Deoxyribonuclease I gene polymorphism in Han Chinese population: frequency and effect on glucose and lipid parameters

DNASE1, the encoding gene of deoxyribonuclease I (DNase I), exhibits polymorphisms, including a single nucleotide polymorphism (SNP A2317G) in exon 8 and a 56 bp variable number of tandem repeat, designated as HumDN1 in intron 4. Several different ethnic population studies have revealed both A2317G and HumDN1 demonstrate genetic heterogeneity in the worldwide distribution. Recently, G2317 allele was proposed as an independent risk factor for myocardial infarction in Japanese population. In the present study, we identified A2317G and HumDN1 genotypes in 402 unrelated healthy Han Chinese individuals. At the same time, the impact of different genotypes and diplotypes of DNase I on plasma lipids levels and fasting blood glucose was also illuminated. Polymerase chain reaction and restriction fragment length polymorphism were used for the detection of HumDN1 and A2317G polymorphisms. Plasma glucose and lipids were measured in fasting state by biochemical methods. Three genotypes of A2317G and 9 genotypes of HumDN1 were detected in Han Chinese population. Among them, the most predominate alleles were A2317 (frequency = 53.6%) and HumDN1*3 (frequency = 47.4%) respectively. Linkage disequilibrium between A2317G and HumDN1 polymorphisms was also observed (D' = 0.717). Haplotype A-3, presented in frequency of 46.5%, was most common. Compared to other ethnic populations, Han Chinese had its own unique DNase I gene distribution characteristics. As for the influence of DNase I gene polymorphisms on lipids and glucose levels, no association was found between either genotype or diplotype and these parameters. (all P > 0.05). Results obtained in this study could be used for anthropological investigation, probing into relations between DNase I gene and diseases.     

The Ca2+-sensing receptor gene (PCAR1) mutation T151M in isolated autosomal dominant hypoparathyroidism

Isolated autosomal dominant hypoparathyroidism is a heterogeneous disorder characterized by parathyroid hormone (PTH) deficiency, hypocalcemia and hyperphosphatemia. The candidate gene approach was used to study a large Norwegian family. The loci for the PTH gene, PTH receptor gene and RET protooncogene were excluded using dinucleotide markers and restriction fragment length polymorphism analysis. Complete cosegregation of this trait was found with the chromosomal region 3q13, using the short tandem repeat markers D3S1267, D3S1269, D3S1303, D3S1518, and RHO. This region contains the candidate locus for the Ca²⁺-sensing receptor (PCAR1). By single-strand conformation polymorphism (SSCP) analysis of all PCAR1 exons followed by automated sequencing, we identified a C to T transition in exon 2 (cDNA position 452) on the mutant allele in the family. The mutation predicts a substitution of Thr to Met in amino acid position 151 (T151M). A StyI restriction site created by the nucleotide substitution was used to confirm the mutation on all alleles, as well as to exclude it among 100 normal alleles (blood donors). SSCP analysis also identified a novel polymorphism of PCAR1 intron 4 (1609–88t→c) on normal alleles.The T151M mutation is located in the extracellular N-terminal domain of PCAR1, which belongs to the superfamily of G protein-coupled receptors. We suggest that this is a gain-of-function mutation that increases the sensitivity of the receptor to [Ca²⁺], thereby decreasing the calcium set point. Received: 29 September 1995 / Revised: 19 January 1996     

New Polymorphic Sites in the Human Phenylalanine Hydroxylase Gene

A previously unknown sequence of the human phelylalanine hydroxylase (PAH) gene intron 7 (GeneBank AN AF204239) has been reported. Screening of the group of phenylketonuria patients from Nobosibirsk region for polymorphic sites within intron 7 revealed single nucleotide substitutions at intron positions 332, 451, 574 and 791. Polymorphic site at intron position 791 corresponds to one of the eight restriction sites (MspI) utilized for haplotype construction. Analysis of the MspI allele frequencies in 29 phenylketonuria patients showed that the frequency of the MspI+ allele in this group was 79.4%. Polymorphic sites at nucleotide position +97 from the beginning of intron 10, and at nucleotide position –54 from the end of intron 5, were also described. The polymorphic sites revealed can be used as markers for identification of the PAH alleles in population genetic studies, and also serve for diagnostics of phenylketonuria (PKU). The presence of numerous nucleotide substitutions within the intronic sequences confirms highly polymorphic structure of the PAH gene.     

A polymorphism in a region with enhancer activity in the second intron of the human apolipoprotein B gene

A 443-base pair fragment (+622 to +1064) from the second intron of the human apolipoprotein B gene was shown to contain a tissue-specific enhancer when placed in front of an apolipoprotein B promoter-chloramphenicol acetyltransferase construct in transfection experiments. To identify potential regulatory mutations in this region of the gene, DNA from various subjects was examined for the presence of point mutations by means of chemical cleavage of mismatched heteroduplexes. An A----G substitution within the second intron of the gene at position +722 was identified in three unrelated subjects and confirmed by DNA sequencing. Although the base substitution was contained within a nuclear protein-binding site, as determined by DNase I footprinting, it did not appear to affect the protein/DNA interaction in its vicinity, as shown by gel retardation experiments. The single base substitution at position +722 abolishes a StyI restriction site, thus creating a StyI polymorphism. Using allele-specific oligonucleotides, we screened the DNA of 172 subjects for the presence of this polymorphism: two other subjects carrying the polymorphism were found. In each of the five unrelated subjects, the polymorphism was associated with the same haplotype.     

Two new polymorphisms in introns 2 and 3 of the human porphobilinogen deaminase gene

Sequencing of the polymerase chain reaction amplified exon 2–4 fragment of the human porphobilinogen deaminase gene revealed a G/T polymorphism (I2G and I2T) in intron 2, and a G/A polymorphism (I3G and I3A) in intron 3 of the gene. The frequencies of these alleles are presented.The nucleotide sequence data reported in this paper (the sequence of introns 2 and 3, and the polymorphic sites) will appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Database with accession nos. D10608 and D12722     

Molecular mapping of the Oregon Wolfe Barleys: a phenotypically polymorphic doubled-haploid population

A phenotypically polymorphic barley (Hordeum vulgare L.) mapping population was developed using morphological marker stocks as parents. Ninety-four doubled-haploid lines were derived for genetic mapping from an F₁ using the Hordeum bulbosum system. A linkage map was constructed using 12 morphological markers, 87 restriction fragment length polymorphism (RFLP), five random amplified polymorphic DNA (RAPD), one sequence-tagged site (STS), one intron fragment length polymorphism (IFLP), 33 simple sequence repeat (SSR), and 586 amplified fragment length polymorphism (AFLP) markers. The genetic map spanned 1,387 cM with an average density of one marker every 1.9 cM. AFLP markers tended to cluster on centromeric regions and were more abundant on chromosome 1 (7H). RAPD markers showed a high level of segregation distortion, 54% compared with the 26% observed for AFLP markers, 27% for SSR markers, and 18% for RFLP markers. Three major regions of segregation distortion, based on RFLP and morphological markers, were located on chromosomes 2 (2H), 3 (3H), and 7 (5H). Segregation distortion may indicate that preferential gametic selection occurred during the development of the doubled-haploid lines. This may be due to the extreme phenotypes determined by alleles at morphological trait loci of the dominant and recessive parental stocks. Several molecular markers were found to be closely linked to morphological loci. The linkage map reported herein will be useful in integrating data on quantitative traits with morphological variants and should aid in map-based cloning of genes controlling morphological traits. Received: 23 August 2000 / Accepted: 15 December 2000     

Restriction fragment length polymorphism at the HLA-C locus

We have used the HLA-C-specific DNA probe pC250 to investigate restriction fragment length polymorphism (RFLP) at the HLA-C locus. Genomic Southern blot hybridization included DNA prepared from a panel of homozygous typing cells representing serological specificities Cw1 to Cw8 and also from samples representing Cw blanks. Although many restriction nucleases failed to reveal any polymorphism, RFLPs were evident with Taq I, Pvu II, Bst XI, Nde 1, and Nci I in addition to the previously reported Eco RI. In the case of Bst XI, a unique RFLP defined a subset of serologically defined Cw blanks. Comparison of RFLP sizes with restriction fragment lengths obtained from the known HLA-Cw3 gene sequence permitted the localization of intragenic C locus RFLLs and the identification of a variable Taq I site in the second intron, a variable Nci I site near the end of the fourth exon, and a variable Pvu lI site in the fifth intron.     

A new marker at DXS 115 useful for carrier detection in hemophilia A

Summary In this brief communication we report a new intergenic polymorphism at DXS115 as a marker for detection of heterozygotes in families at risk for hemophilia A. Total genomic DNA was isolated from white blood cells, double digested by KpnI and XbaI and hybridized with EcoRI/SstI fragment of the genomic probe p482.6. The incidence of the polymorphic 5.1-kb fragment was estimated as 0.069 in a German population. A technical advantage of using the XbaI/KpnI RFLP is that both the intragenic XbaI-RFLP in intron 22 of factor VIII gene and the new intergenic RFLP can be evaluated at the same time.     

HLA-JY328: Mapping studies and expression of a polymorphic HLA class I gene

The JY328 clone was identified in a human genomic library using cDNA corresponding to mRNA for HLA-B7 as a probe. The L/328 cell line was established by cotransformation of mouse Ltk^– cells with the herpes thymidine kinase gene and clone JY328. On Northern blots, RNA from,L/328 strongly hybridized to an HLA class I probe, and an antigen was recognized by an anti-HLA class I framework antibody on the cell surface. A DNA probe corresponding to a segment of intron 7 was developed by comparing the nucleotide sequence of clone JY328 with that of other HLA class I-type genes. Using the radiolabeled probe to screen Southern blots of DNA from families with siblings exhibiting intra-HLA recombinations, a restriction fragment length polymorphism was revealed —a 1.4 kb BstE II band not present in all individuals. A corresponding fragment was apparent in the base sequence of clone JY328. The occurrence of this band on Southern blots established that JY328 maps distinct from and centromeric to the HLA-C locus and near to the HLA-B locus. Antibody absorption studies and cytotoxicity tests indicated that the JY328 gene product was not an HLA-B antigen but that it did specifically absorb CW7-specific antibody. In sum, these results suggest a novel, polymorphic HLA class I gene which expresses a product serologically similar to HLA-Cw7 but which does not map within the corresponding locus.     

A PvuII restriction fragment length polymorphism of the glucose-6-phosphate dehydrogenase gene is an African-specific marker

Summary The site of a PvuII restriction fragment length polymorphism (RFLP) of the human glucose-6-phosphate dehydrogenase (G6PD) gene has been located in intron V, 60 bp upstream of G6PD exon VI. A population survey shows this RFLP to be specific for African populations, with frequencies of the rarer allele (PvuII type 2 site present) of 0.32–0.40 in Kenyans, Nigerians, Zambians, and West Indians. This allele has not been found in the European, Asian and Middle Eastern populations studied. Such population-specific markers may be useful in the study of population affinities and may provide insight into prehistoric migrations of peoples.     

Apolipoprotein B amino acid 3611 substitution from arginine to glutamine creates the Ag (h/i) epitope: the polymorphism is not associated with differences in serum cholesterol and apolipoprotein B levels

Summary A G-to A-DNA sequence change in exon 26 of the human apolipoprotein B (apo B) gene leads to a glutamine substitution for arginine at codon 3611 of the mature apolipoprotein B100 and causes a loss of an MspI site. In 106 Finnish individuals, a complete correspondence exists between this MspI polymorphic site and the Ag (h/i) immunochemical polymorphism. Linkage disequilibrium was found between this MspI polymorphic site and the apo B XbaI and EcoRI variable sites and the Ag (a1/d) and (c/g) epitope pairs; there is apparent linkage equilibrium with the apo B PvuII variable site. Based on three population studies (samples from London, Finland and Italy), no significant association was found between this RFLP and serum cholesterol and apo B levels. These data suggest that the arginine 3611glutamine 3611 substitution has no significant effect on apo B function.     

A new polymorphic site in the G6PD gene

Summary A polymorphic restriction site has been found in intron 11 of the gene for glucose-6-phosphate dehydrogenase (G6PD). This site is produced by a TC substitution 13 bp upstream of exon 12, producing an NlaIII restriction site. In various populations there was a strong association between a T at nt 1311 of the G6PD cDNA and the presence of the NlaIII restriction site. Among African Americans, however, the presence of a C at nt 1311 was sometimes associated with the presence of a polymorphic NlaIII site.     

Microsatellites and a single-nucleotide polymorphism differentiate apparentamylose classes in an extended pedigree of US rice germ plasm

 The Waxy gene (Wx) encodes the granule-bound starch synthase responsible for the synthesis of amylose in rice (Oryza sativa). Recently, a polymorphic microsatellite sequence closely linked to the Wx gene was reported. To determine whether polymorphism in this sequence correlates with variation in apparent amylose content, we tested an extended pedigree of 92 current and historically important long-, medium- and short-grain US rice cultivars representing the efforts of many breeders over more than 80 years. Seven Wx microsatellite alleles were identified which together explained 82.9% of the variation in apparent amylose content of the 89 non-glutinous rice cultivars tested. Similar results were also obtained with 101 progenyof a cross between low- and intermediate-amylose breeding lines. An additional, unique microsatelliteallele, (CT)₁₆, was detected in one glutinous cultivar,CI 5309. However, the other glutinous cultivars,Calmochi 101 and Tatsumi mochi, were in the (CT)₁₇ class along with three other cultivars that contained15–16.5% amylose. We sequenced a 200-bp PCR-amplified fragment containing the CT microsatellite and the putative 5′ splice site of the Wx leader intron from a subset of 42 cultivars representing all eight microsatellite alleles. All of the cultivars with 18% or less amylose had the sequence AGTTATA at the putative leader intron 5′ splice site, while all cultivars with a higher proportionof amylose had AGTTATA. This single nucleotidesubstitution could also be assayed by AccI digestion of the amplified fragment. Overall, this single nucleotide polymorphism could explain 79.7% of the variation in the apparent amylose content of the 89 non-glutinous cultivars tested. Interestingly, cultivars in the (CT)₁₉ microsatellite classes that differed substantially in amylose content still showed the correlation between this G-T polymorphism and apparent amylose content. The G-T polymorphism at this site was not, however, able to explain the very low amylose contents of the three glutinous cultivars tested, all of which had the sequence AGTTATA. Received: 31 July 1996 / Accepted: 22 November 1996     

Identification of a PstI polymorphism in the p21Cip1/Waf1 cyclin-dependent kinase inhibitor gene

A PstI polymorphism in the 3 flanking region of the p21^CiP1/Waf1 cyclin-dependent kinase inhibitor gene is described. DNA sequencing analysis identified a CT base substitution in the 3 flanking region of the gene. This substitution leads to the destruction of a PstI site and results in a biallelic DNA polymorphism. This restriction fragment length polymorphism (RFLP) provides the first known genetic marker for this cell cycle regulatory gene.     

Characterization of a new splicing mutation in the steroid 21-hydroxylase gene

We identified a novel mutation in the CYP21A2 gene, a C to G substitution in the 7-position of the intron 2 acceptor splice site (c.290-7C>G), which causes a steroid 21-hydroxylase deficiency. The effect of the mutation on splicing was checked in the system of CYP21A minigene expression in cultured mammalian cells. The mutation impairs the use of the intron 2 acceptor splice site, resulting in intron retention in mRNA.     

MspI polymorphic site in intron 22 of the factor VIII gene in the Japanese population

Summary A novel MspI DNA polymorphic site has been found in intron 22 of the human factor VIII gene. This site is informative almost exclusively in the Japanese population (heterozygosity 0.45) and will be of considerable importance in carrier detection and prenatal diagnosis of hemophilia A in this population.     

Identification of TaqI polymorphism in the mitochondrial acetoacetyl-CoA thiolase gene and familial analysis of 3-ketothiolase deficiency

We analyzed the mitochondrial acetoacetyl-CoA thiolase gene (T2) by Southern blotting. Fifteen unrelated healthy individuals and members of five families with 3-ketothiolase deficiency (3KTD) were analyzed. We found a TaqI polymorphism, the heterozygosity of which was calculated to be 0.5 among healthy Japanese individuals. This restriction fragment length polymorphism (RFLP) proved to be useful for detecting 3KTD patients and its obligatory carriers, at the DNA level and in two out of five 3KTD families. This polymorphism was found to be generated by the presence/ absence of a TaqI site in intron 9 of the T2 gene. With in vitro amplification of the genomic region around the TaqI site, this RFLP can be detected within 2 days.     

Direct Evidence for Mendelian Inheritance of the Variations in the Ribosomal Protein Gene Introns in Yellowfin Tuna (Thunnus albacares)

Restriction fragment length polymorphism found in the S7 ribosomal protein gene introns of yellowfin tuna (Thunnus albacares) was compared between a single pair of parents and their offspring. The sizes of the first intron (RP1) and second intron (RP2) amplified by polymerase chain reaction were 810 bp and 1400 bp, respectively. The dam and sire had different restriction types from one another in HhaI and RsaI digestions for RP1 and in DdeI, HhaI, and ScrFI digestions for RP2. Putative genotypes in both introns of 64 larvae were found to be segregated in Mendelian proportions. Genotype distributions in a wild yellowfin tuna sample (n= 34) were in Hardy-Weinberg proportions, and observed heterozygosity ranged from 0.149 to 0.388. This study presents novel Mendelian markers, which are feasible for tuna population genetic study and pedigree analysis. Received December 8, 1999; accepted April 11, 2000.     

An RNA splice site mutation in the C1-inhibitor gene causes type I hereditary angio-oedema

Summary Restriction fragment length polymorphism analysis, the polymerase chain reaction and nucleotide sequencing have been used to characterise a single base substitution (GT) at nucleotide 8863 in the C1-inhibitor gene. This destroys the 5 donor splice site recognition motif of the sixth intron. Family studies suggest that the mutation is responsible for type I hereditary angio-oedema in a studied kindred.