Rare SNP Research Articles

Ancestry-Specific Genetic Determinants of Clonal Haematopoiesis: A Comparative Analysis of 136,401 Admixed Americans and 419,228 Europeans

Introduction: Somatic mutations in blood stem cells can drive clonal expansion and result in clonal haematopoiesis (CH). CH is a precursor to myeloid malignancies, and is increasingly also recognised as a risk factor for non-malignant diseases. CH has been investigated in Europeans, but remains understudied in non-European populations. Here, we investigate the causes and consequences of CH in two large prospective cohort studies, specifically the Mexico City Prospective Study (MCPS) and UK Biobank (UKB). MCPS represents the largest CH study in a non-European population to date. Methods: Admixed Americans were identified from MCPS (n=136,401) while Europeans were identified from the UKB (n=419,228) participants. Somatic variant calling was performed using Mutect2 on whole exome sequencing (WES) data across a selected panel of genes to identify putative CH driver mutations. WES was performed at an average depth of 55x, and CH was defined using a variant allele frequency of ≥3%. Genome-wide association studies (GWAS) for CH were performed using imputed array data, and exome-wide association studies (ExWAS) and gene-level association analyses using WES data. Global/Continental-level ancestry was assigned based on peddy score ≥95% while local ancestry was inferred using RFMix (Ziyatdinov et al., 2022). Results: The most recurrently mutated genes in MCPS and UKB were DNMT3A, TET2, ASXL1, PPM1D, TP53, SF3B1 and SRSF2. The prevalence of CH increased progressively with age to approximately 8/22 (36%) detected as carriers at age 100 and above. CH was 40% more prevalent in age-matched Europeans (4.96%) compared to Admixed Americans (3.10%). Inter-population comparison revealed overall CH, DNMT3A-, TET2-, ASXL1-, PPM1D-, TP53-, JAK2-, and SRSF2-mutant CH to be more prevalent in UKB relative to MCPS (Figure A). Intra-population analysis of the Admixed American cohort further revealed that individuals with a higher fraction of European ancestry were at higher risk of overall CH, DNMT3A-, ASXL1-, and SRSF2-mutant CH, but not TET2-, PPM1D-, TP53-, and JAK2-mutant CH. These suggest differences in relative contribution of genetic, lifestyle or environment factors to specific CH genes. CH GWAS performed in Admixed Americans recapitulated previously reported variants in Europeans, and also identified novel, ancestry-specific variants associated with CH risk, including SNPs upstream of TCL1B (rs968294563: OR=1.79, P=2.01x10 -9; rs187319135: OR=1.85, P=2.69x10 -9). Notably, TCL1B variants were associated with an increased risk of TET2- and ASXL1-mutant CH (rs968294563: OR=3.17, P=3.82x10 -16 for TET2; OR=2.36, P=2.40x10 -3 for ASXL1), but a decreased risk of DNMT3A-mutantCH (rs968294563: OR=0.51, P=6.32x10 -4) (Figure B). The minor allele frequency (MAF) of the most common TCL1B variant in Admixed Americans was 1.23% but was virtually absent in Europeans. CH ExWAS can identify rare causal variants not captured by genotyping or imputation, and these variants may be in linkage disequilibrium with common variants detectable via GWAS. Indeed, ExWAS in Admixed Americans identified one rare SNP on the TCL1B promoter (rs774615666: OR=2.24, P=1.94x10 -8) that was associated with an increased risk of TET2-mutantCH (OR=4.19, P=2.77x10 -10) and a decreased risk of DNMT3A-mutantCH (OR=0.13, P=1.65x10 -4), mirroring our GWAS findings. The rs774615666 risk allele was >200-fold more common in Admixed Americans (MAF: 0.33%) compared to Europeans (MAF: 0.0011%) and was not in linkage disequilibrium with the previously reported TCL1A promoter CH risk SNP in Europeans (Weinstock et al., 2023). Meta-analyses were performed using 555,629 individuals, from both MCPS Admixed American CH GWAS and UKB European CH GWAS. Of the 11 loci reported for overall CH, one was novel, namely GACAT3/ CYRIA. Lastly, we investigated the phenotypic associations of CH in Admixed Americans. Gene-specific CH was associated with increased risk of death from haematological malignancies ( TET2, TP53, SF3B1, and SRSF2), other cancer types ( ASXL1, DNMT3A, and SRSF2) and cardiovascular diseases( DNMT3A, TP53). Conclusions: The substantial difference in CH prevalence between populations and the ancestry-specific genetic associations, demonstrate how the analysis of non-European cohorts can generate novel insights and highlights the importance of such analyses in advancing health equality amongst different human populations.

Evidence of a genetic background predisposing to complex regional pain syndrome type 1

BackgroundComplex regional pain syndrome type 1 (CRPS-1) is a rare, disabling and sometimes chronic disorder usually arising after a trauma. This exploratory study examined whether patients with chronic CRPS-1 have...

The complete and fully-phased diploid genome of a male Han Chinese.

Since the release of the complete human genome, the priority of human genomic study has now been shifting towards closing gaps in ethnic diversity. Here, we present a fully phased and well-annotated diploid human genome from a Han Chinese male individual (CN1), in which the assemblies of both haploids achieve the telomere-to-telomere (T2T) level. Comparison of this diploid genome with the CHM13 haploid T2T genome revealed significant variations in the centromere. Outside the centromere, we discovered 11,413 structural variations, including numerous novel ones. We also detected thousands of CN1 alleles that have accumulated high substitution rates and a few that have been under positive selection in the East Asian population. Further, we found that CN1 outperforms CHM13 as a reference genome in mapping and variant calling for the East Asian population owing to the distinct structural variants of the two references. Comparison of SNP calling for a large cohort of 8869 Chinese genomes using CN1 and CHM13 as reference respectively showed that the reference bias profoundly impacts rare SNP calling, with nearly 2 million rare SNPs miss-called with different reference genomes. Finally, applying the CN1 as a reference, we discovered 5.80 Mb and 4.21 Mb putative introgression sequences from Neanderthal and Denisovan, respectively, including many East Asian specific ones undetected using CHM13 as the reference. Our analyses reveal the advances of using CN1 as a reference for population genomic studies and paleo-genomic studies. This complete genome will serve as an alternative reference for future genomic studies on the East Asian population.

Structure-Switchable Hairpin-Powered Exponential Replications for Sensing Attomolar microRNA-Related Single Nucleotide Polymorphisms in Human Cancer Tissues with Zero Background.

MicroRNA (miRNA)-related single-nucleotide polymorphisms (miR-SNPs) are a novel class of genetic variations involved in multiple cellular functions, and they have emerged as promising biomarkers for cancer diagnostics and prognostics. Herein, we demonstrate for the first time the structure-switchable hairpin-powered exponential replications for sensing attomolar miR-SNPs in human cancer tissues with zero background. In the presence of target miR-196a2T, hairpin probes (i.e., HP1 and HP2) are splinted together to construct the dumbbell-shaped probe (DSP) with SplintR ligase catalysis. Once the DSP is formed, the self-primed polymerization extension and linear FIP/BIP-primed strand displacement DNA synthesis (SDS) are automatically repeated to activate self-circulated exponential amplification, producing large amounts of double-stranded DNAs (dsDNAs) which can be real-time monitored using SYBR Green I. This nanodevice can detect miR-196a2T with an ultralow detection limit of 2.46 aM; distinguish rare miR-196a2 SNP with a selectivity factor of 0.001%; and even profile miR-196a2T in human tissues for nonsmall cell lung cancer (NSCLC) diagnosis, risk assessment, and cancer type prediction. Notably, this nanodevice can be rapidly and homogeneously used in one tube in a real-time and label-free manner, providing a powerful point-of-care platform for noninvasive diagnostics and prognostics of miR-SNPs-related human cancers.

The APETALA2 homolog CaFFN regulates flowering time in pepper

Flowering time is an important agronomic trait that contributes to fitness in plants. However, the genetic basis of flowering time has not been extensively studied in pepper. To understand the genetics underlying flowering time, we constructed an F2 population by crossing a spontaneous early flowering mutant and a late-flowering pepper line. Using bulked segregant RNA-seq, a major locus controlling flowering time in this population was mapped to the end of chromosome 2. An APETALA2 (AP2) homolog (CaFFN) cosegregated with flowering time in 297 individuals of the F2 population. A comparison between the parents revealed a naturally occurring rare SNP (SNP2T > C) that resulted in the loss of a start codon in CaFFN in the early flowering mutant. Transgenic Nicotiana benthamiana plants with high CaFFN expression exhibited a delay in flowering time and floral patterning defects. On the other hand, pepper plants with CaFFN silencing flowered early. Therefore, the CaFFN gene acts as a flowering repressor in pepper. CaFFN may function as a transcriptional activator to activate the expression of CaAGL15 and miR156e and as a transcriptional repressor to repress the expression of CaAG, CaAP1, CaSEP3, CaSOC1, and miR172b based on a qRT-PCR assay. Direct activation of CaAGL15 by CaFFN was detected using yeast one-hybrid and dual-luciferase reporter assays, consistent with the hypothesis that CaFFN regulates flowering time. Moreover, the CaFFN gene association analysis revealed a significant association with flowering time in a natural pepper population, indicating that the CaFFN gene has a broad effect on flowering time in pepper. Finally, the phylogeny, evolutionary expansion and expression patterns of CaFFN/AP2 homologs were analyzed to provide valuable insight into CaFFN. This study increases our understanding of the involvement of CaFFN in controlling flowering time in pepper, thus making CaFFN a target gene for breeding early maturing pepper.

Familial Psychosis Associated With a Missense Mutation at MACF1 Gene Combined With the Rare Duplications DUP3p26.3 and DUP16q23.3, Affecting the CNTN6 and CDH13 Genes.

Psychosis is a highly heritable and heterogeneous psychiatric condition. Its genetic architecture is thought to be the result of the joint effect of common and rare variants. Families with high prevalence are an interesting approach to shed light on the rare variant’s contribution without the need of collecting large cohorts. To unravel the genomic architecture of a family enriched for psychosis, with four affected individuals, we applied a system genomic approach based on karyotyping, genotyping by whole-exome sequencing to search for rare single nucleotide variants (SNVs) and SNP array to search for copy-number variants (CNVs). We identified a rare non-synonymous variant, g.39914279 C > G, in the MACF1 gene, segregating with psychosis. Rare variants in the MACF1 gene have been previously detected in SCZ patients. Besides, two rare CNVs, DUP3p26.3 and DUP16q23.3, were also identified in the family affecting relevant genes (CNTN6 and CDH13, respectively). We hypothesize that the co-segregation of these duplications with the rare variant g.39914279 C > G of MACF1 gene precipitated with schizophrenia and schizoaffective disorder.

The Arg-293 of Cryptochrome1 is responsible for the allosteric regulation of CLOCK-CRY1 binding in circadian rhythm

Mammalian circadian clocks are driven by transcription/translation feedback loops composed of positive transcriptional activators (BMAL1 and CLOCK) and negative repressors (CRYPTOCHROMEs (CRYs) and PERIODs (PERs)). CRYs, in complex with PERs, bind to the BMAL1/CLOCK complex and repress E-box-driven transcription of clock-associated genes. There are two individual CRYs, with CRY1 exhibiting higher affinity to the BMAL1/CLOCK complex than CRY2. It is known that this differential binding is regulated by a dynamic serine-rich loop adjacent to the secondary pocket of both CRYs, but the underlying features controlling loop dynamics are not known. Here we report that allosteric regulation of the serine-rich loop is mediated by Arg-293 of CRY1, identified as a rare CRY1 SNP in the Ensembl and 1000 Genomes databases. The p.Arg293His CRY1 variant caused a shortened circadian period in a Cry1-/-Cry2-/- double knockout mouse embryonic fibroblast cell line. Moreover, the variant displayed reduced repressor activity on BMAL1/CLOCK driven transcription, which is explained by reduced affinity to BMAL1/CLOCK in the absence of PER2 compared with CRY1. Molecular dynamics simulations revealed that the p.Arg293His CRY1 variant altered a communication pathway between Arg-293 and the serine loop by reducing its dynamicity. Collectively, this study provides direct evidence that allosterism in CRY1 is critical for the regulation of circadian rhythm.

The Neurotransmitter Receptor Gabbr1 Regulates Proliferation and Function of Hematopoietic Stem and Progenitor Cells

Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells which differentiate to maintain and replenish blood lineages throughout life. Due to these characteristics, HSPC transplants represent a cure for patients with a variety of hematological disorders. HSPC function and behavior is tightly regulated by various cell types and factors in the bone marrow niche. The nervous system has been shown to indirectly influence hematopoiesis by innervating the niche; however, we present a direct route of HSPC regulation via expression of neurotransmitter receptors on HSPC surface. We have identified Gamma Aminobutyric acid (GABA) receptor B subunit 1 (Gabbr1), a hitherto unknown hematopoietic player, as a regulator of HSPC function. GABBR1 is known to be expressed on human HSPCs (Steidl et al., Blood 2004), however its function in their regulation remains unknown. Based on published RNA-seq data (Nestorowa et al., Blood 2016), we discovered that Gabbr1 is expressed on a subset of HSPCs. We confirmed this expression using RT-qPCR to assay hematopoietic populations in the bone marrow (BM). Surface receptor expression analysis showed that Gabbr1 protein is expressed on a subset of BM HSPCs. To detect GABA, the ligand for Gabbr1 in the BM microenvironment, we utilized imaging mass spectrometry (IMS). We detected regionally specific GABA signal in the endosteal region of the BM. We further identified B cells as a cellular source of GABA in the BM. To understand the role of Gabbr1 in hematopoiesis, we generated CRISPR-Cas9 Gabbr1 null mutants on a C57/BL6 background suitable for hematopoietic studies and studied their hematopoietic phenotype. We discovered a decrease in the absolute number of Lin-Sca1+cKit+ (LSK) HSPCs, but the long-term hematopoietic stem cells (LT-HSCs) remain unaffected. Further analysis of peripheral blood of Gabbr1 null mutants showed decreased white blood cells due to reduced B220+ cells. This differentiation defect was confirmed in an in vitro differentiation assay where Gabbr1 null HSPCs displayed an impaired ability to produce B cells. We show that Gabbr1 null HSCs show diminished reconstitution ability when transplanted in a competitive setting. Reduced Gabbr1 null HSC reconstitution persisted in secondary transplant recipients indicating a cell autonomous role for Gabbr1 in regulating reconstitution of HSCs in transplant recipients. Our results show a crucial role for Gabbr1 in HSPC regulation and may translate to human health as a rare human SNP within the GABBR1 locus that correlates with altered leukocyte counts has been reported (Astle et al., Cell 2016). Our studies indicate an important role for Gabbr1 in HSPC reconstitution and differentiation into B cell lineages. Disclosures No relevant conflicts of interest to declare.

Genotype Analysis of Adolescents with Heavy Menstrual Bleeding and Low Von Willebrand Activity - Report of a Multi-Center Study

Introduction: Low von Willebrand factor (VWF) activity is prevalent in adolescents with heavy menstrual bleeding (HMB). There is a need to better genetically characterize these patients and improve our understanding of the pathophysiology of their bleeding risk. Methods: One of the main objectives of this multi-center, single arm, observational cohort study was to genotype adolescent females with HMB and low VWF (≥ 30 and ≤ 50 IU/dL) by means of whole exome sequencing (WES), to identify variants throughout the exome that may modulate risk for bleeding. Post-menarchal females < 21 years with HMB (defined as PBAC score >100) and low VWF were eligible for the study. All patients were enrolled by participating centers where blood samples were collected. Exome data for 86 cases and 900 unrelated pediatric (<21 years) controls taken from a genetic study on Chiari 1 Malformation (CM1) were aligned and sorted, and variants called using the Sentieon software package. Annotation including allele frequencies, function, amino acid change and Clinvar rating among others was obtained using ANNOVAR. Variants were retained if there was a genotype call rate of >0.8, GQ>20, AB between 0.3-0.7 and min DP>8. Case/control analyses included common and rare SNP associations, gene burden analysis of both rare nonsynonymous variants and ClinVar 'pathogenic' variants, and gene-set burden analyses. Variants were considered rare if they had a minor allele frequency of <1% in the Genome Aggregation Database (gnomAD). Count differences between cases/controls were determined by Fisher's Exact test. Results: Of the 113 subjects enrolled, 86 had sufficient blood samples collected for WES. The median age was 16.2 years (range: 11.5-19.6). 36% of cases showed variants in VWF vs. 26% of controls (p=0.14). After multiple test correction, WES revealed a significant common variant association in FERMT2 with an LD block with a frequency of 24% in cases and 6% in controls (p=7.5x10-7). Rare variant analysis showed a significant association with an intronic variant in ABCA13 (p=1.6x10-7). Among the gene burden analysis using rare nonsynonymous variants, 2 genes of interest passed multiple test correction: IL12B and DTNBP1. When using ClinVar 'pathogenic' variants as the input for the gene burden analysis, 4 genes of interest passed multiple test correction: HBM, MYLK, RUNX1 and CD36. Gene-set burden analysis revealed 5 significant pathways of interest, including platelet degranulation, platelet alpha granule lumen and erythrocyte differentiation. We then focused a subset of known risk genes and compared the number of missense, nonsense and ClinVar 'pathogenic' variants between cases and controls. GP6, and MTHFR had significantly more missense variants in controls vs. cases (p=0.003 and 0.01, respectively), and F13B approached significance, with more missense variants in controls than cases (p=0.07). Conclusion: We found VWF variants in 36% of subjects, in accordance with previous reports. We found novel SNP associations with variants in FERMT2 and ABCA13.FERMT2 encodes for the integrin, Kindlin 2, which has been shown to be critical for supporting vascular integrity. Several other relevant genes passed multiple test correction in gene burden analyses, including genes known to cause Hermansky-Pudlak syndrome (DTNBP1), familial platelet disorder (RUNX1), platelet glycoprotein IV deficiency (CD36) and aortic aneurysm (MYLK). This association with platelet disorders was strengthened by our gene-set burden results, which implicate several platelet-related pathways. These data suggest that while a subset of HMB patients may have their bleeding explained by variants in VWF, there is a role for other hemostasis, platelet biology and vascular integrity related gene variants which can contribute to the variation in bleeding severity in adolescents with low-VWF related HMB. Study supported by an investigator-initiated research grant from Shire US Inc., now part of Takeda Disclosures O'Brien: Bristol Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees. Mullins:Takeda, Bayer: Other: Advisory Board. Sidonio:Takeda: Research Funding. Ragni:Sangamo: Consultancy, Research Funding; Takeda: Research Funding; Bioverativ: Consultancy, Research Funding; Spark: Consultancy, Research Funding; BioMarin: Consultancy, Research Funding; Alnylam/Sanofi, ATHN, BioMarin, Bioverativ, Sangamo, Spark: Research Funding; Alnylam/Sanofi, BioMarin, Bioverativ, Spark: Consultancy; Alnylam Pharmaceuticals Inc., Baxalta/Takeda, BioMarin, Bioverativ, and Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees; American Thrombosis Hemostasis Network: Other: Committee work; Baxalta/Takeda, CSL Behring, Genentech, a member of the Roche Group, OPKO Biologics, and Vascular Medicine Institute: Research Funding. Kulkarni:Sanofi/ Bioverativ, Bayer, Biomarin, Shire/Takeda, Novo Nordisk, Freeline: Other: clinical trial research grants ; Bioverativ/Sanofi, BPL, Genentech, Kedrion, Novo Nordisk, Octapharma, Pfizer, Takeda, Catalyst Bioscience Bayer: Membership on an entity's Board of Directors or advisory committees. Srivaths:Shire US Inc., now part of Takeda: Research Funding.

Two Novel Rare Strongly Linked Missense SNPs (P27R and A85G) Within the GDF9 Gene Were Significantly Associated With Litter Size in Shaanbei White Cashmere (SBWC) Goats.

Growth differentiation factor 9 (GDF9) is a high-fertility candidate gene that plays a crucial role in early folliculogenesis in female mammals. In this study, direct sequencing was used to screen possible SNP loci in the goat GDF9 gene. Three SNP loci, p.proline27alanine (P27R), p.leucine61leucine (L61L), and p.alanine85glycine (A85G), were identified in Shaanbei white cashmere (SBWC) goats. Among the three SNPs, two rare missense SNP loci (P27R and A85G) were discovered to be strongly linked with each other (D′ value = 0.926, r2 value = 0.703). Both P27R and A85G loci had two genotypes: wild type and heterozygous type. A85G exerted a significant effect on litter size (P = 0.029) in SBWC goats, and the heterozygous genotype was superior in comparison with the wild type. The heterozygous genotype was also superior in P27R but no significant association was found. However, the combination genotypes of P27R and A85G were identified to have superior effects on litter size (P = 3.8E−15). This information suggested that these two SNPs influenced litter size in goats synergistically. Combining this information with our previous studies, we propose that the GDF9 gene is the principal high-fertility candidate gene and that the A85G locus is a promising SNP that affects litter size in goats. These results may fill a research gap regarding rare mutations as well as provide crucial molecular markers that could be useful in marker-assisted selection (MAS) goat rearing when selecting superior individuals.

R269C variant of ESR1: high prevalence and differential function in a subset of pancreatic cancers

BackgroundEstrogen receptor α (ESR1) plays a critical role in promoting growth of various cancers. Yet, its role in the development of pancreatic cancer is not well-defined. A less studied region of ESR1 is the hinge region, connecting the ligand binding and DNA domains. rs142712646 is a rare SNP in ESR1, which leads to a substitution of arginine to cysteine at amino acid 269 (R269C). The mutation is positioned in the hinge region of ESR1, hence may affect the receptor structure and function. We aimed to characterize the activity of R269C-ESR1 and study its role in the development of pancreatic cancer.MethodsTranscriptional activity was evaluated by E2-response element (ERE) and AP1 –luciferase reporter assays and qRT-PCR. Proliferation and migration were assessed using MTT and wound healing assays. Gene-expression analysis was performed using RNAseq.ResultsWe examined the presence of this SNP in various malignancies, using the entire database of FoundationOne and noted enrichment of it in a subset of pancreatic non-ductal adenocarcinoma (n = 2800) compared to pancreatic ductal adenocarcinoma (PDAC) as well as other tumor types (0.53% vs 0.29%, p = 0.02). Studies in breast and pancreatic cancer cells indicated cell type-dependent activity of ESR1 harboring R269C. Thus, expression of R269C-ESR1 enhanced proliferation and migration of PANC-1 and COLO-357 pancreatic cancer cells but not of MCF-7 breast cancer cells. Moreover, R269C-ESR1 enhanced E2-response elements (ERE) and AP1-dependent transcriptional activity and increased mRNA levels of ERE and AP1-regulated genes in pancreatic cancer cell lines, but had a modest effect on MCF-7 breast cancer cells. Accordingly, whole transcriptome analysis indicated alterations of genes associated with tumorigenicity in pancreatic cancer cells and upregulation of genes associated with cell metabolism and hormone biosynthesis in breast cancer cells.ConclusionsOur study shed new light on the role of the hinge region in regulating transcriptional activity of the ER and indicates cell-type specific activity, namely increased activity in pancreatic cancer cells but reduced activity in breast cancer cells. While rare, the presence of rs142712646 may serve as a novel genetic risk factor, and a possible target for therapy in a subset of non-ductal pancreatic cancers.

MON-514 Detection of RAS Mutations and RET/PTC Fusions in Thyroid Cancer Using Microfluidic Digital PCR

Background:The identification of somatic mutations and gene fusions is crucial for guiding therapeutic decisions in patients with thyroid cancer. Microfluidic digital PCR is currently considered as a technique of choice for highly sensitive detection of gene mutations/fusion. We recently demonstrated that dPCR is a useful tool for detection of BRAFV600E and TERT promoter mutations in thyroid tumors.Objectives:This study aimed to determine the analytic and clinical validity of dPCR for detection of RAS mutations and RET/PTC fusions in thyroid cancer tissue.Material and Methods:Thyroid tissues from 75 patients with PTCs (58 classical PTC (CPTC) and 17 follicular variant (FVPTC) were used for DNA and RNA extraction. The rare mutation SNP genotyping assays which were multiplexed for detection of mutant and wild type NRASQ61; as well as RET/PTC1 and RET/PTC3 were synthesized by Thermo Fisher Scientific. Digital PCR was performed using a QuantStudio 3D Digital PCR platform. QuantStudio Software was used for relative and quantitative data analysis.Results:NRASQ61 was detected in 0/58 CPTC and in 6/17 (35%) FVPTC. The ratios of mutant/total varying from 11.7% to 61.5%. Among patients with FVPTC there were no significant associations between the presence of NRASQ61 and patient’s age, sex, multifocal growth, extra-thyroidal invasion and lymph node metastases. The ratios mutant/total correlated with tumor size in patients harboring NRASQ61. In 23 cases, RET/PTC1 and RET/PTC3 transcripts were examined. RET/PTC1 and RET/PTC3 transcripts were detected in 3 and 1 case, respectively. RET/PTCs were detected in CPTC, but not in FVPTC. RET/PTC positive tumors were characterized by multi-focal patterns of growth, presence of extra-thyroidal invasion, and presence of lymph node metastases (4 of 4 cases with RET/PTC). There were not RET/PTCs positive tumors harboring simultaneously anomalies in RAS oncogene.Conclusions:Microfluidic digital PCR allows specific, sensitive and rapid detection of RAS mutations and RET/PTC fusions in thyroid tissue samples. Implementation of dPCR-based assays may facilitate analysis of thyroid tumors and support research in patients with thyroid cancer.

Microfluidic Droplet Digital PCR Is a Powerful Tool for Detection of BRAF and TERT Mutations in Papillary Thyroid Carcinomas.

We examined the utility of microfluidic digital PCR (dPCR) for detection of BRAF and TERT mutations in thyroid tumors. DNA extracted from 100 thyroid tumors (10 follicular adenomas, 10 follicular cancers, 5 medullary cancers, and 75 papillary thyroid cancer (PTC) were used for detection of BRAF and TERT mutations. Digital PCRs were performed using rare mutation SNP genotyping assays on QuantStudio 3D platform. In PTCs, BRAFV600E was detected by dPCR and Sanger sequencing in 42/75 (56%) and in 37/75 (49%), respectively. BRAFV600E was not detected in other tumors. The ratio of mutant/total BRAF alleles varied from 4.7% to 47.5%. These ratios were higher in classical PTCs (27.1%) as compared to follicular variant PTCs (9.4%) p = 0.001. In PTCs with and without metastases, the ratios of mutant/total BRAF alleles were 27.6% and 18.4%, respectively, (p = 0.03). In metastatic lesions percentages of mutant/total BRAF alleles were similar to those detected in primary tumors. TERTC228T and TERTC250T were found in two and one cases, respectively, and these tumors concomitantly harbored BRAFV600E. These tumors exhibited gross extra-thyroidal extension, metastases to lymph nodes, and pulmonary metastases (one case). Our results showed that dPCR allows quantitative assessment of druggable targets in PTCs and could be helpful in a molecular-based stratification of prognosis in patients with thyroid cancer.

The Neurotransmitter Receptor Gabbr1 Regulates Proliferation and Function of Hematopoietic Stem and Progenitor Cells

Hematopoietic stem and progenitor cells (HSPCs) have multi-lineage potential and can be used in transplants as a curative treatment for various hematopoietic diseases. HSPC function and behavior is tightly regulated by various cell types and factors in the bone marrow niche. One level of regulation comes from the sympathetic nervous system that innervates the niche and releases neurotransmitters to stromal cells. However, the direct regulation of HSPCs via cell surface expression of neural receptors has not been functionally explored. Using imaging mass spectrometry, we detected strong and regionally specific gamma-aminobutyric acid (GABA) neurotransmitter signal in the endosteal region of mouse bone marrow. GABBR1 is known to be expressed on human HSPCs (Steidl et al., Blood 2004), however its function in their regulation has not been investigated. Based on published mouse HSPC single cell RNA-seq data (Nestorowa et al., Blood 2016), we found that a subset of HSPCs expressed the GABA type B receptor subunit 1 (Gabbr1). We confirmed by surface receptor expression that a subset of mouse bone marrow HSPCs express Gabbr1 protein. Using the same single cell RNA-seq data as above, our own gene set enrichment analysis (GSEA) revealed positive correlation of Gabbr1 expression with genes involved in immune system processes, such as response to type I interferons. We generated a CRISPR-Cas9 Gabbr1 mutant mouse model on a C57/BL6 background suitable for hematopoietic studies. Analysis of Gabbr1 mutant bone marrow cells revealed a reduction in the absolute number of Lin-Sca1+cKit+ (LSK) HSPCs, but no change in the number of long-term hematopoietic stem cells (LT-HSCs). With further hematopoietic profiling, we discovered reduced numbers of white blood cells in peripheral blood that was primarily due to fewer B220+ cells. We show that Gabbr1 null HSPCs display reduced proliferative capacity, as well as diminished reconstitution ability when transplanted in a competitive setting. An in vitro differentiation assay revealed the impaired ability of Gabbr1 null HSPCs to produce B cell lineages. We tested our predicted association with type I interferon response by administration of poly(I:C) and found reduced HSPC proliferation in Gabbr1 null mice. Our results may translate well to humans, as a rare human SNP within the GABBR1 locus was found that correlates with altered leukocyte counts (Astle et al., Cell 2016). Our results indicate an important role for Gabbr1 in the regulation of HSPC proliferation and differentiation, highlighting Gabbr1 as an emerging factor in the modulation of HSPC function and behavior. Disclosures No relevant conflicts of interest to declare.

SIRT1 Allele Frequencies in Depressed Patients of European Descent in Russia

Depressive disorder (DD) is a widespread mental disorder. Although DD is to some extent inherited, the genes contributing to the risk of this disorder and its genetic mechanisms remain poorly understood. A recent large-scale genome-wide association Chinese study revealed a strong association between the SIRT1 gene variants and DD. The aim of this study was to analyze the occurrence of heterozygote carriers and search for rare SNP variants of the SIRT1 gene in a cohort of DD patients as compared with a cohort of randomly selected members of the Russian population. The complete coding sequences of the SIRT1 gene from 1024 DNA samples from the general Russian population and from 244 samples from patients with DD were analyzed using targeted sequencing. Four new genetic variants of the SIRT1 were discovered. While no significant differences in the allele frequencies were found between the DD patients and the general population, differences between the frequencies of homozygote carriers of specific alleles and occurrences of heterozygous were found to be significant for rs2236318 (P < 0.0001), and putatively, rs7896005 (P < 0.05), and rs36107781 (P < 0.05). The study found for the first time that two new SNPs (i.e., 10:69665829 and 10:69665971) along with recently reported ones (rs773025707 and rs34701705), are putatively associated with DD. The revealed DD-associated SIRT1 SNPs might confer susceptibility to this disorder in Russian population of European descent.

Role of variant allele fraction and rare SNP filtering to improve cellular DNA repair endpoint association.

BackgroundLarge cancer genome studies continue to reveal new players in treatment response and tumorigenesis. The discrimination of functional alterations from the abundance of passenger genetic alterations still poses challenges and determines DNA sequence variant selection procedures. Here we evaluate variant selection strategies that select homozygous variants and rare SNPs and assess its value in detecting tumor cells with DNA repair defects.MethodsTo this end we employed a panel of 29 patient-derived head and neck squamous cell carcinoma (HNSCC) cell lines, of which a subset harbors DNA repair defects. Mitomycin C (MMC) sensitivity was used as functional endpoint of DNA crosslink repair deficiency. 556 genes including the Fanconi anemia (FA) and homologous recombination (HR) genes, whose products strongly determine MMC response, were capture-sequenced.ResultsWe show a strong association between MMC sensitivity, thus loss of DNA repair function, and the presence of homozygous and rare SNPs in the relevant FA/HR genes. Excluding such selection criteria impedes the discrimination of crosslink repair status by mutation analysis. Applied to all KEGG pathways, we find that the association with MMC sensitivity is strongest in the KEGG FA pathway, therefore also demonstrating the value of such selection strategies for exploratory analyses. Variant analyses in 56 clinical samples demonstrate that homozygous variants occur more frequently in tumor suppressor genes than oncogenes further supporting the role of a homozygosity criterion to improve gene function association or tumor suppressor gene identification studies.ConclusionTogether our data show that the detection of relevant genes or of repair pathway defected tumor cells can be improved by the consideration of allele zygosity and SNP allele frequencies.

Plastin 3 influences bone homeostasis through regulation of osteoclast activity.

Over 200 million people suffer from osteoporosis worldwide, one third of which will develop osteoporotic bone fractures. Unfortunately, no effective cure exists. Mutations in plastin 3 (PLS3), an F-actin binding and bundling protein, cause X-linked primary osteoporosis in men and predisposition to osteoporosis in postmenopausal women. Moreover, the strongest association so far for osteoporosis in elderly women after menopause was connected to a rare SNP in PLS3, indicating a possible role of PLS3 in complex osteoporosis as well. Interestingly, 5% of the general population are overexpressing PLS3, with yet unknown consequences. Here, we studied ubiquitous Pls3 knockout and PLS3 overexpression in mice and demonstrate that both conditions influence bone remodeling and structure: while Pls3 knockout mice exhibit osteoporosis, PLS3 overexpressing mice show thickening of cortical bone and increased bone strength. We show that unbalanced PLS3 levels affect osteoclast development and function, by misregulating the NFκB pathway. We found upregulation of RELA (NFκB subunit p65) in PLS3 overexpressing mice-known to stimulate osteoclastogenesis-but strikingly reduced osteoclast resorption. We identify NFκB repressing factor (NKRF) as a novel PLS3 interactor, which increasingly translocates to the nucleus when PLS3 is overexpressed. We show that NKRF binds to the NFκB downstream target and master regulator of osteoclastogenesis nuclear factor of activated T cells 1 (Nfatc1), thereby reducing its transcription and suppressing osteoclast function. We found the opposite in Pls3 knockout osteoclasts, where decreased nuclear NKRF augmented Nfatc1 transcription, causing osteoporosis. Regulation of osteoclastogenesis and bone remodeling via the PLS3-NKRF-NFκB-NFATC1 axis unveils a novel possibility to counteract osteoporosis.

Functional SNP allele discovery (fSNPd): an approach to find highly penetrant, environmental-triggered genotypes underlying complex human phenotypes

BackgroundSignificant human diseases/phenotypes exist which require both an environmental trigger event and a genetic predisposition before the disease/phenotype emerges, e.g. Carbamazepine with the rare SNP allele of rs3909184 causing Stevens Johnson syndrome, and aminoglycosides with rs267606617 causing sensory neural deafness. The underlying genotypes are fully penetrant only when the correct environmental trigger(s) occur, otherwise they are silent and harmless. Such diseases/phenotypes will not appear to have a Mendelian inheritance pattern, unless the environmental trigger is very common (>50% per lifetime). The known causative genotypes are likely to be protein-altering SNPs with dominant/semi-dominant effect. We questioned whether other diseases and phenotypes could have a similar aetiology.MethodsWe wrote the fSNPd program to analyse multiple exomes from a test cohort simultaneously with the purpose of identifying SNP alleles at a significantly different frequency to that of the general population. fSNPd was tested on trial cohorts, iteratively improved, and modelled for performance against an idealised association study under mutliple parameters. We also assessed the seqeuncing depath of all human exons to determine which were sufficiently well sequenced in an exome to be sued by fSNPd - by assessing forty exomes base by base.ResultsWe describe a simple methodology for the detection of SNPs capable of causing a phenotype triggered by an environmental event. This uses cohorts of relatively small size (30–100 individuals) with the phenotype being investigated, their exomes, and thence seeks SNP allele frequencies significantly different from expected to identify potentially clinically important, protein altering SNP alleles. The strengths and weaknesses of this approach for discovering significant genetic causes of human disease are comparable to Mendelian disease mutation detection and Association Studies.ConclusionsThe fSNPd methodology is another approach, and has potentially significant advantage over Association studies in needing far fewer individuals, to detect genes involved in the pathogenesis of a diseases/phenotypes. Furthermore, the SNP alleles identified alter amino acids, potentially making it easier to devise functional assays of protein function to determine pathogenicity.

A genome-wide association meta-analysis on lipoprotein (a) concentrations adjusted for apolipoprotein (a) isoforms

High lipoprotein (a) [Lp(a)] concentrations are an independent risk factor for cardiovascular outcomes. Concentrations are strongly influenced by apo(a) kringle IV repeat isoforms. We aimed to identify genetic loci associated with Lp(a) concentrations using data from five genome-wide association studies (n = 13,781). We identified 48 independent SNPs in the LPA and 1 SNP in the APOE gene region to be significantly associated with Lp(a) concentrations. We also adjusted for apo(a) isoforms to identify loci affecting Lp(a) levels independently from them, which resulted in 31 SNPs (30 in the LPA, 1 in the APOE gene region). Seven SNPs showed a genome-wide significant association with coronary artery disease (CAD) risk. A rare SNP (rs186696265; MAF ∼1%) showed the highest effect on Lp(a) and was also associated with increased risk of CAD (odds ratio = 1.73, P = 3.35 × 10−30). Median Lp(a) values increased from 2.1 to 91.1 mg/dl with increasing number of Lp(a)-increasing alleles. We found the APOE2-determining allele of rs7412 to be significantly associated with Lp(a) concentrations (P = 3.47 × 10−10). Each APOE2 allele decreased Lp(a) by 3.34 mg/dl corresponding to ∼15% of the population’s mean values. Performing a gene-based test of association, including suspected Lp(a) receptors and regulators, resulted in one significant association of the TLR2 gene with Lp(a) (P = 3.4 × 10−4). In summary, we identified a large number of independent SNPs in the LPA gene region, as well as the APOE2 allele, to be significantly associated with Lp(a) concentrations.

Abstract LB-223: Whole genome sequencing of rhabdomyosarcoma germline cohort identifies low frequency of pathogenic mutations

Abstract Introduction: Rhabdomyosarcoma (RMS) is among the most frequently occurring tumors in Li-Fraumeni syndrome patients and is often observed in patients with a strong family history of cancer. Recent studies suggest that at least 10% of children with cancer harbor an underlying pathogenic germline mutation, although the frequency in children with RMS is not well characterized. In this study we sought to determine the rate of likely pathogenic germline mutations in rhabdomyosarcoma patients, unselected for family history. Methods: Of 275 RMS patients enrolled on one clinical trial (COG ARST0531), whole-genome sequencing was performed on blood-derived DNA of an initial set of 50 using the Illumina HiseqX to an average sequencing depth of 44.9X [34.2-58.4]. Single nucleotide variants (SNVs) and Indels were called and filtered to select only those occurring in exons or splicing regions. Variants were further filtered to include only those with a frequency of &amp;lt;0.5% in the ExAC database and lying in one of 99 genes identified as a cancer predisposition gene in the COSMIC Cancer Gene census. Nonsynonymous SNVs were required to be predicted as deleterious by one or more of Polyphen, mutation assessor or sift. Results: Across the 50 initial samples, a total of 290 variants ([0-17], median of 5) passed the described filters. Notably, no point mutations were identified in TP53 in the initial 50 samples. Two samples contained variants annotated as pathogenic in ClinVar - a frameshift deletion in CHEK2 and a rare SNP in MUTYH. Also notable was a potentially pathogenic frameshift deletion in MSH6 that lacked previous annotation. Conclusions: Our preliminary analysis suggests a lower incidence of causative TP53 mutations in RMS than previously suggested. Whole genome sequencing of the remaining samples in the cohort along with analysis of copy number and structural variants is ongoing. Statistical analysis of the entire cohort of 275 patient samples will allow for the most complete characterization of the germline genomic landscape of rhabdomyosarcoma to date. This information will better inform clinical surveillance and management parameters for RMS patients and families. Citation Format: Nicholas Light, Philip Lupo, Javed Khan, Joshua Schiffman, Douglas Hawkins, Adam Shlien, David Malkin. Whole genome sequencing of rhabdomyosarcoma germline cohort identifies low frequency of pathogenic mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-223. doi:10.1158/1538-7445.AM2017-LB-223