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.

Epigenome-wide association study of lung function level and its change.

Previous reports link differential DNA methylation (DNAme) to environmental exposures that are associated with lung function. Direct evidence on lung function DNAme is, however, limited. We undertook an agnostic epigenome-wide association study (EWAS) on pre-bronchodilation lung function and its change in adults.In a discovery-replication EWAS design, DNAme in blood and spirometry were measured twice, 6-15 years apart, in the same participants of three adult population-based discovery cohorts (n=2043). Associated DNAme markers (p<5×10-7) were tested in seven replication cohorts (adult: n=3327; childhood: n=420). Technical bias-adjusted residuals of a regression of the normalised absolute ß-values on control probe-derived principle components were regressed on level and change of forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC) and their ratio (FEV1/FVC) in the covariate-adjusted discovery EWAS. Inverse-variance-weighted meta-analyses were performed on results from discovery and replication samples in all participants and never-smokers.EWAS signals were enriched for smoking-related DNAme. We replicated 57 lung function DNAme markers in adult, but not childhood samples, all previously associated with smoking. Markers not previously associated with smoking failed replication. cg05575921 (AHRR (aryl hydrocarbon receptor repressor)) showed the statistically most significant association with cross-sectional lung function (FEV1/FVC: pdiscovery=3.96×10-21 and pcombined=7.22×10-50). A score combining 10 DNAme markers previously reported to mediate the effect of smoking on lung function was associated with lung function (FEV1/FVC: p=2.65×10-20).Our results reveal that lung function-associated methylation signals in adults are predominantly smoking related, and possibly of clinical utility in identifying poor lung function and accelerated decline. Larger studies with more repeat time-points are needed to identify lung function DNAme in never-smokers and in children.

Genetic Factors Explain a Major Fraction of the 50% Lower Lipoprotein(a) Concentrations in Finns.

OBJECTIVE: Lp(a) (lipoprotein(a)) concentrations are widely genetically determined by the LPA isoforms and show 5-fold interpopulation differences. Two- to 3-fold differences have been reported even within Europe. Finns represent a distinctive population isolate within Europe and have been repeatedly reported to present lower Lp(a) concentrations than Central Europeans. The significance of this finding was unclear for a long time because of the difficult comparability of Lp(a) assays. Recently, a large standardized study in >50 000 individuals from 7 European populations confirmed this observation but could not provide insights into the causes. APPROACH AND RESULTS: We investigated Lp(a) concentrations, LPA isoforms, and genotypes of established genetic variants affecting Lp(a) concentrations (LPA variants, APOE isoforms, and PCSK9 R46L) in the Finnish YFS (Cardiovascular Risk in Young Finns Study) population (n=2281) and 3 Non-Finnish Central European populations (n=10 003). We observed ≈50% lower Lp(a) concentrations in Finns. The isoform distribution was shifted toward longer isoforms, and the percentage of low-molecular-weight isoform carriers was reduced. Most interestingly, however, Lp(a) was reduced in each single-isoform group. In contrast to the known inverse relationship between LPA isoforms and Lp(a) concentrations, especially very short isoforms presented unexpectedly low Lp(a) concentrations in Finns. The investigated genetic variants, as well as age, sex, and renal function, explained 71.8% of the observed population differences. CONCLUSIONS: The population differences in Lp(a) concentrations between Finnish and Central European populations originate not only from a different LPA isoform distribution but suggest the existence of novel functional variation in the small-isoform range.

A genome-wide association meta-analysis on apolipoprotein A-IV concentrations.

Apolipoprotein A-IV (apoA-IV) is a major component of HDL and chylomicron particles and is involved in reverse cholesterol transport. It is an early marker of impaired renal function. We aimed to identify genetic loci associated with apoA-IV concentrations and to investigate relationships with known susceptibility loci for kidney function and lipids. A genome-wide association meta-analysis on apoA-IV concentrations was conducted in five population-based cohorts (n = 13,813) followed by two additional replication studies (n = 2,267) including approximately 10 M SNPs. Three independent SNPs from two genomic regions were significantly associated with apoA-IV concentrations: rs1729407 near APOA4 (P = 6.77 × 10 - 44), rs5104 in APOA4 (P = 1.79 × 10-24) and rs4241819 in KLKB1 (P = 5.6 × 10-14). Additionally, a look-up of the replicated SNPs in downloadable GWAS meta-analysis results was performed on kidney function (defined by eGFR), HDL-cholesterol and triglycerides. From these three SNPs mentioned above, only rs1729407 showed an association with HDL-cholesterol (P = 7.1 × 10 - 07). Moreover, weighted SNP-scores were built involving known susceptibility loci for the aforementioned traits (53, 70 and 38 SNPs, respectively) and were associated with apoA-IV concentrations. This analysis revealed a significant and an inverse association for kidney function with apoA-IV concentrations (P = 5.5 × 10-05). Furthermore, an increase of triglyceride-increasing alleles was found to decrease apoA-IV concentrations (P = 0.0078). In summary, we identified two independent SNPs located in or next the APOA4 gene and one SNP in KLKB1 The association of KLKB1 with apoA-IV suggests an involvement of apoA-IV in renal metabolism and/or an interaction within HDL particles. Analyses of SNP-scores indicate potential causal effects of kidney function and by lesser extent triglycerides on apoA-IV concentrations.

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.

A genome-wide association meta-analysis on lipoprotein (a) concentrations adjusted for apolipoprotein (a) isoforms.

High lipoprotein (a) [Lp(a)] concentrations are an independent risk factor for cardiovascular outcomes. Concentrations are strongly influenced by apo(a) kringle IV repeat isoforms. We aimed to identify genetic loci associated with Lp(a) concentrations using data from five genome-wide association studies (n = 13,781). We identified 48 independent SNPs in the LPA and 1 SNP in the APOE gene region to be significantly associated with Lp(a) concentrations. We also adjusted for apo(a) isoforms to identify loci affecting Lp(a) levels independently from them, which resulted in 31 SNPs (30 in the LPA, 1 in the APOE gene region). Seven SNPs showed a genome-wide significant association with coronary artery disease (CAD) risk. A rare SNP (rs186696265; MAF ∼1%) showed the highest effect on Lp(a) and was also associated with increased risk of CAD (odds ratio = 1.73, P = 3.35 × 10-30). Median Lp(a) values increased from 2.1 to 91.1 mg/dl with increasing number of Lp(a)-increasing alleles. We found the APOE2-determining allele of rs7412 to be significantly associated with Lp(a) concentrations (P = 3.47 × 10-10). Each APOE2 allele decreased Lp(a) by 3.34 mg/dl corresponding to ∼15% of the population's mean values. Performing a gene-based test of association, including suspected Lp(a) receptors and regulators, resulted in one significant association of the TLR2 gene with Lp(a) (P = 3.4 × 10-4). In summary, we identified a large number of independent SNPs in the LPA gene region, as well as the APOE2 allele, to be significantly associated with Lp(a) concentrations.

Effect of diet-induced weight loss on lipoprotein(a) levels in obese individuals with and without type 2 diabetes.

AIMS/HYPOTHESIS: Elevated levels of lipoprotein(a) [Lp(a)] are an independent risk factor for cardiovascular disease (CVD), particularly in individuals with type 2 diabetes. Although weight loss improves conventional risk factors for CVD in type 2 diabetes, the effects on Lp(a) are unknown and may influence the long-term outcome of CVD after diet-induced weight loss. The aim of this clinical study was to determine the effect of diet-induced weight loss on Lp(a) levels in obese individuals with type 2 diabetes. METHODS: Plasma Lp(a) levels were determined by immunoturbidimetry in plasma obtained before and after 3-4 months of an energy-restricted diet in four independent study cohorts. The primary cohort consisted of 131 predominantly obese patients with type 2 diabetes (cohort 1), all participants of the Prevention Of Weight Regain in diabetes type 2 (POWER) trial. The secondary cohorts consisted of 30 obese patients with type 2 diabetes (cohort 2), 37 obese individuals without type 2 diabetes (cohort 3) and 26 obese individuals without type 2 diabetes who underwent bariatric surgery (cohort 4). RESULTS: In the primary cohort, the energy-restricted diet resulted in a weight loss of 9.9% (95% CI 8.9, 10.8) and improved conventional CVD risk factors such as LDL-cholesterol levels. Lp(a) levels increased by 14.8 nmol/l (95% CI 10.2, 20.6). In univariate analysis, the change in Lp(a) correlated with baseline Lp(a) levels (r = 0.38, p < 0.001) and change in LDL-cholesterol (r = 0.19, p = 0.033). In cohorts 2 and 3, the weight loss of 8.5% (95% CI 6.5, 10.6) and 6.5% (95% CI 5.7, 7.2) was accompanied by a median increase in Lp(a) of 13.5 nmol/l (95% CI 2.3, 30.0) and 11.9 nmol/l (95% CI 5.7, 19.0), respectively (all p < 0.05). When cohorts 1-3 were combined, the diet-induced increase in Lp(a) correlated with weight loss (r = 0.178, p = 0.012). In cohort 4, no significant change in Lp(a) was found (-7.0 nmol/l; 95% CI -18.8, 5.3) despite considerable weight loss (14.0%; 95% CI 12.2, 15.7). CONCLUSIONS/INTERPRETATION: Diet-induced weight loss was accompanied by an increase in Lp(a) levels in obese individuals with and without type 2 diabetes while conventional CVD risk factors for CVD improved. This increase in Lp(a) levels may potentially antagonise the beneficial cardiometabolic effects of diet-induced weight reduction.

Large-scale mitochondrial DNA analysis in Southeast Asia reveals evolutionary effects of cultural isolation in the multi-ethnic population of Myanmar.

BACKGROUND: Myanmar is the largest country in mainland Southeast Asia with a population of 55 million people subdivided into more than 100 ethnic groups. Ruled by changing kingdoms and dynasties and lying on the trade route between India and China, Myanmar was influenced by numerous cultures. Since its independence from British occupation, tensions between the ruling Bamar and ethnic minorities increased. RESULTS: Our aim was to search for genetic footprints of Myanmar's geographic, historic and sociocultural characteristics and to contribute to the picture of human colonization by describing and dating of new mitochondrial DNA (mtDNA) haplogroups. Therefore, we sequenced the mtDNA control region of 327 unrelated donors and the complete mitochondrial genome of 44 selected individuals according to highest quality standards. CONCLUSION: Phylogenetic analyses of the entire mtDNA genomes uncovered eight new haplogroups and three unclassified basal M-lineages. The multi-ethnic population and the complex history of Myanmar were reflected in its mtDNA heterogeneity. Population genetic analyses of Burmese control region sequences combined with population data from neighboring countries revealed that the Myanmar haplogroup distribution showed a typical Southeast Asian pattern, but also Northeast Asian and Indian influences. The population structure of the extraordinarily diverse Bamar differed from that of the Karen people who displayed signs of genetic isolation. Migration analyses indicated a considerable genetic exchange with an overall positive migration balance from Myanmar to neighboring countries. Age estimates of the newly described haplogroups point to the existence of evolutionary windows where climatic and cultural changes gave rise to mitochondrial haplogroup diversification in Asia.

Somatic mutations throughout the entire mitochondrial genome are associated with elevated PSA levels in prostate cancer patients.

The genetic etiology of prostate cancer, the most common form of male cancer in western countries, is complex and the interplay of disease genes with environmental factors is far from being understood. Studies on somatic mitochondrial DNA (mtDNA) mutations have become an important aspect of cancer research because these mutations might have functional consequences and/or might serve as biosensors for tumor detection and progression. We sequenced the entire mitochondrial genome (16,569 bp) from 30 prospectively collected pairs of macrodissected cancerous and benign cells from prostate cancer patients and compared their genetic variability. Given recent concerns regarding the authenticity of newly discovered mtDNA mutations, we implemented a high-quality procedure for mtDNA whole-genome sequencing. In addition, the mitochondrial genes MT-CO2, MT-CO3, MT-ATP6, and MT-ND6 were sequenced in further 35 paired samples from prostate cancer patients. We identified a total of 41 somatic mutations in 22 out of 30 patients: the majority of these mutations have not previously been observed in the human phylogeny. The presence of somatic mutations in transfer RNAs (tRNAs) was found to be associated with elevated PSA levels (14.25 ± 5.44 versus 7.15 ± 4.32 ng/ml; p = 0.004). The level and degree of heteroplasmy increased with increasing tumor activity. In summary, somatic mutations in the mitochondrial genome are frequent events in prostate cancer. Mutations mapping to mitochondrial tRNAs, ribosomal RNAs, and protein coding genes might impair processes that occur within the mitochondrial compartment (e.g., transcription, RNA processing, and translation) and might finally affect oxidative phosphorylation.

Investigation and functional characterization of rare genetic variants in the adipose triglyceride lipase in a large healthy working population.

Recent studies demonstrated a strong influence of rare genetic variants on several lipid-related traits. However, their impact on free fatty acid (FFA) plasma concentrations, as well as the role of rare variants in a general population, has not yet been thoroughly addressed. The adipose triglyceride lipase (ATGL) is encoded by the PNPLA2 gene and catalyzes the rate-limiting step of lipolysis. It represents a prominent candidate gene affecting FFA concentrations. We therefore screened the full genomic region of ATGL for mutations in 1,473 randomly selected individuals from the SAPHIR (Salzburg Atherosclerosis Prevention program in subjects at High Individual Risk) Study using a combined Ecotilling and sequencing approach and functionally investigated all detected protein variants by in-vitro studies. We observed 55 novel mostly rare genetic variants in this general population sample. Biochemical evaluation of all non-synonymous variants demonstrated the presence of several mutated but mostly still functional ATGL alleles with largely varying residual lipolytic activity. About one-quarter (3 out of 13) of the investigated variants presented a marked decrease or total loss of catalytic function. Genetic association studies using both continuous and dichotomous approaches showed a shift towards lower plasma FFA concentrations for rare variant carriers and an accumulation of variants in the lower 10%-quantile of the FFA distribution. However, the generally rather small effects suggest either only a secondary role of rare ATGL variants on the FFA levels in the SAPHIR population or a recessive action of ATGL variants. In contrast to these rather small effects, we describe here also the first patient with "neutral lipid storage disease with myopathy" (NLSDM) with a point mutation in the catalytic dyad, but otherwise intact protein.

Comparison of lipoprotein (a) serum concentrations measured by six commercially available immunoassays.

BACKGROUND AND AIMS: Lipoprotein (a) [Lp(a)] is an established causal risk factor for cardiovascular disease (CVD), independently of low-density lipoproteins (LDL) and other risk factors. The recognition of Lp(a) as an atherogenic molecule has raised the demand for reliable quantification methods in the clinical laboratory. The aim of this work is to compare commercial immunochemical assays. METHODS: We measured Lp(a) serum concentrations using six different assays, providing Lp(a) in mg/dl (Denka Seiken, Abbott Quantia, Beckman, Diasys 21FS, and Siemens N Latex) or in nmol/l (Roche TinaQuant, Diasys 21 FS) in 144 serum samples covering the clinically relevant range of Lp(a) concentrations. All assays relied on five-point calibrations using calibrators provided by the manufacturers. Apolipoprotein(a) phenotyping was performed by sodium dodecyl sulfate-agarose gel electrophoresis (SDS-agarose) followed by immunoblotting. RESULTS: Most bivariate correlation coefficients were greater than 0.90. Compared to an established IFCC-proposed reference material, the results of the different assays diverged from the target values (43.3 mg/dl or 96.6 nmol/l) by -8% (Siemens N Latex) and +22% (Abbott Quantia). Stratification of the samples into five groups with increasing Lp(a) concentrations and difference plots showed that the differences among assays were concentration-dependent. Some assays overestimated Lp(a) at high concentrations compared to the Denka Seiken assay. CONCLUSIONS: Current commercial immunological assays for measuring Lp(a) concentrations are differently calibrated. Their biases differ significantly across the clinically relevant concentration range in a non-linear manner. This is not conclusively explained by apolipoprotein (a) phenotypes. Further international efforts to harmonize assays for Lp(a) are needed.

Statin treatment increases lipoprotein(a) levels in subjects with low molecular weight apolipoprotein(a) phenotype.

BACKGROUND AND AIMS: We aimed to evaluate the effect of statin treatment initiation on lipoprotein(a) [Lp(a)] levels in patients with dyslipidemia, and the interactions with the apolipoprotein(a) [apo(a)] phenotype, LPA single nucleotide polymorphisms (SNPs) and change in LDL cholesterol. METHODS: The study population consisted of patients with dyslipidemia, predominantly familial hypercholesterolemia, who first initiated statin treatment (initiation group; n = 39) or were already on stable statin treatment for at least 4 months (control group; n = 42). Plasma Lp(a) levels were determined with a particle-enhanced immunoturbidimetric assay before and at least 2 months after start of statin treatment in individuals of the initiation group, and at two time points with an interval of at least 2 months in the control group. High and low molecular weight (HMW and LMW, respectively) apo(a) phenotype was determined by immunoblotting, and the common LPA SNPs rs10455872, rs3798220 and rs41272110 by Taqman assay. RESULTS: Plasma Lp(a) levels did not increase significantly in the initiation group (median 20.5 (IQR 10.9-80.7) to 23.3 (10.8-71.8) mg/dL; p = 0.09) nor in the control group (30.9 (IQR 9.2-147.0) to 31.7 (IQR 10.9-164.0) mg/dL; p = 0.61). In patients with the LMW apo(a) phenotype, Lp(a) levels increased significantly from 66.4 (IQR 23.5-148.3) to 97.4 (IQR 24.9-160.4) mg/dL (p = 0.026) in the initiation group, but not in the control group and not in patients characterized by the HMW apo(a) phenotype. Interactions with common LPA SNPs and change in LDL cholesterol were not significant. CONCLUSIONS: Statins affect Lp(a) levels differently in patients with dyslipidemia depending on the apo(a) phenotype. Statins increase Lp(a) levels exclusively in patients with the LMW apo(a) phenotype.

Temporary adhesion of the proseriate flatworm Minona ileanae.

Flatworms can very rapidly attach to and detach from many substrates. In the presented work, we analysed the adhesive system of the marine proseriate flatworm Minona ileanae. We used light-, scanning- and transmission electron microscopy to analyse the morphology of the adhesive organs, which are located at the ventral side of the tail-plate. We performed transcriptome sequencing and differential RNA-seq for the identification of tail-specific transcripts. Using in situ hybridization expression screening, we identified nine transcripts that were expressed in the cells of the adhesive organs. Knock-down of five of these transcripts by RNA interference led to a reduction of the animal's attachment capacity. Adhesive proteins in footprints were confirmed using mass spectrometry and antibody staining. Additionally, lectin labelling of footprints revealed the presence of several sugar moieties. Furthermore, we determined a genome size of about 560 Mb for M. ileanae. We demonstrated the potential of Oxford Nanopore sequencing of genomic DNA as a cost-effective tool for identifying the number of repeats within an adhesive protein and for combining transcripts that were fragments of larger genes. A better understanding of the molecules involved in flatworm bioadhesion can pave the way towards developing innovative glues with reversible adhesive properties. This article is part of the theme issue 'Transdisciplinary approaches to the study of adhesion and adhesives in biological systems'.

Validation of Next-Generation Sequencing of Entire Mitochondrial Genomes and the Diversity of Mitochondrial DNA Mutations in Oral Squamous Cell Carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is mainly caused by smoking and alcohol abuse and shows a five-year survival rate of ~50%. We aimed to explore the variation of somatic mitochondrial DNA (mtDNA) mutations in primary oral tumors, recurrences and metastases. METHODS: We performed an in-depth validation of mtDNA next-generation sequencing (NGS) on an Illumina HiSeq 2500 platform for its application to cancer tissues, with the goal to detect low-level heteroplasmies and to avoid artifacts. Therefore we genotyped the mitochondrial genome (16.6 kb) from 85 tissue samples (tumors, recurrences, resection edges, metastases and blood) collected from 28 prospectively recruited OSCC patients applying both Sanger sequencing and high-coverage NGS (~35,000 reads per base). RESULTS: We observed a strong correlation between Sanger sequencing and NGS in estimating the mixture ratio of heteroplasmies (r = 0.99; p<0.001). Non-synonymous heteroplasmic variants were enriched among cancerous tissues. The proportions of somatic and inherited variants in a given gene region were strongly correlated (r = 0.85; p<0.001). Half of the patients shared mutations between benign and cancerous tissue samples. Low level heteroplasmies (<10%) were more frequent in benign samples compared to tumor samples, where heteroplasmies >10% were predominant. Four out of six patients who developed a local tumor recurrence showed mutations in the recurrence that had also been observed in the primary tumor. Three out of five patients, who had tumor metastases in the lymph nodes of their necks, shared mtDNA mutations between primary tumors and lymph node metastases. The percentage of mutation heteroplasmy increased from the primary tumor to lymph node metastases. CONCLUSIONS: We conclude that Sanger sequencing is valid for heteroplasmy quantification for heteroplasmies ≥10% and that NGS is capable of reliably detecting and quantifying heteroplasmies down to the 1%-level. The finding of shared mutations between primary tumors, recurrences and metastasis indicates a clonal origin of malignant cells in oral cancer.

Polymorphisms in the gene regions of the adaptor complex LAMTOR2/LAMTOR3 and their association with breast cancer risk.

BACKGROUND: The late endosomal LAMTOR complex serves as a convergence point for both the RAF/MEK/ERK and the PI3K/AKT/mTOR pathways. Interestingly, both of these signalling cascades play a significant role in the aetiology of breast cancer. Our aim was to address the possible role of genetic polymorphisms in LAMTOR2 and LAMTOR3 as genetic risk factors for breast cancer. METHODOLOGY/RESULTS: We sequenced the exons and exon-intron boundaries of LAMTOR2 (p14) and LAMTOR3 (MP1) in 50 prospectively collected pairs of cancerous tissue and blood samples from breast cancer patients and compared their genetic variability. We found one single nucleotide polymorphism (SNP) in LAMTOR2 (rs7541) and two SNPs in LAMTOR3 (rs2298735 and rs148972953) in both tumour and blood samples, but no somatic mutations in cancerous tissues. In addition, we genotyped all three SNPs in 296 samples from the Risk Prediction of Breast Cancer Metastasis Study and found evidence of a genetic association between rs148972953 and oestrogen (ER) and progesterone receptor negative status (PR) (ER: OR = 3.60 (1.15-11.28); PR: OR = 4.27 (1.43-12.72)). However, when we additionally genotyped rs148972953 in the MARIE study including 2,715 breast cancer cases and 5,216 controls, we observed neither a difference in genotype frequencies between patients and controls nor was the SNP associated with ER or PR. Finally, all three SNPs were equally frequent in breast cancer samples and female participants (n = 640) of the population-based SAPHIR Study. CONCLUSIONS: The identified polymorphisms in LAMTOR2 and LAMTOR3 do not seem to play a relevant role in breast cancer. Our work does not exclude a role of other not yet identified SNPs or that the here annotated polymorphism may in fact play a relevant role in other diseases. Our results underscore the importance of replication in association studies.

Candidate gene sequencing of SLC11A2 and TMPRSS6 in a family with severe anaemia: common SNPs, rare haplotypes, no causative mutation.

BACKGROUND: Iron-refractory iron deficiency anaemia (IRIDA) is a rare disorder which was linked to mutations in two genes (SLC11A2 and TMPRSS6). Common polymorphisms within these genes were associated with serum iron levels. We identified a family of Serbian origin with asymptomatic non-consanguineous parents with three of four children presenting with IRIDA not responding to oral but to intravenous iron supplementation. After excluding all known causes responsible for iron deficiency anaemia we searched for mutations in SLC11A2 and TMPRSS6 that could explain the severe anaemia in these children. METHODOLOGY/RESULTS: We sequenced the exons and exon-intron boundaries of SLC11A2 and TMPRSS6 in all six family members. Thereby, we found seven known and fairly common SNPs, but no new mutation. We then genotyped these seven SNPs in the population-based SAPHIR study (n = 1,726) and performed genetic association analysis on iron and ferritin levels. Only two SNPs, which were top-hits from recent GWAS on iron and ferritin, exhibited an effect on iron and ferritin levels in SAPHIR. Six SAPHIR participants carrying the same TMPRSS6 genotypes and haplotype-pairs as one anaemic son showed lower ferritin and iron levels than the average. One individual exhibiting the joint SLC11A2/TMPRSS6 profile of the anaemic son had iron and ferritin levels lying below the 5(th) percentile of the population's iron and ferritin level distribution. We then checked the genotype constellations in the Nijmegen Biomedical Study (n = 1,832), but the profile of the anaemic son did not occur in this population. CONCLUSIONS: We cannot exclude a gene-gene interaction between SLC11A2 and TMPRSS6, but we can also not confirm it. As in this case candidate gene sequencing did not reveal causative rare mutations, the samples will be subjected to whole exome sequencing.