Conformation Sensitive Gel Electrophoresis Research Articles

Conformation sensitive gel electrophoresis for the detection of calreticulin mutations in BCR-ABL1-negative myeloproliferative neoplasms.

Calreticulin (CALR) mutations in myeloproliferative neoplasms (MPN) have been reported to be key markers in the molecular diagnosis, particularly in patients lacking JAK2 V617F mutation. In most current reports, CALR mutations were analysed by either allele-specific PCR (AS-PCR), or the more expensive quantitative real-time PCR, pyrosequencing and next-generation sequencing. Hence, we report the use of an alternative method, the conformation sensitive gel electrophoresis (CSGE) for the detection of CALR mutations in BCR-ABL1-negative MPN patients. Forty BCR-ABL1-negative MPN patients' DNA: 19 polycythemia vera (PV), 7 essential thrombocytosis (ET) and 14 primary myelofibrosis (PMF), were screened for CALR mutations by CSGE. PCR primers were designed to amplify sequences spanning between exons 8 and 9 to target the mutation hotspots in CALR. Amplicons displaying abnormal CSGE profiles by electrophoresis were directly sequenced, and results were analysed by BioEdit Sequence Alignment Editor v7.2.6. CSGE results were compared with AS-PCR and confirmed by Sanger sequencing. CSGE identified 4 types of mutations; 2 PMF patients with either CALR type 1 (c.1099_1150del52) or type 2 (c.1155_1156insTTGTC), 1 ET patient with nucleotide deletion (c.1121delA) and insertion (c.1190insA) and 1 PV patient with p.K368del (c.1102_1104delAAG) and insertion (c.1135insA) inframe mutations. Three patients have an altered KDEL motif at the C-terminal of CALR protein. In comparison, AS-PCR only able to detect two PMF patients with mutations, either type 1 and type 2. CSGE is inexpensive, sensitive and reliable alternative method for the detection of CALR mutations in BCR-ABL1-negative MPN patients.

2008P - Large genomic rearrangements in BRCA1 and BRCA2 genes in the Portuguese population

BackgroundBRCA1 and BRCA2 are the genes most frequently associated with hereditary breast and ovarian cancer (HBOC). Besides point mutations, BRCA1/2 large genomic rearrangements (LGR) have been observed, with different frequencies in specific populations. This study aims to update the frequency of BRCA1 and BRCA2 LGR in our population, as well as to characterize the gene distribution of these genetic events. MethodsAll high risk pts referred to our multidisciplinary program between 2000-2018 that consented on genetic testing were reviewed for BRCA1 and BRCA2 point pathogenic variants (several methodologies throughout the years including Conformation Sensitive Gel Electrophoresis; Conformation Sensitive Capillary Electrophoresis; Sanger Sequencing and Next Generation Sequencing) and LGR with Multiplex Ligation dependent Probe Amplification (MLPA). Results3763 of 3901 index pts consented on genetic testing and 473 pts had 476 different pathogenic variants (PV). In 3 cases double germline PV were observed: BRCA1 (c.68_69delAG) and CHEK2 [c.(908+1_909-1)_(1095+1_1096-1)del]; ATM (c.8264_8268delATAAG) and PALB2 (c.751C>T); BRCA2 (c.156_157insAlu) and CHEK2 (c.593-1G>T). BRCA1 PV were detected in 151 pts with 24 LGR of these (15,9%) being detected by MLPA. The most frequent BRCA1 LGR was c. (4185+1_4186-1)_(4357+1_4358-1)dup (n=8). BRCA2 PV were detected in 260 pts with 99 LGR: 92 pts with the Portuguese founder mutation c.156_157insAlu (detected by specific PCR or MLPA) and 7 pts with other LGR (detected by MLPA). The second most frequent BRCA2 LGR was c. (8632+1_8633-1)_(8754+1_8755-1)dup (n=5). Nine CHEK2 PV were detected with the SALSA MLPA probemix P045 BRCA2/CHEK2: c.1100delC (8 pts) and c. (908+1_909-1)_(1095+1_1096-1)del (1pt). The total percentage of BRCA1 and BRCA2 LGR in our cohort was 26%. Excluding the Portuguese founder mutation c.156_157insAlu, the prevalence of LGR was 6,5%. ConclusionsThere is a high prevalence of LGR in the Portuguese population, mostly because of the Portuguese founder mutation c.156_157insAlu. Even if excluding this event, other LRG still account for 6,5% of the BRCA1 and BRCA2 pathogenic variants. These results emphasize the importance of testing LGR in all patients with high risk HBOC. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Molecular-genetic and phenotypic characteristics of desmoid-type fibromatosis

Desmoid-type fibromatosis (DF) is a rare mesenchymal tumor occurring in only 2 to 4 people per 1,000,000 population a year. Desmoid tumors are either seen sporadically or in individuals with familial adenomatous polyposis (FAP). The etiology of sporadic DF is uncertain. The aim of this study was to estimate the potential significance of germline mutations in the APC gene in patients with sporadic DF. APC exons were amplified, studied using conformation sensitive gel electrophoresis and then Sanger-sequenced. The obtained data were processed in Statistica 10. Mutations were detected in 6 (12%) of 51 participants with sporadic DF. Those 6 patients shared a typical DF phenotype characterized by early age of onset (5.8 years on average, in contrast to the patients without APC mutations, who developed DF at 19 years of age; p = 0.02), severe clinical course, multifocal localization on the trunk, and poor prognosis. All of the detected APC mutations were localized to the 3'-end of the gene. For the purpose of comparison, we analyzed a sample of 12 patients with FAP-associated DF. Of those patients, 6 carried mutations in the APC gene. In the analyzed sample, the patients with FAP and the mutant APC gene developed DF at older age (35 years) than the patients with sporadic DF (p = 0.004) and their tumors were not multifocal. This means that sporadic and FAP-associated desmoids have different phenotypes in patients with APC mutations. Patients with sporadic tumors have mutations at the 3'-end of the APC gene more often than individuals with FAP-associated DF. To our knowledge, this is the first study to characterize the subtype of sporadic desmoid fibromatosis phenotypically determined by germline mutations in the APC gene.

High resolution melting for F9 gene mutation analysis in patients with haemophilia B.

Since 2001, we have used conformation sensitive gel electrophoresis (CSGE) as our first choice for F9 gene mutation screening, leading to the identification of about 300 mutations causing haemophilia B (HB). To circumvent the disadvantages of CSGE, we recently evaluated high-resolution melting analysis (HRM), which represents the next-generation mutation scanning technology. In order to explore and validate HRM as a new screening method, we analysed 26 HB patients with previous CSGE-detected mutations, 22 patients with CSGE-undetectable mutations and 13 HB patients who had not been previously investigated. All 61 investigated samples, including the previously investigated and not previously investigated samples, proved to be HRM-positive, with the new screening method showing good efficiency and higher sensitivity than the previously used method. Mixing normal and unknown DNA to generate heterozygote conditions proved an excellent strategy to push the detection performance to its maximum. Mutation scanning by HRM analysis seems to be ideal in our context because it is rapid, cheap and capable of detecting the vast majority of mutations in HB patients. Nevertheless, to improve the detection ability of this scanning technology, it is recommended to start with a good strategy, based on good quality samples and optimised polymerase chain reaction amplification parameters, especially regarding primers and length of the amplicons.

Molecular characterization of Glanzmann's thrombasthenia in Iran: identification of three novel mutations.

: Quantitative and/or qualitative defects of the platelet membrane glycoprotein IIb/IIIa complex lead to the clinical entity of Glanzmann's thrombasthenia. A large variety of mutations and polymorphisms are responsible for the aberrant expression and defective activity of this heterodimeric complex. The current study aimed to determine the pattern of mutations among Iranian population with Glanzmann's thrombasthenia. A total of 20 patients with Glanzmann's thrombasthenia have been evaluated. All exons and splice sites of ITGA2B and ITGB3 genes were amplified using touchdown PCR. Mutation screening was analyzed using conformation sensitive gel electrophoresis heteroduplex PCR, and DNA sequencing. In addition to finding one previously identified mutation and polymorphism, the experimenters explored 3 and 2 novel mutations and polymorphisms, respectively. One substitution mutation, two deletions of a single nucleotide, one insertion of a single nucleotide, two synonymous polymorphisms, and one missense polymorphism were found using Sanger sequencing method. All detected mutations were homozygous which will most likely contribute to the pathogenesis of Glanzmann's thrombasthenia. Furthermore, it suggested ITGB3 as the mainly affected gene impaired in the patients with Glanzmann's thrombasthenia. As expected, the molecular results were consistent with the phenotypic findings so that GPIIb/IIIa complex was disrupted due to mutations in all type-I Glanzmann's thrombasthenia patients. It is concluded that intronic alterations or epigenetic regulations could be responsible for aberrant expression and/or defective activity of GPIIb/IIIa complex among other patients.

A novel MTTT mutation m.15933G\u2009>\u2009A revealed in analysis of mitochondrial DNA in patients with suspected mitochondrial disease

BackgroundMitochondrial diseases present with variable multi-organ symptoms. Common disease-causing mutations in mitochondrial DNA (mtDNA) are regularly screened in diagnostic work-up, but novel mutations may remain unnoticed.MethodsPatients (N = 66) with a clinical suspicion of mitochondrial disease were screened for their mtDNA coding region using conformation sensitive gel electrophoresis and sequencing. Long-PCR was used to detect deletions followed by POLG1 sequencing in patients with multiple deletions.ResultsWe discovered three novel mtDNA variants that included m.8743G > C, m.11322A > G and m.15933G > A. The novel MTTT variant m.15933G > A is suggested to be pathogenic. Analysis revealed also multiple mtDNA deletions in two patients and five nonsynonymous variants that were putatively pathogenic according to in-silico prediction algorithms. In addition, a rare haplogroup H associated m.7585_7586insT variant was discovered.ConclusionsAmong patients with a suspected mitochondrial disease, a novel MTTT variant m.15933G > A was discovered and is suggested to be pathogenic. In addition, several putatively pathogenic nonsynonymous variants and rare variants were found. These findings highlight the importance of coding region mtDNA screening among patients with clinical features suggesting a mitochondrial disease, but who lack the common mitochondrial disease mutations.

WISP3 mutational analysis in Indian patients diagnosed with progressive pseudorheumatoid dysplasia and report of a novel mutation at p.Y198.

ObjectivesTo determine the pattern of mutations of the WISP3 gene in clinically identified progressive pseudorheumatoid dysplasia (PPD) in an Indian population.Patients and MethodsA total of 15 patients with clinical features of PPD were enrolled in this study. Genomic DNA was isolated and polymerase chain reaction performed to amplify the WISP3 gene. Screening for mutations was done by conformation-sensitive gel electrophoresis, beginning with the fifth exon and subsequently proceeding to the remaining exons. Sanger sequencing was performed for both forward and reverse strands to confirm the mutations.ResultsIn all, two of the 15 patients had compound heterozygous mutations: one a nonsense mutation c.156C>A (p.C52*) in exon 2, and the other a missense mutation c.677G>T (p.G226V) in exon 4. All others were homozygous, with three bearing a nonsense mutation c.156C>A (p.C52*) in exon 2, three a missense mutation c.233G>A (p.C78Y) in exon 2, five a missense mutation c.1010G>A (p.C337Y) in exon 5, one a nonsense mutation c.348C>A (p.Y116*) in exon 3, and one with a novel deletion mutation c.593_597delATAGA (p.Y198*) in exon 4.ConclusionWe identified a novel mutation c.593_597delATAGA (p.Y198*) in the fourth exon of the WISP3 gene. We also confirmed c.1010G>A as one of the common mutations in an Indian population with progressive pseudorheumatoid dysplasia.Cite this article: V. Madhuri, M. Santhanam, K. Rajagopal, L. K. Sugumar, V. Balaji. WISP3 mutational analysis in Indian patients diagnosed with progressive pseudorheumatoid dysplasia and report of a novel mutation at p.Y198* Bone Joint Res 2016;5:301–306. DOI: 10.1302/2046-3758.57.2000520.

Genetic analysis of bleeding disorders.

Molecular genetic analysis of inherited bleeding disorders has been practised for over 30years. Technological changes have enabled advances, from analyses using extragenic linked markers to next-generation DNA sequencing and microarray analysis. Two approaches for genetic analysis are described, each suiting their environment. The Christian Medical Centre in Vellore, India, uses conformation-sensitive gel electrophoresis mutation screening of multiplexed PCR products to identify candidate mutations, followed by Sanger sequencing confirmation of variants identified. Specific analyses for F8 intron 1 and 22 inversions are also undertaken. The MyLifeOurFuture US project between the American Thrombosis and Hemostasis Network, the National Hemophilia Foundation, Bloodworks Northwest and Biogen uses molecular inversion probes (MIP) to capture target exons, splice sites plus 5' and 3' sequences and to detect F8 intron 1 and 22 inversions. This allows screening for all F8 and F9 variants in one sequencing run of multiple samples (196 or 392). Sequence variants identified are subsequently confirmed by a diagnostic laboratory. After having identified variants in genes of interest through these processes, a systematic procedure determining their likely pathogenicity should be applied. Several scientific societies have prepared guidelines. Systematic analysis of the available evidence facilitates reproducible scoring of likely pathogenicity. Documentation of frequency in population databases of variant prevalence and in locus-specific mutation databases can provide initial information on likely pathogenicity. Whereas null mutations are often pathogenic, missense and splice site variants often require in silico analyses to predict likely pathogenicity and using an accepted suite of tools can help standardize their documentation.

Leukocyte Adhesion Deficiency-I: Clinical and Molecular Characterization in an Indian Population.

To describe clinical and flow cytometric immunophenotyping details of 26 patients of Leukocyte adhesion deficiency-I (LAD-I) along with molecular characterization of 7 patients. Diagnosis of LAD-I was suspected on the basis of clinical features, white blood cell count and absolute neutrophil counts and flow cytometric assessment of expression of CD18 and CD11(a, b, c) on leukocytes. Mutation analysis was performed using DNA PCR and conformation sensitive gel electrophoresis (CSGE) technique followed by sequencing. All the patients were symptomatic by the age of 6mo, with history of recurrent bacterial infections involving skin, mucosa or umbilical cord (omphalitis) being the most frequent presenting symptoms. White blood cells (WBC) and absolute neutrophil counts (ANC) were markedly elevated, without any specific morphological findings. On flow cytometry, CD11a and CD11c showed moderate correlation with CD18 expression. Mutation analysis was performed in 7 patients and six different mutations (4 missense, 2 nonsense and 1 splice site) were identified, all of which were homozygous in nature. A presentation of repeated bacterial infections during infancy, especially omphalitis, with markedly elevated absolute neutrophil counts should trigger investigations for LAD-I including flow cytometric analysis of CD11/CD18 expression.

Characterisation and Clinical Significance of FLT3-ITD and non-ITD in Acute Myeloid Leukaemia Patients in Kelantan, Northeast Peninsular Malaysia.

Mutations of the FMS-like tyrosine kinase-3 (FLT3) receptor gene may promote proliferation via activation of multiple signaling pathways. FLT3-internal tandem duplication (FLT3-ITD) is the most common gene alteration found in patients diagnosed with acute myeloid leukaemia (AML) and has been associated with poor prognosis. We performed mutational analysis of exons 14-15 and 20 of the FLT3 gene in 54 AML patients using PCR-CSGE (conformational sensitive gel electrophoresis) followed by sequencing analysis to characterise FLT3 mutations in adult patients diagnosed with AML at Hospital USM, Kelantan, Northeast Peninsular Malaysia. FLT3 exon 14-15 mutations were identified in 7 of 54 patients (13%) whereas no mutation was found in FLT3 exon 20. Six ITDs and one non-ITD mutation were found in exon 14 of the juxtamembrane (JM) domain of FLT3. FLT3-ITD mutations were associated with a significantly higher blast percentage (p-value=0.008) and white blood cell count (p-value=0.023) but there was no significant difference in median overall survival time for FLT3-ITD+/FLT3-ITD- within 2 years (p-value=0.374). The incidence of FLT3-ITD in AML patients in this particular region of Malaysia is low compared to the Western world and has a significant association with WBC and blast percentage.

Association of PALB2 sequence variants with the risk of familial and early-onset breast cancer in a South-American population.

BackgroundGermline mutations in PALB2 have been identified in approximately 1% of familial breast cancer (BC) in several populations. Nevertheless its contribution in the South-American population is unknown. The goal of this study was to determine the prevalence of PALB2 mutations in the Chilean population.Methods100 Chilean BRCA1/2-negatives familial BC cases were included for the PALB2 mutation analysis. We use conformational sensitive gel electrophoresis and direct sequencing. Using a case-control design, we studied the identified variants in 436 BC cases and 809 controls to evaluate their possible association with BC risk.ResultsNo pathogenic mutations were detected. We identified three variants, the variant c.1861C > A not previously described was found in one of the 436 cases and none of the 809 controls. The bioinformatic analyses indicate that this variant probably is not pathogenic. PALB2 c.1676A > G (rs152451A/G) and c.2993C > T (rs45551636C/T) variants were significantly associated with increased BC risk only in cases with a strong family history of BC (OR = 1.9 [CI 95% 1.3-2.8] p < 0.01 and OR = 3.3 [CI 95% 1.4-7.3] p < 0.01, respectively). The rs152451A/G-rs45551636C/T composite genotype produce increase of the BC risk in cases with a strong family history of BC (OR = 3.6 [CI 95% 1.7-8.0] p = 0.003). The rs152451-G/rs45551636-C and rs152451-G/rs45551636-T haplotypes were associated with an increased BC risk only in cases with a strong family history of BC (OR = 1.6 [CI 95% 1.0-2.5] p = 0.05 and OR = 3.7 [CI 95% 1.8-7.5] p < 0.001, respectively).ConclusionOur results suggest that PALB2 c.1676A > G and c.2993C > T play roles in BC risk in women with a strong family history of BC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1033-3) contains supplementary material, which is available to authorized users.

Melanocortin-4 receptor (MC4R) polymorphisms are associated with growth and meat quality traits in sheep.

The involvement of melanocortin 4 receptor gene (MC4R) in food intake and body weight regulation is well characterized. MC4R mutations are the most frequent monogenic cause of human obesity. Significant associations have been revealed between MC4R mutations and productive traits in pigs, cattle and poultry. Herein, fluorescence-based conformation sensitive gel electrophoresis was used to identify two single nucleotide polymorphisms (SNPs) in the coding region (93G>A and 292G>A) and two SNPs in the 3'-UTR area (1016G>A and 1240T>C) of MC4R gene in 132 German Merino sheep. We found that the 1016G>A mutation in the 3'-UTR was significantly associated with body weight at 120 and 180 days, average daily gain, back fat thickness and loin-eye area. Allele A located at the 292th position of MC4R gene representing Arg98 was associated with significantly higher loin-eye area in sheep. For the synonymous 93G>A mutation, A allele carrier animals had higher back fat thickness. Our results provide evidence that the MC4R gene may be a candidate gene for growth and meat quality traits with MC4R SNPs being potentially valuable as genetic markers for economic traits in German Merino sheep.

Allelic Variability in the Intronic Region of the Fibroin Heavy-Chain Gene in Silkworm Bombyx mori L. Strains of Brazilian Germplasm Bank.

The domesticated silkworm Bombyx mori L. is currently found only in germplasm banks. Therefore, characterization and conservation of this genetic resource is crucial. Based on previous studies that revealed nucleotide differences in silkworm strains, the intron of the fibroin heavy chain gene (H-fib) can be used for molecular silkworm characterization. The H-fib gene has two exons and a unique intron, and encodes the principal component of the silk fiber, the fibroin heavy chain. Therefore, this study aimed to identify the genetic variability of the unique intron of H-fib gene of 20 silkworm strains maintained at the Universidade Estadual de Maringá Brazilian Germplasm Bank (UBGB) by conformation-sensitive gel electrophoresis (CSGE) and nucleotide sequencing. Genomic DNA extracted from silkworm moths was PCR amplified. CSGE revealed that most of the analyzed silkworm strains had only homoduplex molecules. However, DNA from the Japanese strains B106, B82, and M12-2 had two extra DNA fragments produced by heteroduplex molecules, revealing variation between alleles. Sequencing of the H-fib intron was used to confirm the variation previously detected by CSGE and detected a significant polymorphism characterized by a 17-base pair (bp) deletion, a 2-bp insertion, and eight nucleotide substitutions. Although genetic and allelic variability was detected in some silkworm strains, the intron of the H-fib gene revealed not to be the best molecular marker for the characterization of B. mori strains from UBGB.

Conformational Sensitive Gel Electrophoresis (CSGE) as a method for NPM1 mutational screening in patients with Acute Myeloid Leukaemia

IntroductionNucleophosmin gene (NPM1) exon 12 is the most common mutated gene found in Acute Myeloid Leukaemia (AML), which is present in 25–35%. NPM1 gene encodes for nucleophosmin (NPM) protein, which contains 294 amino acids. The NPM protein is an abundant nucleolar phosphoprotein that shuttles between the nucleus and cytoplasm. It contributes to promotion of ribosome biogenesis, regulation of centrosomal duplication during cell division, interaction with tumor suppressor gene and control of various nuclear proteins. ObjectiveTo screen for NPM1 exon 12 mutationsin AML patients diagnosed at Hospital Universiti Sains Malaysia (HUSM) using Conformational Sensitive Gel Electrophoresis (CSGE). MethodsTotal genomic DNAs were obtained from bone marrow aspirates or peripheral blood samples taken at diagnosis from 67 AML patients diagnosed at HUSM [French-American-British (FAB) subtypes: M0=1, M1=2, M2=10, M3=22, M4=9,M5=12,M6=2,M7=1, Not Otherwise Spesified (NOS)=8]. Patients were screened for NPM1 exon 12 mutations by CSGE. Cases displaying abnormal CSGE profiles were directly sequenced. Results & DiscussionMutation of NPM1exon 12 was present in 7/67 AML cases (10%) and all cases were females. They consisted of two M1, two M5b, one M2, one M3 and one NOS FAB subtypes. Most of the mutations identified as insertional (ins) mutation (5/7), 4 patients having ins 962–963 CTGG (2), CATG (1) and CTGT (1). One patient having ins at 969 CATG and followed with stop codon (TGA). One patient having single base deletion (del) at 966 and became TGA (stop codon) while another patient having ins at 956 TGGA and followed with del 958–969. ConclusionOur data showed high frequency of NPM1 in AML-M1 and M5b, which is highly associated in female patients. The CSGE was found to be an inexpensive, simple and reliable test to be used in screening of NPM1 mutations.

Prenatal diagnosis in severe von Willebrand disease families from India using combination of phenotypic and genotypic assays

This study aimed to offer genetic diagnosis to affected type 3 severe von Willebrand disease families. Thirteen families were referred for prenatal diagnosis during the first and second trimesters of pregnancy. Prenatal diagnosis was offered by chorionic villus sampling between 11 and 12 weeks and by cordocentesis between 18 and 19.5 weeks of gestation. Phenotypic analysis included FVIII:C and von Willebrand factor antigen assays. A combination of molecular biological techniques which included PCR-restriction fragment length polymorphism technique using intron 40 variable number tandem repeat (VNTR) markers, conformation sensitive gel electrophoresis, direct DNA sequencing, and multiple ligation probe amplification (MLPA) were used to offer genotyic diagnosis in the remaining families. Diagnosis was offered by intron 40 VNTR analysis in eight families. In one family, the diagnosis was given by direct mutation detection technique, whereas in another diagnosis was given by MLPA technique as the index case showed the presence of large deletion within von Willebrand factor. In three families, diagnosis was offered by cordocentesis on the basis of phenotypic assays, further confirmed by genotyping. Both first and second-trimester prenatal diagnoses could be successfully offered using a combination of phenotypic and genotypic techniques to all severe von Willebrand disease families.

The correlation between p16 expression and INK4a locus mutation with grades and stages in oral squamous cell carcinoma.

p16INK4a is a tumor suppressor gene playing a critical role. Researches have indicated the gene to be altered in oral squamous cell carcinoma. Present studies have tried to assess the correlation between p16INK4a expression and INK4a locus mutation in relation to grades and stages of this tumor. Expression of p16INK4a was studied immunohistochemically in 58 oral squamous sell carcinoma samples and INK4a locus mutation was determined by polymerase chain reaction (PCR) and conformation sensitive gel electrophoresis (CSGE). Expression of p16INK4a was higher in stage1 compared to stage 2, 3, and 4 (P = 0.234). The difference was not significant in grade 1, 2, and 3 (P = 0.671). The average values of total score (TS) were significantly higher in stage1 compared to stage 2, 3, and 4 (P = 0.035). The average values of complete score (CS) were higher in stage 1 compared to stage 2, 3, and 4 (P = 0.061). The research did not show a significant correlation between lymph node involvement and p16INK4a expression (P = 0.491). It seems that 5.1% (3/58) of samples have mutation in INK4a locus. Loss of p16INK4a expression occurred in initial stages of oral squamous cell carcinoma. Evaluation of TS and CS for p16INK4a might be a useful clinical indicator concerning the tumor. However, gene mutation is believed to have minor rate of genetic alteration in carcinogenesis.

The Canadian “National Program for hemophilia mutation testing” database: A ten‐year review

A reference genotyping laboratory was established in 2000 at Queen's University, Kingston, to provide genetic testing for Hemophilia A (HA) and B (HB) and create a Canadian mutation database. Canadian hemophilia treatment centers and genetics clinics provided DNA and clinical information from November 2000 to March 2011. The factor VIII (F8) gene was analyzed in 1,177 patients (47% of HA population) and 787 female family members and the factor IX (F9) gene in 267 patients (47% of HB population) and 123 female family members, using Southern Blot, PCR, conformation sensitive gel electrophoresis, and/or direct sequencing. The mutation detection rates for HA and HB were 91% and 94%, respectively. 380 different F8 mutations were identified: inversions of intron 22 and intron 1, 229 missense, 45 nonsense, eight deletions, 70 frameshifts, 25 splice site, and one compound mutation with a splice site and intron 1 inversion. Of these mutations, 228 were novel to the Hemophilia A Database (HADB, http://hadb.org.uk/). A total 125 different F9 mutations were identified: 80 missense, 12 frameshift, 12 splice site, nine nonsense and seven promoter mutations, three large deletions, and two compound mutations with both missense and nonsense changes. Of these mutations, 36 were novel to the International Haemophilia B Mutation database (http://www.kcl.ac.uk/ip/petergreen/haemBdatabase.html). The Canadian F8 and F9 mutation database reflects the allelic heterogeneity of HA and HB, and is similar to previously described populations. This report represents the largest and longest duration experience of a national hemophilia genotyping program documented, to date.

Mitochondrial DNA variant m.15218A > G in Finnish epilepsy patients who have maternal relatives with epilepsy, sensorineural hearing impairment or diabetes mellitus.

BackgroundMitochondrial diseases caused by mutations in mitochondrial DNA (mtDNA) affect tissues with high energy demand. Epilepsy is one of the manifestations of mitochondrial dysfunction when the brain is affected. We have studied here 79 Finnish patients with epilepsy and who have maternal first- or second-degree relatives with epilepsy, sensorineural hearing impairment or diabetes mellitus.MethodsThe entire mtDNA was studied by using conformation sensitive gel electrophoresis and PCR fragments that differed in mobility were directly sequenced.ResultsWe found a common nonsynonymous variant m.15218A > G (p.T158A, MTCYB) that occurs in haplogroup U5a1 to be more frequent in patients with epilepsy. The m.15218A > G variant was present in five patients with epilepsy and in four out of 403 population controls (p = 0.0077). This variant was present in two branches in the phylogenetic network constructed on the basis of mtDNA variation among the patients. Three algorithms predicted that m.15218A > G is damaging in effect.ConclusionsWe suggest that the m.15218A > G variant is mildly deleterious and that mtDNA involvement should be considered in patients with epilepsy and who have a maternal history of epilepsy, sensorineural hearing impairment or diabetes mellitus.

Molecular basis of quantitative fibrinogen disorders in 27 patients from India

Congenital fibrinogen deficiency is an extremely rare (1:1 000 000) hereditary bleeding disorder caused by defects in genes coding for fibrinogen Aα-, Bβ- and γ-chains, respectively. We report here the molecular basis of fibrinogen deficiency in a large series of patients from India. Twenty-seven patients with clinical features suggestive of fibrinogen deficiency and with prolonged plasma clotting times and low fibrinogen levels were studied. Genomic DNA was screened for mutations in the fibrinogen alpha (FGA), beta (FGB), gamma (FGG) genes by PCR and conformation sensitive gel electrophoresis. Fourteen different disease-causing mutations including frameshifts (51.9%), splice site (22.2%), missense (18.5%) and nonsense mutation (7.4%) were identified in 27 patients. Thirteen of them were novel, including seven frameshifts (fibrinogen Aα: p.Asp296 fs*59, p.Thr466 fs*17 and p.Lys575 fs*74; fibrinogen Bβ: p.Gly414 fs*2 and fibrinogen γ: p.Ser81 fs*5, p.Lys185 fs*13 and p.Asp278_279 fs*17), three splice site mutations (FGA gene c.364+1G>A; c.510+2 T>G; FGB gene c.851+1G>A), two missense substitutions (fibrinogen Bβ: p.Gly288Ser; p.Arg445Thr) and a nonsense mutation in fibrinogen Aα (p.Tyr127*). Two common mutations (FGA: c.364+1G>A, n = 6, FGG: p.Lys185 fs*13, n = 7) affecting 13 patients were identified in this series, suggesting that these mutations could be screened first in Indian patients with fibrinogen deficiency. The molecular data presented here is the largest series of patients with fibrinogen deficiency reported so far, adding significantly to the mutation database of this condition. It also helps create an algorithm for its genetic diagnosis in India.

Assessment of the F9 genotype-specific FIX inhibitor risks and characterisation of 10 novel severe F9 defects in the first molecular series of Argentinian patients with haemophilia B

In haemophilia B (HB) (factor IX [FIX] deficiency), F9 genotype largely determines clinical phenotype. Aimed to characterise Argentinian families with HB, this study presents F9 genotype frequencies and their specific FIX inhibitor risk and 10 novel F9 mutations. Ninety-one DNA samples from HB patients and relatives were subjected to a new scheme: a primary screen for large deletions, a secondary screen for point mutations using conformation sensitive gel electrophoresis, DNA-sequencing and bioinformatic analysis. Our unbiased HB population (N=52) (77% with severe, 11.5% moderate and 11.5% mild HB) showed 32 missense (61.5%), including three novel mutations predicting specific structural/functional defects in silico , seven nonsense (13.5%) (one novel), five large deletions, four splice including three novel mutations affecting predicted splicing scores, three indels (two novel) and one Leiden mutation. Our comprehensive HB population included five patients with long-lasting FIX inhibitors: three nonsense (p.E35* (novel), p.R75*, p.W240*) and two entire- F9 deletions. Another patient with an indel (p.A26Rfs*14) developed transient inhibitors. A case-control analysis, based on our global prevalence of 3.05% for developing inhibitors in HB revealed that missense mutations were associated with a low risk odds ratio (OR) of 0.05 and a prevalence of 0.39%, whereas nonsense and entire- F9 deletions had significantly higher risks (OR 11.0 and 32.7) and prevalence (14.3% and 44.5%, respectively). Our cost-effective practical approach enabled identification of the causative mutation in all 55 Argentine families with HB, analysis of the molecular pathology of novel F9 defects and determination of mutation-associated FIX inhibitor risks.