New genetic variants in the apoA-I and apoC-III genes and familial combined hyperlipidemia

Linkage and association between the apolipoprotein (apo) A-I/C-III/A-IV gene region on chromosome 11 and familial combined hyperlipidemia (FCHL) has been observed previously. Using sequence analysis two new allelic variants were identified, C(317) -T in intron 2 of the apoA-I gene and C(1100)-T in exon 3 of the apoC-III gene. These variants were studied in 30 FCHL probands, 159 hyperlipidemic relatives, 327 normolipidemic relatives, and 218 spouses. The allele frequencies of both variants were significantly different in probands and spouses (P < 0.002 and P < 0.001, respectively), with increased frequency of the minor alleles in the probands. The minor genotypes (TT) were associated with elevated plasma triglyceride and apoC-III. Both variants were in strong, although not complete, linkage disequilibrium with each other and with the MspI site in the promoter region of the apoA-I gene and the SstI site in the 3' untranslated region of exon 4 of the apoC-III gene. Haplotypes based on these four variants were constructed and the distributions of haplotype combinations were significantly different (P < 0.0001). Two distinct haplotypes predisposing to FCHL were found: 2-2-1-2 and 1-2-2-2 (MspI, C(317) -T; SstI, C(1100)-T). The haplotype combinations carrying one of these high risk alleles are associated with elevated lipid levels in probands and in spouses. While these effects can be attributed to the presence of the M2 and S2 minor alleles, these results suggest that the importance of specific allelic haplotypes may be greater than single genotypic effects.     

Utility and efficiency of linked marker genes for genetic counseling. III. Proportion of informative families under linkage disequilibrium.

A marker locus closely linked to a disease locus is often useful for genetic counseling provided that a counselee is heterozygous at both disease and marker loci. Furthermore, the linkage phase of these genes in the counselee must be known. When the linkage between the disease and marker loci is very close, one often finds linkage disequilibrium between the loci. To evaluate the effect of such nonrandom associations on the utility of linked marker genes for genetic counseling, the proportion of informative families is studied for X-linked recessive and autosomal dominant diseases. This proportion is higher for X-linked genes than for autosomal genes, if other factors are the same. In general, codominant markers are more useful than dominant markers. Also, under appropriate conditions, the proportion of informative families is higher when linkage disequilibrium is present. The results obtained in this paper are useful for evaluating the utility of polymorphic restriction endonuclease cleavage sites as markers in genetic counseling.     

Allelic disequilibrium and allele frequency distribution as a function of social and demographic history.

Allelic disequilibrium between closely linked genes is a common observation in human populations and often gives rise to speculation concerning the role of selective forces. In a previous treatment, we have developed a population model of the expected distribution of rare variants (including private polymorphisms) in Amerindians and have argued that, because of the great expansion of Amerindian numbers with the advent of agriculture, most of these rare variants are of relatively recent origin. Many other populations have similar histories of striking recent expansions. In this treatment, we demonstrate that, in consequence of this fact, a high degree of linkage disequilibrium between two nonhomologous alleles <0.5 cM apart is the "normal" expectation, even in the absence of selection. This expectation is enhanced by the previous subdivision of human populations into relatively isolated tribes characterized by a high level of endogamy and inbreeding. We also demonstrate that the alleles associated with a recessive disease phenotype are expected to exist in a population in very variable frequencies: there is no need to postulate positive selection with respect to the more common disease-associated alleles for such entities as phenylketonuria or cystic fibrosis.     

Linkage disequilibrium among RFLPs at the insulin-receptor locus despite intervening Alu repeat sequences.

Multiple mutations of the insulin receptor (INSR) gene have been identified in individuals with extreme insulin resistance. These mutations have included recombination events between Alu repeat units in the tyrosine kinase-encoding beta-chain region of the gene. To evaluate the influence of Alu and dinucleotide repetitive sequences on recombination events within the insulin receptor gene, I examined the degree of linkage disequilibrium between RFLP pairs spanning the gene. I established 228 independent haplotypes for seven RFLPs (two each for PstI, RsaI, and SstI and one for MspI and 172 independent haplotypes which included an additional RFLP with BglII) from 19 pedigrees. These RFLPs span > 130 kb of this gene, and my colleagues and I previously demonstrated that multiple Alu sequences separate RFLP pairs. Observed haplotype frequencies deviated significantly from those predicted. Pairwise analysis of RFLP showed high levels of linkage disequilibrium among RFLP in the beta-chain region of the insulin receptor, but not between alpha-chain RFLPs and those of the beta-chain. Disequilibrium was present among beta-chain RFLPs, despite separation by one or more Alu repeat sequences. The very strong linkage disequilibrium which was present in sizable regions of the INSR gene despite the presence of both Alu and microsatellite repeats suggested that these regions do not have a major impact on recombinations at this locus.     

Linkage disequilibrium mapping of the bolting gene in sea beet using AFLP markers

The possibility of using linkage disequilibrium mapping in natural plant populations was assessed. In studying linkage disequilibrium among 137 mapped AFLP markers in four populations of sea beet (Beta vulgaris ssp. maritima (L.) Arcang.) it was shown that tightly linked loci could be detected by screening for associations. It was hypothesized that the short distances spanned by linkage disequilibrium enable markers that are very tightly linked to a target gene to be identified. The hypothesis was tested by whole-genome screening of AFLP markers for association with the gene for the annual growth habit, the B gene, in a sample of 106 sea beets. Despite the dominant nature of AFLP, two markers showing significant linkage disequilibrium with the B gene were detected. The results indicate the potential use of linkage disequilibrium for gene mapping in natural plant populations.     

Diagnosis of neurofibromatosis I by using tightly linked, flanking DNA markers.

We tested 132 individuals from 21 families segregating an allele for neurofibromatosis type 1 (NF-1), by using nine RFLPs tightly linked to the NF-1 locus. Family members had requested DNA testing either to determine whether "at risk" children were carrying the NF-1 allele or to determine whether their respective families would be informative for prenatal testing. Predictions about whether a child carries the NF-1 mutation were possible for all 32 at-risk offspring (greater than 98% accuracy based on the recombination estimates currently available for these DNA markers). At least one informative probe was available for all 23 matings in these 21 families; flanking markers were informative for 10 matings. Pairwise analysis showed that several of the polymorphisms were in tight linkage disequilibrium; few recombination events were observed with these markers in the families under study. We conclude that the DNA probes used in this study perform well for diagnostic testing of NF-1 in familial cases. A subset of five probe-enzyme systems (pHHH202/RsaI, p11-3C4.2/MspI, pTH17.19/Bg/II, p11-2C11.7/BamHI, and p11-2F9.8/TaqI) provide reliable linkage information for both clinical testing and prenatal diagnosis.     

The Friedreich ataxia gene is assigned to chromosome 9q13-q21 by mapping of tightly linked markers and shows linkage disequilibrium with D9S15.

Chamberlain et al. have assigned the gene for Friedreich ataxia (FA), a recessive neurodegenerative disorder, to chromosome 9, and have proposed a regional localization in the proximal short arm (9p22-cen), on the basis of linkage to D9S15 and to interferon-beta (IFNB), the latter being localized in 9p22. We confirmed more recently the close linkage to D9S15 in another set of families but found much looser linkage to IFNB. We also reported another closely linked marker, D9S5. Additional families have now been studied, and our updated lod scores are z = 14.30 at theta = .00 for D9S15-FA linkage and z = 6.30 at theta = .00 for D9S5-FA linkage. Together with the recent data of Chamberlain et al., this shows that D9S15 is very likely within 1 cM of the FA locus. We have found very significant linkage disequilibrium (delta Std = .28, chi 2 = 9.71, P less than .01) between FA and the D9S15 MspI RFLP in French families, which further supports the very close proximity of these two loci. No recombination between D9S5 and D9S15 was found in the FA families or Centre d'Etude du Polymorphisme Humain families (z = 9.30 at theta = .00). Thus D9S5, D9S15, and FA define a cluster of tightly linked loci. We have mapped D9S5 by in situ hybridization to 9q13-q21, and, accordingly, we assign the D9S5, D9S15, and FA cluster to the proximal part of chromosome 9 long arm, close to the heterochromatic region.     

Evidence of linkage disequilibrium in the Spanish polycystic kidney disease I population.

Forty-one Spanish families with polycystic kidney disease 1 (PKD1) were studied for evidence of linkage disequilibrium between the disease locus and six closely linked markers. Four of these loci--three highly polymorphic microsatellites (SM6, CW3, and CW2) and an RFLP marker (BLu24)--are described for the first time in this report. Overall the results reveal many different haplotypes on the disease-carrying chromosome, suggesting a variety of independent PKD1 mutations. However, linkage disequilibrium was found between BLu24 and PKD1, and this was corroborated by haplotype analysis including the microsatellite polymorphisms. From this analysis a group of closely related haplotypes, consisting of four markers, was found on 40% of PKD1 chromosomes, although markers flanking this homogeneous region showed greater variability. This study has highlighted an interesting subpopulation of Spanish PKD1 chromosomes, many of which have a common origin, that may be useful for localizing the PKD1 locus more precisely.     

Restriction fragment length polymorphism of the caprine ß-globin gene cluster associated with the Hb ß-globin variants in Norwegian dairy goats

Restriction fragment length polymorphism (RFLP) analysis was used to characterize the beta-globin cluster in Norwegian dairy goats. In 122 individuals, different RFLP patterns were detected after BglII and PstI digestion and hybridization with a caprine beta-globin probe. The location of the polymorphic BglII and PstI sites were determined. The different RFLPs were in linkage disequilibrium with each other and with the beta-globin locus as studied at the protein level. A preferential association between certain RFLP haplotypes and beta-globin variants was observed. Polymorphic DNA fragments in the epsilon-globin genes were detected after MspI digestion and hybridization with a caprine epsilon-probe, and eight different band patterns were observed. Correlation between different MspI haplotypes and the beta-globin variants was determined.     

The Genetics of DROSOPHILA SUBOBSCURA Populations. IX. Studies on Linkage Disequilibrium in Four Natural Populations

Gametic frequencies were obtained in four natural populations of D. sub-obscura by extracting wild chromosomes and subsequently analyzing them for inversions and allozymes. The genes Lap and Pept-1, both located within the same inversions of chromosome O, were found in striking nonrandom associations with them of the same kind and degree in all populations studied. On the contrary, the gene Acph, also located within the previously mentioned inversions, was found in linkage disequilibrium with them only in two populations and of opposite directions. This is also the case for the genes Est-9 and Hk, both located within chromosome E inversions. While the gene Est-9 was in strong linkage disequilibrium with the inversions, of the same kind and degree in all populations studied, Hk was found to be in linkage equilibrium. Allele frequencies for the 29 genes studied do not show geographical variation except for the genes Lap, Pept-1 and Est-9, the ones found in linkage disequilibria with the geographically varying gene arrangements. Although mechanical or historical explanations for these equilibria cannot be ruled out, these data cannot be explained satisfactorily by the "middle gene explanation," which states that loci displaying such linkage disequilibria are the ones located near the break points of inversions, while the ones displaying linkage equilibria with them are located in the middle of them. There is no evidence for consistent linkage disequilibria between pairs of loci, except for the closely linked genes of the complex locus, Est-9. This would imply, if it is not a peculiarity of the Est-9 complex, that the linkage disequilibria are found only between very closely linked loci or that, for less closely linked genes, the associations are too weak to be detected by the usual samples sizes.     

Association of plasma lipids and apolipoproteins with the insulin response element in the apoC-III promoter region in familial combined hyperlipidemia.

The apoAI-CIII-AIV gene cluster, located on chromosome 11, contributes to the phenotype of familial combined hyperlipidemia (FCH), but this contribution is genetically complex. Combinations of haplotypes, based on three restriction enzyme polymorphisms: XmnI and MspI sites, 5' of the start site of the apoA-I gene and SstI polymorphism in the 3' untranslated region of exon 4 of the apoC-III gene, were analyzed to characterize their effect on the expression of severe hyperlipidemia. An epistatic interaction was demonstrated: the S2 allele on one haplotype was synergistic in its hyperlipidemic effect to the X2M2 allele on the other haplotype (Dallinga-Thie, G. M. et al. J. Clin. Invest. 1997. 99: 953-961). In the present study two additional polymorphic sites in the insulin response element (IRE) of the apoC-III gene promoter, T-455C: FokI restriction site, C-482T: MspI restriction site, were studied in 34 FCH pedigrees including 34 probands, 220 hyperlipidemic relatives, 300 normolipidemic relatives, and 236 spouses. In contrast to the earlier data for the other polymorphisms in this gene cluster (XmnI, MspI/AI, and SstI), there were no differences in frequency distributions of the T-455C and the C-482T variants between probands, hyperlipidemic and normolipidemic relatives and spouses. No significant associations between plasma lipid traits and DNA variants in the IRE were observed. Analysis of combinations of haplotypes based on the five polymorphisms in the gene cluster provided further evidence for a dominant role of the SstI polymorphism as a major susceptibility locus in FCH. The inclusion of the IRE markers did not improve genetic informativeness, nor our understanding of the observed synergistic relationship associated with the high risk combination of haplotypes in FCH families.     

Expected Linkage Disequilibrium for a Neutral Locus Linked to a Chromosomal Arrangement

The expected value of the squared linkage disequilibrium is derived for a neutral locus associated with a chromosomal arrangement that is maintained in the population by strong balancing selection. For a given value of recombination, the expected squared linkage disequilibrium is shown to decrease as the intensity of selection maintaining the arrangement increases. The transient behavior of the expected square linkage disequilibrium is also derived. This theory applies to loci that are closely linked to inversions in Drosophila species and to loci closely linked to the differential segments of the translocation complexes in ring-forming species of Oenothera. In both cases the strong linkage disequilibria that have been observed in natural populations can be explained by random drift.     

Linkage disequilibrium fine mapping of quantitative trait loci: A simulation study

Recently, the use of linkage disequilibrium (LD) to locate genes which affect quantitative traits (QTL) has received an increasing interest, but the plausibility of fine mapping using linkage disequilibrium techniques for QTL has not been well studied. The main objectives of this work were to (1) measure the extent and pattern of LD between a putative QTL and nearby markers in finite populations and (2) investigate the usefulness of LD in fine mapping QTL in simulated populations using a dense map of multiallelic or biallelic marker loci. The test of association between a marker and QTL and the power of the test were calculated based on single-marker regression analysis. The results show the presence of substantial linkage disequilibrium with closely linked marker loci after 100 to 200 generations of random mating. Although the power to test the association with a frequent QTL of large effect was satisfactory, the power was low for the QTL with a small effect and/or low frequency. More powerful, multi-locus methods may be required to map low frequent QTL with small genetic effects, as well as combining both linkage and linkage disequilibrium information. The results also showed that multiallelic markers are more useful than biallelic markers to detect linkage disequilibrium and association at an equal distance.     

DNA restriction-site polymorphisms associated with the alpha 1-antitrypsin gene.

Restriction-site variation in and around the alpha 1-antitrypsin gene has been studied using two genomic probes. With use of restriction enzymes SstI, MspI, and AvaII, three polymorphic sites have been described with a 4.6-kb probe in the 5' portion of the gene. With use of a 6.5-kb probe, polymorphisms in the coding and 3' regions of the gene have been detected with AvaII, MaeIII, and TaqI. All of these polymorphisms are of sufficiently high frequency to be useful in genetic mapping studies. The polymorphisms with AvaII and MaeIII (6.5-kb probe) are particularly useful for prenatal diagnosis. PI types and M subtypes tend to be associated with specific DNA haplotypes; there are two different types of DNA haplotypes associated with PI M1. The extent of linkage disequilibrium differs throughout the region of the alpha 1-antitrypsin gene.     

Markers linked to vegetative incompatibility (vic) genes and a region of high heterogeneity and reduced recombination near the mating type locus (MAT) in Cryphonectria parasitica

To find markers linked to vegetative incompatibility (vic) genes in the chestnut blight fungus, Cryphonectria parasitica, we constructed a preliminary linkage map. In general, this map is characterized by low levels of polymorphism, as evident from the more than 24 linkage groups observed, compared to seven expected from electrophoretic karyotyping. Nonetheless, we found markers closely linked to two vic genes (vic1 and vic2) making them candidates for positional cloning. Two markers were found to be linked to vic2: one cosegregated with vic2, i.e., it is 0.0 cM from vic2, the other was at a distance of 4.5 cM; a single marker was found 4.0 cM from vic1. The closest markers linked to three other vic genes (vic4, vic6, and vic7) were >15 cM away; additional markers are needed before efficient positional cloning of these three vic genes can be realized. In contrast to the low levels of polymorphism observed across most of the C. parasitica genome, the linkage group containing the MAT locus appears to harbor an extremely high level of RAPD heterogeneity and reduced recombination. Markers within this highly heterogeneous region are in linkage disequilibrium in some natural populations; however, recombination is clearly evident between this region and the MAT locus.     

Linkage disequilibrium analyses and restriction mapping of four RFLPs at the proα2(I) collagen locus: lack of correlation between linkage disequilibrium and physical distance

Summary Restriction fragment length polymorphisms (RRLPs) located at short distances may demonstrate linkage disequilibrium. Under the assumption that the distances between the loci of the RFLPs are inversely related to the linkage disequilibria, gene order may be deduced. However, if the assumption is invalid, the results may be incorrect. We have studied four different DNA polymorphisms at the COLIA2 locus in 180 unrelated Norwegian individuals. Observed frequencies (presence/absence) for the different polymorphic sites were as follows: site A (EcoRI) 0.30/0.70, site B (MspI) 0.83/0.16, site C (StuI) 0.86/0.14, and site D (RsaI) 0.66/0.34. Of 16 possible haplotypes 12 were demonstrated, and 2 additional were deduced to be present. Restriction mapping of the four polymorphic sites gave the following order of the sites from the 5 to the 3 of the gene: A-D-B-C. Linkage disequilibrium was not found between the sites A and D; strong disequilibrium was found between sites A and C, and B and C; and less strong, between A and B, B and D, and C and D. Analysis of linkage disequilibrium coefficients between all pairs of loci demonstrated that there is no consistent relationship between linkage disequilibrium and physical distance (=-0.07). These results suggest that for a small region of the genome, factors such as deviating mutation rate and gene conversion may add significantly to rearrangements by recombination. Thus, a deduced gene order from linkage disequilibrium data has to be regarded with great caution.     

Isolation of a new DNA marker in linkage disequilibrium with cystic fibrosis, situated between J3.11 (D7S8) and IRP.

A cosmid library of recombinants containing nonmethylated CpG sites for rare-cutter restriction enzymes was used previously to isolate the gene IRP and four polymorphic DNA markers (pPT-3, pXV-2c, pCS.7, and pKM.19) which are close to and in linkage disequilibrium with the cystic fibrosis (CF) mutation. We have analyzed several new clones from the same library and have isolated a further cosmid, cNX.6d, which maps approximately 160 kb from CS.7, in the J3.11 direction. A DNA fragment (pMP6d-9) (D7S399) derived from cosmid cNX.6d detects a frequent polymorphism with MspI. Strong linkage disequilibrium between CF and MP6d-9 is found in European populations. Recombinations in two families suggest that CF is between the MspI polymorphic site recognized by pMP6d-9 and the polymorphism recognized by pJ3.11. The new marker is the closest, to date, to CF and will be useful for prenatal diagnosis and carrier testing.     

Linkage disequilibrium and fingerprinting in sugar beet

It has been suggested that map information for molecular markers can be used to strengthen finterprinting analyses. The success of this strategy depends on the distribution of linkage disequilibrium over the genome. Using 451 mapped AFLP markers, we investigated the occurrence of linkage disequilibrium in nine sugar beet breeding lines. A low but significant level of linkage disequilibrium was found for unlinked markers. Only for very tigthly linked (<3 cM) markers was this level substantially higher. This implies that little is gained in utilising the map position of the markers in fingerprinting applications. Received: 25 May 1999 / Accepted: 18 Oktober 1999     

Random search for shared chromosomal regions in four affected individuals: the assignment of a new hereditary ataxia locus.

Infantile-onset spinocerebellar ataxia (IOSCA) is an autosomal recessively inherited progressive neurological disorder of unknown etiology. This ataxia, identified so far only in the genetically isolated Finnish population, does not share gene locus with any of the previously identified hereditary ataxias, and a random mapping approach was adopted to assign the IOSCA locus. Based on the assumption of one founder mutation, a primary screening of the genome was performed using samples from just four affected individuals in two consanguineous pedigrees. The identification of a shared chromosomal region in these four patients provided the first evidence that the IOSCA gene locus is on chromosome 10q23.3-q24.1, which was confirmed by conventional linkage analysis in the complete family material. Strong linkage disequilibrium observed between IOSCA and the linked markers was utilized to define accurately the critical chromosomal region. The results showed the power of linkage disequilibrium in the locus assignment of diseases with very limited family materials.     

Genetic homogeneity at the Friedreich ataxia locus on chromosome 9

Classical Friedreich ataxia, a progressive, neurodegenerative disorder involving both the central and peripheral nervous systems, has been subclassified according to the observed clinical heterogeneity. The variations in the age at onset and in the spectrum and severity of symptoms have previously been interpreted as evidence of genetic heterogeneity. We have studied the linkage between the disorder and closely linked DNA markers in families of distinct ethnic origins, including the "typical" French-Canadians and the Acadian population of Louisiana. The disease in these two populations, both of continental French origin, has a very similar initial clinical picture. However, a marked difference in the rate of progression of the obligatory symptoms after 10 years of apparent disease is observed. A total of 553 individuals from 80 families with 202 affected members have been typed with the chromosome 9 marker MCT112, which we have previously shown to be closely linked to the disease locus. Evidence for linkage was observed in all families with the generation of a combined total lod score of 25.09 at a recombination fraction of theta = .00, providing strong evidence for genetic homogeneity at this locus for the classical form of this disease.