Analysis of the <Emphasis Type="Italic">Alu</Emphasis>I polymorphism in intron 1 of the human coagulation factor VIII gene: A new marker for the hemophilia a carrier detection

Frequencies of the C/T SNP alleles at position 2403 of the human coagulation factor VIII gene intron 1, containing the AluI restriction endonuclease recognition site, were examined. Genomic DNA samples for the analysis were obtained from the consulted women and their relatives from the families with hemophilia A. A total of 221 unrelated X chromosomes were studied. The two allelic variants were found with similar frequencies of T(Alu+), 0.53 and C(Alu?), 0.47. The heterozygosity index evaluated as equal to 0.50 was correlated with the experimental heterozygote number. The absence of a tight linkage between the AluI SNP and the widely used in the hemophilia A gene diagnostics HindIII polymorphism (C/T SNP at position 103 of intron 19) was demonstrated. Summarized informativity of these two markers for obligate carriers and for those detected in this study constituted 68% (32 out of 47). At the same time using one of the markers, only 40% (HindIII) and 51% (AluI) of the consulted women were informative. The new marker was used in 13 prenatal DNA diagnostics of hemophilia A. A new deletion polymorphism (del TGA, position 2281–2283 of intron 1) was described in close proximity of the AluI SNP with the frequency of about 0.05. among the five other SNP of the factor VIII gene examined (Bme18I, intron 1; HpaII, intron 13; MnlI, exon 14; Bst4CI, exon 25; and MseI, exon 26) no effective diagnostic markers were found. Only the MnlI polymorphism could be recommended for limited usage.     

Molecular genetic study of the factor VIII gene in families from Bashkir with hemophilia A

The distribution of hemophilia A was studied in Bashkortostan. The factor VIII gene of the blood coagulation system was analyzed in 34 patients with hemophilia A and 48 of their close relatives. Inversion of intron 22 of the factor VIII gene was revealed in nine cases, which comprised 30% of the total sample analyzed. The type II and type III of this mutation occurred at a relatively high frequency, which may be explained by the founder effect and genetic drift. The allelic frequencies of the polymorphic locus HindIII at intron 19 were similar; a substantial allelic heterogeneity of both microsatellite (CA)-repeats at intron 13 and the DXS52 locus were found on normal and mutant X chromosomes. The molecular genetic analysis of (CA)-repeats and the loci HindIII and DXS52 in families with hemophilia A makes it possible to reveal up to 89% of the informative families.     

Combined analysis of the MspI and XbaI polymorphisms in intron 22 of the factor VIII gene for detection of hemophilia A in a Korean population

To determine the usefulness of MspI/int22h-1 (intron 22 homologous region-1) polymorphism of the factor VIII gene for molecular genetic diagnosis of hemophilia A in the Korean population, MspI/intron 22 and XbaI/intron 22 polymorphisms were analyzed in 101 unrelated Korean families with severe hemophilia A. The expected heterozygosity rates of MspI/int22h-1 and XbaI/int22h-1 polymorphisms were 49.5 and 43.6%, respectively; these polymorphisms were not in complete linkage disequilibrium. Combined analysis using both polymorphisms provided an informative rate of 66.3%. These results suggest that PCR analysis of the MspI/int22h-1 polymorphism of the factor VIII gene would be useful for carrier detection and prenatal diagnosis of hemophilia A in the Korean population.     

A new polymorphism in the factor VIII gene for prenatal diagnosis of hemophilia A.

A restriction fragment length polymorphism (RFLP) has been found in the gene for clotting factor VIII. Defects in this gene are the cause of hemophilia A. The DNA polymorphism affects an XbaI site in intron 22 of the gene. Two alleles occur in a frequency of 59 and 41 percent of the X chromosomes tested. Furthermore, about 25 percent of females who are homozygous for the previously reported BclI RFLP in the factor VIII gene are heterozygous for the XbaI polymorphism. This new RFLP thus represents a significant addition to available probes for the DNA-based prenatal diagnosis and carrier detection of this disease.     

Molecular genetic analysis of polymorphism of the HindII and (CA)-repeat of the factor VIII gene loci in people of the Volgo-Ural region

Molecular genetic analysis of polymorphism of the factor VIII gene at loci HindIII (intron 19) and (CA)-repeats (intron 13) was performed in seven ethnic groups of the Volga-Ural region (Bashkirs, Tatars, Chuvashes, Maris, Mordovians, Udmurts, and Komis). The allelic frequency of the HindIII locus was shown to be similar in these ethnic groups. The ethnic groups were more heterogeneous with regard to the genotype and allele frequencies of (CA)-repeats, which indicates that this polymorphic system can be used to characterize the genetic structure of the Volga-Ural human populations.     

Association of DNA polymorphisms of the growth hormone and prolactin genes with milk productivity in Yaroslavl and black-and-white cattle

Polymorphisms of the prolactin (bPRL) and growth hormone (bGH) genes were studied comparatively in the Russian and German Black-and-White and Yaroslavl cattle breeds. Two polymorphisms were studied for each gene. In the case of the bPRL gene, the polymorphism of the 5'-untranslated region was examined by microsatellite analysis and the RsaI polymorphism of exon 3, by RFLP analysis. In the case of the bGH gene, the MspI polymorphism of intron III and the AluI polymorphism of exon 5 were assessed by RFLP analysis. Differences in allele and genotype frequencies were observed both between and within breeds. The heterozygosity at the RsaI marker was low (9.4%) in the Russian Black-and-White breed; that at the microsatellite of the bPRL gene was low (3.2-24%) in all breeds examined. Homozygotes BB at the bPRL gene, which had not been reported earlier for European cattle breeds, were detected in the German Black-and-White and Yaroslavl breeds (at frequencies 0.16 and 0.13, respectively). The frequency of allele MspI(-) of the bGH gene in the Yaroslavl breed was extremely low (0.02), comparable only with that of the Holstein cattle (0.02). The heterozygosity at the AluI polymorphism was higher than at the MspI polymorphism of the bGH gene and reached 55% in the Yaroslavl breed. Genotype BB of the RsaI polymorphism of the bPRL gene tended to show a negative association with the fat content in milk. The genotypes of the AluI polymorphism of the bGH gene were associated with the fat content in milk in the Yaroslavl (F = 4.56, P = 0.013) and German Black-and-White (F = 4.1, P = 0.014) breeds: the highest fat content in milk was observed in the subsample of cows with heterozygous genotype VL.     

A HindIII RFLP and a gene lesion in the coagulation factor VIII gene

Summary The presence and inheritance of restriction fragment length polymorphisms (RFLPs) and gene lesions in the coagulation factor VIII gene were investigated in 15 hemophilia families. An abnormal HindIII 2.6-kb band, previously detected in a severe hemophiliac, was observed in a not severely affected patient and also in the normal gene of a woman carrying a hemophilic gene in which the lesions was found. The TaqI site in exon 24 of this defective gene was removed by a C to T transition causing an amino acid change (ArgGln). Very low amounts of factor VIII activity and antigen were detected in the severely affected grandson. The presence of the HindIII 2.6-kb fragment in both normal and patholgoical genes indicates that a factor VIII RFLP without functional meaning was found. Its frequency, determined in 60 chromosomes, is 0.18. Double digestions enabled us to map the polymorphic site 3 to the exon 19.     

Detection of carriers of hemophilia A by testing for HindIII polymorphism in the factor VIII gene by PCR

Representatives of 62 families from Moscow and Leningrad with haemophilia A observed in the pedigree were tested for HindIII polymorphism in the factor VIII gene. The proposed scheme of investigation was based on intron 19 of the FVIII gene amplification by the PCR technique followed by restriction analysis with the inner control of hydrolysis. 207 unrelated X-chromosomes were analysed, the frequency of the incidence of the polymorphic HindIII site in the given population found to be 0.29. The frequency of incidence of the HindIII heterozygotes calculated according to Hardy-Weinberg equation was 0.41. This value evidences for relatively high informativity of this polymorphism for carrier detection and prenatal diagnosis of haemophilia A. 23 families (37%) out of 62 examined in the study were informative for this criteria. The new scheme proved to be effective in testing HindIII polymorphism for haemophilia A carrier detection and prenatal diagnosis. The whole procedure takes one day, the radiolabelled probes are not used. The scheme described was inculcated in the All-Union Research Center for Haematology, Ministry of Health, USSR, Moscow, Research Institute for Obstetrics and Gynecology, Leningrad, Institute of Medical Genetics, Greifswald, DDR.     

A SNP in GmBADH2 gene associates with fragrance in vegetable soybean variety ��Kaori�� and SNAP marker development for the fragrance

Fragrance in soybean is due to the presence of 2-acetyl-1-pyrroline (2AP). BADH2 gene coding for betaine aldehyde dehydrogenase has been identified as the candidate gene responsible for fragrance in rice (Oryza sativa L.). In this study, using the RIL population derived from fragrant soybean cultivar "Kaori" and non-fragrant soybean cultivar "Chiang Mai 60" (CM60), STS markers designed from BADH2 homolog were found associating with 2AP production. Genetic mapping demonstrated that QTL position of fragrance and 2AP production coincides with the position of GmBADH2 (Glycine max betaine aldehyde dehydrogenase 2). Sequence comparison of GmBADH2 between Kaori and non-fragrant soybeans revealed non-synonymous single-nucleotide polymorphism (SNP) in exon 10. Nucleotide substitution of G to A in the exon results in an amino acid change of glycine (GGC; G) to aspartic acid (GAC; D) in Kaori. The amino acid substitution changes the conserved EGCRLGPIVS motif of GmBADH2, which is essential for functional activity of GmBADH2 protein, to EGCRLDPIVS motif, suggesting that the SNP in GmBADH2 is responsible for the fragrance in Kaori. Five single nucleotide-amplified polymorphism (SNAP) markers which are PCR-based allele specific SNP markers were developed for fragrance based on the SNP in GmBADH2. Two markers specific to A allele produced a band in only Kaori, while three markers specific to G alleles produced a band in only CM60. The simple PCR-based allele specific SNAP markers developed in the present study are useful in marker-assisted breeding of fragrant soybean.     

兔成纤维细胞生长因子5(FGF5)基因SNP及其与产毛量的相关分析

通过PCR产物直接测序的方法, 对荥经长毛兔、天府黑兔以及加利福尼亚兔的FGF5基因外显子1和外显子3进行单核苷酸多态性分析。在外显子1的217位(位点A)检测到由TCT三碱基插入引起的移码突变, 在外显子3的59位(位点B)和3位(位点C)分别发生了错义突变由T→C和同义突变由T→C。通过计算发现各位点不同的基因型和等位基因频率在3个兔品种中存在较大的差异, 位点A、B在长毛兔和肉兔中均有各自的优势基因型和等位基因。各位点基因型与产毛量的最小二乘分析表明, 位点A各基因型的个体在产毛量上差异不显著(P＞0.05), 位点B各基因型个体产毛量的差异极显著(P＜0.01), 位点C各基因型个体产毛量的差异显著(P＜0.05)。初步推断FGF5基因可能是影响长毛兔产毛量潜在的主效基因或者与主效基因连锁, 可作为长毛兔产毛性状连锁分析的候选遗传标记。     

Genetic screening of the lipoprotein lipase gene for mutations associated with high triglyceride/low HDL-cholesterol levels

The lipoprotein lipase (LPL) enzyme plays a major role in lipid metabolism, primarily by regulating the catabolism of triglyceride (TG)-rich lipoprotein particles. The gene for LPL is an important candidate for affecting the risk of atherlosclerosis in the general population. Previously, we have shown that the HindIII polymorphism in intron 8 of the LPL gene is associated with plasma TG and HDL-cholesterol variation in Hispanics and non-Hispanic whites (NHWs). However, this polymorphism is located in an intron and hence may be in linkage disequilibrium with a functional mutation in the coding region or intron-exon junctions of the LPL gene. The aim of this study was to initially screen the LPL coding region and the intron-exon junctions by single-strand conformation polymorphism (SSCP) analysis for mutation detection in a group of 86 individuals expressing the phenotype of high TG/low HDL, followed by association studies in a population-based sample of 1,014 Hispanics and NHWs. Four sequence variations were identified by SSCP and DNA sequencing in the coding region of the gene, including two missense mutations (D9N in exon 2 and N291S in exon 6), one samesense mutation (V108V in exon 3), and one nonsense mutation (S447X in exon 9). Multiple regression analyses, including these four mutations and the HindIII polymorphic site, indicate that the association of the HindIII site with plasma TG (P=0.001 in NHWs and P=0.002 in Hispanics) and HDL-cholesterol (P=0.007 in NHWs and P=0.127 in Hispanics) is independent of all other LPL variable sites examined. These observations reinforce the concept that the intronic 8 HindIII site is functional by itself and provide a strong rationale for future comprehensive functional studies to delineate its biological significance.     

An insertion within the factor IX gene: hemophilia BEl Salvador.

A patient with moderate to severe hemophilia B has been found to have a large insertion within his factor IX gene. The site of insertion is located in a DNA segment of approximately 0.8 kb between exon IV and an EcoRI site within intron D. The size of the DNA insertion is approximately 6 kb, and it contains at least two TaqI sites, two EcoRI sites, and one HindIII site. The insert probably originates from outside the FIX gene and does not represent an internal duplication. We propose that this abnormal FIX gene be called FIX El Salvador in recognition of the birthplace of the patient.     

Further studies on the molecular systematics of Biomphalaria snails from Brazil

The polymerase chain reaction and restriction fragment length polymorphism (RFLP) of the internal transcribed spacer (ITS) region of the rRNA gene, using the enzyme DdeI were used for the molecular identification of ten species and one subspecies of Brazilian Biomphalaria. Emphasis is given to the analysis of B. oligoza, B. schrammi and B. amazonica. The RFLP profiles obtained using this enzyme were highly distinctive for the majority of the species and exhibited low levels of intraspecific polymorphism among specimens from different regions of Brazil. However, B. peregrina and B. oligoza presented very similar profiles that complicated their identification at the molecular level and suggested a very close genetic similarity between the two species. Others enzymes including HaeIII, HpaII, AluI and MnlI were tested for their ability to differentiate these species. For B. amazonica three variant profiles produced with DdeI were observed. The study demonstrated that the ITS contains useful genetic markers for the identification of these snails     

Study on the Relationship Between TSHR Gene and Thyroid Diseases

Thyroid stimulating hormone receptor (TSHR) is thought to play a critical role in the pathogenesis of certain thyroid diseases, including Graves' disease (GD), multinodular thyroid goiter (MTG), and Hashimoto's thyroiditis (HT). In order to understand whether single nucleotide polymorphisms in the TSHR gene contribute to thyroid diseases, we have conducted a case-control study in which, we examined 8 TSHR gene single-nucleotide polymorphisms in introns 1, 4, 5, 6 and exons 7 and 8, respectively, among patients with thyroid diseases. These included one family with GD (3 patients and 9 healthy members); 60 patients with familiar thyroid diseases (30 with GD, 20 with MTG, and 10 with HT patients), 48 sporadic patients with GD and 96 healthy control individuals. Direct sequencing of all 10 exons and part of introns of TSHR gene, in these patients as well as healthy controls revealed eight polymorphisms. A novel polymorphism in exon 8 AGA(Arg) → CGA(Arg). However, there were no significant differences between patients and controls in the incidence of these polymorphisms. These results suggest that the polymorphisms (polymorphism in intron 1 at 81 bp upstream of exon 2; polymorphism in intron 4 at 135 bp upstream of exon 5; polymorphism in intron 4 at 365 bp upstream of exon 5; polymorphism in intron 5 at 69 bp upstream of exon 6; means polymorphism in intron 6 at 13 bp downstream of exon 6; polymorphism in intron 6 at 187 bp upstream of exon 7; E7+16: polymorphism in 16 bp of exon 7; polymorphism in 40 bp of exon 8) of the TSHR gene may not contribute to the pathogenesis of thyroid diseases.     

First report of molecular diagnosis of Tunisian hemophiliacs A: identification of 8 novel causative mutations

ABSTRACT: INTRODUCTION: Hemophilia A is an X linked recessive hemorrhagic disorder caused by mutations in the F8 gene that lead to qualitative and/or quantitative deficiencies of coagulation factor VIII (FVIII). Molecular diagnosis of hemophilia A is challenging because of the high number of different causative mutations that are distributed throughout the large F8 gene. Molecular studies of these mutations are essential in order to reinforce our understanding of their pathogenic effect responsible for the disorder. Aim In this study we have performed molecular analysis of 28 Tunisian hemophilia A patients and analyzed the F8 mutation spectrum. METHODS: We screened the presence of intron 22 and intron 1 inversion in severe hemophilia A patients by southern blotting and polymerase chain reaction (PCR). Detection of point mutations was performed by dHPLC/sequencing of the coding F8 gene region. We predict the potential functional consequences of novel missense mutations with bioinformatics approaches and mapping of their spatial positions on the available FVIII 3D structure. RESULTS: We identified 23 different mutations in 28 Tunisian hemophilia A patients belonging to 22 unrelated families. The identified mutations included 5 intron 22 inversions, 7 insertions, 4 deletions and 7 substitutions. In total 18 point mutations were identified, of which 9 are located in exon 14, the most mutated exonic sequence in the F8 gene. Among the 23 mutations, 8 are novel and not deposited in the HAMSTeRS database nor described in recently published articles. CONCLUSION: The mutation spectrum of Tunisian hemophilia A patients is heterogeneous with the presence of some characteristic features. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1693269827490715.     

Polymorphism of PCR-based markers targeting exons, introns, promoter regions, and SSRs in maize and introns and repeat sequences in oat.

Sequence databases could be efficiently exploited for development of DNA markers if it were known which gene regions reveal the most polymorphism when amplified by PCR. We developed PCR primer pairs that target specific regions of previously sequenced genes from Avena and Zea species. Primers were targeted to amplify 40 introns, 24 exons, and 23 promoter regions within 54 maize genes. We surveyed 48 maize inbred lines (previously assayed for simple-sequence repeat (SSR) polymorphism) for amplification-product polymorphism. We also developed primers to target 14 SSRs and 12 introns within 18 Avena genes, and surveyed 22 hexaploid oat cultivars and 2 diploid Avena species for amplification-product polymorphism. In maize, 67% of promoter markers, 58% of intron markers, and 13% of exon markers exhibited amplification-product polymorphisms. Among polymorphic primer pairs in maize, genotype diversity was highest for SSR markers (0.60) followed by intron markers (0.46), exon markers (0.42), and promoter markers (0.28). Among all Avena genotypes, 64% of SSR markers and 58% of intron markers revealed polymorphisms, but among the cultivars only, 21% of SSR markers and 50% of intron markers were polymorphic. Polymorphic-sequence-tagged sites for plant-breeding applications can be created easily by targeting noncoding gene regions.     

New efficient extragenic microsatellite markers for hemophilia A carrier state diagnostics

In search of new efficient markers for genetic diagnostics of hemophilia A, two tri-nucleotide microsatellite repeats (STR) at chromosome X loci, which flank coagulation factor VIII gene (F8), namely STR HA472--CTT-repeat, which is localized adjacent to the GAB3 gene 163 bp apart from the 3' end of the F8 gene and STR HA544--repeat (CTT)x(ATT)y located at a distance of 375 bp from the 5' end of the F8 gene were discovered. Detailed analysis using PCR and sequencing has shown that STR HA472 contains two long variable CTT-blocks separated by small spacer CCTCCC. The location of recognition site of restriction endonuclease Mnl1 (CCTC) in the spacer permits to test differentially the polymorphic blocks and thus to increase the analysis informativity. STR HA544 is also represented by two polymorphic blocks (CTT and ATT), for separate amplification of which highly informative PCR amplification assays were elaborated. The study has been done using DNA samples of 212 individuals (125 women) from 48 families with hemophilia A carriers. Our results point to Mendelian inheritance of the markers studied, a high number of allelic variants and high heterozygosity, which was 90% and 100% for HA544 and HA472, respectively. This permitted us to use these data for practical gene diagnostics of the carriers and prenatal diagnostics of hemophilia A. In addition to high informativity STR HA472 and HA544 are highly important for diagnostics as they are located at a shorter distance than other known extragenic polymorphisms of the F8 gene. In contrast to dinucleotide repeats, trinucleotide repeats are readily tested, not requiring high-resolution electrophoretic systems. In addition, they are located on the opposite sites of the F8 gene. This permits to control homologous recombination events in the locus and thus to prevent diagnostic mistakes.