Rare SNP Research Articles

Dysfunction of the Heteromeric KV7.3/KV7.5 Potassium Channel is Associated with Autism Spectrum Disorders

Heterozygous mutations in the KCNQ3 gene on chromosome 8q24 encoding the voltage-gated potassium channel KV7.3 subunit have previously been associated with rolandic epilepsy and idiopathic generalized epilepsy (IGE) including benign neonatal convulsions. We identified a de novo t(3;8) (q21;q24) translocation truncating KCNQ3 in a boy with childhood autism. In addition, we identified a c.1720C > T [p.P574S] nucleotide change in three unrelated individuals with childhood autism and no history of convulsions. This nucleotide change was previously reported in patients with rolandic epilepsy or IGE and has now been annotated as a very rare SNP (rs74582884) in dbSNP. The p.P574S KV7.3 variant significantly reduced potassium current amplitude in Xenopus laevis oocytes when co-expressed with KV7.5 but not with KV7.2 or KV7.4. The nucleotide change did not affect trafficking of heteromeric mutant KV7.3/2, KV7.3/4, or KV7.3/5 channels in HEK 293 cells or primary rat hippocampal neurons. Our results suggest that dysfunction of the heteromeric KV7.3/5 channel is implicated in the pathogenesis of some forms of autism spectrum disorders, epilepsy, and possibly other psychiatric disorders and therefore, KCNQ3 and KCNQ5 are suggested as candidate genes for these disorders.

Deregulation of TLR2-JMJD3 Innate Immunity Signaling, Including a Rare TLR2 SNP As a Potential Somatic Mutation, in Myelodysplastic Syndromes (MDS)

Abstract 1700To better understand molecular bases of MDS pathogenesis, we performed a genome-wide CHIP-Seq analysis of H3K4me3, a histone mark associated with gene activation. In MDS CD34+ cells (N=4), 36 genes showed higher levels of promoter-H3K4me3 compared to controls. 10 of 11 randomly selected genes from these 36 showed increased mRNA expression in MDS CD34+ cells (N=100), supporting the positive correlation between expression and increased promoter H3K4me3. Pathway Analysis indicated that a majority of these genes are involved in innate immunity signal and NF-kB activation. This was validated by increased phospho-p65 in MDS bone marrow CD34+ specimens (N=15). Knock-down of 4 of these genes (C5AR1, FPR2, PTAFR and TYROBP) in OCI-AML3 cells resulted in reduction of p-p65. The observation of innate immunity signal activation and epigenetic deregulation led us screen key innate immunity activators, the Toll-like Receptor (TLR) family genes, and histone methylation regulators in MDS CD34+ cells. Among 8 TLRs and 24 histone methylation regulators, TLR1, 2 and 6, and Jmjc-domain histone demethylase JMJD3 were found to be significantly overexpressed in MDS CD34+ cells compared to control counterparts. This is of biological significance because TLR1 and 6 form functional hetero-dimmers with TLR2. Also, JMJD3 expression can be activated by TLR-NFkB in macrophages. To study TLR2 activation in HSC, we treated CD34+ cells with TLR2 agonists. This led to increased expression of JMJD3, supporting the biological function of TLR2 signal in HSC. We observed that JMJD3 knockdown in OCI-AML3 cells led to reduced expression of innate immunity genes (N=7), accompanied with changes of promoter H3K27me3 and H3K4me3, suggesting that JMJD3 forms a positive feedback to perpetuate innate immunity pathway. To further study the role of TLR-JMJD3 in MDS, we performed capture deep sequencing in 40 MDS bone marrow mononuclear cells (TLR1, 2, 4 and 6, JMJD3, UTX, UTY and JMJD1A). Seven different rare SNP in the coding regions of JMJD3, UTY, JMJD1A, and TLR2 were identified in 5 patients. Among them, one SNP of TLR2 causes a missense mutation, changing a conserved hydrophobic Phe217 to a hydrophilic Ser. We then analyzed the presence of TLR2 F217S in 150 MDS samples by Sanger sequencing. TLR2 F217S was observed in 17 (11%) patients. To evaluate the somatic nature of this alteration, CD3+ cells from 15 corresponding patients were sequenced and only two (13%) CD3+ cell samples carried TLR2 F217S. In the available 9 CD34+ cDNA samples, TLR2 F217S was observed in 8 (90%). We then expressed wild-type or F217S TLR2 in 293T cells, a cell line without endogenous TLR2 expression. Reporter assays indicated that in the absence of TLR2 agonist wildtype and F217S mutant TLR2 led to similar levels of NF-kB activation, whereas F217S led to an increased NF-kB activation compared to wildtype at the presence of TLR2 agonists. F217S also led to increased activation (phospho- and polyubiquitin-) of IRAK1, a key signal mediator for TLR signaling. These results suggest that TLR2 F217S led to more robust innate immunity signal activation when stimulated by agonists. We further studied the impact of TLR2 activation on hematopoietic differentiation. Colony formation of CD34+ cells indicated that TLR2 agonists led to decreased number of erythroid colonies, which was confirmed by flowcytometry that demonstrated TLR2 agonist treatments could reduce the number of CD71 high/HLADR low featured erythroid precursors. To examine the effect of targeting TLR2-JMJD3 in primary MDS cells, we transduced MDS CD34+ cells with shRNAs. In 4 CD34+ cases isolated from lower risk MDS, 3 transduced with JMJD3 shRNA and 4 transduced with TLR2 shRNA had increased ratio of erythroid colonies. In average, JMJD3 and TLR2 shRNA transduction led to a 50% increase in erythroid colonies. This was accompanied by increased expression of genes positively involved in differentiation of erythroid lineage (GYPA, GATA1 and EPOR). Finally, CD34+ cell mRNA expression levels of four genes in this study (NCF2, AQP9, MEFV and TLR1) were associated with overall survival of patients. Taken together, these studies highlight the implication of the deregulation of TLR2-JMJD3 mediated innate immunity signals in MDS pathogenesis and suggest that intervention of this pathway may have therapeutic potential in MDS. Disclosures:No relevant conflicts of interest to declare.

DNMT3A rs36012910 A>G polymorphism and gastric cancer susceptibility in a Chinese population

DNA-methyltransferase (DNMT)-3A plays a crucial role in embryonic development and aberrant DNA methylation in carcinogenesis. Polymorphisms of the DNMT3A gene may influence its enzymatic activity and its contribution to susceptibility to cancer. This study evaluated the association of DNMT3A rs36012910 A>G with susceptibility to gastric cancer (GC) in a Chinese population. Genomic DNA was extracted from samples taken from 340 patients with GC and 251 healthy control subjects. The genotype frequency of DNMT3A rs36012910 A>G in all subjects was detected by polymerase chain reaction-restriction fragment length polymorphism and confirmed by sequencing. Stratification analyses were used to study subgroups by age and gender and to evaluate the association of rs36012910 A>G polymorphism with genetic susceptibility to GC. All patients and control individuals were successfully genotyped for the DNMT3A rs36012910 A>G polymorphism. The frequency of DNMT3A rs36012910 allele G is 3.39 % in healthy individuals and 7.78 % in GC patients, respectively. The rs36012910 AG genotype was significantly more common in the GC group than in the controls, although the rs36012910 GG genotype was only one case in GC patients. Further stratification indicated that AG+GG genotypes were associated with susceptibility to GC in males older than 60, but this polymorphism has no significant association with GC susceptibility in females. Male individuals who carried AG+GG genotypes had a 2.362-fold increased risk of GC compared to those who carried the AA genotype. The rs36012910 allele G was associated with an increased risk of GC compared to the rs36012910 allele A. This is the first report to investigate the distribution and evaluate the association of a rare SNP in DNMT3A with genetic susceptibility to GC. DNMT3A rs36012910 A>G might become a potential biomarker for use in GC prediction, although further studies in larger groups and different populations are needed for confirmation.

CYP2C19 Genetic Polymorphism in Saudi Arabians

The main objective of this study was to evaluate CYP2C19 genetic polymorphism in a Saudi Arabian population by determining the frequencies of CYP2C19*2, *3, *4, *6, *7 and *17 alleles and their relevant genotypes. Genomic DNA was isolated from 192 healthy Saudi Arabians, representing different geographical regions, and genotyping of the selected CYP2C19 variants was carried out by direct sequencing after PCR amplification. The allelic frequency of heterozygous CYP2C19*2 was 8.2% with only one individual found to carry the homozygous genotype of this defective allele. None of the other investigated poor metabolizer alleles (i.e. CYP2C19*3, *4, *6 and *7) was detected in the study population. About 46% of the examined volunteers were found to carry CYP2C19*17 genotype (37.5% heterozygous and 8.1% homozygous of the defective allele) with an overall CYP2C19*17 allelic frequency of 26.9%. In addition, a novel CYP2C19 SNP (G356A) and another very rare SNP (C336T) have been identified in this study with a frequency of about 50% for each. Further studies are required to evaluate the metabolic and clinical relevance of CYP2C19*17, G356A and C336T in the Saudi Arabian population.

RETRACTED ARTICLE: Frequency of TP53 Mutations and its Impact on Drug Sensitivity in Acute Myeloid Leukemia?

The purpose of this study is to find out the frequency of TP53 mutations in acute myeloid leukemia (AML) patients and correlate sensitivity of drug response with TP53 mutations. In AML more than 90% of cases comprise of wild type TP53. 94.2% of TP53 mutations are found within exon 5-8 of which 73% are point mutations. TP53 mutations were analysed with high resolution melting curve analysis. We analysed 106 AML samples of which we found nine mutations which represents 8.5% mutation rate and found one rare SNP. The effect of TP53 mutations were studied on the chemosensitivity of two new drugs AZD115 and RHPS4, an Aurora Kinase B inhibitor and Telomerase inhibitor respectively. Four mutations were found out of 17 for RHPS4 stating significant (p=0.002) increase in sensitivity and no mutation found in AZD1152 database, but need more study to get definite conclusion.

Reference genome-independent assessment of mutation density using restriction enzyme-phased sequencing.

BackgroundThe availability of low cost sequencing has spurred its application to discovery and typing of variation, including variation induced by mutagenesis. Mutation discovery is challenging as it requires a substantial amount of sequencing and analysis to detect very rare changes and distinguish them from noise. Also challenging are the cases when the organism of interest has not been sequenced or is highly divergent from the reference.ResultsWe describe the development of a simple method for reduced representation sequencing. Input DNA was digested with a single restriction enzyme and ligated to Y adapters modified to contain a sequence barcode and to provide a compatible overhang for ligation. We demonstrated the efficiency of this method at SNP discovery using rice and arabidopsis. To test its suitability for the discovery of very rare SNP, one control and three mutagenized rice individuals (1, 5 and 10 mM sodium azide) were used to prepare genomic libraries for Illumina sequencers by ligating barcoded adapters to NlaIII restriction sites. For genome-dependent discovery 15-30 million of 80 base reads per individual were aligned to the reference sequence achieving individual sequencing coverage from 7 to 15×. We identified high-confidence base changes by comparing sequences across individuals and identified instances consistent with mutations, i.e. changes that were found in a single treated individual and were solely GC to AT transitions. For genome-independent discovery 70-mers were extracted from the sequence of the control individual and single-copy sequence was identified by comparing the 70-mers across samples to evaluate copy number and variation. This de novo "genome" was used to align the reads and identify mutations as above. Covering approximately 1/5 of the 380 Mb genome of rice we detected mutation densities ranging from 0.6 to 4 per Mb of diploid DNA depending on the mutagenic treatment.ConclusionsThe combination of a simple and cost-effective library construction method, with Illumina sequencing, and the use of a bioinformatic pipeline allows practical SNP discovery regardless of whether a genomic reference is available.

Rare coding SNP in DZIP1 gene associated with late-onset sporadic Parkinson's disease.

An association between a rare, coding, non-synonymous SNP variant in the gene DZIP1 and Parkinson's disease was found, based on an analysis of the existing NGRC genome-wide association study dataset. The statistical analysis utilized the hypothesis-rich, targeted search unbiased assessment approach, rather than the hypothesis-free, genome-wide agnostic search paradigm. The association of DZIP1 with Parkinson's disease is discussed in the context of a Parkinson's disease stem-cell ageing theory.

Lack of EGFR-activating mutations in European patients with triple-negative breast cancer could emphasise geographic and ethnic variations in breast cancer mutation profiles

IntroductionTriple-negative breast cancers (TNBCs) are characterised by lack of expression of hormone receptors and epidermal growth factor receptor 2 (HER-2). As they frequently express epidermal growth factor receptors (EGFRs), anti-EGFR therapies are currently assessed for this breast cancer subtype as an alternative to treatments that target HER-2 or hormone receptors. Recently, EGFR-activating mutations have been reported in TNBC specimens in an East Asian population. Because variations in the frequency of EGFR-activating mutations in East Asians and other patients with lung cancer have been described, we evaluated the EGFR mutational profile in tumour samples from European patients with TNBC.MethodsWe selected from a DNA tumour bank 229 DNA samples isolated from frozen, histologically proven and macrodissected invasive TNBC specimens from European patients. PCR and high-resolution melting (HRM) analyses were used to detect mutations in exons 19 and 21 of EGFR. The results were then confirmed by bidirectional sequencing of all samples.ResultsHRM analysis allowed the detection of three EGFR exon 21 mutations, but no exon 19 mutations. There was 100% concordance between the HRM and sequencing results. The three patients with EGFR exon 21 abnormal HRM profiles harboured the rare R836R SNP, but no EGFR-activating mutation was identified.ConclusionsThis study highlights variations in the prevalence of EGFR mutations in TNBC. These variations have crucial implications for the design of clinical trials involving anti-EGFR treatments in TNBC and for identifying the potential target population.

Identification of Venous Thromboembolism (VTE)-Associated Novel Variants in the ABO Gene Using Targeted Deep Sequencing

Abstract 709 Background:A recent analysis of merged genome-wide and candidate gene genotypes in VTE cases and controls identified multiple tag SNPs that were strongly associated with VTE. Objective:To identify rare and/or novel functional variants by sequencing the implicated genes. Methods:Cases (n=1488) were Mayo Clinic European-American patients of non-Hispanic ancestry with objectively-diagnosed VTE in the absence of active cancer, venous catheter or antiphospholipid antibodies. Controls (n=1439) were Mayo Clinic outpatients without VTE who were frequency-matched on case age, gender, race, MI/stroke status and state of residence. For this analysis, we selected a subset of these cases and controls for sequencing to take advantage of the joint configuration of two ABO SNPs of primary interest, rs8176719 (ABO exon 6 deletion determining type O blood group) and rs2519093 (ABO intron 1 tag SNP), which were previously shown to be strongly associated with VTE (p=5.7E-12 and p=3.0E-16, respectively). We randomly sampled 82 cases and 14 controls within 3 of the 9 potential allele frequency cells (Figure). The rs8176719 alleles are -−/−- (double deletion is the common allele), –/G, and G/G (the rare allele). The rs2519093 alleles are GG (G is the common allele), AG, and AA (A is the rare allele). For each SNP, the genotypes are represented as 0, 1, or 2 copies of the minor allele. We represented the joint allelic configuration of the two SNPs with the number of copies of the rs8176719 given first as 0/0 (both with 0 copies of the minor allele), 0/1, 0/2 (0 copies of the rs8176719 SNP), 1/0 (1 copy of the rare rs8176719 SNP), 1/1/, and 1/2, and 2/0 (2 copies of the rare allele for the rs8176719 SNP and 0 copies of the rare allele of the rs2519093 SNP), 2/1, and 2/2. From the Figure one observes discrepancies between cases and controls at the 0/0, 1/1 and 2/2 combinations. We randomly sampled from these three combinations, taking one third of the case series. For each SNP, we had 28 cases with 0/0 copies of the rare allele, 27 cases with 1/1 copies of the rare allele; and 27 cases with the combination of 2/2 copies of the rare allele. We compared these 82 cases with 14 controls that do not have any of these combinations. Sixteen genes were selected for deep sequencing, including 5 genes harboring SNPs significantly associated with VTE (F5, SLC19A2, ABO, NME7, ATP1B1), 10 genes with SNPs marginally associated with VTE (C1orf114, KLKB1, SELP, F11, SCUBE1, PRKCB1, CD44, ITPR1, GFRA1, BLZF1), and CYP4V2 which reportedly confounds F11 and KLKB1. Agilent SureSelect probes were designed to capture and enrich the ∼2 Mb genomic regions of these 16 genes. Samples were multiplexed (12-plex) and sequenced using Illumina HiSeq 2000. The sequence reads were aligned to the human genome build 36 using Burrows-Wheeler Aligner, and the single nucleotide variants (SNVs) and small INDELs were called using SNVMix and GATK, respectively. For this analysis, novel ABO SNVs were tested for an association with VTE using age-, sex-adjusted logistic regression and Fisher’s Exact Test. [Display omitted] Results:98% of the targeted regions were sequenced with > 20X coverage. On average, ∼2500 SNVs and ∼200 INDELs were detected in each sample. Fifteen novel SNVs in intron 6 and 3’ of the ABO gene were associated with VTE (p<E-06) and belonged to 3 distinctive LD blocks; none were in LD with the coding or tag ABO SNPs (rs8176719; rs2519093). SNVs inside the middle LD block at the 3’ of ABO are located within an enhancer and promoter histone marked with putative transcription factor binding sites. In addition, strong evidence from both ENCODE and dbEST support the middle LD block as lying within a novel transcript, probably an extension of the 3’ of ABO. In addition, we discovered a novel, significant, protective, frame-shifting single base (G) deletion at ABO chr9:135120877. Conclusion:Novel ABO functional variants that are associated with VTE were identified by deep sequencing. Disclosures:Heit:Daiichi Sankyo: Consultancy, Honoraria.

Mutations in CDON, Encoding a Hedgehog Receptor, Result in Holoprosencephaly and Defective Interactions with Other Hedgehog Receptors

Holoprosencephaly (HPE), a common human congenital anomaly defined by a failure to delineate the midline of the forebrain and/or midface, is associated with diminished Sonic hedgehog (SHH)-pathway activity in development of these structures. SHH signaling is regulated by a network of ligand-binding factors, including the primary receptor PTCH1 and the putative coreceptors, CDON (also called CDO), BOC, and GAS1. Although binding of SHH to these receptors promotes pathway activity, it is not known whether interactions between these receptors are important. We report here identification of missense CDON mutations in human HPE. These mutations diminish CDON's ability to support SHH-dependent gene expression in cell-based signaling assays. The mutations occur outside the SHH-binding domain of CDON, and the encoded variant CDON proteins do not display defects in binding to SHH. In contrast, wild-type CDON associates with PTCH1 and GAS1, but the variants do so inefficiently, in a manner that parallels their activity in cell-based assays. Our findings argue that CDON must associate with both ligand and other hedgehog-receptor components, particularly PTCH1, for signaling to occur and that disruption of the latter interactions is a mechanism of HPE.

Deep Sequencing of the Nicastrin Gene in Pooled DNA, the Identification of Genetic Variants That Affect Risk of Alzheimer's Disease

Nicastrin is an obligatory component of the γ-secretase; the enzyme complex that leads to the production of Aβ fragments critically central to the pathogenesis of Alzheimer's disease (AD). Analyses of the effects of common variation in this gene on risk for late onset AD have been inconclusive. We investigated the effect of rare variation in the coding regions of the Nicastrin gene in a cohort of AD patients and matched controls using an innovative pooling approach and next generation sequencing. Five SNPs were identified and validated by individual genotyping from 311 cases and 360 controls. Association analysis identified a non-synonymous rare SNP (N417Y) with a statistically higher frequency in cases compared to controls in the Greek population (OR 3.994, CI 1.105–14.439, p = 0.035). This finding warrants further investigation in a larger cohort and adds weight to the hypothesis that rare variation explains some of genetic heritability still to be identified in Alzheimer's disease.

Mutations of the TGFBR2 gene in Chinese patients with Marfan-related syndrome

Transforming growth factor beta receptors II gene (TGFBR2) mutations associated with Marfan syndrome and Marfan-associated disorders have been investigated. However, such studies are limited in China. To obtain more information about TGFBR2 mutations, we analyzed 6 unrelated Chinese patients with Marfan-associated disorders and without ocular manifestation. The genomic DNA from blood leukocytes of these 6 patients and their relatives was isolated, and the entire coding region of TGFBR2 was amplified using PCR. We determined the sequence of TGFBR2 with the ABI 3100 Genetic Analyzer. Three mutations were identified in TGFBR2. Two mutations were associated with Loeys-Dietz syndrome (LDS), which were distributed as following: one missense mutation R528C (caused by a 1582C > T substitution) and one polymorphism T315M (a rare SNP). The third mutation was a novel silent mutation associated with MFS2, which was K291K caused by an 873 C > T substitution. The TGFBR2 gene missense mutations are possibly causative mutations of Loeys-Dietz syndrome. This result suggests an increase in the mutation spectrum of Marfan-related disorders in China and possibly world-wide.

Association between a rare SNP in the second intron of human Agouti related protein gene and increased BMI

BackgroundThe agouti related protein (AGRP) is an endogenous antagonist of the melanocortin 4 receptor and is one of the most potent orexigenic factors. The aim of the present study was to assess the genetic variability of AGRP gene and investigate whether the previously reported SNP rs5030980 and the rs11575892, a SNP that so far has not been studied with respect to obesity is associated with increased body mass index (BMI).MethodsWe determined the complete sequence of the AGRP gene and upstream promoter region in 95 patients with severe obesity (BMI > 35 kg/m2). Three polymorphisms were identified: silent mutation c.123G>A (rs34123523) in the second exon, non-synonymous mutation c.199G>A (rs5030980) and c.131-42C>T (rs11575892) located in the second intron. We further screened rs11575892 in a selected group of 1135 and rs5030980 in group of 789 participants from the Genome Database of Latvian Population and Latvian State Research Program Database.ResultsThe CT heterozygotes of rs11575892 had significantly higher mean BMI value (p = 0.027). After adjustment for age, gender and other significant non-genetic factors (presence of diseases), the BMI levels remained significantly higher in carriers of the rs11575892 T allele (p = 0.001). The adjusted mean BMI value of CC genotype was 27.92 ± 1.01 kg/m2 (mean, SE) as compared to 30.97 ± 1.03 kg/m2 for the CT genotype. No association was found between rs5030980 and BMI.ConclusionThis study presents an association of rare allele of AGRP polymorphism in heterozygous state with increased BMI. The possible functional effects of this polymorphism are unclear but may relate to splicing defects.

P01-192 Tryptophan hydroxylase 2 (TPH2) gene in bipolar I disorder in the romanian population

Objective:Since the discovery of the tryptophan hydroxylase 2 gene (TPH2) several studies reported association of TPH2 genetic variation with bipolar I disorder (BPI). Our objectives were to replicate in the Romanian population the recently described association of a rare functional SNP (rs17110563) and of a haplotype covering the 5′ region of TPH2 with BPI (Cichon et al., 2008) and to investigate the influence of the phenotypic traits age-of-onset, family history and parent-of-origin”, defined according to clinical criteria, on the degree of association between TPH2 and BPI.Method:Sixteen TPH2 SNPs were genotyped in a Romanian sample of 198 BPI patients and 180 controls screened for psychiatric disorders. Statistical analysis of the data was performed with Haploview3.32 and FAMHAP.Results:The functional SNP rs17110563 (encoding a Pro206Ser substitution) was present in Romanian BPI patients and absent in controls. SNPs located in the 5′-region (rs11178997, rs11178998, rs7954758), significantly associated with BPI in German patients were not associated with BPI in Romanian patients at single-marker level, but gave evidence for association at haplotypic level in a subgroup of patients with paternal transmission of BPI. Evidence for association was identified between haplotypes located in the 3′-region of TPH2 and BPI in the overall sample as well as in the subgroups of familial cases, the subgroup with paternal transmission, and the subgroup with AO≤25 years.Conclusion:Our data provide support for the involvement of TPH2 in the etiology of BPI.

High-Density Genomic Scan with 50K SNP Arrays Reveals Existence of Cryptic Chromosomal Lesions and Germ Line Allelic Polymorphism That Might Determine Predisposition to MDS.

Chromosomal damage is a hallmark of MDS. Inability to detect abnormal karyotype in a portion of MDS patients is consistent with the theory that large lesions may represent the extreme of possible DNA damage. Predisposition to MDS may be due to inherited defects; possibly, genes coding for detoxifying enzymes, DNA repair genes and immunogenetic factors could be involved and the risk can be multifactorial. To date studies dealing with complex pathogenesis of MDS have been based on empiric approaches allowing for very limited insights into the possibly complex genetic traits and DNA changes during evolution of the disease. Gene array technology facilitates detailed genomic analysis. We have applied 50K SNP Affymetrix arrays to the analysis of the MDS genome. The goal of our study was to establish the feasibility of this technology to study 2 separate aspects: i) to detect acquired cryptic chromosomal damage, ii) to investigate germ line allelic variants in MDS that could constitute predisposition factors. Our MDS cohort included 22 patients 15 RA/RS, 7 RAEB; 11 with typical cytogenetic abnormalities and 11 normal karyotypes. Using a threshold value 6 -log10(pval) we confirmed previously known changes in 8/11 patients. The remaining 3 showed multiple scattered lesions throughout affected chromosomes; false negatives were likely due to dilution by non-clonal cells. However, using a high density SNP scan, novel previously cryptic lesions contiguous over various chromosomal portions were found in 5/11 patients with normal metaphase cytogenetics and some patients with previously established defects. Chromosomes 5, 7 and 8 showed a higher number of smaller defects consistent with the pathogenesis of MDS; deletions within 7p14, 7p12.2 and 7q21.3 were found in 4 patients without obvious mononosomy 7. Similarly, deletions of various size within 5q12.3–35.5 were seen in 4/15 patients with low-grade MDS. Analysis of such defects within phenotypically defined subsets of patients may reveal possible consensus lesions or commonly affected genes.We also investigated the presence of putatively MDS-specific genotypes. Overrepresentation of otherwise rare SNP may suggest existence of cryptic changes involving adjacent portions of the genome. Globally SNP array allowed for classification of 55953 SNP in 22 patients. 28% were heterozygous, while 36% were homozygous for either variant. Complex analysis of such a large number of genotypes may allow for determination of potential associations between genetic makeup and clinical sub-entities. Based on the allelic frequencies of individual SNP in MDS group and controls, we have identified significantly overrepresented SNPs occurring in either homo- or heterozygous form. For example, FOXL1 SNP occurring at a frequency of 2.4% in homo- and heterozygote form in normals was found in 25% of patients, and 10% of patients were homozygous for a Semaphorin SNP present in only 0.015% of controls. Similarly, PARP variant showed an allelic frequency of 2.7 vs. 14% in controls and patients, respectively. In general, our study show the potential value of high density SNP arrays in precise analysis of clonal genomic lesions and/loss heterozygozity as well as complex genotypic profiles that may potentially contribute to inherited predisposition traits.

High-Density Genomic Scan with 50k SNP Arrays May Reveal Existence of Germline Allelic Polymorphisms That Determine Predisposition to Aplastic Anemia.

Although immune mechanisms are likely responsible for most cases of acquired idiopathic AA, the pathogenesis of this disease is not known and various causes, including viral infections and chemical exposures have been implicated. Irrespective of the cause it appears clear that, in analogy to other autoimmune conditions, only certain individuals are susceptible to the development of AA. Theoretically, inherited complex traits may, in combination with exogenous factors, determine the risk to AA. A spectrum of such possible genetic determinants ranges from immunogenetic factors, detoxifying enzymes to DNA repair genes. Overrepresentation of otherwise rare SNP may suggest existence of cryptic changes involving adjacent portions of the genome. The goals of our study were: i) to establish the feasibility of high density SNP scan in the study of genetic factors predisposing to AA and ii) to develop principles for efficient identification of putative disease-associated variants in a large number of AA patients. First, we have studied individual patients with typical AA to identify globally overrepresented alleles. Second, we have examined ability of detection of overrepresented SNPs in pooled DNA samples derived from the cohort of patients. We have applied 50K SNP Affymetrix arrays to the analysis of genome in AA. Lymphocytes from 8 patients were used as a source of germline DNA. DNA was digested with Xba III; hybridization to SNP arrays resulted in average 96.2% genotype call rate. Subsequent analysis concentrated on the detection of polymorphic genotypes rather than changes in gene copy number as seen in MDS. Globally, SNP array allowed for classification of 56725 SNPs in 8 patients (>0.453x106 genotypes). Of them 28% showed heterozygozity, while 36% were homozygous for either genetic variant. Complex analysis of such a large number of genotypes may allow for determination of potential associations between the genetic makeup and disease risk. Based on the sampling size and comparison with control population, we have identified 370 and 88 polymorphisms (in hetero- or homozygous form) that were observed at higher than the expected control frequency (p<.05 and p<.001, respectively). For some variants, SNP were present in >80% of patients. Examples of 70 overrepresented SNPs (>62% in AA;<30% in controls) include IL20R, IL22R, CD109, CD51 and catenin. For some of the SNPs, a homozygous configuration was observed e.g., for DEK (bb in 4/8) and CBP2 (bb in 3/8); for both genes the homozygote frequency in general population was <.0001.The principle of pooling DNA samples relies on the theory that randomly distributed SNP will be diluted out. To determine the possible sensitivity of a significant SNP overrepresentation, individually determined polymorphic frequencies were compared to the results obtained in a pooled sample (equimolar DNA concentrations). Individually detected allelic frequencies of ≥50 were correctly genotyped with the pooled array. Thus, our technique may be helpful in eliminating variants that show lower prevalence within the patient population. While investigations of higher numbers of patients with various clinical, including PNH are under way, our study reveals feasibility of application of the high density SNP arrays in the study genotypic profiles that may potentially contribute to inherited predisposition traits in AA.

Geographic distribution and recombination of genomic fragments on the short arm of chromosome 2 of Arabidopsis thaliana.

Range expansion from Pleistocene refugia and anthropogenic influences contribute to the present distribution pattern of Arabidopsis thaliana. We scored a genome-wide set of CAPSs and found two markers with an east-west geographic distribution across the Eurasian range of the species. Regions around the two SNPs were sequenced in 98 accessions, including newly collected plants from Middle Asia and Western Siberia. These regions correspond to a gene ( approximately 1500 bp) and a non-coding region ( approximately 500 bp) 300 kbp apart on chromosome 2. Nucleotide diversities, pi, of the two sequenced fragments were 0.0032 and 0.0130. The haplotypes of both sequences belonged to one of two groups: a rather uniform "Asian" and a more variable "European" haplotype group, on the basis of non-disjunct clusters of SNPs. Recombination between "Asian" and "European" haplotypes occurs where they meet. Especially in the "European" haplotype, many rare SNP variants representing independent mutations are scattered among the shared haplotype-specific SNPs. This agrees with previous suggestions of two large haplotype groups in A. thaliana and the post-glacial colonization of central Europe from the east and the west. A clear correlation between climatic factors and the haplotype distribution may reflect the dispersal history rather than local climate adaptation. The pattern of SNP variation within the contiguous sequences explains why only a minority of SNPs selected across the genome show evidence of this geographic pattern.