PCR-based Single-strand Conformation Polymorphism Research Articles

Molecular Differentiation of Four Species of Oropsylla (Siphonaptera: Ceratophyllidae) Using PCR-Based Single Strand Conformation Polymorphism Analyses and DNA Sequencing.

It is often difficult to distinguish morphologically between closely related species of fleas (Siphonaptera). Morphological identification of fleas often requires microscopic examination of internal structures in specimens cleared using caustic solutions. This process degrades DNA and/or inhibits DNA extraction from specimens, which limits molecular-based studies on individual fleas and their microbiomes. Our objective was to distinguish between Oropsylla rupestris (Jordan), Oropsylla tuberculata (Baker), Oropsylla bruneri (Baker), and Oropsylla labis (Jordan & Rothschild) (Ceratophyllidae) using PCR-based single strand conformation polymorphism (SSCP) analyses and DNA sequencing. A 446 bp region of the nuclear 28S ribosomal RNA (rRNA) gene was used as the genetic marker. The results obtained for 36 reference specimens (i.e., fleas that were morphologically identified to species) revealed no intraspecific variation in DNA sequence, whereas the DNA sequences of the four species of Oropsylla differed from one another at two to six nucleotide positions. Each flea species also had a unique SSCP banding pattern. SSCP analyses were then used to identify another 84 fleas that had not been identified morphologically. DNA sequencing data confirmed the species identity of fleas subjected to SSCP. This demonstrates that PCR-SSCP combined with DNA sequencing of the 28S rRNA gene is a very effective approach for the delineation of four closely related species of flea.

Identification of bacteria in the Rocky Mountain wood tick, Dermacentor andersoni, using single-strand conformation polymorphism (SSCP) and DNA sequencing.

PCR-based single-strand conformation polymorphism (SSCP) analyses combined with DNA sequencing of the prokaryotic 16S ribosomal (r) RNA gene encompassing the hypervariable V4 region was used to determine the bacterial composition of Rocky Mountain wood ticks (Dermacentor andersoni) attached to Richardson's ground squirrels (Urocitellus richardsonii) and questing on vegetation in southern Saskatchewan, Canada. The bacteria present in questing adult ticks from Saskatchewan Landing Provincial Park included Rickettsia peacockii, a Francisella-like endosymbiont (FLE) and an Arsenophonus-like endosymbiont. Bacteria in the adult and nymphal ticks attached to U. richardsonii collected from Beechy included R. peacockii, a FLE, and several other genera (e.g., Ralstonia, Sphingobium, Comamonas and Pseudomonas). The bacteria detected in D. andersoni in the present study are consistent with the findings of other studies that have characterized the microbiome of this tick species in the USA using next generation sequencing. This result demonstrates that the SSCP-based approach used in this study is cost- and time-effective for examining bacterial composition in ticks.

Association analysis of polymorphisms in six keratin genes with wool traits in sheep.

ObjectiveThe purpose of this study was to investigate the genetic effects of six keratin (KRT) genes on the wool traits of 418 Chinese Merino (Xinjiang type) (CMXT) individuals.MethodsTo explore the effects and association of six KRT genes on sheep wool traits, The polymerase chain reaction-based single-strand conformation polymorphism (PCR-SSCP), DNA sequencing, and the gene pyramiding effect methods were used.ResultsWe report 20 mutation sites (single-nucleotide polymorphisms) within the six KRT genes, in which twelve induced silent mutations; five induced missense mutations and resulted in Ile→Thr, Glu→Asp, Gly→Ala, Ala→Ser, Se→His; two were nonsense mutations and one was a same-sense mutation. Association analysis showed that two genotypes of the KRT31 gene were significantly associated with fiber diameter (p<0.05); three genotypes of the KRT36 gene were significantly associated with wool fineness score and fiber diameter (p<0.05), three genotypes of the KRT38 gene were significantly associated with the number of crimps (p< 0.05); and three genotypes of the KRT85 gene were significantly associated with wool crimps score, body size, and fiber diameter (p<0.05). Analysis of the gene pyramiding effect between the different genotypes of the gene loci KRT36, KRT38, and KRT85, each genotype in a gene locus was combined with all the genotypes of another two gene loci and formed the different three loci combinations, indicated a total of 26 types of possible combined genotypes in the analyzed population. Compared with the other combined genotypes, the combinations CC-GG-II, CC-HH-IJ, CC-HH-JJ, DD-HH-JJ, CC-GH-IJ, and CC-GH-JJ at gene loci KRT36, KRT38, and KRT85, respectively, had a greater effect on wool traits (p<0.05).ConclusionOur results indicate that the mutation loci of KRT31, KRT36, KRT38, and KRT85 genes, as well as the combinations at gene loci KRT36, KRT38, and KRT85 in CMXT have significant effects on wool traits, suggesting that these genes are important candidate genes for wool traits, which will contribute to sheep breeding and provide a molecular basis for improved wool quality in sheep.

Further evidence from SSCP and ITS DNA sequencing support Trypanosoma evansi and Trypanosoma equiperdum as subspecies or even strains of Trypanosoma brucei

The subgenus Trypanozoon includes three species Trypanosoma brucei, Trypanosoma evansi and Trypanosoma equiperdum, which are morphologically identical and indistinguishable even using some molecular methods. In this study, PCR-based single strand conformation polymorphism (PCR-SSCP) was used to analyze the ribosomal DNA of the Trypanozoon species. Data indicate different patterns of ITS2 fragments between T. brucei, T. evansi and T. equiperdum by SSCP. Furthermore, analysis of total ITS sequences within these three members of the subgenus Trypanozoon showed a high degree of homology using phylogenetic analysis but were polyphyletic in haplotype networks. These data provide novel nuclear evidence to further support the notion that T. evansi and T. equiperdum should be subspecies or even strains of T. brucei.

Culture -independent Pathogenic Bacterial Communities in Bottled Mineral Water

Bottled mineral water (BMW) is an alternative to mains water and consider it to be better and safer. Access to safe BMW from the bacteria involving potential health hazard is essential to health. Cultivation-independent technique PCR-based single-strand conformation polymorphism (SSCP) for genetic profiling of PCR-amplified 16S rRNA genes was performed using Com primer set targeting the 16S rRNA genes for detection of pathogenic bacteria in bottled mineral water from the final product of six factories for bottled mineral drinking water in Wadi El-natron region- Egypt. These factories use often ozone technology to treat large quantities of water because of its effectiveness in purifying and conditioning water. A total of 27 single products were isolated from the profiles by PCR re-amplification and cloning. Sequence analysis of 27 SSCP bands revealed that the 16S rRNA sequences were clustered into seven operational taxonomic units (OTUs) and the compositions of the communities of the six samples were all common. The results showed that most communities from phyla Alphaproteobacteria and certainly in the Sphingomonas sp . Culture-independent approaches produced complementary information, thus generating a more accurate view for the bacterial community in the BMW, particularly in the disinfection step, as it constitutes the final barrier before BMW distribution to the consumer

Mutation scanning-based identification of larval and nymphal ticks (Acari: Ixodidae) from Richardson's ground squirrels (Spermophilus richardsonii)

The objective of this study was to identify the tick species parasitizing Richardson's ground squirrels (Spermophilus richardsonii) in southern Saskatchewan (Canada). Morphological examination of the adult ticks revealed the presence of three tick species, Ixodes sculptus, Ixodes kingi and Dermacentor andersoni. However, given the difficulties in identifying some of the larval and nymphal (immature) ticks using this approach, PCR-based single-strand conformation polymorphism (SSCP) and DNA sequence analyses of a portion of the mitochondrial (mt) 16S rRNA gene were used to determine their species identity. The results showed that each tick species had a unique set of SSCP profiles and DNA sequences using this mt marker. The species identity of larval and nymphal ticks was determined based on a comparison of these profiles and sequences with those of morphologically-identified adults. The detection of three tick species, which are known vectors of disease-causing agents, on the same host has important implications for understanding the ecology of vector-borne diseases, and provides an opportunity to examine fundamental questions regarding the structure and composition of the bacterial communities (i.e., both endosymbiotic and pathogenic species) in these ticks. This study shows the utility and benefits of using the present molecular method for the accurate identification of ticks at any stage of development.

Molecular-based investigation of Cryptosporidium and Giardia from animals in water catchments in southeastern Australia

There has been no large-scale systematic molecular epidemiological investigation of the waterborne protozoans, Cryptosporidium or Giardia, in southeastern Australia. Here, we explored, for the first time, the genetic composition of these genera in faecal samples from animals in nine Melbourne Water reservoir areas, collected over a period of two-years. We employed PCR-based single-strand conformation polymorphism (SSCP) and phylogenetic analyses of loci (pSSU and pgp60) in the small subunit (SSU) of ribosomal RNA and 60-kDa glycoprotein (gp60) genes to detect and characterise Cryptosporidium, and another locus (ptpi) in the triose-phosphate isomerase (tpi) gene to identify and characterise Giardia. Cryptosporidium was detected in 2.8% of the 2009 samples examined; the analysis of all amplicons defined 14 distinct sequence types for each of pSSU and pgp60, representing Cryptosporidium hominis (genotype Ib – subgenotype IbA10G2R2), Cryptosporidium parvum (genotype IIa – subgenotypes IIaA15G2R1, IIaA19G2R1, IIaA19G3R1, IIaA19G4R1, IIaA20G3R1, IIaA20G4R1, IIaA20G3R2 and IIaA21G3R1), Cryptosporidium cuniculus (genotype Vb – subgenotypes VbA22R4, VbA23R3, VbA24R3, VbA25R4 and VbA26R4), and Cryptosporidium canis, Cryptosporidium fayeri, Cryptosporidium macropodum and Cryptosporidium ubiquitum as well as six new pSSU sequence types. In addition, Giardia was identified in 3.4% of the samples; all 28 distinct ptpi sequence types defined were linked to assemblage A of Giardia duodenalis. Of all 56 sequence types characterised, eight and one have been recorded previously in Cryptosporidium and Giardia, respectively, from humans. In contrast, nothing is known about the zoonotic potential of 35 new genotypes of Cryptosporidium and Giardia recorded here for the first time. Future work aims to focus on estimating the prevalence of Cryptosporidium and Giardia genotypes in humans and a wide range of animals in Victoria and elsewhere in Australia. (Nucleotide sequences reported in this paper are available in the GenBank database under accession nos. KC282952–KC283005).

An in‐situ baiting bioassay for detecting Phytophthora species in irrigation runoff containment basins

Experiments were conducted in irrigation runoff containment basins to assess the effects of bait species (Camellia japonica, Ilex crenata or Rhododendron catawbiense), bait type (whole leaf vs. leaf disc), baiting duration (1, 2, 7 or 14 days), baiting depth and growth media (modified PARP‐V8 or PARPH‐V8) on the recovery of Phytophthora species. A two‐rope, flexible bait‐deployment system was compared with a one‐rope fixed system for bait stability at designated locations and depths. A total of 907 Phytophthora isolates were subjected to PCR‐based single‐strand conformation polymorphism (PCR‐SSCP) analysis to identify to species level. Seven distinct SSCP patterns representing six morphospecies: P. citricola (Cil I), P. citrophthora (Cip I), P. hydropathica (Hyd), P. insolita (Ins), P. megasperma (Meg I &amp; II) and an unidentified Phytophthora species were identified. Irrespective of culture medium, 7 days of baiting with rhododendron leaves consistently resulted in the recovery of the greatest diversity and populations of Phytophthora species with minimum interference from Pythium species. The flexible bait‐deployment system was superior to the fixed system, minimizing the risk of bait loss and dislocation of baiting units and allowing baits to remain at designated depths from the surface under inclement weather.

Atrial Natriuretic Peptide Gene and Plasma Pro-ANP Concentration in Patients with Primary Open-Angle Glaucoma

Atrial Natriuretic Peptide Gene and Plasma Pro-ANP Concentration in Patients with Primary Open-Angle GlaucomaAtrial natriuretic peptide (ANP) appears to have a physiological role in volume and pressure homeostasis. Increased cardiac expression and synthesis of ANP suggest a possible local paracrine function in a number of tissues including the eye. Therefore, the identification of genetic markers may prove to be an important advance in the diagnosis of patients with glaucoma and hypertension. Plasma pro-ANP concentration was measured in 30 clinical patients. A significant elevated level of prehormone was observed in glaucoma patients with blood hypertension. Also, the distribution of the genotypes and alleles of the HpaII, SmaI and ScaI polymorphisms of the ANP gene was examined in 20 hypertensive patients with glaucoma and normotensive controls. The frequencies of the ANP genotypes and alleles did not differ significantly between controls and hypertensive patients. PCR-RFLP (polymerase chain reaction—restriction fragment length polymorphism) analysis shows a T2238→C transition in three hypertensive patients within the stop codon leading to the translation of ANP with an additional arginine. In the current study we also searched for any alterations in the 5' proximal promoter region of the ANP gene (-595 bp - -384 bp) in 20 glaucoma patients with hypertension using PCR-based SSCP (single-strand conformation polymorphism) analysis. No significant alterations in the 5' proximal promoter region of the ANP gene were observed among hypertensive patients. The structure of the ANP hormone encoded gene suggests potential importance in various diseases, but the regulatory function of ANP in the eye requires further investigations.

Targeting c-KIT, PDGFR in cancer of unknown primary: a screening study for molecular markers of benefit

In view of available targeted therapies, we investigated the presence of c-kit, PDGFR gene mutations and protein expression in cancer of unknown primary (CUP) in order to study their contribution in pathogenesis, their prognostic value and potential as therapeutic targets. Mutations in hot spots c-kit exon 11 and PDGFR exons 12 and 18 were studied in paraffin-embedded tumour samples from 50 patients with CUP by means of PCR-based single-strand conformational polymorphism and protein expression by means of streptavidin-biotin immunoperoxidase assays. Molecular markers were screened for possible correlations with patient outcome. No shifted band was detected in any of the polyacrylamide gel electrophoreses, indicating absence of c-kit exon 11 and PDGFR exon 12, 18 mutations. Immunohistochemical analysis in 37 tumours revealed positive membranous CD117 expression in 30 samples (81%) of which five exhibited strong (+3), four moderate (+2) and 21 weak (+1) staining. PDGFRa protein staining was seen in 15 out of 30 (50%) cases, mostly weak (13) and rarely moderate (1) or strong (1). The expression of KIT or PDGFRa protein did not correlate with the clinical outcome of the patients in our cohort. In a moderate-sized CUP patient cohort, KIT or PDGFRa protein overexpression is rare, does not have gross prognostic significance for survival and is not associated with presence of activating mutations.

Classification of Mycoplasma synoviae strains using single-strand conformation polymorphism and high-resolution melting-curve analysis of the vlhA gene single-copy region

Mycoplasma synoviae is an economically important pathogen of poultry worldwide, causing respiratory infection and synovitis in chickens and turkeys. Identification of M. synoviae isolates is of critical importance, particularly in countries in which poultry flocks are vaccinated with the live attenuated M. synoviae strain MS-H. Using oligonucleotide primers complementary to the single-copy conserved 5' end of the variable lipoprotein and haemagglutinin gene (vlhA), amplicons of approximately 400 bp were generated from 35 different M. synoviae strains/isolates from chickens and subjected to mutation scanning analysis. Analysis of the amplicons by single-strand conformation polymorphism (SSCP) revealed 10 distinct profiles (A-J). Sequencing of the amplicons representing these profiles revealed that each profile related to a unique sequence, some differing from each other by only one base-pair substitution. Comparative high-resolution melting (HRM) curve analysis of the amplicons using SYTO 9 green fluorescent dye also displayed profiles which were concordant with the same 10 SSCP profiles (A-J) and their sequences. For both mutation detection methods, the Australian M. synoviae strains represented one of the A, B, C or D profiles, while the USA strains represented one of the E, F, G, H, I or J profiles. The results presented in this study show that the PCR-based SSCP or HRM curve analyses of vlhA provide high-resolution mutation detection tools for the detection and identification of M. synoviae strains. In particular, the HRM curve analysis is a rapid and effective technique which can be performed in a single test tube in less than 2 h.

Mutational analysis of PTPRT phosphatase domains in common human cancers

A recent report revealed that the protein-tyrosine phosphatase, receptor-type, T (PTPRT) gene is somatically mutated in several types of human cancer, suggesting that the mutated PTPRT gene is a tumor suppressor gene in human cancers. However, because previously the mutational search has focused primarily on colon cancers, data on PTPRT mutations in other types of human cancer have largely been lacking. Here, we performed a mutational analysis of the PTPRT phosphatase domain by polymerase chain reaction-based single-strand conformation polymorphism (PCR-SSCP) assay in 345 cases of common human cancers, including colon carcinomas, hepatocellular carcinomas, acute leukemias, gastric carcinomas, breast carcinomas and non-small cell lung cancers. We detected PTPRT phosphatase domain mutations in 1 of 105 colon carcinomas (1%) and 1 of 48 gastric carcinomas (2%), but none in acute leukemias, hepatocellular carcinomas, breast carcinomas and non-small cell lung cancers. The PTPRT mutation detected in the colon carcinoma was a missense mutation and the mutation in the gastric carcinomas was a splice-site mutation. Contrary to the previous report on the frequent PTPTR phosphatase domain mutations in colon cancers, this study demonstrated that the somatic mutation of the PTPRT phosphatase domain rarely occurred in common human cancers. The data suggested that alterations of the PTPRT-mediated signaling pathway by PTPRT phosphatase domain mutation may not play a critical role in the development of common human cancers.

The relationship between frameshift mutations of transforming growth factor-beta type II receptor, insulin growth factor II receptor, bcl-2 associated X protein, E2F4 and microsatellite instability in gastric carcinoma

To determine the microsatellite instability in gastric carcinomas, examine the frameshift mutations of transforming growth factor-beta type II receptor (TGFbetaRII), insulin growth factor II receptor (IGFIIR), bcl-2 associated X protein (BAX) and E2F4, and investigate whether or how alterations of the TGFbetaRII, IGFIIR, BAX and E2F4 gene are associated with MSI in gastric cancer. Formalin-fixed, paraffin-embedded gastrectomy specimens and matching normal tissues of 65 cases of gastric carcinomas were retrieved from shanghai Ruijin Hospital and Shanghai East Hospital. DNA was extracted from tissue sections using phenol chloral isoamyl alcohol. Sections with no more than 50% of tumor cell areas were isolated by microdissection. DNA was amplified by PCR-based single strand conformation polymorphism (SSCP) for microsatellite analysis and was sequenced directly. Frameshift mutations in the coding regions, repetitive mononucleotide tracts of (A)10 for TGFbetaRII, (G)8 for IGFIIR, (G)8 for BAX, and trinucleotide repeats of (AGC)13 for transcription factors E2F4 were detected using PCR. Tumors were classified as being microsatellite stable (MSS) or having a low frequency of MSI (MSI-L, one of markers different in the tumor) or a high frequency of MSI (MSI-H, two or more of markers different). Eleven cases (18.0%) showed MSI-L, 12 (19.7%) showed MSI-H and 38 (62.3%) cases showed MSS. The mutation rates of TGFbetaRII, IGFIIR, BAX and E2F4 gene were 19.7%, 4.9%, 6.6% and 16.4% respectively. Among the 12 MSI-H gastric cancers, there were 10 TGFbetaRII mutations, 3 IGFIIR mutations, 4 BAX mutations and 10 E2F4 gene mutations. The alterations in the repeats of the related genes presented polymorphisms. Associations of MSI-H status and mutations of the 4 genes were highly significant (P < 0.01, respectively). No repeat tracts mutations in TGFbetaRII, IGFIIR, BAX and E2F4 gene were found in MSS tumors. The repeat coding regions within TGFbetaRII, IGFIIR, BAX and E2F4 gene are the targets of microsatellite instability. Frameshift mutations of the 4 genes play an important role in the development and progression of gastric cancers with microsatellite instability.

Analysis of genetic variability within Thelazia callipaeda (Nematoda: Thelazioidea) from Europe and Asia by sequencing and mutation scanning of the mitochondrial cytochrome c oxidase subunit 1 gene

This study investigated genetic variability within the 'eyeworm'Thelazia callipaeda (Nematoda: Thelazioidea) from Europe and Asia by polymerase chain reaction (PCR)-coupled sequencing and mutation scanning of the mitochondrial cytochrome c oxidase subunit 1 gene (cox 1). Eight different sequence variants of cox 1 (haplotypes) were determined for the 50 individual adult specimens of T. callipaeda (from dogs, foxes or cats from Italy, Germany and the Netherlands and from dogs from China and Korea). Nucleotide variation (0.3--2%) was detected at 23 of 649 positions in the cox 1. Six of these positions were invariable among all 37 individuals from Europe and among the 13 individuals from Asia (irrespective of host origin) but differed (five G<-->A and one C<-->T changes) between Europe and Asia. PCR-based single-strand conformation polymorphism (SSCP) analysis of the most variable portion (v-cox 1) of the cox 1 was validated (for a subset of samples) as a tool to rapidly screen for genetic (haplotypic) variability. The results for the SSCP analysis and sequencing were concordant, indicating that the mutation scanning approach provides a useful tool for investigating the population genetics and molecular ecology of T. callipaeda.

PCR-SSCP as a molecular tool for the identification of Benedeniinae (Monogenea: Capsalidae) from marine fish

PCR-based single strand conformation polymorphism (SSCP) analysis was used to characterize monogenean specimens of the subfamily Benedeniinae of morphologically uncertain specific status from different marine fish species, using Neobenedenia melleni, N. girellae and Entobdella corona for comparison. The first internal transcribed spacer (ITS-1) and the 5' terminal variable region (D1-D3 domains) of the large subunit ribosomal DNA (lsrDNA) were amplified separately from individual monogeneans, and the amplicons were subjected to PCR-SSCP analyses, followed by direct sequencing. Both SSCP patterns and the ITS-1 sequences data allowed specimens representing Entobdella spp. from the host Taeniura melanospilos to be unequivocally distinguished from those representing Neobenedenia spp. and those representing Benedenia spp. Neobenedenia girellae, a morphologically controversial species, had identical SSCP banding pattern and ITS-1 sequence to that of N. melleni, supporting the proposal that N. girellae is a synonym of N. melleni. Neobenedenia spp. and Benedenia spp. had identical SSCP patterns and ITS-1 sequences. These findings and the PCR-SSCP approach taken should have implications for the accurate identification and assessment of taxonomic validity of other important monogenean groups of the marine fish.

An improved ‘cold SSCP’ method for the genotypic and subgenotypic characterization of Cryptosporidium

A simple, non-isotopic PCR-based single-strand conformation polymorphism (‘cold SSCP’) method is described which allows the efficient detection of genetic variation among and within genotypes of Cryptosporidium parvum. This low cost approach has important advantages over other ‘genotyping’ methods and is applicable to a wide range of genetic loci and organisms.

An electrophoretic tool for the genetic characterisation and delineation of lungworms

In the present study, PCR-based single-strand conformation polymorphism (SSCP) analysis of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) was applied to the genetic characterisation of Dictyocaulus from red deer from New Zealand and to its differentiation from species of lungworm from cattle and other hosts. Based on SSCP profiles, Dictyocaulus individuals from red deer from different geographical localities in New Zealand could be readily distinguished from those representing other lungworms examined, irrespective of low-level sequence variability in the ITS-2 (0.4–2.6%) detectable among individuals. The ITS-2 of Dictyocaulus from red deer differed in sequence by ∼7–35% from congeners from other cervid hosts, demonstrating that this parasite is genetically distinct from other species of Dictyocaulus for which ITS-2 sequence data are presently available. The results emphasize the need for a large-scale molecular systematic study of Dictyocaulus specimens from various species of cervid and other ruminant hosts and the usefulness of mutation scanning for taxonomic, epidemiological and population genetic investigations.

Association analyses between polymorphisms of the phase II detoxification enzymes (GSTM1, NQO1, NQO2) and alcohol withdrawal symptoms.

NRH-quinone oxidoreductase 2 (NQO2) along with glutathione S-transferase M1 (GSTM1) and NAD(P)H-quinone oxidoreductase 1 (NQO1), which is involved in phase II detoxification reactions, is thought to be important for detoxification of catechol o-quinones in the central nervous system. Our previous study revealed that the human NQO2 gene is highly polymorphic. In this study, we investigated a possible association between polymorphisms of the GSTM1, NQO1, and NQO2 genes and alcohol withdrawal symptoms such as delirium tremens, hallucination, and seizure. A total of 247 Japanese male alcoholic patients with alcohol withdrawal symptoms or without the symptoms, and 134 age-matched Japanese male controls (nonhabitual drinkers), were examined by using polymerase chain reaction (PCR), PCR restriction fragment length polymorphism, PCR-based single-strand conformational change polymorphism, and PCR direct sequencing analyses. RESULTS A significant difference was found between alcoholic patients and controls in genotype frequency at an insertion/deletion site in the promoter region of the NQO2 gene (p = 0.0014). The frequency of the homozygous genotype for the D allele at this locus was significantly higher in delirium tremens-positive patients (p = 0.0004) and in hallucination-positive patients (p = 0.0001), and in patients displaying both delirium tremens and hallucination (p = 0.0002), than in controls. The values were still significant after Bonferroni correction. On the other hand, no significant difference was detected for allele frequencies or genotype frequencies for the other polymorphic loci of the NQO2 gene. Moreover, GSTM1 gene deletion and missense mutation (Pro187Ser) of the NQO1 gene showed no significant association with alcohol withdrawal symptoms. Present data suggest that an insertion/deletion polymorphism in the promoter region of the NQO2 gene plays an important role in the pathogenesis of alcoholism and alcohol withdrawal symptoms.

Survey of Cryptosporidium parvum genotypes in humans from the UK by mutation scanning analysis of a heat shock protein gene region

A mutation scanning–selective sequencing approach was employed for the genotypic identification and differentiation of Cryptosporidium parvum isolates. Genomic DNA samples ( n=158) from Cryptosporidium oocysts from humans with clinical cryptosporidiosis (following recent foreign travel or during different outbreaks) in the UK were subjected to PCR-based single-strand conformation polymorphism (SSCP) analysis of a heat shock protein 70 gene region ( p-hsp70; 448 bp). Samples representing different SSCP profiles were then subjected to sequencing. The analysis allowed the classification of 149 of the 158 samples as type-1 (∼49%) or type-2 (∼46%); amplicons from the remaining nine samples were consistent in size with p-hsp70 but represented non-specific, faecal contaminants. The percentages reflected those of a previous study in the UK for autochtonous, sporadic cases of cryptosporidiosis (∼49% for type-1 and ∼47% for type-2; n≈4000), but contrasted another survey of sporadic cases where type-2 dominated (∼62%; n≈1000). The ability of the present SSCP-sequencing approach to accurately screen for C. parvum genotypes and to reliably discern erroneous amplicons has significant implications for the accurate diagnosis and monitoring of cryptosporidiosis and for population genetic studies.

Germline mutations of the MEN1 gene in Korean families with multiple endocrine neoplasia type 1 (MEN1) or MEN1-related disorders.

Multiple endocrine neoplasia type 1 (MEN1) is a familial cancer syndrome characterized by the combined occurrence of tumours of the parathyroid glands, pancreatic islet cells and anterior pituitary gland. Mutation analysis of the MEN1 gene has enabled the genetic diagnosis of patients with MEN1. Two MEN1-related disorders - familial isolated hyperparathyroidism (FIHP) and familial pituitary adenoma - are considered to be variants of MEN1, or at least to be incompletely expressed variants. Germline mutations of the MEN1 gene have been reported in some with FIHP, but familial pituitary adenoma usually lacks the MEN1 mutation and has been described as a genetically distinct disorder. In this work, we investigated five Korean families with MEN1, one family with FIHP and one family with familial pituitary adenoma. Polymerase chain reaction (PCR)-based single-strand conformation polymorphism (PCR-SSCP) analysis, denaturing high-performance liquid chromatography (DHPLC) and sequencing were used to detect the MEN1 mutations. Screening of the genetic variations of the MEN1 gene revealed four germline mutations in five typical MEN1 families. All four germline mutations led to truncated proteins or a change in the amino acids of the functional domains. In this study, we identified three novel MEN1 germline mutations (969C >A, 973G >C and 1213C >T) and one previously reported mutation (200-201insAGCCC). The frequency of the MEN1 germline mutation in Korean MEN1 families (four of five; 80%) was similar to those reported previously. In accordance with previous studies, no MEN1 germline mutation was detected in two families with FIHP or familial pituitary adenoma.