A deep intronic mutation in the RB1 gene leads to intronic sequence exonisation

Familial forms of retinoblastoma, an embryonic neoplasm of retinal origin, are caused by constitutional mutations of the RB1 gene. In this paper, we describe a family with retinoblastoma affecting two brothers with no previous family history of cancer. Complete RB1 mutational screening including point mutation and large rearrangement screening failed to demonstrate any mutation. The whole coding sequence was therefore investigated at the cDNA level, demonstrating a 103 bp intronic insertion between exons 23 and 24, leading to subsequent frameshift and premature termination of translation. This intronic exonisation was caused by a deep intronic mutation in intron 23 generating a cryptic 3' splice site. This is the first report of a deep intronic mutation in RB1 and is a proof of concept that some undetected RB1 mutations should be investigated at the cDNA level, particularly in hereditary forms of retinoblastoma.     

A comprehensive scanning method for rapid detection of beta-globin gene mutations and polymorphisms.

We describe a scanning procedure for the detection of beta-globin gene mutations and the prenatal diagnosis of beta-thalassemias. The method is based on the combined use of PCR and denaturing gradient gel electrophoresis (DGGE) of six amplified fragments encompassing the whole beta-globin coding region and splice junctions, as well as the promoter and 3' untranslated regions. The whole beta-globin gene can be rapidly scanned for the presence of deleterious mutations. The proposed diagnostic strategy provides a major improvement over current approaches to beta-globin gene analysis in both research and clinical laboratories, especially those which analyse DNA samples from individuals belonging to various ethnic or population groups. The use of this procedure has enabled us to detect six novel sequence changes in the beta-globin gene, including two deleterious mutations and four polymorphisms.     

DNA sequence analysis of the apocytochrome b gene of Podospora anserina: a new family of intronic open reading frame

Summary The 5,969 by (base pair) DNA sequence of the apocytochrome b mitochondrial (mt) gene of race A Podospora anserina was located in a 8.5 Kbp region. This gene contained a 2,499 by subgroup IB and a 1,306 by subgroup ID intron as well as a 990 bp subgroup IB intron which is present in race A but not race s. The large subgroup IB intron and the race A specific IB intron both contained potential alternate splice sites which brought their open reading frames into phase with their upstream exon sequences. All three introns were compared with regard to their secondary structures and open reading frames to the other 30 group I introns in Podospora anserina, as well as to other fungal introns. We detected a new family of intronic ORFs comprising seven P. anserina introns, several N. crassa introns, as well as the T4td bacteriophage intron. Sequence similarities to intron-encoded endonucleases were noteworthy. The DNA sequences reported here and in the accompanying paper complete the analysis of race s and race A mitochondrial DNA.     

Improved typing procedure for the polymorphic single-copy RLA-DQA gene of the rabbit reveals a new allele

The DQA gene of the rabbit major histocompatibility complex (MHC, RLA) is highly polymorphic and, in contrast to those reported for other mammalian species, is present as a single copy. These properties allow use of this gene in a method to type the class II locus of RLA by a combination of single-stranded conformational polymorphism (SSCP) and heteroduplex (HD) analysis. Familial segregation of RLA-DQA was shown and RLA class II types for rabbits of unknown pedigree were determined using migration patterns of amplified genomic DNA. Typing results were confirmed in experiments where unknown samples were mixed with products from rabbits of RLA types defined by sequence analysis. These analyses detected an RLA-DQA allele in addition to the five previously described; this new allele is designated RLA-DQA-F.     

A survey of long-range DNA polymorphisms on the human Y chromosome

The human Y chromosome is poor in conventional DNA polymorphisms,and this has hindered studies of the paternal lineage. However,three large hypervariable arrays exist, and haplotyping at theseloci defines two groups in Caucasian and Asian populations,reflecting the existence of two ancestral Y chromosomes. Inthis study, the Y was systematically surveyed for further long–rangepolymorphisms, by the hybridization of 33 probes to Sfi digestsof DNA from males of different ethnic origins and from the twogroups. Five novel polymorphisms were identified, all showingvariability consistent with a changing number of tandem repeatswithin an array. A search for conventional polymorphisms wasalso done, using 41 probes and the enzyme Taq three novel variantsand one polymorphism with a frequency of 18% (n = 66) were found.The novel polymorphisms were typed in 66 Y chromosomes, includingthe set of 42 in which the two groups were originally defined.Known long–range and conventional polymorphisms were alsoextended to cover the whole set, yielding compound haplotypescomprising the states of twelve polymorphisms. This haplotypingdistinguishes between all 66 chromosomes, and should distinguishbetween most in the population. The existence of the two groupsis supported, and a third group can be defined; six of the eightmembers of this group are known to be from India. Twelve chromosomesdo not fall into any of these groups, and are likely to be representativesof further groups.     

Validation of the performance of a comprehensive genotyping assay panel of single nucleotide polymorphisms in drug metabolism enzyme genes

A class of genes, known as drug metabolism enzymes (DMEs) are responsible for the metabolism and transport of drugs and other xenobiotics. Variation in DME genes most likely accounts for a proportion of the variability in drug response in humans, and may contribute to complex diseases such as cancer (Nebert DW, Dieter MZ. Pharmacology 2000;61:124-135). To date, assessing the extent of this variation has proven difficult, especially because of sequence paralogy issues that cause difficulty when attempting to genotype polymorphisms in very closely-related gene families (Murphy MP. Pharmacogenomics 2000;1:115-123; Ingelman-Sundberg M. Drug Metab Rev 1999;31:449-459). We have developed and genotyped a panel of N=2,325 individual TaqMan genotyping assays for polymorphisms in >200 DME genes; many of the variants in the panel are single nucleotide polymorphisms (SNPs) that are of known or putative function (e.g., missense, nonsense or frameshift). Using these assays, we have examined genetic variation among several groups of populations, including: 1) the two SNP500 Cancer population panels (http://snp500cancer.nci.nih.gov; last accessed: 11 December 2007); and 2) the panel used in the International HapMap Project panel (www.hapmap.org; last accessed: 11 December 2007). We have developed a comprehensive validation strategy to ensure reproducibility and accuracy of the assays and estimated minor allele frequencies. Here, we present the results of these analyses, which strongly suggest that this panel of DME assays are of extremely high quality and produce robust, accurate, and reproducible results.     

Possible role of intronic polymorphisms in the PHACTR1 gene on the development of cardiovascular disease

Cardiovascular disease (CVD) is a complex multifactorial and polygenetic disease in which the interaction of numerous genes, genetic variants, and environmental factors plays a major role in its development. In an attempt to demonstrate the association between certain genetic variants and CVD, researchers have run large genomic wild association studies (GWAS) in recent decades. These studies have correlated several genomic variants with the presence of CVD. Recently, certain polymorphisms in the phosphatase and actin regulator 1 (PHACTR1) gene have been shown to be associated with CVD (i.e., coronary artery disease, coronary artery calcification, early onset myocardial infarction, cervical artery dissection and hypertension) in different ethnic groups. It is important to state that all of the described PHACTR1 genetic variants associated with CVD are located in non-translating gene regions known as introns. Thus, the purpose of this article is to hypothesize the effect of certain intronic polymorphisms in the PHACTR1 gene on pathological processes in the cardiovascular system. In addition, we present compelling evidence that supports this hypothesis as well as a methodology that could be used to assess the allelic effect using in vitro and in vivo models, which will ultimately demonstrate the pathophysiological contribution of PHACTR1 intronic polymorphisms to the development of CVD.     

Genomic localization and sequence analysis of the putative bovine herpesvirus-1 DNA polymerase gene

Summary The bovine herpesvirus-1 (BHV-1) genome was analysed by Southern blot hybridization using the herpes simplex virus type 1 (HSV-1) DNA polymerase gene as a probe. A 2.5 kilobase region which hybridized specifically to the HSV-1 DNA polymerase gene was identified within the Hind III G fragment at approximate map units 0.334–0.352. In order to provide further evidence that this is the location of the BHV-1 DNA polymerase gene, the 2.5 kilobase region was cloned and part of it sequenced. An uninterrupted stretch of over 800 nucleotides was obtained and an open reading frame spanning the entire sequence was identified. The amino acid sequence that it encodes shows striking homology to a region within the C-terminal half of other herpesvirus DNA polymerases.     

RMRP gene sequence analysis confirms a cartilage-hair hypoplasia variant with only skeletal manifestations and reveals a high density of single-nucleotide polymorphisms

Mutations in the RMRP gene that codes for an RNA subunit of the MRP RNAse complex are the cause of cartilage-hair hypoplasia (CHH; MIM 250250). We tested the hypothesis that recessive metaphyseal dysplasia without hypotrichosis (M1M 250460), a disorder presenting with short stature and metaphyseal dysplasia similar to CHH, but lacking hair anomalies, immunodeficiency and other extra skeletal features, might be allelic to CHH. We identified four mutation-carrying alleles segregating with the skeletal phenotype in two unrelated boys and their parents. One allele carried the common Finnish mutation +70A--> G; the remaining three carried +195C--> T, +238C--> T, and dupAAGCTGAGGACG at -2. Sequencing 120 alleles from a control group revealed an unusually high density of single-nucleotide polymorphisms in and around the RMRP gene: the biological significance of this finding is unclear. We conclude that recessive metaphyseal dysplasia without hypotrichosis is a variant of CHH, manifesting only as short stature and metaphyseal dysplasia. Precise diagnosis of this form of metaphyseal dysplasia is not without importance because of recessive inheritance with corresponding recurrence risk, as well as because of potential complications such as anaemia, susceptibility to infections and the increased likelihood of developing cancer. The short stature and metaphyseal changes associated with cone-shaped epiphyses of the hands should raise the diagnostic possibility of a CHH-related disorder that can then be confirmed by mutation analysis.     

Development and clinical evaluation of a highly sensitive DNA microarray for detection and genotyping of human papillomaviruses

Human papillomavirus (HPV) has been found in cervical cancer, tonsillar cancer, and certain types of head and neck cancers. We report on a DNA microarray-based method for the simultaneous detection and typing of HPVs. The genotype spectrum discriminated by this HPV DNA microarray includes 15 high-risk HPV genotypes and 12 low-risk HPV genotypes. The HPV DNA microarray showed high degrees of specificity and reproducibility. We evaluated the performance of the HPV DNA microarray by application to three HPV-positive cell lines (HeLa, Caski, and SiHa cells) and two HPV-negative cell lines (C33A and A549 cells). The HPV DNA microarray successfully identified the known types of HPV present in the cell lines. The detection limit of the HPV DNA microarray was at least 100-fold higher than that of PCR. To assess the clinical applicability of the HPV DNA microarray, we performed the HPV genotyping assay with 73 nonmalignant and malignant samples from 39 tonsillar cancer patients. Twenty-five of the 39 (64.1%) malignant samples were positive for HPV, whereas 3 of 34 (8.8%) nonmalignant samples were positive for HPV. This result shows a preferential association of HPV with tonsillar carcinomas. The correlations of the presence of HPV with the grade of differentiation and risk factors were not significant. Our data show that the HPV DNA microarray may be useful for the diagnosis and typing of HPV in large-scale epidemiological studies.     

Genetic linkage between Becker muscular dystrophy and a polymorphic DNA sequence on the short arm of the X chromosome.

A study of DNA restriction fragment polymorphisms and Becker muscular dystrophy has shown eight families informative for the cloned sequence L1.28, which is located on the short arm of the X chromosome between Xp110 and Xp113. Analysis of these families reveals linkage between the two loci, with the maximum likelihood estimate of the genetic distance being 16 centiMorgans (95% confidence limits between 7 and 32 centiMorgans). Since a study of DNA polymorphisms in Duchenne muscular dystrophy has shown a comparable linkage distance with L1.28, our results suggest that the locus for Becker muscular dystrophy, like that for Duchenne dystrophy, is on the short arm of the X chromosome, and further that these two loci may be closely linked or possibly allelic.     

TNF-alpha and -beta gene polymorphisms in multiple sclerosis: a highly significant role for determinants in the first intron of the TNF-beta gene

Tumor necrosis factor (TNF)-alpha and TNF-beta are proinflammatory cytokines that have been implicated in the pathogenesis of several autoimmune diseases. The aim of this investigation was to determine whether a determinant in the first intron of the TNF-beta gene (TNF-beta(+252)) and two promoter-region polymorphisms of the TNF-alpha gene (TNF-alpha(-308) and TNF-alpha(-238)) affect susceptibility to multiple sclerosis (MS). DNA samples from 133 Caucasian MS patients and 148 healthy controls from Norway were genotyped for several polymorphic determinants, using polymerase chain reaction-based restriction fragment length polymorphisms (PCR-RFLP) methods. TNF-beta(+252) genotypes were significantly associated with MS: The frequency of TNF-beta 2,2 was increased (p = 0.00009) while the frequency of TNF-beta 1,2 was decreased (p = 0.0012) in MS patients as compared to controls. TNF-alpha genotypes were not associated with MS. These results suggest that the TNF-beta gene plays a significant role in the etiopathogenesis of MS.     

Detection of DNA sequence polymorphisms in carcinogen metabolism genes by polymerase chain reaction.

The glutathione transferase mu gene (GST1) and the debrisoquine hydroxylase gene (CYP2D6) are known to be polymorphic in the human population and have been associated with increased susceptibility to cancer. Smokers with low lymphocyte GST mu activity are at higher risk for lung cancer, while low debrisoquine hydroxylase activity has been correlated with lower risk for lung and bladder cancer. Phenotypic characterization of these polymorphisms by lymphocyte enzyme activity (GST) and urine metabolite ratios (debrisoquine) is cumbersome for population studies. Recent cloning and sequencing of the mutant alleles of these genes has allowed genotyping via the polymerase chain reaction (PCR). Advantages of PCR approaches are speed, technical simplicity, and minimal sample requirements. This article reviews the PCR-based methods for detection of genetic polymorphisms in human cancer susceptibility genes.