6p21 Region Research Articles

Expert consensus on the standardization and clinical application of genotyping for human leukocyte antigens

The genes encoding human leukocyte antigens (HLA) are located in the region of 6p21.31 of the human chromosome, accounting for 1/3 000 of the human genome. The area is seen as a structurally complex, with the characteristics of multi-gene inheritance, high polymorphism, haplotype inheritance, linkage disequilibrium, racial diversity, and regional distribution. In recent years, with the continuous innovation of HLA genotyping technology, new issues and challenges have emerged in terms of the standardization of the technology platform, the implementation of the performance validation and verification plan for clinical testing projects, quality control of the entire testing process, analysis and interpretation of results, consultation and selection of donors, and clinical application. This consensus, in combination with the current status of HLA genotyping technology in clinical practice in China, systematically sorts out the key contents in the standardization and clinical application of the HLA genotyping technology platform. The first part proposes the basic requirements for the standardization of the HLA typing technology platform from the perspectives of personnel, instrumentation, reagents and consumables, experimental methods, environmental settings, and introduces the methods and performance indicators for confirming the performance of HLA genotyping with next-generation sequencing (NGS) technology. The second part elaborates on the key points of quality control during the entire process of HLA genotyping procedure, quality control of result reporting and releasing, and management of result interpretation and bioinformatics systems. The last part describes the clinical consultation and application promotion of HLA genotyping. After widely soliciting revisions and discussions from domestic experts in the field of HLA and transplantation, the consensus aims to promote the development and clinical application of the HLA gene in related medical field.

Contribution from MHC-Mediated Risk in Schizophrenia Can Reflect a More Ethnic-Specific Genetic and Comorbid Background.

The immune system seems to play a significant role in the development of schizophrenia. This becomes more evident with the emerging role of MHC complex and cytokines in schizophrenia. In the recent past, several GWAS have implied that the 6p21 region was associated with schizophrenia. However, the majority of these studies were performed in European populations. Considering tremendous variations in this region and the probability of South Indian populations being quite different from the European gene-pool from an immunogenetic point, the present study was initiated to screen SNPs in the 2.28 MB region, spanning the extended MHC locus, in 492 cases and controls from a South Indian population. We found a very strong association of rs3815087 with schizophrenia at both allelic and genotypic levels with a 7.3-fold increased risk in the recessive model. Interestingly, the association of none of the earlier reported GWAS hits, such as rs3130375, rs3131296, rs9272219, or rs3130297 were found to be replicable in our study population. rs3815087 lies in the 5′UTR region of the psoriasis susceptibility 1 candidate 1 (PSORS1C1) gene, which further suggests that inflammatory processes might be an important common pathogenic pathway leading to both schizophrenia and psoriasis. The study hints at ethnic specific gene–environment interaction in determining the critical threshold for disease initiation and progression.

Effect of 6p21 region on lung function is modified by smoking: a genome-wide interaction study

Smoking is a major risk factor for chronic obstructive pulmonary disease (COPD); however, more than 25% of COPD patients are non-smokers, and gene-by-smoking interactions are expected to affect COPD onset. We aimed to identify the common genetic variants interacting with pack-years of smoking on FEV1/FVC ratios in individuals with normal lung function. A genome-wide interaction study (GWIS) on FEV1/FVC was performed for individuals with FEV1/FVC ratio ≥ 70 in the Korea Associated Resource cohort data, and significant SNPs were validated using data from two other Korean cohorts. The GWIS revealed that rs10947231 and rs8192575 met genome-wide significant levels; For {text{H}}_{0} :beta_{SNP} = beta_{SNP*pack - years} = 0{text{ vs H}}_{1} : not {text{H}}_{0} , the likelihood ratio (LR) test was conducted, and its P values, PLR, for rs10947231 and rs8192575 were 2.23 × 10–12 and 1.18 × 10–8, respectively. Interaction between rs8192575 and smoking is significantly replicated with two additional data (PINT = 0.0454, 0.0131). Expression quantitative trait loci, topologically associated domains, and PrediXcan analyses revealed that rs8192575 is significantly associated with AGER expression. SNPs on the 6p21 region are associated with FEV1/FVC, and the effect of smoking on FEV1/FVC differs among the associated genotypes.

Genetic variants of chromosome 9p21.3 region associated with coronary artery disease and premature coronary artery disease in an Asian Indian population

IntroductionAsian Indians have a propensity for premature, severe, and diffuse coronary artery disease (CAD). Several single-nucleotide polymorphisms (SNPs) in the ‘core CAD’ region of the chromosomal region 9p21.3 are known to be strongly associated with CAD. ObjectivesWe aimed to study SNPs in the 9p21.3 region associated with CAD and premature CAD and identify their association with demographic and clinical characteristics in an Asian Indian population. MethodsSNP genotyping was performed for 30 SNPs of the 9p21.3 region using MassARRAY® technology. Along with demographic and SNP data analysis, we also performed multivariate logistic regression analysis and multifactor dimensionality reduction analysis to study SNP–SNP and SNP–demographic/clinical variable interactions. ResultsOur results suggest that females are at a higher risk of premature CAD. We found that SNPs rs1333045 (CC), rs16905599 (AA), rs2383206 (GG), rs2383208 (AG), and rs4977574 (GG) were significantly associated with premature CAD. When adjusted for covariates/confounders, we found that rs2383206 showed the strongest risk association with CAD followed by rs16905599 and rs2383208. Further, SNPs rs1333049 (CC) and rs4977574 (GG) were found to be exclusively associated with premature CAD cases, suggesting their potential as genetic markers for premature CAD in the local population. Upon gender-based stratification, it was found that rs10757272 (TT and TC) is significantly associated with eightfold to ninefold CAD risk specifically among females. SNP rs7865618 (GG) is significantly associated with more than 2.5-fold CAD risk specifically among males. ConclusionOur study suggests that SNPs at the 9p21 risk locus may be used to generate a reliable genetic risk score along with markers at other loci.

Genetic Variation in 9p21 and the Plasma Proteome.

Single nucleotide polymorphisms (SNPs) in the noncoding region of 9p21 have been associated with cardiovascular disease (CVD), but the mechanisms by which these genetic variants contribute to the pathogenesis of CVD remain unknown since no annotated proteins are present in this region of DNA. The objective of the current study was to determine if 9p21 genotypes are associated with distinct plasma proteins in young adults. Subjects were 1611 young adults aged 20-29 years from the Toronto Nutrigenomics and Health Study (1098 females and 513 males). DNA was isolated from fasting blood to analyze four SNPs in 9p21 (rs2383206, rs10757274, rs10757278, and rs1333049). Abundant plasma proteins ( n = 54) were measured using liquid chromatography multiple reaction monitoring mass spectrometry. Analysis of covariance was used to determine differences in plasma proteins between genotypes. In Caucasians ( n = 524), four SNPs were associated with apolipoprotein E and two with haptoglobin-β-chain concentration. In East Asians ( n = 388), three SNPs were associated with α-1b-Glycoprotein, two with apolipoprotein B-100,one with apolipoprotein E and one withhaptoglobin-β-chain concentration. In South Asians ( n = 117), one SNP was associated with apolipoprotein B-100 concentration. Our findings suggest that 9p21 genotypes may play a role in various pathophysiological pathways that contribute to risk of CVD in early adulthood that might be distinct among different ethnocultural groups.

OR6 Next generation sequencing HLA typing in patients with copy neutral loss of heterozygosity

Aim Next Generation Sequencing (NGS) technology is well documented as a highly sensitive and specific method for HLA typing. Here we investigate the possibility that copy neutral loss of Heterozygosity (cnLOH) involving the HLA complex located at 6p21 is a potential confounder of interpreting NGS HLA typing results. Methods HLA typing for 11 cases with cnLOH of 6p21 identified by Chromosomal Genomic Array Testing (CGAT), were evaluated by NGS and rSSOP. Tumor burden (TB) estimates for CGAT are based on standard practice. The TB estimate for NGS is based on allele frequency (AF) and calculated using the formula: TB = 100–2 * AF. The baseline of AF is established by evaluating 36 controls without LOH. Results One of 11 patients was a post-transplant case with mixed chimerism. The remaining 10 patients were not transplanted and had confirmed 20%–80% cnLOH at 6p21 by CGAT and Flow Cytometry (FC). In NGS HLA typing, the average baseline AF for HLA-A, B, C, DRB1, DQA1, DQB1, DPA1 and DPB1 loci was 43% ± 5%. In patients who had less than 50% cnLOH by CGAT, the average HLA AF and estimated TB were 34.3% ± 5.2% and 29.8% ± 10.5%, respectively. However, patients who had over 51% of cnLOH, the average HLA AF and TB were 18.8% ± 6.2% and 62.6% ± 12.6%, respectively; suggesting that the higher TB results in greater allele imbalance. Although cnLOH involved the HLA region of 6p21 in all patients, both NGS and rSSOP methods accurately typed HLA alleles except in two patients. In the first case, the patient had received related haplo-identical transplant and was known to be chimeric with 65% of donor cells; this case demonstrated loss of the non-shared haplotype. In the second patient, the specimen had a TB of greater than 80% by CGAT and FC, and HLA typing was not possible for the B and C loci by both NGS and rSSOP. In this patient, the border of cnLOH detected by CGAT involved only the HLA class I genes with the proximal border resting close to the HLA-C and B loci. Conclusions Our findings suggest that HLA locus specific cnLOH could be closely tied with exact molecular location of cnLOH detected by CGAT. The calculated AF by the TruSight Assign software in NGS typing decreases proportionally to increasing TB in cnLOH at 6p21. Our findings indicate that caution is warranted in cases with low frequency alleles and in cases with multiple contiguous homozygous genes.

Nonparametric Risk and Nonparametric Odds in Quantitative Genetic Association Studies.

The coefficient in a linear regression model is commonly employed to evaluate the genetic effect of a single nucleotide polymorphism associated with a quantitative trait under the assumption that the trait value follows a normal distribution or is appropriately normally distributed after a certain transformation. When this assumption is violated, the distribution-free tests are preferred. In this work, we propose the nonparametric risk (NR) and nonparametric odds (NO), obtain the asymptotic normal distribution of estimated NR and then construct the confidence intervals. We also define the genetic models using NR, construct the test statistic under a given genetic model and a robust test, which are free of the genetic uncertainty. Simulation studies show that the proposed confidence intervals have satisfactory cover probabilities and the proposed test can control the type I error rates and is more powerful than the exiting ones under most of the considered scenarios. Application to gene of PTPN22 and genomic region of 6p21.33 from the Genetic Analysis Workshop 16 for association with the anticyclic citrullinated protein antibody further show their performances.

Increased angiogenesis is associated with a 32-gene expression signature and 6p21 amplification in aggressive endometrial cancer.

Angiogenesis is a hallmark of cancer. The aim of this study was to explore whether microvessel proliferation is associated with gene expression profiles or copy number alterations in endometrial cancer. A prospective series of endometrial carcinomas was studied for angiogenesis markers, gene expression profiles, and gene copy number data. For validation, an independent series of endometrial carcinomas as well as an external cohort of endometrial cancer patients were examined by gene expression microarrays. Increased microvessel proliferation (MVP) was associated with aggressive tumor features and reduced survival, and a 32-gene expression signature was found to separate tumors with high versus low MVP. An increased 32-gene signature score was confirmed to associate with high-grade tumor features and reduced survival by independent cohorts. Copy number studies revealed that amplification of the 6p21 region was significantly associated with MVP, a high 32-gene score, as well as reduced survival. Increased MVP was significantly associated with aggressive endometrial cancer and reduced survival. Integrated analyses demonstrated significant associations between increased vascular proliferation, amplification of the 6p21 region, VEGF-A mRNA expression, and the 32-gene angiogenesis signature. Our findings indicate amplification of 6p21 as a possible driver of tumor vascular proliferation in endometrial cancer.

Genome‐wide association studies identify locus on 6p21 influencing lung function in the Korean population

Loss of lung function is an important chronic obstructive pulmonary disease phenotype and decreased forced expiratory volume in 1 s (FEV₁) is an independent risk factor of morbidity and mortality. Genome-wide association studies (GWAS) identifying genetic variants underlying lung function have been performed mostly in Caucasian populations. In this study, we aimed to identify genetic variants influencing lung function in a Korean population. GWAS on lung function (FEV₁ and FEV₁/forced vital capacity (FVC) ratio) were performed in two cohort studies. A population-based cohort, the Korean Association Resource phase 3 (KARE3) (6223 subjects), served as a discovery set. The replication analysis was performed in a family-based cohort, the Healthy Twin Study (HTS; 2730 subjects). Dense single-nucleotide polymorphism array data from each study were imputed and used for genetic analysis. At the discovery phase, variants in 6p21 and 17q24 showed the strongest association with FEV₁/FVC ratio and FEV₁. Several variants in FAM13A on 4q22 locus exhibited positive association with FEV₁/FVC ratio. In the replication set, PPT2 in the 6p21 region showed significant association with lung function in the HTS, although the 4q22 locus and the 17q24 locus were not replicated. We identified that PPT2 on chromosome 6p21 is associated with loss of lung function in the Korean population.

Cell Cycle Deregulation Determines Acute Transformation In Chronic Type Adult T-Cell Leukemia/Lymphoma

IntroductionAdult T-cell leukemia/lymphoma (ATL) is a human T-cell leukemia virus type-1-induced neoplasm with four clinical subtypes; acute, lymphoma, chronic and smoldering. Although chronic and smoldering subtypes are regarded as indolent ATL, about half of these cases progress to acute type ATL and subsequent death. Therefore, cases of indolent ATL also have poor prognosis and acute transformation is a predictive indicator for patients with indolent ATL. However, the molecular pathogenesis of acute transformation remains unknown. In the present study, oligo-array comparative genomic hybridization (CGH) and comprehensive gene-expression profiling (GEP) were applied to 27 and 35 cases of chronic and acute type ATL, respectively, in an effort to delineate the molecular pathogeneses of ATL, and especially the molecular mechanism of acute transformation. Materials and MethodsAll DNA and RNA used in this study were extracted from purified CD4-positive cells. Oligo-array CGH analyses and comprehensive GEP analyses were performed on 27 and 35 cases of chronic and acute type ATL, respectively. Subsequently, we established Tet-OFF ATL cell lines for functional analyses. ResultsOligo-array CGH revealed that genomic loss of 9p21.3 was significantly characteristic of acute type ATL, but not chronic type ATL (p-value= 0.039). Although the minimal common deleted region of 9p21.3 contained MTAP, CDKN2A and CDKN2B, the expression level of only CDKN2A was reduced with genomic loss of 9p21.3 (Figure 1). Moreover, analysis of serial samples of a chronic type ATL patient showing acute transformation also revealed that reduction of CDKN2A expression by 9p21.3 loss was associated with acute transformation in this case. CDKN2A contains two known variants, INK4a and ARF. Re-expression of INK4a and ARF suppressed proliferation of Tet-OFF ATL cell lines, while the suppression efficiency of INK4a was stronger than that of ARF (Figure 2). In cell-cycle assays, the induction of INK4a and ARF decreased the proportion of S-phase cells. Additionally, re-expression of INK4a also increased the amount of apoptotic cells in induced cell lines, while re-expression of ARF did not have this effect. Since CDKN2A is a well-known cell cycle regulator, deregulation of the cell-cycle might be involved in acute transformation of chronic type ATL. In fact, deregulation of the cell-cycle pathway has been reported as a predictive indicator for the outcome in diffuse large B-cell lymphoma patients (Cancer Cell, 22:359-372). Therefore, we examined whether chronic ATL patients had alterations in cell-cycle related genes and found that chronic ATL patients could be divided into two groups. The group possessing alterations in these genes (referred to as “Cell cycle Alteration”) showed poorer prognosis compared with the group lacking such alterations (referred to as “Clean”) (p-value= 0.037) (Figure 3). Additionally, patients with such alterations tended to have earlier progression to acute type ATL. [Display omitted] [Display omitted] [Display omitted] ConclusionThese findings indicated that cell cycle-related genes play an important role in acute transformation and should serve as good prognostic markers for chronic type ATL. Disclosures:No relevant conflicts of interest to declare.

Association between 9p21.3 genomic markers and coronary artery disease in East Asians: a meta-analysis involving 9,813 cases and 10,710 controls

Recently, genome-wide association studies on Coronary Artery Disease (CAD) identified a series of associated Single-Nucleotide Polymorphisms (SNPs) in an intergenic region of chromosome 9p21.3, near the CDKN2A and CDKN2B genes. We investigated the association of this locus with CAD in 12 case–control studies of East Asians and undertook a meta-analysis for effect size, heterogeneity, publication bias, and strength of evidence. English and Chinese language articles were tested for 9p21.3 SNPs with coronary heart/artery disease or myocardial infarction as primary outcomes. Included articles also provided race, numbers of participants, and data to compute an Odds Ratio (OR). Articles were excluded if reporting other outcomes (e.g., stroke). Thirty-five articles were initially identified and 12 were included. Independent extraction was performed by two reviewers and consensus was reached. SNP rs1333049, rs2383206 and rs10757278 representing the 9p21.3 locus, were genotyped in 12 case–control studies involving a total of 9,813 patients and 10,710 controls. For rs1333049 (8 data sets), using a fixed-effects model, the summary OR was 1.29 (95% CI, 1.23–1.36, P = 0.001). For rs2383206 (6 data sets), using a fixed-effects model, the summary OR was 1.24 (95% CI, 1.18–1.31, P = 0.001). For rs10757278 (6 data sets), using a random-effects model, the summary OR was 1.34 (95% CI, 1.21–1.50, P = 0.001). In addition, we defined the haploblock structure of SNPs within the region of 9p21.3 in China population in one study. This broad replication provides unprecedented evidence for association between genetic variants at chromosome 9p21.3 and risk of CAD in East Asians.

Germline Genetic Variation and Risk of Follicular Lymphoma Transformation in the Modern Treatment Era

AbstractAbstract 149 Background.There are few established factors that predict risk of follicular lymphoma (FL) transformation, and only two studies have evaluated the role of germline genetic variation and risk of transformation. One study reported an association of a BCL6 SNP (397C>G) with risk of transformation, and a second study reported an association with rs6457327, which is in the HLA region of 6p21. This study evaluated the risk of FL transformation in the modern treatment era with germline SNPs that were previously reported to be associated with FL transformation (BCL6SNPs and rs6457327), along with SNPs (N=45) previously associated with either risk of FL or FL prognosis. Methods.Newly diagnosed FL patients were prospectively enrolled from 2002–2009 into the Molecular Epidemiology Resource of the University of Iowa/Mayo Clinic Lymphoma SPORE. Peripheral blood for genotyping was collected at enrollment, and clinical and treatment data were abstracted from medical records. All patients were actively followed for disease progression, retreatment, transformation and death. For this analysis, only FL patients with grade I-IIIa were included. Patients with composite DLBCL, FL grade IIIb, or other evidence of transformation at diagnosis were excluded. Transformation was based on either 1) biopsy confirmation of FL grade IIIb, DLBCL, or higher grade B-cell lymphoma; or 2) clinical indicators of transformation based on the published literature, including sudden rise in LDH, rapid discordant localized nodal growth, new involvement of unusual extranodal sites, new B-symptoms, or hypercalcemia. Genotyping was conducted using a custom Illumina iSelect panel. Cox regression was used to estimate Hazard Ratios (HRs) and 95% confidence intervals (CI) for individual SNPs with time to transformation, with death modeled as a competing risk. Each SNP was modeled with the most prevalent homozygous genotype used as the reference group, and each SNP was modeled as having a log-additive (per minor allele) effect in the regression model. An ordinal test was used to assess the trend, and p<0.05 was considered statistically significant. Results.There were 631 newly diagnosed grade I-IIIa FL patients, of whom 532 had DNA and were successfully genotyped. The median age at diagnosis was 60 years (range 23–93) and 53% were male. FLIPI distribution was 42% 0–1, 34% 2 and 24% 3–5. Initial therapies included observation (32%), rituximab (R) monotherapy (13%), alkylator-based chemotherapy +/− R (20%), and anthracycline-based chemotherapy +/− R (19%). At a median of 59 months of follow-up (range, 11–110), 58 patients died, 254 had an event (death, progression or retreatment) and 54 patients (10%) transformed. Transformation was biopsy proven in 43 of the 54 patients (80%). The SNP rs6457327 on chromosome 6p that was previously reported to be associated with transformation was positively associated with risk of transformation in our data (HR=1.40; 95%CI 0.97–2.04; p=0.07), although this association was weaker but still consistent with the prior report. Further, we evaluated additional typed SNPs from the HLA region of 6p, and found additional associations (p<0.05) with HLA-DRA (3 SNPs), HLA-DQA2 (2 SNPs), HLA-DOA (2 SNPs) and HLA-DPB1/HLA-DPA2 (2 SNPs), of which rs10484569 from HLA-DPB1/HLA-DPA2 was the most strongly associated with risk of transformation (HR=3.01; 95%CI 1.48–6.09; p=0.002). None of the BCL6 or other previously reported risk SNPs were associated with transformation. Of the SNPs previously associated with FL prognosis, only the FCGR2A rs1801274 (HR=1.41; 95%CI 0.94–2.10; p=0.09) and CD46 (CD46 molecule, complement regulatory protein) rs2466571 (HR=1.72; 95%CI 1.16–2.55; p=0.007) were associated with risk of transformation. For CD46, one additional SNP (rs2796269) was also associated with transformation (HR=0.61; 95%CI 0.40–0.95; p=0.03). Further adjustment for FLIPI score and treatment (observation versus all other) did not alter these associations. Conclusions.The previously reported association of rs6457327 with risk of transformation in the modern treatment era was replicated. In addition, several HLA loci and two FL prognosis SNPs were linked to transformation risk. The remaining published FL risk or prognosis SNPs were not associated with transformation. Our results support a role for germline genetic variation in the HLA region, FCRGR2A and CD46 in transformation of FL. Disclosures:No relevant conflicts of interest to declare.

Association between 9p21.3 genomic markers and coronary artery disease in East Asians: a meta-analysis involving 9,813 cases and 10,710 controls

Recently, genome-wide association studies on coronary artery disease (CAD) identified a series of associated single-nucleotide polymorphisms (SNPs) in an intergenic region of chromosome 9p21.3, near the CDKN2A and CDKN2B genes. We investigated the association of this locus with CAD in 12 case-control studies of East Asians and undertook a meta-analysis for effect size, heterogeneity, publication bias, and strength of evidence. English and Chinese language articles were tested for 9p21.3 SNPs with coronary heart/artery disease or myocardial infarction as primary outcomes. Included articles also provided race, numbers of participants, and data to compute an odds ratio (OR). Articles were excluded if reporting other outcomes (e.g., stroke). Thirty-five articles were initially identified and 12 were included. Independent extraction was performed by two reviewers and consensus was reached. SNP rs1333049, rs2383206 and rs10757278 representing the 9p21.3 locus, were genotyped in 12 case-control studies involving a total of 9,813 patients and 10,710 controls. For rs1333049 (8 data sets), using a fixed-effects model, the summary OR was 1.29 (95 % CI, 1.23-1.36, P = 0.001). For rs2383206 (6 data sets), using a fixed-effects model, the summary OR was 1.24 (95 % CI, 1.18-1.31, P = 0.001). For rs10757278 (6 data sets), using a random-effects model, the summary OR was 1.34 (95 % CI, 1.21-1.50, P = 0.001). In addition, we defined the haploblock structure of SNPs within the region of 9p21.3 in China population in one study. This broad replication provides unprecedented evidence for association between genetic variants at chromosome 9p21.3 and risk of CAD in East Asians.

Motor sequencing deficit as an endophenotype of speech sound disorder

The aim of this pilot study was to investigate a measure of motor sequencing deficit as a potential endophenotype of speech sound disorder (SSD) in a multigenerational family with evidence of familial SSD. In a multigenerational family with evidence of a familial motor-based SSD, affectation status and a measure of motor sequencing during oral motor testing were obtained. To further investigate the role of motor sequencing as an endophenotype for genetic studies, parametric and nonparametric linkage analyses were carried out using a genome-wide panel of 404 microsatellites. In seven of the 10 family members with available data, SSD affectation status and motor sequencing status coincided. Linkage analysis revealed four regions of interest, 6p21, 7q32, 7q36, and 8q24, primarily identified with the measure of motor sequencing ability. The 6p21 region overlaps with a locus implicated in rapid alternating naming in a recent genome-wide dyslexia linkage study. The 7q32 locus contains a locus implicated in dyslexia. The 7q36 locus borders on a gene known to affect the component traits of language impairment. The results are consistent with a motor-based endophenotype of SSD that would be informative for genetic studies. The linkage results in this first genome-wide study in a multigenerational family with SSD warrant follow-up in additional families and with fine mapping or next-generation approaches to gene identification.

Recurrent Rearrangement of the PHF1 Gene in Ossifying Fibromyxoid Tumors

Ossifying fibromyxoid tumor (OFMT) is a soft tissue tumor of unknown lineage. Although most cases are histologically and clinically benign, some show malignant morphological features and local recurrences are not uncommon; a few may even metastasize. In the present study, cytogenetic analysis identified different structural rearrangements of chromosome band 6p21 in tumor cells from three cases of OFMT, including one with typical, one with atypical, and one with malignant morphological features. Mapping of the 6p21 breakpoint by fluorescence in situ hybridization (FISH) indicated that the PHF1 gene was rearranged in all three cases. Further FISH, 5'-rapid amplification of cDNA ends, and RT-PCR analyses disclosed an EP400/PHF1 fusion transcript in one of the cases. Interphase FISH on tumor sections from 13 additional cases of OFMT showed rearrangement of the PHF1 locus in four of four typical, two of three atypical, and one of six malignant lesions. Thus, the PHF1 gene, previously shown to be the 3'-partner of fusion genes in endometrial stromal tumors, is also recurrently involved in the pathogenesis of OFMTs, irrespective of whether they are diagnosed as typical, atypical, or malignant lesions. The PHF1 protein interacts with the polycomb-repressive complex 2 (PRC2), which, in turn, regulates the expression of a variety of developmental genes. Thus, the results indicate that deregulation of PRC2 target genes is crucial for OFMT development.

Replication of results of genome‐wide association studies on lung cancer susceptibility loci in a Korean population

Genome-wide association studies (GWAS) have identified the three chromosomal regions, 5p15, 6p21 and 15q25, as being associated with lung cancer risk in European populations. This study was performed to confirm these associations in Korean patients with lung cancer. The genotypes at rs2736100, rs402710, rs401681 and rs31489 at 5p15, rs9295740 at 6p22, which is in extensive linkage disequilibrium with the 6p21 region, as well as rs2036534 and rs6495309 at 15q25, were determined in 1094 patients with lung cancer and 1100 healthy control subjects, who were frequency matched for age and gender. The single-nucleotide polymorphisms (SNP) at 5p15 (rs2736100, adjusted odds ratio [aOR] 1.32, 95% confidence interval [CI] 1.03-1.67, P = 0.025; rs402710, aOR 0.82, 95% CI 0.69-0.98, P = 0.025; rs401681, aOR 0.82, 95% CI 0.69-0.98, P = 0.026) and at 15q25 (rs2036534, aOR 0.75, 95% CI 0.61-0.93, P = 0.01; rs6495309, aOR 0.81, 95% CI 0.65-1.00, P = 0.052) were significantly associated with lung cancer risk. The magnitude of the effect was similar to that reported in previous studies, and the association was in the same direction. The effect of SNP in the 5p15 region on the risk of lung cancer was significant only for adenocarcinoma. The two SNP in the 15q25 region were significantly associated with lung cancer risk in ever-smokers and in patients with squamous-cell carcinoma. However, there was no association between the SNP at 6p22 and lung cancer risk. The association between SNP in the 5p15 and 15q25 regions and the risk of lung cancer was confirmed in a Korean population.

Abstract IA2: Navigating the balance of INK4/ARF expression in cancer: ncRNAs as sensors and regulators of transcription

Abstract The INK/ARF (CDKN2A) locus expresses p15Ink4B, p16Ink4A, p14Arf and is under control of Polycomb Repressive Complexes PRC1 and PRC2 to impose transcriptional silencing to bypass cellular senescence upon oncogenic stress. Previously, we had identified a constituent member of human PRC1, known as the chromobox protein 7 (CBX7), is endowed the capacity to bind specifically to elements of the long non-coding RNA identified as the non-coding anti-sense RNA of the INK locus (ANRIL) RNA transcript as a mature processed polyadenylated RNA transcript. ANRIL represent a large heterogeneous population of individual transcripts and their isoforms arising from the localized genomic region overlapping INK/ARF. These transcripts reveal independent patterns of expression dependent on the state of cell growth and differentiation. Moreover, specific transcripts overlapping the ANRIL region of chromosome 9p21.3 provide strict correlation to human embryonic stem cell pluripotency and self-renewal versus differentiation. We recently identify specific ANRIL transcripts and isoforms that are correlative with human prostate tumor stages and states of malignancy. We had previously shown that certain transcripts correlate with PRC1 is organized on the INK/ARF locus through the interaction between CBX7 and the nascent ANRIL transcript to help establish and maintain repressive histone H3K27 methylation and H2AK119 monoubiquitination. Both PRC1 and PRC2 can interact with different ANRIL transcript regions and it remains unclear whether this activity is cooperative. This activity is abrogated in the absence of RNA polymerase II activity thereby requiring nascent transcription of anti-sense transcripts of INK/ARF by RNA polymerase II (RNA POLII). Interestingly, TAF15/TAFII68 can be tethered to CBX7 in prostate carcinoma cells where the ANRIL ncRNA transcripts are elevated. Our recent finding is that specific ANRIL transcripts directed by RNAPOLII are influenced by RNA polymerase III (RNA POLIII) as measured through the depletion of the core RNA POLIII factor BRF1. This evidence suggests that RNA POLIII may populate RNA synthesis of shorter transcripts to influence PRC1 and PRC2 binding thereby instructing the behavior of RNA POLII synthesis. This provides a rational mechanism whereby RNA POLIII with RNAPOLIII provide the genomic RNA context to instruct PRC1 and PRC2 to exert antisense transcription at the expense of transcriptional activity of INK/ARF. Citation Format: Martin J. Walsh, Francesca Aguilo, SiDe Li, Kyoko Yap, Jesus Gil, Ming-Ming Zhou. Navigating the balance of INK4/ARF expression in cancer: ncRNAs as sensors and regulators of transcription [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr IA2.

A C9orf72 promoter repeat expansion in a Flanders-Belgian cohort with disorders of the frontotemporal lobar degeneration-amyotrophic lateral sclerosis spectrum: a gene identification study

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are extremes of a clinically, pathologically, and genetically overlapping disease spectrum. A locus on chromosome 9p21 has been associated with both disorders, and we aimed to identify the causal gene within this region. We studied 305 patients with FTLD, 137 with ALS, and 23 with concomitant FTLD and ALS (FTLD-ALS) and 856 controls from Flanders (Belgium); patients were identified from a hospital-based cohort and were negative for mutations in known FTLD and ALS genes. We also examined the family of one patient with FTLD-ALS previously linked to 9p21 (family DR14). We analysed 130 kbp at 9p21 in association and segregation studies, genomic sequencing, repeat genotyping, and expression studies to identify the causal mutation. We compared genotype-phenotype correlations between mutation carriers and non-carriers. In the patient-control cohort, the single-nucleotide polymorphism rs28140707 within the 130 kbp region of 9p21 was associated with disease (odds ratio [OR] 2·6, 95% CI 1·5-4·7; p=0·001). A GGGGCC repeat expansion in C9orf72 completely co-segregated with disease in family DR14. The association of rs28140707 with disease in the patient-control cohort was abolished when we excluded GGGGCC repeat expansion carriers. In patients with familial disease, six (86%) of seven with FTLD-ALS, seven (47%) of 15 with ALS, and 12 (16%) of 75 with FTLD had the repeat expansion. In patients without known familial disease, one (6%) of 16 with FTLD-ALS, six (5%) of 122 with ALS, and nine (4%) of 230 with FTLD had the repeat expansion. Mutation carriers primarily presented with classic ALS (10 of 11 individuals) or behavioural variant FTLD (14 of 15 individuals). Mean age at onset of FTLD was 55·3 years (SD 8·4) in 21 mutation carriers and 63·2 years (9·6) in 284 non-carriers (p=0·001); mean age at onset of ALS was 54·5 years (9·9) in 13 carriers and 60·4 years (11·4) in 124 non-carriers. Postmortem neuropathological analysis of the brains of three mutation carriers with FTLD showed a notably low TDP-43 load. In brain at postmortem, C9orf72 expression was reduced by nearly 50% in two carriers compared with nine controls (p=0·034). In familial patients, 14% of FTLD-ALS, 50% of ALS, and 62% of FTLD was not accounted for by known disease genes. We identified a pathogenic GGGGCC repeat expansion in C9orf72 on chromosome 9p21, as recently also reported in two other studies. The GGGGCC repeat expansion is highly penetrant, explaining all of the contribution of chromosome 9p21 to FTLD and ALS in the Flanders-Belgian cohort. Decreased expression of C9orf72 in brain suggests haploinsufficiency as an underlying disease mechanism. Unidentified genes probably also contribute to the FTLD-ALS disease spectrum. Full funding sources listed at end of paper (see Acknowledgments).

Cdkn2a Is an Atherosclerosis Modifier Locus That Regulates Monocyte/Macrophage Proliferation

Common genetic variants in a 58-kb region of chromosome 9p21, near the CDKN2A/CDKN2B tumor suppressor locus, are strongly associated with coronary artery disease. However, the underlying mechanism of action remains unknown. We previously reported a congenic mouse model harboring an atherosclerosis susceptibility locus and the region of homology with the human 9p21 locus. Microarray and transcript-specific expression analyses showed markedly decreased Cdkn2a expression, including both p16(INK4a) and p19(ARF), but not Cdkn2b (p15(INK4b)), in macrophages derived from congenic mice compared with controls. Atherosclerosis studies in subcongenic strains revealed genetic complexity and narrowed 1 locus to a small interval including Cdkn2a/b. Bone marrow (BM) transplantation studies implicated myeloid lineage cells as the culprit cell type, rather than resident vascular cells. To directly test the role of BM-derived Cdkn2a transcripts in atherogenesis and inflammatory cell proliferation, we performed a transplantation study using Cdkn2a(-/-) cells in the Ldlr(-/-) mouse model. Cdkn2a-deficient BM recipients exhibited accelerated atherosclerosis, increased Ly6C proinflammatory monocytes, and increased monocyte/macrophage proliferation compared with controls. These data provide a plausible mechanism for accelerated atherogenesis in susceptible congenic mice, involving decreased expression of Cdkn2a and increased proliferation of monocyte/macrophages, with possible relevance to the 9p21 human locus.

Variant on 9p21 Is Strongly Associated with Coronary Artery Disease but Lacks Association with Myocardial Infarction and Disease Severity in a Population in Western India

Coronary artery disease (CAD) is the leading cause of death worldwide, especially so in Indians. Recently, genome-wide studies have implicated SNPs in the 58 kb region of chromosome 9p21 to be associated with CAD. In the current study we evaluated the association of single nucleotide polymorphism (SNP) rs10757278 at the 9p21 locus with CAD in a population from Western India. Genotyping for rs10757278 A/G was done by direct sequencing in 215 cases with confirmed CAD and 150 controls. A significantly higher frequency of the G allele was seen in cases as compared to controls (0.64 vs. 0.53). In the current study the G allele showed association with risk of CAD (OR 1.832 per G allele 95% 1.035-3.242, P 0.042; OR 2.452 GG vs. AA 95% 1.358-4.4431, P 0.004). Addition of the 9p21 allele to Framingham risk score (FRS) resulted in a shift of 17% of individuals from the low-risk category to the intermediate-low (>5-<10% 10-year risk) and 7% from intermediate-low to intermediate-high (>10-<20% 10-year risk) categories. The rs10757278 G variant at the 9p21 locus is significantly associated with the risk of CAD in our population of Western India, similar to the observed trend in other populations; however, the association is much stronger in the present cohort and, considering the high propensity of Indians to develop CAD, it is an important marker even in terms of risk classification.