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At least 631 records · Page 35Linked to original sources
PubMed2000

Screening for mutations of Bcl10 in leukaemia.

The Bcl10 gene was identified through characterization of the t(1;14)(p22;q32) associated with mucosa-associated lymphoid tissue (MALT) lymphoma. Bcl10 is implicated in the regulation of apoptosis and has been reported to be mutated in other subtypes of non-Hodgkin's B-cell lymphoma (B-NHL) and leukaemic cell lines, raising the possibility that its deregulation could be implicated in other forms of haematological malignancy. We screened 226 cases, including 123 acute myeloid leukaemia (AML), 50 acute lymphoblastic leukaemia (ALL), 20 chronic myeloid leukaemia (CML), 10 chronic lymphocytic leukaemia-prolymphocytic leukaemia (CLL/PLL) and 23 cases with 1p abnormalities, for Bcl10 mutations by reverse transcription polymerase chain reaction-single-stranded conformation polymorphism (RT-PCR/SSCP). Three known polymorphisms and two common splice variants were identified; however, no mutations were detected. One splice variant led to a 33-bp in frame deletion, whereas the other caused a 16-bp deletion predicting C-terminal truncation of Bcl10. However, both splice variants were also detected in normal bone marrow, suggesting that they are unlikely to be of pathogenetic significance. Furthermore, Southern blot analysis revealed no rearrangements of Bcl10 among 16 ALL and 11 cases of haematological malignancy with 1p abnormalities. Our results suggest that mutation of the Bcl10 gene as a mechanism of tumorigenesis is not associated with leukaemia.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Autosomal dominant pattern dystrophy: identification of a novel splice site mutation in the peripherin/RDS gene.

PURPOSE: To describe the clinical features of and identify the mutation responsible for an autosomal dominant pattern dystrophy occurring in a three-generation family. METHODS: Five affected family members underwent clinical examination and additional testing including intravenous fluorescein angiography where indicated. Mutation screening of the peripherin/RDS gene was performed. RESULTS: Visual acuity ranged from 20/20 to counting fingers. All patients who reported vision loss noted the onset after the age of 40 years. Predominantly perifoveal, discrete, retinal pigment epithelial changes were present in all patients. Two patients had extensive peripheral yellowish flecks, and one had an atrophic macular scar. Mutation screening of the complete peripherin/RDS coding sequence and exon/intron boundaries revealed a novel splice site mutation. CONCLUSION: A three-generation family with an autosomal dominant pattern dystrophy arising from a previously unreported splice site mutation in the RDS gene is described.

Adult↗
PubMed2005

Detection of an MEN1 gene mutation depends on clinical features and supports current referral criteria for diagnostic molecular genetic testing.

OBJECTIVE: Diagnostic molecular genetic testing for multiple endocrine neoplasia type 1 (MEN1) has been available since the identification of the MEN1 gene in 1997. Mutation screening of the MEN1 gene has been recommended for patients who meet clinical criteria for MEN1 (at least two of the following: parathyroid hyperplasia, pancreatic endocrine tumour or pituitary adenoma) and those in whom a diagnosis of MEN1 is suspected. We examined the appropriateness of these clinical criteria. PATIENTS AND METHODS: A total of 292 patients were referred for diagnostic testing. The coding region of the MEN1 gene was sequenced in 186 index cases and mutation testing was requested for 106 subjects, including 83 asymptomatic relatives. RESULTS: MEN1 gene mutations were identified in 68/186 index cases (37%). Twenty-nine of the 60 MEN1 mutations reported are novel. The likelihood of finding a mutation was correlated with the number of MEN1-related tumours (mutation detection rate of 79%, 37% and 15% in patients with three, two and one main MEN1-related tumours; P < or = 0.00001) and increased in the presence of a family history (mutation detection rate of 91%, 69% and 29%vs. 69%, 23% and 0% in sporadic cases with three, two or one main MEN1-related tumours, respectively; P < or = 0.00001). The pick-up rate in the 83% of subjects who met proposed criteria for diagnostic testing was 42%, but in those who did not meet these criteria this fell to 0%. CONCLUSIONS: The likelihood of finding an MEN1 mutation depends on the clinical features of the patient and their family. This large series supports present referral criteria for diagnostic mutation screening, but suggests that patients with sporadic isolated tumours rarely have MEN1 mutations.

Adolescent↗
PubMed2005

A GFP-based genetic screen reveals mutations that disrupt the architecture of the zebrafish retinotectal projection.

The retinotectal projection is a premier model system for the investigation of molecular mechanisms that underlie axon pathfinding and map formation. Other important features, such as the laminar targeting of retinal axons, the control of axon fasciculation and the intrinsic organization of the tectal neuropil, have been less accessible to investigation. In order to visualize these processes in vivo, we generated a transgenic zebrafish line expressing membrane-targeted GFP under control of the brn3c promoter/enhancer. The GFP reporter labels a distinct subset of retinal ganglion cells (RGCs), which project mainly into one of the four retinorecipient layers of the tectum and into a small subset of the extratectal arborization fields. In this transgenic line, we carried out an ENU-mutagenesis screen by scoring live zebrafish larvae for anatomical phenotypes. Thirteen recessive mutations in 12 genes were discovered. In one mutant, ddl, the majority of RGCs fail to differentiate. Three of the mutations, vrt, late and tard, delay the orderly ingrowth of retinal axons into the tectum. Two alleles of drg disrupt the layer-specific targeting of retinal axons. Three genes, fuzz, beyo and brek, are required for confinement of the tectal neuropil. Fasciculation within the optic tract and adhesion within the tectal neuropil are regulated by vrt, coma, bluk, clew and blin. The mutated genes are predicted to encode molecules essential for building the intricate neural architecture of the visual system.

Animals↗
PubMed2002

TP53 alterations as a potential diagnostic marker in superficial bladder carcinoma and in patients serum, plasma and urine samples.

Molecular markers are needed for better distinguishing of non-invasive papillary (pTa) and minimally invasive (pT1) bladder carcinomas and for identifying individual tumors with a high risk of recurrence or disease progression. First aim of our study was to evaluate TP53 microsatellite and mutation analysis as an effective concept for the characterization of superficial bladder tumors with different biological aggressiveness. Mutation screening in the TP53 hot spot region was performed in 55 microdissected superficial bladder tumor samples by direct genomic sequencing. PCR based LOH analysis was done with two markers at 17p13. Second, there is considerable interest in the development of non-invasive techniques that would detect recurrent bladder neoplasia. In order to evaluate TP53 alterations as a potential marker for a non-invasive diagnosis of recurrences or residuals and to determine whether tumor-specific DNA exhibiting LOH or sequences harbouring a mutation, can be detected in body fluids, mutation screening was performed in urine, plasma and serum of patients with a mutated primary tumor. LOH analysis with two markers at 17p was done in the corresponding urine and blood samples of 31 primary tumors. As seen from our results, TP53 inactivation by mutation seems to characterize higher malignant superficial bladder tumors which tend to recur and in which the probability is higher that the rezidives progress to muscle invasive growth pattern. Only in 2/8 cases, the TP53 mutation from the primary tumor could be re-detected in patients urine and blood. 17p microsatellite changes with at least one marker were found in 30/31 body fluids of the tumor patients (97%). Correlating the 17p status found in body fluids to the status of the primary tumor, the concordance is only about 52%. We conclude that TP53 genotyping as a non-invasive diagnostic tool in outpatient samples is of limited value for clinical practice.

Aged↗
PubMed1995

HPV DNA and p53 alterations in oropharyngeal carcinomas.

We have examined a series of 37 oropharyngeal squamous cell carcinomas for the presence of HPV 6/11, 16, and 18 DNA by polymerase chain reaction (PCR)/Southern blotting and for p53 alterations by immunohistochemistry and mutation screening with temperature gradient gel electrophoresis (TGGE). HPV sequences were found in a total of 26 of 37 cancers (70.3%), most frequently HPV 16 (20/37) followed by HPV 18 (11/37). Double infections with HPV 16 and 18 were present in 5 tumours. p53 accumulation was detectable immunohistochemically in 21 of 37 carcinomas (56.8%). There were remarkable differences in the distribution of immunoreactive tumour cells in relation to the tumour grade. A mutation screening for p53 by TGGE, directed to the amplified exons 5-8, revealed p53 mutations in 14 of 37 carcinomas (37.8%). Mutations in two different exons were present in 3 tumours, 11 tumours being hit once. Exon 7 was mutated in 6 carcinomas, exons 5 and 8 in 4 cases, and exon 6 in 3 cases. When grouping the tumours with p53 mutation according to their HPV state, HPV-positive cases showed slightly more mutations (11/26) than HPV-negative cases (3/11). Only 5 of 37 carcinomas (13.5%) contained neither HPV DNA nor p53 alterations. Our results indicate that high-risk HPV and p53 mutations frequently coexist in oropharyngeal carcinomas, in contrast to genital tumours, notably carcinomas of the cervix uteri. This may reflect different pathways in carcinogenesis in squamous cell epithelium from different sites.

Adult↗
PubMed1997

Diagnostic strategy, genetic diagnosis and identification of new mutations in intermittent porphyria by denaturing gradient gel electrophoresis.

Acute intermittent porphyria (AIP) is an autosomal dominant inherited disease of heme metabolism caused by mutations in the hydroxymethylbilane synthase gene. Diagnosing AIP during an acute attack using traditional biochemical markers is unproblematic, but it can be difficult to obtain a definite diagnosis in asymptomatic carriers. These limitations may, however, be solved through a genetic approach for diagnosing AIP carrier status. A mutation screening assay based on the denaturing gradient gel electrophoresis (DGGE) principle was established in a setup that allows within 24 hr to pinpoint which of the 15 exons of the hydroxymethylbilane synthase gene carries the underlying mutation, and thereby reduces subsequent sequencing, needed to determine the specific mutation, to this particular gene region. To evaluate sensitivity and specificity of the DGGE assay, samples from 22 AIP patients with known mutations and six healthy controls were examined in a blinded design. Following unblinding, it was revealed that in all 22 AIP samples the correct mutation carrying region had been pointed out. In two samples containing a previously undescribed polymorphism, this additional region was also pointed out. All controls were correctly characterized as normal in the DGGE assay. Subsequently, to evaluate the assay in the clinical setting, samples from six previously uncharacterized Danish AIP probands were examined and the underlying mutation detected in all six. In conclusion, a simple and sensitive mutation screening assay based on the DGGE principle allows genetic diagnosis of AIP in a routine setting and may be used as an additional tool in genetic counseling of AIP families.

DNA Mutational Analysis↗
PubMed2001

Identification of a common variant in the lipoprotein lipase gene in a large Utah kindred ascertained for coronary heart disease: the -93G/D9N variant predisposes to low HDL-C/high triglycerides.

Defects in the lipoprotein lipase (LPL) gene are associated with dyslipidemia in the general population. Several rare mutations in the gene, as well as two common coding region polymorphisms, D9N and N291S, exhibit deleterious effects on circulating lipid levels. Using a linkage-based approach, we have identified a large Utah kindred segregating the D9N variant in the LPL gene. The kindred was ascertained for premature coronary heart disease and was expanded based on familial dyslipidemia. A genomic scan identified a region of linkage including LPL, and mutation screening identified the segregating variant. In the kindred, the variant shows high penetrance for a hypoalphalipoproteinemia phenotype, but is also associated with hypertriglyceridemia and elevated insulin levels. The strength of linkage was dependent on the combination of phenotype definition and model parameters, favoring the use of a MOD score approach. Most other studies of LPL have proceeded by mutation screening of randomly chosen individuals or selected affected probands; this is the first example identifying a segregating LPL mutation using direct linkage.

Aged↗
PubMed2000

Mutation detection in the breast cancer gene BRCA1 using the protein truncation test.

About 400 distinct mutations have been defined in the BRCA1 gene, and these are spread fairly evenly through the 5592 bp of coding DNA. This circumstance presents a formidable challenge for mutation screening. Apart from total direct sequencing, the preferred screening method has been single-strand conformation polymorphism (SSCP) gels, with a smaller input from constant denaturant gradient electrophoresis (CDGE), heteroduplex (HD) analysis, and mismatch cleavage. The protein truncation test (PTT) was used early in BRCA1 mutation screening but has not been widely adopted, perhaps because a straightforward analysis of the whole BRCA1 gene requires working with RNA and all its perceived problems. The present work was undertaken to assess the practicality of using the PTT under routine conditions for the screening of long genes such as BRCA1 that are not highly expressed in lymphocytes. We conclude that, provided RNA preparation is carried out effectively and consistently, the PTT approach has significant advantages over other methodologies such as SSCP gels.

Base Sequence↗
PubMed2006

Optimization of capillary array electrophoresis single-strand conformation polymorphism analysis for routine molecular diagnostics.

Mutation screening is widely used for molecular diagnostics of inherited disorders. Furthermore, it is anticipated that the present and future identification of genetic risk factors for complex disorders will increase the need for high-throughput mutation screening technologies. Capillary array electrophoresis (CAE) SSCP analysis is a low-cost, automated method with a high throughput and high reproducibility. Thus, the method fulfills many of the demands to be met for application in routine molecular diagnostics. However, the need for performing the electrophoresis at three temperatures between 18 degrees C and 35 degrees C for achievement of high sensitivity is a disadvantage of the method. Using a panel of 185 mutant samples, we have analyzed the effect of sample purification, sample medium and separation matrix on the sensitivity of CAE-SSCP analysis to optimize the method for molecular diagnostic use. We observed different effects from sample purification and sample medium at different electrophoresis temperatures, probably reflecting the complex interplay between sequence composition, electrophoresis conditions and sensitivity in SSCP analysis. The effect on assay sensitivity from three different polymers was tested using a single electrophoresis temperature of 27 degrees C. The data suggest that a sensitivity of 98-99% can be obtained using a 10% long chain poly-N,N-dimethylacrylamide polymer.

Acrylamides↗
PubMedDate not supplied

Screening of CFTR mutations in an isolated population: identification of carriers and patients.

One important application of the identification of disease-causing mutations is carrier screening in the general population. Such a project requires a simple accurate test by which a large proportion of the mutations can be identified. This study describes screening for CFTR mutations in an isolated Israeli Arab village. Two mutations, G85E and delta F508, accounted for all the CF alleles of these patients. The screening program tested for these two mutations, as well as the 5T allele, which has recently been shown to down-regulate the CFTR expression and cause variable phenotype. The screened population comprised 497 students from one school, which all the children of the village attend. The results revealed high carrier frequency, 8.5%, for the two CFTR mutations, G85E and delta F508, and a carrier frequency of 12% for the 5T allele. Two compound heterozygotes for the CFTR mutations, delta F508/G85E and G85E/5T, were identified. Both of these students had not been diagnosed previously as having CF since their disease presentation was not typical of CF. The CF incidence in this village was found to be extremely high, 1:72 life births. The screening results were reported to the physicians of the village to be used, upon request, for genetic counselling. This study emphasizes the importance of such programs for the identification of non-classical patients and for carrier detection.

Arabs↗
PubMed2004

Molecular analysis of the putative tumour-suppressor gene EXTL1 in neuroblastoma patients and cell lines.

Although neuroblastoma is the most common extracranial solid tumour of childhood, little is known about its aetiology. Together with MYCN amplification and chromosome 17q gain, chromosome 1p deletion is one of the most frequently occurring genetic abnormalities in neuroblastoma. Based upon mapping of deletion breakpoints, putative tumour suppressor gene loci have been assigned to the distal part of the short arm of chromosome 1. Recently, the EXTL1 gene was suggested as a candidate neuroblastoma-suppressor gene and to evaluate this hypothesis, we performed 1p deletion analysis and mutation screening of the EXTL1-coding region on DNA from 22 primary neuroblastomas and 21 neuroblastoma cell lines. Deletions of the chromosome region 1p36.1, including the EXTL1 gene, were detected in several neuroblastoma cell lines and primary tumours. EXTL1 mutation screening resulted in the detection of one unclassified variant (Ser28Cys) but could not provide additional evidence of EXTL1 being involved in the aetiology of neuroblastoma.

Cell Line, Tumor↗
PubMedDate not supplied

Comparison of conformation-sensitive gel electrophoresis and single-strand conformation polymorphism analysis for detection of mutations in the BRCA1 gene using optimized conformation analysis protocols.

In order to develop a selective mutation screening strategy for BRCA1, one of the gene responsible for hereditary predisposition to breast cancer, we analysed by single-strand conformation polymorphism (SSCP) and conformation-sensitive gel electrophoresis (CSGE) a cohort of 20 Bulgarian breast cancer patients, prescreened for nonsense mutations by the protein truncation test. By assaying the complete coding sequence of the gene applying both methods, we were able to detect 12 sequence alterations: 11 nucleotide substitutions and one deletion. Two of the alterations are intronic polymorphisms, the rest are exon sequence variants. Of the 12 polymorphisms identified, 11 are described and one is new. All sequence changes were detected by CSGE and eight of them were also shown by SSCP analysis. There was no sequence alterations which could be detected by SSCP analysis only. We propose that because of the specificity of most sequence variants detected (nucleotide substitutions) and the comparatively high percentage of AT content of the BRCA1 gene (58.4%), CSGE turned out to be the more sensitive technique in our assay. This observation is in agreement with other accepted analysis strategies for BRCA1 and it may prove useful for mutation screening of AT-rich, multi-exon genes.

Adult↗
PubMed2005

Screening of mutations in the CFTR gene in 1195 couples entering assisted reproduction technique programs.

Genetic testing of the cystic fibrosis transmembrane conductance (CFTR) gene is currently performed in couples undergoing assisted reproduction techniques (ART), because of the high prevalence of healthy carriers in the population and the pathogenic relationship with congenital bilateral absence of vas deferens (CBAVD). However, discordant data have been reported concerning the usefulness of this genetic test in couples with no family history of cystic fibrosis (CF). In this study, we report the results of CFTR molecular screening in 1195 couples entering ART. Genetic testing was initially carried out in a single partner of each couple. CFTR mutations were detected in 55 subjects (4.6%), a percentage that overlaps with the one reported in the general population. However, significantly higher frequencies of were found in CBAVD individuals (37.5%) and in males with nonobstructive azoospermia (6.6%). The 5T allele was found in 78 patients (6.5%). This figure was again significantly different in males with nonobstructive-azoospermia (9.9%) and in those with CBAVD (100%). All together, 139 subjects (11.6%) had either a CFTR mutation or the 5T allele. Subsequent molecular analysis of their partners disclosed a CFTR mutation or 5T allele in nine cases (6.5%). However, none of these couples had CFTR alterations in both members, a CFTR mutation being invariably present in one partner and the 5T allele in the other. In order to improve genetic counselling of these couples, the TG-M470V-5T association was analyzed, and a statistically significant relationship between 12TG-V470 and CBAVD was detected.

Alleles↗
PubMed1996

Systematic screening for mutations in the coding region of the human serotonin transporter (5-HTT) gene using PCR and DGGE.

Dysfunctions in serotonergic pathways may underlie several psychiatric disorders. The reuptake of serotonin (5-HT) from synaptic terminals is mediated by a specific transporter (5-HTT). Genetic variation in the gene coding for the 5-HTT protein might be involved in the predisposition to psychiatric disorders. A systematic screening of the whole coding sequence of the 5-HTT gene in mood disorder (MD) and obsessive-compulsive disorder (OCD) patients, as well as in healthy controls, using PCR and denaturing gradient gel electrophoresis (DGGE) revealed the presence of two mutations. The first was in intron 4, and the second was a C-->A transversion leading to an amino-acid exchange (Leu-->Met) in position 255 of the deduced protein sequence. No further occurrence of this substitution was found in an extended sample of patients and controls. Therefore, structural modifications of the 5-HTT gene do not seem to play either a major or minor role in the genetic predisposition to MD or OCD.

Adult↗
PubMed2004

Preliminary data suggest that mutations in the CgRP pathway are not involved in human sporadic cryptorchidism.

In testicular descent to the scrotum, a multistep process, many anatomical and hormonal factors play a role. Cryptorchidism occurs in about 1-2% of males and may cause secondary degeneration of the testes. Animal models have shown that abnormalities, in the calcitonin gene-related peptide (CgRP) activity, could be relevant in the pathogenesis of cryptorchidism. We performed a mutation screening by PCR exon amplification, single-strand conformation polymorphism (SSCP) and sequencing in four candidate genes, CgRPs (alphaCgRP, betaCgRP), their receptor (CgRPR) and the receptor component protein (CgRP-RCP), in 90 selected cases of idiopathic unilateral or bilateral cryptorchidism. Mutation screening of the coding regions and intron-exon boundaries revealed some polymorphic variants but no pathogenic sequence changes. These preliminary data suggest that these genes are not major factors for cryptorchidism in humans.

Adult↗
PubMed2005

Periodontal treatment of two siblings with juvenile hyaline fibromatosis.

BACKGROUND AND AIM: Juvenile hyaline fibromatosis (JHF) is an autosomal recessive disease that presents with multiple subcutaneous nodular tumours, gingival fibromatosis, flexion contractures of the joint and hyaline material accumulation in extracellular area. Recently, the causative gene for JHF, capillary morphogenesis protein 2 (CMG2) was identified. In this case report, periodontal status, treatment and follow-up together with histopathologic evaluation of gingival tissue specimens and mutation screening of two JHF cases are presented. CASE REPORTS: A 10-year-old female (case 1) and her 3-year-old brother (case 2) were first examined in our department with a complaint of gingival hyperplasia in 1991. Symptoms of the disease were detected in two of four siblings in the family. Several gingivectomy operations were carried out over 11 years with hygiene motivation and initial phase therapy. After the last gingivectomy operation in 2002, the patients were reviewed frequently. RESULTS AND CONCLUSIONS: Although there was linear marginal gingival inflammation, no remarkable enlargement was noted at last appointment. Histopathological findings showed increased amounts of subepithelial nodular connective tissue, thinned epithelial mucosa, separated inter-cellular bridges and decreased numbers of connective tissue cells in gingival tissue samples. Electron microscopic examinations supported the histopathological findings. Mutation screening of CMG2 demonstrated that the siblings were homozygous for a pathogenic missense mutation, V386F. Our clinical findings demonstrate that gingivectomy is useful and frequent periodontal visits are important for maintaining oral hygiene and decreasing growth rate of gingiva in JHF.

Child↗
PubMed2005

Clinical consequences of PKHD1 mutations in 164 patients with autosomal-recessive polycystic kidney disease (ARPKD).

BACKGROUND: ARPKD is associated with mutations in the PKHD1 gene on chromosome 6p12. Most cases manifest peri-/neonatally with a high mortality rate in the first month of life while the clinical spectrum of surviving patients is much more variable than generally perceived. METHODS: We examined the clinical course of 164 neonatal survivors (126 unrelated families) over a mean observation period of 6 years (range 0 to 35 years). PKHD1 mutation screening was done by denaturing high-performance liquid chromatography (DHPLC) for the 66 exons encoding the 4074 aa fibrocystin/polyductin protein. RESULTS AND CONCLUSION: This is the first study that reports the long-term outcome of ARPKD patients with defined PKHD1 mutations. The 1- and 10-year survival rates were 85% and 82%, respectively. Chronic renal failure was first detected at a mean age of 4 years. Actuarial renal survival rates [end point defined as start of dialysis/renal transplantation (RTX) or by death due to end-stage renal disease (ESRD)] were 86% at 5 years, 71% at 10 years, and 42% at 20 years. All but six patients (92%) had a kidney length above or on the 97th centile for age. About 75% of the study population developed systemic hypertension. Sequelae of congenital hepatic fibrosis and portal hypertension developed in 44% of patients and were related with age. Positive correlations could further be demonstrated between renal and hepatobiliary-related morbidity suggesting uniform disease progression rather than organ-specific patterns. PKHD1 mutation analysis revealed 193 mutations (70 novel ones; 77% nonconservative missense mutations). No patient carried two truncating mutations corroborating that one missense mutation is indispensable for survival of newborns. We attempted to set up genotype-phenotype correlations and to categorize missense mutations. In 96% of families we identified at least one mutated PKHD1 allele (overall detection rate 76.6%) indicating that PKHD1 mutation screening is a powerful diagnostic tool in patients suspected with ARPKD.

Adolescent↗