Mass Screening for Non-Synonymous SNPs Using Custom Cancer Microarrays Directly Reveals Possible Pathogenic Predisposition Factors in AA

Abstract Abstract 1333 While immune mechanisms are involved in the pathogenesis of idiopathic aplastic anemia (AA), identification of heritable predisposition/susceptibility traits has been difficult due to the impact of exogenous factors and the low prevalence of AA. The seemingly sporadic and likely complex and heterogeneous traits leading to AA are not easily amenable to genetic studies. With the advent of whole genome scanning (WGS) technologies such as single nucleotide polymorphism arrays (SNP-A), large scale investigations in various disorders have been conducted. A systems level understanding of a particular disease may allow for the identification of candidate genetic variants as prognostic and diagnostic biomarkers. Previous studies utilized arrays with mostly tagging SNPs and thereby the identification of causative polymorphisms was only indirectly possible through the narrowing of LD intervals. We have applied a custom cancer chip (Illumina) containing 211,155 SNP probes designed for non-synonymous SNPs, allowing for a more direct discovery of pathogenic genes with the aim of identifying low prevalence genetic variants that contribute to the development, risk and therapy responsiveness in idiopathic AA. Our study involved 116 cases used for discovery and 120 cases to be used for confirmatory studies, as well as a cohort of 1964 controls to improve the power of detection. After exclusion of SNPs with a GenTrain score of <0.65 and those in violation of Hardy Weinberg equilibrium, 202,905 SNPs (96.0% of the initial set) were passed for further investigation. Single allele 2 statistics for all autosomal markers were performed; 853 SNPs with p<1×10-7 were selected. Subsequently, all 853 SNPs were screened for functional prediction (transcription factor binding site, affecting splicing, detrimental non-synonymous variant of proteins such as cytokines or cytokine receptors). An initial group of SNPs was expanded by all the SNPs being in linkage disequilibrium (>0.8) to a total of 7445 loci. Remarkably, informative LD blocks were identified, represented by multiple markers pointing to the presence of informative polymorphisms in the corresponding regions. A total of 3 SNPs were prioritized for final investigation based on the frequency differential between patients/controls. Of great interest was rs2544773 located in MYT1L represented directly by a singular marker. The frequency of the heterozygous variant among patients was 41.9% vs. 6.5% in controls and 15.0% vs. 0.4% for the minor homozygous variant (p<1×10–17). Our results suggest that carriers of at least one copy of the G allele (GA/AA) are at a higher risk of developing AA (OR 17.6). Another interesting variant identified was a non-synonymous SNP (rs13405539) located in DPYSL5 represented directly by a singular marker. This gene was recently associated with autoimmune myelopathy and cancer. Our analysis showed that minor allele has a protective potential and was present at a frequency in patients; occurring at a homozygous (AA) frequency of 1.8% vs. 16.4% in patients and controls, respectively and a heterozygous (GA) frequency of 14.6% vs. 49.2% in patients and controls respectively (p<1×10–20). Comparison of AA patients with healthy individuals has been a primary focus of GWAS. However, we have also compared subgroups defined by clinical criteria. We subdivided patients based on responsiveness to immunosuppressive therapy. CEBPZ was represented by rs3213746 through LD with rs12469082 (p<.0001). The heterozygous (CT) frequency observed amongst refractory patients was 12.4% vs. 1.4% and 8.2% vs. 0% for the minor homozygous (TT) variant, in refractory and responder, respectively. Odds ratio: Patients carrying at least one copy of the minor allele (CT/TT) are at much higher risk being a non-responder to immunosuppressive treatment (OR=26.3). Genotypic frequencies of patients that responded to immunosuppressive treatment were similar to the frequencies observed in healthy population. CEBPZ belongs to a family of CCAAT/enhancer proteins. It has been shown to interact with TP53 and therefore may play role in modulation of apoptosis. In sum, our study represents novel, whole genome approach using custom designed, high density cancer microarray that unravels new gene targets responsible for disease susceptibility as well as response to immunosuppressive therapy. Disclosures: No relevant conflicts of interest to declare.

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Whole Genome Scanning in Aplastic Anemia Indicates That Polymorphisms in CD33 and Other Loci Can Constitute Predisposition Factors in This Disease

Blood ◽

10.1182/blood.v112.11.3095.3095 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3095-3095

Author(s):

Bartlomiej P Przychodzen ◽

Monika Jasek ◽

Sandra P Smieszek ◽

Anna Malgorzata Jankowska ◽

Christine O’Keefe ◽

...

Keyword(s):

Bone Marrow ◽

Aplastic Anemia ◽

Allele Frequency ◽

Minor Allele Frequency ◽

Minor Allele ◽

Whole Genome ◽

Genome Scanning ◽

A Minor ◽

Low Prevalence ◽

The Impact

Abstract While immune mechanisms are involved in the pathogenesis of idiopathic aplastic anemia (AA), the discovery of telomerase machinery mutations in a minority of patients with a not easily discernable phenotype illustrates that polygenic, low penetrance traits may exist and contribute to increased risk for this disease in certain individuals. However, due to the impact of exogenous factors and the low prevalence of AA, this disease is not easily amenable to genetic studies. With the advent of whole genome scanning (WGS) technologies such as SNP arrays (SNP-A), large scale investigations in various disorders have been conducted. We have applied SNP-A to conduct the first GWAS in AA with the goal to identify possible low prevalence genetic variants that contribute to the pathogenesis and explain individual disease risk. We have studied 128 patients with AA and PNH and 119 controls using SNP-A. Affymetrix GeneChip 6.0 (924644 SNP probes covering most known LD blocks) was designed to capture 67%–89% of SNP variation among Caucasians. Following exclusion of SNPs with a call rate of <95%, and those with serious violation of Hardy-Weinberg equilibrium, single allele χ2 statistics for all autosomal markers was performed. For the purpose of this study, SNPs with a minor allele frequency (MAF) <10% and p<0.001 after false discovery rate correction were selected, whereby all SNP were used multi-variedly to predict disease association. Application of the Benjamini-Hochberg strategy more closely reflects complex polygenic traits. The top 85 SNPs (ranked by p-value) were chosen for further analysis. Our investigations focused on 5 SNPs which pointed directly to 2 genes or indirectly to informative loci through LD, including CD33 and TCF7. For instance, a minor allele (rs3972624, rs17167302, p<5.69×10−5) of TCF7 is present in a heterozygous and homozygous constellation in patients at 19% and 3% (vs. 3.7% and 0% of controls). TCF7 can bind the enhancer element of TCRα and is preferentially expressed in cells polarized to the Th1 direction. Polymorphism in the TCF7 gene was reported to be associated with type-1 diabetes, which is a disease with dominating Th1 phenotype. Furthermore, TCF7 is a member of the WNT-β-catenin LEF1/TCF7 pathway with a known role in the maintenance of hematopoietic stem cells. Increased minor allele frequency of rs1803254, rs9676731, rs1501449 in the CD33 locus, already part of a LD block, was homozygous and heterozygous at 3.4% vs. 0% and 24.8% vs. 7.9% (p<7.1×10−5) in patients and controls, respectively. To further confirm this genetic link, we have investigated the frequency of this polymorphism using an independent set of controls (N=1658) and an overlapping group of patients (12 additional and 52 6.0 array-studied patients). This analysis provided technical validation of genotyping calls in 100% repetitive samples and confirmed the allelic frequency of this SNP in AA at 14.9% vs. 4.5% in controls (p<4×10−5). This marker (rs1803254), representing itself as a genetic proxy, points toward an informative locus in exon 7 of the CD33 gene. CD33 is cell-surface glycoprotein that is specific for the myeloid lineage. It was reported that expression frequencies of CD33 were significantly reduced in AA bone marrow, in comparison to normal bone marrow. In vitro experiments indicate that CD33 may act as an inhibitory receptor and anti-CD33 antibody can induce apoptosis in AML cells. In sum, our study constitutes one of highest resolution investigations into susceptibility loci in AA. Our results point towards a number of interesting genetic disease-associations in AA which are currently being investigated through sequencing of corresponding loci in larger independent cohorts of patients.

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GWAS Indicates the Presence of Rare Immunogenetic Polymorphisms as Possible Predisposition Factors in Aplastic Anemia.

Blood ◽

10.1182/blood.v114.22.1093.1093 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1093-1093

Author(s):

Bartlomiej P Przychodzen ◽

Monika Jasek ◽

Sandra P Smieszek ◽

Ron Paquette ◽

Rainer Richter ◽

...

Keyword(s):

Aplastic Anemia ◽

Genetic Variants ◽

Large Scale ◽

Disease Risk ◽

Conflicts Of Interest ◽

New Paradigm ◽

Genetic Traits ◽

Network Analyses ◽

Low Prevalence ◽

The Impact

Abstract Abstract 1093 Poster Board I-115 While immune mechanisms are involved in the pathogenesis of idiopathic aplastic anemia (AA), due to the impact of exogenous factors and the low prevalence of AA, this disease is not easily amenable to genetic studies. With the advent of whole genome scanning (WGS) technologies such as single nucleotide polymorphism arrays (SNP-A), large scale investigations in various disorders have been conducted. A systems level understanding of particular disease can allows for identification of candidate genetic variants as prognostic and diagnostic markers. We have applied 6.0 SNP-A containing 924644 SNP probes to conduct a comprehensive GWAS in AA with the aim of identifying low prevalence genetic variants that contribute to the pathogenesis of this condition and contribute to individual disease risk. We studied 124 AA patients and significant cohort of 2230 controls that increase detection power using SNP-A. After exclusion of SNP's with call rate of <95% and those with violation of Hardy Weinberg equilibrium (p<.01), 809.802 SNPs (87.5% of initial set) were passed for further investigation. Single allele χ2 statistics for all autosomal markers were performed. 1935 SNP's pointing towards genes with minor allele frequency (MAF) <10% and p<.001 after Bonferroni correction (more stringent than False Discovery Rate) were selected. Of great interest was the top scoring non synonymous SNP (1/38) rs1028180 located in BLZF1 (OR 6.62) involved in cell proliferation and growth. It was represented by singular marker (p<1×10–4) occurring at a heterozygous frequency of 15% vs. 3.5% in controls, and a homozygous frequency of 3.7% vs. 0% in controls. A total of 1 non-synonymous and 3 strongest intronic SNPs were prioritized for final investigation. These included rs9566991, rs1773557 and rs1495963 and directed to informative genes TNFSF11 (OR 6.24), CD247 (OR 3.52) and IL12RB2 (OR 7.04), respectively. Remarkably, several informative LD blocks were identified represented by multiple markers pointing to the presence of informative polymorphisms in the corresponding regions. TNFSF1 gene was represented by marker rs9566991 (p<1×10–3) occurring at the heterozygous frequency of 15.4% vs. 2.7% in controls. The corresponding MAF was 7.6% vs. 1.3%. A second potential locus identified in our study (CD247) was represented by rs1773557 marker (p<1×10–20) occurring at a heterozygous frequency of 19.6% vs. 5.9% in controls, and in homozygous frequency of 0% vs. 0% in controls. Other SNPs including rs1737501, rs1737502 pointed to the same locus. IL12RB2 was represented by a singular marker rs1495963 (p<1×10–6) occurring at the heterozygous frequency of 24% vs. allelic frequency of 3.2% in controls. MAF were 12% versus 1.9%. Another potential loci marked by rs17131583 was TGFBR3. Analysis targeting individual SNP has been the primary focus of GWAS but such an approach offers only limited understanding of the complex diseases as not an individual SNP, and rather a joint action of several SNPs results in particular outcomes. Consequently, in study of AA, we applied the network gene association analysis as a new paradigm incorporating both “operator OR” and “operator AND” thereby allowing for dependence and independence testing. Consequently, the proposed paradigm may lead to identification of meaningful pathways. We performed a simulation study, where genotypes were randomly drawn including homozygous reference, heterozygous and homozygous variant for each SNP Si = 1, 50 where the MAF of SNP is chosen uniformly at random. Of great interest was a pair consisting of rs1737501 CD247 and rs1495963 IL12RB2 both in heterozygous variant, involving operator AND at p<1×10–23. It was reported with occurrence of 12.2% in patients and 0.005% in controls giving a specificity score of 99.995%. In addition to the described pair, SNP in strong LD within CD247 (rs1737502) was scored again together with rs1495963. Functionally, both genes are involved in regulation response. Another pair rs16908086 and rs1773557 that pointed together to CD247 and MRVI2 created a pair with occurrence 21% versus and 3% in controls. In sum, our study constituted the first network analyses of predisposing factors and complex genetic traits enriched in informative loci in immunoregulatory genes. Rare polymorphic variants of these genes may constitute risk factors for development of AA. Disclosures No relevant conflicts of interest to declare.

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The Effect of SARS-CoV-2 Virus Infection on the Course of Atopic Dermatitis in Patients

Medicina ◽

10.3390/medicina57060521 ◽

2021 ◽

Vol 57 (6) ◽

pp. 521

Author(s):

Martyna Miodońska ◽

Agnieszka Bogacz ◽

Magdalena Mróz ◽

Szymon Mućka ◽

Andrzej Bożek

Keyword(s):

Atopic Dermatitis ◽

Immunosuppressive Therapy ◽

Systemic Treatment ◽

Immunosuppressive Treatment ◽

Skin Lesions ◽

Skin Condition ◽

Severe Form ◽

Immune Mediated ◽

Skin Symptoms ◽

The Impact

Background and objectives: Atopic dermatitis (AD) is a disease with a complex pathophysiology involving immune-mediated reactions that lead to skin lesions that are typically localized and recurrent. Following the outbreak of the COVID-19 (coronavirus disease 2019) pandemic, attempting to assess the impact of SARS-CoV-2 infection on diseases caused by complex immune mechanisms has become important. The aim of this study was to assess the impact of SARS-CoV-2 infection on the course of AD, including immunosuppressive therapy, in patients with a severe form of the disease. Materials and methods: A retrospective analysis of 21 adults aged 18 to 52 years with AD diagnosed with COVID-19, including patients requiring hospitalization, was performed. Results: During SARS-CoV-2 infection, temporary exacerbation of skin lesions and/or skin pruritus was observed in nine (43%) patients but without the need for systemic treatment intervention. Patients with severe AD who received immunosuppressive therapy most often manifested mild exacerbation of skin symptoms. The skin condition improved in three patients. There was no significant effect of disease severity on the risk of severe COVID-19 (HR = 0.45; 95% CI: 0.32–0.65). Conclusions: The course of atopic dermatitis during SARS-CoV-2 infection may be different from the severity of its symptoms due to the lack of a significant influence. The immunosuppressive treatment used in patients with severe AD did not significantly affect the course of SARS-CoV-2 infection.

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Common Genetic Variants in Trypsin Regulating Genes Are Associated with AsparAginase-Associated Pancreatitis in Children with Acute Lymphoblastic Leukemia: A Ponte Di Legno Toxicity Working Group Study

Blood ◽

10.1182/blood.v130.suppl_1.885.885 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 885-885

Author(s):

Benjamin Ole Wolthers ◽

Thomas Leth Frandsen ◽

Andishe Attarbaschi ◽

Shlomit Barzilai ◽

Antonella Colombini ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Genetic Variants ◽

Working Group ◽

Risk Allele ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Minor Allele ◽

European Ancestry ◽

Common Genetic Variants ◽

Increased Risk

Abstract Background: Asparaginase-associated pancreatitis (AAP) is a well-known toxicity of childhood acute lymphoblastic leukemia (ALL) therapy. Recent multi-trial group phenotyping of 465AAP caseshas documented severe complications to AAP, including 8% risk of needing assisted mechanical ventilation, 26% risk of developing pancreatic pseudocysts and 9% risk of developing persisting diabetes (Wolthers et al. Lancet Oncology, 2017) . Investigation of host genome variation associated with AAP has been limited by varying phenotype definition, inclusion criteria and small study sizes. Objectives and Methods: To investigate genetic variants associated with risk of developing AAP, this genome-wide association study reports data on 1544 children (1.0−17.9 years) from 10 ALL trial groups treated with ALL from January 2000−January 2016. The Ponte di Legno toxicity working group consensus definition (Schmiegelow et al. Lancet Oncology, 2016) was used to diagnose AAP: At least two of i) amylase, pancreatic amylase, or pancreatic lipase >3x upper normal limit (UNL), ii) abdominal pain, iii) imaging compatible with AAP. Controls included children treated for ALL with verified completion of intended asparaginase therapy, 78% of whom (1024/1320) received at least 8 injections of PEG-asparaginase without developing AAP. Germline DNA obtained after clinical remission was genotyped on Illumina Infinium Omni2.5exome-8 BeadChip arrays. Association analyses were done in PLINK and annotation in Ensembl. Results: Of 1564 patients passing genotype quality control, 244 had AAP. 205 of 244 (84%) of cases and 1185/1320 (90%) of controls were of European ancestry. Median age was 8.1 years (IQR 4.3−13.1) and 5 (IQR: 3−9) for cases and controls, respectively. After filtering, 1401908 single nucleotide polymorphisms (SNPs) with a minor allele frequency above 1% were analyzed. In logistic regression analysis, adjusting for age and ancestry, the variant rs62228256 (reference allele=C, minor allele=T (C>T)) on 20q13.2 had the strongest association to AAP (OR=3.75; 95% CI 2.33−6.04; p=5.2∙10-8). rs62228256 is located in a non-coding region without known regulatory effects. rs13228878 (A>G; OR=0.61; 95% CI 0.5−0.76; p=7.1∙10-6) and rs10273639 (C>T; OR=0.62; 95% CI 0.5−0.77; p =1.1∙10-5) were among the top 30 SNPs most significantly associated to AAP. They are in high linkage disequilibrium (R2=0.94) and located in the PRSS1-PRSS2 locus on chromosome 7. The rs13228878 A risk allele was not associated with level of amylase (p=0.1) or lipase (p=0,68) at diagnosis of AAP, age at diagnosis of AAP (p=0.63), or risk of pseudocysts (p=0.78). Using identical diagnostic criteria for pancreatitis, the major C allele in rs10273639 has been associated with pancreatitis risk in adults (Whitcomb et al. Nature Genetics, 2012; Masson et al. Gut, 2017) with identical risk allele and similar odds ratios. PRSS1 and PRSS2 encode cationic and anionic trypsinogen, respectively. rs10273639 is an expressive quantitative locus for PRSS1 and the C risk-variant is associated with elevated expression of trypsinogen in pancreatic tissue. Gain of function mutations in PRSS1, known from hereditary pancreatitis, lead to increased autoactivation, increased intra-acinar trypsin levels, and increased risk of auto-digestion leading to pancreatitis. Further investigation of previously validated SNPs known to regulate trypsin activation gave the following results for associations with AAP; rs17107315 in pancreatic secretory trypsin inhibitor (SPINK1; OR=2.87; 95% CI 1.36−5.8; p=4∙10-3), rs10436957 in chymotrypsin C (CTRC ; OR=0.69; 95% CI 0.53−0.89; p=5∙10-3) and rs4409525 in Claudin-2 (CLDN2 ; OR=1.41; 95% CI 1.08−1.83; p=1∙10-2). In total, 207 out of 244 cases were homozygous for the risk allele in rs13228878 (n=104), rs17107315 (n=1), rs10436957 (n=165) and/or rs4409525 (n=16). However, no significant additive effect of having more than one risk allele was found. Conclusion: Children who develop AAP possess the same pancreatitis risk variants as adults with non-asparaginase associated pancreatitis. This shared genetic disposition may facilitate research into pathogenesis and identification of effective interventions towards AAP. Disclosures No relevant conflicts of interest to declare.

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Identification of Genetic Polymorphisms Contributing to Risk in MDS Using Innovative GWAS Approaches.

Blood ◽

10.1182/blood.v114.22.734.734 ◽

2009 ◽

Vol 114 (22) ◽

pp. 734-734

Author(s):

Sandra P Smieszek ◽

Bartlomiej P Przychodzen ◽

Ron Paquette ◽

Rainer Richter ◽

Manuel Afable ◽

...

Keyword(s):

Genetic Variants ◽

Large Scale ◽

Genome Wide Association Study ◽

Disease Risk ◽

Conflicts Of Interest ◽

Snp Array ◽

Genetic Traits ◽

Synonymous Snps ◽

Homozygous Variant ◽

Network Analyses

Abstract Abstract 734 Genetic predisposition to MDS and AML is likely polygenic and may involve several low penetrance alleles which in concert with exogenous factors result in highly variable phenotype and late presentation, not easily amenable to genetic studies. With the advent of whole genome scanning (WGS) technologies utilizing various SNP array (SNP-A) platforms, large scale investigations in various disorders have been conducted. A systems level understanding of particular disease allows for identification of candidate genetic variants as prognostic or diagnostic markers. We have applied 6.0 SNP-A containing 924.644 SNP probes to conduct a comprehensive genome-wide association study (GWAS) in MDS including sAML with the aim of identifying low prevalence genetic variants that contribute to individual disease risk. We have studied 189 patients with MDS and sAML and a cohort of 2230 controls. After exclusion of SNP's with call rate of <95% and those with violation of Hardy Weinberg equilibrium (p<.01), 809.802 SNPs (87.5% of initial set) were passed for further investigation. Single allele χ2 for all autosomal markers was performed. A set of 3600 SNP's pointing towards genes with minor allele frequency (MAF) <10% and p<.001 after Bonferroni correction (more conservative multiple hypothesis correction than False Discovery Rate) were selected. Out of 38 significant non-synonymous SNPs 3 were selected, while 3/64 exonic non synonymous SNPs were prioritized for final investigation. These included rs805267, rs2499953 and rs2681417 pointing towards LY6G5B with (OR 4.9), MMP26 (OR 4.7) and CD86 (OR2.9), respectively. LY6G5B gene was represented by marker rs4656334 (p<1×10–12) occurring at the heterozygous frequency of 16.3% vs. 5.4% in controls, and in homozygous frequency of 4.1% vs. 0.04% in controls resulting in MAF of 12.2% vs. 2.7% in controls (p<1×10-12). Rs2499953 and rs2499956 pointed towards MMP26 and was found in the heterozygous variant in 17.0% vs. 3.7% in controls. CD86 gene was represented by singular rs2681417 marker (p<1×10–7) occurring at the heterozygous frequency of 29.0% vs. 13.6% frequency in controls, and in homozygous frequency of 4.0% vs. 0.4% in controls. The 3/3536 strongest intronic SNPs included rs4656334, rs4647493 and rs700060 and directed via LD to informative genes ATF6 with odds 3.15, FANCC (OR 135.3) and RABGAP1 (OR 37.1) respectively. Of interest is that ATF6/αaRheb-mTOR signalling promotes survival of dormant tumour cells in vivo. ATF6 gene was represented by marker rs4656334 (p<1×10–7) and occurred at a heterozygous frequency of 7% vs. 8.3% in controls, while the homozygous constellation was 7 × higher in patients (14.6% vs. 2.4% in controls) with the corresponding MAF of 18.0 vs. 6.5%. Another 2 SNPs within this locus were highly significant (rs16860777, p<1×10–6; and rs12401299, p<1×10–6). Second potential locus identified in our study (FANCC gene) was represented by singular rs4647493 marker (p<1×10–20) occurring at the heterozygous frequency of 16.9% vs. 0.05% in controls. RABGAP1 gene was represented by singular rs700060 marker (p<1×10–17) occurring at the heterozygous frequency of 13.9% vs. allelic frequency of 0.3% in controls. While these results of analysis targeting individual SNP provide intriguing research avenues, such an approach offers only limited understanding of the complex genetic traits as not an individual SNP, but rather a joint action of several SNPs results in particular outcomes. Consequently, in study of MDS, we applied the network gene association analysis as a new paradigm incorporating both “operator OR” and “operator AND” thereby allowing for dependence and independence testing and possibly, to identification of meaningful pathways. We performed a simulation study, where genotypes were drawn including homozygous reference, heterozygous and homozygous variant for each SNP Si = 1,… 64 where the MAF of SNP is chosen uniformly at random. We have identified rs236113 and rs2499953 (MCM8 and MMP26), both in homozygous variant with occurrence of 19% in patients and 1.8% in controls at a specificity score 98.2% and p<1×10–32. Accordingly the presence of both SNP's increases relative risk given the specificity when both SNPs are present. In sum, our study constituted the first network analyses of predisposing factors taken in consideration as groups and identified informative loci that can lead to delineation of causative genetic profiles. Disclosures: No relevant conflicts of interest to declare.

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Genetic Polymorphisms of the RANKL and OPG Genes Associated with Myeloma

Blood ◽

10.1182/blood.v118.21.2911.2911 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2911-2911

Author(s):

Gunter Assmann ◽

Stefanie Keller ◽

Mei Fang Ong ◽

Sandra Grass ◽

Michael Pfreundschuh ◽

...

Keyword(s):

Conflicts Of Interest ◽

Regulatory Region ◽

Minor Allele ◽

Taqman Assay ◽

Nucleotide Polymorphisms ◽

Accessory Cells ◽

Increased Risk ◽

Receptor Activator ◽

Smoldering Myeloma ◽

The Impact

Abstract Abstract 2911 The bone disease in myeloma is the result of the complex interactions between myeloma cells and the bone marrow osteoclasts plus other accessory cells and microenvironmental components. The receptor activator of NfkappaB (RANK) and osteoprotegerin (OPG) cascade system have been reported to be an essential pathway in the osteoclastogenesis. Genetic variations in the genes coding for RANK, RANK ligand (RANKL), and OPG are supposed to play a role in the susceptibility of myeloma. In the present case control study genomic DNA was obtained in 217 myeloma patients including smoldering myeloma (age: mean 62, 33–88) and 516 healthy controls (healthy blood donors, HC; age mean: 44, 18–65). We studied six single-nucleotide-polymorphisms (SNPs) in the genes of RANK (2 SNPs, rs1805034, rs35211496), OPG (2 SNPs, rs3102735, rs2073618), and of RANKL (2 SNPs, rs9533156, rs2277438) using TaqMan assay guided polymerase chain reaction for the respective SNPs. The genotype and allelic frequencies comparing myeloma patients with HC were analyzed with χ2 test for 2×3 and 2×2 tables, respectively. The genotype distributions of the SNP rs2277438 in the RANKL gene and SNP rs3102735 in the OPG gene differed highly significantly (p=0.0001 and p=0.005, respectively) between myeloma patients and HC (table 1). No significantly increased risk was detected in the other SNP analyzed in the study. A subgroup analysis of the myeloma patients stratified into beta2 microglobulin levels lower or higher than 3.5 mg/l showed lower values to be significantly associated with the minor allele of the SNP rs3102735 in the OPG gene (major vs. minor allele in myeloma vs. HC: 77/23% vs. 89/11%; OR 2.49; CI 1.38–4.49). Further stratification into gender, serum calcium levels, hemoglobin values at diagnosis showed no differences.Table 1Myeloma patientsHealthy controls (HC)OR (CI)pRANKL rs2277438Allelen = 434 (%)n = 1024 (%)1324 (75)860 (84)1.7790.00012110 (25)164 (16)(1.373–2.341)Genotypen = 217 (%)n = 512 (%)11132 (61)360 (70)0.00011260 (28)140 (27)2225 (12)12 (2)OPG rs 3102735Allelen = 420 (%)n = 1028 (%)167 (16)109 (11)1.6000.0052353 (84)919 (89)(1.153–2.222)Genotypen = 210 (%)n = 514 (%)119 (4)5 (1)0.0051249 (23)99 (19)22152 (72)410 (80)RANK = receptor activator of NfkappaB, RANKL = receptor activator of NfkappaB ligand, OPG = osteoprotegerin, OR = odds ratio, CI = confidence intervals 95% To the best of our knowledge, this is the first study reporting a highly significant association of the SNP rs2277438 of the RANKL gene and the SNP rs3102735 in the OPG gene, respectively, with the susceptibility to develop myeloma in the Caucasian population. Both SNPs are associated with an increased risk for myeloma. However, myeloma patients carrying the minor allele of OPG SNP rs3102735 developed lower levels of the proliferation marker beta2 microglobulin. The impact of the OPG SNP rs3102735 (location: promoter region, chromosome 8q24) or of the SNP rs2277438 located in intron 1 (chromosome 13q14.11), a potentially regulatory region of the RANKL gene, on the regulation of RANKL expression is unclear. Disclosures: No relevant conflicts of interest to declare.

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Treatment of Relapse After Allogeneic Hematopoietic Stem-Cell Transplantation for Myelodysplastic Syndrome: A Large-Scale Study On Behalf of the Société Française De Greffe De Moelle Et De Thérapie Cellulaire (SFGM-TC)

Blood ◽

10.1182/blood.v120.21.593.593 ◽

2012 ◽

Vol 120 (21) ◽

pp. 593-593

Author(s):

Romain Guièze ◽

Gandhi Damaj ◽

Marie Robin ◽

Mohamad Mohty ◽

Mauricette Michallet ◽

...

Keyword(s):

Myelodysplastic Syndrome ◽

Supportive Care ◽

Large Scale ◽

Conflicts Of Interest ◽

Immunosuppressive Treatment ◽

Tumor Burden ◽

Hematopoietic Stem ◽

Satisfactory Outcome ◽

Cytoreductive Treatment ◽

The Impact

Abstract Abstract 593 Background: Treatment of patients with myelodysplastic syndrome (MDS) relapsing after allo-SCT remains disappointing and prognostic factors for outcome are still unclear. Several therapeutic approaches are offered to those patients including: palliative and supportive care, intensive chemotherapy (ICT), demethylating agents (DMA) and immunotherapy with either donor lymphocyte infusion (DLI) or second allo-SCT. The aim of this study was to identify predictive factors for outcome and to investigate the impact of different treatment groups on survival. Methods: We report a retrospective study on 137 consecutive MDS patients who relapsed after allo-SCT between Jul 1999 and March 2011 in 19 French and Belgian centers. Data quality was ensured using computerized discrepancy errors and vigorous on-site data verification of every single file. HLA matching was double-checked by the French Bone Marrow Donor Registry. Results: At diagnosis, 61 patients presented RA/RARS/RCMD, 27 RAEB1 and 49 RAEB2 according the WHO classification. Cytogenetic was unfavorable in 47 patients (35%) while 76 patients (57%) had IPSS Int-2 or High and 52 (39%) had progressed to a more advanced disease before allo-SCT. At transplant, 62 patients (47%) were considered responders (in CR or PR), while 70 were transplanted with progressive disease (untreated, stable without hematological improvement, relapsed or refractory disease). Patients received either myeloablative (MAC) (n=38) or nonmyeloablative (RIC) (n=99) conditioning. Source of stem cells was BM (n=40) or PBSC (n=97) from sibling (n=89) or allele well-matched unrelated (10/10) (n=48) donors. Median age at relapse was 57 years (range, 19–70). Patients aged <18 years and those who received graft from either cord blood or mismatched donor were excluded. Post-transplant relapse occurred after a median time of 6 months (range, 0.8–102). Of the 109 evaluable patients 62 (57%) had >= 10% of marrow blasts, of whom 38 had >= 20%. At the time of relapse, 61 patients were still under immunosuppressive treatment which was quickly stopped in all of them. For the analysis, patients were divided into three groups according to ultimate salvage received treatment as follows: palliative and supportive care (PSC-group) (n=42, 31%), cytoreductive treatment alone (CRT-group) [DMA (n=18, 13%) and ICT (n= 11, 8%)] and immunotherapy preceded or not by a CRT (IT-group) [DLI (n=48, 35%) and second allo-SCT (n=18, 13%)]. With a median follow-up of 33 months from the relapse, estimated 2-year overall survival was 2.4%, 6.7% and 30.3% for the PSC-group, CRT-group and IT-group, respectively (p<.0001). In multivariate analysis, 3 independent factors have been identified: the absence of immunotherapy HR= 1.45 [95% IC= 1.25–1.69, p<.0001], early relapse within 6 months after transplant, HR=2.71 [95% IC= 1.66–4.45, p<.0001] and marrow blasts at relapse >= 10%, HR=2.56 [95% IC= 1.55–4.22, p<.0001]. Conclusion: This study shows that salvage immunotherapy (DLI or second allo-SCT) provides the best results and should, whenever possible, be offered to patients with MDS who relapse after allo-SCT especially those with low tumor burden. Patients who received cytoreductive treatment alone (be it chemotherapy or DMA) had less satisfactory outcome. Our results emphasize the need to perform prospective protocols combining cytoreductive treatments and immunotherapy. Disclosures: No relevant conflicts of interest to declare.

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Next-Generation Sequencing: A Discovery Tool for Blood Disorders

Blood ◽

10.1182/blood.v120.21.sci-10.sci-10 ◽

2012 ◽

Vol 120 (21) ◽

pp. SCI-10-SCI-10

Author(s):

Elaine R. Mardis ◽

Li Ding ◽

Peter Westervelt ◽

John S. Welch ◽

Jeffery M. Klco ◽

...

Keyword(s):

Conflicts Of Interest ◽

Massively Parallel Sequencing ◽

Tumor Biology ◽

Cancer Genome ◽

Driver Mutations ◽

Tumor Evolution ◽

Whole Genome ◽

Next Generation ◽

Hematopoietic Stem ◽

The Impact

Abstract Abstract SCI-10 The advent and evolution of next-generation massively parallel sequencing (MPS) has radically altered our approaches to studying the cancer genome and transcriptome. By using unbiased and comprehensive MPS in the context of clinically annotated samples from leukemia cases, rapid progress has resulted in our understanding of the mutational spectrum of hematopoietic malignancies, the heterogeneity of disease presentation, and the impact of chemotherapy on the cancer genome, among others. The complexity of the transcriptome, while daunting from an analytical standpoint, further reveals the nuances of gene expression changes in leukemia that often cannot be predicted simply by studying the genome. By applying the digital nature of MPS to explore tumor heterogeneity and tumor evolution, we have shown that de novo acute myeloid leukemia (AML) presents either as a mono- or multiclonal disease, and that the relapse presentation in the same patient is an evolved genetic derivation of the de novopresentation, often with novel driver mutations that have been acquired during the course of chemotherapy (1). New data from whole genome sequencing of hematopoietic stem cells in healthy volunteers indicates that somatic mutations largely are acquired during aging and occur randomly, carrying forward in the transformed cell. This baseline is important for the further comparison of AML subtypes, and provides a context for understanding tumor biology. Last, by studying 200 AML cases using whole-genome and exome sequencing, RNA-seq, miRNA-seq and array-based methylation, we have begun an integrated characterization of AML in an effort to inform tumor biology. These studies and the accompanying technologies set the stage for precision treatment of each AML patient according to the additional information provided by the person's integrated “omic” profile. Disclosures: No relevant conflicts of interest to declare.

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Abstract P331: Upregulated TBX1 by genetic variants are associated with human congenital heart disease

Circulation Research ◽

10.1161/res.129.suppl_1.p331 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Liwei Yu ◽

Binbin Li ◽

Hongyan Wang

Keyword(s):

Congenital Heart Disease ◽

Heart Disease ◽

Genetic Variants ◽

Heart Development ◽

Congenital Heart ◽

Minor Allele ◽

Expression Level ◽

Cardiac Progenitors ◽

Rare Mutation ◽

The Impact

Congenital heart disease (CHD) is the most common human birth defect worldwide. The cause of CHD is so far not well understood. Uncovering genetic factors leading to CHD is still a pressing task to be solved. TBX1 is one of the transcription factors early expressed in embryonic cardiac progenitors. In animal models, imbalanced TBX1 activity leads to cardiac defects. Given the dosage effect of TBX1, it is possible that genetic variant altering TBX1 function or expression level would affect heart development and contribute to CHD. In order to study the association of genetic variants of TBX1 and CHD susceptibility, we performed genetic screening in 409 CHD patients and 213 healthy controls. Bioinformatic and in vitro functional studies were performed to evaluate the impact of genetic variants. One single nucleotide polymorphism (SNP), rs41260844, in TBX1 promoter region was identified to be associated with CHD. The minor allele of rs41260844 is associated with higher CHD risk and shows increased TBX1 promoter activity (Fig A). Further study showed the minor allele attenuates TBX1 promoter binding affinity with nuclear protein(s) (Fig B). In addition, a novel case-specific missense rare mutation, p.P164L, in T-box domain was identified and predicted as a deleterious mutation. Functional analysis showed a trend of increased TBX1 function with the rare mutation. In summary, we concluded that a higher TBX1 expression level or activity is associated with CHD susceptibility, which could affect TBX1 downstream targets and thus disrupt the balance of the complex regulation network during cardiogenesis.

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Influence of Paroxysmal Nocturnal Hemoglobinuria Clone Positivity on Outcome of Childhood Acquried Aplastic Anemia: A Multicenter Center Study

Blood ◽

10.1182/blood-2018-99-118858 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5101-5101

Author(s):

Sule Unal Cangul ◽

Deniz Yilmaz Karapinar ◽

Arzu Yazal Erdem ◽

Husniye Nese Yarali ◽

Hamiyet Hekimci Ozdemir ◽

...

Keyword(s):

Aplastic Anemia ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Conflicts Of Interest ◽

Accurate Determination ◽

Immunosuppressive Treatment ◽

Indirect Bilirubin ◽

Significant Difference ◽

Causes Of Mortality ◽

The Impact ◽

Pnh Clone

Abstract Introduction: Paroxysmal nocturnal hemoglobinuria(PNH) in the classical hemolytic form is rare among childhood and children may present with aplastic anemia (AA) phenotype or with an overlapping phenotype of both aplastic and hemolytic components. In the recent years the introduction of FLAER methods prompted a more accurate determination of PNH clones in the suspected patients. Among the patients with aplastic anemia, PNH clone positivity has been detected in 18-59% of these patients with more sensitive FLAER methods. There is limited data related to the effect of PNH clone positivity on treatment outcomes in pediatric AA. Methods: A total of 142 patients, who were diagnosed with acquired AA, from 12 centers were included. All of the patients were tested for PNH clone positivity with either CD55/CD59, FLAER. None of the patients were DEB positive. Results: PNH clone was tested initially at diagnosis with CD55/CD59 flow cytometric analyses in 64 (45%) of the ppatients 66 received IST.atients and with FLAER method in 78 (55%) of the patients. Thirthy three (23.2%) were found to be PNH clone positive, two of them were negative with CD55/CD59 at diagnosis, but were found positive with FLAER during follow-up.Of the PNH clone positive patients 71% were male, compared to 43% in PNH clone nnegative group (p=0.001). The mean ages of patients in PNH clone positive and negative groups were 146±50 vs 121±53 months, respectively(p=0.016). There was no statistically significant difference between clone postive and negative patients in terms of mean Hb, WBC, platelet, AST, ALT, indirect bilirubin and haptoglobin levels measured at diagnosis of AA. However serum creatinin level was significantly higher at diagnosis in PNH clone postive patients compared to negative patients (0.57±0.18 vs 0.49±0.15 mg/dl); whereas MCV levels were significantly higher in clone postive group (93.2±9.7 vs 87±11.5 fl). Of 33 patients with PNH clone positivity, 19 (57.6%) were treated with immunosuppressive treatment (IST), as first line treatment, whereas of the clone negative 109 patients, 66 received IST (60.6%) (p=0.76). Of the 19 clone postive patients 11 (57.9%) had treatment response; whereas 36/65 (55.4%) of clone negative patients responded to IST. There was no statistically significant difference between PNH clone positive and negative groups in terms of IST response. IST response was not affected according to gender, age, ALT, AST, WBC, Hb, thrombocyte count, size of the erythrocyte and granulocyte clone at diagnosis. Thrombosis developed in 2 of clone positive (6.1%) and 2 of clone gative patients (1.8%) (p=0.23). Three year survival was 97.6±2.4% and 37.2±8.7% among IST responders and IST non-responders, respectively (p=0.0). Of the 142 patients, 59 underwent HSCT, five received eculizimab and 1 received eltrombopag treatments. Three patients developed clonality during follow-up. Thirty-seven (26%) of the patients deceased among the all study group and the causes of mortality were fungal infection, sepsis, pneumonia, leukemia progression, HSCT related complications, thrombosis and bleeding. Conclusions: PNH clone positivity was found in 23.2% of our patients with pediatric acquired AA. We found no impact of clone positivity on outcome in terms of IST response. This is one of the largest cohort of pediatric patients which investigated the impact of PNH clone positivity on AA outcome. Disclosures No relevant conflicts of interest to declare.

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