Four DNA polymorphisms in the LDL-receptor gene and their use in diagnosis of familial hypercholesterolemia

Summary To examine the potential usefulness of restriction fragment length polymorphisms (RFLPs) for diagnosis of familial hypercholesterolemia (FH), we determined the genotype of FH patients and their relatives for the ApalI, NcoI, PvuII and StuI RFLP of the LDL-receptor gene in a sample of German patients attending the Lipid Clinic in Munich. There was no significant difference in the relative allele frequency between the group of FH patients and controls for any of the four polymorphisms. Using linkage analysis, we could determine the four-RFLP haplotypes of 39 defective and 90 normal LDL-receptor genes in 38 FH families. In our sample, defective LDL-receptor genes occur on 6 different chromosomes determined by the four RFLPs. This suggests that at least 6 different genetic defects may cause FH in this sample. RFLPs of the LDL-receptor gene cannot be used to detect FH in individuals; however, appropriate diagnosis can be carried out in more than 90% of families using linkage analysis and these RFLPs.     

Haplotype frequencies of the collagen type-I genes in the Italian population

Summary We have determined the frequencies of six restriction fragment length polymorphisms (RFLPs) of type-I collagen genes in a random sample of 100 subjects. Alpha 1 gene (COL1A1) DNA polymorphisms, FG2/MspI, 2FC6/RsaI, and NST70/RsaI, had polymorphism information content (PIC) values of 0.35, 0.32, and 0.26, respectively. Alpha 2 gene (COL1A2) RFLPs, NJ3/EcoRI, Hf32/RsaI, and Hf32/MspI had PIC values of 0.36, 0.35, and 0.25, respectively. The combined haplotype PIC values were 0.71 at the COL1A1 locus and 0.73 for COL1A2. Two COL1A1 and two COL1A2 RFLPs were more polymorphic than in the English population, making them better markers for the analysis of Italian families affected by osteogenesis imperfecta and some other inherited collagen diseases.     

Mucopolysaccharidosis IVA: polymorphic haplotypes and informative RFLPs in the Japanese population

Seven different restriction fragment length polymorphisms (RFLPs) at the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) locus were analyzed using Southern blotting and polymerase chain reaction based techniques to search for the frequency of each RFLP produced by StyI, SphI, HaeIII, StuI, HapII, XhoI, and BamHI restriction endonucleases, respectively, in 36 mutant alleles, including two sibling cases and 100 normal alleles. Calculation of heterozygosity indexes showed that these RFLPs were polymorphic, ranging from 0.31 to 0.69 in mucopolysaccharidosis IVA (MPS IVA) patients compared with 0.21 to 0.65 in normal individuals. There was some significant difference in several RFLPs and in the combination with four kinds of RFLPs (SphI, StuI, HapII, XhoI polymorphisms). The normal alleles were composed of 13 different RFLPs haplotypes; the most common among the Japanese population carrying normal alleles was haplotype 8 (bDEF1) (31.3%), the others being dispersed. The same haplotype 8 was the most frequent in the mutant alleles (44.4%), with seven further haplotypes. These findings revealed the striking variety of polymorphic haplotypes in the MPS IVA gene. By using these five kinds of RFLPs, we examined the theoretical informativity of haplotype analysis in heterozygote detection in nine unrelated MPS IVA families and ten unrelated normal families. All the members of the MPS IVA families studied were diagnosed as a patient, carrier, or noncarrier. We propose that prenatal diagnosis or family analysis in cases in which mutations have not been characterized is now feasible.     

DNA polymorphisms within the porphobilinogen deaminase gene in two Swedish families with acute intermittent porphyria

Summary Two unrelated families with acute intermittent porphyria (AIP), an autosomal dominant disease related to a defect in porphobilinogen deaminase (PBG-D, EC 4.1.3.8.), were studied with regard to three restriction fragment length polymorphisms (RFLPs) (MspI, PstI, BstNI) within the PBG-D gene. The results indicate that linkage analysis of RFLPs within the gene can be used as a complement to PBG-D analysis for the diagnosis of gene carriers in families with AIP.     

Two missense mutations causing tyrosinemia type 1 with presence and absence of immunoreactive fumarylacetoacetase

Hereditary tyrosinemia type 1, due to a deficiency of fumarylacetoacetase (FAH), is characterized by progressive liver damage and renal tubular dysfunction and may occur in an acute or a chronic form. An Ala 134 to Asp (GCT to GAT) transition was found in one Turkish and two Norwegian patients with chronic tyrosinemia. SphI digestion of polymerase chain reaction (PCR) amplified genomic DNA identified the mutation and showed that the patients were heterozygous. All these patients had immunoreactive FAH protein in fibroblasts. Another Norwegian patient with chronic disease, without FAH immunoreactive material in fibroblasts, had a Pro 342 to Leu mutation (CCG to CTG). This mutation was identified by MspI digestion of PCR amplified genomic DNA, and the patient was heterozygous. Northern blotting showed FAH mRNA of normal size and amounts in all patients. Site directed mutagenesis and translation in a rabbit reticulocyte lysate demonstrated that both mutations abolished FAH activity.     

The human tyrosine aminotransferase gene: characterization of restriction fragment length polymorphisms and haplotype analysis in a family with tyrosinemia type II

Summary Deficiency in hepatic tyrosine aminotransferase (TAT) causes tyrosinemia type II, an autosomal recessively inherited disorder. Using a TAT cosmid clone, we have identified an MspI restriction fragment length polymorphism (RFLP) 5 to the TAT gene, with allele frequencies of 0.63 and 0.37. Analysis of the cloned maternal and paternal TAT alleles from patient with tyrosinemia type II led to the identification of a HaeIII RFLP at the 3 end of the TAT gene, with allele frequencies of 0.94 and 0.06. The two RFLPs are 27 kb apart and in no allelic association. From haplotype frequencies, a polymorphism information content (PIC) value of 0.44 was obtained. The two RFLPs have allowed the unambiguous identification of the mutant TAT alleles in the patient's pedigree by haplotype analysis.     

Identification of three RFLPs at the HCK locus on chromosome 20

New HindIII, RsaI and TaqI restriction fragment length polymorphisms (RFLPs) within the haemopoietic cell kinase gene in chromosome band 20q11.2 are described. These RFLPs provide a useful marker for linkage analysis in proximal 20q.     

No association between RFLPs at the porphobilinogen deaminase gene and schizophrenia

Summary An association study of restriction fragment length polymorphisms (RFLPs) in the porphobilinogen deaminase (PBGD) gene and schizophrenia was conducted. RFLPs detected by MspI, PstI, ApaLI and BstNI in intron 1 of the gene were studied in 49 patients and 79 controls. There were no significant differences between the groups in allele frequencies, genotype counts or haplotype distribution.     

Improved carrier detection of haemophilia A using novel RFLPs at the DXS115 (767) locus

Summary Two novel restriction fragment length polymorphisms (RFLPs) around the DXS115 (767) locus, detectable with the restriction enzymes MspI, are described. Since DXS115 is closely linked to the factor VIII gene (F8C), the MspI RFLP was employed in haemophilia A carrier detection. The utility of these RFLPs lies in the increased applicability and accuracy of diagnoses carried out in cases where available intragenic markers are uninformative.     

Type I interferon genes in cattle: restriction fragment length polymorphisms,gene numbers and physical organization on bovine chromosome 8

Multiple, superimposed Type I interferon (IFN) restriction fragments were resolved following 72–92 h of horizontal electrophoresis. Restriction fragment length polymorphisms (RFLPs) for α IFN (IFNA), β IFN (IFNB), ωIFN (IFNW) and trophoblast IFN (IFNT) genes were identified in Hin dill, Eco RI and Taql digestions from 313 cattle. RFLPs with codominant segregation in cattle pedigrees were considered alleles, and 19 distinct polymorphic Type I IFN loci (5 IFNA, 4 IFNB, 8 IFNW and 2 IFNT) were identified. Allele frequencies and observed heterozygosity values were calculated for each locus and several loci were considered highly informative for linkage analysis. Bovine IFN gene numbers (10 IFNA, 6 IFNB, 20 IFNW and 6 IFNT) were estimated from the number of polymorphic loci plus additional monomorphic hybridizing bands present in Eco RI and Hindlll digestions. Physical linkage of the Type I IFN gene families on bovine chromosome 8 was demonstrated by pulsed field gel electrophoresis (PFGE). Hybridization of two or more IFN probes to similarly sized PFGE fragments suggested the tentative gene family order: IFNA/IFNW-IFNT-IFNB. These studies provide a basis for the development of more detailed genetic and physical maps of the bovine Type I IFNs.     

Use of three DNA polymorphisms of the LDL receptor gene in the diagnosis of familial hypercholesterolemia

Summary Familial hypercholesterolemia (FH) is an autosomal dominant metabolic disorder caused by several different mutations in the low density lipoprotein (LDL) receptor gene. This large number of different mutations, often undetectable in Southern blotting, makes it impossible directly to diagnose the disease. However, restriction fragment length polymorphisms (RFLPs) can be used to follow the inheritance of the defective gene in FH families. In the present study, we report the use of three RFLPs, detected by PvuII, ApaLI and AvaII restriction enzymes, to determine the haplotypes of normal and defective LDL receptor genes in 61 families with FH and in 128 normal individuals. Two of the nine haplotypes were significantly more often associated with the affected genes, whereas one was significantly less frequent. Although none of the associations was strong enough to allow diagnosis in individuals, it was possible, using the three RFLPs, to identify the haplotype of the affected gene in 57 families and to carry out unequivocal diagnosis in 67% of the cases. In four families, PvuII and AvaII detected an abnormal fragment co-segregating with the disease, thus increasing the percentage of diagnosis to 73.4% of the cases.     

Structural organization and analysis of the human fumarylacetoacetate hydrolase gene in tyrosinemia type I

Fumarylacetoacetate hydrolase (FAH) is a metabolic enzyme functioning at the last steo of tyrosine catabolism. Deficiency in this enzyme activity is associated with tyrosinemia type I, characterized by hypertyrosinemia, liver dysfunction, renal tubular dysfunction, liver cirrhosis, and hepatic tumors. We isolated from a human gene library a chromosomal gene related to FAH. The human FAH gene is 30 kilobases long and is split into 14 exons. All of the splice donor and acceptor sites conform to the GT/AG rule. We also analyzed findings in a patient with tyrosinemia type I with respect to the mutation responsible for detects in the enzyme. A nucleotide change from T to G was found in the exon 2 of the gene and this change was accompanied by an amino acid substitution (Phe62Cys). Transfection and expression analysis of the cDNA is cultured BMT-10 cells with the nucleotide substitution demonstrated that the substitution was indeed responsible for the decreased activity of the enzyme in the patient. These results confirmed that the T to G mutation was one of the causes of tyrosinemia type I. Structure of the FAH gene and tests for expression of the mutant FAH will facilitate further understanding of various aspects of FAH.     

Alcohol dehydrogenase genes: restriction fragment length polymorphisms for ADH4 (π-ADH) and ADH5 (χ-ADH) and construction of haplotypes among different ADH classes

Of the five human alcohol dehydrogenase (ADH) genes located in the region q21–25 of chromosome 4, genetic markers have been reported previously only for class I enzymes, ADH1-3. Here, new restriction fragment length polymorphisms (RFLPs) are described for the genes of two other classes, ADH4 () and ADH5 ( or formaldehyde dehydrogenase, FDH). The frequencies and modes of inheritance of these RFLPs were determined with DNA both from unrelated individuals and from families. A polymorphic PstI site is assigned to the fourth intron of the ADH4 gene. Pairwise linkage disequilibrium calculations for these new RFLPs and already known RFLPs at the ADH2 and ADH3 loci establish strong linkage disequilibria between polymorphic MspI and BstXI sites in the ADH5 gene as well as between XbaI and MspI sites in the ADH3 gene. Furthermore, linkage disequilibria were detected between RFLPs of the ADH2 and ADH3 genes as well as between those of the ADH4 and ADH5 genes. The latter disequilibrium implies a hitherto unknown physical proximity of two genes belonging to different ADH classes. The RFLPs were used to construct chromosomal haplotypes that include three ADH classes. Of the 16 possible haplotypes for four RFLP markers used here, 10 were experimentally detected. The potential application of the ADH RFLPs and haplotypes in linkage or association studies of inherited diseases such as familial alcoholism is discussed.     

Hereditary tyrosinemia type I: strong association with haplotype 6 in French Canadians permits simple carrier detection and prenatal diagnosis.

Hereditary tyrosinemia type 1 (HT1), a severe inborn error of tyrosine catabolism, is caused by deficiency of the terminal enzyme, fumarylacetoacetate hydrolase (FAH). The highest reported frequency of HT1 is in the French Canadian population, especially in the Saguenay-Lac-St-Jean region. Using human FAH cDNA probes, we have identified 10 haplotypes with TaqI, KpnI, RsaI, BglII, and MspI RFLPs in 118 normal chromosomes from the French Canadian population. Interestingly, in 29 HT1 children, a prevalent haplotype, haplotype 6, was found to be strongly associated with the disease, at a frequency of 90% of alleles, as compared with approximately 18% in 35 control individuals. This increased to 96% in the 24 patients originating from Saguenay-Lac-St-Jean. These results suggest that one or only a few prevailing mutations are responsible for most of the HT1 cases in Saguenay-Lac-St-Jean. Since most patients were found to be homozygous for a specific haplotype in this population, FAH RFLPs have permitted simple carrier detection in nine different informative HT1 families, with a confidence level of 99.9%. Heterozygosity rate values obtained from 52 carriers indicated that approximately 88% of families at risk from Saguenay-Lac-St-Jean are fully or partially informative. Prenatal diagnosis was also achieved in an American family. Analysis of 24 HT1 patients from nine countries gave a frequency of approximately 52% for haplotype 6, suggesting a relatively high association, worldwide, of HT1 with this haplotype.     

Linkage disequilibrium relationships among four polymorphisms within the human fibrinogen gene cluster

The extent of linkage equilibrium was estimated among four recently characterized human fibrinogen restriction fragment length polymorphisms (RFLPs) using a randomly selected group of 110 individuals from California. Two coding region RFLPs, RsaI and MnlI (FGA codon 312 and FGB codon 448, respectively), and two RFLPs located in the 5 flanking region of the FGB gene, AluI (HindIII) and HaeIII, were analyzed. Maximum likelihood estimates based on genotypic data indicated that the RsaI polymorphism in the FGA gene was at apparent linkage equilibrium with the MnlI, AluI, and HaeIII sites in the FGB gene, but strong linkage disequilibrium was noted for the MnlI-AluI, MnlI-HaeIII, and AluI-HaeIII RFLP pairs within the latter gene. The discrepancy in disequilibrium relationships among these closely linked RFLPs may indicate a region of increased recombination between the FGA and FGB RFLP loci. The FGA RsaI polymorphism, when used in conjunction with any of the FGB sites examined, will provide more detailed linkage or association data than analyses that would utilize only FGB sites. Effective use of polymorphisms within the fibrinogen locus will aid analysis of the relationships between fibrinogen genotype, plasma fibrinogen levels, and risk of cardiovascular disease.     

Multiple restriction fragment length polymorphisms associated with the Vc determinant of the MN blood group-related chimpanzee V-A-B-D system

Twelve restriction fragment length polymorphisms (RFLPs) were detected in common chimpanzee using two restriction enzymes (HindIII andMspI) and four DNA probes to the coding regions of the human glycophorin A (GPA) and glycophorin B (GPB) genes and their 3-untranslated regions. Seven RFLPs correlated with red cell expression of the V^c determinant of the MN blood group-related V-A-B-D system and five RFLPs correlated with nonexpression of this antigen. Animals heterozygous for theV allele that encodes the V^c determinant had all 12 polymorphic restriction fragments and appeared to show reduced intensity of probe hybridization to these fragments, consistent with the presence of aV and a non-V allele. No RFLPs were detected withEcoRI,SstI, orBamHI, in spite of the relatively large segment of DNA (at least 20 kb) involved in the polymorphisms. The RFLPs were chimpanzee specific and were not found in man, gorilla, orangutan, or gibbon. Multiple RFLPs distinguishing primate species are rare and may be useful markers for molecular evolution.This work was supported in part by National Institutes of Health Grants AM 33463 and CA 33000.     

Restriction Fragment Length Polymorphism Analysis of the Mitochondrial DNA of Fusarium oxysporum f. sp. ciceris

Seven isolates of Fusarium oxysporum f. sp. ciceris, representing pathogenic races 1 , 2, 3, and 4 from India and 0, 5, and 6 from Spain, were assayed for restriction fragment length polymorphisms (RFLPs) in the mitochondrial DNA,(mt DNA). The mt DNA fraction of total fungal DNA was purified and digested with the restriction endonucleases Bam HI, Bgl II, Eco RI. Kpn I, Sac I, Sal I, Sma I, and Xho I. The mt DNA is a circular molecule of 40.5 kb. No RFLP in the mt DNA was detected among the seven races of F. o. ciceris. The identical restriction patterns of mt DNA indicates an extensive conservation in the gene composition of mt DNA without sequence variation, and suggests that mt DNA of F. o. ciceris may not be responsible for pathogenic diversity. The restriction map of mt DNA from the race 6 isolate Fo 8272 was constructed by digestion of the mt DNA with five restriction enzymes: Eco RI, Kpn I, Sac I, Sal I, and Xho I, either singly or in selected pairs.     

Restriction fragment length polymorphisms detected by anonymous DNA probes mapped to defined intervals of human chromosome 16

Summary Three anonymous DNA probes ACH207, ACH224, and ACH202, isolated from a flow-purified chromosome 16 library and mapped to defined intervals of human chromosome 16, detected restriction fragment length polymorphisms (RFLPs). The RFLPs were of simple two allele types. The ACH207 (D16S4) probe detected a TaqI and an MspI RFLP with polymorphism information content (PIC) values of 0.30 and 0.27; the ACH224 (D16S5) probe detected and RsaI RFLP, PIC value of 0.34; and the ACH202 (D16S14) probe detected an XbaI RFLP, PIC value of 0.22.     

DNA polymorphism of the human complementC8β gene: formal genetics and intragenic localization

The eighth component of human complement consists of three subunits of different molecular mass, which are coded for by three separate genetic loci. Polymorphisms have been described at the protein level for the alpha and beta subunits by means of sodium dodecyl sulfate gel electrophoresis and isoelectric focusing. Using a full-length humanC8β cDNA probe, we have studied more than 100 individuals by Southern blot analysis to detect DNA polymorphisms. We have found two restriction fragment length polymorphisms (RFLPs) with the enzymesTaqI andBam HI. TheTaq I polymorphism is defined by two alleles, i.e., a single 4.9 kb fragment or two 2.8/2.1 kb fragments. The allele frequencies are 0.68 and 0.32, respectively. The second RFLP withBam HI is correlated with theTaq I variants: 3 kbBam HI; 4.9 kbTaq I and 3.3 kbBam HI; 2.8/2.1 kbTaqI. Both RFLPs could be mapped to the 3′ portion of theC8β gene. Based on the size of genomic restriction fragments, theC8β gene can be estimated to have a size of 32–36 kb. Because of the even frequency distribution, theC8β DNA polymorphisms may be useful in gene mapping and disease association studies.     

Inheritance of plastids in interspecific hybrids of blue spruce and white spruce

Summary Chloroplast DNA (cpDNA) was purified from blue spruce (Picea pungens Engelm.) and white spruce [P. glauca (Moench) Voss], and was digested with several different restriction endonucleases. Restriction fragment length polymorphisms (RFLPs) were identified that differentiated the cpDNA of both species. Intraspecific conservation of the RFLPs that differentiated each species was confirmed by examining trees from across the natural range of each species. Ten F₁ hybrids were examined, and the cpDNA from each showed the banding pattern of the paternal species. Cloned Petunia cpDNA containing part of the rbcL gene hybridized to polymorphic bands, while a cloned maize mtDNA probe of the coxII gene failed to hybridize to any band.