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PubMed2005

Mutation analysis of the SDHD gene in four kindreds with familial paraganglioma: description of one novel germline mutation.

The familial paraganglioma syndrome is an autosomal dominant disorder characterized by the presence of carotid body paragangliomas and, less frequently, paragangliomas of the glomus jugulare, glomus vagale, and adrenal pheochromocytomas. Germline mutations of the genes for succinate dehydrogenase subunits D, B, or C (SDHD, SDHB, SDHC) have been identified in some kindreds with familial paraganglioma. In this study, we report the clinicopathologic features of four different kindreds with familial paraganglioma, which were screened for germline mutations in the SDHD gene. DNA was obtained from tumor and normal tissue, as well as from peripheral blood. Mutation analysis was performed by single-strand conformation polymorphism analysis and DNA sequencing. SDHD germline mutations were detected in the affected family members of the four families, as well as in several asymptomatic carriers. An identical mutation in exon 4 of SDHD (334-337delACTG) was identified in two apparently unrelated kindreds. The third family showed a germline mutation in exon 2 (W43X). The mutations present in these three families had been previously described in Spanish families, suggesting a founder effect. The fourth family exhibited a mutation in exon 2 of SDHD (170-171delTT), which had not been previously identified. The affected family members of the four kindreds showed paragangliomas, located in the head and neck region, and all of them were benign. These results confirm that genetic testing of SDHD may be a powerful tool for the identification of the syndrome in patients with multiple or bilateral paragangliomas.

DNA Mutational Analysis↗

PubMed1998

GNAS1 mutational analysis in pseudohypoparathyroidism.

OBJECTIVE: Mutations of the GNAS1 gene, which is located on chromosome 20q13.11 and encodes the alpha-subunit of the stimulatory GTP-binding protein, have been identified in patients with pseudohypoparathyroidism type Ia (PHPIa) and pseudopseudohypoparathyroidism (PPHP). We have undertaken studies to determine the prevalence of GNAS1 mutations and to explore methods for their more rapid detection. METHODS: Thirteen unrelated families (8 with PHPIa and PPHP patients, and 5 with PPHP patients only) were investigated for GNAS1 mutations in the 1050 base-pair (bp) region spanning exons 2-13 by single-stranded conformational polymorphism (SSCP) and DNA sequence analysis. RESULTS: GNAS1 mutations were detected in 4 of the 8 families with PHPIa patients. These consisted of: two novel de novo missense mutations (Pro115Ser and Glu259Val) in two families and an identical 4 bp deletion of codons 189 and 190 resulting in a frame-shift in two unrelated families. These results expand the spectrum of GNAS1 mutations associated with this disorder and confirm the presence of a mutational hot-spot involving codons 189 and 190. SSCP analysis was found to be a specific and sensitive method that detected all 4 mutations. GNAS1 mutations were not detected in any of the PPHP only families. CONCLUSIONS: The pseudohypoparathyroid disorders appear to represent a heterogeneous group with GNAS1 mutations forming the molecular aetiology in approximately 50% of pseudohypoparathyroidism type Ia families. Such mutations can be reliably identified by single-stranded conformational polymorphism and this will help to supplement the clinical evaluation of some patients and their families, particularly as the disease may not be fully penetrant.

Adolescent↗

PubMed1999

Mutation analysis in patients of Mediterranean descent with Wilson disease: identification of 19 novel mutations.

In this study, we report further results of mutation analysis of the ATP7B gene in Wilson disease (WD) patients of Mediterranean origin. A total of 136 WD chromosomes, 73 of which were of Italian, 43 of Turkish, 18 of Sardinian, and two of Spanish origin, were analysed and the mutation characterised in 84.5% of them. We found 50 different mutations of which 19 are novel, including three nonsense, one frameshift, and 15 missense mutations. The mutations detected were rare and mostly found in the compound heterozygous state together with other mutations and only rarely in homozygosity. Most of these mutations lie in the transmembrane and ATP binding loop regions. These data expand our knowledge of both the structure-function relationships of the WD protein and the molecular pathology of WD, thus improving our capability of prevention and genetic counselling.

Adenosine Triphosphatases↗

PubMed2004

Mutation analysis of KIF21A in congenital fibrosis of the extraocular muscles (CFEOM) patients.

PURPOSE: CFEOM type 1 refers to a group of congenital eye movement disorders that is characterized by nonprogressive ophthalmoplegia affecting all the extraocular muscles. Individuals with the classic form of CFEOM are born with bilateral ptosis, infraducted eyes, and impossibility to raise their eyes above midline. This phenotype is often inherited as an autosomal dominant trait. CFEOM1 maps to the FEOM1 locus on chromosome 12 and is the consequence of mutations in the KIF21A gene. We analyzed three families and one sporadic case for potential genetic heterogeneity. METHODS: Blood samples were collected from members of three families (Swiss, Turkish, and French origin) and one sporadic case (Iranian origin). In families, haplotype was tested for linkage to the autosomal dominant CFEOM1 locus on chromosome 12. Linkage studies were conducted using 2 polymorphic DNA microsatellite markers, D12S331 and D12S1048. Mutation analysis was performed by PCR amplification and bidirectional direct sequencing. RESULTS: Haplotype analysis was compatible with linkage to the CFEOM1 locus in all affected members. Mutation analysis revealed the classical mutation R954W in all affected cases, including the sporadic case, regardless of their ethnic origin. The c.2860C>T base change was not observed in 100 individuals from various ethnic origins. CONCLUSIONS: As reported, the classical c.2860C>T mutation represents a hotspot for mutation in various ethnic groups, including Swiss, Turkish, French, and Iranian patients. Sporadic cases are often due to neo-mutations as in our case. Mutation analysis is important, especially in sporadic cases, to correctly evaluate recurrence and transmission risks.

Chromosomes, Human, Pair 12↗

PubMed2006

[Clinical analysis of dopa-responsive dystonia and mutation analysis of the GCH I gene].

OBJECTIVE: To investigate the clinical characteristics and GCH I gene mutations in patients with dopa-responsive dystonia (DRD). METHODS: The clinical features of 3 families with 6 affected members and 8 sporadic cases were analyzed to determine the clinical characteristics, and 2 families with 4 affected members and 2 sporadic cases were screened for mutations of the GCH I gene. RESULTS: Age at onset was (10 +/- 3) years. Onset occurred earlier in female (9 +/- 4) years than in male (12 +/- 1) years. The initial symptom was a gait disorder, dystonia or tremor in most patients and nine patients (64%) presented with diurnal fluctuation. Thirteen patients (93%) were cured and one was improved after administration of low doses of levodopa for 3 months and no long-term side effects of levodopa had occurred. Two independent mutations were found in three patients. Gln161Pro, a new missense mutation, was found in a sporadic case, leading to a relatively severe phenotype. The two patients with mild phenotype in one family were found to have Lys224Arg mutation, as previously described. CONCLUSIONS: DRD patients have diverse phenotypes and diurnal fluctuation is an important feature. They have dramatic and sustained response to levodopa. There may be a correlation between genotype and phenotype. The detection of GCH I mutations is helpful in early diagnosis of non-typical cases.

Age of Onset↗

PubMed2005

VSX1 mutational analysis in a series of Italian patients affected by keratoconus: detection of a novel mutation.

PURPOSE: Keratoconus is a noninflammatory corneal disorder that is clinically and genetically heterogeneous. Mutations in the VSX1 (visual system homeobox 1) gene have been identified for two distinct, inherited corneal dystrophies: posterior polymorphous corneal dystrophy and keratoconus. To evaluate the possible role of the VSX1 gene in a series of Italian patients, 80 keratoconus-affected subjects were screened for mutations. METHODS: The diagnosis of keratoconus was made on the basis of clinical examination and corneal topography. The whole coding region and the exon-intron junctions of the VSX1 gene were analyzed by direct sequencing. RESULTS: Three already-described changes, D144E, G160D, and P247R, and a novel L17P mutation were found in 7 of 80 unrelated patients (8.7%). Two undescribed intronic polymorphisms are also reported. CONCLUSIONS: Mutational analysis of the VSX1 gene in a series of Italian patients revealed one novel mutation and confirmed an important role played by this gene in a significant proportion of patients affected by keratoconus, when it is inherited as an autosomal dominant trait with variable expressivity and incomplete penetrance.

Adolescent↗

PubMed2005

Mutation analysis of TBX22 reveals new mutation in Tunisian CPX family.

Cleft palate with ankyloglossia (CPX; OMIM 303400) is inherited as a Mendelian semidominant X-Linked disorder. Linkage studies resulted in mapping CPX to Xq13-q 21-31 region. TBX22 was identified as causing CPX. We report a new mutation in a Tunisian family and the first Arab family with X-Linked cleft palate and ankyloglossia. The family includes 6 affected members, 4 males and 2 females. Linkage study was performed using 9 microsatellite markers surrounding the CPX locus with a maximum lod score 1.81 at theta=0 with several markers. Sequence analysis of TBX22 gene revealed a novel change c.358C>T in exon 3 (R120W) located in the T-BOX domain; this change was present in all affected members and none of the 100 controls. A second modification in exon 4 (c.559G>A) predicted to result in a nonconservative substitution (E187 K) was present in the affected members but also in 2 controls, suggesting a polymorphism which functional role cannot be excluded without further study.

Cleft Palate↗

PubMed1999

Utility of RET mutation analysis in multiple endocrine neoplasia type 2.

OBJECTIVE: To review the role of RET mutation analysis in the diagnosis of multiple endocrine neoplasia type 2 (MEN 2) and in presymptomatic screening for this disorder. DATA SOURCES: Review of the medical literature and current clinical practice. CONCLUSIONS: RET mutation analysis is a sensitive and specific test for MEN 2. It plays a pivotal role in the diagnosis and management of patients and families with MEN 2 and in the individual who presents with an apparently sporadic medullary thyroid carcinoma or pheochromocytoma. These disorders may first come to the attention of either the anatomic or clinical pathologist, who has the opportunity to see that appropriate testing is done. As with any familial disease, professional genetic counseling is an important part of the care of these patients.

Adrenal Gland Neoplasms↗

PubMed1999

Mutation analysis in adenylosuccinate lyase deficiency: eight novel mutations in the re-evaluated full ADSL coding sequence.

The deficiency of adenylosuccinate lyase (ADSL, also termed adenylosuccinase) is an autosomal recessive disorder characterized by the accumulation in body fluids of succinylaminoimidazole-carboxamide riboside (SAICA-riboside) and succinyladenosine (S-Ado). Most ADSL-deficient children display marked psychomotor delay, often accompanied by epilepsy or autistic features, or both, although some patients may be less profoundly retarded. Occasionally, growth retardation and muscular wasting are also present. Up to now, nine missense mutations of the ADSL gene had been reported in six apparently unrelated sibships. In the present study of 10 additional patients with ADSL deficiency, nine point mutations, among which seven unreported missense mutations, and the first splicing error reported in this disorder, have been identified. These mutations have been characterized, taking into account the finding that the cDNA of human ADSL is 75 nucleotides longer at its 5'-end, and encodes a protein of 484 rather than 459 amino acids as previously reported. Five apparently unrelated patients were found to carry a R426H mutation. With the exceptions of the latter mutation, of a R190Q mutation that had been reported previously, and of a K246E mutation that was found in two unrelated patients, all other mutations were found only in a single family.

Adenylosuccinate Lyase↗

PubMed2000

Molecular staging of colorectal cancer: K-ras mutation analysis of lymph nodes upstages Dukes B patients.

PURPOSE: Multiple attempts have been made to improve the clinical/pathologic staging system of Dukes to focus adjuvant therapy decisions. The purpose of this study was to determine whether K-ras mutational status of regional nodes in patients with Dukes B2 colorectal cancer could be used to stage their disease more accurately. METHODS: Using formalin-fixed, paraffin-embedded archival material, tumor samples were screened for K-ras mutations using a mutation-specific polymerase chain reaction method, followed by gel electrophoresis in a 96-well array. Patients with Dukes B2 tumors that have mutations in codon 12 or 13 of the K-ras gene were identified. RESULTS: Mutational analysis of the lymph nodes from these patients revealed an 80 percent (16/20) incidence of the same mutations in regional lymph nodes. None of the four patients with mutation-free nodes developed recurrence compared with 37.5 percent (6/16) with K-ras positive lymph nodes. CONCLUSIONS: The data suggest that patients with Dukes B2 colorectal cancers that have mutations in codon 12 or 13 of the K-ras gene are at high risk for the development of nodal metastases. Mutational analysis of the lymph nodes identifies high-risk patients who should be considered for adjuvant chemotherapy. Therefore, K-ras mutational analysis should be considered for molecular staging of colorectal cancer.

Adenocarcinoma↗

PubMed2006

Mutational analysis of RPGR and RP2 genes in Japanese patients with retinitis pigmentosa: identification of four mutations.

PURPOSE: To identify mutations in RPGR and RP2 genes in a series of Japanese retinitis pigmentosa (RP) families and to determine the association between the phenotypic changes in patients/carriers and the mutations. METHODS: A total of 37 unrelated RP families were recruited, three of which were with typical X-linked RP (XLRP), and other 34 families included 29 multiplex families and 5 simplex RP cases with no family history of RP. In addition, At least one RP patient had myopia >-3.0D in these families. RPGR and RP2 genes were comprehensively screened by using the direct polymerase chain reaction-sequencing method. Detailed phenotypes of the families with confirmed mutations were assessed by routine ophthalmic examinations, Goldmann perimetry, electroretinography and color fundus photography. RESULTS: Four mutations in RPGR and RP2 genes were identified. Of the three XLRP families, one had an ORF15 mutation and another had an RP2 mutation. Two ORF15 mutations were found in three of the other 34 RP families, with two families sharing a same mutation, g.ORF15+652-653delAG. All the three ORF15 mutations were first reported in the Japanese population. Affected males showed relatively severe symptoms while female carriers showed a wide spectrum of severity. A tapetal-like reflex was observed in two young females, indicating clinically the carrier status. CONCLUSIONS: We identified three ORF15 mutations and one RP2 mutation in five Japanese RP families. Moderate or severe myopia might be an indicator for the XLRP status in multiplex RP families which pedigree data are insufficient to allow accurate subtyping. It is suggested that mutational analysis of RPGR and RP2 may help to identify the causative mutation in a proportion of multiplex RP patients with myopia.

Adult↗

PubMed2005

Mutation analysis of POUF-1, PROP-1 and HESX-1 show low frequency of mutations in children with sporadic forms of combined pituitary hormone deficiency and septo-optic dysplasia.

OBJECTIVES: Mutations in the genes encoding the transcription factors PROP1 and POUF-1 (Pit-1) have been reported as common causes of combined pituitary hormone deficiency (CPHD), and HESX1 mutations have been identified in children with septo-optic dysplasia (SOD). There are few data on UK children. We have performed mutation analysis in a large cohort of affected children within the West Midlands region to assess the feasibility of a screening strategy for molecular diagnosis in CPHD and SOD. DESIGN AND PATIENTS: The three coding exons of PROP1, and six exons of POUF-1 in 27 children from 26 families with CPHD, and three exons of HESX1 in 23 children from 22 families with SOD were directly sequenced from a well-characterized regional cohort. RESULTS: We identified a C to T transition in exon 6 of POUF-1, resulting in a known missense mutation (R271W) in a mother and daughter from one family with CPHD. We also found a novel homozygous T to C transition in exon 6 of POUF-1, resulting in a missense mutation (F233L) in a twin with CPHD. This mutation was excluded in 100 ethnically matched control alleles. We did not identify any mutations in the PROP1 gene or HESX1. The median maternal age at delivery for the CPHD children was 27 years, compared to 21 years for the mothers of SOD children (P = 0.04). CONCLUSIONS: Mutations in POUF-1, PROP1 and HESX1 are rare causes of CPHD and SOD, respectively, in children from the West Midlands. In particular, we did not confirm the reported 'hotspot' in PROP1. A screening strategy that targets familial cases is highly likely to increase the mutation yield. The young maternal age at conception of children with SOD and potential teratogen exposure indicate the predominance of environmental factors in this condition compared with CPHD.

Child↗

PubMed2002

Molecular analysis, mutation screening, and association study of adenylate cyclase type 9 gene (ADCY9) in mood disorders.

Chromosome 16p13 has been shown to display modest linkage signals for mood disorders in a number of studies. An interesting candidate gene in this region is the adenylate cyclase (AC) type 9 gene (ADCY9). ACs are critical in neuronal signaling, and perturbation of brain AC activity has been reported in mood disorder postmortem brains. ACs may also act as targets of antidepressants. Two distinct length transcripts for the ADCY9 gene have been reported, but molecular mechanisms are unknown. To investigate the potential role of ADCY9 in mood disorders, we clarified alternative poly(A) sites for the two mRNA species, delineated the exon-intron structure, and screened the gene for genetic variants. The two transcripts encoded by ADCY9 shared the first 10 exons, but exon 11 was shorter in one of the mRNA species. Seven single nucleotide polymorphisms, including a missense mutation and one polymorphic microsatellite repeat in the 3'-UTR, were identified. However, a case-control study using the missense polymorphism, 2316A>G (Ile772Met), and the tetranucleotide repeat (TTTA)n showed no significant association with mood disorders in Japanese samples. The DNA polymorphisms detected in this study can be tested in other ethnic samples and/or other psychiatric diseases.

Adenylyl Cyclases↗

PubMed1984

Control of pT181 replication II. Mutational analysis.

We describe the isolation and analysis of mutations affecting the regulation of Staphylococcus aureus plasmid pT181 replication. Previous results suggested that regulation is achieved by control of the synthesis of RepC, a plasmid-coded replication protein and that the primary negative control element is CopA RNA, which consists of two transcripts that are complementary to the 5' region of the repC mRNA leader. CopA inhibition probably involves a base pairing interaction with the complementary region of the RepC mRNA leader which would facilitate the formation of a downstream stem-loop in the leader that occludes the repC ribosome binding site. RepC is freely diffusible so that regulation of pT181 replication is indirect. Both CopA RNA-sensitive (recessive) and -insensitive (dominant) mutants were isolated. The recessives have defects in CopA RNA structure or activity, the dominants have defects in the site of action (target) of the inhibitor. Some dominants were located within the copA coding sequence. These therefore affect the structure of CopA RNA as well as that of its target. Other dominant mutations mapped outside of the copA gene and therefore produced wild-type CopA RNA. In contrast to directly regulated plasmids, pT181 copy mutants producing wild-type inhibitor could be co-maintained with the wild-type plasmid and mutational changes in inhibitor-target specificity did not change incompatibility specificity.

Bacillus subtilis↗

PubMed1998

Structure and mutation analysis of the glycogen storage disease type 1b gene.

Glycogen storage disease (GSD) 1b is the deficiency of endoplasmic reticulum glucose-6-phosphate (G6P) transport. We here report the structure of the gene encoding a protein likely to be responsible for G6P transport, and its mapping to human chromosome 11q23.3. The gene is composed of nine exons spanning a genomic region of approximately 4 kb. Primers based on the genomic sequence were used in single strand conformation polymorphism (SSCP) analysis and mutations were found in six out of seven GSD 1b patients analysed.

Antiporters↗

PubMed1998

Epithelial p53 gene expression and mutational analysis, combined with growth fraction assessment, in oral lichen planus.

The immunohistochemical detection of epithelial p53 protein expression in oral lichen planus (OLP) biopsies was supplemented with molecular analysis for mutations of the p53 gene using the polymerase chain reaction - single stranded conformational polymorphism (PCR-SSCP) technique. p53 protein expression, in the basal epithelial cell layer, as detected by the DO7 and 1801 antibodies, was significantly more frequent in OLP compared with other oral keratoses and normal mucosa, as was the growth fraction. The 10 OLP biopsies that had the most frequent p53 staining (plus a case of OLP found in continuity with a SCC) were screened by the PCR-SSCP technique, but no mutations were detected in the p53 gene (exons 5 9). The p53 overexpression in the OLP samples may be a physiological response to the hyper-proliferative state, as revealed by the growth fraction determination. This may usefully serve to protect against mutagenesis, and so be a factor in the low incidence of carcinoma associated with OLP.

Adult↗

PubMed1992

The role of the N terminus in Tet repressor for tet operator binding determined by a mutational analysis.

The N-terminal residues preceding the alpha-helix-turn-alpha-helix motif on the Tn10 Tet repressor protein were probed by oligonucleotide-directed deletion mutagenesis for their role in protein activity. All deletion mutants showed decreased repression in vivo, emphasizing the importance of the N terminus for tet operator binding. Only two of the mutants, TetR delta 2-23 and TetR delta 3-8 displayed a reduced intracellular protein level. The remaining deletion mutants showed either reduced binding to tet operator and inducibility by tetracycline or transdominance. We conclude that these deletions do not affect stability and overall protein structure. DNA binding activities of residue-wise increasing deletions, TetR delta 9 through TetR delta 9-13, reveal a pattern consistent with an alpha-helical structure of the affected residues. This conclusion is supported by the helical wheel projection and the hydrophobic moment profile calculated for the protein segment ranging from residues S7-V20. We propose that these residues form an amphipathic alpha-helix which packs closely against the alpha-helix-turn-alpha-helix motif and is essential for Tet repressor activity. The residues preceding this putative alpha-helix contribute to DNA binding, but no direct interactions with base pairs of tet operator were revealed in a loss of contact analysis. Individual mutation of the 4 charged residues to alanine at the N terminus shows that no single residue can account for the reduction in repression observed for the deletion mutants.

Alleles↗

PubMed2001

Mutation analysis in mitochondrial fatty acid oxidation defects: Exemplified by acyl-CoA dehydrogenase deficiencies, with special focus on genotype-phenotype relationship.

Mutation analysis of metabolic disorders, such as the fatty acid oxidation defects, offers an additional, and often superior, tool for specific diagnosis compared to traditional enzymatic assays. With the advancement of the structural part of the Human Genome Project and the creation of mutation databases, procedures for convenient and reliable genetic analyses are being developed. The most straightforward application of mutation analysis is to specific diagnoses in suspected patients, particularly in the context of family studies and for prenatal/preimplantation analysis. In addition, from these practical uses emerges the possibility to study genotype-phenotype relationships and investigate the molecular pathogenesis resulting from specific mutations or groups of mutations. In the present review we summarize current knowledge regarding genotype-phenotype relationships in three disorders of mitochondrial fatty acid oxidation: very-long chain acyl-CoA dehydrogenase (VLCAD, also ACADVL), medium-chain acyl-CoA dehydrogenase (MCAD, also ACADM), and short-chain acyl-CoA dehydrogenase (SCAD, also ACADS) deficiencies. On the basis of this knowledge we discuss current understanding of the structural implications of mutation type, as well as the modulating effect of the mitochondrial protein quality control systems, composed of molecular chaperones and intracellular proteases. We propose that the unraveling of the genetic and cellular determinants of the modulating effects of protein quality control systems may help to assess the balance between genetic and environmental factors in the clinical expression of a given mutation. The realization that the effect of the monogene, such as disease-causing mutations in the VLCAD, MCAD, and SCAD genes, may be modified by variations in other genes presages the need for profile analyses of additional genetic variations. The rapid development of mutation detection systems, such as the chip technologies, makes such profile analyses feasible. However, it remains to be seen to what extent mutation analysis will be used for diagnosis of fatty acid oxidation defects and other metabolic disorders.

Acyl-CoA Dehydrogenase↗