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PubMed2006

Genetic heterogeneity in patients with pantothenate kinase-associated neurodegeneration and classic magnetic resonance imaging eye-of-the-tiger pattern.

We performed a detailed molecular study in two unrelated families with pantothenate kinase-associated neurodegeneration (PKAN) and the specific magnetic resonance imaging (MRI) eye-of-the-tiger pattern. In the first family with classic PKAN, linkage analysis using polymorphic markers from the PANK2 region ruled out linkage with this locus, and no mutation of the PANK2 gene was found. In the second family with atypical PKAN, we identified a novel homozygous C-to-T transition at nucleotide 1069 of the PANK2 gene, which resulted in an arginine to tryptophane substitution at codon 357. As far as we are aware, this is the first case of classic PKAN with the specific MRI eye-of-the-tiger pattern not carrying a PANK2 mutation. Therefore, the present observation reinforces the notion of the phenotypic and genetic heterogeneity in PKAN.

Adult↗
PubMed1999

Variegate porphyria in Western Europe: identification of PPOX gene mutations in 104 families, extent of allelic heterogeneity, and absence of correlation between phenotype and type of mutation.

Variegate porphyria (VP) is a low-penetrance, autosomal dominant disorder characterized clinically by skin lesions and acute neurovisceral attacks that occur separately or together. It results from partial deficiency of protoporphyrinogen oxidase encoded by the PPOX gene. VP is relatively common in South Africa, where most patients have inherited the same mutation in the PPOX gene from a common ancestor, but few families from elsewhere have been studied. Here we describe the molecular basis and clinical features of 108 unrelated patients from France and the United Kingdom. Mutations in the PPOX gene were identified by a combination of screening (denaturing gradient gel electrophoresis, heteroduplex analysis, or denaturing high-performance liquid chromatography) and direct automated sequencing of amplified genomic DNA. A total of 60 novel and 6 previously reported mutations (25 missense, 24 frameshift, 10 splice site, and 7 nonsense) were identified in 104 (96%) of these unrelated patients, together with 3 previously unrecognized single-nucleotide polymorphisms. VP is less heterogeneous than other acute porphyrias; 5 mutations were present in 28 (26%) of the families, whereas 47 mutations were restricted to 1 family; only 2 mutations were found in both countries. The pattern of clinical presentation was identical to that reported from South Africa and was not influenced by type of mutation. Our results define the molecular genetics of VP in western Europe, demonstrate its allelic heterogeneity outside South Africa, and show that genotype is not a significant determinant of mode of presentation.

Alleles↗
PubMed2000

[Molecular biological methods and their consequences in taxonomy and diagnosis of dermatophytes].

Trichophyton rubrum, T. mentagrophytes, T. verrucosum, Microsporum canis, M. gypseum and Epidermophyton flocoosum represent the cause of human dermatomycoses isolated most often at the University Hospital of Dermatology in Graz, Austria, between 1991 and 1998. So far, identification was mainly based on the cultivation of fungal isolates on special media as well as on the analysis of their microscopic characters. Restriction enzyme length polymorphisms (RFLPs) were now used to identify these human fungal parasites. For this purpose, internal transcribed spacers (ITS) which are located between ribosomal RNA genes have been amplified by using PCR and have afterwards been used to generate species specific RFLP patterns. By this method, a fast and reliable identification of these species was made possible. Nucleotide sequence data of this region not needed for identification have been worked out to show RFLPs in more detail.

Arthrodermataceae↗
PubMed2004

Bioinformatics tools for single nucleotide polymorphism discovery and analysis.

Single nucleotide polymorphisms (SNPs) are a valuable resource for investigating the genetic basis of disease. These variants can serve as markers for fine-scale genetic mapping experiments and genome-wide association studies. Certain of these nucleotide polymorphisms may predispose individuals to illnesses such as diabetes, hypertension, or cancer, or affect disease progression. Bioinformatics techniques can play an important role in SNP discovery and analysis. We use computational methods to identify SNPs and to predict whether they are likely to be neutral or deleterious. We also use informatics to annotate genes that contain SNPs. To make this information available to the research community, we provide a variety of Internet-accessible tools for data access and display. These tools allow researchers to retrieve data about SNPs based on gene of interest, genetic or physical map location, or expression pattern.

Chromosome Mapping↗
PubMed2004

Global patterns of human mitochondrial DNA and Y-chromosome structure are not influenced by higher migration rates of females versus males.

Global-scale patterns of human population structure may be influenced by the rate of migration among populations that is nearly eight times higher for females than for males. This difference is attributed mainly to the widespread practice of patrilocality, in which women move into their mates' residences after marriage. Here we directly test this hypothesis by comparing global patterns of DNA sequence variation on the Y chromosome and mitochondrial DNA (mtDNA) in the same panel of 389 individuals from ten populations (four from Africa and two each from Europe, Asia and Oceania). We introduce a new strategy to assay Y-chromosome variation that identifies a high density of single-nucleotide polymorphisms, allows complete sequencing of all individuals rather than relying on predetermined markers and provides direct sequence comparisons with mtDNA. We found the overall proportion of between-group variation (Phi(ST)) to be 0.334 for the Y chromosome and 0.382 for mtDNA. Genetic differentiation between populations was similar for the Y chromosome and mtDNA at all geographic scales that we tested. Although patrilocality may be important at the local scale, patterns of genetic structure on the continental and global scales are not shaped by the higher rate of migration among females than among males.

Alu Elements↗
PubMed2005

Detection of single nucleotide polymorphism in the FUT2 gene by temperature gradient gel electrophoresis.

A simple and rapid analysis system for single nucleotide polymorphisms (SNPs) was investigated for the FUT2 gene using the temperature gradient gel electrophoresis (TGGE) method. The 426-bp or 259-bp FUT2 fragments were amplified from heterozygous samples using primers, and the heteroduplex and homoduplex bands were detected by TGGE. The FUT2 fragments amplified from homozygous samples were denatured and re-annealed with a known sequence fragment, forming heteroduplex bands which were analyzed by TGGE. The fragment patterns of homoduplex and heteroduplex bands in TGGE were specific to the genotypes, and more specifically, the number of heteroduplex bands and the migration of the homoduplex bands corresponded to the number of nucleotide substitutions and the change in GC content due to the substitutions, respectively. Thus, the TGGE technique can act as a high-throughput method for the detection and the presumption of sequences of known and unknown SNPs in the FUT2 gene.

DNA Primers↗
PubMed2003

The intronic prothrombin 19911A>G polymorphism influences splicing efficiency and modulates effects of the 20210G>A polymorphism on mRNA amount and expression in a stable reporter gene assay system.

The common prothrombin gene cleavage site mutation 20210G>A is associated with elevated prothrombin levels and thrombosis. The pathomechanism of the 20210G>A mutation was explained by increased mRNA formation and/or more efficient translation. Human studies also showed an influence of the intronic 19911A>G polymorphism on prothrombin activity. We established HepG2 cell lines stably transfected with prothrombin mini-genes containing the last 2 prothrombin exons, the last intron, 3' untranslated region (UTR), and flanking sequence. The highest mRNA expression and protein activity resulted from the mutant haplotype 19911A-20210A. Haplotypes with wild-type cleavage site (19911A-20210G, 19911G-20210G) also differed significantly as a consequence of the intronic 19911 mutation; the 19911G-20210G haplotype showed lower expression than the 19911A-20210G haplotype, whereas previous clinical studies have reported elevated prothrombin activity with the 19911G-20210G haplotype. The cleavage site pattern was homogeneous with 20210A, which may cause a favorable intracellular processing, and heterogeneous with 20210G. In an independent assay for splicing efficiency, 19911G showed about 30% higher efficiency than 19911A. We conclude that the intronic 19911A>G single nucleotide polymorphism is itself functional and changes splicing efficiency by altering a known functional pentamer motif. Further studies are needed to define the value of additional prothrombin 19911 genotyping for thrombophilia screening, especially in cases heterozygous for 20210G>A.

Alternative Splicing↗
PubMed2005

Rapid investigation of hepatitis A virus outbreak by single strand conformation polymorphism analysis.

Investigation of hepatitis A virus (HAV) outbreaks often implies nucleotide sequence analysis. As an alternative method for the identification of related strains, single strand conformation polymorphism method (SSCP) was compared to sequence analysis. Twenty-three strains from sporadic and outbreak cases were studied retrospectively. SSCP, sequence identity and phylogenetic analyses were conducted on a 267 bp fragment of the VP1-2A variable region. The results of SSCP pattern comparison and sequence identity were highly correlated (r = 0.92, P < 0.001). If SSCP showed similar patterns, the VP1-2A fragments had a high and significant probability to have a sequence identity over 99.6%. Results were concordant for outbreak strains. The only discordant result concerned a cluster of three sporadic cases evidenced by phylogenetic analysis while SSCP showed similar patterns for only two of these three cases. A prospective SSCP analysis of a recent HAV outbreak confirmed the reliability of this technique. SSCP may thus provide a rapid and cost-effective tool for preliminary investigation of HAV outbreaks, before undertaking exhaustive nucleotide sequence analysis.

Adolescent↗
PubMed2001

Application of denaturing high-performance liquid chromatography for mapping of single nucleotide polymorphisms in barley (Hordeum vulgare L.).

Recent advances in DNA sequence analysis and the establishment of high-throughput assays have provided the framework for large-scale discovery and analysis of DNA sequence variation. In this context, single nucleotide polymorphisms (SNPs) are of particular interest. To initiate a systematic approach to develop an SNP map of barley (Hordeum vulgare L.), we have employed denaturing high-performance liquid chromatography (DHPLC) to analyse segregating SNP patterns in a doubled-haploid (DH) mapping population. To this end, SNPs between the parental genotypes were identified using a direct sequencing approach. Once a SNP was established between the parents, the optimal melting temperature of the PCR fragment containing the SNP was predicted for its analysis by DHPLC. Following the detection of the optimal temperature, the DH lines were analysed for the presence of either of the alleles. To test the utility of the analysis, data from previously mapped RFLP markers from which these SNPs were derived were compared. Results from these experiments indicate that DHPLC can be efficiently employed in analysing SNPs on a high-throughput scale.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

Molecular characterization of nuclear small subunit (18S)-rDNA pseudogenes in a symbiotic dinoflagellate (Symbiodinium, Dinophyta).

For the dinoflagellates, an important group of single-cell protists, some nuclear rDNA phylogenetic studies have reported the discovery of rDNA pseudogenes. However, it is unknown if these aberrant molecules are confined to free-living taxa or occur in other members of the group. We have cultured a strain of symbiotic dinoflagellate, belonging to the genus Symbiodinium, which produces three distinct amplicons following PCR for nuclear small subunit (18S) rDNA genes. These amplicons contribute to a unique restriction fragment length polymorphism pattern diagnostic for this particular strain. Sequence analyses revealed that the largest amplicon was the expected region of 18S-rDNA, while the two smaller amplicons are Symbiodinium nuclear 18S-rDNA genes that contain single long tracts of nucleotide deletions. Reverse transcription (RT)-PCR experiments did not detect RNA transcripts of these latter genes, suggesting that these molecules represent the first report of nuclear 18S-rDNA pseudogenes from the genome of Symbiodinium. As in the free-living dinoflagellates, nuclear rDNA pseudogenes are effective indicators of unique Symbiodinium strains. Furthermore, the evolutionary pattern of dinoflagellate nuclear rDNA pseudogenes appears to be unique among organisms studied to date, and future studies of these unusual molecules will provide insight on the cellular biology and genomic evolution of these protists.

Animals↗
PubMed2005

Genetic evidence of an Asian background in heteroplasmic Iberian cattle (Bos taurus): effect on food authentication studies based on polymerase chain reaction-restriction fragment length polymorphism analysis.

This work was aimed at identifying nucleotide polymorphic sites in a 359 bp region of the cytochrome b (cytb) mitochondrial gene of Iberian cattle (Bos taurus). This region is widely used as target in polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) species identification studies in foodstuffs destined for human and animal consumption. Two different coexisting restriction patterns were observed in four of the six animals studied when the 359 bp DNA fragment was cleaved with PalI, HinfI, MvaI, RsaI, or MboI. The amplification of both genotypes with the mitochondrial-specific primers L14735 and H15149 revealed the absence of nuclear pseudo-cytb genes, confirming the existence of mitochondrial heteroplasmy. The two coexisting mtDNA fragments were selectively sequenced in PCR extracts in which one genotype predominated over the other, both exhibiting a sequence variation of 10.4%. From the 37 nucleotide mismatches observed between genotypes, 32 were transitions and five were transversions. While 31 of the nucleotide mismatches between genotypes resulted to be conservative at the amino acid level, six changes implied amino acid substitutions, five of them being located in the variable transmembrane region. Genetic analysis suggests the presence of an Asian background in the mitochondria of Iberian cattle: while one of the genotypes matched the published sequence for Bos taurus, the other genotype clustered with a B. primigenius indicus animal and close to an Asian Bos taurus animal. These results also suggest that a number of current PCR-RFLP species identification methods based on cytb sequences may not be reliable for the accurate detection and identification of bovine material: an alternative battery of enzymes consisting of MmeI, NlaIV, and AluI is proposed.

Animals↗
PubMed2003

Identification and functional analysis of single nucleotide polymorphism in the tandem repeat sequence of thymidylate synthase gene.

The variable number of tandem repeat (VNTR) of thymidylate synthase (TS) gene, mainly 2 repeat (2R) and 3 repeat (3R), is one of the genetic variations that can potentially predict the effectiveness of 5-fluorouracil-based chemotherapy. In this study we identified an additional single nucleotide polymorphism (SNP) in the VNTR of TS, followed by functional and clinical analysis of the SNP. Two-hundred fifty eight tumor samples were obtained from patients with primary colorectal adenocarcinoma. We observed three different patterns of electrophoresis by analysis of the VNTR with 2R/3R heterozygote. The sequencing results revealed a SNP, G/C polymorphism, within the 28-bp repeat component of TS VNTR. Each polymorphic allele was assigned as 2G, 2C, 3G, or 3C according to the combination of SNP and VNTR. Functional analysis showed that the plasmid construct with 3G sequence had three to four times greater efficiency of translation than other polymorphic sequences. 3R allele in colorectal cancer was subdivided into around half by the SNP, indicating its commonness among Japanese. TS genotypes of the patients with colorectal cancer were classified into high expression type (2R/3G, 3C/3G, and 3G/3G) and low expression type (2R/2R, 2R/3C, and 3C/3C). The patients who received oral fluoropyrimedines survived longer than the patients with no treatment in the group of low expression type. No benefit of oral fluoropyrimedines was observed in the group of high expression type. These results suggest that the double polymorphism in the TS tandem repeat sequence, the SNP and the VNTR, may provide a potential for more effective prediction of the clinical outcome of 5-fluorouracil-based chemotherapy.

Adenocarcinoma↗
PubMed1996

Detection of heterozygous mutations in BRCA1 using high density oligonucleotide arrays and two-colour fluorescence analysis.

The ability to scan a large gene rapidly and accurately for all possible heterozygous mutations in large numbers of patient samples will be critical for the future of medicine. We have designed high-density arrays consisting of over 96,600 oligonucleotides 20-nucleotides (nt) in length to screen for a wide range of heterozygous mutations in the 3.45-kilobases (kb) exon 11 of the hereditary breast and ovarian cancer gene BRCA1. Reference and test samples were co-hybridized to these arrays and differences in hybridization patterns quantitated by two-colour analysis. Fourteen of fifteen patient samples with known mutations were accurately diagnosed, and no false positive mutations were identified in 20 control samples. Eight single nucleotide polymorphisms were also readily detected. DNA chip-based assays may provide a valuable new technology for high-throughput cost-efficient detection of genetic alterations.

BRCA1 Protein↗
PubMed2001

Human DNA sequence variation in a 6.6-kb region containing the melanocortin 1 receptor promoter.

An approximately 6.6-kb region located upstream from the melanocortin 1 receptor (MC1R) gene and containing its promoter was sequenced in 54 humans (18 Africans, 18 Asians, and 18 Europeans) and in one chimpanzee, gorilla, and orangutan. Seventy-six polymorphic sites were found among the human sequences and the average nucleotide diversity (pi) was 0.141%, one of the highest among all studies of nuclear sequence variation in humans. Opposite to the pattern observed in the MC1R coding region, in the present region pi is highest in Africans (0.136%) compared to Asians (0.116%) and Europeans (0.122%). The distributions of pi, theta, and Fu and Li's F-statistic are nonuniform along the sequence and among continents. The pattern of genetic variation is consistent with a population expansion in Africans. We also suggest a possible phase of population size reduction in non-Africans and purifying selection acting in the middle subregion and parts of the 5' subregion in Africans. We hypothesize diversifying selection acting on some sites in the 5' and 3' subregions or in the MC1R coding region in Asians and Europeans, though we cannot reject the possibility of relaxation of functional constraints in the MC1R gene in Asians and Europeans. The mutation rate in the sequenced region is 1.65 x 10(-9) per site per year. The age of the most recent common ancestor for this region is similar to that for the other long noncoding regions studied to date, providing evidence for ancient gene genealogies. Our population screening and phylogenetic footprinting suggest potentially important sites for the MC1R promoter function.

Animals↗
PubMed1997

AFLP markers for DNA fingerprinting in cattle.

This work reports on use of the recently described amplified fragment length polymorphism (AFLP) technology for DNA fingerprinting in cattle. The AFLP technology produces molecular markers through the high-stringency polymerase chain reaction (PCR)-amplification of restriction fragments that are ligated to synthetic adapters and amplified using primers, complementary to the adapters, which carry selective nucleotides at their 3' ends. While, for plants, the double digestion of genomic DNA with EcoRI and MseI is suggested, in mammals the enzyme combination EcoRI/TaqI produces clearer and more polymorphic AFLP patterns. In a sample of 47 Italian Holstein genotypes, 16 EcoRI/TaqI primer combinations identified 248 polymorphic bands in a species known for its low level of restriction polymorphism. In spite of the low information content carried by each AFLP polymorphism (average polymorphism information content = 0.31), the number of fragments revealed by each primer combination increased significantly the level of genetic information gained in each experiment. AFLP patterns are reproducible in independent experiments and polymorphic fragments segregate in cattle families according to Mendelian rules.

Animals↗
PubMed2004

Enteroaggregative Escherichia coli: an emerging enteric pathogen.

Enteroaggregative Escherichia coli (EAEC) represents an emerging pathogen that causes enteric and food-borne infectious diseases. Subgroups in many populations throughout the world are susceptible to EAEC infection. EAEC pathogenesis involves adherence to the intestinal mucosa; increased production and deposition of a mucus biofilm; and mucosal toxicity due to inflammation and cytokine release. Due to the heterogeneity of EAEC strains and differing host immune responses, not all EAEC infections are symptomatic. Recent data suggest that individuals with a homozygous genotype -251 AA single nucleotide polymorphism (SNP), in the IL-8 promoter region, are more susceptible to EAEC diarrhea. The HEp-2 cell adherent assay allows identification of EAEC's characteristic aggregative or "stacked brick" adherence pattern. Antimicrobial treatment of individuals who develop EAEC diarrhea should be individually based. Ciprofloxacin and rifaximin, compared to placebo, have been shown to significantly shorten the course of diarrhea in patients who developed EAEC infection. The objective of this review is to increase awareness of this important emerging pathogen and to discuss the epidemiology, pathogenesis, and host-pathogen factors associated with EAEC infection.

Adult↗
PubMed1997

A novel mitochondrial G8313A mutation associated with prominent initial gastrointestinal symptoms and progressive encephaloneuropathy.

We describe a childhood mitochondrial disorder in which the clinical symptoms began and remained confined to the gastrointestinal (GI) system during the first 4 y. Seizures heralded the onset of progressive encephalopathy at age 7. Peripheral neuropathy, retinitis pigmentosa, and neural deafness developed subsequently. Laboratory investigations disclosed elevated levels of plasma lactate, and a muscle biopsy revealed ragged red fibers lacking cytochrome c oxidase activity and diminished levels of respiratory chain enzyme complexes. Molecular genetic tests failed to show any of the previously reported pathogenic mitochondrial DNA (mtDNA) mutations. We therefore screened the whole mitochondrial genome by coupling restriction digestions with single-strand conformational polymorphism (SSCP) patterns. We identified a unique SSCP in the segment that encompassed the tRNA(Lys) gene, and direct sequencing of this segment revealed a G-->A transition at an evolutionarily conserved nucleotide at mtDNA position 8313. This G8313A transition was heteroplasmic in muscle and fibroblasts of the patient, but was absent in the white blood cells and platelets from his maternal relatives. This report illustrates how GI symptoms can be the initial manifestation in a mitochondrial disorder and suggests that mitochondrial dysfunction should be considered in differentials of unexplained chronic GI symptoms, especially when lactic acidosis or other unrelated clinical signs or symptoms are present.

Adolescent↗
PubMed1990

Chromosomal and molecular evolution in Asiatic wild asses.

Chromosomal studies of Tibetan wild ass, Equus kiang holdereri are reported for the first time. A Robertsonian polymorphism resulting in diploid numbers of 2n = 51 and 2n = 52 was identified. This polymorphism involves the same elements that participate in the polymorphic Robertsonian rearrangement producing diploid numbers of 54-56 in E. hemionus kulan and E. hemionus onager. Comparison of mitochondrial DNA restriction fragment patterns among three unrelated kiang individuals and between kiangs and a Transcaspian kulan, E. hemionus kulan, indicates an approximately 1% nucleotide sequence divergence of mitochondrial DNAs between the two taxa. The chromosomal divergence between E. hemionus and E. kiang probably originated less than 500,000 years ago. Studies of additional Asiatic wild ass taxa are indicated in order to gain further insights into the speciation and systematics of this threatened group of mammals.

Animals↗