All the 961 insC mutation were detected by direct sequencing. Results： The percent age of A1555G mutation and mt961C insertion were 6.4% and 2.6% in the hearing-impaired Chinese subjects respectively. Conclusion： A1555G and 961insC mutations in mitochondrial DNA 12S rRNA gene regions may play a role in the pathogenesis of hearing loss in the sporadic cases.     

GJB2 (Cx26) gene mutations in Chinese patients with congenital sensorineural deafness and a report of one novel mutation

Background Mutations in GJB2 gene are a major cause of autosomal recessive congenital hearing loss and the cause in some rare cases of the autosomal dominant form. The purpose of this study was to investigate the frequency and the features of GJB2 mutations in the Chinese patients with congenital sensorineural deafness.Methods Using PCR amplifying the entire coding region of GJB2 gene and direct DNA sequencing to analyze mutations in this gene among unrelated 69 cases with autosomal recessive congenital nonsyndromic deafness and 27 cases of dominant congenital deafness and 35 sporadic cases. We also detected mutations in GJB2 in 100 control subjects with normal hearing.Results 17. 4% (12/69) of the probands in the autosomal recessive, 7. 4% (2/27) of dominant families and 5.7% ( 2/35 ) of the sporadic congenital deafness patients had deafness-causing mutations in GJB2, respectively. Nine types of the mutations in GJB2 were detected in the recessive and sporadic group. They consisted of five types of polymorphism, and four types of deafness-causing mutation with homozygous 35delG in 1 sporadic (1/35), and 235delC frameshift mutation in 1 sporadic (homozygotes) and 10 recessive patients (2 heterozygotes and 8 homozygotes), and homozygous 442G→A missense mutation and homozygous 465T→A nonsense mutation in 1 different recessive proband, respectively. The 465T→A that related to recessive deafness was a novel mutation found by this study. The homozygous (10/69, 14. 5%) and the heterozygous (2/69,2.9%) GJB2 mutation in the recessive patients (12/69, 17.4%) and the homozygotes in the sporadic patient (2/35, 5. 7%) all had congenital severe to profound sensorineural hearing loss.511G→A missense mutation and 299 -300delAT frameshift mutation were found in two autosomal dominant congenital deafness families (2/27, 7.4%). The total mutation frequency of GJB2 was 12.2% (16/131) in the Chinese patients with congenital sensorineural deafness and 235delC was the most common deafness-causing mutation. Six types of mutation-5 types of polymorphism and 1 type of heterozygous deletion (235delC) mutation were found in the 100 control subjects. The carry rate of the most frequent type of mutation 235delC was 0. 5% in the controls(1/200 alleles). 109G→A was the most frequent (15/100, 15%) and 79G→A was the second common (8/100, 8%) polymorphism in this population.Conclusions The general mutation rate of GJB2 is 12.2% (16/131) and the 235delC is the most common type of deafness-causing mutation in Chinese patients with congenital hearing loss. 465T→A nonsense mutation that is associated to autosomal recessive deafness is a novel mutation found by this screening. 511G→A and 299-300delAT mutations contribute to autosomal dominant hearing loss. The study further supports the view that the common types of mutation in GJB2 according to different ethnic background and that the mutation prevalence in the East Asian deafness population is lower than that in the white population.     

bcl 10 gene mutation in hepatocellular carcinoma

Objective To detect the mutation frequency of the bcl 10 gene in the early and advanced st ages of hepatocellular carcinoma (HCC).Methods Genome DNA samples were extracted from 46 cases of fresh HCC tumor tissues and t heir non-tumor adjacent tissues. Polymerase chain reaction-single strand conf ormation polymorphism method was used to detect point mutations of t he three exons of the bcl 10 gene. For each individual exon, six random samples from those showing abnormal DNA bands were sequenced to verify those mutations . The relationship between serum alpha-fetoprotein (AFP) level and bcl 10 muta tion, between the tumor size and bcl 10 mutation was also analyzed.Results Among the 46 samples, 26 cases (56.5%) were found to have mutations in exon 1, 5 out of the 6 cases were shown to have 5744 C→G mutation by sequencing; 25 cas es (54.3%) were found to have mutations in exon 2, 4 out of the 6 cases were sh own to have 11 311 T deletion mutation by sequencing. Twenty-one cases (4 5.7%) were found to have mutations in exon 3, all of the 6 cases selected for sequenc ing were shown to have 14 116 C→T mutation. Statistical analysis showed t hat ne ither serum alpha-fetoprotein level nor the size of hepatocellular carcino ma has a significant relationship with bcl 10 mutation.Conclusion The bcl 10 gene has a high mutation frequency in liver cancer.     

R555W mutation of TGFβI related to granular corneal dystrophy in Chinese patients

Background Mutations in the transforming growth factor beta I （TGFBI） gene cause several types of autosomal-dominant corneal dystrophies. We investigated the role of this gene in a Chinese family affected by granular corneal dystrophy （GCD）.
Methods Family history and phenotypic data were recorded. The diagnosis of GCD was made on the basis of clinical evaluation. The genomic DNA was extracted from peripheral blood leukocytes. All the exons and flanking intron-exon boundary sequences of TGFβ1 were amplified by polymerase chain reaction （PCR） and screened for mutation by direct DNA sequencing.Results A heterozygous C to T transition at nucleotide c.1663 （CGG to TGG R555W） of TGFβ1 gene was present in two affected members but was absent in the rest of the family members. Conclusion A recurrent pathogenic R555W of TGFβ1 gene mutation is identified, which appears to be the predominant mutations causing GCD in different populations.     

A novel mis-sense mutation (G1381A) in the G6PD gene identified in a Chinese man

Objective　To detect new mutations among 29 glucose-6-phosphate dehydrogenase (G6PD) deficient individuals from Yunnan province. Methods　The nitroblue tetrazolium (NBT) method was used to screen G6PD deficient individuals. Mutation was identified by single strand conformation polymorphism (SSCP), amplification created restriction site (ACRS), amplification refractory mutation system (ARMS) and DNA sequencing. Results　Among 29 cases, 18 cases of G1388A, 1 case of C1004A, and 1 case of G1381A were identified. Nine cases remained to be defined. The G1381A mutation is a novel mis-sense mutation, with a substitution of threonine for alanine (A461T). The resultant G6PD had reduced enzymatic activity. In addition, G1381A caused a restriction site of Stu I to disappear, providing a rapid method for the detection of this mutation. Conclusion　A novel mis-sense mutation G1381A was found. This mutation results in a substitution of threonine for alanine, producing enzyme with reduced activity. The loss of the Stu I restriction site offers a rapid method for the detection of this mutation.     

Comparative study of mutation spectrums of MT-RNR1 m.1555A>G, GJB2, and SLC26A4 between familial and sporadic patients with nonsyndromic sensorineural hearing loss in Chinese Han

Background The mutation frequencies of three common deafness genes （MT-RNR1 m.1555A〉G,GJB2,and SLC26A4） among patients with nonsyndromic sensorineural hearing loss （NSHL） were different in previous studies.Inconsistent selection criteria for recruiting patients could have led to differences in estimating the frequencies of genetic mutations thus resulting in different mutation frequencies among these studies.The aim of this study was to reveal the differences in the mutation spectrums of the three common genes between familial and sporadic Chinese Han patients.Methods Totally,301 familial probands and 703 sporadic patients with NSHL were enrolled in this study.Three genes,MT-RNR1 m.1555A〉G,GJB2,and SLC26A4,were screened for mutation in our study cohort.A X2 test was performed to compare the mutation frequencies between the two groups.Results The study showed that the disease-causing mutation frequencies of MT-RNR1 m.1555A〉G,GJB2,and SLC26A4 were 12.29%,14.62%,and 18.27% in familial probands and 3.56%,18.63%,and 18.92% in sporadic patients,respectively.The mutation frequency of MT-RNR1 m.1555A〉G in familial probands was significantly higher than in sporadic patients （X2 test,P=0.000）,while there were no significant differences in the mutation frequencies of GJB2 and SLC26A4 between the familial and sporadic groups （X2 test,P 〉0.05）.Conclusions It is necessary to reveal the differences in gene mutation frequencies between patients of different sources or characteristics by comparative studies in order to avoid selection bias.The mutations of GJB2,SLC26A4,and MTRNR1 m.1555A〉G are the most important etiological factors in Chinese Han patients,among which SLC26A4 might be the most frequent.     

A novel CFTR mutation found in a Chinese patient with cystic fibrosis

Background Cystic fibrosis (CF) is rare in Chinese. We investigated the mutations in the gene of cystic fibrosis transmembrane conductance regulator ( CFTR ) in a Chinese CF patient and reviewed the clinical features, gene mutations in Chinese CF cases. Methods Blood samples were collected from a previously reported CF girl and her parents. The 24 coding exons of CFTR of the proband were amplified and sequenced. Results A Chinese girl of 16 years old was diagnosed as CF at the age of 14. She had recurrent productive cough with bronchiectasis in bilateral upper lobes, parasinusitis and otitis media, but without pancreatic involvement. Her sweat chloride was (108.9 ±3.3) mmol/L. A heterozygous novel missense mutation of 699 C → A which results in the amino acid change of N189K was identified in exon 5. In addition, a heterozygous 3821-3823 delT mutation in exon 19 was found in CFTR . The mutation 699C → A was inherited from her father, and the 3821-3823delT mutation was from her mother. Twenty patients with CF in Chinese reported from 1974 to 2004 were also reviewed. DelF508 mutation was not found in the nine cases whose CFTR mutations were analyzed. Conclusions The CF proband carries two heterozygous mutations (699C → A and 3821-3823delT) in CFTR . 699C → A mutation is a novel mutation which is not reported previously. Review of reported Chinese cases suggests that the genotype of Chinese CF may be different from those of white cases. More studies are needed to understand the spectra of CFTR and clinical CF features in Chinese.     

Relationship between mutations of mitochondrial DNA ND1 gene and type 2 diabetes

Background Recent studies have indicated that many mutations in mitochondrial (mt)DNA NDI gene region are related to diabetes mellitus. In this study we explored the relationship between various mtDNA ND1 gene mutations and type 2 diabetes mellitus (DM) among Chinese. Methods Using PCR restriction fragment length polymorphism (PCR-RFLP) analysis and gene sequencing, 4 spots of mtDNA (nt3243, nt3316, nt3394, nt3426) were screened in 478 diabetics and 430 non-diabetic subjects.Results In diabetic group, there were 13 carriers (2.72%)of 3316 G→A mutation,12 (2.51%) of 3394 T→C mutation and 2 (0.42%) of 3426A→G mutation. In controls, only 3394 T→C mutation was observed in 2 subjects (0.47%). There was significant difference in the frequency of 3316 and 3394 mutation between two groups (P<0.05, respectively). More subjects with mitochondrial DNA ND1 gene mutations had DM family history and greater tendency of maternal inheritance when compared to those patients without mutation in diabetic group（P<0.01）. A 3426 mutation diabetic pedigree was studied, and we found 12 maternal members in the family had the same mutation. Conclusion mtDNA ND1 gene mutations at nt3316 (G→A), nt3394 (T→C) and 3426 (A→G) might contribute to the pathogenesis of DM with other genetic factors and environment factors.     

Fluoroquinolone resistance and mutation patterns in <Emphasis Type="Italic">gyrA</Emphasis> and <Emphasis Type="Italic">parC</Emphasis> genes in <Emphasis Type="Italic">Neisseria gonorrhoeae</Emphasis> isolates from Shanghai,China

In order to study the resistance of Neisseria （N.） gonorrhoeae to the fluoroquinolone and detect mutation patterns of quinolone resistance-determining regions （QRDRs） of clinical isolates in Shanghai, China, a total of 80 clinical isolates of N. gonorrhoeae were consecutively collected from Shanghai. The MIC of fluoroquinolone for the isolates was examined by using the agar dilution method and the mutation profiles of the QRDRs of gyrA and parC were analyzed by sequencing and restriction fragment length polymorphism （RFLP）. Chi-square test was used for comparison of the t：nutation patterns. The results showed that： （1） High percentages of the 8 isolates were resistant to ciprofloxacin （95.0%）, ofloxacin （95.0%） and lomefloxacin （97.5%）, only one strain was susceptible to the ciprofloxacin. （2） Sensitive strains had a substitute of Asp95→Ala in the gyrA, and all isolates that were resistant or intermediated to the ciprofloxacin, had a double mutation in the gyrA （Ser91, Ala 92 and Asp95）. Some strains also had a mutation in the parC. （3） The MICs of these isolates were significantly associated with the mutation patterns in the gyrA and parC. A double mutation of gyrA combined with parC87 mutation was a predominant pattern in Shanghai and could mediate high level resistance to ciprofloxacin. It suggests that mutations in the QRDRs of gyrA and parC may be responsible for the fluoroquinolone resistance. And fluoroquinolone could not be used as the first line antibiotics for gonorrhea treatment any more in Shanghai, China.     

乳腺肿瘤发生与线粒体DNA体细胞突变的初步探讨

目的 基于线粒体DNA(mtDNA)的全基因组信息研究体细胞突变与乳腺肿瘤发生的相关性.方法 对来自云南省昆明市第一人民医院乳腺科2009年8-12月收治的4例乳腺纤维腺瘤患者的肿瘤组织及外周血mtDNA全基因组序列进行聚合酶链反应(PCR)扩增及DNA测序,比较其突变分布差异,揭示可能存在的肿瘤组织特有的体细胞突变.结果 研究获得了患者肿瘤组织mtDNA全基因组的突变图谱,并构建了系统发育关系树,结果揭示,4例患者的mtDNA分别归属为单倍型类群D4i、G1、R9b及N9a.通过在外周血mtDNA中检测肿瘤样本中发现的私有突变,研究显示仅在单倍型类群归属为R9b的患者中存在16292位置的体细胞变异.结论 基于4例良性乳腺肿瘤患者的肿瘤组织的mtDNA全基因组信息,仅发现1个mtDNA控制区体细胞变异,未能发现存在于mtDNA编码区中的可能具有功能性的体细胞突变.

Abstract：

Objective To investigate the correlation of somatic mutations in whole genome of mitochondrial DNA ( mtDNA ) in patients with breast tumor. Methods The DNA of tumor tissue and peripheral blood in 4 benign breast tumor patients from August 2009 to December 2009 in our hospital were extracted. The mtDNA whole genomes were amplified by polymerase chain reaction (PCR) and the mutations of products screened by sequencing to compare the difference of mutation distribution between tumor tissue and peripheral blood. The likelihood of somatic mutations in tumor tissue was determined. Results The mutation spectrum of mtDNA genomes was obtained by PGR and sequencing. Then a phylogenetic tree was constructed to identify their haplogroups (D4i, G1, R9b, N9a). Their private mutations in peripheral blood were detected and the somatic mutation at 16292 position was found in one patient ( haplogroups: R9b).Conclusion Based on the extensively study on the mtDNA genomes from the tumor issues of 4 patients, our current report observed only a single somatic variation from the control region, no any further mutations with potential function from the coding region were observed.     

Birth of healthy children after preimplantation diagnosis of β-thalassemia

Background Clinical programs for preventing β-thalassemia are presently based on prospective carrier screening and prenatal diagnosis. This paper report an achievement of a pregnancy with unaffected embryos using in vitro fertilization and embryo transfer (IVF-ET), in combination with preimplantation genetic diagnosis (PGD), for a couple at risk of having children with β-thalassemia.Methods A couple carrying different thalassemia mutations, both a codon 41-42 mutation and the IVS Ⅱ 654 mutation, received standard IVF treatment, with intracytoplasmic sperm injection, embryo biopsiy, single cell polymerase chain reaction (PCR) and DNA analysis. Only unaffected or carrier embryos were transferred to the uterine cavity. After confirmation of pregnancy, a prenatal diagnosis was performed.Results Of a total of 13 embryos analyzed for β-globin mutations, PGD indicated that 2 were normal,3 were affected, and 6 were carriers. Diagnosis could not be made in the other 2 embryos. Three embryos were transferred to the uterus on the third day after oocyte retrieval. Ultrasonography revealed a twin pregnancy with one blighted ovum. The prenatal genetic diagnosis revealed that both fetuses were unaffected, and two healthy boys were born, confirming the results of PGD.Conclusions We developed a single-cell based primer extension preamplification (PEP)-PCR assay for the detection of β-thalassemia mutations. The assays were efficient and accurate at all stages of the procedure, and resulted in the birth of PGD-confirmed β-thalassemia free children in China. PEP was used here in PGD for β-thalassemia.     

FBN1 mutation in Chinese patients with Marfan syndrome and its gene diagnosis using haplotype linkage analysis

Objectives To analyze the FBN1 mutations in Chinese patients with Marfan syndrome (MFS) and to make a genetic diagnosis based on haplotype linkage analysis for MFS.Methods Nine MFS families (17 patients) were analyzed with single strand conformation polymorphism (SSCP) and sequencing. Four primers were designed for the flanking sequences of FBN1 gene and used for haplotype-segregation analysis of MFS (B).Results SSCP band alteration was detected in the PCR products for exon 25 in MFS(A) Ⅱ : 1.Direct sequencing revealed a small 13 bp deletion; the deleted sequence is gccTcTgcaccca at bases 3243 -3456 of the cDNA in exon 25. This mutation was novel. MFS(B) families were analyzed using the haplotype linkage technique. The data suggested that MFS(B) families were linked to the FBN1 gene. The proband‘ s daughter was an asymptomatic patient.Conclusion The combination of mutation detection and chromosome haplotype analysis can provide better evidence for a genetic diagnosis of MFS.     

Clinical characteristics of patients with congenital long QT syndrome and bigenic mutations

Background Congenital long QT syndrome （LQTS） is an ion channelopathy associated with genetic mutations. It is well known that most LQTS patients （91%） have a single mutation. The purpose of this study was to investigate the clinical characteristics of congenital LQTS patients with bigenic mutations in Taiwan, China. Methods Congenital LQTS patients were recruited consecutively at Taiwan University Hospital in Taiwan from 2003 to 2009. The diagnosis of LQTS was defined by an LQTS Schwartz score greater than 4. Mutation screening in KCNQ1, KCNH2, KCNE1, and SCN5A was performed using direct sequencing. Results Three of 16 LQTS patients （18.7%） were identified with bigenic mutations. One patient had missense mutations in KCNQ1 and KCNH2, the second in KCNQ1 and KCNE1, and the third in KCNH2 and SCN5A. The mean age at onset of LQTS for patients with bigenic mutations was （17±3） years, and all of these patients were female. Two of them experienced seizure and one presented with syncope, although one of them had a family history of syncope. The mean QTc interval was （515＋17） ms, similar to those with single mutation or SNPs （（536~74） ms, P=-0.63）. Compared to those LQTS patients with single mutation or SNPs, a significantly higher percentage of LQTS patients with bigenic mutations presented with seizure and were younger at onset of the first index event （P=0.03 and 0.001, respectively）, but lower percentage of them presented with sudden cardiac death （P=0.03）. Conclusions Although the percentage of bigenic mutations in LQTS is less than 10% in Caucasian populations, we identified 3 of 16 LQTS patients （18.7%, 95% confidence interval： 0.04-0.46） with bigenic mutations in Taiwan. However, the sevedtv of their clinical presentations was not hioher than those patients with sinele mutation or SNPs.