Patterns of genomic loss of heterozygosity predict homologous recombination repair defects in epithelial ovarian cancer.

BACKGROUND: Defects in BRCA1, BRCA2, and other members of the homologous recombination pathway have potential therapeutic relevance when used to support agents that introduce or exploit double-stranded DNA breaks. This study examines the association between homologous recombination defects and genomic patterns of loss of heterozygosity (LOH). METHODS: Ovarian tumours from two independent data sets were characterised for defects in BRCA1, BRCA2, and RAD51C, and LOH profiles were generated. Publically available data were downloaded for a third independent data set. The same analyses were performed on 57 cancer cell lines. RESULTS: Loss of heterozygosity regions of intermediate size were observed more frequently in tumours with defective BRCA1 or BRCA2 (P=10(-11)). The homologous recombination deficiency (HRD) score was defined as the number of these regions observed in a tumour sample. The association between HRD score and BRCA deficiency was validated in two independent ovarian cancer data sets (P=10(-5) and 10(-29)), and identified breast and pancreatic cell lines with BRCA defects. CONCLUSION: The HRD score appears capable of detecting homologous recombination defects regardless of aetiology or mechanism. This score could facilitate the use of PARP inhibitors and platinum in breast, ovarian, and other cancers.

Evidence for HTR1A and LHPP as interacting genetic risk factors in major depression.

The HTR1A -1019C>G genotype was associated with major depression in the Utah population. Linkage analysis on Utah pedigrees with strong family histories of major depression including only cases with the HTR1A -1019G allele revealed a linkage peak on chromosome 10 (maximum HLOD=4.4). Sequencing of all known genes in the linkage region revealed disease-segregating single-nucleotide polymorphisms (SNPs) in LHPP. LHPP SNPs were also associated with major depression in both Utah and Ashkenazi populations. Consistent with the linkage evidence, LHPP associations depended on HTR1A genotype. Lhpp or a product of a collinear brain-specific transcript, therefore, may interact with Htr1a in the pathogenesis of major depression.

Genomic DNA pooling for whole-genome association scans in complex disease: empirical demonstration of efficacy in rheumatoid arthritis.

A pragmatic approach that balances the benefit of a whole-genome association (WGA) experiment against the cost of individual genotyping is to use pooled genomic DNA samples. We aimed to determine the feasibility of this approach in a WGA scan in rheumatoid arthritis (RA) using the validated human leucocyte antigen (HLA) and PTPN22 associations as test loci. A total of 203 269 single-nucleotide polymorphisms (SNPs) on the Affymetrix 100K GeneChip and Illumina Infinium microarrays were examined. A new approach to the estimation of allele frequencies from Affymetrix hybridization intensities was developed involving weighting for quality signals from the probe quartets. SNPs were ranked by z-scores, combined from United Kingdom and New Zealand case-control cohorts. Within a 1.7 Mb HLA region, 33 of the 257 SNPs and at PTPN22, 21 of the 45 SNPs, were ranked within the top 100 associated SNPs genome wide. Within PTPN22, individual genotyping of SNP rs1343125 within MAGI3 confirmed association and provided some evidence for association independent of the PTPN22 620W variant (P=0.03). Our results emphasize the feasibility of using genomic DNA pooling for the detection of association with complex disease susceptibility alleles. The results also underscore the importance of the HLA and PTPN22 loci in RA aetiology.

Variants in Apaf-1 segregating with major depression promote apoptosome function.

APAF1, encoding the protein apoptosis protease activating factor 1 (Apaf-1), has recently been established as a chromosome 12 gene conferring predisposition to major depression in humans. The molecular phenotypes of Apaf-1 variants were determined by in vitro reconstruction of the apoptosome complex in which Apaf-1 activates caspase 9 and thus initiates a cascade of proteolytic events leading to apoptotic destruction of the cell. Cellular phenotypes were measured using a yeast heterologous expression assay in which human Apaf-1 and other proteins necessary to constitute a functional apoptotic pathway were overexpressed. Apaf-1 variants encoded by APAF1 alleles that segregate with major depression in families linked to chromosome 12 shared a common gain-of-function phenotype in both assay systems. In contrast, other Apaf-1 variants showed neutral or loss-of-function phenotypes. The depression-associated alleles thus have a common phenotype that is distinct from that of non-associated variants. This result suggests an etiologic role for enhanced apoptosis in major depression.

Analysis of missense variation in human BRCA1 in the context of interspecific sequence variation.

INTRODUCTION: Interpretation of results from mutation screening of tumour suppressor genes known to harbour high risk susceptibility mutations, such as APC, BRCA1, BRCA2, MLH1, MSH2, TP53, and PTEN, is becoming an increasingly important part of clinical practice. Interpretation of truncating mutations, gene rearrangements, and obvious splice junction mutations, is generally straightforward. However, classification of missense variants often presents a difficult problem. From a series of 20,000 full sequence tests of BRCA1 carried out at Myriad Genetic Laboratories, a total of 314 different missense changes and eight in-frame deletions were observed. Before this study, only 21 of these missense changes were classified as deleterious or suspected deleterious and 14 as neutral or of little clinical significance. METHODS: We have used a combination of a multiple sequence alignment of orthologous BRCA1 sequences and a measure of the chemical difference between the amino acids present at individual residues in the sequence alignment to classify missense variants and in-frame deletions detected during mutation screening of BRCA1. RESULTS: In the present analysis we were able to classify an additional 50 missense variants and two in-frame deletions as probably deleterious and 92 missense variants as probably neutral. Thus we have tentatively classified about 50% of the unclassified missense variants observed during clinical testing of BRCA1. DISCUSSION: An internal test of the analysis is consistent with our classification of the variants designated probably deleterious; however, we must stress that this classification is tentative and does not have sufficient independent confirmation to serve as a clinically applicable stand alone method.

Linkage of body mass index to chromosome 20 in Utah pedigrees.

Several linkage studies have hinted at the existence of an obesity predisposition locus on chromosome 20, but none of these studies has produced conclusive results. Therefore, we analyzed 48 genetic markers on chromosome 20 for linkage to severe obesity (BMI> or =35) in 103 extended Utah pedigrees (1,711 individuals), all of which had strong aggregation of severe obesity. A simple dominant model produced a maximum multipoint heterogeneity LOD score of 3.5 at D20S438 (55.1 cM). Two additional analyses were performed. First, a one-gene, two-mutation model (with one dominant mutation and one recessive mutation) increased the LOD score to 4.2. Second, a two-locus model (with one locus dominant and one recessive) generated a multipoint LOD score of 4.9. We conclude that one or more severe obesity predisposing genes lie within an interval of approx. 10 cM on chromosome 20. This study generated significant LOD scores which confirm suggestive linkage reports from previous studies. In addition, our analyses suggest that the predisposing gene(s) is localized very near the chromosome 20 centromere.

Identification of a common variant in the lipoprotein lipase gene in a large Utah kindred ascertained for coronary heart disease: the -93G/D9N variant predisposes to low HDL-C/high triglycerides.

Defects in the lipoprotein lipase (LPL) gene are associated with dyslipidemia in the general population. Several rare mutations in the gene, as well as two common coding region polymorphisms, D9N and N291S, exhibit deleterious effects on circulating lipid levels. Using a linkage-based approach, we have identified a large Utah kindred segregating the D9N variant in the LPL gene. The kindred was ascertained for premature coronary heart disease and was expanded based on familial dyslipidemia. A genomic scan identified a region of linkage including LPL, and mutation screening identified the segregating variant. In the kindred, the variant shows high penetrance for a hypoalphalipoproteinemia phenotype, but is also associated with hypertriglyceridemia and elevated insulin levels. The strength of linkage was dependent on the combination of phenotype definition and model parameters, favoring the use of a MOD score approach. Most other studies of LPL have proceeded by mutation screening of randomly chosen individuals or selected affected probands; this is the first example identifying a segregating LPL mutation using direct linkage.

A new nonparametric linkage statistic for mapping both qualitative and quantitative trait loci.

We describe an alternative nonparametric linkage (NPL) statistic to that of Kruglyak et al. [Am. J. Hum. Genet. 58:1347-63, 1996] that can be used with qualitative phenotypes, and is easily extended for use with quantitative phenotypes. We analyzed the Genetic Analysis Workshop 12 simulated isolated population data, replicate 1, using two phenotypes; affected status (AFF) a dichotomous phenotype and quantitative trait Q5, which was chosen since it was the most strongly associated with AFF. One false positive significant NPL score was observed for the AFF phenotype. For Q5 a single region on chromosome 1 reached genome-wide significance. The peak of this signal was for marker D01G137 at 135.1 cM with a quantitative trait locus (QTL)-NPL score of 4.19. The nearest marker to the true location of the major gene (MG5 at 137.1 cM) was D01G139 at position 135.8 cM, where the QTL-NPL score was still high at 4.08.

A robust multipoint linkage statistic (tlod) for mapping complex trait loci.

Classical parametric two-point linkage analysis is a powerful analysis tool, however there are clear disadvantages too, including the sensitivity to allele frequency mis-specification. Conversely, multipoint linkage analysis is not sensitive to allele frequency mis-specification, but it is sensitive to genetic model mis-specification. Göring and Terwilliger [Am J Hum Genet 66:1095-106, 2000] proposed a new robust multipoint statistic that increased the robustness of multipoint analyses. In this paper we have referred to this new statistic as the tlod. We applied this new statistic to the Genetic Analysis Workshop (GAW) 12 data using affected status (AFF) as the phenotype of interest. The heterogeneity tlod and two-point hlod scores correlated highly across the genome (p < 0.0001), as expected, but the het-tlod had a lower number false positives. In addition, the tlod analysis handled missing data better, as would be expected for a multipoint method. When one-third of the genotype data was removed (dead people) the tlod analysis was less affected than the two-point analysis. When tlod scores were compared with multipoint lod scores in true gene locations, the robustness of the tlod to model mis-specification was clearly evident. When the "best" replicate from the general population was analyzed, a borderline genome-wide significant two-point hlod result (3.6) was found 4 cM from MG6 and MG7 on chromosome 6. The heterogeneity tlod score was lower than the two-point hlod score (1.8), but greater than the heterogeneity multipoint lod score (0.4). However, when replicate 1 of the isolated population was analyzed none of the true gene locations were identified with either statistic.

Allelic association in large pedigrees.

We subjected the first replication of the simulated isolated population data set to a novel analysis for association between marker alleles and either disease phenotypes or quantitative variable. The analysis depends on being able to reliably reconstruct all haplotypes in the pedigree. This was achieved using the MCLINK blocked Gibbs sampling program. We observed a highly significant association between the variable Q5 and marker D01G138, and suggestive associations between the disease trait and markers D03G056 and D07G004.

What can disulfide bonds tell us about protein energetics, function and folding: simulations and bioninformatics analysis.

We study the impact of disulfide bonds on protein stability and folding. Using lattice model simulations, we show that formation of a disulfide bond stabilizes a protein to an extent that depends on the distance along the chain between linked cysteine residues. However, the impact of disulfide bonds on folding kinetics varies broadly, from acceleration when disulfides are introduced in or close to the folding nucleus, to slowing when disulfides are introduced outside the nucleus. Having established the effect of disulfide bonds on stability, we study the correlation between the number of disulfide bonds and the composition of certain amino acid classes with the goal to use it as a statistical probe into factors that contribute to stability of proteins. We find that the number of disulfides is negatively correlated with aliphatic hydrophobic but not aromatic content. It is surprising that we observe a strong correlation of disulfide content with polar (Q,S,T,N) amino acid content and a strong negative correlation with charged (E,D,K,R) content. These findings provide insights into factors that determine protein stability and principles of protein design as well as possible relations of disulfide bonds and protein function.

Evidence of linkage of familial hypoalphalipoproteinemia to a novel locus on chromosome 11q23.

Coronary heart disease (CHD) accounts for half of the 1 million deaths annually ascribed to cardiovascular disease and for almost all of the 1.5 million acute myocardial infarctions. Within families affected by early and apparently heritable CHD, dyslipidemias have a much higher prevalence than in the general population; 20%-30% of early familial CHD has been ascribed to primary hypoalphalipoproteinemia (low HDL-C). This study assesses the evidence for linkage of low HDL-C to chromosomal region 11q23 in 105 large Utah pedigrees ascertained with closely related clusters of early CHD and expanded on the basis of dyslipidemia. Linkage analysis was performed by use of 22 STRP markers in a 55-cM region of chromosome 11. Two-point analysis based on a general, dominant-phenotype model yielded LODs of 2.9 for full pedigrees and 3.5 for 167 four-generation split pedigrees. To define a localization region, model optimization was performed using the heterogeneity, multipoint LOD score (mpHLOD). This linkage defines a region on 11q23.3 that is approximately 10 cM distal to-and apparently distinct from-the ApoAI/CIII/AIV gene cluster and thus represents a putative novel localization for the low HDL-C phenotype.

Factors that affect the folding ability of proteins.

The folding ability of a heteropolymer model for proteins subject to Monte Carlo dynamics on a simple cubic lattice is shown to be strongly correlated with the stability of the native state. We consider a number of estimates of the stability that can be determined without simulation, including the energy gap between the native state and the structurally dissimilar part of the spectrum (Z score) and, for sequences with fully compact native states, the gap in energy between the native and first excited fully compact states. These estimates are found to be more robust predictors of folding ability than a parameter sigma that requires simulation for its evaluation: sigma = 1 - Tf/Ttheta, where Tf is the temperature at which the fluctuation of an order parameter is at its maximum and Ttheta is the temperature at which the specific heat is at its maximum. We show that the interpretation of Ttheta as the collapse transition temperature is not correct in general and that the correlation between sigma and the folding ability arises from the fact that sigma is related to the energy gap (Z score).

How evolution makes proteins fold quickly.

Sequences of fast-folding model proteins (48 residues long on a cubic lattice) were generated by an evolution-like selection toward fast folding. We find that fast-folding proteins exhibit a specific folding mechanism in which all transition state conformations share a smaller subset of common contacts (folding nucleus). Acceleration of folding was accompanied by dramatic strengthening of interactions in the folding nucleus whereas average energy of nonnucleus interactions remained largely unchanged. Furthermore, the residues involved in the nucleus are the most conserved ones within families of evolved sequences. Our results imply that for each protein structure there is a small number of conserved positions that are key determinants of fast folding into that structure. This conjecture was tested on two protein superfamilies: the first having the classical monophosphate binding fold (CMBF; 98 families) and the second having type-III repeat fold (47 families). For each superfamily, we discovered a few positions that exhibit very strong and statistically significant "conservatism of conservatism"-amino acids in those positions are conserved within every family whereas the actual types of amino acids varied from family to family. Those amino acids are in spatial contact with each other. The experimental data of Serrano and coworkers [Lopez-Hernandez, E. & Serrano, L. (1996) Fold. Des. (London) 1, 43-55]. for one of the proteins of the CMBF superfamily (CheY) show that residues identified this way indeed belong to the folding nucleus. Further analysis revealed deep connections between nucleation in CMBF proteins and their function.

A protein engineering analysis of the transition state for protein folding: simulation in the lattice model.

BACKGROUND: Protein engineering has been used extensively to evaluate the properties of transition states in protein folding. Although the method has proved useful, its limitations and the details of interpretation of the obtained results remain largely unexplored. RESULTS: Lattice model simulations are used to test and verify the protein engineering analysis of the transition state in protein folding. It is shown that in some cases - but not always - this method is able to determine the transition state with reasonable accuracy. Limitations of protein engineering are revealed and analyzed. In particular, the change in non-native interactions as a result of mutations is shown to influence the results of the protein engineering analysis. Furthermore, the temperature dependencies of phi values (which are a measure of the participation of a residue in the transition state) and the character of the transition state ensemble are studied. It is shown that as a general trend phi values decrease when the temperature decreases, a finding consistent with recent experimental results. Our analysis suggests that this trend results primarily from the formation of some contacts (native and non-native) in the unfolded state at a lower temperature, when the barrier for folding is energetic. CONCLUSIONS: Our analysis helps to interpret the results of protein engineering and allows observed φ values to be directly related to structural features of the unfolded state, the transition state and the native state.

Theory of kinetic partitioning in protein folding with possible applications to prions.

This study focuses of the phenomenon of kinetic partitioning when a polypeptide chain has two ground-state conformations, one of which is kinetically more reachable than the other. We designed sequences for lattice model proteins with two different conformations of equal energy corresponding to the global energy minimum. Folding simulations revealed that one of these conformations was indeed much more kinetically accessible than the other. We found that the number and strength of local contacts in the ground-state conformation are the major factors that determine which conformation is reached faster; the greater the number of local contacts, the more kinetically reachable a conformation is. We present simple statistical-mechanical arguments to explain these findings. Our results may be relevant in explaining the phenomenology of such proteins as human plasminogen activator inhibitor-1 (PAI-1), photosystem II, and prions.

Computer simulations of prebiotic evolution.

This paper is a review of our previous work on the field of possible ways of prebiotic evolution. We propose an algorithm providing sequences of model proteins with rapid folding into a given native conformation. Thermodynamical analysis shows that the increase in speed is matched by an increase in stability: the evolved sequences are much more stable in their native conformation than the initial random sequence. We discuss a possible origin of the first biopolymers, having stable unique structure. We suggest that at the prebiotic stage of evolution, long organic polymers had to be compact in order to avoid hydrolysis and had to be soluble and thus must not be exceedingly hydrophobic. We present an algorithm that generates such sequences of model proteins. The evolved sequences turn out to have a stable unique structure, into which they quickly fold. This result illustrates the idea that the unique three-dimensional native structure of first biopolymers could have evolved as a side effect of a nonspecific physico-chemical factors acting at the prebiotic stage of evolution.

How the first biopolymers could have evolved.

In this work, we discuss a possible origin of the first biopolymers with stable unique structures. We suggest that at the prebiotic stage of evolution, long organic polymers had to be compact to avoid hydrolysis and had to be soluble and thus must not be exceedingly hydrophobic. We present an algorithm that generates such sequences for model proteins. The evolved sequences turn out to have a stable unique structure, into which they quickly fold. This result illustrates the idea that the unique three-dimensional native structures of first biopolymers could have evolved as a side effect of nonspecific physicochemical factors acting at the prebiotic stage of evolution.

Conserved residues and the mechanism of protein folding.

Experimental and simulation studies show that small monomeric proteins fold in one kinetic step, which entails overcoming the free-energy barrier between the unfolded and the native protein through a transition state. Two models of transition state formation have been proposed: a 'nonspecific' one in which it depends on the formation of a sufficient number of native-like contacts regardless of what amino acids are involved, and a 'specific' one, in which it depends on formation of a specific subset of the native structure (a folding nucleus). The latter requires that some amino acids form most of their contacts in the transition state, whereas others only do so on reaching the native conformation. If so, mutations affecting the stability of the transition state nucleus should have a greater effect on the folding kinetics than mutations elsewhere, and the residues involved should be evolutionarily conserved. Lattice-model simulations and experiments suggest that such mutations exist. Here we present a method for determining the folding nucleus of a protein with known structure with two-state folding kinetics. This method is based on the alignment of many sequences designed to fold into the native conformation of a protein to identify the positions where amino acids are most conserved in designed sequences. The method is applied to chymotrypsin inhibitor 2 (CI2), a protein whose transition state has been previously studied by protein engineering. The involvement of residues in folding nucleus of CI2 is clearly correlated with their conservation in design, and the residues forming the nucleus are highly conserved in 23 natural sequences homologous to CI2.

Simulations of chaperone-assisted folding.

We investigated a chaperone mechanism of protein folding using a 36-mer model on a cubic lattice. The mechanism simulates folding, which proceeds with repetitive cycles of binding, unfolding, and releasing of misfolded metastable states. We measured the yield enhancement due to this mechanism for sequences selected by evolutionary design and showed that the binding and releasing mechanism is efficient for the yield enhancement of folding for sequences that are poorly designed, i.e., where selection is not adequately strong. From this it follows that the chaperone mechanism can be considered as the evolutionary alternative to compensate for poor sequence design. On the other hand, random sequences show a decrease in yield and no effect on the total mean first passage time when the proposed chaperone mechanism is implemented, thus implying that sequence optimization is a necessary condition for the efficiency of the proposed mechanism. We qualitatively reproduced experimental results for folding in the presence of GroEL/GroES, fit our results with the aid of a double-exponential model of folding kinetics, and characterized the conditions under which this mechanism of chaperone action affects folding.