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At least 217 records · Page 12Linked to original sources
PubMed1998

Systematic mutation screening of the pro-opiomelanocortin gene: identification of several genetic variants including three different insertions, one nonsense and two missense point mutations in probands of different weight extremes.

Pro-opiomelanocortin (POMC) is the precursor of melanocortins (adrenocorticotropin: ACTH, beta-endorphin, beta-lipotropin: beta-LPH, corticotropin like intermediate peptide, alpha-, beta- and gamma-melanocyte-stimulating hormone: alpha-, beta- and gamma-MSH) some of which act in the brain to reduce food intake and are potential mediators of leptin action. Recently, three different mutations in the POMC gene (POMC) were identified in two unrelated children that lead to early-onset extreme obesity, adrenal insufficiency, and red hair pigmentation. In the present study we systematically screened the coding region of POMC in 96 extremely obese children and adolescents, 60 healthy underweight individuals and 46 patients with anorexia nervosa (AN) and identified several variants. a) A 9 and an 18 base pair insertion (9bp and 18bp: AGC AGC GGC and AGC AGC GGC AGC AGC GGC, respectively, between codon 73 and 74; 1,2). These in-frame variants lead to the insertion of three or six amino acids (Ser-Ser-Gly; Ser-Ser-Gly-Ser-Ser-Gly) carboxy-terminal to gamma-MSH. Frequencies of the 9bp insertion allele varied between 3 and 5% among the different study groups (Pearson's chi2 P>0.5). b) Both an out-of-frame 6 bp insertion (within codon 176: GGG CCC) leading to the insertion of two amino acids (Arg-Ala) and a premature stop-codon (G-7316-T: Glu-180-Stop) within the gamma-LPH sequence were maternally inherited in an obese female proband. This proband inherited another missense mutation from her father (Glu-188-Gly). c) A missense mutation (G-7016-A; Asp-80-Asn) was observed in a single patient with AN who also harboured the 9bp insertion on a paternally derived haplotype. d) The allelic co-occurence of two silent mutations (C-6982-T and C-7285-T) was detected in two obese subjects. e) Two further silent mutations (C-3832-T; C-7111-G) were detected in an underweight and an obese subject, respectively. We conclude that the POMC gene harbors several different polymorphisms and mutations, none of which can readily be associated with the phenotypes under study.

Adolescent↗
PubMed1999

Hepatocyte nuclear factor-4gamma: cDNA sequence, gene organization, and mutation screening in early-onset autosomal-dominant type 2 diabetes.

The aim of this study was to investigate whether mutations in hepatocyte nuclear factor (HNF)-4gamma, a transcription factor homologous to HNF-4alpha, contribute to the etiology of early-onset type 2 diabetes. Linkage between diabetes and two polymorphic markers at the HNF-4gamma locus (D8S286 and D8S548) was evaluated in 32 multigenerational families with early-onset autosomal-dominant type 2 diabetes unlinked to known maturity-onset diabetes of the young genes. Total logarithm of odds (LOD) scores were strongly negative (-50.3 at D8S286 and -46.2 at D8S548), but linkage could not be excluded in 15 families having LOD scores >-2.0. To screen these pedigrees for HNF-4gamma mutations, the gene structure was defined. Because reverse transcriptase-polymerase chain reaction experiments indicated that the first 1,674 bp of the published cDNA sequence (3,248 bp) were a cloning artifact, the correct cDNA sequence was determined by 5' rapid amplification of cDNA ends (RACE) and primer extension assay. Based on the new cDNA sequence (1,731 bp), 11 exons were found. After screening the 5' flanking region and all coding exons for mutations, we identified several polymorphisms, one of which affected the amino acid sequence (M190I). However, no mutations segregating with diabetes could be found in these families. We conclude that genetic variability in the HNF-4gamma gene is unlikely to play a major role in the etiology of early-onset autosomal-dominant type 2 diabetes.

Base Sequence↗
PubMed2000

Molecular diagnosis of Stickler syndrome: a COL2A1 stop codon mutation screening strategy that is not compromised by mutant mRNA instability.

We have developed a novel strategy for screening families with type 1 Stickler syndrome due to COL2A1 nonsense mutations, using a modified RNA-based protein truncation test. To overcome the problem of the unavailability of collagen II-producing cartilage cells, reverse transcription polymerase chain reaction (RT-PCR) was performed on the illegitimate transcripts of accessible cells (lymphoblasts and fibroblasts), which were pre-incubated with cycloheximide to prevent nonsense-mutation-induced mRNA decay. The five overlapping RT-PCR fragments covering the COL2A1 coding region were then transcribed and translated in vitro to identify smaller truncated protein products which result from a premature stop codon. This method was used to screen a 4-generation Stickler family and a protein truncating mutation was identified, which was present in all affected individuals. Targeted sequencing identified the mutation as a G(+1) to A substitution at the 5' splice donor site of intron 25, which led to the activation of a cryptic splice site 8-bp upstream causing aberrant mRNA splicing and a translational frameshift that introduced a premature stop codon. Mutant mRNA was undetectable without cycloheximide protection, demonstrating that the mutant mRNA was subjected to nonsense-mediated mRNA decay. As well as providing further evidence that type 1 Stickler syndrome results from COL2A1 premature stop codon mutations, this study suggests mutant mRNA instability leading to haploinsufficiency may also be an important, but previously unrecognized, molecular basis of Stickler syndrome. This rapid new test for COL2A1 nonsense mutations is of particular clinical importance to Stickler syndrome families, where the identification of individuals who are at risk of this potentially preventable form of blindness will allow them to undergo regular ophthalmological surveillance and preventative or early ameliorative treatment.

Abnormalities, Multiple↗
PubMed2001

Routine mutation screening of HNF-1alpha and GCK genes in MODY diagnosis: how effective are the techniques of DHPLC and direct sequencing used in combination?

AIMS/HYPOTHESIS: Mutations in the hepatocyte nuclear factor (HNF)-1alpha and glucokinase (GCK) genes are the major causes of monogenic forms of Type II (non-insulin-dependent) diabetes mellitus (Maturity-Onset Diabetes of the Young subtypes, MODY). We evaluated the effectiveness of fluorescent single-strand conformation polymorphism (F-SSCP), denaturing high-performance liquid chromatography (DHPLC) and sequencing based mutation detection in the molecular diagnosis of MODY. Our goal is to identify a rapid, efficient and cost effective mutation detection method for the molecular diagnosis of MODY and other human genetic disorders. METHODS: We evaluated the accuracy of DHPLC in screening for MODY 2 and 3 mutations. In addition, we compared the sensitivity, specificity, cost, handling time and analysis time of fluorescent single-strand conformation polymorphism, denaturing high-performance liquid chromatography and direct sequencing screening methods. RESULTS: Denaturing high-performance liquid chromatography is a recently developed method for mutation detection. It is cost effective, powerful and reliable and quite suitable for 22 out of the 24 fragments required for MODY 2 and 3 testing. However, exons 1 and 7 of the HNF-1alpha gene are very polymorphic and so direct sequencing is faster as well as more efficient and reliable. CONCLUSION/INTERPRETATION: Our results suggest that combining denaturing high-performance liquid chromatography and direct sequencing is a good approach for the routine detection of HNF-1alpha and GCK mutations in MODY families. Denaturing high-performance liquid chromatography appears to be a powerful tool in genetic testing and the method could be applied to the molecular diagnosis of other human genetic diseases.

Base Sequence↗
PubMed2004

Mutational screening of SCN5A linked disorders in Polish patients and their family members.

Mutations in SCN5A lead to a broad spectrum of phenotypes, including the Long QT syndrome, Brugada syndrome, Idiopathic ventricular fibrillation (IVF), Sudden infant death syndrome (SIDS) (probably regarded as a form of LQT3), Sudden unexplained nocturnal death syndrome (SUNDS) and isolated progressive cardiac conduction defect (PCCD) (Lev-Lenegre disease). Brugada Syndrome (BS) is a form of idiopathic ventricular fibrillation characterized by the right bundle-branch block pattern and ST elevation (STE) in the right precordial leads of the ECG. Mutations of the cardiac sodium channel SCN5A cause the disorder, and an implantable cardioverter-defibrillator is often recommended for affected individuals. In this study sequences of the coding region of the SCN5A gene were analysed in patients with the LQT3, Brugada Syndrome and other arrythmogenic disorders. Different mSSCP patterns are described with no disease-related SSCP conformers in any sample. Direct sequencing of the SCN5A gene confirmed the absence of mutations. This suggests that the analysed region of the SCN5A gene is not commonly involved in the pathogenesis of the Brugada Syndrome and associated disorders.

Adult↗
PubMed1999

Allele loss and mutation screen at the Peutz-Jeghers (LKB1) locus (19p13.3) in sporadic ovarian tumours.

Germline mutations in the LKB1 (STK11) gene (chromosome sub-band 19p13.3) cause characteristic hamartomas and pigmentation to develop in patients with Peutz-Jeghers syndrome. Peutz-Jeghers syndrome carries an overall risk of cancer that may be up to 20 times that of the general population and Peutz-Jeghers patients are at increased risk of benign and malignant ovarian tumours, particularly granulosa cell tumours. Loss of heterozygosity (allele loss, LOH) has been reported in about 50% of ovarian cancers on 19p13.3. LKB1 is therefore a candidate tumour suppressor gene for sporadic ovarian tumours. We found allele loss at the marker D19S886 (19p13.3) in 12 of 49 (24%) sporadic ovarian adenocarcinomas. Using SSCP analysis, we screened ten ovarian cancers with LOH, 35 other ovarian cancers and 12 granulosa cell tumours of the ovary for somatic mutations in LKB1. No variants were detected in any of the adenocarcinomas. Two mutations were detected in one of the granulosa cell tumours: a mis-sense mutation affecting the putative 'start' codon (ATG --> ACG, M1T); and a silent change in exon 7 (CTT --> CTA, leucine). Like BRCA1 and BRCA2, therefore, it appears that LKB1 mutations can cause ovarian tumours when present in the germline, but occur rarely in the soma. The allele loss on 19p13.3 in ovarian cancers almost certainly targets a different gene from LKB1.

AMP-Activated Protein Kinase Kinases↗
PubMed2001

Factor V Leiden mutation screened by PCR and detected with lanthanide-labeled probes.

The Factor V Leiden mutation is an important human polymorphism, responsible for increased risk of venous thrombosis in heterozygotes as well as homozygotes. Therefore, screening is a useful possibility, and many detection systems have been described for PCR products. We have developed a simplified and robust assay using oligonucleotide probes for normal and mutant sequences, labeled with europium and samarium, respectively, and measured by time-resolved fluorescence. Populations consisting of 233 Welsh and 148 Irish subjects were examined by both restriction fragment length polymorphism (RFLP) analysis and our assay. The allele frequency was 14/466 in the Welsh and 5/296 in the Irish population, in line with other surveys of European populations. Results were not obtained in 2/381 samples by RFLP, compared with 1/381 with our method. We conclude that our method represents an improved system capable of considerable throughput at reasonable cost.

Factor V↗
PubMed1999

Mutation screening of the entire coding regions of the TSC1 and the TSC2 gene with the protein truncation test (PTT) identifies frequent splicing defects.

Mutation analyses in tuberous sclerosis (TSC) have reported a wide variety of disease-causing aberrations in the two known predisposing genes, TSC1 and TSC2 on chromosomes 9q34 and 16p13, comprising mainly small mutations distributed over the entire genes. So far, all known TSC1 mutations as well as the majority of TSC2 mutations truncate the proteins hamartin and tuberin, respectively. We describe for the first time an RNA-based screening of the entire coding regions of both TSC genes for truncating mutations applying the protein truncation test (PTT). Simultaneous investigation of both TSC genes in a group of 48 unassigned TSC patients, which were previously tested to exclude large intragenic TSC2 rearrangements, revealed aberrant migrating polypeptides resulting from truncating mutations in nine TSC1 cases and in 16 TSC2 cases while three TSC2 cases showed enlarged proteins. TSC1 mutations include two nonsense mutations, four insertions, and three splice mutations. Nineteen mutations identified in TSC2 were composed of four different nonsense mutations in five patients, one deletion, one insertion, and seven different splicing aberrations due to at least eight different mutations found in 12 patients. Additional predicted truncating mutations according to PTT without possible identification of the causative alteration allowed assignment to TSC1 in one and TSC2 in seven cases. Twelve patients without abnormalities in the PTT are assumed to harbor missense mutations, probably in TSC2. The high proportion of TSC2 splicing aberrations strengthens the importance of intronic disease-causing mutations and the application of RNA-based screening methods to confirm their consequences.

Adolescent↗
PubMed1998

A systematic mutation screen of 10 nuclear and 25 mitochondrial candidate genes in 21 patients with cytochrome c oxidase (COX) deficiency shows tRNA(Ser)(UCN) mutations in a subgroup with syndromal encephalopathy.

COX deficiency is believed to be the most common defect in neonates and infants with mitochondrial diseases. To explore the causes of this group of disorders, we examined 25 mitochondrial genes (three COX subunit genes and 22 tRNA genes) and 10 nuclear COX subunit genes for disease associated mutations using PCR-SSCP and direct sequencing of polymorphic SSCP fragments. DNA from one patient with severe COX deficiency and with consanguineous parents was entirely sequenced. The patient population consisted of 21 unrelated index patients with mitochondrial disorders and predominant (n=7) or isolated (n=14) COX deficiency. We detected two distinct tRNA(Ser)(UCN) mutations, which have been recently described in single kindreds, in a subgroup of four patients with COX deficiency, deafness, myoclonic epilepsy, ataxia, and mental retardation. Besides a number of nucleotide variants, a single novel missense mutation, which may contribute to the disease phenotype, was found in the mitochondrial encoded COX 1 gene (G6480A). Mutations in nuclear encoded COX subunit genes were not detected in this study.

Adolescent↗
PubMed2000

Mutation screening in Rett syndrome patients.

Rett syndrome (RTT) was first described in 1966. Its biological and genetic foundations were not clear until recently when Amir et al reported that mutations in the MECP2 gene were detected in around 50% of RTT patients. In this study, we have screened the MECP2 gene for mutations in our RTT material, including nine familial cases (19 Rett girls) and 59 sporadic cases. A total of 27 sporadic RTT patients were found to have mutations in the MECP2 gene, but no mutations were identified in our RTT families. In order to address the possibility of further X chromosomal or autosomal genetic factors in RTT, we evaluated six candidate genes for RTT selected on clinical, pathological, and genetic grounds: UBE1 (human ubiquitin activating enzyme E1, located in chromosome Xp11.23), UBE2I (ubiquitin conjugating enzyme E2I, homologous to yeast UBC9, chromosome 16p13.3), GdX (ubiquitin-like protein, chromosome Xq28), SOX3 (SRY related HMG box gene 3, chromosome Xq26-q27), GABRA3 (gamma-aminobutyric acid type A receptor alpha3 subunit, chromosome Xq28), and CDR2 (cerebellar degeneration related autoantigen 2, chromosome 16p12-p13.1). No mutations were detected in the coding regions of these six genes in 10 affected subjects and, therefore, alterations in the amino acid sequences of the encoded proteins can be excluded as having a causative role in RTT. Furthermore, gene expression of MECP2, GdX, GABRA3, and L1CAM (L1 cell adhesion molecule) was also investigated by in situ hybridisation. No gross differences were observed in neurones of several brain regions between normal controls and Rett patients.

Adolescent↗
PubMed2001

Prenatal diagnosis of respiratory chain deficiency by direct mutation screening.

Respiratory chain deficiency (RCD) is responsible for a clinically heterogeneous group of early-onset untreatable disorders. Enzymological prenatal diagnosis (PD) can only be offered to a fraction of families. Moreover, due to the two-fold genetic origin of the respiratory chain (nuclear and mitochondrial DNA) and owing to the large number of nuclear genes involved in the respiratory chain assembly, maintenance and functioning, the identification of the disease causing gene in a given family remains challenging. Here, we report on PD of RCD by direct screening of NDUFV1, SDH-Fp, SCO1 and SURF1 mutations in five unrelated families with complex I, II and IV deficiency, respectively. The identification of the disease-causing gene in a given family with RCD is a major issue to provide both adequate genetic counselling and early, reliable PD.

Electron Transport↗
PubMed1995

PKU in Slovakia: mutation screening and haplotype analysis.

The restriction fragment length polymorphism haplotypes and seven common mutations in the phenylalanine hydroxylase gene were analysed in 49 unrelated Slovak phenylketonuria (PKU) families of Caucasian origin. The predominant mutation in this population sample is R408W, with a frequency of 45.9%. In addition, four other mutations have been identified at relatively high frequencies: IVS12nt1, 10.2%; R158Q, 7.1%; R261Q, 7.1%; R252W, 2.0%. The mutation-haplotype associations correspond to those described in other European populations. The high proportion of mutations (72.4%) amenable to simple rapid detection based on the polymerase chain reaction provides a good basis for direct DNA-diagnosis of PKU in the Slovak population.

Haplotypes↗
PubMed2000

Genomic structure and mutation screening of the E2F4 gene in human tumors.

E2F4, a member of the E2F family of transcription factors, is abundant in non-proliferating and differentiated cells where it plays an important role in the suppression of proliferation-associated genes. The E2F4 gene spans 6 kb and has 10 exons. It contains a serine (CAG) repeat tract in exon 7, which is unstable in gastrointestinal tumors. To further investigate a possible role of this gene in tumorigenesis we performed mutational analysis and expression studies in different tumors. Primary human tumor tissue of the stomach, colon, breast and lung (28), metastatic tumors of the colon (3) and small cell lung tumor cell lines (18) were screened for somatic mutations in the coding region of E2F4. No mutation was found. Microsatellite instability of the CAG repeat, however, was documented in primary stomach and colon tumors. Northern blot analysis revealed upregulated E2F4 transcript levels in tumor cell lines. Our data suggest that a direct involvement of E2F4 in tumorigenesis is unlikely, although increased E2F4 expression may be associated with human cancer.

Base Sequence↗
PubMed2005

[Mutation screening of GCK gene in Chinese early-onset diabetes population].

OBJECTIVE: To investigate the prevalence of mutations and sequence variations of glucokinase gene GCK in Chinese early-onset diabetes population. METHODS: The study was conducted in 174 unrelated Chinese residents, including 80 nondiabetic controls, 94 probands of early-onset diabetes pedigree. Direct sequencing was performed to screen all 10 exons of glucokinase gene, including promoter and exon/intron junctions. RESULTS: No mutations were identified in coding region, but several previously reported sequence variants were identified. 5'-untranslated region of exon 1a, 84 bp upstream of the translation initiation site GGCGG to GGGGG(early-onset diabetes group G allele frequency 0.106 vs control group 0.075, P=0.355); IVS1b+12 (A-->T) (early-onset diabetes group T allele frequency 0.005 vs non-identity of this variation in control group); IVS 5+29 (G-->T) (early-onset diabetes group T allele frequency 0.027 vs control group 0.019, P=0.731); IVS 9+8 (T-->C) (early-onset diabetes group C allele frequency 0.585 vs 0.694, P=0.044). A novel variation IVS 9+49 (G-->A) (early-onset diabetes group A allele frequency 0.011 vs control 0.006, P=1.000) was identified. There were no significant relationships of the exon 1a 5'-untransted region -84 bp(C-->G), IVS 5+29 (G-->T), IVS 9+8 (T-->C) and IVS 9+49 (G-->A) variants of GCK gene to the clinical variables such as plasma glucose, insulin, C-peptide and fasting lipid profile. CONCLUSION: The prevalence of structural mutations in glucokinase gene responsible for early-onset diabetes appears to be rare among Chinese patients.

Adult↗
PubMed1998

Mutation screening of the dopamine D1 receptor gene in Tourette's syndrome and alcohol dependent patients.

We report a single stranded conformational polymorphism (SSCP) analysis of the coding region of the dopamine D1 receptor (DRD1) in Tourette's syndrome (n = 50) and control (n = 50) subjects. Tourette's syndrome populations with comorbidity for attention deficit-hyperactivity disorder (AD-HD) (n = 35) and obsessive compulsive disorder (OCD) (n = 30) were also screened. As a related study, we also screened patients diagnosed with alcohol dependence (n = 72). The present study discovered no DRD1 coding region mutations in any of the Tourette's syndrome or alcohol dependent patients. One silent mutation, a C for a T at Ile49, was discovered in one control subject. The non-polymorphic structure of the DRD1 gene among the Tourette's syndrome, Tourette's syndrome comorbid with AD-HD and OCD and the alcohol dependent populations screened by SSCP suggests that coding region mutations of the DRD1 gene are unlikely to contribute to the inheritance of these disorders.

Alcoholism↗
PubMed2004

Mutation screen of the glutamate decarboxylase-67 gene and haplotype association to unipolar depression.

Abnormally low concentrations of brain gamma-aminobutyric acid (GABA) have been reported in unipolar depression. Almost all of the brain GABA is synthesized by glutamate decarboxylase (GAD) enzymes (GAD67 and GAD65). These enzymes, therefore, play a central role in brain GABA homeostasis. We screened all the 17 exons of the GAD67 gene for mutations using single strand conformation polymorphism (SSCP) or denaturing high pressure liquid chromatography (dHPLC) in a sample of 43 individuals diagnosed with major unipolar depression or other disorders with putative GABAergic dysfunction. We identified eight novel variants (five synonymous base substitutions, two insertion/deletions and one tandem repeat). Three relatively common (minor allele frequency >20%) single nucleotide polymorphisms (SNPs), located in the 5' non-coding region (exon 0), intron 8, and the 3' non-coding region (exon 16) of the gene, were genotyped in 103 European-American (EA) subjects with depression and 125 EA psychiatrically screened controls. Linkage disequilibrium (LD) and haplotype frequencies were estimated using the 3LOCUS program. Significant LD was observed between the intron 8 SNP and the exon 16 SNP and between the exon 0 SNP and the exon 16 SNP. Three common GAD67 haplotypes were observed in this population, which accounted for >90% of the possible GAD67 three-locus haplotypes. Comparison of SNP and haplotype frequencies between individuals with depression and controls revealed no differences. These results demonstrate a significant within-gene LD for GAD67 in the EA population and begin to establish a haplotype map for this gene. Furthermore, these results suggest that common genetic variation within the GAD67 gene does not play a major role in the predisposition to unipolar depression.

DNA↗
PubMed2004

Mutation screening of the phosducin gene PDC in patients with retinitis pigmentosa and allied diseases.

PURPOSE: To search for a phenotype associated with mutations in the phosducin gene PDC. METHODS: We screened 853 patients with retinitis pigmentosa or an allied disease diseases, including groups of 61 to 212 patients, each with dominant retinitis pigmentosa (RP), recessive RP, Leber congenital amaurosis, or cone-rod degeneration, for mutations in the PDC gene using direct genomic sequencing of the three coding exons and their flanking intron splice sites. RESULTS: We found one polymorphism in the 5' untranslated region (minor allele frequency of 0.149) and three rare single-base sequence variants (one missense change, one isocoding change, and one in the 3' untranslated region). The rare variants were found in one heterozygous patient each and none was interpreted as pathogenic. CONCLUSIONS: Phosducin mutations are not a major cause of dominant or recessive RP, Leber congenital amaurosis, or cone-rod degeneration. The human phenotype associated with phosducin defects remains unknown.

DNA Mutational Analysis↗
PubMed2005

COMP mutation screening as an aid for the clinical diagnosis and counselling of patients with a suspected diagnosis of pseudoachondroplasia or multiple epiphyseal dysplasia.

The skeletal dysplasias are a clinically and genetically heterogeneous group of conditions affecting the development of the osseous skeleton and fall into the category of rare genetic diseases in which the diagnosis can be difficult for the nonexpert. Two such diseases are pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED), which result in varying degrees of short stature, joint pain and stiffness and often resulting in early onset osteoarthritis. PSACH and some forms of MED result from mutations in the cartilage oligomeric matrix protein (COMP) gene and to aid the clinical diagnosis and counselling of patients with a suspected diagnosis of PSACH or MED, we developed an efficient and accurate molecular diagnostic service for the COMP gene. In a 36-month period, 100 families were screened for a mutation in COMP and we identified disease-causing mutations in 78% of PSACH families and 36% of MED families. Furthermore, in several of these families, the identification of a disease-causing mutation provided information that was immediately used to direct reproductive decision-making.

Adolescent↗