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PubMed2002

Exon concatenation to increase the efficiency of mutation screening by DGGE.

For genes that have a substantial number of exons and long intronic sequences, mutation screening by denaturing gradient gel electrophoresis (DGGE) requires the amplification of each exon from genomic DNA by PCR. This results in a high number of fragments to be analyzed by DGGE so that the analysis of large sample sets becomes labor intensive and time consuming. To address this problem, we have developed a new strategy for mutation analysis, lexon-DGGE, which combines the joining of different exons by PCR (also known as lexons) with a highly sensitive technique such as DGGE to screen for mutations. The lexon technique is based on the concatenation of several exons, adjacent or not, from genomic DNA into a single DNA fragment so that this approach could simultaneously be used to check the mutational status of several small genes. To show the feasibility of the approach, we have used the lexon-DGGE technique to analyze all coding exons, intron-exon junctions, noncoding exon 1, and part of the noncoding region of exon 11 of the TP53 gene. The validity and performance of the technique were confirmed by using negative and positive controls for each of the DNAfragments analyzed.

DNA Mutational Analysis↗
PubMed2005

Diagnostic DHPLC Quality Assurance (DDQA): a collaborative approach to the generation of validated and standardized methods for DHPLC-based mutation screening in clinical genetics laboratories.

Genetic testing in a clinical diagnostic environment must be subject to rigorous quality control procedures, in order to ensure consistency and accuracy of results. Denaturing high performance liquid chromatography (DHPLC) has become a standard prescreening tool for mutation detection, offering very high efficiency and sensitivity of detection. Despite the relatively simple software-assisted assay setup, DHPLC is a complex assay, and quality control is reliant on ensuring optimal instrument performance, excellent assay design and validation, and sufficient user training and proficiency to interpret results. We describe here a unique collaborative effort by a group of diagnostic clinical genetics laboratories with DHPLC expertise who, together with the manufacturer of one of the most widely used DHPLC platforms, have generated standard operating procedures (SOPs) for instrument operation and maintenance, and for mutation detection by DHPLC. We also describe the validation of a disease-specific SOP for DHPLC assisted mutation screening of the MECP2 gene associated with Rett syndrome. The proposed SOP was validated, and used independently in two laboratories to introduce MECP2 testing. In addition, we provide empirically derived normal ranges for the WAVE System Mutation Standards, which are essential for optimal instrument performance. This effort was initiated to try to standardize DHPLC-based mutation screening procedures across laboratories, and so increase the overall quality of this testing method. This endeavor will thus save each laboratory from having to generate SOPs on their own, which is a lengthy and laborious task. In this respect, we define "generic" SOPs as procedures that are easily adaptable to the individual laboratories' quality systems.

Chromatography, High Pressure Liquid↗
PubMed2004

The usefulness of buccal swabs for mutation screening in patients with suspected corneal dystrophies.

PURPOSE: To make ophthalmologists aware of the usefulness of buccal swabs for the collection of cells from which DNA may be extracted to be used in genetic screening of patients with known or suspected inherited ocular disorders, such as corneal dystrophies. DESIGN: Illustrative interventional case report. METHODS: Buccal epithelial swabs were collected from a 3-year-old boy with a presumed corneal dystrophy and his unaffected parents. Swabs were mailed to a laboratory where transforming growth factor-beta-induced gene (TGFBI) mutation screening was performed. MAIN OUTCOME MEASURES: Results of sequencing TGFBI exons 4 and 12. RESULTS: Transforming growth factor-beta-induced gene mutation screening in a child with bilateral anterior corneal stromal opacification revealed the following changes in exon 12: 1667T>C (Phe540Phe), 1684C>A (Ala546Asp), and 1699C>A (Pro551Gln). The child's father demonstrated the 1667T>C nucleotide substitution, but neither parent demonstrated the 1684T>C or the 1699C>A changes. CONCLUSIONS: Buccal swabs provide the ophthalmologist with a simple, inexpensive means of collecting and transporting cells from which DNA may be extracted to be used to either confirm or refute diagnoses solely on the basis of clinical features. In this case, the diagnosis of an atypical variant of lattice corneal dystrophy was made on the basis of the identification of previously reported mutations in TGFBI.

Child, Preschool↗
PubMed2000

Mutation screening using automated bidirectional dideoxy fingerprinting.

The need continues to grow for mutation identification in genetic disease in both research and clinical settings. We have developed a rapid nonradioactive bidirectional dideoxy fingerprint mutation screening procedure that is performed using an automated DNA analyzer. This technique features standardized primers and easily interpreted results from separate, but simultaneously collected, images for coding and noncoding strands. Another advantage is simplified mutation verification by sequencing using the same amplified DNA templates and also application to large multi-exon genes. We demonstrate the efficiency and reproducibility of the method in which we screen a DNA fragment encompassing exon 5 of the PTCH gene (in which mutations cause Gorlin Syndrome) in a panel of 22 patients.

Basal Cell Nevus Syndrome↗
PubMed2005

Algorithm for efficient PKHD1 mutation screening in autosomal recessive polycystic kidney disease (ARPKD).

Autosomal recessive polycystic kidney disease (ARPKD) is an important cause of childhood renal- and liver-related morbidity and mortality with variable disease expression. While most cases manifest peri-/neonatally with a high mortality rate in the first month of life, others survive to adulthood. ARPKD is caused by mutations in the Polycystic Kidney and Hepatic Disease 1 (PKHD1) gene on chromosome 6p12. PKHD1 is an exceptionally large gene (470 kb) with a longest open reading frame transcript of 67 exons predicted to encode a 4,074-amino acid (aa) (447 kDa) multidomain integral membrane protein (fibrocystin/polyductin) of unknown function. Recent DHPLC-based mutational studies have reported detection rates of about 80% and a minimum of one PKHD1 mutation in more than 95% of families. Thus far, a total of 263 different PKHD1 mutations (639 mutated alleles) are included in the locus-specific database (www.humgen.rwth-aachen.de). Except for a few population-specific founder alleles and the common c.107C>T (p.Thr36Met) missense change, PKHD1 is characterized by significant allelic diversity, making mutation screening time-consuming and labor-intensive. Mutations are distributed throughout the gene's coding sequence; however, they are not equally scattered. Thus, we aimed to set up an algorithm for efficient molecular genetic diagnostics in ARPKD. A total of 80% of known PKHD1 mutations can be identified if a subset of 27 out of 77 DHPLC fragments is screened. The current study provides an essential platform for PKHD1 mutation screening in a routine setting that will largely alleviate molecular genetic diagnostics in patients suspected to have ARPKD.

Algorithms↗
PubMed2003

Mutation screening of Pakistani families with congenital eye disorders.

To map the disease loci several Pakistani families suffering from autosomal recessive retinitis pigmentosa with preserved para-arteriolar retinal pigment epithelium and Leber congenital amaurosis (LCA) were analyzed. Analysis revealed close genetic linkage between the disease phenotype of some of the families (3330RP, 111RP and 010LCA) and the microsatellite markers on chromosome 1q31. Mutation screening of the candidate gene CRB1 revealed a G to A transversion in exon 7 in arRP family 330RP and a T to C substitution in another arRP family, 111RP. In exon 9 of the CRB1 gene a T to C transversion was found in the family suffering from LCA (010LCA). The LCA phenotype of another family (011LCA) in which the CRB1 locus was excluded, showed linkage with microsatellite markers D17S1294 and D17S796 on chromosome 17p13.1. The association of the candidate gene GUCY2D (17p13.1) with the disease phenotype was excluded as no disease-associated mutation was found in any of its exons. Mutation screening of another candidate gene, AIPL1 located in the same region, showed a novel homozygous C to A substitution in exon 2. These sequence changes are unique for the Pakistani families and some of these have not been reported previously.

Adolescent↗
PubMed2003

Evaluation of DLC1 as a prostate cancer susceptibility gene: mutation screen and association study.

A gene or genes on chromosome 8p22-23 have been implicated in prostate carcinogenesis by the observation of frequent deletions of this region in prostate cancer cells. More recently, two genetic linkage studies in hereditary prostate cancer (HPC) families suggest that germline variation in a gene in this region may influence prostate cancer susceptibility as well. DLC1 (deleted in liver cancer), a gene in this interval, has been proposed as a candidate tumor suppressor gene because of its homology (86% similarity) with rat p122 RhoGAP, which catalyzes the conversion of active GTP-bound rho complex to the inactive GDP-bound form, and thus suppresses Ras-mediated oncogenic transformation. A missense mutation and three intronic insertions/deletions in 126 primary colorectal tumors have been previously identified. However, there are no reports of DLC1 mutation screening in prostate tumors or in germ line DNA of prostate cancer patients. In this study, we report the results of the first mutation screen and association study of DLC1 in genomic DNA samples from hereditary and sporadic prostate cancer patients. The PCR products in the 5' UTR, all 14 exons, exon-intron junctions, and 3' UTR were directly sequenced in 159 HPC probands. Eight exonic nucleotide polymorphisms (SNPs) were identified, only one of which resulted in an amino acid change. Twenty-three other SNPs were identified in intronic regions. Seven informative SNPs that spanned the complete DLC1 gene were genotyped in an additional 249 sporadic cases and 222 unaffected controls. No significant difference in the allele and genotype frequencies were observed among HPC probands, sporadic cases, and unaffected controls. These results suggest that DLC1 is unlikely to play an important role in prostate cancer susceptibility.

GTPase-Activating Proteins↗
PubMed1997

Dystrophin point mutation screening using a multiplexed protein truncation test.

We report here the first use of a multiplexed protein truncation test for the high throughput screening of dystrophin point mutations. We have developed a substantially more robust and efficient procedure incorporating large savings in cost which uses muscle biopsy or lymphocyte total RNA as the template. The entire dystrophin open reading frame is screened in only five overlapping fragments using a long RT-PCR strategy to amplify dystrophin cDNA in excess of 3.7 kb. These five fragments are uniquely transcribed and translated in vitro in a single multiplexed reaction containing magnesium ions to reduce nonspecific internal initiation of translation. We have used this system to analyze mutations in 11 Duchenne muscular dystrophy patients (10 unrelated) with previously uncharacterized mutations. A single truncating mutation was identified in all patients, which was confirmed at the genomic level. Multiplex PTT provides the most efficient method for point mutation screening in this large gene and has potential applications to several disease genes with a significant proportion of truncating mutations.

Amino Acid Sequence↗
PubMed2006

Mutational screening of the CYP26A1 gene in patients with caudal regression syndrome.

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 chi(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 (447 C>G, 1134 G>A, IVS 1+10 G>C, and IVS 4+8 AG>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.

Cytochrome P-450 Enzyme System↗
PubMed2001

Recent developments in high-throughput mutation screening.

Screening of large sample materials for the presence of known or unknown mutations is a key element in pharmacogenomics. Although automated DNA sequencing has developed rapidly during the last decade, the technology is not well suited for projects involving analysis of hundreds of thousands of mutations. Consequently, a number of methods for high-throughput mutation screening have been developed. DNA microarrays and high-density oligonucleotide chips have proven to be well suited for parallel hybridisation-based analysis of hundreds or thousands of known mutations. Methods based on detection using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) have been developed. MALDI-TOF MS detection is limited to analysis of small DNA fragments but has a large potential for high-throughput single nucleotide polymorphism (SNP) analysis, due to a very fast analysis time and possibilities for automation. Currently, the best suited methods for high-throughput screening for unknown mutations are probably methods like single strand conformation polymorphism (SSCP) analysis or conformation sensitive gel electrophoresis (CSGE), combined with capillary array electrophoresis or denaturing high-performance liquid chromatography. This is due to a relatively short analysis time, potential for automation and a high sensitivity. The recent development of capillary array electrophoresis chips suggests that the analysis time for some of these methods may be reduced by one order of magnitude in the near future.

Chromatography, High Pressure Liquid↗
PubMed2003

SPG3A mutation screening in English families with early onset autosomal dominant hereditary spastic paraplegia.

Mutations in the SPG3A gene encoding the novel GTPase atlastin have recently been implicated in causing autosomal dominant hereditary spastic paraplegia (ADHSP) in six unrelated families. The phenotype of affected individuals in all cases has been of an early onset uncomplicated form of the disease. One particular missense mutation, R239C, in exon 7 of SPG3A has been identified in three of these families. We performed mutation screening by direct sequencing of all 14 exons and flanking sequences of the SPG3A gene in affected individuals from 12 unrelated English families, all with an early onset uncomplicated ADHSP in whom spastin mutations had previously been excluded. The R239C mutation was found to co-segregate with the disease in one English ADHSP family confirming a widespread prevalence for this commonly occurring mutation. No additional SPG3A mutations were identified in the remaining 11 families suggesting that even within this specific sub-set of early onset uncomplicated ADHSP patients atlastin mutations are relatively rare.

Adolescent↗
PubMed2000

Hierarchical mutation screening protocol for the BRCA1 gene.

The identification of mutations in the BRCA1 gene poses difficulties in achieving a screening outcome that satisfies the twin needs of speed and accuracy. These needs must also take into account the patient's family history and the statistical evaluation of the probability of detecting a mutation. Given the above, we present here a hierarchical mutation screening strategy that comprises two tiers: first, multiplex heteroduplex and exon 13 duplication analysis; second, exon amplification and direct sequencing using a 96-well tray format. The advantages of this strategy are two-fold: first, the division of analytical tools in order to achieve low and high-resolution mutation screening, respectively; second, a streamlined sequencing approach that leads to a sensitive and rapid assay that reduces labor costs and handling errors. The success of this approach is shown by the identification of a novel deletion mutation in exon 14 of the BRCA1 gene, which was not detected by the more conventional protein truncation assay due to the small size of the predicted truncated protein.

Adult↗
PubMed1999

Evaluation of mutation screening by heteroduplex analysis in acute intermittent porphyria: comparison with denaturing gradient gel electrophoresis.

Acute intermittent porphyria is the major autosomal dominant form of acute hepatic porphyrias. The disease is due to mutations in the gene encoding for porphobilinogen deaminase (PBGD). Many different strategies have been developed to screen for mutations. However the high prevalence (0.6 per thousand) of PBGD gene defect, the large allelic heterogeneity of mutations (n = 130), and the limitations of the PBGD enzymatic assay for asymptomatic patients' detection, require for diagnosis an efficient and easy to handle strategy for locating mutations within the PBGD gene. In a recent study the sensitivity of the denaturing gradient gel electrophoresis (DGGE) technique was 100%. However DGGE requires the preparation of gradient gels and the use of primers with long GC-clamps; thus alternative methods should be preferable in the clinical laboratory. We have compared the detection rate of DGGE with heteroduplex analysis (HA) using 16 characterized PBGD gene mutations. Six different HA conditions were used to determine the efficiency of the method, including: (1) MDE (mutation detection enhancement) gel concentration; (2) addition of urea and sodium dodecyl sulfate (SDS); (3) radioactive labelling. The sensitivity of each HA condition varied from 31 to 81% vs. 100% in DGGE analysis. HA using 1 x MDE with 15% urea with or without 0.55% SDS was the most sensitive condition. This first comparative study of DGGE and HA mutation screening methods suggests that DGGE is a more sensitive screening assay than optimized HA. However, because of its simplicity HA should be considered as an efficient alternative mutation screening method.

DNA↗
PubMed2006

High-efficiency multiplex capillary electrophoresis single strand conformation polymorphism (multi-CE-SSCP) mutation screening of SCN5A: a rapid genetic approach to cardiac arrhythmia.

Mutations in the SCN5A gene coding for the alpha-subunit of the cardiac Na(+) ion channel cause long QT syndrome, Brugada syndrome, idiopathic ventricular fibrillation, sick sinus node syndrome, progressive conduction disease, dilated cardiomyopathy and atrial standstill. These diseases exhibit variable expressivity, and identification of gene carriers is clinically important, particularly in sudden infant and adult death syndromes. The SCN5A gene comprises 28 exons distributed over 100 kbp of genomic sequence at chromosome 3p21. Disease-causing mutations are private and scattered over the DNA sequence, making it difficult to screen for specific mutations. We developed a multiplex capillary-electrophoresis single-strand conformation polymorphism (Multi-CE-SSCP) mutation screening protocol on the ABI 3100 platform and applied it to 10 previously slab-gel SSCP identified mutations and SNPs and used it to identify one novel deletion. The method is highly efficient, with a turnover of 23 patients per 24 h and a false positive rate of 0.5% of the analyzed amplicons. Each variant has a particular elution pattern, and all 20 carriers of the H558R polymorphism out of 57 persons were correctly identified. We suggest that the method could become part of routine work-up of patients with suspicious syncope and of members of families with sudden unexplained death.

Amino Acid Substitution↗
PubMed2002

Mutational screening of patients with nonalcoholic chronic pancreatitis: identification of further trypsinogen variants.

OBJECTIVES: Mutations of the cationic trypsinogen (CT) and the serine protease inhibitor, Kazal type 1 (SPINK 1) are associated with chronic pancreatitis. After mutational screening of a cohort of patients with nonalcoholic chronic pancreatitis, we report three novel variants of the trypsinogen molecule and the clinical characteristics of the carriers. METHODS: The coding region of the exon 2 and 3 of the CT gene of 523 patients with chronic nonalcoholic pancreatitis (108 patients with suspected hereditary pancreatitis (HP) and 415 patients with "idio pathic" pancreatitis [IP]) and 82 controls was analyzed after polymerase chain reaction amplification. Clinical characteristics were obtained by questioning the patients and their relatives and physicians. HP was suspected when two members of a family had chronic pancreatitis. A restriction digestion was used to analyze the N34S mutation SPINK1. RESULTS: The mutation R122H of the cationic trypsinogen was found in 21 index patients, N291 in six index patients, and A16V and D22G in one index patient, all from HP families. The N34S mutation of SPINK1 was found in two index patients with a family history of HP. In three patients, the novel point mutations L104P, R116C, and C139F of the cationic trypsinogen were found. A clear autosomally dominant inheritance of chronic pancreatitis was not present in these families. In 75 index patients from HP families (69.4%), no mutation could be found. The SPINK 1-mutation N34S was detected in only one patient carrying a CT mutation, and was found in 68 (16.4%) of patients with IP. CONCLUSIONS: The R122H and N291 mutations of CT are the most common disease-associated mutations in HP; the N34S mutation of SPINK I is the most frequent genetic risk factor associated with IP. The CT gene carries several variations that could be associated with chronic pancreatitis. To avoid overestimating the pathogenetic impact of novel trypsinogen variants, a detailed clinical characterization of all patients with early onset chronic pancreatitis is mandatory.

Adult↗
PubMed2001

Population-based risk estimates of Wilms tumor in sporadic aniridia. A comprehensive mutation screening procedure of PAX6 identifies 80% of mutations in aniridia.

Aniridia is a severe eye disease characterized by iris hypoplasia; both sporadic cases and familial cases with an autosomal dominant inheritance exist. Mutations in the PAX6 gene have been shown to be the genetic cause of the disease. Some of the sporadic cases are caused by large chromosomal deletions, some of which also include the Wilms tumor gene (WAGR syndrome), resulting in an increased risk of developing Wilms tumor. Based on the unique registration of both cancer and aniridia cases in Denmark, we have made the most accurate risk estimate to date for Wilms tumor in sporadic aniridia. We have found that patients with sporadic aniridia have a relative risk of 67 (confidence interval: 8.1-241) of developing Wilms tumor. Among patients investigated for mutations, Wilms tumor developed in only two patients out of 5 with the Wilms tumor gene (WT1) deleted. None of the patients with smaller chromosomal deletions or intragenic mutations were found to develop Wilms tumor. Our observations suggest a smaller risk for Wilms tumor than previous estimates, and that tumor development requires deletion of WT1. We report a strategy for the mutational analysis of aniridia cases resulting in the detection of mutations in 68% of sporadic cases and 89% of familial cases. We also report four novel mutations in PAX6, and furthermore, we have discovered a new alternatively spliced form of PAX6.

Alternative Splicing↗
PubMed1996

Mutational screening of APP gene in patients with early-onset Alzheimer disease utilizing mismatched PCR-RFLP.

To elucidate the frequency of mutations of the beta/A4 amyloid protein precursor (APP) gene in early-onset Alzheimer disease, we designed a mismatched PCR-RFLP that can identify all kinds of missense mutations at codon 717 in addition to the seven kinds of known mutations at exon 17. When we screened mutations at exon 17 utilizing this method and the double missense mutations at exon 16 of the APP gene by PCR-RFLP, no cases revealed mutations of the APP gene among 13 familial and 54 sporadic cases, except one family (OS-1) that had previously been reported and used as a positive control of APP717(Val-->Ile). Our results support the hypothesis that mutations in the APP gene are not major causes in early-onset Alzheimer disease.

Adult↗