Genetic variation among individual humans occurs on many different scales, ranging from gross alterations in the human karyotype to single nucleotide changes. Here we explore variation on an intermediate scale--particularly insertions, deletions and inversions affecting from a few thousand to a few million base pairs. We employed a clone-based method to interrogate this intermediate structural variation in eight individuals of diverse geographic ancestry. Our analysis provides a comprehensive overview of the normal pattern of structural variation present in these genomes, refining the location of 1,695 structural variants. We find that 50% were seen in more than one individual and that nearly half lay outside regions of the genome previously described as structurally variant. We discover 525 new insertion sequences that are not present in the human reference genome and show that many of these are variable in copy number between individuals. Complete sequencing of 261 structural variants reveals considerable locus complexity and provides insights into the different mutational processes that have shaped the human genome. These data provide the first high-resolution sequence map of human structural variation--a standard for genotyping platforms and a prelude to future individual genome sequencing projects.

/pdf/mapping-and-sequencing-of-structural-variation-from-eight-29e9yonfd0.pdf

Mapping and sequencing of structural variation from eight human genomes

Inversions, deletions and insertions are important mediators of disease and disease susceptibility. We systematically compared the human genome reference sequence with a second genome (represented by fosmid paired-end sequences) to detect intermediate-sized structural variants >8 kb in length. We identified 297 sites of structural variation: 139 insertions, 102 deletions and 56 inversion breakpoints. Using combined literature, sequence and experimental analyses, we validated 112 of the structural variants, including several that are of biomedical relevance. These data provide a fine-scale structural variation map of the human genome and the requisite sequence precision for subsequent genetic studies of human disease.

Fine-scale structural variation of the human genome.

The human genome contains numerous blocks of highly homologous duplicated sequence. This higher-order architecture provides a substrate for recombination and recurrent chromosomal rearrangement associated with genomic disease. However, an assessment of the role of segmental duplications in normal variation has not yet been made. On the basis of the duplication architecture of the human genome, we defined a set of 130 potential rearrangement hotspots and constructed a targeted bacterial artificial chromosome (BAC) microarray (with 2,194 BACs) to assess copy-number variation in these regions by array comparative genomic hybridization. Using our segmental duplication BAC microarray, we screened a panel of 47 normal individuals, who represented populations from four continents, and we identified 119 regions of copy-number polymorphism (CNP), 73 of which were previously unreported. We observed an equal frequency of duplications and deletions, as well as a 4-fold enrichment of CNPs within hotspot regions, compared with control BACs (P 4-fold within regions of CNP. Almost without exception, CNPs were not confined to a single population, suggesting that these either are recurrent events, having occurred independently in multiple founders, or were present in early human populations. Our study demonstrates that segmental duplications define hotspots of chromosomal rearrangement, likely acting as mediators of normal variation as well as genomic disease, and it suggests that the consideration of genomic architecture can significantly improve the ascertainment of large-scale rearrangements. Our specialized segmental duplication BAC microarray and associated database of structural polymorphisms will provide an important resource for the future characterization of human genomic disorders.

https://www.cell.com/ajhg/pdf/S0002-9297(07)60903-3.pdf

Segmental Duplications and Copy-Number Variation in the Human Genome

Plasma lipoprotein(a) [Lp(a)], a low density lipoprotein particle with an attached apolipoprotein(a) [apo(a)], varies widely in concentration between individuals. These concentration differences are heritable and inversely related to the number of kringle 4 repeats in the apo(a) gene. To define the genetic determinants of plasma Lp(a) levels, plasma Lp(a) concentrations and apo(a) genotypes were examined in 48 nuclear Caucasian families. Apo(a) genotypes were determined using a newly developed pulsed-field gel electrophoresis method which distinguished 19 different genotypes at the apo(a) locus. The apo(a) gene itself was found to account for virtually all the genetic variability in plasma Lp(a) levels. This conclusion was reached by analyzing plasma Lp(a) levels in siblings who shared zero, one, or two apo(a) genes that were identical by descent (ibd). Siblings with both apo(a) alleles ibd (n = 72) have strikingly similar plasma Lp(a) levels (r = 0.95), whereas those who shared no apo(a) alleles (n = 52), had dissimilar concentrations (r = -0.23). The apo(a) gene was estimated to be responsible for 91% of the variance of plasma Lp(a) concentration. The number of kringle 4 repeats in the apo(a) gene accounted for 69% of the variation, and yet to be defined cis-acting sequences at the apo(a) locus accounted for the remaining 22% of the inter-individual variation in plasma Lp(a) levels. During the course of these studies we observed the de novo generation of a new apo(a) allele, an event that occurred once in 376 meioses.

/pdf/apolipoprotein-a-gene-accounts-for-greater-than-90-of-the-5cix0arhtf.pdf

Apolipoprotein(a) gene accounts for greater than 90 % of the variation in plasma lipoprotein(a) concentrations

Subjects with diabetes have a greatly increased risk of CHD, which is only partially related to their elevated glucose. Other factors such as insulin resistance and dyslipidemia are likely to be important. The type of dyslipidemia that is most characteristic of type 2 diabetic subjects is elevated triglycerides and decreased HDL cholesterol levels, although all lipoproteins have compositional abnormalities. Surprisingly few good prospective studies of lipoprotein levels in relation to CHD have been done in diabetic subjects. Available studies suggest that low HDL cholesterol may be the most important risk factor for CHD in observational studies. In studies in which total cholesterol and triglyceride were done, cholesterol and triglycerides were risk factors for CHD, although triglycerides were often a stronger predictor. However, the strength of triglyceride as a risk factor for CHD may depend partially on its association with other variables (e.g., hypertension, plasminogen activator inhibitor 1 [PAI-1], etc.). In clinical trials in diabetic subjects, LDL reduction with statins has led to significant reductions in CHD incidence. In addition, overall mortality was reduced with statin therapy, although the results were not statistically significant. Gemfibrozil has led to reductions in CHD incidence in diabetic subjects, although the results were not statistically significant perhaps because of low sample size. Regarding lipoproteins and CHD risk in diabetic patients, the very positive results of statin trials point to LDL cholesterol being more important than previous realized. Apparently, having a borderline high LDL cholesterol (between 130 and 160 mg/dl) in a diabetic patient is equivalent to a much higher LDL cholesterol in terms of CHD risk for a nondiabetic subject. Therefore, the primary target of therapy in diabetic patients is lowering LDL cholesterol (or possibly, non-HDL cholesterol). Statins are the preferred pharmacological agent in this situation. Once LDL cholesterol levels have been lowered, attention can be given to treatment of residual hypertriglyceridemia and low HDL. The goal here is weight reduction and increased exercise. However, for selected patients, combining a fibric acid (or low-dose nicotinic acid) with a statin also can be considered. Reduction of LDL levels should take priority over reduction of triglycerides in combined hyperlipidemia because of the proven safety of the statin class of drugs as well as greater reduction in CHD incidence.

Management of Dyslipidemia in Adults With Diabetes

Lipoprotein(a) [Lp(a)] is a cholesterol-rich lipoprotein that is distinguished by its content of a glycoprotein called apolipoprotein(a) [apo(a)]. Apo(a) varies in size among individuals owing to different numbers of cysteine-rich sequences that are homologous to kringle 4 of plasminogen. The genetic basis for this variation is not understood at the genomic level. In this study we used pulsed-field gel electrophoresis and genomic blotting to identify a highly polymorphic restriction fragment from the apo(a) gene. The fragment contains multiple tandem repeats of a kringle 4-encoding sequence and varies in length from 48 to 190 kb depending on the number of kringle 4-encoding sequences. A total of 19 different alleles were identified among 102 unrelated Caucasian Americans. 94% of individuals studied had two different alleles which could be distinguished by size on pulsed-field gel electrophoresis. The degree of size heterogeneity was much greater than had been previously appreciated based on the analysis of the apparent molecular mass of the protein. The size of the apo(a) gene correlated directly with the size of the apo(a) protein, and inversely with the concentration of Lp(a) in plasma. Segregation analysis of the apo(a) gene was performed in families; siblings with identical apo(a) genotypes had similar plasma levels of Lp(a). These results suggest that in the normal population, the level of plasma Lp(a) is largely determined by alleles at the apo(a) locus.

/pdf/molecular-basis-of-apolipoprotein-a-isoform-size-iao0wsvtlf.pdf

Molecular basis of apolipoprotein (a) isoform size heterogeneity as revealed by pulsed-field gel electrophoresis.

Lipoprotein(a) [Lp(a)], an atherogenic lipoprotein, consists of an LDL particle complexed with a large glycoprotein, apolipoprotein(a) [apo(a)]. Apo(a) is one of the most polymorphic expressed sequences in the human genome. The glycoprotein varies in size over a approximately 500 kDa range due to length polymorphisms in the apo(a) gene. The region of the apo(a) gene responsible for this size polymorphism was cloned and characterized. Apo(a) alleles of different length were found to contain varying numbers of a subset of a tandemly-repeated, 5.5 kilobase, kringle IV-encoding sequences. A total of 34 apo(a) alleles and glycoproteins could be distinguished using pulsed-field gel electrophoresis and genomic blotting and immunoblotting. Molecular analysis of a newly-generated apo(a) allele of different length suggests that the high degree of length polymorphism is in part due to recombination between sister chromatids.

Molecular definition of the extreme size polymorphism in apolipoprotein(a)

https://www.amjmed.com/article/0002-9343(94)90087-6/pdf

Influence of intranasal cocaine on plasma constituents associated with endogenous thrombosis and thrombolysis

▪Objective:To determine the influence of diabetes control on serum lipoprotein (a) concentrations. ▪Setting:Diabetes clinic of a large metropolitan public hospital, with primary- and secon...

Carolin Lackner

Papers

Molecular basis of apolipoprotein (a) isoform size heterogeneity as revealed by pulsed-field gel electrophoresis.

Molecular definition of the extreme size polymorphism in apolipoprotein(a)

Influence of intranasal cocaine on plasma constituents associated with endogenous thrombosis and thrombolysis

Lipoprotein (a) Levels in Diabetes Mellitus: Relationship to Metabolic Control