Whole genome amplification on DNA from filter paper blood spot samples: an evaluation of selected systems.

As the number of single-nucleotide polymorphism (SNP) screening and other mutation scanning studies have increased explosively, following the development of high-throughput instrumentation, it becomes even more important to have sufficient template DNA. The source of DNA is often limited, especially in epidemiological studies, which require many samples as well as enough DNA to perform numerous SNP screenings or mutation scannings. Therefore, the aim is to solve the problem of stock DNA limitation. This need has been an important reason for the development of whole genome amplification (WGA) methods. Several systems are based on Phi29 polymerase multiple displacement amplification (MDA) or on DNA fragmentation (OmniPlex). Using TaqMan SNP genotyping assays, we have tested four WGA systems -- AmpliQ Genomic Amplifier Kit, GenomiPhi, Repli-g, and GenomePlex -- on DNA extracted from Guthrie cards to evaluate the amplification bias, concordance- and call rates, cost efficiency, and flexibility. All systems successfully amplified picograms of DNA from Guthrie cards to micrograms of product without loss of heterozygosity and with minimal allelic bias. A modified AmpliQ set up was chosen for further evaluation. In all, 2,000 SNP genotyping results from amplified and nonamplified samples were compared and the concordance rates between the samples were 99.7%. The call rate using the TaqMan system was 99.8%. DNA extracted from Guthrie cards and amplified with one of the four evaluated WGA systems is applicable in epidemiological genetic screenings. System choice should be based on requirements for system flexibility, product yield, and use in subsequent analysis.

[Screening for fragile X syndrome. International experiences]. / Screening for fragilt X-syndrom. Internationale erfaringer.

Fragile X syndrome (FXS) is the most prevalent cause of inherited mental retardation, and calculations show that there are approximately 700 non-diagnosed cases in Denmark. Since the disease is severe, screening for FXS should be considered in order to improve genetic counselling. International experience indicates that efforts should initially be on active case finding among persons with learning difficulties and subsequently on cascade screening to identify carriers. Prenatal screening programmes may be an option when reliable high-throughput diagnostic methods are available.

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.

P53 mutations in tissue from Danish ovarian cancer patients: from the Danish "MALOVA" ovarian cancer study.

OBJECTIVES: The p53 gene, a tumor suppressor gene located on the short arm of chromosome 17 (17p13), has been found mutated in 30-80% of epithelial ovarian cancers (OC), with the most frequently detected mutations in the conserved regions of the gene. A small number of studies investigated the survival of patients with p53 mutations in OC, but their conclusions are not in agreement. METHODS: We analyzed the frequency of p53 mutations in 124 Danish women with OC, using Single-Stranded Conformation Polymorphism analysis in addition with DNA sequencing and evaluated if mutations correlated with clinicopathological parameters and with patient survival. RESULTS: Thirty-five (28%) ovarian tumors were found to contain one or more p53 variations, two of which were considered polymorphisms. Twenty-seven (82%) mutations were single nucleotide substitutions of which 23 (85%) were missense mutations and therefore led to amino acid substitutions. Significantly shorter survival was found for stage III/IV patients with a p53 missense mutation compared to stage III/IV OC patients with wild type p53 (P = 0.0018). Multivariate Cox regression analysis restricted to 107 OC patients with a p53 missense mutation or p53 wild type in the tumor tissue and with information on radicality of primary surgery showed that missense p53 mutation (HR = 2.5, 95% CI: 1.21-4.98), radicality after primary surgery (HR = 1.7, 95% CI: 1.04-2.88), tetranectin (mg/l: HR = 0.78, 95% CI: 0.67-0.91) and stage (I vs. III: HR = 0.30, 95% CI: 0.10-0.92, II vs. III: HR = 0.24, 95% CI: 0.05-1.05, IV vs. III: HR = 2.70, 95% CI: 1.22-5.98) were independent prognostic factors. CONCLUSION: Missense mutations in the conserved regions of p53 may be of prognostic value in Danish OC patients.

Adverse reactions to injectable soft tissue permanent fillers.

BACKGROUND: Synthetic injectable facial fillers with a permanent effect are widely atoxic and nonimmunogenic, but they differ with respect to composition and in chemical and biologic characteristics. Yet, they all act as foreign bodies in the tissues eliciting a host response that try to remove the gel. Inflammatory nodules may develop at the sites of injection-for some fillers, many years later, for others, not. Why is that? METHODS: Biopsies were contributed by various plastic surgeons from Europe and Australia after requests were made at international congresses and workshops. The study was based on (a) 5 biopsies from unreactive tissue obtained at different times after injection of polyacrylamide hydrogel (Aquamid); (b) 28 biopsies from intermediate or late inflammatory nodules after injection of polyacrylamide hydrogel (Aquamid) (20 cases), a hyaluronic acid-polyhydroxyethylmethacrylate/ethylmethacrylate gel (Dermalive) (2 cases), and a gel consisting of polylactic acid in mannitol/carbomethoxycellulose (New-Fill) (6 cases); and (c) a review of the literature on adverse reactions after injection with permanent fillers. RESULTS: Clinically unreactive tissues after injection with Aquamid showed modest or no host reaction. Inflammatory nodules showed an increased foreign body reaction and a bacterial infection after injection with Aquamid, and a combination of moderate foreign body reaction, fibrosis, and in some cases also bacterial infection after injection with Dermalive and New-Fill. According to the literature, inflammatory nodules occur no later than 1 year after injection with polyacrylamide hydrogel, but up to 6 years after injection of combination gels (Artecol), and up to 28 years after injection of silicone gel. CONCLUSIONS: Inflammatory nodules are likely to be caused by a low-grade infection maintained within a biogfilm surrounding the hydrophobic silicone gel and the combination gels. Aquamid gel may prevent formation of a biofilm through its high water-binding capacity, explaining why late inflammatory nodules are not seen after injection of this polyacrylamide hydrogel product.

One third of Danish hypertrophic cardiomyopathy patients with MYH7 mutations have mutations [corrected] in MYH7 rod region.

Familial hypertrophic cardiomyopathy (FHC) is, in most cases, a disease of the sarcomere, caused by a mutation in one of 10 known sarcomere disease genes. More than 266 mutations have been identified since 1989. The FHC disease gene first characterized MYH7, encodes the cardiac beta-myosin heavy chain, and contains more than 115 of these mutations. However, in most studies, only the region encoding the globular head and the hinge region of the mature cardiac beta-myosin heavy chain have been investigated. Furthermore, most studies carries out screening for mutations in the most prevalent disease genes, and discontinues screening when an apparent disease-associated mutation has been identified. The aim of the present study was to screen for mutations in the rod region of the MYH7 gene in all probands of the cohort, regardless of the known genetic status of the proband. Three disease-causing mutations were identified in the rod region in four probands using capillary electrophoresis single-strand conformation polymorphism as a screening method. All mutations were novel: N1327K, R1712W, and E1753K. Two of the probands had already been shown to carry other FHC-associated mutations. In conclusion, we show that in the Danish cohort we find one third of all MYH7 mutations in the rod-encoding region and we find that two of the patients carrying these mutations also carry mutations in other FHC disease genes stressing the need for a complete screening of all known disease genes in FHC-patients.

Identification and molecular characterization of the gene encoding coli surface antigen 20 of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea among children living in developing countries and of travelers' diarrhea. Current ETEC vaccine designs aim to induce an anti-colonizing immunity by including the ETEC surface colonization factor antigens. We isolated and characterized the structural gene of the coli surface antigen 20 (CS20). CS20 has an N-terminal amino acid sequence similar to that of CS18. We therefore used a DNA fragment carrying the CS18 fotA gene as a probe in a hybridization assay to detect the corresponding gene in a CS20-positive strain isolated from an Indian child. Cross hybridizing DNA was isolated and found to contain an open reading frame encoding a polypeptide of 195 amino acids, including a 22 amino acid signal peptide. The gene, which we named csnA, shows a high degree of identity to the major fimbrial subunits of CS12, CS18 and F6 (also referred to as 987P), a CS of porcine ETEC. The coding region of csnA was inserted into an expression system to generate a polypeptide confirmed to be CS20 by Western blot. A CS20 colony hybridization assay using a DNA probe derived from csnA was developed.

Genetic and phenotypic characterization of mutations in myosin-binding protein C (MYBPC3) in 81 families with familial hypertrophic cardiomyopathy: total or partial haploinsufficiency.

Mutations in the MYBPC3 gene, encoding the sarcomere protein myosin-binding protein C, are among the most frequent causes of autosomal dominant familial hypertrophic cardiomyopathy (FHC). We studied the frequency, type, and pathogenetic mechanism of MYBPC3 mutations in an unselected cohort of 81 FHC families, consecutively enrolled at a tertiary referral center. Nine mutations, six of which were novel, were found in 10 (12.3%) of the families using single-strand conformation polymorphism and DNA sequencing. A frameshift mutation in exon 2 clearly suggests that haploinsufficiency is a pathogenetic mechanism in FHC. In addition, splice site mutations in exon 6 and intron 31, a deletion in exon 13, and a nonsense mutation in exon 25, all lead to premature termination codons, most likely causing loss of function and haploinsufficiency. Furthermore, there were two missense mutations (D228N and A833 T) and one in-frame deletion (DeltaLys813). A considerable intrafamilial variation in phenotypic expression of MYBPC3-based FHC was noted, and we suggest that mutations influencing stability of mRNA could play a role in the variable penetrance and expressivity of the disease, perhaps via partial haploinsuffciency.

Evaluation of a polymorphism in intron 2 of the p53 gene in ovarian cancer patients. From the Danish "Malova" Ovarian Cancer Study.

BACKGROUND: The p53 gene is frequently mutated in various human tumours. In addition, single nucleotide polymorphisms are often observed in exons and introns of the p53 gene in normal tissues and tumours. MATERIALS AND METHODS: A total of 210 blood and tissue samples from 182 ovarian cancers (OC) and 28 ovarian borderline tumours, in addition to blood samples from 72 healthy women, were analysed. The used analyses were PCR and SSCP. The distinguishable SSCP patterns were confirmed by DNA sequencing. RESULTS: A polymorphism located at position 38 in intron 2 of the p53 gene was studied in blood and tumour tissues from Danish ovarian tumour patients and in blood from controls. Significant differences were found between the distributions of the genotypes in blood samples compared to the corresponding tissue samples (p = 0.0002). A tendency towards a significant difference in survival was observed between OC stage II patients with a shift from one genotype in the blood to another genotype in the tissue and patients with no shift (p = 0.05). In multivariate COX regression analysis restricted to stage III OC patients, the only independent factors found were shift, serum-tetranectin and age. CONCLUSION: A shift from one p53 intron 2 genotype in the blood to another genotype in the tissue may be a prognostic factor in ovarian cancer patients.

K-ras alterations in Danish ovarian tumour patients. From the Danish "Malova" Ovarian Cancer study.

OBJECTIVE: Activation of ras oncogenes has been demonstrated in ovarian tumours. All the reported studies are based on a relatively small number of patients and the results therefore remain a subject of debate. METHODS: In this study, we analyzed the presence of mutations at codons 12 and 13 of the K-ras gene in 165 Danish women with ovarian tumours, including 138 invasive ovarian cancers and 27 borderline ovarian tumours, using a restriction fragment length polymorphism-polymerase chain reaction technique and evaluated whether such alterations were associated with the clinicopathological parameters of the patients and survival. RESULTS: K-ras codon 12 gene mutations were found in 8.7% of ovarian cancer patients and in 14.8% of the borderline ovarian tumour patients. A K-ras codon 13 gene mutation was found in 1.5% of ovarian cancer patients. K-ras mutations were found with a significantly higher frequency in mucinous tumours compared to serous tumours (P = 0.011). CONCLUSIONS: Mutation frequency was correlated with the histological type of tumour, but not with stage, radicality of operation, and age. Furthermore, no significant difference in survival was demonstrated between patients with or without K-ras mutation, neither in the univariate nor in the multivariate survival analyses.

Capillary electrophoresis-based single strand DNA conformation analysis in high-throughput mutation screening.

The generation of the draft human genome sequence has created new possibilities for diagnosis, prevention, and treatment of human disease. One consequence of these new possibilities is an increasing need for methods and technology that can be used for high-throughput screening for mutations in large DNA sample materials. In recent years, a number of mutation screening methods have emerged that are based on the analysis of sequence-dependent changes in the conformation of single- and double-stranded DNA using capillary electrophoresis. Common features of these methods are high sensitivity and reproducibility as well as the possibility for automation and massive parallelization. Thus, at present they are among the most attractive technologies for high-throughput mutation screening. This review describes the recent advances in capillary electrophoresis-based single strand conformation polymorphism (CE-SSCP) for detection of unknown mutations, and assesses its practical usability for high-throughput mutation screening based on the available literature. In addition, future prospects are outlined in light of the recent advances in microchip-based capillary electrophoresis.

Outcome of clinical versus genetic family screening in hypertrophic cardiomyopathy with focus on cardiac beta-myosin gene mutations.

OBJECTIVE: Familial hypertrophic cardiomyopathy (FHC) is caused by mutations in genes encoding cardiac sarcomere proteins. Although available, genetic analyses are generally not used clinically. In the present study, we evaluated the outcome of clinical vs. genetic screening of family members with specific focus on mutations in the cardiac beta-myosin heavy chain (MYH7) gene. METHODS: A consecutive cohort of 68 FHC probands and their families (395 persons) of Danish origin was evaluated including patient- and family histories, physical examinations, electrocardiogram and echocardiography. Mutation screening was performed by a combination of single strand conformation/heteroduplex analysis and direct sequencing. RESULTS: Eight different MYH7 gene mutations were identified in nine (13%) families (96 persons). In eight (89%) of the families, major cardiac events had occurred. Myectomy or percutaneous septal alcohol ablation had been performed in a higher number of MYH7 probands i.e. in five of nine (56%) as compared to 10 of 59 (17%) (P<0.05) non-MYH7 mutation probands. Neither echocardiographic nor ECG findings were useful to distinguish MYH7 from non-MYH7 probands. Between adult MYH7 mutation-carriers (n=38) and their non-carrier relatives (n=39), low sensitivity and specificity of the clinical diagnostic criteria tested were observed and minor clinical diagnostic criteria alone were not useful for identification of mutation carriers. By genetic screening of relatives with no or only minor hypertrophy on echocardiography, i.e. a priori possible mutation-carriers normally recommended clinical follow-up-the diagnosis was excluded in 52 (83%) persons. In addition, six relatives with secondary hypertrophy were identified as non-carriers. CONCLUSION: Neither echocardiographic nor ECG findings were useful to distinguish MYH7 from non-MYH7 probands. Extension of screening to include genetic analyses offered a marked diagnostic advantage as compared to clinical screening alone in FHC families.

High-throughput single strand conformation polymorphism mutation detection by automated capillary array electrophoresis: validation of the method.

Capillary array electrophoresis (CAE) is a novel technique, which allows for high throughput analysis of DNA fragments. When screening for mutations in whole populations or large patient groups it is necessary to have robust and well-characterized setups for high throughput analysis. For large-scale mutation screening, we have developed procedures for single strand conformation polymorphism (SSCP) assays using CAE (CAE-SSCP) whereby we may increase both the sensitivity and the throughput compared to conventional SSCP analysis. In this study we have validated CAE-SSCP by 1) comparing detection by slab-gel based SSCP with CAE-SSCP of mutations in the MYH7, MYL2, and MYL3 genes encoding sarcomere proteins from patients suffering from hypertrophic cardiomyopathy; and 2) by constructing a series of 185 mutants having substitution mutations, as well as insertion/deletion mutations, or some combinations of these, in different sequence contexts in four exons and different positions relative to the end of the amplicon (three from the KCNQ1 gene, encoding a cardiac potassium channel, and one from the TNNI3 gene encoding cardiac troponin I). The method identified 181 out of 185 mutations (98%), and the data suggest that the position of mutation in the fragment had no effect on the sensitivity. Analysis of the specificity of the method showed that only very few mutants could not be distinguished from each other and there were no false positives.

Single-strand conformation polymorphism analysis using capillary electrophoresis.

Single-strand conformation polymorphism (SSCP) is one of the most frequently used mutation detection methods. This unit describes a method of SSCP with automated analysis by capillary electrophoresis in order to increase the capacity and throughput. A protocol is provided for sample preparation. For a medium throughput laboratory, a single capillary instrument, as described in this unit, may be quite sufficient. In many cases, however, screening for mutations in large population groups requires a high throughput, and this is best obtained through the use of a multi-capillary instrument, as discussed.

Distribution of p53 codon 72 polymorphisms in ovarian tumour patients and their prognostic significance in ovarian cancer patients.

BACKGROUND: The p53 gene is frequently mutated in various human tumours. In addition, single nucleotide polymorphisms are often observed in exons and introns of the p53 gene in normal tissues and tumours. MATERIALS AND METHODS: The prevalence of a polymorphism involving codon 72 of exon 4 in the p53 gene was studied in peripheral white blood cells and tumour tissues from Danish ovarian tumour patients and in peripheral white blood cells from controls. The analyses were performed by Polymerase Chain Reaction (PCR), Restriction Fragment Length Polymorphism (RFLP) and DNA sequencing. The amino acid residue at this position is either arginine (p53-Arg) or proline (p53-Pro). RESULTS: There was no correlation between the frequency of the three genotypes (Pro/Pro, Arg/Arg and Arg/Pro) and age, stage or histological type of the tumour. A significant difference in survival was observed between ovarian cancer stage III patients with a shift from one genotype in the blood to another genotype in the tissue and patients with no shift (p=0.0216). CONCLUSION: Kaplan-Meier survival analyses and multivariate Cox regression analysis stratified to stage III ovarian cancer patients showed that a shift from one genotype in the blood to another genotype in the tissue is a prognostic factor in Danish ovarian cancer patients.