Sequence variation within the KIV-2 copy number polymorphism of the human LPA gene in African, Asian, and European populations

Abstract

Amazingly little sequence variation is reported for the kringle IV 2 copy number variation (KIV 2 CNV) in the human LPA gene. Apart from whole genome sequencing projects, this region has only been analyzed in some detail in samples of European populations. We have performed a systematic resequencing study of the exonic and flanking intron regions within the KIV 2 CNV in 90 alleles from Asian, European, and four different African populations. Alleles have been separated according to their CNV length by pulsed field gel electrophoresis prior to unbiased specific PCR amplification of the target regions. These amplicons covered all KIV 2 copies of an individual allele simultaneously. In addition, cloned amplicons from genomic DNA of an African individual were sequenced. Our data suggest that sequence variation in this genomic region may be higher than previously appreciated. Detection probability of variants appeared to depend on the KIV 2 copy number of the analyzed DNA and on the proportion of copies carrying the variant. Asians had a high frequency of so-called KIV 2 type B and type C (together 70% of alleles), which differ by three or two synonymous substitutions respectively from the reference type A. This is most likely explained by the strong bottleneck suggested to have occurred when modern humans migrated to East Asia. A higher frequency of variable sites was detected in the Africans. In particular, two previously unreported splice site variants were found. One was associated with non-detectable Lp(a). The other was observed at high population frequencies (10% to 40%). Like the KIV 2 type B and C variants, this latter variant was also found in a high proportion of KIV 2 repeats in the affected alleles and in alleles differing in copy numbers. Our findings may have implications for the interpretation of SNP analyses in other repetitive loci of the human genome.

Fig 1. LPA gene structure.

The LPA gene contains 27 non-repetitive exons including the 5’UTR (grey), one copy each of kringle (K) domains KIV-1, KIV-3 to KIV-10 (black), KV (green), all comprising two exons, followed by six exons encoding the protease-like domain (purple) and the 3’UTR (grey). The KIV-2 domain (red) varies in number from 1 to >40 copies, thereby forming the KIV-2 CNV. Each KIV-2 repeat is approximately 5.5 kb long, composed of the first exon (160 bp), a long intron (4 kb), the second exon (182 bp), and a short intron (1.2 kb). Depicted is the general exon/intron structure of the LPA gene, with one KIV-2 repeat, and an example containing six KIV-2 repeats and, hence, 15 KIV repeats (as is the case for LPA in the human reference sequence).

Fig 2. Examples for the possible distribution of sequence variations in KIV-2 CNV alleles.

Sequence variants affecting KIV-2 copies (shown as filled circles) can be present in single or several KIV-2 copies within alleles of different sizes. (A) Low and high intra-allelic frequencies of a variant on short (e.g. 5 KIV-2) alleles. (B) Low and high intra-allelic frequencies of a variant for longer (limited to 10 KIV-2 for graphic display) alleles. (C) The same number of KIV-2 copies harbors the variant on a short and a longer allele, i.e. the intra-allelic frequency of the variant is higher on the short allele. Thus detection of the variant would be more likely if present on the short allele. (D) Both alleles have the same intra-allelic frequency (20%), though the number of copies carrying the variant is different. Hence the probability of detection would be the same. (E) The order of variant carrying KIV-2 copies within the allele might vary. These different scenarios cannot be distinguished by present methods. (F) Different variants can be allocated in cis, shown for a genotype with one short and one longer allele or (G) in trans. While scenarios F and G cannot be distinguished in analyses based on diploid samples, this is possible in our analysis based on separated alleles.

Fig 3. Positions of the PCR primers and regions amplified for cloning and batchwise screening.

Below a stretch of KIV-2 domains showing the positions of the exons and the short and long introns in the KIV-2 CNV, the regions amplified by the different PCRs are depicted by horizontal lines with the names of the PCRs written above and the primers below. PCRs “421” and “422” are spanning the first and second exon of the KIV-2 respectively, and also contain the flanking intron sequences. These two PCRs were used in the batchwise screening and cloning. PCR “412to422” amplifies both exons and the long intron, while PCR “422to421” includes both exons and the short intron. The locations of the different primers used for sequencing are shown above the depicted stretch of KIV-2 CNV. The figure is drawn to scale (2.5 cm = 1 kb).

Fig 4. Distribution of sequence variation within KIV-2 across KIV CNV alleles and populations.

The frequency distribution of the KIV CNV alleles is given for all samples, and by population. Alleles carrying variants within the screened KIV-2 exon 1 and KIV-2 exon 2 domains are colored according to the variation found in the batchwise screening. Alleles harboring several variants are striped. Note that for the individual alleles, the number of KIV-2 copies carrying the depicted variants cannot be derived from this figure. See S6 Table for these intra-allelic frequencies. Inserts show the mean KIV CNV sizes (i.e. number of KIV-2 repeats plus the nine non-repetitive KIVs), and the mean allele-associated Lp(a) concentrations in mg/dl for the populations.

Fig 5. Donor splice site K421 +1G>A and its association with a null allele.

Southern and Western blots for the Khoi San sample (K5) harboring the donor splice site mutation K421 +1G>A on its short allele with 15 KIV repeats are shown in context with family members of the individual (family members are not included in the analyses of the present study). The electropherogram from the batchwise sequencing for this allele is shown on the left. KIV CNV sizes for the Southern Blot are given for all family members, and for the Western blot the sizes of the apo(a) isoforms size marker is shown on the left. While both alleles of the sample K5 are clearly visible in the Southern blot (15 and 23 KIV copies), the corresponding isoform for the short allele is missing in the Western blot, indicating a direct effect of the donor splice site mutation on the phenotype by introducing a null allele. Note that this Western Blot is exposed for a long time, so as not to miss low expressed isoforms.