LPA gene: interaction between the apolipoprotein(a) size ('kringle IV' repeat) polymorphism and a pentanucleotide repeat polymorphism influences Lp(a) lipoprotein level.

OBJECTIVES: In order to search for factors influencing the Lp(a) lipoprotein level, we have examined the apolipoprotein(a) (apo(a)) size polymorphism as well as a pentanucleotide (TTTTA) repeat polymorphism in the 5' control region of the LPA gene. DESIGN: Lp(a) lipoprotein levels were compared between individuals with different genotypes as defined by pulsed field gel electrophoresis of DNA plugs, and PCR of DNA samples followed by polyacrylamide gel electrophoresis. DNA plugs and DNA were prepared from blood samples collected from blood donors. RESULTS: Twenty-seven different K IV repeat alleles were observed in the 71 women and 92 men from which apo(a) size polymorphism results were obtained. Alleles encoding 26-32 Kringle IV repeats were the most frequent. Alleles encoding seven to 11 TTTTA repeats were detected in the 84 women and 122 men included in the pentanucleotide polymorphism study, and homozygosity for eight TTTTA repeats was the most common genotype. The eight TTTTA repeat allele occurred with almost any apo(a) allele. An inverse relationship between number of K IV repeats and Lp(a) concentration was confirmed. The contributions of the apo(a) size polymorphism and the pentanucleotide repeat polymorphism to the interindividual variance of Lp(a) lipoprotein concentrations were 9.7 and 3.5%, respectively (type IV sum of squares). Nineteen per cent of the variance in Lp(a) lipoprotein level appeared to be the result of the multiplication product (interaction) between the apo(a) size polymorphism and the pentanucleotide repeat polymorphism. CONCLUSIONS: The contribution of the apo(a) size polymorphism alone to the variation in Lp(a) lipoprotein level was lower than previously reported. However, the multiplicative interaction effect between the K IV repeat polymorphism and the pentanucleotide repeat polymorphism may be an important factor explaining the variation in Lp(a) lipoprotein levels among the populations.

Sequence conservation in kringle IV-type 2 repeats of the LPA gene.

We have studied the homology of repeating kringle IV-type 2 (K IV-type 2) elements of the LPA gene. Two K IV-type 2 genomic polymerase chain reaction (PCR) fragment libraries were constructed, one from an individual with high and one from an individual with low Lp(a) lipoprotein level. Only minor K IV-type 2 repeat length heterogeneity was observed. Sequence analysis data from the cloned K IV-type 2 repeats revealed a high degree of LPA sequence conservation in exons as well as in introns both within and between the two libraries. This sequence conservation of the IV-type 2 kringles is in agreement with our previously reported results of simultaneous 'batch' DNA sequence analyses of all the K IV-type 2 repeats from single individuals. Sequence data from the clones, combined with genomic DNA sequencing, revealed that the K IV-type 2 reading frame of exons 1 and 2 are extended into the conserved flanking introns by 519 base pairs (bp) and 312 bp, respectively. The theoretical coding capacity of the exon 1 extended open reading frame (ORF I) is three times larger (173 amino acids, aa) than the translated exon 1, and that of the extended open reading frame of exon 2 (ORF II) is about twice (104 aa) the length of exon 2. A central portion of the intron separating exons 1 and 2 also exhibited a high degree of sequence conservation, with the exception of a polymorphic CA repeat. Within the 61 K IV repeat clones analysed, 19 different CA repeat patterns with 12-18 CA dinucleotide repeats were observed. A comparison between the 37 clones from the individual with high Lp(a) lipoprotein level and the 24 clones from the individual with low Lp(a) lipoprotein level, revealed that seven of the CA repeat variants were present in both clone libraries. The observed high level of sequence conservation in K IV-type 2 exons and introns matches relevant areas of the plasminogen gene, and our findings fit with recent K IV-type 2 duplications and evolutionary selection pressure theories, although gene conversion events could also explain the findings. DNA sequences within K IV-type 2 appeared to have no influence on Lp(a) lipoprotein level.

High-degree sequence conservation in LPA kringle IV-type 2 exons and introns.

In the search for factors contributing to the regulation of the Lp(a) lipoprotein concentration, we have sequenced the kringle IV-type 2 encoding exons 1 and 2 together with the flanking intron sequences of the LPA gene in individuals with different serum concentrations of Lp(a) lipoprotein. The high degree of sequence identity between the kringle IV-type 2 repeats made it possible to analyse all the 3-42 kringles simultaneously by polymerase chain reaction and direct DNA sequencing. The strategy used allowed us to determine approximately 700 bp from each kringle IV-type 2 repeat, resulting in a rapid screen of on average 28,000 bp of the LPA gene from each individual. Comparing these bipartite kringle IV-type 2 repeat sequences from 12 individuals with high and 11 individuals with low Lp(a) lipoprotein level revealed that: 1. no sequence polymorphism could be detected in the exons examined; 2. no sequence polymorphism could be detected in the consensus GT/AG splicing signals of exon/intron junctions; and 3. the proximal intron sequences seemed almost completely conserved in the 76-135 bp analysed. Only one position in the intron sequences exhibited the pattern of a G/A polymorphism. We observed no differences between the group with high and the group with low Lp(a) lipoprotein level. The very high conservation of intron sequences could support the hypothesis that the LPA gene evolved relatively recently. The contradictory finding of a corresponding sequence conservation between the human LPA and the plasminogen gene suggests that an evolutionary pressure has preserved these intron sequences over the last 40-90 million years.

Unilateral cleft lip in a boy with Angelman syndrome.

We report on a mentally retarded boy with epileptic seizures, microcephaly, ataxia, and developmental delay. His clinical features were consistent with Angelman syndrome. Fluorescent in situ hybridization and DNA analysis showed a deletion of chromosome 15 q11-13 and thus confirmed the diagnosis. In addition, the patient had a unilateral, incomplete cleft lip, a feature which has not previously been reported in Angelman syndrome.

Identification of the apo B-3500 mutation in the Norwegian population.

Familial defective apolipoprotein B-100 (FDB) is caused by a mutation in codon 3500 of the apo B gene. It is inherited in a co-dominant fashion and is characterized by hypercholesterolaemia. Thus, FDB has similar features to familial hypercholesterolaemia (FH). In order to investigate whether some of the Norwegian subjects diagnosed as having FH actually have FDB, we have screened 208 Norwegian FH heterozygotes for the apo B-3500 mutation. One of the subjects possessed the mutation which was on a haplotype compatible with the mutation-bearing haplotype found in other populations. Although, hypercholesterolaemia segregated with haplotypes both at the apolipoprotein B and low density lipoprotein (LDL) receptor loci in the proband's family, LDL receptor analysis revealed that the proband was not doubly heterozygous for FDB and FH.

Haplotype analysis at the low density lipoprotein receptor locus in normal and familial hypercholesterolemia Norwegian subjects.

We have performed haplotype analysis at the low density lipoprotein receptor (LDLR) locus in order to investigate the molecular genetics of familial hypercholesterolemia (FH) in Norway. Haplotypes were constructed using 7 restriction fragment length polymorphisms (RFLPs) in 194 subjects from 48 unrelated Norwegian FH families. Hypercholesterolemia co-segregated with haplotypes at the LDLR locus in all 48 families. Unambiguous haplotypes could be established for 190 independent chromosomes from 51 FH heterozygotes and 44 healthy normal subjects. A total of 20 different haplotypes was found. The most frequent haplotype was haplotype 3, which accounted for 32.4% or 43.1% of the normal and defective haplotypes, respectively. Haplotype 2 was significantly more frequent among the defective alleles than among the normal alleles (33.3% and 5.8%, respectively, p < 0.0001). Thus, haplotypes 2 and 3 accounted for 76.4% of the defective haplotypes. More data are needed to determine the possible existence of founder genes in the Norwegian population. Haplotypes 1, 2, 3, 5 and 8 accounted for 88.2% of the normal haplotypes. Based upon the cumulative heterozygosity index, the SphI, NcoI and 3' ApaLI RFLPs are the most informative markers in the Norwegian population.

Screening for point mutations in exon 10 of the low density lipoprotein receptor gene by analysis of single-strand conformation polymorphisms: detection of a nonsense mutation-FH469-->Stop.

DNA from 40 unrelated familial hypercholesterolemia (FH) heterozygotes were subjected to analyses of single-strand conformation polymorphisms (SSCPs) of exon 10 of the low density lipoprotein receptor (LDLR) gene. Four different SSCP patterns were observed. The underlying mutations were characterized by DNA sequencing. Three of the patterns represented the three genotypes of a recently described sense mutation in codon 450. A method based upon the polymerase chain reaction (PCR) was developed to analyze this mutation. The frequencies of the wild-type (G at nucleotide 1413) and mutant (A at nucleotide 1413) alleles were 0.56 and 0.44, respectively. The fourth pattern was found in only one FH heterozygote and was caused by heterozygosity at nucleotide 1469 (G/A). Nucleotide 1469 is the second base of codon 469Trp(TGG). The G-->A mutation changes this codon into the amber stop codon, and is referred to as FH469-->Stop. The mutant receptor consists of the amino terminal 468 amino acids. Because the truncated receptor has lost the membrane-spanning domain, it will not be anchored in the cell membrane. FH469-->Stop destroys an AvaII restriction site, and this characteristic was used to develop a PCR method to establish its frequency in Norwegian FH subjects. Two out of 204 (1%) unrelated FH heterozygotes possessed the mutation.

Screening for point mutations by semi-automated DNA sequencing using sequenase and magnetic beads.

We have established an improved method for detecting point mutations by semi-automated DNA sequencing of PCR fragments generated from genomic DNA. The method employs magnetic beads to create immobilized single-stranded DNA templates, and the sequencing reaction is performed with Sequenase. This method is superior to sequencing with Taq DNA polymerase because the uniform peak height with Sequenase makes heterozygosity easily detectable as double peaks that are half the normal height. Detection of heterozygosity by this method is illustrated by sequencing a 180-bp fragment of the human apolipoprotein B gene. This fragment contains codon 3500, where a point mutation (3500CGG-->CAG) is found in subjects with the autosomal dominant disease familial defective apolipoprotein B. The nonuniform peak height with Taq DNA polymerase makes it more difficult to detect heterozygosity. This is also illustrated by sequencing a 278-bp fragment of the low-density lipoprotein receptor gene.

A 9.6 kilobase deletion in the low density lipoprotein receptor gene in Norwegian familial hypercholesterolemia subjects.

Haplotype analysis of the low density lipoprotein receptor (LDLR) gene was performed in Norwegian subjects heterozygous for familial hypercholesterolemia (FH). Southern blot analysis of genomic DNA, using an exon 18 specific probe and the restriction enzyme NcoI, showed that two out of 57 unrelated FH subjects had an abnormal 3.6 kb band. Further analyses revealed that this abnormal band was due to a 9.6 kb deletion that included exons 16 and 17. The 5' deletion breakpoint was after 245 bp of intron 15, and the 3' deletion breakpoint was in exon 18 after nucleotide 3390 of cDNA. Thus, both the membrane-spanning and cytoplasmatic domains of the receptor had been deleted. A polymerase chain reaction (PCR) method was developed to identify this deletion among other Norwegian FH subjects. As a result of this screening one additional subject was found out of 124 subjects screened. Thus, three out of 181 (1.7%) unrelated Norwegian FH subject possessed this deletion. The deletion was found on the same haplotype in the three unrelated subjects, suggesting a common mutagenic event. The deletion is identical to a deletion (FH-Helsinki) that is very common among Finnish FH subjects. However, it is not yet known whether the mutations evolved separately in the two countries.

StyI polymorphism in an enhancer region of the second intron of the apolipoprotein B gene in hyper- and hypocholesterolemic subjects.

The regulation of the human apolipoprotein (apo) B gene that plays a crucial role in lipid metabolism is apparently very complex, with multiple cis- and trans-acting regulatory factors. One of these factors is an enhancer region in the second intron. In this region a point mutation at position + 722 has been found that is detectable by the restriction enzyme StyI. The report of Levy-Wilson et al. (1991) could suggest that the mutant allele (abolished StyI site) is associated with hypocholesterolemia. To investigate further the possible effect of this mutation on plasma cholesterol levels, we have compared the frequency of the mutant allele between 206 hypercholesterolemic Norwegian or Czech subjects on one hand, and 165 hypocholesterolemic Norwegian or Czech subjects on the other hand. No significant difference in frequency was found between the hypercholesterolemic and the hypocholesterolemic groups. This finding indicates either that the mutation at position + 722 does not affect the enhancer activity or that this in vitro enhancer activity is of little or no clinical significance. One of the Norwegian hypercholesterolemic subjects who was of Czech descent possessed the apoB 3500 mutation that leads to defective binding of low density lipoprotein (LDL) to the LDL receptors. Haplotype analysis of the apoB gene in her family showed that the mutation-bearing allele was identical to that reported in other countries, indicating a common gene source.

A new polymorphism in exon 11 of the LDL receptor gene in healthy people and in familial hypercholesterolemia subjects.

We have screened exon 11 of the low density lipoprotein receptor (LDLR) gene from familial hypercholesterolemia (FH) heterozygotes for point mutations by using analysis of single strand conformation polymorphisms (SSCP). A variant pattern was observed in three out of 39 subjects. By DNA sequencing, this variant pattern was found to be due to a C-->T transition at nucleotide 1617 that affects the third base of codon 518. A PCR method was developed to screen FH heterozygotes and normal subjects for this mutation. The gene frequencies in FH heterozygotes and normal subjects were 4% and 4.5%, respectively. Thus, the mutation cannot be in linkage disequilibrium with a mutation that causes FH. Rather, the mutation may be a useful genetic marker at the LDLR locus. Haplotype analysis at the LDLR locus in two FH families where the proband possessed the mutation revealed that the mutation was on two different haplotypes. This finding is consistent with the mutation occurring at a mutational hot spot.