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PubMed2006

[Human pot1 gene exon12 mutation screening in cultured human carcinoma cell strains (lines)].

OBJECTIVE: To screen the exon12 mutation of pot1 gene in cultured human carcinoma cell strains (lines). METHODS: The chromosomal DNA was extracted from 27 cultured carcinoma cell strains (lines). The exon 12 of pot1 gene was amplified by PCR, and the product was purified and screened. The screening results were compared with the data of GenBank and NCBI and the exon 12 mutations in cultured human carcinoma cell strains (lines) analyzed. RESULTS: The exon12 sequence of pot1 could be specifically amplified using the designed primers. Direct sequence analysis of the PCR products after purification showed that 4 of the 5 carcinoma cell lines of the female genital system such as Hela and HO8910-PM cells shared the same transition (G17722-->C) in exon12 of human pot1 gene resulting in a conversion of G1385-->C in the cDNA and amino acid change of Leu454-->Phe in the translated polypeptide. The rest of the 23 cell strains (lines) from different origins showed no such mutation. CONCLUSION: The exon12 (17,722 bp) is a mutant region specific for female genital system tumor.

Amino Acid Sequence↗
PubMed1999

Evaluation of a mutation screening strategy for sporadic cases of ATR-X syndrome.

We report on the evaluation of a strategy for screening for XNP/ATR-X mutations in males with mental retardation and associated dysmorphology. Because nearly half of the mutations in this gene reported to date fall into a short 300 bp region of the transcript, we decided to focus in this region and to extend the mutation analysis to cases with a negative family history. This study includes 21 mentally retarded male patients selected because they had severe mental retardation and a typical facial appearance. The presence of haemoglobin H or urogenital abnormalities was not considered critical for inclusion in this study. We have identified six mutations which represents a mutation detection rate of 28%. This figure is high enough for us to propose this strategy as a valid first level of screening in a selected subset of males with mental retardation. This approach is simple, does not require RNA preparation, does not involve time consuming mutation detection methods, and can thus be applied to a large number of patients at a low cost in any given laboratory.

Adolescent↗
PubMed2004

Mutation screening of X-chromosomal neuroligin genes: no mutations in 196 autism probands.

Autism, a childhood neuropsychiatric disorder with a strong genetic component, is currently the focus of considerable attention within the field of human genetics as well many other medical-related disciplines. A recent study has implicated two X-chromosomal neuroligin genes, NLGN3 and NLGN4, as having an etiological role in autism, having identified a frameshift mutation in one gene and a substitution mutation in the other, segregating in multiplex autism spectrum families (Jamain et al. [2003: Nat Genet 34:27-29]). The function of neuroligin as a trigger for synapse formation would suggest that such mutations would likely result in some form of pathological manifestation. Our own study, screening a larger sample of 196 autism probands, failed to identify any mutations that would affect the coding regions of these genes. Our findings suggest that mutations in these two genes are infrequent in autism.

Autistic Disorder↗
PubMed2006

Mutational screening of phospholamban gene in hypertrophic and idiopathic dilated cardiomyopathy and functional study of the PLN -42 C>G mutation.

BACKGROUND: Phospholamban is an endogenous sarcoplasmic reticulum calcium ATPase inhibitor with a regulatory effect on cardiac contraction/relaxation coupling. Mutations in the phospholamban gene (PLN) have been associated with primary cardiomyopathies. AIMS: To screen for PLN mutations in our population of patients with primary cardiomyopathies and to perform functional analysis of the mutations identified. METHODS: We performed SSCP mutational screening and DNA sequencing of the PLN gene in 186 patients with either hypertrophic or dilated cardiomyopathy. To study promoter strength we constructed reporter plasmids containing the luciferase gene and performed transient transfection analysis in C6 and C2C12 cell lines. RESULTS: The PLN -42 C>G mutation was found in one patient with late onset familial apical hypertrophic cardiomyopathy. This mutation decreased phospholamban promoter activity by 43% and 47%, in C6 and C2C12 cell lines respectively. One son had mild apical hypertrophic cardiomyopathy and carried the mutation, another son with normal ECG and echocardiogram also had the mutation. CONCLUSION: The PLN -42 C>G mutation is associated with a benign form of apical hypertrophic cardiomyopathy in this family, though the presence of a healthy adult carrier suggests that other genetic and environmental factors could be involved. Otherwise, mutations in the PLN gene are not a frequent cause of cardiomyopathies in our population.

Calcium-Binding Proteins↗
PubMed2006

BRCA1 and BRCA2 mutation screening using SmartCycler II high-resolution melt curve analysis.

CONTEXT: Real-time polymerase chain reaction technologies have replaced many of the more labor-intense methods in the molecular diagnostics laboratory. Similarly, melt curve analysis can provide a rapid means of mutation screening. OBJECTIVE: To determine if real-time polymerase chain reaction and melt curve analysis using the SmartCycler II could be used as a screening tool for 3 common mutations in BRCA1 and BRCA2. DESIGN: Real-time polymerase chain reaction amplification with SYBR Green I detection was performed on DNA from cell lines known to carry the 185delAG or 5382insC mutation in BRCA1 or the 6174delT mutation in BRCA2. The melting temperatures and the melt curves were analyzed for differences between wild-type DNA and cell lines that were heterozygous for each mutation. RESULTS: Significant differences were present in the melt curves for each of the mutations compared with those of the wild-type sequences. The melt curve for the 185delAG mutation showed a separate peak at a lower temperature, which represented the melting temperature of the heteroduplex. For the 6174delT mutation, the melt curve had a shoulder at a lower temperature, while the melt curve for the 5382insC mutation was shifted to the left and was broader than that for the wild-type sequences. CONCLUSIONS: High-resolution melt curve analysis is a quick, reliable method for identifying mutations due to small deletions or insertions. As a proof of principle, we used this assay to identify the 3 most common BRCA1 and BRCA2 mutations in the Ashkenazi Jewish population.

Cell Line, Tumor↗
PubMed1995

Point mutation screening for 16 exons of the dystrophin gene by multiplex single-strand conformation polymorphism analysis.

We have developed a rapid and nonradioactive method to screen for point mutations using the Pharmacia PhastSystem. In an SSCP analysis, we applied the two multiplex exon PCR kits, commonly used for the detection of deletions in Duchenne and Becker muscular dystrophy patients. The different exon bands in the multiplex SSCP pattern could be identified by running well-characterised deletion patients in this system. Two common polymorphisms were easily identifiable and are helpful in the haplotype analysis in families. Screening of 70 patients in which no gross rearrangement was detectable with the multiplex PCR and Southern blot, resulted in the identification of 6 patients with a band shift after SSCP analysis. Of these 6 band shifts, 5 were the result of a frame shift or termination mutation. The other band shift was found to be a rare polymorphism unlikely to be the cause of the patient's phenotype. Application of this technique enabled us to improve diagnosis in the families involved and will allow us to extend the search for point mutations in the remaining exons of the dystrophin gene.

Blotting, Southern↗
PubMed2003

Evaluation of multiplex capillary heteroduplex analysis: a rapid and sensitive mutation screening technique.

Bardet-Biedl syndrome (BBS) is a heterogeneous disease; to date seven loci have been mapped and five identified (BBS1, BBS2, BBS4, BBS6, and BBS7). Inheritance in some families is complex with multiallelic participation making linkage analysis difficult. Previous mutation screens have been carried out by direct sequencing but with an increasing number of patients to be screened for five relatively large genes, a more rapid and cost-effective mutation assay for BBS was required. We have adapted the technique of heteroduplex analysis for use on the MegaBACE 1000, a capillary-based DNA fragment analyser, to improve the resolution and sensitivity of the system. Twelve known alterations (insertions, deletions, missenses, and SNPs) in BBS1, BBS2, BBS4, and BBS6 were used to test the sensitivity of the assay and subsequently used to screen new patients for mutations. We achieved a 100% detection rate while dramatically increasing the sample throughput by virtue of multiplexing up to six PCR products in each capillary. In addition, four novel variants were identified: two in BBS2 [c.522T>A (p.D174E) and c.805-20A>G] and two in BBS4 [c.332+27_28insA and c.1414A>G (p.M472V)]. Compared with sequencing and alternative screening methods, multiplex capillary heteroduplex analysis (MCHA) is extremely cost effective. Hum Mutat 22:151-157, 2003.

Bardet-Biedl Syndrome↗
PubMed1999

Fibroblast growth factor receptor mutational screening in newborns affected by metopic synostosis.

A number of craniosynostotic disorders have recently been ascribed to mutations in genes coding for the fibroblast growth factor receptors(FGFRs). The common feature of these FGFR-associated conditions is the unilateral or bilateral premature ossification of the coronal suture. One distinct craniosynostotic condition is trigonocephaly, which results from the premature fusion of the metopic suture. Trigonocephaly mostly occurs as isolated cranial defect; however, the premature closure of the metopic suture may represent a feature of more complex craniosynostotic conditions in which a progressive involvement of other cranial sutures with age is observed. The possible involvement of mutated FGFRs in trigonocephaly was investigated in nine newborns affected by isolated premature synostosis of the metopic suture. All except one of these cases carried no mutations in the FGFR1-3 domains indicated as hot spots for craniosynostosis-associated mutations. A T(978)C transition in the FGFR2 exon IIIa was found in a patient who had a phenotype that apparently fitted the trigonocephalic condition at birth, but showed additional facial anomalies, which worsened progressively with age towards a Crouzon-like profile. The present finding points out the importance, from both diagnostic and prognostic points of view, of early FGFR mutational screening in craniosynostotic conditions, even in forms that apparently do not involve closure of the coronal suture at birth.

Craniofacial Abnormalities↗
PubMed2001

Mutational screening of the cationic trypsinogen gene in a large cohort of subjects with idiopathic chronic pancreatitis.

Several missense mutations, including R122H, N29I, K23R, A16V and D22G, in the cationic trypsinogen gene (PRSS1), have been associated with certain forms of hereditary pancreatitis (HP). Their occurrence in the idiopathic chronic pancreatitis (ICP) and whether novel mutations could be identified in PRSS1 remain to be further evaluated. These were addressed by the mutational screening of the entire coding sequence and the intronic/exonic boundaries of the PRSS1 gene in 221 ICP subjects, using a previously established denaturing gradient gel electrophoresis technique. Among the known PRSS1 mutations, only the R122H was detected in a single subject and the A16V in two subjects in the cohort, strengthening that HP-associated PRSS1 mutations are rare in ICP. Additional missense mutations, including P36R, E79K, G83E, K92N and V123M, were identified once separately. By analogy with the known PRSS1 mutations, predisposition to pancreatitis by some of them, particularly the V123M autolysis cleavage site mutation, is suspected. Functional analysis is expected to clarify their possible medical consequences.

Amino Acid Sequence↗
PubMed1999

Mutational-screening in the factor VIII gene resulting in the identification of three novel mutations, one of which is a donor splice mutation. Mutations in brief no. 245. Online.

Hemophilia A is an X-linked bleeding disease caused by mutations in the coagulation factor VIII gene. The identification and characterization of pathogenic mutations allows the recognition of new mechanisms of functional disturbances of factor VIII. To screen for mutations exons 1-26 of the factor VIII gene have been amplified genomically and analyzed by SSCP followed by direct sequencing of respective exons showing abnormal electrophoretic mobility on SSCP analysis. In the present study we report the detection of four mutations in the factor VIII gene, of which three are novel. The mutational analysis of a patient with severe hemophilia A has revealed that the ac transversion at position 3 of the donor-splice-site of intron 23 results in the skipping of exon 23. A novel nonsense mutation Q1778X in exon 16 of factor VIII gene has been identified in a second hemophilia A case. Furthermore two missense mutations have been ascertained: a novel, S183R, causing a mild phenotype of hemophilia A and R282H, previously described in association with severe hemophilia A.

Exons↗
PubMed2003

Gene structure, chromosomal localization, and mutation screening of the human gene for the inner ear protein otospiralin.

Otospiralin is a novel protein of unknown function that is produced by non-sensory cells (fibrocytes) of the inner ear (cochlea and vestibule). We showed that downregulation of otospiralin in guinea pigs leads to deafness and we therefore hypothesized that genetic defects in the otospiralin gene could also cause deafness in humans. In this study, we cloned and localized OTOSP, the human gene for otospiralin. OTOSP spans 1630 nucleotides, contains four exons and codes for a 567-nucleotide cDNA. By fluorescence in situ hybridization and hybrid panel mapping we localized OTOSP on chromosome 2 at position q37.3. There is currently no deafness family linked to this region. We screened OTOSP for mutations in 410 unrelated patients exhibiting various levels of hearing loss. Beside intronic polymorphisms, a rare variant (Pro7Leu) was found in 4 deafness patients and 3 control individuals, indicating that this change is not involved in this condition and excluding OTOSP as a major gene for genetic deafness.

Amino Acid Substitution↗
PubMed1995

A quality control study of CFTR mutation screening in 40 different European laboratories. The European Concerted Action on Cystic Fibrosis.

A quality control study was performed to determine the accuracy of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) mutation screening in 40 different genetic screening laboratories throughout Europe. A total of 9 different samples were investigated blindly by the participating laboratories. Only 25/40 laboratories, i.e. 62.5%, were able to type all samples correctly for the mutations for which they routinely screened. Only 2 of the 9 samples were correctly typed in all 40 laboratories. The lowest accuracy rate was 80% for 1 sample. 12.5% of the participating laboratories interpreted the F508C polymorphism as a true CF disease mutation and 23.5% interpreted the delta I507 mutation as a delta F508 mutation. For the delta F508 mutation, a false-negative result of 3.75% was obtained. It is clear that the accuracy of CFTR typing should be improved.

Cystic Fibrosis Transmembrane Conductance Regulato↗
PubMed2003

BRCA1/2 mutation screening and LOH analysis of lung adenocarcinoma tissue in a multiple-cancer patient with a strong family history of breast cancer.

BACKGROUND: Germline mutations in BRCA1/2 greatly elevate risks of breast and ovarian cancers, but the role of these genes in tumourigenesis of other cancer types is still being investigated. OBJECTIVE: We report on an investigation of BRCA1/2 mutations and their loss of heterozygosity (LOH) in a patient with a strong family history of breast cancer who was diagnosed with consecutive primary cervical, ovarian and lung carcinomas. METHODS AND RESULTS: BRCA1/2 mutation screening of the proband revealed a common familial breast- and ovarian cancer-associated germline BRCA2 mutation (3034del4bp). We then performed LOH analysis for BRCA2 in lung adenocarcinoma tissue of the patient. Using the laser-capture microdissection (LCM) technique, we obtained pure populations of neoplastic cells from which DNA could be extracted. Mutation analysis by denaturing high-performance liquid chromatography (DHPLC) and direct sequencing revealed loss of the mutant allele in the adenocarcinoma tumour tissue. CONCLUSION: To our knowledge, this is the first report of investigation for LOH for BRCA2 in primary lung adenocarcinoma tissue of a patient with multiple primary tumours related to a familial germline BRCA2 mutation. Interestingly, it was the mutant, not the wild-type, allele which was lost in the lung adenocarcinoma tissue.

Journal Article↗
PubMed2005

Mutation screening in dilated cardiomyopathy: prominent role of the beta myosin heavy chain gene.

AIMS: Familial dilated cardiomyopathy (FDCM) is associated with mutations in more than 10 genes, but genes mutation frequencies and associated clinical features remain largely unknown. Here, we performed a mutation analysis of four genes involved in FDCM in a population of idiopathic DCM. METHODS AND RESULTS: A SSCP and sequencing mutation screening of all the exons coding for beta myosin heavy chain (MYH7 gene), cardiac T troponin (TNNT2 gene), phospholamban (PLN gene), and the cardio-specific exon of metavinculin (VCL gene) were performed in 96 independent patients (54 familial and 42 sporadic). It led to the identification of eight heterozygous mutations, seven new ones in MYH7, and the already described R141W mutation in TNNT2. MYH7 mutations (in five familial and two sporadic cases) substitute residues located either in the head (I201T, T412N, A550V) or tail domains (T1019N, R1193S, E1426K, R1634S) of the protein. DCM was not associated with skeletal myopathy or conduction defects in any patients. Contrasting clinical features were observed between MYH7 and TNNT2 mutations carriers. In MYH7 vs. TNNT2, mean age at diagnosis was late (P<0.03), penetrance was incomplete in adults (56 vs. 100%), and mean age at major cardiac event was higher (P<0.04). CONCLUSION: We have identified seven mutations in MYH7, one in TNNT2, and none in PLN or in the VCL cardio-specific exon. MYH7 appears as the most frequently mutated gene in our FDCM population (approximately 10%), and mutation carriers present with delayed onset, in contrast to TNNT2.

Adult↗
PubMed2004

[Clinical features and hMSH2/hMLH1 germline mutation screening of Chinese hereditary nonpolyposis colorectal cancer patients].

OBJECTIVE: To analyze the clinical features of hereditary nonpolyposis colorectal cancer (HNPCC) among Chinese and report the results of screening of hMSH2 and hMLH1 gene mutations. METHODS: The data concerning sex, site of colorectal cancer (CRC), age of diagnosis, history of synchronous and/or metachronous colorectal cancer, instance of extracolonic cancers, and histopathology of tumors of 126 patients from 28 independent families of HNPCC in China were collected, of which 15 met the Amsterdam criteriaI and 13 met the Japanese clinical diagnosis criteria. The genomic DNA was extracted from the peripheral lymphocytes. Polymerase chain reaction (PCR) and denaturing high-performance liquid chromatography (DHPLC) were used to screen the coding region of hMSH2 and hMLH1 genes. Samples showing abnormal DHPLC profiles were sequenced by a 377 DNA sequencer. RESULTS: One hundred and seventy malignant neoplasms were found in the 126 patients, in which 23 of multiple cancers were found. Ninety-eight of the 126 patients (77.8%) had colorectal cancers, with an average age of onset of 45.9 years and a right-sided predominance. Eight hMSH2 or hMLH1 gene sequence variations were found in 12 families, and a germline G204X nonsense mutation in the third exon of hMSH2 was found for the first time, the first mismatch repair gene (MMR) mutation ever found in Chinese Mongolian people. CONCLUSIONS: HNPCC is a typical auto-dominant hereditary disease, characterized by early onset, proximal predominance of colorectal cancer, multiple synchronous and metachronous colorectal cancers, and an excess of extra-colonic cancers. Frequent gastric cancer occurrence and less synchronous colorectal cancers are notable features in Chinese HNPCC patients. DHPLC is a powerful tool in hMSH2 and hMLH1 gene mutation screening. Three novel mutations have been found. hMLH1 gene mutations, especially those of the first nine exons, are more common than hMSH2 gene mutations in Chinese patients.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Mutation screening of the LDLR gene and ApoB gene in patients with a phenotype of familial hypercholesterolemia and normal values in a functional LDL receptor/apolipoprotein B assay.

Mutations in the LDL receptor (LDLR) or the apolipoprotein B-100 genes causing familial hypercholesterolemia (FH) and familial defective apolipoprotein B-100 (FDB), two of the most frequent inherited diseases, are the underlying genetic defects in a small proportion of patients suffering from premature atherosclerotic heart disease. Consequently, secure diagnostic tools for these conditions allowing early preventive measures are needed. Since clinical and biochemical diagnosis often is inaccurate, assays analyzing patient LDLR function and LDL affinity have been established. These assays are, however, not able clearly to differentiate between suspected FH/FDB samples and normal controls. To evaluate if this may be caused by other hitherto undescribed genetic defects or to failure of the functional assays, we undertook denaturing gradient gel electrophoresis based mutation screening of the LDLR gene and the codon 3456 3553 region of the apolipoprotein B gene in six French FH/FDB patients with normal outcomes on functional assays. In all six patients, pathogenic LDLR mutations were found, including three previously undescribed mutations, suggesting that failure of the functional assays explains the normal results found in some phenotypic FH/FDB patients and illustrating the need for DNA based screening techniques for routine genetic diagnosis in FH/FDB.

Apolipoprotein B-100↗
PubMed2005

Concomitant neutrophil JAK2 mutation screening and PRV-1 expression analysis in myeloproliferative disorders and secondary polycythaemia.

Polycythaemia vera (PV) is closely associated with both an acquired activating mutation of the JAK2 tyrosine kinase (JAK2(V617F)) in granulocyte-derived DNA and increased granulocyte polycythaemia rubra vera-1 (PRV-1) expression. In order to explore the correlation between these two biological markers and compare their diagnostic utility, mutation analysis for JAK2(V617F) and quantitative measurement of granulocyte PRV-1 expression were performed on the same study sample from 100 participants: 38 with PV, 22 with essential thrombocythaemia (ET), 10 with agnogenic myeloid metaplasia (AMM), 19 with secondary polycythaemia (SP) and 11 healthy volunteers. The respective overall (homozygous) JAK2(V617F) mutational frequencies were 95% (26%), 55% (0%), 30% (0%), 0% and 0%. The corresponding figures for increased PRV-1 expression were 89%, 18%, 20%, 21% and 9%. In patients with either ET or AMM, the likelihood of detecting JAK2(V617F) was significantly higher in the presence of an increased PRV-1 expression (83% vs. 38%; P = 0.05). Similarly, in patients with PV, homozygous as compared with heterozygous JAK2(V617F) correlated with higher levels of PRV-1 expression (P = 0.11). The present study suggests an allele dose-dependent effect of JAK2(V617F) on granulocyte PRV-1 expression. However, compared with the PRV-1 assay, mutation screening for JAK2(V617F) displayed greater accuracy in distinguishing PV from SP.

Adolescent↗
PubMed2005

Identification of three novel mutations in Japanese patients with Menkes disease and mutation screening by denaturing high performance liquid chromatography.

BACKGROUND: Menkes disease is an X-linked recessive disorder resulting in a connective-tissue disturbance and profound neurodegeneration in early childhood. The gene for Menkes disease has been isolated and predicted to code for copper transporting ATPase. In this study, a mutation analysis in Japanese patients with Menkes disease was performed, as was a mutation screening by denaturing high performance liquid chromatography (DHPLC). METHODS: A mutation analysis on five Japanese patients with Menkes disease was performed using a direct sequencing method and DHPLC. RESULTS: Two nonsense mutations, two missense mutations and one splice donor site mutation were found. The DHPLC analysis showed differences in the peaks between the DNA fragments of wild type and heteroduplex (wild type and mutant). CONCLUSIONS: Three novel mutations (Asp1044Gly, Pro1279Leu and IVS21+1 g to a) were detected. The Asp1044Gly mutation destroys the highly conserved phosphorylation domain in exon 16. The splice site abnormality leads to a skipping of exon 21 coding for part of the seventh transmembrane domain. These two mutations could cause a severe protein dysfunction. Another missense mutation, Pro1279Leu, in exon 20 was found in a patient with a mild type of Menkes disease. It is speculated that this mutation partially maintains the ATP7A function is. A DHPLC analysis could detect these mutations. It is concluded that the best way to make a molecular diagnosis for Menkes disease is to first screen DNA samples for all exons using DHPLC, and thereafter perform direct sequencing for exons which have an abnormal elution profile in order to rapidly detect such mutations.

Case-Control Studies↗