Genetic linkage between lipoprotein(a) phenotype and a DNA polymorphism in the plasminogen gene

Coronary heart disease risk correlates directly with plasma concentrations of lipoprotein(a) (Lp(a)), a low-density lipoprotein-like particle distinguished by the presence of the glycoprotein apolipoprotein(a) (apo(a)), which is bound to apolipoprotein B-100 (apoB-100) by disulfide bridges. Size isoforms of apo(a) are inherited as Mendelian codominant traits and are associated with variations in the plasma concentration of lipoprotein(a). Plasminogen and apo(a) show striking protein sequence homology, and their genes both map to chromosome 6q26-27. In a large family with early coronary heart disease and high plasma concentrations of Lp(a), we found tight linkage between apo(a) size isoforms and a DNA polymorphism in the plasminogen gene; plasma concentrations of Lp(a) also appeared to be related to genetic variation at the apo(a) locus. We found free recombination between the same phenotype and alleles of the apoB DNA polymorphism. This suggests that apo(a) size isoforms and plasma lipoprotein(a) concentrations are each determined by genetic variation at the apo(a) locus.     

A DNA marker closely linked to the factor IX (haemophilia B) gene

Summary We have isolated a DNA segment, pX58dIIIc, from an X-chromosome library which identifies an SstI restriction fragment length polymorphism (RFLP) at locus DXS99. Linkage analysis in six informative families has shown that the DXS99 locus lies close to the factor IX gene (F9). No recombination was detected between these loci in 39 informative meioses (Z=9.79, =0.0). Therefore, DXS99 will be useful as a DNA marker for the assessment of carrier status in families with haemophilia B where intragenic markers are not informative. Heterozygosity at DXS99 is approximately 50% and, in conjunction with the RFLPs at F9, 90% of females at risk for being haemophilia B carriers should be diagnosed.     

Linkage analysis with RFLPs in families with androgen resistance syndromes: evidence for close linkage between the androgen receptor locus and the DXS1 segment

Summary Three families with androgen resistance syndromes — two with testicular feminization and one with Reifenstein syndrome — have been studied for linkage analysis. Using three cloned DNA sequences from the centromere region and the proximal long arm of the X chromosome (p8, pDP34, and S9, which define respectively the chromosomal segments DXS1, DXYS1, and DXS17), we found no recombination between the DXS1 locus and the mutant genes in the three families. Assuming that these disorders are the result of allelic mutations at the same locus for the androgen receptor, we can conclude that there is a close linkage between DXS1 and the androgen receptor locus, with a maximum lod score =3.5 at a recombination fraction =0.0 using the LIPED program (Ott 1974).     

A single base pair polymorphism in the WT1 gene detected by single-strand conformation polymorphism analysis

The Wilms' tumor predisposition gene, WT1, was analysed exon-by-exon in a variety of tumours using the single-strand conformation polymorphism (SSCP) technique. A consistent variation in the usual band pattern for exon 7 was detected in this survey. On sequencing, a silent mutation was noted in codon 313 resulting in an AG transition in an arginine codon. The AG transition destroys an AflIII restriction enzyme recognition site, which provides a rapid means of identifying heterozygotes at this locus. Analysis of the segregation of this polymorphism in families demonstrated a co-dominant inheritance pattern. In an analysis of 21 randomly selected individuals 25% were heterozygous at this locus, which makes this polymorphism useful in a variety of genetic analyses.     

Linkage studies of the Wiskott-Aldrich syndrome: polymorphisms at TIMP and the X chromosome centromere are informative markers for genetic prediction

Summary Eleven families segregating for the X-linked recessive immune deficiency disorder, Wiskott-Aldrich syndrome (WAS), were studied by linkage analysis with an alpha satellite DNA probe, pBamX-7, which detects polymorphism at the X chromosome centromere, locus DXZ1, as well as three other polymorphic markers defining loci on the proximal short arm of the X chromosome. Linkage has been established between WAS and DXZ1 ( ()=7.08 at =0.03) and WAS and the TIMP gene locus ( ()=5.09 at =0.0). We have also confirmed close linkage between DXZ1 and two marker loci, DXS14 and DXS7, previously shown to be linked to the WAS locus. The probe pBamX-7 detected allelic variation in all females tested, reflecting the high frequency of polymorphism at the centromere. One WAS carrier revealed a recombination between WAS and both marker loci DXZ1 and DXS14, indicating that WAS does not map between these loci. In conjunction with previous data from genetic mapping studies of WAS, these results confirm the pericentromerix Xp localization of WAS and demonstrate the usefulness of alpha satelite DNA probes as tools for genetic prediction in WAS as well as other pericentric X-linked diseases.     

A third gene locus for tuberous sclerosis is closely linked to the phenylalanine hydroxylase gene locus

Summary Following the observation of a patient suffering from tuberous sclerosis (TSC) with a de novo reciprocal translocation t(3;12)(p26.3;q23.3), we have undertaken a linkage study in 15 TSC families using polymorphic DNA markers neighbouring the chromosome breakpoints. Significant lod scores have been obtained for markers D12S7 (z _max=2.34, =0.14) and PAH (phenylalanine hydroxylase) (z _max=4.34, =0.0). In multipoint linkage analysis, the peak lod score was 4.56 at the PAH gene locus. These data suggest the existence of a third gene locus for TSC (TSC3) on chromosome 12q22-24.1. The regions that have been found to be linked to TSC in different families map to the positions of three enzymes, phenylalanine hydroxylase (12q22-24), tyrosinase (11q14-22), and dopamine-beta-hydroxylase (9q34), all of which are involved in the conversion of phenylalanine to catecholamine neurotransmitters or melanin. Disorders of these biochemical pathways might be involved in the pathogenesis of TSC.     

No evidence for linkage of long QT syndrome and chromosome 11p15.5 markers in a Chinese family: evidence for genetic heterogeneity

Recently the defective gene locus in seven Caucasian families with the Romano-Ward form of long QT syndrome (LQT) has been mapped to chromosome 11p. To understand the molecular basis of LQT in Chinese, a three-generation family was investigated. Fourteen family members were studied and five individuals were diagnosed to be affected, according to electrocardiographic criteria. Two genomic DNA probes (c-Ha-ras-3-HVR and insulin-5-HVR) and one tetranucleotide repeat polymorphism (THZ) derived from chromosome 11p15.5 loci and previously demonstrated to be closely linked to LQT were used as probes to analyze this family. A lod score of less than -2 was noted for all three polymorphisms. Our data show that there was no evidence of linkage between these three loci and the gene for LQT in this studied family. We believe that this result provides additional evidence for genetic heterogeneity of LQT.     

Molecular genotyping of human T-cell antigen receptor variable gene segments

The gene complex encoding the chain of the T-cell antigen receptor (Tcr) in man was previously reported to contain a restriction fragment length polymorphism (RFLP) involving a single Bgl II site adjacent to the second constant region gene. This RFLP allowed assignment of Tcr genotypes in certain human families. In the present study, two different RFLP in a V gene family were detected using the murine probe V8.1 in genomic DNA samples digested with the restriction endonucleases Hind III and Bam HI. Use of these RFLP to mark the V gene complex allowed complete haplotype assignment in four of seven families studied and provided support for linkage of the V gene complex to the constant region genes. Different combinations of the C and two V region markers can result in eight possible distinct haplotypes. The observation of all but one of the eight possible haplotypes in parents of the families studied suggests that recombination events occur between the C and V region and among members of the V region subfamily marked by the V8.1 probe. These markers can be used for mapping studies of the V gene complex in man and will allow an appraisal of possible associations between Tcr genes and disease susceptibility.Abbreviations used in this paper: Tcr T-cell antigen receptor - RFLP restriction fragment length polymorphism - C2 second Tcr constant region gene - V Variable - C constant - J joining - D diversity     

Mapping dystrophin gene recombinants in Greek DMD/BMD families: low recombination frequencies in the STR region

A systematic study of 42 Greek DMD/BMD families using 14 polymorphic markers that span the dystrophin gene was performed in order to assess the position and frequency of recombinants in the Greek population and to test whether hot spots of recombination and deletions coincide when exclusively studying DMD/BMD families. We report a low percentage of recombination between markers STR44 and STR50; otherwise, the distribution of recombination events in other parts of the gene is largely in agreement with previously published data on Centre d'Etude du Polymorphisme Humaine families. We therefore propose that recombination frequencies and the correlation between recombination and deletion hot spots should be evaluated on DMD/BMD families exclusively.     

DNA linkage analysis of X-linked retinoschisis

Summary Four families with juvenile retionoschisis (RS) have been studied by linkage analysis utilizing eleven polymorphic X-chromosomal markers. The results suggest a close linkage between DXS43, DXS41, and DXS208 and the RS locus at Xp22. The RS locus is distal to the OTC locus, DXS84, and the DMD locus but proximal to DXS85. No recombination events were observed between the RS locus and DXS43 and DXS41. The maximum likelihood estimate of the recombination fraction () was thus zero and the peak lod scores () were 4.98 (DXS43) and 4.09 (DXS41). The linkage data suggest that the gene order on Xp is DXS85-(DXS43, RS, DXS41)-DMD-DXS84-OTC.     

Linkage of aspartylglucosaminuria (AGU) to marker loci on the long arm of chromosome 4

Summary Aspartylglucosaminuria (AGU) is caused by deficient activity of the enzyme aspartylglucosaminidase (AGA). The structural gene for AGA has been assigned to the region 4q21-qter of chromosome 4. We have studied the map position of the AGU locus in relation to other marker loci on the long arm of chromosome 4 using linkage analyses. Restriction fragment length polymorphism alleles for the ADH2, ADH3, EGF, FG and FG loci and blood group antigenes for the MNS locus were determined in a panel of 12 Finnish AGU families. The heterozygous family members were identified by reduced activity of AGA in lymphocytes. Linkage studies were performed using both pairwise and multipoint analyses. Loose linkage of the AGU locus to the FG and MNS loci was observed ( = 1.16, = 1.39, respectively). Multipoint analysis to the fixed map [ADH-(0.03)-EGF-(0.35)-FG-(0.11)-MNS] suggests that the location of the AGU locus is 0.05–0.30 recombination units distal to MNS ( = 3.03). The order cen-ADH-EGF-FG-MNS-AGU is 35 times more likely than the next best order cen-ADH-EGF-AGU-FG-MNS.     

A PvuII polymorphism of the low density lipoprotein receptor gene is not associated with plasma concentrations of low density lipoproteins including LP(a)

Lipoprotein(a) [Lp(a)] is a low density lipoprotein (LDL), in which apolipoprotein B-100 (apo B-100) is attached to apolipoprotein(a) [apo(a)], a glycoprotein of variable size. Lp(a) may be as atherogenic as LDL. In normal populations, Lp(a) concentrations in plasma are largely determined by the apo(a) gene locus on chromosome 6, but regulation of synthesis and catabolism of Lp(a) is poorly understood. In some studies, a PvuII restriction fragment length polymorphism (RFLP) in the LDL receptor gene seems to affect concentrations of LDL in plasma, and other studies have indicated that Lp(a) catabolism could be mediated by the LDL receptor. We therefore expected that the PvuII polymorphism in the LDL receptor gene might be associated with Lp(a) levels in 170 Caucasian men aged 40 years, selected to have a high representation of low molecular weight apo(a) phenotypes. However, plasma concentrations of cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides and Lp(a) were all unrelated to the LDL receptor gene PvuII polymorphism both in the group as a whole and when it was subgrouped by apo(a) phenotype. Therefore our data do not support the concept that this particular LDL receptor gene polymorphism is associated with LDL receptor function, and our data therefore neither support nor rule out a role for the LDL receptor in Lp(a) catabolism.     

Duplications and deletions of Vh genes in inbred strains of mice

evolution of variable region (Vh) gene family copy number and polymorphism was investigated by the analysis of the immunoglobulin heavy chain variable region (Igh-V) locus in 74 inbred strains and substrains of mice. Several strains were found to have slight differences from Igh-V haplotypes previously identified, usually of a single Vh gene family. These results indicate that the evolution of copy number in the mouse Igh-V locus proceeds largely by the accumulation of incremental changes, reflecting the clustered organization of the mouse Igh-V locus. We have found no evidence of very large or frequent duplication or deletion events indicative of rapid expansion or contraction processes. The existence of one or more particularly large Vh gene families most likely reflects random copy number variation, rather than selection for the amplification of their members. The identification of strains with recombinant Vh gene arrays demonstrates that recombination, both within and between haplotypes, appears to be the predominant mechanism generating the high restriction fragment length polymorphism in the Igh-V locus.Abbreviations used in this paper Igh-V immunoglobulin heavy chain variable region locus - Vh heavy chain variable region gene - Dh heavy chain diversity region gene - V immunoglobulin kappa light chain variable region gene - V T-cell receptor beta chain variable region gene     

Frequent recombination in the human T-cell receptor beta gene complex

Although individual TCRVBV gene segments exhibit limited polymorphism, human T-cell receptor beta (TCRB) haplotypes are characterized by multiple different combinations of allelic markers. This observation suggests that genetic recombination may have played a role in the generation of these haplotypes. Meiotic recombination in a region spanning 250 kilobases (kb) at the 3 end of the TCRB gene complex was investigated by extended family studies and by analysis of single sperm. Segregation patterns of polymorphic TCRB markers in families allowed the assignment of TCRB alleles to parental haplotypes and detection of recombinants among the offspring. Among the 178 informative paternal meioses, four (2%) were recombinant, whereas no recombinants were found in the 199 maternal meioses. In addition, segregation of two allelic markers was examined in a total of 1101 individual sperm from two heterozygous donors to detect exchange events in this region. The results revealed a similar rate of recombination, 1.3%, which, along with the family data, suggests that at, least in males, meiotic recombination in this 250 kb region may be six times higher than the average rate of 1% per 10⁶ bases that has been estimated for the human genome.     

Assignment of the gene for dyskeratosis congenita to Xq28

Summary Dyskeratosis congenita is an X-linked recessive disorder with diagnostic dermatological features, bone marrow hypofunction, and a predisposition to neoplasia in early adult life. Linkage analysis was undertaken in an extensive family with the condition using the Xg blood group and 17 cloned X chromosomal DNA sequences which recognise restriction fragment length polymorphisms (RFLPs). No recombination was observed between the locus for dyskeratosis congenita (DKC) and the RFLPs identified by DXS52 (St 14-1) (Z_max=3.33 at _max=0 with 95% confidence limits of 0 to 14 cM). Similarly no recombination was observed for the disease locus and F8 (Z_max=1.23 at _max=0) nor for DXS15 (Z_max=1.62 at _max=0), but both of these markers were only informative in part of the family whereas DXS52 was fully informative. DXS52, DXS15, and F8 are known to be tightly linked and have previously been assigned to Xq28. Thus the gene for dyskeratosis congenita can be assigned to Xq28. These DNA sequence polymorphisms will be of clinical value for carrier detection and prenatal diagnosis.     

Exclusion of two candidate pigment loci,c and b,part of chromosome 11p,and 33 random polymorphic markers as the locus for tyrosinase-positive oculocutaneous albinism

The locus for Tyrosinase-Positive Oculocutaneous Albinism (ty-pos OCA) has not yet been localised. The search for the ty-pos OCA locus has included a search for linkage to candidate pigment loci and a candidate chromosomal region, as well as a random search using highly polymorphic markers in 42 families, including 271 individuals of whom 79 are affected. The lod scores for the tyrosinase (TYR) locus (11q14–q21), homologous to the albino locus, c, in the mouse and the CAS2/TRP1 locus (9p22-pter), homologous to the brown locus, b, in the mouse were -5.89 and -7.22, respectively, at a recombination fraction of =0.01, thus excluding them from being the ty-pos OCA locus. In the candidate chromosomal region, 11p, four loci (probes) were tested, SAA (pSAA82), CALC (pHC36), HBB (Gamma-globin haplotype) and an AC repeat polymorphism at the Wilm's Tumour locus (WT1). A portion of 11p was excluded with the following lod scores: pSAA82 lod=-2.05 at =0.10; pHC36 lod=-3.87 at =0.05; gamma-globin haplotype lod=-2.80 at =0.10; and WT1 lod=-2.34 at =0.10. Thirty-three polymorphic markers randomly distributed on 13 different chromosomes were all excluded from close linkage to ty-pos OCA.     

Genetics of the quantitative Lp(a) lipoprotein trait

Summary Lp(a) glycoprotein exhibits an apparent size polymorphism that is associated with genetically controlled Lp(a) lipoprotein concentrations in plasma (Utermann et al. 1988). We have tested the hypothesis that this polymorphism is genetically controlled by studying 15 matings with a total of 44 offspring. This confirmed our conclusion that Lp(a) types are controlled by a series of codominant alleles Lp^F, Lp^B, Lp^S1, Lp^S2, Lp^S3 and Lp^S4 and by a null allele Lp^o. Together with the data from the accompanying paper this indicates that the structural gene for the Lp(a) protein is the major gene locus determining Lp(a) lipoprotein concentrations in plasma.     

Reevaluation of the chromosome 4q candidate region for early onset periodontitis

Evidence of linkage (lod=3.1, =0.05) was reported previously in one large kindred (the Brandywine genetic isolate) for an autosomal dominant form of early onset periodontitis (EOP) with a protein polymorphism in the vitamin D binding protein (GC) located on chromosome 4q12-q13. To evaluate the generality of this finding, 19 unrelated families (228 individuals), each with two or more EOP affected individuals, were ascertained and sampled. A restriction fragment length polymorphism (RFLP) at the GC locus and eight other polymorphic DNA markers and two red blood cell antigens located on proximal chromosome 4q in the vicinity of the GC locus were typed. Twelve genetic models of EOP were evaluated, which varied in diagnostic classification, penetrance, and mode of disease transmission. Results for all models strongly exclude linkage between an EOP susceptibility gene and this chromosomal region assuming locus homogeneity. Our data statistically exclude (lod -2.0) the possibility that more than 40% of our families are linked to this candidate region for one model tested. Linkage under heterogeneity was excluded less strongly for other models, but no significant evidence in support of linkage was obtained for any model. Our results indicate that either the previous report of linkage was a false positive, or that there are two or more unlinked forms of EOP, with the form located in 4q12-q13 being less common.     

The apolipoprotein CII gene: Subchromosomal localisation and linkage to the myotonic dystrophy locus

Summary The human apolipoprotein CII gene probe detects a restriction fragment length polymorphism located on chromosome 19. We have investigated the linkage of this polymorphism to the myotonic dystrophy locus in families. The two lici are closely linked with a maximum Lod score of 7.877 at 4% recombination. The close linkage and informativeness of the APOC2 polymorphism suggest that this probe may be of use for presymptomatic diagnosis of the myotonic dystrophy gene. The APOC2 gene was localised to the region 19p13–19q13 using somatic cell hybrids, providing further evidence that the myotonic dystrophy locus is situated in the central region of chromosome 19.     

Parathyroid hormone gene analysis in autosomal hypoparathyroidism using an intragenic tetranucleotide (AAAT) n polymorphism

We have identified a polymorphic tetranucleotide consisting of (AAAT) _n within the first intron of the parathyroid hormone (PTH) gene, and have used this to investigate the segregation of the PTH gene and idiopathic hypoparathyroidism in 7 affected and 21 unaffected members from three families. An association between the PTH locus and autosomal dominant idiopathic hypoparathyroidism in one family was excluded by observing recombination between the two loci. In the remaining two families with autosomal recessive idiopathic hypoparathyroidism, the PTH locus was not similarly excluded. We had previously demonstrated a donor splice site mutation of the PTH gene in one of these families, and PTH gene abnormalities were therefore sought in the second of these families. DNA sequence analysis of the three exons, together with 4 exon-intron boundaries and the promoter region of the PTH gene revealed no abnormalities, thereby indicating molecular pathology at another locus. Thus, our analysis of idiopathic hypoparathyroidism reveals genetic heterogeneity for this disorder. In addition, our indentification of a microsatellite polymorphism of the PTH gene should help further segregation studies of this locus in families with parathyroid disorders.