Discovery of potential new gene variants and inflammatory cytokine associations with fibromyalgia syndrome by whole exome sequencing.

Fibromyalgia syndrome (FMS) is a chronic musculoskeletal pain disorder affecting 2% to 5% of the general population. Both genetic and environmental factors may be involved. To ascertain in an unbiased manner which genes play a role in the disorder, we performed complete exome sequencing on a subset of FMS patients. Out of 150 nuclear families (trios) DNA from 19 probands was subjected to complete exome sequencing. Since >80,000 SNPs were found per proband, the data were further filtered, including analysis of those with stop codons, a rare frequency (<2.5%) in the 1000 Genomes database, and presence in at least 2/19 probands sequenced. Two nonsense mutations, W32X in C11orf40 and Q100X in ZNF77 among 150 FMS trios had a significantly elevated frequency of transmission to affected probands (p = 0.026 and p = 0.032, respectively) and were present in a subset of 13% and 11% of FMS patients, respectively. Among 9 patients bearing more than one of the variants we have described, 4 had onset of symptoms between the ages of 10 and 18. The subset with the C11orf40 mutation had elevated plasma levels of the inflammatory cytokines, MCP-1 and IP-10, compared with unaffected controls or FMS patients with the wild-type allele. Similarly, patients with the ZNF77 mutation have elevated levels of the inflammatory cytokine, IL-12, compared with controls or patients with the wild type allele. Our results strongly implicate an inflammatory basis for FMS, as well as specific cytokine dysregulation, in at least 35% of our FMS cohort.

Colorectal cancer linkage on chromosomes 4q21, 8q13, 12q24, and 15q22.

A substantial proportion of familial colorectal cancer (CRC) is not a consequence of known susceptibility loci, such as mismatch repair (MMR) genes, supporting the existence of additional loci. To identify novel CRC loci, we conducted a genome-wide linkage scan in 356 white families with no evidence of defective MMR (i.e., no loss of tumor expression of MMR proteins, no microsatellite instability (MSI)-high tumors, or no evidence of linkage to MMR genes). Families were ascertained via the Colon Cancer Family Registry multi-site NCI-supported consortium (Colon CFR), the City of Hope Comprehensive Cancer Center, and Memorial University of Newfoundland. A total of 1,612 individuals (average 5.0 per family including 2.2 affected) were genotyped using genome-wide single nucleotide polymorphism linkage arrays; parametric and non-parametric linkage analysis used MERLIN in a priori-defined family groups. Five lod scores greater than 3.0 were observed assuming heterogeneity. The greatest were among families with mean age of diagnosis less than 50 years at 4q21.1 (dominant HLOD = 4.51, α = 0.84, 145.40 cM, rs10518142) and among all families at 12q24.32 (dominant HLOD = 3.60, α = 0.48, 285.15 cM, rs952093). Among families with four or more affected individuals and among clinic-based families, a common peak was observed at 15q22.31 (101.40 cM, rs1477798; dominant HLOD = 3.07, α = 0.29; dominant HLOD = 3.03, α = 0.32, respectively). Analysis of families with only two affected individuals yielded a peak at 8q13.2 (recessive HLOD = 3.02, α = 0.51, 132.52 cM, rs1319036). These previously unreported linkage peaks demonstrate the continued utility of family-based data in complex traits and suggest that new CRC risk alleles remain to be elucidated.

Three ways of combining genotyping and resequencing in case-control association studies.

We describe three statistical results that we have found to be useful in case-control genetic association testing. All three involve combining the discovery of novel genetic variants, usually by sequencing, with genotyping methods that recognize previously discovered variants. We first consider expanding the list of known variants by concentrating variant-discovery in cases. Although the naive inclusion of cases-only sequencing data would create a bias, we show that some sequencing data may be retained, even if controls are not sequenced. Furthermore, for alleles of intermediate frequency, cases-only sequencing with bias-correction entails little if any loss of power, compared to dividing the same sequencing effort among cases and controls. Secondly, we investigate more strongly focused variant discovery to obtain a greater enrichment for disease-related variants. We show how case status, family history, and marker sharing enrich the discovery set by increments that are multiplicative with penetrance, enabling the preferential discovery of high-penetrance variants. A third result applies when sequencing is the primary means of counting alleles in both cases and controls, but a supplementary pooled genotyping sample is used to identify the variants that are very rare. We show that this raises no validity issues, and we evaluate a less expensive and more adaptive approach to judging rarity, based on group-specific variants. We demonstrate the important and unusual caveat that this method requires equal sample sizes for validity. These three results can be used to more efficiently detect the association of rare genetic variants with disease.

Missense mutations in the MEFV gene are associated with fibromyalgia syndrome and correlate with elevated IL-1beta plasma levels.

BACKGROUND: Fibromyalgia syndrome (FMS), a common, chronic, widespread musculoskeletal pain disorder found in 2% of the general population and with a preponderance of 85% in females, has both genetic and environmental contributions. Patients and their parents have high plasma levels of the chemokines MCP-1 and eotaxin, providing evidence for both a genetic and an immunological/inflammatory origin for the syndrome (Zhang et al., 2008, Exp. Biol. Med. 233: 1171-1180). METHODS AND FINDINGS: In a search for a candidate gene affecting inflammatory pathways, among five screened in our patient samples (100 probands with FMS and their parents), we found 10 rare and one common alleles for MEFV, a gene in which various compound heterozygous mutations lead to Familial Mediterranean Fever (FMF). A total of 2.63 megabases of genomic sequence of the MEFV gene were scanned by direct sequencing. The collection of rare missense mutations (all heterozygotes and tested in the aggregate) had a significant elevated frequency of transmission to affecteds (p = 0.0085, one-sided, exact binomial test). Our data provide evidence that rare missense variants of the MEFV gene are, collectively, associated with risk of FMS and are present in a subset of 15% of FMS patients. This subset had, on average, high levels of plasma IL-1beta (p = 0.019) compared to FMS patients without rare variants, unaffected family members with or without rare variants, and unrelated controls of unknown genotype. IL-1beta is a cytokine associated with the function of the MEFV gene and thought to be responsible for its symptoms of fever and muscle aches. CONCLUSIONS: Since misregulation of IL-1beta expression has been predicted for patients with mutations in the MEFV gene, we conclude that patients heterozygous for rare missense variants of this gene may be predisposed to FMS, possibly triggered by environmental factors.

A risk variant in an miR-125b binding site in BMPR1B is associated with breast cancer pathogenesis.

MicroRNAs regulate diverse cellular processes and play an integral role in cancer pathogenesis. Genomic variation within miRNA target sites may therefore be important sources for genetic differences in cancer risk. To investigate this possibility, we mapped HapMap single nucleotide polymorphisms (SNP) to putative miRNA recognition sites within genes dysregulated in estrogen receptor-stratified breast tumors and used local linkage disequilibrium patterns to identify high-ranking SNPs in the Cancer Genetic Markers of Susceptibility (CGEMS) breast cancer genome-wide association study for further testing. Two SNPs, rs1970801 and rs11097457, scoring in the top 100 from the CGEMS study, were in strong linkage disequilibrium with rs1434536, an SNP that resides within a miR-125b target site in the 3' untranslated region of the bone morphogenic receptor type 1B (BMPR1B) gene encoding a transmembrane serine/threonine kinase. We validated the CGEMS association findings for rs1970801 in an independent cohort of admixture-corrected cases identified from families with multiple case histories. Subsequent association testing of rs1434536 for these cases and CGEMS controls with imputed genotypes supported the association. Furthermore, luciferase reporter assays and overexpression of miR-125b-mimics combined with quantitative reverse transcription-PCR showed that BMPR1B transcript is a direct target of miR-125b and that miR-125b differentially regulates the C and T alleles of rs1434536. These results suggest that allele-specific regulation of BMPR1B by miR-125b explains the observed disease risk. Our approach is general and can help identify and explain the mechanisms behind disease association for alleles that affect miRNA regulation.

Evolutionary signatures of common human cis-regulatory haplotypes.

Variation in gene expression may give rise to a significant fraction of inter-individual phenotypic variation. Studies searching for the underlying genetic controls for such variation have been conducted in model organisms and humans in recent years. In our previous effort of assessing conserved underlying haplotype patterns across ethnic populations, we constructed common haplotypes using SNPs having conserved linkage disequilibrium (LD) across ethnic populations. These common haplotypes cluster into a simple evolutionary structure based on their frequencies, defining only up to three conserved clusters termed 'haplotype frameworks'. One intriguing preliminary finding was that a significant portion of reported variants strongly associated with cis-regulation tags these globally conserved haplotype frameworks. Here we expand the investigation by collecting genes showing stringently determined cis-association between genotypes and expression phenotypes from major studies. We conducted phylogenetic analysis of current major haplotypes along with the corresponding haplotypes derived from chimpanzee reference sequences. Our analysis reveals that, for the vast majority of such cis-regulatory genes, the tagging SNPs showing the strongest association also tag the haplotype lineages directly separated from ancestry, inferred from either chimpanzee reference sequences or the allele frequency-derived haplotype frameworks, suggesting that the differentially expressed phenotypes were evolved relatively early in human history. Such evolutionary signatures provide keys for a more effective identification of globally-conserved candidate regulatory haplotypes across human genes in future epidemiologic and pharmacogenetic studies.

Strong signature of natural selection within an FHIT intron implicated in prostate cancer risk.

Previously, a candidate gene linkage approach on brother pairs affected with prostate cancer identified a locus of prostate cancer susceptibility at D3S1234 within the fragile histidine triad gene (FHIT), a tumor suppressor that induces apoptosis. Subsequent association tests on 16 SNPs spanning approximately 381 kb surrounding D3S1234 in Americans of European descent revealed significant evidence of association for a single SNP within intron 5 of FHIT. In the current study, re-sequencing and genotyping within a 28.5 kb region surrounding this SNP further delineated the association with prostate cancer risk to a 15 kb region. Multiple SNPs in sequences under evolutionary constraint within intron 5 of FHIT defined several related haplotypes with an increased risk of prostate cancer in European-Americans. Strong associations were detected for a risk haplotype defined by SNPs 138543, 142413, and 152494 in all cases (Pearson's chi(2) = 12.34, df 1, P = 0.00045) and for the homozygous risk haplotype defined by SNPs 144716, 142413, and 148444 in cases that shared 2 alleles identical by descent with their affected brothers (Pearson's chi(2) = 11.50, df 1, P = 0.00070). In addition to highly conserved sequences encompassing SNPs 148444 and 152413, population studies revealed strong signatures of natural selection for a 1 kb window covering the SNP 144716 in two human populations, the European American (pi = 0.0072, Tajima's D = 3.31, 14 SNPs) and the Japanese (pi = 0.0049, Fay & Wu's H = 8.05, 14 SNPs), as well as in chimpanzees (Fay & Wu's H = 8.62, 12 SNPs). These results strongly support the involvement of the FHIT intronic region in an increased risk of prostate cancer.

Matchmaking, metrics and money: a pathway to progress in translational research.

In the 24 years since the founding of BioEssays, the level of translational research, as well as the expectations for its success, have burgeoned. Based on our analysis of current and projected US efforts to establish effective centers of translational research, our own institutional experience and discussions with academic research center leaders and institutional research executives, we have arrived at several critical conclusions about how best to foster disease-based research on the institutional, national and international level, what summary statistics may best serve as a measurement of successful practice, roughly how much more money will be required to fund the ongoing venture in the US and how to organize and promote this vision.

SUMO conjugation to the matrix attachment region-binding protein, special AT-rich sequence-binding protein-1 (SATB1), targets SATB1 to promyelocytic nuclear bodies where it undergoes caspase cleavage.

SATB1 (special AT-rich sequence-binding protein-1) provides a key link between DNA loop organization, chromatin modification/remodeling, and association of transcription factors at matrix attachment regions (MARs). To investigate the role of SATB1 in cellular events, we performed a yeast two-hybrid screen that identified SUMO-1, Ubc9, and protein inhibitor of activated STAT (PIAS) family members as SATB1 interaction partners. These proteins, working in concert, enhanced SUMO conjugation to lysine-744 of SATB1. Overexpression of SUMO or PIAS in Jurkat cells, which express high levels of endogenous SATB1, exhibited enhanced caspase cleavage of this MAR-associating protein. Sumoylation-deficient SATB1 (SATB1(K744R)) failed to display the characteristic caspase cleavage pattern; however, fusion of SUMO in-frame to SATB1(K744R) restored cleavage. A SUMO-independent interaction of inactive caspase-6 and SATB1 was noted. A subset of total cellular SATB1 localized into promyelocytic leukemia nuclear bodies where enhanced SATB1 cleavage was detected subsequent to caspase activation. These results reveal a novel sumoylation-directed caspase cleavage of this key regulatory molecule. The role of regulated proteolysis of SATB1 may be to control transcription in immune cells during normal cell functions or to assist in efficient and rapid clearance of nonfunctional or potentially damaging immune cells.

Identification and functional significance of SNPs underlying conserved haplotype frameworks across ethnic populations.

BACKGROUND: The study of genetic variation will promote our understanding of the differential predisposition to common diseases and variation in drug responses of individuals and ethnic populations. Such genetic variation is intrinsically structured into blocks of haplotypes in populations. Therefore, a comprehensive haplotype map based on the most abundant form of genetic variation, single nucleotide polymorphisms, will be useful. At the present time, however, our knowledge of the similarities and differences of haplotype structure among different ancestral populations is still inadequate. METHODS: To determine whether common underlying haplotype patterns existed across ethnic populations, we analyzed data derived from African and European Americans for twenty-two genes spanning a total of 516 kb and the HapMap ENCODE data across 500 kb on chromosome 2p16.3 from three major world populations. RESULTS AND CONCLUSIONS: We observed that strong pairwise linkage disequilibrium (LD) between SNPs selected from populations having African ancestry was highly conserved across other non-African populations. Common haplotypes described by these LD-selected SNPs demonstrated a simple evolutionary structure with up to three major frameworks, which were likely ancestral backgrounds upon which more recent mutations have been superimposed. Also, haplotype block boundaries defined in populations having African ancestry revealed completely concordant recombinant haplotypes across all populations, providing a consistent definition of block structure. Finally, a large fraction of regulatory polymorphisms described in the literature appeared to tag these conserved haplotype frameworks, strongly suggesting their significance for disease association and pharmacogenetic studies.

Genetic linkage of prostate cancer risk to the chromosome 3 region bearing FHIT.

We conducted linkage analysis of 80 candidate genes in 201 brother pairs affected with prostatic adenocarcinoma. Markers representing two adjacent candidate genes on chromosome 3p, CDC25A and FHIT, showed suggestive evidence for linkage with single-point identity-by-descent allele-sharing statistics. Fine-structure multipoint linkage analysis yielded a maximum LOD score of 3.17 (P = 0.00007) at D3S1234 within FHIT intron 5. For a subgroup of 38 families in which three or more affected brothers were reported, the LOD score was 3.83 (P = 0.00001). Further analysis reported herein suggested a recessive mode of inheritance. Association testing of 16 single nucleotide polymorphisms (SNP) spanning a 381-kb interval surrounding D3S1234 in 202 cases of European descent with 143 matched, unrelated controls revealed significant evidence for association between case status and the A allele of single nucleotide polymorphism rs760317, located within intron 5 of FHIT (Pearson's chi(2) = 8.54, df = 1, P = 0.0035). Our results strongly suggest involvement of germline variations of FHIT in prostate cancer risk.

Identification of a SUMO-binding motif that recognizes SUMO-modified proteins.

Posttranslational modification by the ubiquitin homologue, small ubiquitin-like modifier 1 (SUMO-1), has been established as an important regulatory mechanism. However, in most cases it is not clear how sumoylation regulates various cellular functions. Emerging evidence suggests that sumoylation may play a general role in regulating protein-protein interactions, as shown in RanBP2/Nup358 and RanGAP1 interaction. In this study, we have defined an amino acid sequence motif that binds SUMO. This motif, V/I-X-V/I-V/I, was identified by NMR spectroscopic characterization of interactions among SUMO-1 and peptides derived from proteins that are known to bind SUMO or sumoylated proteins. This motif binds all SUMO paralogues (SUMO-1-3). Using site-directed mutagenesis, we also show that this SUMO-binding motif in RanBP2/Nup358 is responsible for the interaction between RanBP2/Nup358 and sumoylated RanGAP1. The SUMO-binding motif exists in nearly all proteins known to be involved in SUMO-dependent processes, suggesting its general role in sumoylation-dependent cellular functions.

Polymorphism in the androgen receptor and mammographic density in women taking and not taking estrogen and progestin therapy.

There is some evidence that women with a higher number of CAG repeat lengths on the androgen receptor (AR) gene have increased breast cancer risk. We evaluated the association between AR-CAG repeat length and mammographic density, a strong breast cancer risk factor, in 404 African-American and Caucasian breast cancer patients. In postmenopausal estrogen progestin therapy users, carriers of the less active AR-CAG had statistically significantly higher mean percentage of density (41.4%) than carriers of the more active AR-CAG (25.7%; P = 0.04). Our results raise the question of whether the number of AR-CAG repeats predicts breast cancer risk in estrogen progestin therapy users.

The HRAS1 variable number of tandem repeats and risk of breast cancer.

Rare alleles at the HRAS1 variable number of tandem repeats (VNTRs) locus have been implicated in breast cancer risk. We assessed the association of rare HRAS1 alleles and breast cancer in a case-control study nested within the Nurses' Health Study cohort. Using PCR-based methods, 717 incident breast cancer cases and 798 controls were genotyped for the HRAS1 VNTRs. The prevalence of the rare alleles in breast cancer cases was not different compared with controls (10.7 versus 12.0%, respectively; P = 0.45, two-sided Cochran-Mantel-Haenzel chi(2) test). There was no evidence that women heterozygous (multivariate odds ratio, 0.97; 95% confidence interval, 0.73-1.27) or homozygous (multivariate odds ratio, 0.83; 95% confidence interval, 0.32-2.14) for rare alleles were at an increased risk of breast cancer or that a positive gene-dose effect existed. The results did not vary by menopausal status. Although as a group the rare alleles were not associated with breast cancer, one class of rare alleles between the common alleles of a3 and a4 was associated with a significantly increased risk. These results suggest that there is no overall association between rare alleles of the HRAS1 VNTR and breast cancer.

Specific interaction of PML bodies with the TP53 locus in Jurkat interphase nuclei.

PML bodies play an important role in multiple pathways of growth control, such as transformation suppression, apoptosis, and Ras-induced senescence. However, the molecular and biological bases for these physiological phenomena are not well understood. The findings that viruses transcribe their genomes adjacent to PML bodies and that nascent RNA accumulates at their periphery have suggested that PML bodies are transcription-permissible domains. To investigate the role of PML bodies in regulation of cell transformation and apoptosis-related gene transcription, we employed the immuno-FISH method to examine the relationship between PML bodies and the TP53 and BCL2 gene loci. PML bodies were found to localize specifically with the TP53 locus in about 50% of Jurkat interphase nuclei, but never in proximity with the BCL2 locus. We speculate that PML bodies may interact directly with the TP53 DNA sequence to regulate TP53 gene expression.

The thymocyte-specific MAR binding protein, SATB1, interacts in vitro with a novel variant of DNA-directed RNA polymerase II, subunit 11.

A yeast two-hybrid screen of a Jurkat (T cell) derived cDNA library, using SATB1 (a matrix attachment region binding protein) as the bait, yielded four independent isolates of a novel variant of the DNA directed RNA polymerase II, subunit 11 (RPB11). Absence of lysine-17 from the amino terminus of this variant cannot be explained by alternative mRNA splicing. Instead, allele-specific PCR, combined with GenBank database searches, suggests that a recent gene duplication event has resulted in distinct loci encoding three variant forms of RPB11. Differential splicing of mRNA transcripts accounts for unique carboxy termini among the RPB11 proteins. The exclusive association of SATB1 with one form of RPB11 is influenced primarily by the N-terminal amino acid disparity, as deletion of the entire C terminus does not alter interaction affinity. Association of RPB11 with SATB1 maps between amino acids 58 and 222 of SATB1, a region that includes a PDZ-like dimerization motif.