LPA is the major locus controlling Lp(a) plasma levels which vary extensively within and across populations and are associated with coronary heart disease. Identified causal genetic polymorphisms, including the KIV-2 CNV and other sequence variants, explain only a fraction of the variation in Lp(a) levels attributable to the LPA locus. Therefore additional sequence variation in LPA is expected to explain more of the observed variation in Lp(a) levels. One region of LPA not yet fully screened for such sequence variants is the KIV-2 CNV. Some variants in this region have been reported by the large re-sequencing projects but for those variants any data on the Lp(a) levels and the KIV-2 CNV size are missing.
In one projects of this thesis, coding regions of the KIV 2 CNV were screened in individuals from six different populations (Gabonese Bantu, South African Bantu, Khoi San, Egyptians, Hong Kong Chinese and Austrians). LPA alleles of each individual were separated by PFGE and used for batchwise amplification of PCR fragments specific for the two exons of KIV-2. Despite a restricted sample size (in total 90 alleles) and limited sensitivity of the screening method used, several synonymous and non-synonymous variants as well as two previously unreported splice site variants were identified. Most of these variants were detected in African alleles. Among the variants found, an African specific acceptor splice site variant (K422 6T>G) associated with small KIV-2 CNV size was present in all African populations at high population frequencies (10% to 40%). This variant appears to be associated with low Lp(a) levels than expected considering the KIV-2 CNV size of the alleles carrying it. In contrast to the frequent African specific acceptor splice site variant, a rare donor splice site variant (K421 +1G>A) was found which was shared among Africans and Europeans and associated with non-detectable Lp(a). In Asians, only the synonymous variants that define the KIV 2 type B and type C were found at very high frequencies (70%) and observed with a broad range of different KIV 2 CNV sizes. These variants were rare in Europeans and Africans. A strong bottleneck suggested to have occurred during the migration of modern humans to East Asia might explain the enrichment of these variants in Asians. A key observation was that the variants which were frequent were shared among the KIV-2 copies of the alleles carrying them. The acceptor splice site variant as well as type B/C defining variants were observed at high intra allelic frequencies (relative proportion of the variant vs wild type carrying KIV-2 copies), i.e. they are shared among KIV-2 copies of an allele. Similar intra allelic frequencies were observed for the same variants carried on different sized KIV-2 allele.
Given the association between elevated Lp(a) and risk for coronary heart disease (CHD), an accurate and cost-effective screening of individuals at high risk for CHD has become a target of research. Assessment of apo(a) isoform size, which is an important factor for risk evaluation, is laborious and costly. rs3798220 which is a non-synonymous SNP in the protease like domain of LPA has been shown to tag high risk LPA alleles in Europeans. This led to the proposition of its use as a cost-efficient risk marker. Whether it could serve the purpose to identify high risk LPA alleles in other populations appears doubtful given a negative result from a recent GWAS in Japanese. In a second project, the association of rs3798220 with Lp(a) levels and KIV-2 CNV size was investigated in different non-European populations from Africa (Khoi San, Gabonese Bantu and Egyptians), South Asia (North Indians and South Indians), and Southeast Asia (Hong Kong Chinese, Japanese, Indonesians, Thai and Trobriand Islanders), and a possible contribution to the Lp(a) attributed risk in CAD patients from North and South India was assessed. The variant was found to be very rare in Asian Indians, and could not explain the Lp(a) attributed risk for CAD in that population. In accordance with 1000G data, the variant was absent in autochthonous Africans but it was frequent in East and Southeast Asians with MAFs ranging from 2.9% to 11.6%. However, no association with either higher Lp(a) or short apo(a) isoforms/ KIV-2 CNV size was found. This is in contrast to the findings in Europeans where it was observed with short apo(a) isoforms and with high Lp(a). This finding suggests that rs379220 itself is not causal and its association with high Lp(a) could most likely be explained by the LD with short isoform size which come with very high Lp(a) levels in Europeans. The background haplotype of the variant carrying alleles was shared among Europeans and Asians.
The findings reported in this dissertation suggest that the LPA locus harbors more sequence variation in the KIV-2 region, which might explain an additional fraction of variation in Lp(a) levels across ethnicities. Population specific differences exist in the association of SNPs with the KIV-2 CNV size, and the associated Lp(a) levels could be influenced by the differences in the LD patterns across populations. The SNP markers associated with Lp(a) levels or predicting the risk for CHD in one population may not be applicable to other populations. On the other hand, population specific SNPs in LD with a narrow range of KIV-2 CNV sizes can be causally associated with Lp(a) levels. One example could be the frequent African specific acceptor splice variant site, detected in the screening of the KIV-2 region, which is associated with short KIV-2 alleles and comparatively low Lp(a). This variant has potential clinical relevance for investigating the association between CHD risk and apo(a) isoform size and might provide some explanation for the missing association of the short apo(a) size with CHD in individuals of African descent.
The nature of LD between SNPs and KIV-2 CNV size across populations can depend on the mode and the rate of size changing mutations at the CNV. The rs3798220 variant allele is observed with vastly different KIV-2 CNV sizes between Europeans and non-Europeans. The shared haplotype background of the variant carrying alleles in the two continental groups argues against it originating from a recurrent point mutation, but rather indicates that the CNV size has changed since the split of the populations. It remains to be determined whether CNV size changes can also explain the observation that the same variants are found on multiple KIV-2 copies on the same allele. Here, inter-locus gene conversion might also play an essential role. The same applies for the observation that similar intra allelic frequencies of specific KIV-2 variants are kept even between alleles of different KIV-2 CNV sizes. More extensive SNP haplotype data within the KIV-2 CNV may provide further insights into the mechanism of KIV-2 CNV size changes and their role in the spreading of variants across the KIV-2 copies.