Frequent LPA KIV-2 Variants Lower Lipoprotein(a) Concentrations and Protect Against Coronary Artery Disease.

BACKGROUND: Lipoprotein(a) (Lp(a)) concentrations are a major independent risk factor for coronary artery disease (CAD) and are mainly determined by variation in LPA. Up to 70% of the LPA coding sequence is located in the hypervariable kringle IV type 2 (KIV-2) region. It is hardly accessible by conventional technologies, but may contain functional variants. OBJECTIVES: This study sought to investigate the new, very frequent splicing variant KIV-2 4733G>A on Lp(a) and CAD. METHODS: We genotyped 4733G>A in the GCKD (German Chronic Kidney Disease) study (n = 4,673) by allele-specific polymerase chain reaction, performed minigene assays, identified proxy single nucleotide polymorphisms and used them to characterize its effect on CAD by survival analysis in UK Biobank (n = 440,234). Frequencies in ethnic groups were assessed in the 1000 Genomes Project. RESULTS: The 4733G>A variant (38.2% carrier frequency) was found in most isoform sizes. It reduces allelic expression without abolishing protein production, lowers Lp(a) by 13.6 mg/dL (95% CI: 12.5-14.7; P < 0.0001) and is the strongest variance-explaining factor after the smaller isoform. Splicing of minigenes was modified. Compound heterozygosity (4.6% of the population) for 4733G>A and 4925G>A, another KIV-2 splicing mutation, reduces Lp(a) by 31.8 mg/dL and most importantly narrows the interquartile range by 9-fold (from 42.1 to 4.6 mg/dL) when compared to the wild type. In UK Biobank 4733G>A alone and compound heterozygosity with 4925G>A reduced HR for CAD by 9% (95% CI: 7%-11%) and 12% (95% CI: 7%-16%) (both P < 0.001). Frequencies in ethnicities differ notably. CONCLUSIONS: Functional variants in the previously inaccessible LPA KIV-2 region cooperate in determining Lp(a) variance and CAD risk. Even a moderate but lifelong genetic Lp(a) reduction translates to a noticeable CAD risk reduction.

Analyzing Low-Level mtDNA Heteroplasmy-Pitfalls and Challenges from Bench to Benchmarking.

Massive parallel sequencing technologies are promising a highly sensitive detection of low-level mutations, especially in mitochondrial DNA (mtDNA) studies. However, processes from DNA extraction and library construction to bioinformatic analysis include several varying tasks. Further, there is no validated recommendation for the comprehensive procedure. In this study, we examined potential pitfalls on the sequencing results based on two-person mtDNA mixtures. Therefore, we compared three DNA polymerases, six different variant callers in five mixtures between 50% and 0.5% variant allele frequencies generated with two different amplification protocols. In total, 48 samples were sequenced on Illumina MiSeq. Low-level variant calling at the 1% variant level and below was performed by comparing trimming and PCR duplicate removal as well as six different variant callers. The results indicate that sensitivity, specificity, and precision highly depend on the investigated polymerase but also vary based on the analysis tools. Our data highlight the advantage of prior standardization and validation of the individual laboratory setup with a DNA mixture model. Finally, we provide an artificial heteroplasmy benchmark dataset that can help improve somatic variant callers or pipelines, which may be of great interest for research related to cancer and aging.

Profiling of Mitochondrial DNA Heteroplasmy in a Prospective Oral Squamous Cell Carcinoma Study.

While a shift in energy metabolism is essential to cancers, the knowledge about the involvement of the mitochondrial genome in tumorigenesis and progression in oral squamous cell carcinoma (OSCC) is still very limited. In this study, we evaluated 37 OSCC tumors and the corresponding benign mucosa tissue pairs by deep sequencing of the complete mitochondrial DNA (mtDNA). After extensive quality control, we identified 287 variants, 137 in tumor and 150 in benign samples exceeding the 1% threshold. Variant heteroplasmy levels were significantly increased in cancer compared to benign tissues (p = 0.0002). Furthermore, pairwise high heteroplasmy frequency difference variants (∆HF% > 20) with potential functional impact were increased in the cancer tissues (p = 0.024). Fourteen mutations were identified in the protein-coding region, out of which thirteen were detected in cancer and only one in benign tissue. After eight years of follow-up, the risk of mortality was higher for patients who harbored at least one ∆HF% > 20 variant in mtDNA protein-coding regions relative to those with no mutations (HR = 4.6, (95%CI = 1.3-17); p = 0.019 in primary tumor carriers). Haplogroup affiliation showed an impact on survival time, which however needs confirmation in a larger study. In conclusion, we observed a significantly higher accumulation of somatic mutations in the cancer tissues associated with a worse prognosis.

A comprehensive map of single-base polymorphisms in the hypervariable LPA kringle IV type 2 copy number variation region.

Lipoprotein (a) [Lp(a)] concentrations are among the strongest genetic risk factors for cardiovascular disease and present pronounced interethnic and interindividual differences. Approximately 90% of Lp(a) variance is controlled by the LPA gene, which contains a 5.6-kb-large copy number variation [kringle IV type 2 (KIV-2) repeat] that generates >40 protein isoforms. Variants within the KIV-2 region are not called in common sequencing projects, leaving up to 70% of the LPA coding region currently unaddressed. To completely assess the variability in LPA, we developed a sequencing strategy for this region and report here the first map of genetic variation in the KIV-2 region, a comprehensively evaluated ultradeep sequencing protocol, and an easy-to-use variant analysis pipeline. We sequenced 123 Central-European individuals and reanalyzed public data of 2,504 individuals from 26 populations. We found 14 different loss-of-function and splice-site mutations, as well as >100, partially even common, missense variants. Some coding variants were frequent in one population but absent in others. This provides novel candidates to explain the large ethnic and individual differences in Lp(a) concentrations. Importantly, our approach and pipeline are also applicable to other similar copy number variable regions, allowing access to regions that are not captured by common genome sequencing.

A novel but frequent variant in LPA KIV-2 is associated with a pronounced Lp(a) and cardiovascular risk reduction.

AIMS: Lp(a) concentrations represent a major cardiovascular risk factor and are almost entirely controlled by one single locus (LPA). However, many genetic factors in LPA governing the enormous variance of Lp(a) levels are still unknown. Since up to 70% of the LPA coding sequence are located in a difficult to access hypervariable copy number variation named KIV-2, we hypothesized that it may contain novel functional variants with pronounced effects on Lp(a) concentrations. We performed a large scale mutation analysis in the KIV-2 using an extreme phenotype approach. METHODS AND RESULTS: We compiled an discovery set of 123 samples showing discordance between LPA isoform phenotype and Lp(a) concentrations and controls. Using ultra-deep sequencing, we identified a splice site variant (G4925A) in preferential association with the smaller LPA isoforms. Follow-up in a European general population (n = 2892) revealed an exceptionally high carrier frequency of 22.1% in the general population. The variant explains 20.6% of the Lp(a) variance in carriers of low molecular weight (LMW) apo(a) isoforms (P = 5.75e-38) and reduces Lp(a) concentrations by 31.3 mg/dL. Accordingly the odds ratio for cardiovascular disease was reduced from 1.39 [95% confidence interval (CI): 1.17-1.66, P = 1.89e-04] for wildtype LMW individuals to 1.19 [95%CI: 0.92; 1.56, P = 0.19] in LMW individuals who were additionally positive for G4925A. Functional studies point towards a reduction of splicing efficiency by this novel variant. CONCLUSION: A highly frequent but until now undetected variant in the LPA KIV-2 region is strongly associated with reduced Lp(a) concentrations and reduced cardiovascular risk in LMW individuals.