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Mutational Screening (mutational + screening)
Selected AbstractsMutational and expression analysis of CDK1, cyclinA2 and cyclinB1 in epilepsy-associated glioneuronal lesionsNEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 2 2007V. Schick Gangliogliomas and focal cortical dysplasias (FCDs) constitute glioneuronal lesions, which are frequently encountered in biopsy specimens of patients with pharmacoresistant focal epilepsy and relate to impaired differentiation and migration of neural precursors. However, their molecular pathogenesis and relationship are still largely enigmatic. Recent data suggest several components of the insulin-pathway, including TSC1 and TSC2 mutated in tuberous sclerosis complex (TSC), to be altered in gangliogliomas and FCD with Taylor type balloon cells (FCDIIb). The proteins tuberin (TSC2) and hamartin (TSC1) constitute a tumour suppressor mechanism involved in cell-cycle control. Hamartin and/or tuberin were reported to colocalize and/or interact with CDK1, cyclinB1 and cyclinA2 that are critically involved in cell-size and cell-growth control. Here, we have carried out mutational and expression analyses of CDK1, cyclinB1 and cyclinA2 in gangliogliomas and FCDIIb. Mutational screening was performed by single-strand conformation polymorphism analysis in gangliogliomas (n = 20), FCDIIb (n = 35) and controls. CyclinB1 revealed a polymorphism (G to A, cDNA Position 966, GenBank: NM_031966) in exon 7 with similar frequencies in FCDIIb, gangliogliomas and control specimens (FCD n = 9/35; gangliogliomas n = 5/20; control n = 20/100). We used real-time reverse transcription polymerase chain reaction to determine expression levels of CDK1, cyclinB1 and cyclinA2 in 10 FCDIIb and nine gangliogliomas compared with unaffected adjacent control tissue of the same patients. We observed significantly lower expression of CDK1 and cyclinA2 in FCDIIb vs. controls whereas no significant expression differences were present for CDK1, cyclinB1 and cyclinA2 in gangliogliomas. Our data strongly argue against mutational events of CDK1, cyclinB1 and cyclinA2 to play a role in gangliogliomas or FCDIIb. However, a potential functional significance of lower expression for the cell-size and cell-cycle regulators CDK1 and cyclinA2 in FCDIIb composed of large dysplastic neurones and balloon cells needs to be further resolved. [source] Investigating the etiology of multiple tooth agenesis in three sisters with severe oligodontiaORTHODONTICS & CRANIOFACIAL RESEARCH, Issue 1 2008S Swinnen Structured Abstract Authors,,, Swinnen S, Bailleul-Forestier I, Arte S, Nieminen P, Devriendt K, Carels C Objectives,,, To describe the dentofacial phenotypes of three sisters with severe non-syndromic oligodontia, to report on the mutation analysis in three genes, previously shown to cause various phenotypes of non-syndromic oligodontia and in two other suspected genes. Based on the phenotypes in the pedigree of this family, the different possible patterns of transmission are discussed. Methods,,, Anamnestic data and a panoramic radiograph were taken to study the phenotype of the three sisters and their first-degree relatives. Blood samples were also taken to obtain their karyotypes and DNA samples. Mutational screening was performed for the MSX1, PAX9, AXIN2, DLX1 and DLX2 genes. Results,,, The probands' pedigree showed evidence for a recessive or multifactorial inheritance pattern. Normal chromosomal karyotypes were found and , despite the severe oligodontia present in all three sisters , no mutation appeared to be present in the five genes studied so far in these patients. Conclusions,,, In the three sisters reported, their common oligodontia phenotype is not caused by mutations in the coding regions of MSX1, PAX9, AXIN2, DLX1 or DLX2 genes, but genetic factors most probably play a role as all three sisters were affected. Environmental and epigenetic factors as well as genes regulating odontogenesis need further in vivo and in vitro investigation to explain the phenotypic heterogeneity and to increase our understanding of the odontogenic processes. [source] Mutational screening of the CYP26A1 gene in patients with caudal regression syndrome,BIRTH DEFECTS RESEARCH, Issue 2 2006Patrizia De Marco Abstract BACKGROUND The retinoic acid (RA),catabolizing enzyme Cyp26a1 plays an important role in protecting tailbud tissues from inappropriate exposure to RA. Cyp26a1 -null animals exhibit caudal agenesis and spina bifida, imperforate anus, agenesis of the caudal portions of the digestive and urogenital tracts, and malformed lumbosacral skeletal elements. This phenotype closely resembles the most severe form of caudal agenesis in humans. In view of these findings, we investigated a potential involvement of the human CYP26A1 gene in the pathogenesis of caudal regression syndrome (CRS). METHODS Mutational screening of 49 CRS patients and 132 controls was performed using denaturing high-performance liquid chromatography and sequencing. Differences in the genotype and allele frequency of each SNP were evaluated by ,2 analysis. The biological significance of the intronic variants was investigated by transfection assays of mutant constructs and by analysis of the splicing patterns with RT-PCR. RESULTS Mutational screening allowed us to identify 6 SNPs, 4 of which (447C>G, 1134G>A, IVS1+10G>C, and IVS4+8AG>GA) are new. In addition, we describe a novel 2-site haplotype consisting of the 2 intronic SNPs. Both single-locus and haplotype analyses revealed no association with increased risk for CRS. The consequences of the 2 intronic polymorphisms on the mRNA splicing process were also investigated. Moreover, using functional and computational methods we demonstrated that both of these intronic polymorphisms affect the intron splicing efficiency. CONCLUSIONS Our research did not provide evidence that CYP26A1 has implications for the pathogenesis of human CRS. However, the relationship between CRS risk and the CYP26A1 genotype requires further study with a larger number of genotyped subjects. Birth Defects Research (Part A), 2006. © 2006 Wiley-Liss, Inc. [source] Mutational screening of the cationic trypsinogen gene in a large cohort of subjects with idiopathic chronic pancreatitisCLINICAL GENETICS, Issue 3 2001Jm Chen 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. [source] Current antiviral therapies for chronic hepatitis BHEPATOLOGY RESEARCH, Issue 6 2008Mari Inada Among current treatment options for chronic hepatitis B, nucleoside/nucleotide analog therapy has better tolerability and most patients respond to the therapy, while interferon (IFN) therapy has rather severe side-effects and a lower response rate. However, nucleoside/nucleotide analog therapies have problems of the emergence of drug resistance and poor sustainability of response after discontinuation. After the first nucleoside/nucleotide analog lamivudine, adefovir and entecavir are now utilized in many countries. Adefovir has efficacy for lamivudine resistant patients and current data suggests that adding adefovir to ongoing lamivudine is better than switching to adefovir in terms of viral suppression and the occurrence of resistance. Entecavir can be the first choice for naïve patients, although cross-resistance has been known for lamivudine resistant patients and mutational screening should take place before using entecavir with such patients. Many other new nucleoside/nucleotide analogs are being developed such as telbivudine, clevudine and tenofovir; the details of each drug will be disclosed in near future. [source] Myoclonus-dystonia: significance of large SGCE deletions,,HUMAN MUTATION, Issue 2 2008A. Grünewald Abstract Myoclonus-dystonia (M-D) is an autosomal-dominant movement disorder caused by mutations in SGCE. We investigated the frequency and type of SGCE mutations with emphasis on gene dosage alterations and explored the associated phenotypes. We tested 35 M-D index patients by multiplex ligation-dependent probe amplification (MLPA) and genomic sequencing. Mutations were found in 26% (9/35) of the cases, all but three with definite M-D. Two heterozygous deletions of the entire SGCE gene and flanking DNA and a heterozygous deletion of exon 2 only were detected, accounting for 33% (3/9) of the mutations found. Both large deletions contained COL1A2 and were additionally associated with joint problems. Further, we discovered one novel small deletion (c.771_772delAT, p.C258X) and four recurrent point mutations (c.289C>T, p.R97X; c.304C>T, p.R102X; c.709C>T, p.R237X; c.1114C>T, p.R372X). A Medline search identified 22 articles on SGCE mutational screening. Sixty-four unrelated M-D patients were described with 41 different mutations. No genotype,phenotype association was found, except in patients with deletions encompassing additional genes. In conclusion, a rigorous clinical preselection of patients and careful accounting for non-motor signs should precede mutational tests. Gene dosage studies should be included in routine SGCE genetic testing. © 2007 Wiley-Liss, Inc. [source] Novel high-throughput SNP genotyping cosegregation analysis for genetic diagnosis of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis,HUMAN MUTATION, Issue 5 2007Esther Pomares Abstract Retinitis pigmentosa (RP), the major cause of blindness in adults, is an extremely heterogeneous monogenic disorder. More than 32 causative genes have been identified, 18 of which are involved in autosomal recessive RP (arRP); however, more than 50% of the cases remain unassigned. There are no major causative genes identified for arRP nor any prevalent mutations, which make mutational screening of the already reported RP genes extremely time consuming and costly. Nonetheless, this step is unavoidable for genetic diagnosis of patients and potential carriers, and it is a prerequisite before approaching the identification of new RP genes and loci. We have designed an innovative high-throughput time- and cost-effective strategy for cosegregation analysis of 22 genes of arRP and Leber congenital amaurosis (LCA; an autosomal recessive retinal dystrophy that shares some of the RP genes and traits) by SNP genotyping. This novel indirect method has been validated in a panel of 54 consanguineous and nonconsanguineous arRP families. In a single and fast genotyping step: 1) we discarded all the 22 candidate genes in 13% of the pedigrees, highlighting the families of choice to search for novel arRP genes/loci; 2) we excluded an average of 18,19 genes per family, thus diminishing the number of genes to screen for pathogenic mutations; and 3) we identified CERKL as the causative RP gene in a family in which this candidate had been previously discarded by microsatellite cosegregation analysis. This type of approach can also be applied to other nonretinal diseases with high genetic heterogeneity, such as hereditary deafness or Parkinson disease. Hum Mutat 28(5), 511,516, 2007. © 2007 Wiley-Liss, Inc. [source] Erratum: mutational screening of the RB1 gene in Indian patients with retinoblastoma reveals eight novel and several recurrent mutations ,,HUMAN MUTATION, Issue 6 2003Velamakanni Saroj Kiran The original article to which this Erratum refers was published in Human Mutation 22:339 Human Mutation (2003) 22(4) 339 The authors have noted several errors in their manuscript that they were unable to correct prior to publication. They regret the errors and list corrections below: 1.Abstract line 9,10: "Eight novel mutations were identified, including 4 single base changes," This should read: "Eight novel mutations were identified, including 5 single base changes," 2.Page 3 2nd line from bottom: ",(g.76940del12; IVS15del+20-33) extending from +18 to +32 of the intron (RB72)." This should be ",(g.76940del14; IVS15del+20-33) extending from +20 to +33 of the intron (RB72)." 3.Page 4 line 7: ",were also to recur," This should read ",were also found to recur," [source] Mutations of the Wnt antagonist AXIN2 (Conductin) result in TCF-dependent transcription in medulloblastomasINTERNATIONAL JOURNAL OF CANCER, Issue 2 2007Arend Koch Abstract Medulloblastomas (MBs) represent the most common malignant brain tumors in children. Most MBs develop sporadically in the cerebellum, but their incidence is highly elevated in patients with familial adenomatous polyposis coli. These patients carry germline mutations in the APC tumor suppressor gene. APC is part of a multiprotein complex involved in the Wnt signaling pathway that controls the stability of ,-catenin, the central effector in this cascade. Previous genetic studies in MBs have identified mutations in genes coding for ,-catenin and its partners, APC and AXIN1, which cause activation of Wnt signaling. The pathway is negatively controlled by the tumor suppressor AXIN2 (Conductin), a scaffold protein of this signaling complex. To investigate whether alterations in AXIN2 may also be involved in the pathogenesis of sporadic MBs, we performed a mutational screening of the AXIN2 gene in 116 MB biopsy samples and 11 MB cell lines using single-strand conformation polymorphism and sequencing analysis. One MB displayed a somatic, tumor-specific 2 bp insertion in exon 5, leading to carboxy-terminal truncation of the AXIN2 protein. This tumor biopsy showed nuclear accumulation of ,-catenin protein, indicating an activation of Wnt signaling. In 2 further MB biopsies, mutations were identified in exon 5 (Glu408Lys) and exon 8 (Ser738Phe) of the AXIN2 gene, which are due to predicted germline mutations and rare polymorphisms. mRNA expression analysis in 22 MBs revealed reduced expression of AXIN2 mRNA compared to 8 fetal cerebellar tissues. Promoter hypermethylation could be ruled out as a major cause for transcriptional silencing by bisulfite sequencing. To study the functional role of AXIN2 in MBs, wild-type AXIN2 was overexpressed in MB cell lines in which the Wnt signaling pathway was activated by Wnt-3a. In this assay, AXIN2 inhibited Wnt signaling demonstrated in luciferase reporter assays. In contrast, overexpression of mutated AXIN2 with a deleted C-terminal DIX-domain resulted in an activation of the Wnt signaling pathway. These findings indicate that mutations of AXIN2 can lead to an oncogenic activation of the Wnt pathway in MBs. © 2007 Wiley-Liss, Inc. [source] Exclusion of coding region mutations in MSX1, PAX9 and AXIN2 in eight patients with severe oligodontia phenotypeORTHODONTICS & CRANIOFACIAL RESEARCH, Issue 3 2006A Gerits Structured Abstract Authors,,, Gerits A, Nieminen P, De Muynck S, Carels C Purpose,,, This paper describes the screening of eight patients with severe oligodontia for PAX9 and AXIN2 mutations. Subjects and Methods,,, Anamnestic data and a panoramic radiograph were collected to study the phenotype of eight patients with oligodontia and their first-degree relatives. A blood sample was taken for a mutational screening for PAX9 and AXIN2 mutations. Results,,, No mutations were discovered, but a unique nucleotide change in a conserved 5, flanking region of PAX9 was revealed. Earlier screening of the same patients for MSX1 mutations also had a negative outcome. Conclusions,,, Considering the discrepancy between the high incidence rate of agenesis and the relatively small number of reported causative mutations in PAX9, MSX1 and AXIN2 genes, the genetic contribution to oligodontia probably is much more heterogeneous than expected so far. Therefore negative results, like the present exclusion data, should be published more often in order to get a better appreciation of the relative contribution of these specific mutations causing oligodontia. In this context the exact number of tested probands also should be mentioned at all cases. Recent evidence of PAX9,MSX1 protein interactions in odontogenesis as well as other genes and developmental factors should receive more attention. [source] MECP2 mutations in Serbian Rett syndrome patientsACTA NEUROLOGICA SCANDINAVICA, Issue 6 2007A. Djarmati Background,,, Rett syndrome is a severe neurodevelopmental X-linked dominant disorder affecting 1/15,000 girls worldwide. Eight years ago, the MECP2 gene was associated with the devastating clinical features observed in Rett syndrome patients. Objectives,,, To investigate the spectrum and the frequency of MECP2 mutations in Serbian Rett syndrome patients. Patients and methods,,, We screened the MECP2 coding region by conventional mutational screening (single-strand conformation polymorphism/sequencing) in 24 patients of Serbian origin and in their 41 unaffected family members. In search for gene dosage alterations in seemingly mutation-negative girls, we developed a new, specific quantitative PCR method. Results,,, Nineteen patients (79%) carried MECP2 mutations, five of which were novel (one nonsense mutation, one duplication and three deletions). Fourteen previously described disease-causing sequence changes and one polymorphism were also detected. Detailed case reports are given for the carriers of the novel mutations. Large MECP2 rearrangements cause Rett syndrome in a significant number of girls without ,classic' mutations in this gene. Therefore, we developed a specific quantitative PCR method, covering MECP2 exons 3 and 4, which previously has not been used for screening. No dosage alterations of the two exons were found in the four tested mutation-negative girls. Conclusions,,, This is the first genetic study of Rett syndrome in Serbian patients describing the MECP2 mutational and phenotypic spectrum in this population. Detailed clinical descriptions of this ethnically homogeneous patient population add to our knowledge of genotype/phenotype correlations in this severe condition. [source] Definition of three minimal deleted regions by comprehensive allelotyping and mutational screening of FHIT,p16INK4A, and p19ARF genes in nasopharyngeal carcinomaCANCER, Issue 7 2002Jenq-Yuh Ko M.D. Abstract BACKGROUND Recurrent deletion on a chromosomal location in tumor cells can be detected by frequent allelic loss and generally is considered to be an indication of the existence of a tumor suppressor gene (TSG) in the region. In the current study, using fluorescent-labeled, high-density microsatellite markers for allelotyping, the authors pinpointed three minimal deleted regions (MDRs) and screened mutations of putative TSGs on chromosomes 3, 9, and 11 in nasopharyngeal carcinoma (NPC) cases occurring in Taiwan. METHODS A total of 133 informative microsatellite markers were used on chromosomes 3, 9, and 11 with an average marker density of 4 centimorgans (cM) for the allelotyping of genomic DNAs isolated from NPC tissues and their corresponding lymphocytes in 48 patients. The correlation between allelic loss and the clinicopathologic parameters of NPC tissues was examined. In addition, putative TSGs including FHIT, p16INK4a, and p19ARF were selected for mutation screening to investigate their potential participation in NPC tumorigenesis. RESULTS Of 3787 informative allelotyping data, 25 frequent allelic losses (or loss of heterozygosity [LOH]) in 13 cytogenetic loci were identified based on a deletion frequency that was greater than the average of allelic loss on that particular chromosome. Several significant associations were determined after statistical analysis of the correlation between allelic loss and clinicopathologic parameters. The allelic losses by D9S318 and D11S1304 were associated with N2/N3 (P = 0.035 and P = 0.005, respectively), and those by D9S905 and D11S1304 were associated with grouped American Joint Committee on Cancer (AJCC) Stage III/IV samples (P = 0.022 and P = 0.017, respectively) of NPC tissues. In addition, three MDRs were revealed on 3p25.3-24.1 (< 19 cM), 3p23-21.31 (< 9 cM), and 11q22.1-23.2 (< 8 cM). To examine somatic mutations in previously reported TSGs located near these frequent LOH loci, three candidate genes, p16INK4a, p19ARF, and FHIT, were analyzed. Point mutations in the coding region of FHIT and in the intron 1 splicing acceptor site of both p16INK4a and p19ARF were detected in NPC cell lines. Sequence analysis of both p16INK4a and p19ARF transcripts revealed that the point mutation resulted in skipping of exon 2 and the generation of shorter transcripts. CONCLUSIONS High-density allelotyping permitted the discovery of 3 MDRs on 3p25.3-24.1 (< 19 cM), 3p23-21.31 (< 9 cM), and 11q22.1-23.2 (< 8 cM) and a correlation was determined between allelic loss and clinicopathologic parameters of NPC tissues. More important, one somatic mutation in NPC cell lines on the intron 1/exon 2 splicing acceptor site of the INK4a/ARF locus was found to result in exon 2 skipping both p16INK4a and p19ARF transcripts, which presumably inactivates the functions of both the p16INK4a and p19ARF proteins. Cancer 2002;94:1987,96. © 2002 American Cancer Society. DOI 10.1002/cncr.10406 [source] 2245: Electrodiagnosis in inherited retinal diseaseACTA OPHTHALMOLOGICA, Issue 2010GE HOLDER Purpose To describe the roles of electrophysiology in the diagnosis and counselling of patients with inherited retinal disease. Methods Electrophysiological testing performed to incorporate and extend the recommendations of the International Society for Clinical Electrophysiology of Vision. Results Using a case-based presentation, it will be shown that electrophysiological testing can objectively assess the function of the different cell types and layers within the retina of the patient with inherited retinal dysfunction, which enables accurate diagnosis and counselling when placed in clinical context. The roles of pattern and multi-focal ERG in the assessment of macular function will be discussed. The electrophysiological findings will be discussed in relation to imaging studies when appropriate. It will shown that distinctive electrophysiological findings can direct appropriate and therefore cost-effective mutational screening in patients with atypical fundus changes. Conclusion Electrophysiological testing is fundamental to the successful management of patients with inherited disorders of retinal function. [source] Mutational screening of the cationic trypsinogen gene in a large cohort of subjects with idiopathic chronic pancreatitisCLINICAL GENETICS, Issue 3 2001Jm Chen 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. [source] | |||||||||||||