The human fumarylacetoacetase gene: characterisation of restriction fragment length polymorphisms and identification of haplotypes in tyrosinemia type 1 and pseudodeficiency

Summary Deficiency of human fumarylacetoacetase (FAH) activity results in hereditary tyrosinemia type I. Using the restriction enzymes BglII, KpnI and StuI and a 1.3-kb cDNA probe for the FAH gene, we have found 6 restriction fragment length polymorphisms (RFLPs). These RFLPs were utilised in 3 tyrosinemia families in which one or both parents are carriers of both a tyrosinemia and a pseudodeficiency gene for FAH. Full information was obtained in two of these families. The polymorphisms identified 6 haplotypes. The haplotype distribution was significantly different in 32 unrelated tyrosinemia patients compared with a reference population of 100 individuals. The combined polymorphism information content was 0.77.     

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.     

Linkage disequilibrium detected between myotonic dystrophy and the anonymous marker D19S63 in the Spanish population

Summary We used the following polymorphic markers: APOC2 (BanI, BglI, TaqI), CKMM (NcoI, TaqI), and D19S63 (PstI) to haplotype 33 Spanish myotonic dystrophy (DM) families. We analysed the allele and haplotype frequencies of our sample, and the possible association of alleles or haplotypes with the disease. We found a slight linkage disequilibrium between APOC2 (BanI) and DM, but no disequilibrium when using all other APOC2 and CKMM RFLPs; this agrees with data previously reported. In addition, we found a very strong linkage disequilibrium when using D19S63 (PstI), the + allele being associated with the DM locus. This disequilibrium in the Spanish population indicates that D19S63 is very close to the DM locus.     

Polymorphisms at the GLUT1 (HepG2) and GLUT4 (muscle/adipocyte) glucose transporter genes and non-insulin-dependent diabetes mellitus (NIDDM)

Summary In order to determine the possible contribution of the GLUT1 (HepG2) glucose transporter gene to the inheritance of non-insulin-dependent diabetes mellitus (NIDDM), two restriction fragment length polymorphisms (RFLPs) and the related haplotypes at this locus were studied in 48 Italian diabetic patients and 58 normal subjects. Genotype frequencies for the XbaI polymorphism were significantly different between patients and controls (XbaI: ² = 9.80, df= 2, P < 0.0079). A significant difference was also found in the allele frequencies between NIDDM patients and controls (² =9.39, df = 1, P < 0.0022), whereas no differences were found for the StuI RFLP. No linkage disequilibrium was detected between the XbaI and StuI RFLPs in this sample. The analysis of the four haplotype frequencies (X1S1, X1S2, X2S1, X2S2) revealed a significant difference between diabetic patients and controls (² = 14.26, df =3, P < 0.002). By comparing single haplotype frequencies, a significant difference between the two groups was found for the X1S1 and X2S2 haplotypes. A two-allele RFLP at the GLUT4 (muscle/adipocyte) glucose transporter gene, detected with the restriction enzyme KpnI, was also examined; no differences were found between patients and controls for this RFLP. The finding of an association between polymorphic markers at the GLUT1 transporter and NIDDM suggests that this locus may contribute to the inherited susceptibility to the disease in this Italian population.     

Mucopolysaccharidosis IVA: structural gene alterations identified by Southern blot analysis and identification of racial differences

Ninety-six alleles (36 alleles of Japanese and 60 of Caucasian origin) from forty-eight patients with mucopolysaccharidosis IVA were investigated for structural gene alterations using Southern blot analysis. All patients had a previously demonstrated deficiency of N-acetylgalactosamine-6-sulfate-sulfatase and exhibited a wide spectrum of clinical severity. Initially, using the fulllength cDNA as a probe, five of 36 chromosomes from the Japanese patients revealed similar rearrangements with respect to DNA digested with BamHI, SacI, and XhoI. Subsequent analysis using seven genomic fragments, covering the entire gene, enhanced the detection of aberrant fragments produced by the above restriction enzymes. Conversely, the 60 chromosomes of Caucasian origin revealed no evidence of large structural rearrangements when analyzed by these methods. There was a statistically significant difference between the two populations (P < 0.01). A severely affected Japanese patient showed structural rearrangements on both chromosomes by means of BamHI blots. An 8.0-kb fragment and a highly polymorphic 7.0-kb to 11.0-kb fragment present in normal individuals disappeared and two aberrant fragments of 11.5 kb and 12.0 kb were observed. Three other Japanese patients also showed these two aberrant fragments, in addition to the normal fragment pattern, and were thus heterozygous for this rearrangement. Interpretation of Southern blots was difficult because of the complexity of polymorphic bands resulting from variable number of tandem repeat elements. However, by utilizing these aberrant fragments or polymorphic bands, carrier detection was effective, even in families with poorly characterized mutations. Hybridization with probe MG-A (5end genomic probe in intron 1) showed a 8.4-kb fragment in BamHI blots of one Japanese and one Caucasian patient; XhoI, SacI, and EcoRI blots were normal. Since this BamHI alteration was also observed in one normal control, it appears to be a rare nonpathological polymorphism.     

Polymorphic haplotypes and recombination rates at the LDL receptor gene locus in subjects with and without familial hypercholesterolemia who are from different populations.

RFLPs at the low-density lipoprotein (LDL) receptor locus for TaqI, StuI, HincII, AvaII, ApaLI (5' and 3'), PvuII, and NcoI were studied in Swiss and German families with familial hypercholesterolemia (FH). A total of 1,104 LDL receptor alleles were analyzed using Southern blotting and new PCR-based techniques for detection of the TaqI, StuI, HincII, AvaII, NcoI RFLPs. Two hundred fifty-six independent haplotypes from 368 individuals of 61 unrelated Swiss families, as well as 114 independent haplotypes from 184 subjects of 25 unrelated German families, were constructed. In 76 families, clinical diagnosis of FH was confirmed by cosegregation analysis. Of the 43 unique haplotypes consisting of seven RFLPs detected in the Swiss and Germans, only 9 were common in both population samples. Analysis of linkage disequilibrium revealed nonrandom associations between several of the investigated RFLPs. ApaLI (5'), NcoI, PvuII, TaqI, and AvaII or HincII were particularly informative (cumulative informativeness .85). Relative frequencies, heterozygosity indexes, and PICs of the RFLPs from the Swiss and Germans were compared with values calculated from reported haplotype data for Italians, Icelanders, North American Caucasians, South African Caucasians, and Japanese. Pairwise comparisons of population samples by common RFLPs demonstrated unexpected differences even between geographically adjacent populations (e.g., the Swiss and Germans). Furthermore, genetic distances from the Germans to the other Caucasians were larger than to the Japanese. An unexpected lack of correlation between linkage disequilibria and physical distances was detected for the German and Japanese data, possibly because of nonuniform recombination with excessively high rates between exon 13 and intron 15. Hence, the present study revealed a striking variety of polymorphic haplotypes and heterogeneity of RFLP frequencies and recombination rates among the seven population samples.     

Diversity of MHC class I haplotypes in cynomolgus macaques

Cynomolgus macaques are widely used as a primate model for human diseases associated with an immunological process. Because there are individual differences in immune responsiveness, which are controlled by the polymorphic nature of the major histocompatibility (MHC) locus, it is important to reveal the diversity of MHC in the model animal. In this study, we analyzed 26 cynomolgus macaques from five families for MHC class I genes. We identified 32 Mafa-A, 46 Mafa-B, 6 Mafa-I, and 3 Mafa-AG alleles in which 14, 20, 3, and 3 alleles were novel. There were 23 MHC class I haplotypes and each haplotype was composed of one to three Mafa-A alleles and one to five Mafa-B alleles. Family studies revealed that there were two haplotypes which contained two Mafa-A1 alleles. These observations demonstrated further the complexity of MHC class I locus in the Old World monkey.     

Linkage disequilibrium between phenylketonuria and RFLP haplotype 1 at the phenylalanine hydroxylase locus in Portugal

Summary RFLPs of 36 normal and 41 mutant alleles at the phenylalanine hydroxylase locus were determined in 31 Portuguese kindreds. A total of 14 haplotypes including 10 normal and 7 mutant alleles were observed. Almost 75% of all mutant alleles were confined within only two haplotypes, namely haplotype 9 (17.1%) and haplotype 1 (56.1%). This frequency of mutant haplotype 1 in Portugal is, to our knowledge, the highest for this mutant haplotype in all studies reported to date. Other mutant haplotypes were either rare (haplotype 2, 9.7%) or totally absent (haplotype 3, 0%). Only 24.5% of all mutant alleles were found to consistently carry identified mutations, particularly R261Q (9.8%), R252W (3.3%), R408W (1.6%) and I94 (3.3%). A new mutation, L249F, located in the seventh exon of the gene, accounted for 6.5% of all mutant alleles in our series. Interestingly, this mutant genotype was consistently associated with mutant haplotype 1 (P<0.01), as also observed for the R261Q mutation. It appears, therefore, that mutant haplotype 1 is genotypically heterogeneous in Portugal and that more than two mutations account for its prevalence in this country.     

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.     

DNA haplotypes of the human apoprotein B gene in coronary atherosclerosis

Summary Haplotypes of the apoprotein B gene, localised to chromosome 2, were identified using restriction fragment length polymorphisms (RFLPs) for the enzymes XbaI and EcoRI. Four haplotypes were identified at this locus, X1R1 (H1), X1R2 (H2), X2R1 (H3) and X2R2 (H4); where the X1 and X2 alleles were characterised by gene-related fragments of 5.0 and 8.6 kb respectively and the R1 and R2 alleles by fragments of 13.0 and 11.0 kb respectively. Although the polymorphic sites are less than 10 kb apart, they were found to be in linkage equilibrium. The value of the disequilibrium parameter (D) was 0.0042, approximately 7.5% of the theoretical maximum (D_max=0.054). No disease association could be demonstrated between either apoB RFLP, or haplotype, and coronary athersclerosis in our population from south-east England. This was in accordance with a study of apoB RFLPs for a population from the West Coast of the United States, but in contrast to a study of an East-Coast population. There are no previous data for the association between apoB haplotypes and coronary atherosclerosis.     

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.     

Linkage disequilibrium between DNA polymorphisms within the porphobilinogen deaminase gene

Summary Three restriction fragment length polymorphisms (RFLPs) (MspI, PstI, ScrFI/BstNI) within the human porphobilinogen deaminase (PBG-D) gene have been studied in 47 unrelated patients with the autosomal dominant disorder, acute intermittent porphyria (AIP), and in 92 control subjects. Each enzyme identified a two-allele polymorphism with allele frequencies close to 0.50; however, marked linkage disequilibrium limited the number of observed haplotypes to four, of which one is uncommon. No association was detected between any haplotype and AIP.     

The most common mutation causing medium-chain acyl-CoA dehydrogenase deficiency is strongly associated with a particular haplotype in the region of the gene

Summary RFLP haplotypes in the region containing the medium-chain acyl-CoA dehydrogenase (MCAD) gene on chromosome 1 have been determined in patients with MCAD deficiency. The RFLPs were detected after digestion of patient DNA with the enzymes BanII, PstI and TaqI and with an MCAD cDNA-clone as a probe. Of 32 disease-causing alleles studied, 31 possesed the previously publised AG point-mutation at position 985 of the cDNA. This mutation has been shown to result in inactivity of the MCAD enzyme. In at least 30 of the 31 alleles carrying this G985 mutation a specific RFLP haplotype was present. In contrast, the same haplotype was present in only 23% of normal alleles (P3.4×10^-18). These findings are consistent with the existence of a pronounced founder effect, possibly combined with biological and/or sampling selection.     

Molecular genetic assay of mucopolysaccharidosis IVA in South China

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Molecular mutational analysis was performed by PCR product sequencing for fourteen exons and exon–intron boundaries of GALNS gene in 21 patients from 19 unrelated families with severe MPS IVA in South China. We identified fifteen different mutations, including 10 reported mutations (p.P125L, p.G290S, p.M318R, p.G340D, p.L366P, p.R386C, p.A392V, c.1243-1G>C, p.L440RfsX54 and p.X523E) and five novel mutations (p.N177S, p.G290R, p.F306S, p.W403_T404delinsCS, p.W520X). All five novel mutations were inherited from parents of the patients and not found in 100 normal control alleles. Three mutations, p.M318R, p.L366P and p.R386C were common, accounting for 36.8% of mutant alleles investigated. One patient homozygous of p.A392V and the other two unrelated patients homozygous of p.L366P presented classical disease course. The results show that the GALNS gene has a different mutational spectrum in South China as compared to other regions. The p.A392V and p.L366P mutations were associated with severe phenotype of MPS IVA.     

Analysis of the Polymorphic Markers within the CFTR Gene in Cystic Fibrosis Patients and Healthy Donors from Bashkortostan

The differences in the polymorphic allele frequency distribution patterns of the biallelic (M470V and TUB20) and microsatellite (IVS6aGATT, IVS8CA, and IVS17CA) markers within the CFTR gene between normal and delF508 chromosomes have been established. For most of the marker loci similar distribution of the allele frequencies on normal and mutant chromosomes without delF508 was demonstrated. Certain polymorphic alleles displayed substantial linkage disequilibrium with the delF508 mutation. Analysis of the IVS6aGATT-IVS8CA-M470V-IVS17CA-TUB20 haplotypes association on normal and mutant chromosomes provided identification of the delF508 ancestral haplotype. It was suggested that delF508 mutant chromosomes were introduced into the modern Bashkir gene pool as a result of Slavic migrations from the Eastern Europe. The IVS6aGATT-IVS8CA-M470V-IVS17CA-TUB20major haplotype (77272) revealed was statistically significantly most frequently found on the mutant chromosomes without the delF508 mutation. This finding suggests that the Bashkir cystic fibrosis patients, mostly belonging to the Turkic-speaking families, possessed specific CFTR gene defect associated with the given haplotype.     

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 type I (Hurler syndrome): Linkage disequilibrium indicates the presence of a major allele

Summary Two polymorphisms exist in the -l-iduronidase (IDUA) gene, the gene that is defective in mucopolysaccharidosis type I (MPS I), viz. aKpnI polymorphism and a variable number of tandem repeats (VNTR) polymorphism with three common alleles. The analysis of allele and haplotype frequencies for these two polymorphisms in the normal population and in MPS I patients revealed the presence of linkage disequilibrium. The frequency of the 2,2 (VNTR,KpnI) allele in MPS I patients was 57% compared with only 37% in the normal population. The implications for the presence of a major MPS I allele and the ability to predict patient phenotype are discussed.     

Angiotensin I-converting enzyme (ACE): estimation of DNA haplotypes in unrelated individuals using denaturing gradient gel blots

The angiotensin I-converting enzyme (ACE) gene (17q23) is a candidate gene for essential hypertension and related diseases, but investigation of its role in human pathology is hampered by a lack of identified polymorphisms. Currently, a 287-bp insertion/deletion (I/D) RFLP in intron 16 represents the only one known. Additional polymorphisms for the ACE gene would make most families informative for linkage studies and would allow haplotypes to be assigned in association studies. To increase the information provided by the ACE gene, we used a sensitive screening technique, denaturing gradient gel electrophoresis (DGGE) blots, to identify polymorphisms and combined this with gene counting to identify haplotypes. Five independent polymorphisms, restriction fragment melting polymorphisms (RFMPs), were identified by four probes (encompassing half of the ACE cDNA) in digests produced by three restriction enzymes (DdeI, RsaI, and AluI). One RFMP has three alleles while the others have two alleles. In a sample of 67 unrelated control subjects, minor allele frequencies ranged from 0.12 to 0.49. A significant level of linkage disequilibrium was found for all pairs of markers. The four most informative RFMPs, taken in combination, define 24 potential haplotypes. Based on gene counting, 11 of the 24 are rare or nonexistent in this population, and the estimated heterozygosity of the remaining 13 haplotypes approaches 80%. Under these conditions for the ACE locus, phase-unknown genotypes could be assigned to haplotype pairs in unrelated subjects with reasonable certainty. Thus, using DGGE blot technique for identifying numerous DNA polymorphisms in a candidate locus, in combination with gene counting, one can often identify DNA haplotypes for both related and unrelated study subjects at a candidate locus. These markers in the ACE gene should be useful for clinical and epidemiologic studies of the role of ACE in human disease.     

Association of polymorphisms in the HLA-B region with extended haplotypes

Genomic probes from the HLA-B region of the major histocompatibility complex (MHC) were used to study the association of restriction fragment length polymorphisms (RFLPs) with various MHC alleles, complotypes, and extended haplotypes. The two DNA probes, M20A and R5A, were derived from previously cloned cosmids and are located 38 and 110 kilobases (kb) centromeric to HLa-B, respectively. Five different RFLP variants occuring in five different haplotypic combinations were detected within a panel of 40 homozygous-typing cells and cells from 21 families using Bst EII. In two informative families with HLA-B/DR recombinations the inheritance of the RFLP variants was consistent with their mapping between HLA-B and complotypes. The R5A/M20A haplotypic pattern 6.5 kb/3.0 kb (A) had a normal Caucasian frequency of approximately 0.43 and was found in all independent examples of the extended haplotypes [HLA-B8,SC01,DR3], [HLA-B18,F1C30, DR3], [HLa-Bw62,SC33,Dr4], [HLa-B44,SC30,Dr4], and [HLA-Bw47,FC91,0DR7]. The patterns of 6.9 kb/ 3.0 kb (B), 6.5 kb/4.7 kb (C), 1.45 kb/3.0 kb (D), and 6.9 kb/4.7 kb (E) had normal Caucasian frequencies of approximately 0.23, 0.15, 0.15, and 0.04 and were found on all independent examples of [HLA-B38,SC21, DR4], [HLA-Bw57,SC61,DR7], [HLA-B7,SC31,DR2], and [HLA-B44,FC31,DR7], respectively. Individual complotypes or HLA-B alleles which were markers of extended haplotypes showed variable associations. For example, HLA-B7 and the complotype SC31 were associated with all R5A/M20A RFLP haplotypes except haplotype E, although [HLA-B7,SC31,DR7] was associated exclusively with haplotype D. HLA-B27, not known to be part of an extended haplotype, was suprisingly exclusively associated with the 6.5 kb/4.7 kb or C haplotypic pattern in all five instances tested. These findings support the concept of regional conservation of DNA on independent examples of extended haplotypes. The results also further characterize these haplotypes.     

Marker haplotype association with growth in German cystic fibrosis patients

Summary In 84 families with 101 children with cystic fibrosis (CF) and 103 unaffected siblings, the haplotype of CF chromosomes was determined with six restriction fragment length polymorphism (RFLP) markers that span the CF gene locus. Patient groups with different genotypes in the more distant flanking marker loci MET D, MET H, and D7S8 differed significantly from each other with respect to percentile height and weight, and percentage of weight for height. Patients homozygous 1-1 in met D (TaqI) and met H (TaqI) were thin and tall when homozygous 1-1 in J3.11 (MspI), and small when homozygous 2-2 in J3.11. Heterozygosity in 3.11 and met H and homozygosity 1-1 in met D segregated with the most severe growth retardation. In contrast, growth was normal in patients who were heterozygous in met D and/or had an uncommon KM.19/XV-2c haplotype. Most patients with pancreatic sufficiency and/or borderline sweat test values were carrying rare haplotypes on their CF chromosomes. Adult patients clustered in genotype groups with normal height percentile distributions. This association between haplotype and clinical severity of CF in the German population provides evidence for genetic microheterogeneity of the CF locus, either because of the existence of multiple alleles of the CF gene itself and/or because of the existence of closely linked polymorphic genes that control growth and development and hence modulate the clinical course and prognosis of CF.