Cloning of canine rom-1 and its investigation as a candidate gene for generalized progressive retinal atrophies in dogs

Generalized progressive retinal atrophy (gPRA) represents a genetically heterogenous group of retinal degenerations affecting pedigree dogs. Currently, we are using a candidate gene approach in an attempt to identify mutations causing gPRA in dogs. Here we report the cloning, sequencing and analysis of canine rom-1 , a structural gene of the rod photoreceptor. Single-stranded conformation polymorphism (SSCP) analysis was used to look for polymorphisms segregating with gPRA in the English cocker spaniel, Labrador retriever, miniature poodle, miniature long-haired dachshund, Tibetan terrier, miniature schnauzer, Cardigan Welsh corgi and Irish wolfhound. Further investigation involved DNA sequencing and restriction fragment length polymorphism (RFLP) analysis. Our studies revealed the presence of three polymorphisms, none of which segregated with disease phenotype. Haplotype analysis identified four rom-1 alleles. Our results indicate that rom-1 is unlikely to be a cause of gPRA in the breeds of dog examined.     

Origin and colonization history of the white-clawed crayfish,Austropotamobius pallipes,in Ireland

The presence of the white-clawed crayfish Austropotamobius pallipes complex in Ireland is suspected to be a result of human translocations. Two hypotheses have been formulated about the origin of the crayfish: from British populations or from western French populations. In order to resolve this question, nine Irish crayfish populations (a total of 124 individuals) were sampled along a south-north cline and investigated by combining two molecular markers: mtDNA and RAPDs. The mtDNA marker, analysed by RFLP on the entire molecule, showed an absence of polymorphism within and among Irish populations. The RFLP haplotype found in Irish populations was only recorded in western French populations and was different from those found in English populations. This result may be explained by a human introduction of crayfish to Ireland from western French populations. RAPD analysis showed a clinal reduction of genetic variability within Irish populations from south to north, associated with an increase in their genetic differentiation. A stepwise model of translocation from the south to the north of Ireland is proposed and discussed.     

Identification of a DNA methylation point in the promoter region of the bovine CYP21 gene

The CYP21 (steroid 21-hydroxylase) gene is involved in the synthesis of steroid hormones. Bov-A2 is a retroposon that is common in ruminant genomes. The promoter region of bovine CYP21 contains a short interspersed nucleotide element of Bov-A2, which overlaps a putative Sp1 binding site. We looked for RFLP/HpaII polymorphism in the Bov-A2 element in bovine Zebu breeds by PCR-RFLP, and examined whether polymorphism in this element is associated with methylation. Among DNA samples from 135 Brazilian Zebu breed cattle, we identified an RFLP/HpaII polymorphism (T/C), which, based on a restriction methylation-sensitive assay employing HpaII and isoschizomer MspI enzymes (methylation-sensitive and -non-sensitive enzymes, respectively), appears to be a DNA methylation point. This is the first report of this polymorphism and on DNA methylation in the bovine CYP21 promoter region in Brazilian Zebu cattle.     

The effects of endothelial lipase gene (LIPG) variants on inflammation marker levels and atherosclerosis development

Atherosclerosis is a major pathological process related with several important adverse vascular events including coronary artery disease, stroke, and peripheral arterial disease. Endothelial lipase is an enzyme the activity of which affects all of lipoproteins, whereas HDL is the main substrate. The purpose of our study was to investigate the effects of endothelial lipase gene polymorphism and inflammation markers (CRP, IL-1β, IL-6, IL-8 and TNF-α) in the atherosclerosis. 104 patients with atherosclerosis and 76 healthy individuals were included in the study. LIPG ?584C/T polymorphism gene polymorphisms were assessed with PCR–RFLP method. The serum CRP levels were measured by turbidimetric method using a biochemistry autoanalyzer, whereas serum IL-1β, IL-6, IL-8, TNF-α levels were determined by enzyme-linked immunosorbent assay. In this study, we found that the frequencies of TC genotype are more prevalent in patients than controls. We found a statistically significant difference of IL-6 levels between patient and control group. Our findings suggest that T allele might play a potential role in the susceptibility to atherogenesis in the Turkish population.     

Genotyping of the lactase-phlorizin hydrolase c/t-13910 polymorphism by means of a new rapid denaturing high-performance liquid chromatography-based assay in healthy subjects and colorectal cancer patients

Adult-type hypolactasia results from the progressive decline of lactase-phlorizin hydrolase activity in enterocytes after weaning. Lactase nonpersistence may determine a primary lactose intolerance with reduced diary product consumption, which is possibly related to an increased risk of colon cancer. Recently, a genetic variant C/T(-13910) upstream of the lactase-phlorizin hydrolase (LCT) gene has been strongly correlated with the lactase persistence/nonpersistence trait in both family and case-control studies. The authors validate a denaturing high-performance liquid chromatography (dHPLC)-based assay versus conventional genotype sequencing in detecting the C/T(-13910) polymorphism of LCT and evaluate its prevalence in 2 different Italian geographical areas and in colorectal cancer patients. DNA samples of 157 healthy subjects and 124 colon cancer patients from Apulia and of 97 healthy subjects from Sardinia were evaluated for the C/T(-13910) polymorphism by dHPLC, sequencing, and restriction fragment length polymorphism (RFLP). Under optimized conditions, dHPLC was as sensitive as DNA sequencing and detected a new genetic variant (T/C(-13913)) in 2 individuals that was not identified by RFLP assay. Frequency of lactase nonpersistence genotype (C/C(-13910)) was similar in healthy subjects from 2 different Italian geographical areas and not increased in patients with colorectal cancer. The results indicate that the dHPLC method may be used as a rapid, noninvasive, and labor-saving screening tool for genotyping C/T(-13910) polymorphism, with high success, low cost, and reproducibility.     

Heterozygosity for Tay-Sachs and Sandhoff diseases in non-Jewish Americans with ancestry from Ireland, Great Britain, or Italy

Previous reports have found that non-Jewish Americans with ancestry from Ireland have an increased frequency of heterozygosity for Tay-Sachs disease (TSD), although frequency estimates are substantially different. Our goal in this study was to determine the frequency of heterozygosity for TSD and Sandhoff diseases (SD) among Irish Americans, as well as in persons of English, Scottish, and/or Welsh ancestry and in individuals with Italian heritage, who were referred for determination of their heterozygosity status and who had no known family history of TSD or SD or of heterozygosity for these conditions. Of 610 nonpregnant subjects with Irish background, 24 TSD heterozygotes were identified by biochemical testing, corresponding to a heterozygote frequency of 1 in 25 (4%; 95% CI, 1/39-1/17). In comparison, of 322 nonpregnant individuals with ancestry from England, Scotland, or Wales, two TSD heterozygotes were identified (1 in 161 or 0.62%; 95% CI, 1/328-1/45), and three TSD heterozygotes were ascertained from 436 nonpregnant individuals with Italian heritage (1 in 145 or 0.69%; 95% CI, 1/714-1/50). Samples from 21 Irish heterozygotes were analyzed for HEXA gene mutations. Two (9.5%) Irish heterozygotes had the lethal + 1 IVS-9 G --> A mutation, whereas 9 (42.8%) had a benign pseudodeficiency mutation. No mutation was found in 10 (47.6%) heterozygotes. These data allow for a frequency estimate of deleterious alleles for TSD among Irish Americans of 1 in 305 (95% CI, 1/2517-1/85) to 1 in 41 (95% CI, 1/72-1/35), depending on whether one, respectively, excludes or includes enzyme-defined heterozygotes lacking a defined deleterious mutation. Pseudodeficiency mutations were identified in both of the heterozygotes with ancestry from other countries in the British Isles, suggesting that individuals with ancestry from these countries do not have an increased rate of TSD heterozygosity. Four SD heterozygotes were found among individuals of Italian descent, a frequency of 1 in 109 (0.92%; 95% CI, 1/400-1/43). This frequency was higher than those for other populations, including those with Irish (1 in 305 or 0.33%; 95% CI, 1/252-1/85), English, Scottish, or Welsh (1 in 161 or 0.62%; 95% CI, 1/1328-1/45), or Ashkenazi Jewish (1 in 281 or 0.36%; 95% CI, 1/1361-1/96) ancestry. Individuals of Irish or Italian heritage might benefit from genetic counseling for TSD and SD, respectively.     

A combined allele-specific PCR and RFLP assay to detect the 35delG mutation in the Connexin 26 gene

Mutations in the Connexin-26 (specified GJB2) gene have been shown to be a major cause of nonsyndromic recessive deafness (NSRD), and a single mutation 35delG in the GJB2 gene accounts for the majority of cases of NSRD. For diagnostic analyses and for scientific studies of large numbers of patients, fast and economic assays that can be performed with standard polymerase chain reaction (PCR) instruments are highly desirable. We have developed an allele-specific amplification (ASA)-based restriction fragment length polymorphism (RFLP) assay. We evaluated the multiplex method for its ability to 35delG mutation. Our method is a stable, reproducible and concordend with previously reported PCR-RFLP assays.     

HDAPD: a web tool for searching the disease-associated protein structures

Background

PCR-restriction fragment length polymorphism (RFLP) assay is a cost-effective method for SNP genotyping and mutation detection, but the manual mining for restriction enzyme sites is challenging and cumbersome. Three years after we constructed SNP-RFLPing, a freely accessible database and analysis tool for restriction enzyme mining of SNPs, significant improvements over the 2006 version have been made and incorporated into the latest version, SNP-RFLPing 2.

Results

The primary aim of SNP-RFLPing 2 is to provide comprehensive PCR-RFLP information with multiple functionality about SNPs, such as SNP retrieval to multiple species, different polymorphism types (bi-allelic, tri-allelic, tetra-allelic or indels), gene-centric searching, HapMap tagSNPs, gene ontology-based searching, miRNAs, and SNP500Cancer. The RFLP restriction enzymes and the corresponding PCR primers for the natural and mutagenic types of each SNP are simultaneously analyzed. All the RFLP restriction enzyme prices are also provided to aid selection. Furthermore, the previously encountered updating problems for most SNP related databases are resolved by an on-line retrieval system.

Conclusions

The user interfaces for functional SNP analyses have been substantially improved and integrated. SNP-RFLPing 2 offers a new and user-friendly interface for RFLP genotyping that can be used in association studies and is freely available at http://bio.kuas.edu.tw/snp-rflping2.     

Detection and discrimination of Cryptosporidium parvum and C. hominis in water samples by immunomagnetic separation-PCR

Cryptosporidium parvum and C. hominis have been the cause of large and serious outbreaks of waterborne cryptosporidiosis. A specific and sensitive recovery-detection method is required for control of this pathogen in drinking water. In the present study, nested PCR-restriction fragment length polymorphism (RFLP), which targets the divergent Cpgp40/15 gene, was developed. This nested PCR detected only the gene derived from C. parvum and C. hominis strains, and RFLP was able to discriminate between the PCR products from C. parvum and C. hominis. To evaluate the sensitivity of nested PCR, C. parvum oocysts inoculated in water samples of two different turbidities were recovered by immunomagnetic separation (IMS) and detected by nested PCR and fluorescent antibody assay (FA). Genetic detection by nested PCR and oocyst number confirmed by FA were compared, and the results suggested that detection by nested PCR depends on the confirmed oocyst number and that nested PCR in combination with IMS has the ability to detect a single oocyst in a water sample. We applied an agitation procedure with river water solids to which oocysts were added to evaluate the recovery and detection by the procedure in environmental samples and found some decrease in the rate of detection by IMS.     

PCR-restriction fragment length polymorphism method for detection of Cyclospora cayetanensis in environmental waters without microscopic confirmation

We developed an alternative nested-PCR-restriction fragment length polymorphism (RFLP) protocol for the detection of Cyclospora cayetanensis in environmental samples that obviates the need for microscopic examination. The RFLP method, with the restriction enzyme AluI, differentiates the amplified target sequence from C. cayetanensis from those that may cross-react. This new protocol was used to reexamine a subset (121 of 180) of surface water samples. Samples previously positive when the CYCF3E and CYCR4B primers (33) and RFLP with MnlI (20) were used were also PCR positive with the new primers; however, they were RFLP negative. We verified, by sequencing these amplicons, that while two were most likely other Cyclospora species, they were not C. cayetanensis. We can detect as few as one oocyst seeded into an autoclaved pellet flocculated from 10 liters of surface water. This new protocol should be of great use for environmental microbiologists and public health laboratories.     

A rapid PCR-SSP assay for the hemochromatosis-associated Tyr250Stop mutation in the TFR2 gene

Several genes associated with hemochromatosis and primary iron overload have been identified. Mutations in the HFE gene have been detected in 60-100% of hemochromatosis patients of northern, central, and western European descent, although the frequencies of these mutations vary among racial and ethnic groups. Recently, a mutation in the gene encoding transferrin receptor-2 (exon 6, nucleotide 750 C --> G; Y250X) was detected by a PCR-restriction fragment length polymorphism (RFLP) method in Sicilians with hemochromatosis. We describe a modification of the original assay in which the sequence-specific priming PCR assay does not require the use of restriction endonuclease. The modified assay is robust and cost-efficient, and may be more useful for large-scale population studies because it can be performed rapidly on DNA extracted from buccal swabs.     

Non-phenotypic selection of N-methyl-N-nitrosourea-induced mutations in human cells.

The distribution of mutations in a particular gene as detected by a selective mutation assay could be affected by the structural properties of the target protein. To investigate this, we have analysed N-methyl-N-nitrosourea (MNU)-induced mutations in two restriction recognition sequences of a target gene for mutation analysis and compared these data with what previously observed in a phenotypic mutation assay. DNA base changes in the Ncil and EcoRV sites of the gpt gene maintained in human cells by a shuttle vector system were measured by restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) technique. After MNU-treatment of human cells, mutations were detected in the Ncil recognition sequence but not in the EcoRV site. DNA sequencing analysis revealed that all Ncil-resistant mutations were GC to AT transitions located over four bases of the Ncil recognition sequence. Only one of these mutations drastically affected the functionality of the GPT protein. The Ncil-resistant mutations were randomly distributed in both DNA strands of the gpt gene and were preferentially targeted at guanine residues flanked 5' by a guanine. Our results indicate that the structure of the GPT protein is the main contributor to the strand-specificity of MNU-induced mutations previously reported by using a phenotypic mutation assay. The potential use of the RFLP/PCR technique as a general tool for mutation detection is also discussed.     

The 5178C/A and 16189T/C polymorphisms of mitochondrial DNA in Korean men and their associations with blood iron metabolism

Several studies reported that there were the associations between the genetic polymorphisms in the mitochondrial DNA (mtDNA) and some blood iron markers. Thus, we tried to investigate the relationship between two genetic polymorphisms (5178C/A and 16189T/C) in the mtDNA and several blood iron markers in Korean men. A total of unrelated 131 Korean men were participated in this study. Two genetic polymorphisms in the mtDNA was determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) method, and hematological or biochemical assay performed by autoanalyzer. Although the 16189T/C polymorphism was not significantly associated with any iron parameters measured in this study, we found that the 5178C/A polymorphism was significantly associated with red blood cell (RBC) count and hematocrit (HCT) value in Korean men (P < 0.05). Therefore, our data suggest that the 5178C/A polymorphism in the mtDNA might be useful as a genetic marker with respect to blood iron metabolism.     

HLA-DR genotyping by restriction fragment length polymorphism analyses

We have established unique restriction fragment length polymorphism (RFLP) patterns characteristic of homozygous typing cells (HTCs) for HLA-DR-1 through HLA-DR-8 haplotypes. These RFLP patterns were found to segregate in family members and correlate 100% with HLA-DR antibody phenotyping. The RFLP patterns were used to type chronic myelocytic leukemic cells which have a Philadelphia translocation from 23 randomly selected Caucasoid patients. The results show an alternative method for the determination of the HLA-DR types without using live cells and to study disease association with the HLA-DR region.     

Angiotensin-converting enzyme insertion/deletion polymorphism genotyping error: the cause and a possible solution to the problem

Rigat and colleagues were the first ones to develop a rapid PCR-based assay for identifying the angiotensin converting enzyme insertion/deletion (I/D) polymorphism. Due to a big difference between the length of the wild-type and mute alleles the PCR method is prone to mistyping because of preferential amplification of the D allele causing depicting I/D heterozygotes as D/D homozygotes. The aim of this study was to investigate whether this preferential amplification can be repressed by amplifying a longer DNA fragment in a so called Long PCR protocol. We also aimed to compare the results of genotyping using five different PCR protocols and to estimate the mistyping rate. The study included 200 samples which were genotyped using standard method used in our laboratory, a stepdown PCR, PCR protocol with the inclusion of 4 % DMSO, PCR with the use of insertion specific primers and new Long PCR method. The results of this study have shown that accurate ACE I/D polymorphism genotyping can be accomplished with the standard and the Long PCR method. Also, as of our results, accurate ACE I/D polymorphism genotyping can be accomplished regardless of the method used. Therefore, if the standard method is optimized more cautiously, accurate results can be obtained by this simple, inexpensive and rapid PCR protocol.     

Amplification of a single-locus variable-number direct repeats with restriction fragment length polymorphism (DR-PCR/RFLP) for genetic typing of Acinetobacter baumannii strains

In search of an effective DNA typing technique for Acinetobacter baumannii strains for hospital epidemiology use, the performance and convenience of a new target sequence was evaluated. Using known genomic sequences of Acinetobacter baumannii strains AR 319754 and ATCC 17978, we developed single-locus variable-number direct-repeat analysis using polymerase chain reaction-restriction fragment length polymorphism (DR-PCR/RFLP) method. A total of 90 Acinetobacter baumannii strains isolated from patients of the Clinical Hospital in Bydgoszcz, Poland, were examined. Initially, all strains were typed using macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis (REA-PFGE). Digestion of the chromosomal DNA with the ApaI endonuclease and separation of the fragments by PFGE revealed 21 unique types. Application of DR-PCR/RFLP resulted in recognition of 12 clusters. The results showed that the DR-PCR/RFLP method is less discriminatory than REA-PFGE, however, the novel genotyping method can be used as an alternative technique for generating DNA profiles in epidemiological studies of intra-species genetic relatedness of Acinetobacter baumannii strains.     

Comparing Techniques for the Identification of the MTHFR A1298C Polymorphism

The restriction fragment length polymorphism (RFLP) technique with the MboII enzyme is used by a number of researchers as a methodology for the identification of the genetic polymorphism MTHFR A1298C. However, the reliability of this enzyme for genotyping this polymorphism has been questioned, since the silent polymorphism T1317C, located close to the polymorphic region A1298C on gene MTHFR, also has a recognition site for MboII. Thus, the fragments formed by the digestion of MboII present similar sizes, making it difficult to differentiate the allele MTHFR 1298A in the presence of the allele MTHFR 1317C. Hence, we investigated the A1298C polymorphism in a Brazilian population of renal transplant patients, using the RFLP technique with digestion by Mbo II and using sequencing, in order to examine the concordance between the two techniques. Our results showed an 8.6% difference in genotyping between RFLP and sequencing, but the statistical concordance test presented a kappa coefficient equal to 0.81 (CI 95% 0.74–88), which indicates a virtually perfect concordance, according to the criterion of Landis and Koch. Therefore, we concluded that the RFLP technique is concordant with automated sequencing in the detection of polymorphism A1298C under our laboratory conditions.     

光敏核不育水稻等位突变系的AFLP分析

通过对NK58S和NK58F这一对光敏核不育水稻等位突变系的AFLP分析,比较了AFLP,RAPD及RFLP检测DNA多态性的相对效率。结果表明,这三种分子标记的DNA多态性检出效率依次为AFLP>RAPD>RFLP;找出了水稻AFLP分析的最适反应条件;通过AFLP和集群混合分析(Bulked segregating analysis,BSA),筛选出了一批与水稻光敏核不育(PGMS)基因连锁的多态性AFLP产物,已完成了对4个多态性AFLP产物的克隆,Southern杂交证明其中2个为单拷贝顺序,另外2个为低拷贝顺序。对上述三种分子标记各自的优缺点及它们在DNA多态性检测中的适用之处进行了分析探讨。     

Isolation of probes detecting restriction fragment length polymorphisms from X chromosome-specific libraries: potential use for diagnosis of Duchenne muscular dystrophy

Summary We have isolated 23 human X chromosome-specific DNA fragments from libraries, prepared from flow-sorted X chromosomes. To increase diagnostic potential for X-linked genetic disorders, including Duchenne muscular dystrophy (DMD), the fragments were tested for restriction fragment length polymorphisms (RFLPs) with six restriction enzymes. All fragments were regionally mapped to segments of the X chromosome with a panel of somatic cell hybrids and with human cell lines carrying unbalanced chromosomal abnormalities. Two of the isolated probes detected a high frequency RFLP. One, 754, maps between Xp11.3 and Xp21 and detects a PstI polymorphism with an allele frequency of 0.38. The other, 782, maps between Xp22.2 and Xp22.3 and reveals an EcoRI polymorphism with an allele frequency of 0.40. According to a pilot linkage study of families at risk for Duchenne muscular dystrophy, 754 gives a maximum Lod score of 7.6 at a recombination fraction of 0.03. Probe 782 lies telomeric to DMD with a maximum Lod score of 2.2 at a recombination fraction of 0.17. Using our X-chromosomal probes and a set of autosomal probes, isolated and examined in an identical way, we found a significantly lower RFLP frequency for the X chromosome as compared to the autosomes.