Affymetrix GeneChip Human Mapping Research Articles

Modified eQTL and Somatic DNA Segment Alterations in Esophageal Squamous Cell Carcinoma for Genes Related to Immunity, DNA Repair, and Inflammation.

Simple SummaryWe applied an integrated approach to analyze expression, genotyping and somatic DNA alterations to find genetic markers (genes and SNPs) related to esophageal squamous cell carcinoma (ESCC). We extended the expression-quantitative trait loci (eQTL) analysis by using tumor vs. normal fold change data. By analyzing both RNA and DNA data from multiple platforms and focusing on the genes in three pathways: inflammation, DNA repair, and immunity, we have found results more relevant to ESCC.We integrated ESCC expression and GWAS genotyping, to investigate eQTL and somatic DNA segment alterations, including somatic copy number alteration, allelic imbalance (AI), and loss of heterozygosity (LOH) in ESCC. First, in eQTL analysis, we used a classical approach based on genotype data from GWAS and expression signals in normal tissue samples, and then used a modified approach based on fold change in the tumor vs. normal samples. We focused on the genes in three pathways: inflammation, DNA repair, and immunity. Among the significant (p < 0.05) SNP-probe pairs from classical and modified eQTL analyses, 24 genes were shared by the two approaches, including 18 genes that showed the same numbers of SNPs and probes and 6 genes that had the different numbers of SNPs and probes. For these 18 genes, we found 28 SNP–probe pairs were correlated in opposite directions in the two approaches, indicating an intriguing difference between the classical and modified eQTL approaches. Second, we analyzed the somatic DNA segment alterations. Across the 24 genes, abnormal gene expression on mRNA arrays was seen in 19–95% of cases and 26–78% showed somatic DNA segment alterations on Affymetrix GeneChip Human Mapping Arrays. The results suggested that this strategy could identify gene expression and somatic DNA segment alterations for biological markers (genes) by combining classical and modified eQTLs and somatic DNA evaluation on SNP arrays. Thus, this study approach may allow us to understand functionality indicative of potentially relevant biomarkers in ESCC.

Mutation detection of Pakistani families with autosomal recessive retinal dystrophies

PurposeHereditary retinal dystrophies (RD) are a group of heterogeneous disorders caused by mutations in over 200 genes. RDs can be subdivided into different groups based on the primary degeneration of rod or cone photoreceptor cells. This study was conducted to investigate the underlying RD genes and mutation in consanguineous families from Pakistan.MethodsFamilies were recruited after informed consent. Peripheral blood was collected and genomic DNA was extracted according to standard procedures. Homozygosity mapping was performed using Affymetrix Gene Chip Human Mapping 250 K‐NspI arrays. The data were analyzed using Homozygosity Mapper software. Primers for amplifying all exons and intron boundaries of all genes were designed with Primer3plus software followed by PCR and Sanger sequencing. Minigene splicing assay and DNA walking were performed on respective samples.ResultsHomozygosity mapping identified a novel locus in family A, one novel gene (C8ORF37) in family B and a large novel genomic deletion of the (LCA5) gene in family C.ConclusionsOur study indicates the heterogeneous nature of retinal dystrophies in Pakistan. Although earlier studies have explored a number of RD families, but underlying genes are still unknown for significant proportion of Pakistani families. Theoretically the traditional screening methods (homozygosity mapping based candidate gene sequencing) were successful, however SNP based genome wide scan were further implemented to improve the detection of underlying variations responsible for Retinal Dystrophies. Therefore the amalgamation of traditional and modern molecular techniques is required for accurate identification of mutations. It is anticipated that these findings will contribute to future genetic testing in Pakistani families to minimize the risk of recessive disorders.

CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays

In five separate families, we identified nine individuals affected by a previously unidentified syndrome characterized by growth retardation, spine malformation, facial dysmorphisms, and developmental delays. Using homozygosity mapping, array CGH, and exome sequencing, we uncovered bi-allelic loss-of-function CDK10 mutations segregating with this disease. CDK10 is a protein kinase that partners with cyclin M to phosphorylate substrates such as ETS2 and PKN2 in order to modulate cellular growth. To validate and model the pathogenicity of these CDK10 germline mutations, we generated conditional-knockout mice. Homozygous Cdk10-knockout mice died postnatally with severe growth retardation, skeletal defects, and kidney and lung abnormalities, symptoms that partly resemble the disease's effect in humans. Fibroblasts derived from affected individuals and Cdk10-knockout mouse embryonic fibroblasts (MEFs) proliferated normally; however, Cdk10-knockout MEFs developed longer cilia. Comparative transcriptomic analysis of mutant and wild-type mouse organs revealed lipid metabolic changes consistent with growth impairment and altered ciliogenesis in the absence of CDK10. Our results document the CDK10 loss-of-function phenotype and point to a function for CDK10 in transducing signals received at the primary cilia to sustain embryonic and postnatal development.

Linkage and Association Analysis Identifies TRAF1 Influencing Common Carotid Intima-Media Thickness.

Carotid intima-media thickness is a marker for subclinical atherosclerosis that predicts subsequent clinical cardiovascular events. The aim of this study was to identify chromosomal loci with linkage or association to common carotid intima-media thickness. Nuclear families were recruited using the single parental proband sib-pair design. Genotype data were available for 546 individuals from 132 nuclear families of the Bonn IMT Family Study using the Affymetrix GeneChip Human Mapping 250K Sty chip. Multipoint logarithm of the odds (LOD) scores were determined with the quantitative trait locus statistic implemented in multipoint engine for rapid likelihood. Linkage analysis and family-based association tests were conducted. Data from 2471 German participants from the HNR (Heinz Nixdorf Recall) Study were used for subsequent replication. Two new genomic regions with suggestive linkage (LOD>2) were identified on chromosome 4 (LOD=2.26) and on chromosome 17 (LOD=2.01). Previously reported linkage findings were replicated on chromosomes 13 and 14. Fifteen single nucleotide polymorhisms, located on chromosomes 4, 6, and 9, revealed P<10-4 in the family-based association analyses. One of these signals was replicated in HNR (rs2416804, 1-sided P=1.60×10-3, located in the gene TRAF1). This study presents the first genome-wide linkage and association study of common carotid intima-media thickness in the German population. Alleles of rs2416804 in TRAF1 were identified as being linked and associated with carotid intima-media thickness. Further studies are needed to evaluate the contribution of this locus to the development of atherosclerosis.

Genetic Risk Factors for the Development of Osteonecrosis in Children Under Age 10 Treated for Acute Lymphoblastic Leukemia

▪Background: Therapy induced osteonecrosis has become a limiting toxicity in the intensification of treatment for pediatric acute lymphoblastic leukemia (ALL), particularly among patients 10 to 20 years of age. Prior studies on the genetic determinants of osteonecrosis have focused primarily on patients older than 10 years, leaving the genetic risk factors for the larger group of children with ALL less than 10 years old incompletely understood. It is hypothesized that genetic risk factors may account for a greater proportion of risk of osteonecrosis or involve differing mechanisms in younger than in older patients.Methods: We performed the first evaluation of genetic risk factors for osteonecrosis in children less than 10 years old using a discovery cohort of 82 cases of osteonecrosis and 287 controls treated on Children's Oncology Group (COG) protocol AALL0331 (NCI standard risk ALL) and tested for replication in 817 children less than 10 treated on COG protocol AALL0232 (high risk ALL). Genotyping was performed using the Affymetrix Gene Chip Human Mapping Array 6.0 and the Illumina Human Exome BeadChip v1.1. A subset of 15 cases in the discovery cohort had coding variant calls verified by whole exome sequencing. Both discovery and replication genome-wide association studies (GWAS) adjusted for demographic and therapy variables known to modify the risk of osteonecrosis. Enhancer enrichment analysis using HaploReg identified tissues affected by the identified single nucleotide polymorphisms (SNPs). Genes associated with the identified SNPs were evaluated using Ingenuity Pathway Analysis for enrichment in biologically relevant pathways.Results: Within the discovery cohort, top ranked variants were rs76599360 and rs77556622 which were in full linkage disequilibrium [P =1.13x10-9, odds ratio (OR) 22.0, 95% confidence interval (95%CI) 8.15-59.6] located near bone morphogenic protein 7 (BMP7). The top replicated SNPs were located near BMP7 [rs75161997, P =5.34x10-8 (OR 15.0; 95%CI 5.64-39.7) and P =0.0498 (OR 8.44; 95%CI 1.002-71.1) in the discovery and replication cohorts, respectively] and PROX1-antisense RNA1 [PROX1-AS1:rs1891059, P =2.28x10-7 (OR 6.48; 95%CI 3.19-13.1) and P =0.0077 (OR 3.78; 95%CI 1.42-10.1) for the discovery and replication cohorts, respectively]. The top replicated non-synonymous SNP, rs34144324, was in a glutamate receptor gene [GRID2, P=8.65x10-6 (OR 3.46; 95%CI 2.00-5.98) and 0.0136 (OR 10.8; 95%CI 1.63-71.4) in the discovery and replication cohorts, respectively], and the genotyping of this variant was verified in the whole exome sequencing data. In a meta-analysis of both cohorts, the replicated BMP7 and PROX1-AS1 variants (rs75161997 and rs1891059, respectively) and a variant in NCRNA00251 (rs141059755) met the genome-wide significance threshold of <5x10-8 (Figure 1). In a meta-analysis of both cohorts, replicated SNPs with meta-analysis P<1 x10-5 showed enrichment in enhancers active in mesenchymal stem cells. Pathway analysis of genes linked to top SNPs (meta-analysis P <0.001) demonstrated enrichment in glutamate receptor signaling and adipogenesis pathways.Conclusions: Variants in genes important to bone and fat differentiation from mesenchymal stem cells were associated with osteonecrosis in children less than 10 years old. The importance of variants in glutamate receptor signaling in children less than 10 also confirms the findings of a recently completed GWAS of osteonecrosis in AALL0232 (Blood 2014 124:367; 2014) including patients of all ages and in which osteonecrosis occurred primarily in older children. These data provide new insights into osteonecrosis with implications for patients of all ages. [Display omitted] DisclosuresHunger:Sigma Tau: Consultancy; Jazz Pharmaceuticals: Consultancy; Merck: Equity Ownership; Spectrum Pharmaceuticals: Consultancy.

Homozygous sequence variants in the FKBP10 gene underlie osteogenesis imperfecta in consanguineous families.

Osteogenesis imperfecta (OI, MIM 610968) is a genetically and clinically heterogeneous disorder characterized by bone fragility. It is one of the rare forms of skeletal deformity caused by sequence variants in at least 14 different genes, including FKBP10 (MIM 607063) encoding protein FKBP65. Here we present three consanguineous families of Pakistani origin segregating OI in an autosomal-recessive pattern. Genotyping using either single-nucleotide polymorphism markers by Affymetrix GeneChip Human Mapping 250K Nsp array or polymorphic microsatellite markers revealed a homozygous region, containing a candidate gene FKBP10, among affected members on chromosome 17q21.2. Sequencing the FKBP10 gene revealed a homozygous novel nonsense variant (c.1490G>A, p.Trp497*) in the family A and two previously reported variants, including a missense (c.344G>A, p.Arg115Gln), in the family B and duplication of a nucleotide C (c.831dupC, p.Gly278ArgfsX295) in the family C. Our findings further extend the body of evidence that supports the importance of FKBP10 gene in the development of skeletal system.

A Pooled Genome-Wide Association Study of Asperger Syndrome.

Asperger Syndrome (AS) is a neurodevelopmental condition characterized by impairments in social interaction and communication, alongside the presence of unusually repetitive, restricted interests and stereotyped behaviour. Individuals with AS have no delay in cognitive and language development. It is a subset of Autism Spectrum Conditions (ASC), which are highly heritable and has a population prevalence of approximately 1%. Few studies have investigated the genetic basis of AS. To address this gap in the literature, we performed a genome-wide pooled DNA association study to identify candidate loci in 612 individuals (294 cases and 318 controls) of Caucasian ancestry, using the Affymetrix GeneChip Human Mapping version 6.0 array. We identified 11 SNPs that had a p-value below 1x10-5. These SNPs were independently genotyped in the same sample. Three of the SNPs (rs1268055, rs7785891 and rs2782448) were nominally significant, though none remained significant after Bonferroni correction. Two of our top three SNPs (rs7785891 and rs2782448) lie in loci previously implicated in ASC. However, investigation of the three SNPs in the ASC genome-wide association dataset from the Psychiatric Genomics Consortium indicated that these three SNPs were not significantly associated with ASC. The effect sizes of the variants were modest, indicating that our study was not sufficiently powered to identify causal variants with precision.

Alterations of LKB1 and KRAS and risk of brain metastasis: Comprehensive characterization by mutation analysis, copy number, and gene expression in non-small-cell lung carcinoma

BackgroundBrain metastases are one of the most malignant complications of lung cancer and constitute a significant cause of cancer related morbidity and mortality worldwide. Recent years of investigation suggested a role of LKB1 in NSCLC development and progression, in synergy with KRAS alteration. In this study, we systematically analyzed how LKB1 and KRAS alteration, measured by mutation, gene expression (GE) and copy number (CN), are associated with brain metastasis in NSCLC. Materials and methodsPatients treated at University of North Carolina Hospital from 1990 to 2009 with NSCLC provided frozen, surgically extracted tumors for analysis. GE was measured using Agilent 44,000 custom-designed arrays, CN was assessed by Affymetrix GeneChip Human Mapping 250K Sty Array or the Genome-Wide Human SNP Array 6.0 and gene mutation was detected using ABI sequencing. Integrated analysis was conducted to assess the relationship between these genetic markers and brain metastasis. A model was proposed for brain metastasis prediction using these genetic measurements. Results17 of the 174 patients developed brain metastasis. LKB1 wild type tumors had significantly higher LKB1 CN (p<0.001) and GE (p=0.002) than the LKB1 mutant group. KRAS wild type tumors had significantly lower KRAS GE (p<0.001) and lower CN, although the latter failed to be significant (p=0.295). Lower LKB1 CN (p=0.039) and KRAS mutation (p=0.007) were significantly associated with more brain metastasis. The predictive model based on nodal (N) stage, patient age, LKB1 CN and KRAS mutation had a good prediction accuracy, with area under the ROC curve of 0.832 (p<0.001). ConclusionLKB1 CN in combination with KRAS mutation predicted brain metastasis in NSCLC.

Identity-by-descent approaches identify regions of importance for genetic susceptibility to hereditary esophageal squamous cell carcinoma.

Worldwide, the highest prevalence of esophageal cancer (EC) occurs in Northern China. High-density SNP arrays allow identification of identity-by-descent (IBD) segments in genomic DNAs representative of shared common ancestral regions. We utilized IBD approaches to map susceptibility loci associated with low-penetrance SNPs in high-risk Henan hereditary esophageal squamous cell carcinoma (ESCC) patients. Affymetrix GeneChip Human mapping SNP array IBD analysis was performed in 32 Henan family history-positive (FH+) ESCC patients, 18 Henan healthy unrelated individuals, and 45 Chinese individuals from a CHB HapMap dataset using PLink (scoring IBD segments individually) and Beagle (scoring of shared IBD segments among case/case vs. control/control pairs) software. Both analyses identified longer IBD segment lengths associated with FH+ ESCC compared to controls. However, there was no strong evidence for a genetic founder effect. Pairing IBD analysis with BEAGLE identified 8 critical IBD segments residing at 2q32.1-q32.2, 3p22.3-p22.2, 4q21.1-q21.21, 7p22.2, 8q23.2-q23.3, 10q23.33-q24.1, 14q24.3 and 16q11.2-q12.1, which were more significantly shared among case/case compared to control/control. The shared IBD segments in FH+ ESCC samples with no overlap with control/CHB Hapmap may encompass potential cancer susceptibility loci. Selected targeted genes, PLCE1, GPT2, SIAH1 and CYP2C-18, residing within the IBD segments at 10q23.33-q24.1 and 16q11.2-q12.1, had statistically significant differential expression in primary ESCC tissues and are likely involved in ESCC carcinogenesis. The importance of these IBD segments to the etiology and development of ESCC in high-risk areas requires further study with expanded sample sizes. This is the first report employing the pairing IBD approach for elucidation of the genetic basis of hereditary ESCC in Henan by applying high throughput SNP array analysis.

A genome wide association study of mathematical ability reveals an association at chromosome 3q29, a locus associated with autism and learning difficulties: a preliminary study.

Mathematical ability is heritable, but few studies have directly investigated its molecular genetic basis. Here we aimed to identify specific genetic contributions to variation in mathematical ability. We carried out a genome wide association scan using pooled DNA in two groups of U.K. samples, based on end of secondary/high school national academic exam achievement: high (n = 419) versus low (n = 183) mathematical ability while controlling for their verbal ability. Significant differences in allele frequencies between these groups were searched for in 906,600 SNPs using the Affymetrix GeneChip Human Mapping version 6.0 array. After meeting a threshold of p<1.5×10−5, 12 SNPs from the pooled association analysis were individually genotyped in 542 of the participants and analyzed to validate the initial associations (lowest p-value 1.14 ×10−6). In this analysis, one of the SNPs (rs789859) showed significant association after Bonferroni correction, and four (rs10873824, rs4144887, rs12130910 rs2809115) were nominally significant (lowest p-value 3.278 × 10−4). Three of the SNPs of interest are located within, or near to, known genes (FAM43A, SFT2D1, C14orf64). The SNP that showed the strongest association, rs789859, is located in a region on chromosome 3q29 that has been previously linked to learning difficulties and autism. rs789859 lies 1.3 kbp downstream of LSG1, and 700 bp upstream of FAM43A, mapping within the potential promoter/regulatory region of the latter. To our knowledge, this is only the second study to investigate the association of genetic variants with mathematical ability, and it highlights a number of interesting markers for future study.

Who Are the Okinawans? Ancestry, Genome Diversity, and Implications for the Genetic Study of Human Longevity From a Geographically Isolated Population

Isolated populations have advantages for genetic studies of longevity from decreased haplotype diversity and long-range linkage disequilibrium. This permits smaller sample sizes without loss of power, among other utilities. Little is known about the genome of the Okinawans, a potential population isolate, recognized for longevity. Therefore, we assessed genetic diversity, structure, and admixture in Okinawans, and compared this with Caucasians, Chinese, Japanese, and Africans from HapMap II, genotyped on the same Affymetrix GeneChip Human Mapping 500K array. Principal component analysis, haplotype coverage, and linkage disequilibrium decay revealed a distinct Okinawan genome-more homogeneity, less haplotype diversity, and longer range linkage disequilibrium. Population structure and admixture analyses utilizing 52 global reference populations from the Human Genome Diversity Cell Line Panel demonstrated that Okinawans clustered almost exclusively with East Asians. Sibling relative risk (λs) analysis revealed that siblings of Okinawan centenarians have 3.11 times (females) and 3.77 times (males) more likelihood of centenarianism. These findings suggest that Okinawans are genetically distinct and share several characteristics of a population isolate, which are prone to develop extreme phenotypes (eg, longevity) from genetic drift, natural selection, and population bottlenecks. These data support further exploration of genetic influence on longevity in the Okinawans.

Genetic Mosaicism and Hemoglobinopathies; Uniparental Isodisomy

BackgroundRecently we discovered three independent cases of “severe late onset beta-thalassemia”, all presenting with the mild phenotype of beta thalassemia minor up to adult age and developing a severe transfusion dependent phenotype in the third and fourth decade of life when a presumed homozygosity for the beta-thalassemia mutation is observed. We demonstrate that uniparental isodisomy (UPD) of part of chromosome 11p, a mechanism also seen in 20% of Beckwith-Wiedemann syndrome patients, accounts for the observed mosaicism in all three independent cases. Clonal selection for hematopoietic stem cells containing the UPD for the mutant beta-globin gene during life may account for the progressive development of the disease. Recently we discovered another case showing a similar mosaic UPD of 11p, presenting as a regular beta-thalassemia carrier. MethodDirect sequencing of the beta-globin genes. Multiplex Ligation dependent Probe Amplification (MLPA) analysis of the HBB (beta-globin) gene cluster. Affymetrix GeneChip Human Mapping 262K NspI array (Santa Clara, CA, USA). Illumina OmniExpress 730K SNP Beads array (San Diego, CA, USA). ResultsIn all cases molecular analysis shows sequences in which a near to complete homozygosity for the beta-gene mutation in DNA extracted from leucocytes is seen. Loss of heterozygosity due to allele drop-out or a deletion of one allele was excluded by direct sequencing and MLPA analysis. Affymetrix and/or Illumina SNP-array analysis revealed incomplete homozygosity for SNP's along almost the entire short arm of chromosome 11 containing the beta-globin gene, indicating mosaicism for a partial uniparental isodisomy of chromosome 11p. Three patients were born asymptomatic as beta-thal carriers and developed a severe blood-transfusion dependent beta-thalassemia major at different ages and with different percentages of mosaicism. The fourth patient however did not develop the clinical severity despite of an almost 50% mosaicism determined from the DNA isolated from leucocytes. ConclusionWe demonstrate that uniparental isodisomy of part of chromosome 11 accounts for the observed mosaicism in all four independent cases. The most probable mechanism seems clonal selection for hematopoietic stem cells containing the uniparental isodisomy for the mutant beta-globin gene during life, this may account for the progressive development of the disease. However, there seems to be no correlation between the percentage of mosaicism measured in the DNA isolated from the white cells and the severity of the clinical phenotype related to the expression in red cells, which strongly suggests hematopoietic tissue heterogeneity for the observed UPD containing cell lineages. This may have serious consequences for disease prediction and counseling, as this is largely dependent upon DNA isolated from leucocytes. Disclosures:No relevant conflicts of interest to declare.

GPSM2 Mutations Cause the Brain Malformations and Hearing Loss in Chudley-McCullough Syndrome

Autosomal-recessive inheritance, severe to profound sensorineural hearing loss, and partial agenesis of the corpus callosum are hallmarks of the clinically well-established Chudley-McCullough syndrome (CMS). Although not always reported in the literature, frontal polymicrogyria and gray matter heterotopia are uniformly present, whereas cerebellar dysplasia, ventriculomegaly, and arachnoid cysts are nearly invariant. Despite these striking brain malformations, individuals with CMS generally do not present with significant neurodevelopmental abnormalities, except for hearing loss. Homozygosity mapping and whole-exome sequencing of DNA from affected individuals in eight families (including the family in the first report of CMS) revealed four molecular variations (two single-base deletions, a nonsense mutation, and a canonical splice-site mutation) in the G protein-signaling modulator 2 gene, GPSM2, that underlie CMS. Mutations in GPSM2 have been previously identified in people with profound congenital nonsyndromic hearing loss (NSHL). Subsequent brain imaging of these individuals revealed frontal polymicrogyria, abnormal corpus callosum, and gray matter heterotopia, consistent with a CMS diagnosis, but no ventriculomegaly. The gene product, GPSM2, is required for orienting the mitotic spindle during cell division in multiple tissues, suggesting that the sensorineural hearing loss and characteristic brain malformations of CMS are due to defects in asymmetric cell divisions during development.

Abstract 2637: Application of identity-by-descent (IBD) approach to genetic mapping of susceptibility locus in hereditary esophageal squamous cell carcinoma (ESCC) by high density SNP array analysis

Abstract Background and aims: Esophageal cancer (EC) is a deadly disease with a very low 5-year survival rate. The incidence of EC shows great geographical variations with high prevalence in Northern China. Although epidemiological studies suggest that environmental factors may be etiologic factors of EC, strong evidence supporting a causative role of genetic factors in familial EC comes from i) familial aggregation of EC in Yangcheng County, ii) the distinct high EC incidence in Chaoshan migrants to Nanao, and iii) molecular genetic studies such as BRCA2 germline mutations. Recent technological advances allow the identification of long regions of homozygosity (known as IBD segments) in genomic DNAs, which represent the sharing of a common ancestor in those regions, using the high density SNP arrays. Previous studies suggest that a high rate of consanguinity, which produces germline genomic homozygosity, is associated with cancers. We aim to use the IBD approach for mapping the susceptibility locus with low-penetrance SNPs in hereditary ESCC patients in Henan, one of the highest ESCC risk regions in the world. Screening programs based on knowledge of founder mutations may reduce cancer mortality by prevention. Thus, our second aim is to assess if a founder effect exists and predisposes individuals to ESCC in Henan. Methods: Genomic DNAs were extracted from blood samples of Henan family history positive (FH+) ESCC patients and healthy normal individuals. The germline homozygosity in 30 Henan FH+ ESCC patients was explored by using Affymetrix GeneChip Human mapping SNP array (∼238K SNPs, Sty I). IBD analysis was performed using the PLink software. Results: There is longer IBD segment length associated with FH+ ESCC compared with control groups. The 27 IBD segments in FH+ ESCC samples having no overlap with control/Hapmap may encompass the potential cancer-susceptibility loci and should be further examined. However, no strong evidence for founder effect was observed by IBD approach for hereditary ESCC in Henan population. Conclusions: The increased length of germline genomic homozygosity association with hereditary ESCC in Henan is observed. Due to the small sample size in the current study, the association is considered hypothesis-generating only. The importance of these IBD segments to the etiology and development of ESCC in high risk areas require further study with an expanded sample size for validation. Acknowledgements: This work was supported by the Research Grants Council of the Hong Kong Special Administrative Region, People's Republic of China (HKU 3/06C to M.L.L.). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2637. doi:1538-7445.AM2012-2637

Clinical and molecular characterization of diffuse large B-cell lymphomas with 13q14.3 deletion

Deletions at 13q14.3 are common in chronic lymphocytic leukemia and are also present in diffuse large B-cell lymphomas (DLBCL) but never in immunodeficiency-related DLBCL. To characterize DLBCL with 13q14.3 deletions, we combined genome-wide DNA profiling, gene expression and clinical data in a large DLBCL series treated with rituximab, cyclophosphamide, doxorubicine, vincristine and prednisone repeated every 21 days (R-CHOP21). Affymetrix GeneChip Human Mapping 250K NspI and U133 plus 2.0 gene were used. MicroRNA (miRNA) expression was studied were by real-time PCR. Median follow-up of patients was 4.9 years. Deletions at 13q14.3, comprising DLEU2/MIR15A/MIR16, occurred in 22/166 (13%) cases. The deletion was wider, including also RB1, in 19/22 cases. Samples with del(13q14.3) had concomitant specific aberrations. No reduced MIR15A/MIR16 expression was observed, but 172 transcripts were significantly differential expressed. Among the deregulated genes, there were RB1 and FAS, both commonly deleted at genomic level. No differences in outcome were observed in patients treated with R-CHOP21. Cases with 13q14.3 deletions appear as group of DLBCL characterized by common genetic and biologic features. Deletions at 13q14.3 might contribute to DLBCL pathogenesis by two mechanisms: deregulating the cell cycle control mainly due RB1 loss and contributing to immune escape, due to FAS down-regulation.

Mutations in C8orf37, Encoding a Ciliary Protein, are Associated with Autosomal-Recessive Retinal Dystrophies with Early Macular Involvement

Cone-rod dystrophy (CRD) and retinitis pigmentosa (RP) are clinically and genetically overlapping heterogeneous retinal dystrophies. By using homozygosity mapping in an individual with autosomal-recessive (ar) RP from a consanguineous family, we identified three sizeable homozygous regions, together encompassing 46 Mb. Next-generation sequencing of all exons, flanking intron sequences, microRNAs, and other highly conserved genomic elements in these three regions revealed a homozygous nonsense mutation (c.497T>A [p.Leu166(∗)]) in C8orf37, located on chromosome 8q22.1. This mutation was not present in 150 ethnically matched control individuals, single-nucleotide polymorphism databases, or the 1000 Genomes database. Immunohistochemical studies revealed C8orf37 localization at the base of the primary cilium of human retinal pigment epithelium cells and at the base of connecting cilia of mouse photoreceptors. C8orf37 sequence analysis of individuals who had retinal dystrophy and carried conspicuously large homozygous regions encompassing C8orf37 revealed a homozygous splice-site mutation (c.156-2A>G) in two siblings of a consanguineous family and homozygous missense mutations (c.529C>T [p.Arg177Trp]; c.545A>G [p.Gln182Arg]) in siblings of two other consanguineous families. The missense mutations affect highly conserved amino acids, and in silico analyses predicted that both variants are probably pathogenic. Clinical assessment revealed CRD in four individuals and RP with early macular involvement in two individuals. The two CRD siblings with the c.156-2A>G mutation also showed unilateral postaxial polydactyly. These results underline the importance of disrupted ciliary processes in the pathogenesis of retinal dystrophies.

SNP-Arrays Provide New Insights Into the Pathogenesis of Richter Syndrome (RS)

Abstract 263RS represents the development of a diffuse large B-cell lymphoma (DLBCL) in the context of chronic lymphocytic leukemia (CLL). The pathogenesis of RS is still largely unknown. Analysis of RS has been often focused on the study of lesions previously identified in de novo DLBCL. However, we have previously shown that RS lacks many of the typical genetic lesions shown in DLBCL (Rossi et al, Blood 2011). Here, we have applied an unbiased, genome-wide approach, searching for DNA copy number alterations in a large series of RS, comparing them with de novo DLBCL, CLL-phase of RS and untransformed CLL.Methods. The study included 50 RS, 30 CLL-phases preceding RS, 100 DLBCL, 318 CLL. All RS were classified as DLBCL. DNA was extracted from frozen biopsies and was processed using the Affymetrix GeneChip Human Mapping SNP6 arrays. Differences in frequencies between subgroups were evaluated using Fisher's exact test. Clinical data were available in half of the cases and genomic lesions were evaluated for their impact on clinical outcome with the log-rank test.Results. In RS, the most common gains were +12 (40%), +4q12 (14%), +2p25.3-25.2 (13%), +8q23.3-qter (12%), +1q32.1, +2p16.1-13.2 (BCL11A/REL), +15q22.31-26.3, +18q21.33 (BCL2) (10%), and +3q24.1-q26.3 (9%). Amplifications were observed at the BCL2 locus and at 1q32.1. The most common losses were at 17p13.1 (TP53; 29%), 8p21.3 (TNFRSF10A/B; 15%), 8p23.3 (16%), 11q22.3 (ATM) (15%), 14q23.3 (13%), 7q33–35 (11%), 14q32.11 (10%), 14q32.31-32.33 (TRAF3), and 9p21.3 (CDKN2A) (9%). The latter locus was also the target of recurrent homozygous deletions.RS appeared intermediate between CLL-phase and de novo DLBCL in terms of number of recurrent lesions. When compared with de novo DLBCL, RS presented less -6q23 (TNFAIP3; P=2.54E-05), -15q15.1-q21.1 (B2M) (P=5.64E-04), +7/7q (P=5.71E-04), -6q21 (PRDM1; P=6.53E-04), +2p16.1 (BCL11A; P=3.22E-03), -1p36 (TNFRSF14; P 9.57E-03), copy-neutral LOH at 6p25.3-21.32 (P =1.01E-02), +5p (P=1.39E-02), +11q14.1-q25 (P=1.63E-02), +1q22-q41 (P 2.13E-02), +6p21 (P 2.48E-02). Conversely, RS presented more often +4q12 (P=8.96E-03), -7q33-35 (P=2.04E-02), -11q22.3 (ATM; P=2.14E-02), -14q23.3 (P=2.69E-02). No statistical differences were observed for lesions such as +3/+3q, +8q, -8p, - 9p21.3 (CDKN2A), +12q, +15q, -17p (TP53). Compared to CLL-phase, RS had significantly more gains at 1q32.1 (P=2.28E-02), 11q23.3 (P=5.38E-03), 11q24 (P=1.10E-02) and 9p21.3 losses (CDKN2A; P=1.10E-02).Compared to the large series of CLL with no history of transformation, CLL-phase samples presented less frequently losses at 13q14.3 (DLEU2, MIR15A, MIR16-1a; 6.58E-03), more commonly gains at 12 (P=2.66E-02), 4q12 (P=1.103E-02), +2p (P=2.97E-02), and loss at TP53 locus (P=2.23E-03).The presence of losses of CDKN2A (P=4.6E-03), TNFRSF10A/B (P=3.2E-02) and BCL2 gains (P=4.0E-02) determined a poor overall survival in RS.Conclusions. DNA copy number changes in RS are intermediate between de novo DLBCL and CLL-phase. Recurrent lesions were identified that appeared more common in RS than in DLBCL, and that seem to be involved in the progression from CLL to an aggressive lymphoma. Also, the acquisition of mono- and bi-allelic losses of CDKN2A appears as one of the most important event in the transition from CLL-phase to RS. CDKN2A and TNFRSF10A/B losses and BCL2 gains were associated with a poor outcome.AR & EC equally contributed to the work. Disclosures:No relevant conflicts of interest to declare.

BLIMP1 Is Commonly Inactivated In Anaplastic Large T-Cell Lymphomas (ALCL)

Abstract 2634ALCL has been separated in two distinct subtypes based on the presence or absence of translocations involving the ALK gene. It is accepted that ALK+ALCL is a distinct subgroup which shares a unique phenotype, with well defined genetic and clinical features. Although the clinical presentations, translocations and genetic events vary between ALK+ and ALK-ALCL, the relationship between these two ALCL subtypes and whether ALK-ALCL may represent a subset of peripheral T-cell lymphomas, not otherwise specified (PTCL, NOS), remains unclear. In this regard, the WHO Classification classifies ALK-ALCL as a provisional entity. A better understanding of the underlying genetics would provide critical explanations to answer some of these questions. With the aim of identifying the genetic events underlying the pathogenesis of ALCL, we studied a series of 69 cases of ALCL (34 ALK-, 35 ALK+) with high-density genome wide SNP-based arrays. Methods.DNA was extracted from frozen biopsies. DNA profiles were obtained using the Affymetrix GeneChip Human Mapping SNP6 arrays. Differences in frequencies between subgroups were evaluated using Fisher's exact test. A subset of cases also had available gene expression profiles. Clinical data were available in half of the cases and genomic lesions were evaluated for their impact on clinical outcome with the log-rank test. Results.The most common losses were at 6q21, 17p13 (19%), 13q22.3 (15%), 3p21.31, 13q32.3 (14%), 1p13.3, 16q23.1 (WWOX) (13%), 16q23.3–24.1 (12%), 1p33 and 16q22.1 (10%). The most common gains occurred at 8q22 (20%), 1q (13%), 7q (10–15%; CDK6, 15%), 8q24 and 9p24.1 (10%). ALK-ALCL displayed a higher number of genomic aberrations in comparison with ALK+ALCL. The lesions presenting major differences included: -6q21 (35% vs 6%; P=0.002), -1p13 (26% vs 3%, P=0.001), -3q22 (26% vs 0%, P=0.001), -4q12-q26 (18% vs 0%; P=0.009), +9p21 (17% vs 0%, P=0.009), -17p13 (TP53, 26% vs 6%, P=0.019). The deletions at 6q21 targeted the gene PRDM1, coding for BLIMP1. The whole coding sequence of PRDM1 has been sequenced in 33 ALK- ALCL samples. Only one somatic mutation, inducing a stop codon, was identified, in one case bearing copy neutral loss of heterozygosity (cnLOH) spanning PRDM1 locus, suggesting a loss of functional protein in this patient. As a whole, 38% of ALK-ALCL presented loss of at least one allele of PRDM1. Only two cases were observed with complete gene loss: the ALK-case with somatic mutation plus cnLOH, and one ALK+ case with homozygous deletion. The presence of 6q21 deletion had an impact on progression free survival among all ALCL (P=0.048), likely reflecting its association with ALK-ALCL, but not when considering ALK- patients only. Xenografts derived from primary ALCL samples bearing 6q21 loss presented decreased BLIMP1 expression level. The detection of PRDM1 loss was present also in cell lines, in which also a decreased level of BLIMP1 RNA and protein was observed. Additional genes, members of PRDM1 pathway, were identified as targets of focal deletions. Conclusions.A series of recurrent lesions has been identified in ALCL. Alongside TP53 loss, inactivation of PRDM1 by genomic losses or somatic mutations was the most common detected lesion, and was more frequently inactivated in ALK-ALCL. PRDM1, encoding BLIMP1, a master regulator of T-cells differentiation, appears as a central gene in ALCL pathogenesis. Other genes, belonging to the same pathway, were found to have focal genomic aberrations in a smaller number of cases. Disclosures:No relevant conflicts of interest to declare.

Adult-onset autosomal dominant leukodystrophy due to LMNB1 gene duplication

Adult-onset autosomal dominant leukodystrophy (ADLD, OMIM accession number 169500) is a slowly progressive neurological disorder involving early autonomic dysfunction, cognitive impairment, pyramidal lesions, and cerebellar dysfunction. Onset is usually in the fourth or fifth decade of life. The disease is caused by a duplication of a region on chromosome 5q23.2 including the Lamin B1 gene (LMNB1) [1]. Neuroradiological characteristics are symmetrical extensive white matter changes predominantly in the frontal and parietal lobes, middle cerebellar peduncles, and specific brain stem tracts. Furthermore, atrophy of the corpus callosum and the brain stem develop. Sparing or less severe alteration of the periventricular white matter is a typical finding [2]. We report on a 47-year-old male patient with a 2-year history of gait disturbance and micturition problems with imperative strangury and, as mentioned by family members, personality changes and depression. On examination he had moderately severe gait ataxia, symmetrical appendicular ataxia, mild spastic paraparesis, brisk deep tendon reflexes, and bilaterally positive Babinski signs. MR imaging of the brain showed extensive bilateral T2hyperintense lesions in the subcortical and deep cerebral white matter with relative sparing of a periventricular rim and the corpus callosum. The middle cerebellar peduncles, the pyramidal tracts, and medial lemniscus in the midbrain, pons, and medulla, and the decussation of the superior cerebellar peduncles were also affected (Fig. 1). Neuropsychological testing yielded results below average regarding the following domains: verbal fluency and flexibility, verbal memory, verbal recall, working memory, several executive functions, and general working velocity. Lumbar puncture, evoked potentials, nerve conduction studies, and electromyography were without abnormal findings. Blood and urine analysis of amino acids, long-chain fatty acids, and lysosomal enzymes (arylsulfatase A, galactocerebrosidase, alpha-galactosidase) were normal. The pedigree showed numerous affected members of father’s family within three generations. Four out of five uncles and aunts and in the grandparents’ generation, six out of eight family members were affected, mainly by gait disturbance but also by neuropsychiatric symptoms (Fig. 2). Seven affected siblings had died; age at death varied between 46 and 71 years. Because of the mode of inheritance and the clinical and MRI findings, ADLD was supposed and the lamin B1 gene was analyzed. Genomic DNA was extracted from venous blood and hybridized on Affymetrix GeneChip Human Mapping SNP6.0 arrays, following the manufacturer’s instructions. M. M. Dos Santos (&) A. J. Grau Department of Neurology, Klinikum Ludwigshafen, Bremserstrasse 79, 67063 Ludwigshafen, Germany e-mail: santosm@klilu.de

Copy Number Variation in CNP267 Region May Be Associated with Hip Bone Size

Osteoporotic hip fracture (HF) is a serious global public health problem associated with high morbidity and mortality. Hip bone size (BS) has been identified as one of key measurable risk factors for HF, independent of bone mineral density (BMD). Hip BS is highly genetically determined, but genetic factors underlying BS variation are still poorly defined. Here, we performed an initial genome-wide copy number variation (CNV) association analysis for hip BS in 1,627 Chinese Han subjects using Affymetrix GeneChip Human Mapping SNP 6.0 Array and a follow-up replicate study in 2,286 unrelated US Caucasians sample. We found that a copy number polymorphism (CNP267) located at chromosome 2q12.2 was significantly associated with hip BS in both initial Chinese and replicate Caucasian samples with p values of 4.73E-03 and 5.66E-03, respectively. An important candidate gene, four and a half LIM domains 2 (FHL2), was detected at the downstream of CNP267, which plays important roles in bone metabolism by binding to several bone formation regulator, such as insulin-like growth factor-binding protein 5 (IGFBP-5) and androgen receptor (AR). Our findings suggest that CNP267 region may be associated with hip BS which might influence the FHL2 gene downstream.