Linkage disequilibrium predicts physical distance in the adenomatous polyposis coli region.

To test the reliability of linkage-disequilibrium analysis for gene mapping, we compared physical distance and linkage disequilibrium among seven polymorphisms in the adenomatous polyposis coli (APC) region on chromosome 5. Three of them lie within the APC gene, and two lie within the nearby MCC (mutated in colon cancer) gene. One polymorphism lies between the two genes, and one is likely to be 5' of MCC. Five of these polymorphisms are newly reported. All polymorphisms were typed in the CEPH kindreds, yielding 179-205 unrelated two-locus haplotypes. Linkage disequilibrium between each pair of polymorphisms is highly correlated with physical distance in this 550-kb region (correlation coefficient -.80, P < .006). This result is replicated in both the Utah and non-Utah CEPH kindreds. There is a tendency for greater disequilibrium among pairs of polymorphisms located within the same gene than among other pairs of polymorphisms. Trigenic, quadrigenic, three-locus, and four-locus disequilibrium measures were also estimated, but these measures revealed much less disequilibrium than did the two-locus disequilibrium measures. A review of 19 published disequilibrium studies, including this one, shows that linkage disequilibrium nearly always correlates significantly with physical distance in genomic regions > 50-60 kb but that it does not do so in smaller genomic regions. We show that this agrees with theoretical predictions. This finding helps to resolve controversies regarding the use of disequilibrium for inferring gene order. Disequilibrium mapping is unlikely to predict gene order correctly in regions < 50-60 kb in size but can often be applied successfully in regions of 50-500 kb or so in size. It is convenient that this is the range in which other mapping techniques, including chromosome walking and linkage mapping, become difficult.     

Genotyping of PCR-based polymorphisms and linkage-disequilibrium analysis at the NF1 locus.

Six polymorphisms across the NF1 gene have been adapted for genotyping through application of PCR-based assays. Three exon-based polymorphisms--at positions 702, 2034, and 10647 in the NF1 cDNA--were genotyped by mutagenically separated PCR (MS-PCR). A fourth polymorphism, DV1.9, is an L1 insertion element in intron 30, and the other two polymorphisms, GXAlu and EVI-20, are short tandem repeats in intron 27b. All the polymorphisms were evaluated in a cohort of 110 CEPH individuals who previously had been analyzed by use of eight RFLPs at the NF1 locus. Pairwise linkage-disequilibrium analyses with the six PCR-based polymorphisms and their flanking markers demonstrated disequilibrium between all tested loci. Genotypes of the four diallelic polymorphisms (702, 2034, 10647, and DV1.9) were also evaluated in cohorts from the CEPH, African, and Japanese populations. The CEPH and Japanese cohorts showed similar heterozygosities and linkage-disequilibrium coefficients. The African cohort showed a higher degree of heterozygosity and lower linkage-disequilibrium values, compared with the CEPH and Japanese cohorts.     

The polymorphism of the plasma inter-α-trypsin inhibitor (ITI) and its relationship to the heavy chain H1 subunit gene (ITIH1) at 3p211-212

We investigated the ITI protein polymorphism in linkage analysis, usingDraI andSstI as restriction fragment length polymorphism (RFLP) markers for the ITIH1 gene. Isoelectric focusing (IEF) classification from 76 individual plasma samples and RFLP analysis from the corresponding DNA preparations disclosed linkage disequilibrium between the phenotypic IEF patterns of the two common ITI alleles, ITI^*1 and ITI^*2, and the diallelic DNA polymorphisms of two ITIH1 RFLPs, represented byDraI 4.0 kb andDraI 2.4 + 1.6 kb, and bySstI 6.7 kb andSstI 6.0 + 0.7 kb, for the ITI 1 and ITI 2 IEF phenotypes, respectively, and byDraI 4.0/2.4 + 1.6 kb andSstI 6.7/6.0 + 0.7 kb for the heterozygous ITI 1–2 IEF phenotype. Linked segregation between either of the RFLPs and the polymorphic ITI plasma protein locus has been established in nine informative family pedigrees. The less frequent allele in Europeans, ITI^*3, is not represented by a further allelic restriction fragment in either RFLP. The significant linkage disequilibrium observed in this genetic study indicates that the ITI locus, with the alleles ITI^*1 and ITI^*2, must be close to, or reside within, the ITIH1 gene. The diallelic ITI protein polymorphism therefore provides an informative phenotypic marker system for chromosome 3p211-212.     

Sporadic—but Not Variant—Creutzfeldt-Jakob Disease Is Associated with Polymorphisms Upstream of PRNP Exon 1

Human prion diseases have inherited, sporadic, and acquired etiologies. The appearance of the novel acquired prion disease, variant Creutzfeldt-Jakob disease (vCJD), and the demonstration that it is caused by the same prion strain as that causing bovine spongiform encephalopathy, has led to fears of a major human epidemic. The etiology of classical (sporadic) CJD, which has a worldwide incidence, remains obscure. A common human prion-protein-gene (PRNP) polymorphism (encoding either methionine or valine at codon 129) is a strong susceptibility factor for sporadic and acquired prion disease. However, a quantitative-trait-locus study of prion incubation periods in mice has demonstrated an important factor that is close to Prnp but is independent of its coding sequence or that of the nearby prion-like doppel gene (Prnd). We have analyzed the PRNP locus for such tightly linked susceptibility factors. Fifty-six polymorphic sites have been identified within 25 kb of the PRNP open reading frame, including sites within the PRNP promoter and the PRNP 3' untranslated region. These have been characterized in 61 Centre d'Etude du Polymorphisme Humain (CEPH) families, demonstrating extensive linkage disequilibrium around PRNP and the existence of 11 major European PRNP haplotypes. Haplotype frequencies estimated in healthy U.K. control individuals were very similar to those deduced in the CEPH families. A common haplotype was overrepresented in patients with sporadic CJD (sCJD). Through use of a log-linear modeling approach to simultaneously model Hardy-Weinberg and linkage disequilibria, a significant independent association was found between sCJD and a polymorphism upstream of PRNP exon 1 (P=.005), in addition to the strong susceptibility conferred by codon 129 (P=2x10(-8)). However, although our sample size was necessarily small, no association was found between these polymorphisms and vCJD or iatrogenic CJD, in keeping with their having distinct disease mechanisms. In addition, there was no evidence of a PRNP founder effect in the first reported geographical cluster of vCJD.     

Linkage mapping of the AML1 gene on human chromosome 21 using a DNA polymorphism in the 3' untranslated region.

We have detected a polymorphism in the 3' untranslated region of the AML1 gene, which is located at the breakpoint on chromosome 21 in the t(8;21)(q22;q22.3) translocation often associated with patients with acute myeloid leukemia. Informative CEPH families were genotyped for this polymorphism and used to localize the gene on the linkage map of human chromosome 21. The AML1 gene is located between the markers D21S216 and D21S211, in chromosomal band 21q22.3.     

Chromosomal localization to 3q21----qter and two TaqI RFLPs of the human prostate-specific acid phosphatase gene (ACPP)

The chromosomal location of the gene encoding human prostate-specific acid phosphatase (ACPP) was determined by Southern blotting analysis of panels of human x rodent (mouse or Chinese hamster) somatic cell hybrids, using the PAP-1007 and PAP-1004EP ACPP cDNA probes. The ACPP gene was assigned to chromosome 3, which was confirmed by screening a chromosome 3-specific genomic library. Sublocalization of this gene was carried out using hybrids that had retained only various portions of human chromosome 3. The ACPP gene was found to segregate specifically with the chromosomal segment 3q21----qter. Analysis of Southern blots of TaqI-digested DNAs from unrelated individuals and members of large families from northern Finland revealed two simultaneous diallelic restriction fragment length polymorphisms (RFLPs), A and B, when using either PAP-1004EP or PAP-1006A ACPP cDNA probes, but not the 5' flanking PAP-1007 probe. Allele frequencies for polymorphism A were .09 (A1) and .91 (A2), and for polymorphism B, .38 (B1) and .62 (B2). There appears to be only a very minor linkage disequilibrium (chi 2 = 1.12, 0.35 greater than P greater than 0.25) between the two TaqI RFLPs at the ACPP locus. For reasons presently unknown, homozygotes for polymorphism B appear to be overrepresented. These polymorphisms could be of importance in characterizing human prostate cancer.     

Nonsense mutations at Arg-1947 in two cases of familial neurofibromatosis type 1 in Japanese

We report two familial cases of NF1 presenting as C to T transitions changing an Arg-1947 codon to a stop codon. In one of the two families, cosegregation of the mutation with NF1 was demonstrated, indicating this mutation causes the disease in this family. As the same mutation at Arg-1947 has been reported previously in three cases of unrelated Caucasians (two are sporadic; the origin of the other is not reported), the codon at Arg-1947 (CGA) in the NF1 gene is considered to be a hotspot common among different ethnic groups and also among familial and sporadic cases.     

Genetic analysis of carbamyl phosphate synthetase I deficiency

Summary Carbamyl phosphate synthetase I deficiency (CPSD) is an autosomal recessive disorder of ureagenesis characterized by hyperammonemic coma in the neonatal period. To study the genetic basis of CPSD we have performed a molecular analysis of the CPS I genes in CPSD patients from six unrelated families. Using a cDNA probe for the human CPS I gene and restriction endonuclease mapping techniques, we observed no abnormality in the number or size of the hybridizing DNA fragments from the seven affected individuals examined. These findings suggest that no gross alteration affected the CPS I genes. We did detect a frequent restriction fragment length polymorphism (RFLP) at the CPS I locus which we employed as a linkage marker. Our results suggest the polymorphic CPS I restriction fragments cosegregate with the CPSD phenotype, and that linkage disequilibrium exists between the CPSI RFLPs studied and the affected alleles. The RFLPs described may enable prenatal detection of CPSD in families where the coupling phases between CPSD alleles and RFLPs can be determined.A preliminary report of these studies was presented at the Society for Pediatric Research meetings, San Francisco, May 1984 and appeared in abstract form in Pediatric Research 18:296A (1984)     

Restriction analysis of the structural α-L-fucosidase gene and its linkage to fucosidosis

Human alpha-L-fucosidase is a lysosomal enzyme responsible for hydrolysis of alpha-L-fucoside linkages in fucoglycoconjugates. A single gene, FUCA 1, located on chromosome 1p34.1-1p36.1 encodes for alpha-L-fucosidase activity. To gain insight into the nature of the molecular defects leading to fucosidosis, we have characterized the genomic structure of FUCA 1. Restriction-endonuclease analysis suggests that at least seven exons dispersed over 22 kb are present in genomic FUCA 1. Two restriction-fragment-length polymorphisms (RFLPs) have been identified in the Caucasian population. The PvuII and BglI RFLPs each have two codominant alleles in Hardy-Weinberg equilibrium. Allele frequencies for the PvuII RFLP are .70/.30, and those for the BglI RFLP .63/.37. Both RFLPs are in strong linkage disequilibrium with each other, with a correlation coefficient of .94. The polymorphism information content (PIC) of the combined DNA markers is .38, high enough to be useful in the prenatal diagnosis of fucosidosis. The combined lod score for linkage between the fucosidosis mutation and FUCA 1 markers in two families was significant at a recombination fraction of 0. This suggests that the fucosidosis mutation resides in FUCA 1.     

Nonradioactive assay for new microsatellite polymorphisms at the 5'' end of the dystrophin gene, and estimation of intragenic recombination.

Indirect tracking of mutation by DNA polymorphisms is still essential for carrier and prenatal diagnosis of Duchenne/Becker muscular dystrophy, at least in the families where no deletion can be detected. Because of the relatively high level of intragenic recombination, informative and easily testable markers at both ends of the gene are necessary for efficient and accurate diagnosis. We report the characterization of two polymorphic microsatellite sequences (TG repeats) at the 5' end of the dystrophin gene, within 40 kb of the muscle-specific promoter. The most useful one (5' DYS MSA) has 10 alleles with a 57% heterozygosity and can be tested on small polyacrylamide gels in a nonradioactive PCR-based assay. Despite its large number of alleles, this microsatellite shows strong linkage disequilibrium with a two-allele polymorphism reported by Roberts et al., an indication of the stability of this type of sequences. We have used the new microsatellites at the 5' end, along with one we reported previously for the 3' end, to type the families in the CEPH (Centre d'Etude du Polymorphisme Humain) panel. While the number of informative families has increased by a factor of about two with respect to the study of Abbs et al., the estimates of the recombination fractions are in good agreement with this previous report, suggesting a 11% recombination across the gene (3% between the 5' end and the pERT87 region, 8% between pERT87 and the 3' end), which is about fivefold more than expected. However, these estimates still have wide confidence limits.     

Type of mutation in the neurofibromatosis type 2 gene (NF2) frequently determines severity of disease.

The gene predisposing to neurofibromatosis type 2 (NF2) on human chromosome 22 has revealed a wide variety of different mutations in NF2 individuals. These patients display a marked variability in clinical presentation, ranging from very severe disease with numerous tumors at a young age to a relatively mild condition much later in life. To investigate whether this phenotypic heterogeneity is determined by the type of mutation in NF2, we have collected clinical information on 111 NF2 cases from 73 different families on whom we have performed mutation screening in this gene. Sixty-seven individuals (56.2%) from 41 of these kindreds revealed 36 different putative disease-causing mutations. These include 26 proposed protein-truncating alterations (frameshift deletions/insertions and nonsense mutations), 6 splice-site mutations, 2 missense mutations, 1 base substitution in the 3' UTR of the NF2 cDNA, and a single 3-bp in-frame insertion. Seventeen of these mutations are novel, whereas the remaining 19 have been described previously in other NF2 individuals or sporadic tumors. When individuals harboring protein-truncating mutations are compared with cases with single codon alterations, a significant correlation (P < .001) with clinical outcome is observed. Twenty-four of 28 patients with mutations that cause premature truncation of the NF2 protein, schwannomin, present with severe phenotypes. In contrast, all 16 cases from three families with mutations that affect only a single amino acid have mild NF2. These data provide conclusive evidence that a phenotype/genotype correlation exists for certain NF2 mutations.     

Effects of intragenic variability at 3 polymorphic sites of the apolipoprotein B gene on serum lipids and lipoproteins in a multiethnic Asian population.

We determined the allelic (X+/X-, M+/M-, and E+/E-) distribution frequencies of the XbaI, MspI, and EcoRI restriction fragment length polymorphisms (RFLPs) in the apolipoprotein B gene in a control group of 374 healthy Chinese, Malays, and Indians and in a hyperlipidemic cohort of 131 Chinese patients. Covariability between the RFLPs and serum lipid, lipoprotein, and apolipoprotein concentrations was also studied. We found a lower frequency (average 0.0829) of the X+ allele and higher frequencies of the E+ (average 0.9452) and M+ (average 0.9772) alleles in our study population compared with frequencies reported in other populations. The 3 polymorphic sites did not contribute to significant variations in lipid levels (p > 0.1 in all cases). Also, there was no significant variation in genotype frequencies between the control subjects and the hyperlipidemic subjects. Despite their relative close proximity within the APOB gene sequence, the 3 polymorphic sites did not show any significant linkage disequilibrium. However, the presence of the X+ cutting site was in linkage disequilibrium with the Del allele of the 5' insertion-deletion polymorphism and the E-allele was in linkage disequilibrium with the 3' VNTR located near the 3' end of the coding region of the APOB gene.     

Approaches to localizing disease genes as applied to cystic fibrosis.

Using chromosome jumping and walking and restriction fragment length polymorphism (RFLP) analysis, we have defined the region which must contain the cystic fibrosis gene. DNA segments spanning approximately 250 kb in the direction of the gene were isolated and used to identify several new polymorphisms informative in cystic fibrosis families. These RFLPs include a highly polymorphic, CA/GT repeat, and a 10 bp insertion uncovered using the polymerase chain reaction. By analyzing a family with a recombination near the gene, we can exclude this region as containing the mutation. Data on the extent of linkage disequilibrium of these markers provides additional information on where the gene is located.     

COL5a1: fine genetic mapping and exclusion as candidate gene in families with nail-patella syndrome, tuberous sclerosis 1, hereditary hemorrhagic telangiectasia, and Ehlers—Danlos syndrome type II

COL5A1, the gene for the α1 chain of type V collagen, has been considered a candidate gene for certain diseases based on chromosomal location and/or disease phenotype. We have employed 3′-untranslated region RFLPs to exclude COL5A1 as a candidate gene in families with tuberous sclerosis 1, Ehlers—Danlos syndrome type II, and nail-patella syndrome. In addition, we describe a polymorphic simple sequence repeat (SSR) within a COL5A1 intron. This SSR is used to exclude COL5A1 as a candidate gene in hereditary hemorrhagic telangiectasia (Osler—Rendu—Weber disease) and to add COL5A1 to the existing map of “index” markers of chromosome 9 by evaluation of the COL5A1 locus on the CEPH 40-family reference pedigree set. This genetic mapping places COL5A1 between markers D9S66 and D9S67.     

DNA polymorphism and linkage disequilibrium within the apolipoprotein CII locus on human chromosome 19.

The gene for human apolipoprotein CII (APOCII) is located on the proximal long arm of chromosome 19. It has been established as a closely linked marker for myotonic dystrophy (DM), the most common form of adult muscular dystrophy. In the present linkage study, we have analysed 6 APOCII RFLPs in 213 haplotypes: TaqI, 3.8/3.5 kb; BgII, 12.0/9.0 kb; BanI, 2.5/1.6 kb; BamHI, 6.0/4.9 kb; NcoI, 14.5/11.5 kb, and AvaII, 0.6/0.4 kb. The polymorphic enzyme sites were determined to be present at the following frequencies: TaqI, 0.43; BglI, 0.51; BanI, 0.25; BamHI, 0.99; NcoI, 0.51, and AvaII, 0.52. Ordering of the polymorphic sites, 5'----3', has been determined to be (NcoI-BglI)-AvaII-BanI-TaqI. Significant disequilibrium was seen between 5 of the APOCII RFLPs.     

Human fibronectin gene (FN1) RFLPs: Mapping and linkage disequilibrium analysis

We report a mapping and linkage disequilibrium analysis of six restriction fragment length polymorphisms (RFLPs) of the human fibronectin gene. The polymerase chain reaction conditions are described for four of the RFLPs.     

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.     

Genetics of neurofibromatosis 1 in Japan: mutation rate and paternal age effect

Summary We have performed formal genetic studies on 26 patients (14 males, 12 females) with neurofibromatosis 1 (von Recklinghausen's disease, NF1) in Japan. Family studies of 74 members of 18 kindreds revealed that 50% of the cases were caused by a new mutation; the mutation rate was assumed to be 7.3–10.5 × 10^-5. A tendency of paternal age effect, which was not accounted for by the maternal age effect, was observed, but live-birth order had no significant effect. Genetic linkage of neurofibromatosis 1 to the NF1 gene or the genetic marker in the pericentric region of chromosome 17 was established in 3 informative families.     

Localization of the FGR protooncogene on the genetic linkage map of human chromosome 1p

A restriction fragment length polymorphism (RFLP) at the human FGR gene, a member of the src family of protooncogenes, has been identified and used to locate FGR on the genetic linkage map of human chromosome 1p. Single-copy sequences subcloned from a cosmid containing the human FGR gene were used to screen a panel of genomic DNAs for RFLPs. One plasmid, designated pB8, detected a high-frequency EcoRI RFLP (allele frequencies, 0.57/0.43). Analysis of a panel of somatic cell hybrids demonstrated that pB8 maps to the region 1p31-pter. Genetic linkage analysis of the 40 families provided by the Centre d'Etude du Polymorphisme Humain (CEPH) showed that FGR maps to a location 3.1 cM from the Rh blood group locus (RH), and falls in the 17.5-cM gap between alpha-fucosidase (FUCA1) and D1S57. The relative gene order of RH and FGR could not be determined unequivocally, but the most favored gene order was 1pter-PND-ALPL-FUCA1-FGR-RH-D1S57-MYCL.