Allele Counts Research Articles

Complementary value of molecular, phenotypic, and functional aging biomarkers in dementia prediction.

DNA methylation age (MA), brain age (BA), and frailty index (FI) are putative aging biomarkers linked to dementia risk. We investigated their relationship and combined potential for prediction of cognitive impairment and future dementia risk using the ADNI database. Of several MA algorithms, DunedinPACE and GrimAge2, associated with memory, were combined in a composite MA alongside BA and a data-driven FI in predictive analyses. Pairwise correlations between age- and sex-adjusted measures for MA (aMA), aBA, and aFI were low. FI outperformed BA and MA in all diagnostic tasks. A model including age, sex, and aFI achieved an area under the curve (AUC) of 0.94 for differentiating cognitively normal controls (CN) from dementia patients in a held-out test set. When combined with clinical biomarkers (apolipoprotein E ε4 allele count, memory, executive function), a model including aBA and aFI predicted 5-year dementia risk among MCI patients with an out-of-sample AUC of 0.88. In the prognostic model, BA and FI offered complementary value (both βs 0.50). The tested MAs did not improve predictions. Results were consistent across FI algorithms, with data-drivenhealth deficitselection yielding the best performance. FI had a stronger adverse effect on prognosis in males, while BA's impact was greater in females. Our findings highlight the complementary value of BA and FI in dementia prediction. The results support a multidimensional view of dementia, including an intertwined relationship between the biomarkers, sex, and prognosis. The tested MA's limited contribution suggests caution in their use for individual risk assessment of dementia.

Characterization of fine geographic scale population genetics in sugar kelp (Saccharina latissima) using genome-wide markers

BackgroundKelps are not only ecologically important, being primary producers and habitat forming species, they also hold substantial economic potential. Expansion of the kelp cultivation industry raises the interest for genetic improvement of kelp for cultivation, as well as concerns about genetic introgression from cultivated to wild populations. Thus, increased understanding of population genetics in natural kelp populations is crucial. Genotyping-by-sequencing (GBS) is a powerful tool for studying population genetics. Here, using Saccharina latissima (sugar kelp) as our study species, we characterize the population genetics at a fine geographic scale, while also investigating the influence of marker type (biallelic SNPs versus multi-allelic short read-backed haplotypes) and minor allele count (MAC) thresholds on estimated population genetic metrics.ResultsWe examined 150 sporophytes from 10 locations within a small area in Mid-Norway. Employing GBS, we detected 20,710 bi-allelic SNPs and 42,264 haplotype alleles at 20,297 high quality GBS loci. We used both marker types as well as two MAC filtering thresholds (3 and 15) in the analyses. Overall, higher genetic diversity, more outbreeding and stronger substructure was estimated using haplotypes compared to SNPs, and with MAC 15 compared to MAC 3. The population displayed high genetic diversity (HE ranging from 0.18–0.37) and significant outbreeding (FIS ≤ − 0.076). Construction of a genomic relationship matrix, however, revealed a few close relatives within sampling locations. The connectivity between sampling locations was high (FST ≤ 0.09), but subtle, yet significant, genetic substructure was detected, even between sampling locations separated by less than 2 km. Isolation-by-distance was significant and explained 15% of the genetic variation, while incorporation of predicted currents in an “isolation-by-oceanography” model explained a larger proportion (~ 27%).ConclusionThe studied population is diverse, significantly outbred and exhibits high connectivity, partly due to local currents. The use of genome-wide markers combined with permutation testing provides high statistical power to detect subtle population substructure and inbreeding or outbreeding. Short haplotypes extracted from GBS data and removal of rare alleles enhances the resolution. Careful consideration of marker type and filtering thresholds is crucial when comparing independent studies, as they profoundly influence numerical estimates of population genetic metrics.

Association of NTRK2 gene with suicidality: a meta-analysis

Background Previous studies have shown that genes in brain development pathways may have important roles in affecting risk of suicidal behaviors, with our previous meta-analysis supporting a role of the brain-derived neurotrophic factor (BDNF) gene. NTRK2 is a gene that encodes the neurotrophic receptor tyrosine kinase 2, which is a receptor for BDNF. In the current study, we aim to examine the potential association between NTRK2 single nucleotide polymorphism (SNPs) and suicidal ideation/behaviors. Methods We first conducted a literature search using keywords like ‘NTRK2’, ‘TRKB’, and ‘suicid*’ to identify papers on NTRK2 SNPs and suicidal ideation/behaviors. In addition, we have individual-level genotype data for all the identified SNPs in literature search. We used the R meta package to perform meta-analyses on both the genotype count and the allele count data. Moreover, we performed meta-analyses on specific haplotypes within each haplotype block. Main results Following our literature search and meta-analyses on 20 NTRK2 SNPs across up to 8467 samples, we found three SNPs, rs10868235 [N = 5,318, odds ratio (OR) = 1.34, P = 0.02], rs1867283 (N = 5,134, OR = 0.73, P = 0.04), and rs1147198 (N = 5,132, OR = 1.36, P = 0.03) to be nominally associated with suicidal attempts. Those three findings, however, did not survive multiple-testing corrections. Also, none of the haplotype blocks showed significant involvement in suicidality. Conclusion Our results suggest that the NTRK2 gene may not have a major role in suicidality. Future efforts, however, should explore gene–gene interaction and pathway analyses.

Combining evidence from human genetic and functional screens to identify pathways altering obesity and fat distribution.

Overall adiposity and body fat distribution are heritable traits associated with altered risk of cardiometabolic disease and mortality. Performing rare variant (minor allele frequency<1%) association testing using exome-sequencing data from 402,375 participants in the UK Biobank (UKB) for nine overall and tissue-specific fat distribution traits, we identified 19 genes where putatively damaging rare variation associated with at least one trait (Bonferroni-adjusted P<1.58×10-7) and 52 additional genes at FDR≤1% (P≤4.37×10-5). These 71 genes exhibited higher (P=3.58×10-18) common variant prioritisation scores than genes not significantly enriched for rare putatively damaging variation, with evidence of monotonic allelic series (dose-response relationships) among ultra-rare variants (minor allele count≤10) in 22 genes. Five of the 71 genes have cognate protein UKB Olink data available; all five associated (P<3.80×10-6) with three or more analysed traits. Combining rare and common variation evidence, allelic series and proteomics, we selected 17 genes for CRISPR knockout in human white adipose tissue cell lines. In three previously uncharacterised target genes, knockout increased (two-sided t-test P<0.05) lipid accumulation, a cellular phenotype relevant for fat mass traits, compared to Cas9-empty negative controls: COL5A3 (fold change [FC]=1.72, P=0.0028), EXOC7 (FC=1.35, P=0.0096), and TRIP10 (FC=1.39, P=0.0157); furthermore, knockout of SLTM resulted in reduced lipid accumulation (FC=0.51, P=1.91×10-4). Integrating across population-based genetic and in vitro functional evidence, we highlight therapeutic avenues for altering obesity and body fat distribution by modulating lipid accumulation.

Uncertainty in the number of contributor estimation methods applied to a Y-STR profile

Maximum allele count (MAC) and total allele count (TAC) methods are widely used for estimating the number of contributors (NoC) of autosomal short tandem repeat (STR) profile in many forensic laboratories. In this study, we applied NoC estimation methods to mixed Y-STR profiles and evaluated its uncertainty and performance. For the MAC method, as recent Y-STR typing kits involve single- and multi-copy loci, we defined “MAC-single” for use across only single-copy loci and “MAC-multi” for use across only multi-copy loci. We generated a dataset containing 120,000 Y-STR profiles for a one to six-person mixture in silico based on previously reported haplotype frequencies of 27 Y-STR loci in Yfiler Plus for the U.S. population (reported by NIST) and the Henan Han population. The dataset was randomly split into a training set and a test set. The training set was used to construct a TAC distribution (TAC curve), whereas the test set was used to calculate the performance metrics (accuracy, precision, recall, and F1-score). In addition, the effect of the upper limit of NoC considered for estimation on overall accuracy was evaluated. The overall accuracies of MAC-single, MAC-multi, and TAC methods when the upper limit of NoC was set to six-person were 0.7920, 0.4329, and 0.7877 for the U.S. population and 0.8207, 0.4609, and 0.8385 for the Henan Han population. Our results suggest that the MAC-single and TAC methods can estimate the NoC for mixed Y-STR profiles with high levels of accuracy.

Analysis of C-reactive protein omics-measures associates methylation risk score with sleep health and related health outcomes.

DNA methylation (DNAm) predictors of high sensitivity C-reactive protein (CRP) offer a stable and accurate means of assessing chronic inflammation, bypassing the CRP protein fluctuations secondary to acute illness. Poor sleep health is associated with elevated inflammation (including elevated blood CRP levels) which may explain associations of sleep insufficiency with metabolic, cardiovascular and neurological diseases. Our study aims to characterize the relationships among sleep health phenotypes and CRP markers -blood, genetic, and epigenetic indicators-within the Hispanic Community Health Study/Study of Latinos (HCHS/SOL). In HCHS/SOL, methylation risk scores (MRS)-CRP and polygenetic risk score (PRS)-CRP were constructed separately as weighted sums of methylation beta values or allele counts, respectively, for each individual. Sleep health phenotypes were measured using self-reported questionnaires and objective measurements. Survey-weighted linear regression established the association between the multiple sleep phenotypes (obstructive sleep apnea (OSA), sleep duration, insomnia and excessive sleepiness symptom), cognitive assessments, diabetes and hypertension with CRP markers while adjusting for age, sex, BMI, study center, and the first five principal components of genetic ancestry in HCHS/SOL. We included 2221 HCHS/SOL participants (age range 37-76 yrs, 65.7% female) in the analysis. Both the MRS-CRP (95% confidence interval (CI): 0.32-0.42, p = 3.3 × 10-38) and the PRS-CRP (95% CI: 0.15-0.25, p = 1 × 10-14) were associated with blood CRP level. Moreover, MRS-CRP was associated with sleep health phenotypes (OSA, long sleep duration) and related conditions (diabetes and hypertension), while PRS-CRP markers were not associated with these traits. Circulating CRP level was associated with sleep duration and diabetes. Associations between OSA traits and metabolic comorbidities weakened after adjusting for MRS-CRP, most strongly for diabetes, and least for hypertension. MRS-CRP is a promising estimate for systemic and chronic inflammation as reflected by circulating CRP levels, which either mediates or serves as a common cause of the association between sleep phenotypes and related comorbidities, especially in the presence of diabetes.

Segregation GWAS to linearize a non-additive locus with incomplete penetrance: an example of horn status in sheep

BackgroundThe objective of this study was to introduce a genome-wide association study (GWAS) in conjunction with segregation analysis on monogenic categorical traits. Genotype probabilities calculated from phenotypes, mode of inheritance and pedigree information, are expressed as the expected allele count (EAC) (range 0 to 2), and are inherited additively, by definition, unlike the original phenotypes, which are non-additive and could be of incomplete penetrance. The EAC are regressed on the single nucleotide polymorphism (SNP) genotypes, similar to an additive GWAS. In this study, horn phenotypes in Merino sheep are used to illustrate the advantages of using the segregation GWAS, a trait believed to be monogenic, affected by dominance, sex-dependent expression and likely affected by incomplete penetrance. We also used simulation to investigate whether incomplete penetrance can cause prediction errors in Merino sheep for horn status.ResultsEstimated penetrance values differed between the sexes, where males showed almost complete penetrance, especially for horned and polled phenotypes, while females had low penetrance values for the horned status. This suggests that females homozygous for the ‘horned allele’ have a horned phenotype in only 22% of the cases while 78% will be knobbed or have scurs. The GWAS using EAC on 4001 animals and 510,174 SNP genotypes from the Illumina Ovine high-density (600k) chip gave a stronger association compared to using actual phenotypes. The correlation between the EAC and the allele count of the SNP with the highest –log10(p-value) was 0.73 in males and 0.67 in females. Simulations using penetrance values found by the segregation analyses resulted in higher correlations between the EAC and the causative mutation (0.95 for males and 0.89 for females, respectively), suggesting that the most predictive SNP is not in full LD with the causative mutation.ConclusionsOur results show clear differences in penetrance values between males and female Merino sheep for horn status. Segregation analysis for a trait with mutually exclusive phenotypes, non-additive inheritance, and/or incomplete penetrance can lead to considerably more power in a GWAS because the linearized genotype probabilities are additive and can accommodate incomplete penetrance. This method can be extended to any monogenic controlled categorical trait of which the phenotypes are mutually exclusive.

A genetic method to infer ploidy and aberrant inheritance in triploid organisms.

Polyploidy occurs naturally across eukaryotic lineages and has been harnessed in the domestication of many crops and vertebrates. In aquaculture, triploidy can be induced as a biocontainment strategy, as it creates a reproductive barrier preventing farm-to-wild introgression, which is currently a major conservation issue for the industry. However, recent work suggests that triploidisation protocols may, on occasion, produce 'failed triploids' displaying diploidy, aneuploidy and aberrant inheritance. The potentially negative consequences for conservation and animal welfare motivate the need for methods to evaluate the success of ploidy-manipulation protocols early in the production process. We developed a semi-automated version of the MAC-PR (microsatellite DNA allele counting - peak ratios) method to resolve the allelic configuration of large numbers of individuals across a panel of microsatellite markers that can be used to infer ploidy, pedigree and inheritance aberrations. We demonstrate an application of the approach using material from a series of Atlantic salmon (Salmo salar) breeding experiments where ploidy was manipulated using a hydrostatic pressure treatment. We validated the approach to infer ploidy against blood smears, finding a > 99% agreement between these methods, and demonstrate its potential utility to infer ploidy as early as the embryonic stage. Furthermore, we present tools to assign diploid and triploid progeny to families and to detect aberrant inheritance, which may be useful for breeding programmes that utilise ploidy manipulation techniques. The approach adds to the ploidy verification toolbox. The increased precision in detecting ploidy and inheritance aberrations will facilitate the ability of triploidisation programmes to prevent farm-to-wild introgression.

O-270 Clinical-grade whole genome sequencing-based haplarithmisis enables all forms of preimplantation genetic testing

Abstract Study question Can a simple, and scalable assay be developed to concurrently perform all forms of preimplantation genetic testing (PGT), effectively addressing the increasing demand? Summary answer Whole genome sequencing-based haplarithmisis offers a single-assay solution for all forms of PGT, enhancing resolution and diagnosis in complex cases. What is known already State-of-the-art preimplantation genetic testing (PGT) methods, including genotyping-by-sequencing (GBS)-based haplotyping, enable PGT for monogenic disorders (PGT-M), structural rearrangements (PGT-SR), and aneuploidies (PGT-A) in a single assay. However, they are labour intensive, only partially cover the genome, require a close relative to determine meiotic and mitotic origin of aneuploidies, and are troublesome for consanguineous families and in complex genomic contexts. Furthermore, the prevailing PGT approach for mitochondrial DNA disorders (PGT-MT) involves a PCR-based method. This underscores the need to develop a universal PGT method that streamlines laboratory protocols and provides genome-wide coverage to address genetic disorders, even in complex genomic loci. Study design, size, duration In a retrospective pilot study, we compared whole genome sequencing (WGS) at 30-40X with GBS (6 embryos, 2 families, 3 indications). We carried out subsampling of WGS data and determined 10X as the optimal diagnostic depth-of-coverage. We clinically validated our approach by comparing WGS to GBS for PGT-M and PGT-SR (31 embryos, 16 families, 22 indications), to shallow sequencing for PGT-SR (10 embryos, 3 families) and to PCR-based PGT for PGT-MT (10 embryos, 2 families). Participants/materials, setting, methods DNA from parents and close relative(s) and whole genome amplified (WGAed) DNA from embryos, underwent WGS with subsequent haplarithmisis. Quality metrics, such as Mendelian inconsistency rates, number of informative SNPs, and haplotype concordance were assessed. Translocation breakpoints were identified utilizing aberrantly aligned paired sequencing reads. Additionally, single-nucleotide variants (SNVs), including mitochondrial DNA SNVs, were detected by allele counting. Finally, segregational origin was determined via a parents-only haplarithmisis approach that uses the embryo as phasing reference. Main results and the role of chance WGS-PGT alleviates technical limitations by decreasing WGA artifacts by 68.4%, increasing breadth of coverage by 4-fold, and decreasing wet-lab turnaround time by 2.5-fold. Owing to its inherent higher resolution and improved genomic coverage, 5 out of 13 – previously inconclusive – embryos with genetic disorders in complex genomic regions could be assigned a diagnosis. Furthermore, we show the ability to directly detect single- and few-base pair genetic variants in any region of interest. Importantly, we show trio-based PGT-A for aneuploidy origin, an approach we term PGT-AO that – using the parents-only genotype calls – can detect the segregational origin of aneuploidies without the need of a close relative as reference for phasing. The segregational origin detected by parents-only haplarithmisis was concordant with the segregational origin determined from haplarithmisis when using a close relative. Unbalanced translocations could be detected indirectly via haplotyping or directly at single base resolution using a structural variant caller. Mitochondrial heteroplasmy level calculation from WGS yielded comparable results to PCR-based methods, both in blastomere and trophectoderm biopsy samples. Limitations, reasons for caution Direct variant detection with an average coverage of 10X may occasionally be inadequate, as certain locations may lack sufficient coverage (&amp;lt;5 reads). Inherently to the limited sample size of PGT-MT families, WGS-PGT needs further validation. The need for invasive trophectoderm biopsies of preimplantation embryos remains the key limitation of WGS-PGT. Wider implications of the findings Our approach enables all forms of PGT in a single assay. Direct variant detection complements haplarithmisis, addressing inconclusive findings and aiding families with a de novo variant or lacking a close relative for phasing. Finally, PGT-AO facilitates research on the origin of aneuploidies in preimplantation embryos and their clinical outcome. Trial registration number not applicable

Improved individual identification in DNA mixtures of unrelated or related contributors through massively parallel sequencing

DNA mixtures are a common sample type in forensic genetics, and we typically assume that contributors to the mixture are unrelated when calculating the likelihood ratio (LR). However, scenarios involving mixtures with related contributors, such as in family murder or incest cases, can also be encountered. Compared to the mixtures with unrelated contributors, the kinship within the mixture would bring additional challenges for the inference of the number of contributors (NOC) and the construction of probabilistic genotyping models. To evaluate the influence of potential kinship on the individual identification of the person of interest (POI), we conducted simulations of two-person (2 P) and three-person (3 P) DNA mixtures containing unrelated or related contributors (parent-child, full-sibling, and uncle-nephew) at different mixing ratios (for 2 P: 1:1, 4:1, 9:1, and 19:1; for 3 P: 1:1:1, 2:1:1, 5:4:1, and 10:5:1), and performed massively parallel sequencing (MPS) using MGIEasy Signature Identification Library Prep Kit on MGI platform. In addition, in silico simulations of mixtures with unrelated and related contributors were also performed. In this study, we evaluated 1): the MPS performance; 2) the influence of multiple genetic markers on determining the presence of related contributors and inferring the NOC within the mixture; 3) the probability distribution of MAC (maximum allele count) and TAC (total allele count) based on in silico mixture profiles; 4) trends in LR values with and without considering kinship in mixtures with related and unrelated contributors; 5) trends in LR values with length- and sequence-based STR genotypes. Results indicated that multiple numbers and types of genetic markers positively influenced kinship and NOC inference in a mixture. The LR values of POI were strongly dependent on the mixing ratio. Non- and correct-kinship hypotheses essentially did not affect the individual identification of the major POI; the correct kinship hypothesis yielded more conservative LR values; the incorrect kinship hypothesis did not necessarily lead to the failure of POI individual identification. However, it is noteworthy that these considerations could lead to uncertain outcomes in the identification of minor contributors. Compared to length-based STR genotyping, using sequence-based STR genotype increases the individual identification power of the POI, concurrently improving the accuracy of mixing ratio inference using EuroForMix. In conclusion, the MGIEasy Signature Identification Library Prep kit demonstrated robust individual identification power, which is a viable MPS panel for forensic DNA mixture interpretations, whether involving unrelated or related contributors.

Comparison of primary versus metastatic tumor tissue sources when designing panels for whole genome–based tumor-informed ctDNA assays in clear cell renal cell carcinoma.

3039 Background: Due to low shedding and low tumor-mutation burden, metastatic clear cell renal cell carcinoma (ccRCC) is poorly modeled by current cell-free circulating tumor DNA (ctDNA) assays. Second generation tumor-informed ctDNA approaches may demonstrate improved sensitivity in ccRCC, where somatic variants identified by whole-genome sequencing (WGS) of tumor tissue are tracked in probe panels to monitor molecular residual disease (MRD) in patient plasma. However, intertumoral genomic heterogeneity of input tissue may hinder optimal panel design. Here we evaluated the concordance of ctDNA results using panels designed from primary nephrectomy or metastatic tumors in ccRCC. Methods: In five patients with oligometastatic ccRCC (≤5 lesions of metastatic disease; median of 1, range 1-4), 13 primary tumor, 24 metastatic lesion subsites and matched buffy coat samples were collected. Baseline plasma was collected prior to radiotherapy. Samples were tested with Myriad Genetics High-Definition MRD assay. Briefly, tumor and normal DNA were whole-genome sequenced (median coverage 30x). A sub-panel with up to 1000 somatic variants was designed for each lesion subsite; variants could be common across sub-panels. Sites were enriched using hybridization capture from patient plasma-derived cfDNA and sequenced to high depth. A statistical model of variant allele counts was used to assess the presence or absence of ctDNA (MRD status) and infer quantitative tumor fraction. Results: Median time from nephrectomy or most recent metastasis sample collection to baseline plasma collection was 146 months (71-169) and 32 months (7-48), respectively. Median panel probe count per patient was 4,311 (3,400-5,000) and per tumor sample was 1,545 (102-2,158). Confident calls were emitted for tumor fractions ranging from 0.0004% to 0.027%. Baseline MRD status was fully concordant across sub-panels derived from primary tumor lesion subsites and metastases in three of five patients (3/5; 60%). Of the patients with discordant calls, a single metastasis-derived sub-panel (comprising 20%-25% of metastatic sub-panels) called MRD discordantly in each. Of the three patients MRD positive at baseline, only one was alive at last follow up. All patients MRD negative at baseline are still alive. Conclusions: A tumor-informed, WGS MRD assay for ccRCC showed comparable performance between probe capture panels designed from primary tumor and metastatic lesion subsites despite differences in anatomical location and time from plasma collection. This result suggests that intertumoral genomic heterogeneity may not be consequential in MRD assays that leverage large panels to detect ctDNA variants, obviating the need for repeat biopsies of metastatic sites. Work is underway to validate this MRD assay in a larger cohort of patients with ccRCC before and after treatment.

#2709 Genome wide association study regarding disturbed AVF patency

Abstract Background and Aims Veins are frequently exposed to arterial circulation, as coronary artery bypass grafts (CABG) and as a peripheral venous bypass to treat atherosclerotic disease, or in the case of arterio-venous fistulas (AVF) to provide a vascular access for hemodialysis. Therapeutic success is dependent on proficient venous remodeling after introduction in the arterial circulation to sustain the increased hemodynamic pressure. Frequently observed adverse outcomes after the aforementioned procedures include the stenosis or total occlusion of the vein, requiring surgical or interventional treatment or leading to bypass/ AVF failure. Hereditary factors associated with disturbed venous patency have been previously investigated in all three conditions. For example, distinct VEGF-A as well as TGF-beta polymorphisms have been identified to correlate with AVF failure. In the present analysis we aimed at investigating whether genetic polymorphisms associated with disturbed venous patency after AVF creation are similar to findings in CABG and peripheral bypass patients in a genome wide association study. Method The analyses were conducted in White Europeans (N= 425,406) in the UK Biobank using regenie software. This software employs a linear mixed model that accounts for the impact of population structure and individual relatedness. Out of approximately 805,000 variants, we selected 606,201 high-quality variants to construct a relatedness model for regenie. These selected variants were identified by a genotyping rate exceeding 80%, minor allele frequencies greater than 1%, and Hardy-Weinberg equilibrium p-values below 10^-8. We adjusted for age, sex, BMI, smoking, and history venous thromboembolism. We assessed the association of 9,757,417 imputed SNPs, each having a minimum allele count of 12 and a minimum info score of 0.3. Results In total, 768 patients underwent at least 1 surgery for AVF creation. Out of those, 341 (44.4%) had a recorded procedure or diagnosis associated with disturbed fistula patency. The controls included 6067 CABGs and 384 peripheral bypasses with 76 (1.3%) and 67 (17.4%) recorded events, respectively. We found no genome wide significant associations for the tested SNPs and patency-related AVF complications (Fig. 1). Additionally, we also found no genome wide significant associations after combining the phenotypes (Fig. 2). Conclusion Multiple studies have evaluated genetic associations and adverse clinical outcomes after AVF creation. Nevertheless, the generalizability of these findings might be limited at least partially due to small sample sizes. While the sample size of the present analysis is similar to previous research efforts and therefore also comparably low, the lack of consistency in results might be suggestive of the difficulties research in this field faces. Additionally, we found no indication that impaired venous patency after AVF creation is related to complications after coronary or peripheral bypass surgery regarding genetic patient profiles. However, whether this is due to differences in pathophysiology and related genetic predispositions or due to the limited sample size and incidence rates of the present study remains to be determined in follow-up investigations.

GARCOM: A user-friendly R package for genetic mutation counts

Next-generation sequencing (NGS) has enabled analysis of rare and uncommon variants in large study cohorts. A common strategy to overcome these low frequencies and/or small effect sizes relies on collapsing strategies, i.e. to bin variants within genes/regions. Several tools are now available for advanced statistical analyses; however, tools to perform basic tasks such as obtaining allelic counts within defined gene/region boundaries are unavailable or require complex coding. GARCOM (“Gene And Region Count Of Mutations”) library, an open-source freely available package in R language, returns a matrix with allelic counts within genes/regions per sample. GARCOM accepts input data in PLINK or VCF formats, with additional options to subset data for refined analyses.

Abstract PO1-15-12: Circulating tumor DNA (ctDNA) for prediction of pathologic complete response (pCR) after 12 weeks of pembrolizumab + trastuzumab + pertuzumab in HER2-enriched early breast cancer: WSG-Keyriched-1 trial

Abstract Background ctDNA testing is emerging as an important biomarker in early breast cancer (eBC). However, its value in prediction of tumor response to de-escalated, chemotherapy-free neoadjuvant therapy (NAT) remains underexplored. In WSG-Keyriched-1 (NCT03988036), a single-arm phase 2 trial, we investigated for the first time a chemotherapy-free NAT with dual HER2 blockade and pembrolizumab in HER2- enriched HER2+ eBC. In this pre-specified translational analysis, we evaluated whether ctDNA measurement could predict pCR. Methods 48 patients (pts) with HER2 2+ (ISH+) or 3+ eBC (stage I-III) and HER2-enriched subtype by PAM50 were included. Pts received pembrolizumab (200 mg), trastuzumab biosimilar (loading dose (LD) 8 mg/kg bodyweight (BW), maintenance dose (MD) 6 mg/kg BW), and pertuzumab (LD 840 mg/kg BW, MD 420 mg/kg BW) q3w for 12 weeks. Primary objective was pCR (ypT0/is ypN0). ctDNA was analyzed in 92 plasma samples collected from 31 pts at baseline (BL) and week 3 of NAT and 30 pts at end of treatment (EOT). Sequencing libraries with unique molecular identifiers were constructed from cell-free DNA and hybridization panels with ≤50 patient-specific somatic mutations from tumor sequencing were used for enrichment. Libraries were sequenced to an ultra-high depth of 100,000×. Sequencing data was analyzed using a combination of public pipelines (megSAP and umiVar2 for mapping and deduplication) and custom in-house scripts (to extract the allele counts for patient-specific variants). To reduce the error rate, only corrected reads with ≥4 duplicates were used. p-value for ctDNA detection was calculated using the total variant count, depth and sample-specific error. Association between ctDNA with pCR and other clinical parameters were assessed with Chi-square or Fisher’s exact test and univariable logistic regression. Additionally, bivariable logistic regressions for pCR were performed with ctDNA and either cT, cN, or grade. Results ctDNA was detected (ctDNA+) in 58.0% (n=18/31) of pts at BL, 9.7% (n=3/31) at week 3, and 10% (n=3/30) at EOT. ctDNA was cleared by week 3 in 83.3% of pts (n=15/18). Compared with ctDNA-negative cases (ctDNA-) at BL, those ctDNA+ more often had cT2-3 disease (94.4%, n=17/18, vs 38.5%, n=5/13; p=.001) and lymph node involvement (61.1%, n=11/18, vs 0%, n=0/13; p&amp;lt; .001). 72.2% (n=13/18) of ctDNA+ and 61.5% (n=8/13) of ctDNA- cases at BL were grade 3 (p=.53). Each of the 3 pts who remained ctDNA+ at week 3 was node-positive; all pts remaining ctDNA- at week 3 were node-negative. pCR rate was 38.9% (n=7/18) in ctDNA+ vs 76.9% (n=10/13) in ctDNA- pts at BL (p=.067), 0% (n=0/3) in ctDNA+ vs 60.7% (n=17/28) in ctDNA- at week 3 (p=.081), and 33.3% (n=1/3) in ctDNA+ vs 59.3% (n=16/27) in ctDNA- at EOT (p=.565). pCR rate was highest in pts who remained ctDNA- throughout week 3 (76.9%, n=10/13), compared to pts with ctDNA cleared by week 3 (46.7%, n=7/15), and pts remaining ctDNA+ (0%, n=0/3, p=.024). ctDNA at BL was predictive for pCR in univariable analysis (odds ratio, OR 0.22, 95%CI 0.04- 0.93, area under curve, AUC=0.69). Bivariable models including ctDNA and grade yielded the highest accuracy for analyses with ctDNA at BL (AUC=0.77), week 3 (AUC=0.79), and with ctDNA change from BL to week 3 (AUC=0.87). Conclusions Absence of ctDNA prior to and during NAT, as well as early clearance of ctDNA associates with a higher pCR rate in eBC pts with HER2-enriched intrinsic subtype treated with chemotherapy-free pembrolizumab plus dual HER2 blockade. These results are in line with prior data with more intensive therapy from I-SPY 2 and NeoALTTO trials in HER2+ eBC and indicate that ctDNA analysis during NAT appears feasible for real- time monitoring of response to treatment and could be used to guide escalation/de-escalation strategies. Further trials with a larger number of pts and pre-designed follow-up are needed to confirm the role of ctDNA for prediction of tumor response and relapse. Citation Format: Monika Graeser, Sherko Kuemmel, Oleg Gluz, Christopher Schroeder, Leon Schuetz, Olga Kelemen, Stephan Ossowski, Katarzyna Jozwiak, Mattea Reinisch, Athina Kostara, Iris Scheffen, Kerstin Lüdtke-Heckenkamp, Felix Hilpert, Angela Kentsch, Carsten Ziske, Reinhard Depenbusch, Michael Braun, Jens-Uwe Blohmer, Matthias Christgen, Stephan Bartels, Hans-Heinrich Kreipe, Enrico Pelz, Ulrike Nitz, Peter Schmid, Nadia Harbeck, Andreas Hartkopf. Circulating tumor DNA (ctDNA) for prediction of pathologic complete response (pCR) after 12 weeks of pembrolizumab + trastuzumab + pertuzumab in HER2-enriched early breast cancer: WSG-Keyriched-1 trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-15-12.

Association of the COL4A2 Gene Polymorphisms with Primary Intracerebral Hemorrhage Risk and Outcome in Chinese Han Population.

The relationship of single nucleotide polymorphisms (SNPs) in COL4A2 gene with risk and outcome of primary intracerebral hemorrhage (ICH) in the Chinese Han population remains unclear, which was investigated in this study. Primary ICH patients and non-stroke controls of Chinese Han ethnicity were enrolled. The genotypes of 8 tag-SNPs were determined using a custom-by-design 48-Plex SNPscan Kit. Poor 3-month outcome was defined as modified Rankin Scale score 4-6. Logistic regression was employed toexamine association between COL4A2 variants and risk and poor outcome of primary ICH. 323 patients with primary ICH and 376 stroke-free controls were included. Compared to controls, the rs1049931 G and rs1049906 C alleles were associated with increased ICH risk (p = 0.027 and 0.033), and these two allele counts increased this risk after adjustments respectively (additive model: adjusted OR [aOR] 1.41, 95% CI 1.03-1.94, corrected p = 0.043; aOR 1.37, 95% CI 1.01-1.86, corrected p = 0.043). The rs1049931 AG/GG and rs1049906 CT/CC genotypes showed increased susceptibility to non-lobar hemorrhage (aOR 1.63, 95% CI 1.06-2.50, p = 0.025; aOR 1.63, 95% CI 1.07-2.47, p = 0.022). Haplotype analysis revealed an association between rs1049906-rs1049931 haplotype CG and ICH risk (OR 1.36, 95% CI 1.05-1.78, p = 0.021). Regarding clinical outcome, the rs3803230 C allele (dominant model: aOR 1.94, 95% CI 1.04-3.63, p = 0.037) and haplotype AC of rs7990214-rs3803230 (OR 1.98, 95% CI 1.13-3.46, p = 0.015) contributed to 3-month poor outcome. The COL4A2 polymorphisms are associated with an increased risk of primary ICH, mainly non-lobar hemorrhage, and with long-term poor outcome after ICH in Chinese Han population.

Abstract 6148: Multiple new Ewing sarcoma susceptibility loci expand knowledge of germline genetic etiology and nominate mechanisms of risk

Abstract Ewing sarcoma (EwS) is a rare and aggressive pediatric cancer with peak incidence in the second decade of life. EwS occurs primarily in children of European ancestry suggesting a genetic component to susceptibility. Prior investigations identified six susceptibility loci associated with EwS, with evidence for additional signals not reaching the commonly accepted threshold for genome-wide significance (i.e., p&amp;lt;5×10−8). At least two of these loci demonstrate evidence of tagging variation in length of nearby GGAA microsatellites, which alter binding of the pathognomonic EWSR1::FLI1 fusion resulting in downstream dysregulation of target genes promoting sarcomagenesis. To identify additional susceptibility loci, we performed the largest genome-wide association study (GWAS) to date consisting of 1,640 EwS cases and 8,457 cancer-free individuals of European ancestry from 8 studies and matched by principal components. Genotype imputation was carried out by study/array using TOPMed data as the reference and each set was analyzed using PLINK 1.9 adjusting for significant principal components. Variants were included in each study if minor allele count was greater than five in each set of cases. Individual study results were combined using fixed effects meta-analysis in Metasoft with variants demonstrating evidence for heterogeneity excluded.For previously reported EwS loci (i.e., 1p36.22, 6p25.1, 10q21.3, 15q15.1, 20p11.22 and 20p11.23), odds ratios (OR) remained high for GWAS (OR=1.5-2.4) and the -log10 p-values grew by several orders of magnitude, providing strong evidence for replication in the additional 907 EwS cases and 7,111 cancer-free controls. Our analysis identified five novel loci reaching genome-wide significance (P-valuemeta&amp;lt; 5×10−8) at 5q32, 7q32.3, 8q24.2, 13q32.3, and 17q23.2. Risk allele frequency of newly identified loci ranged from 0.001 to 0.75with estimated ORs ranging from 1.31-4.30. eQTL analyses nominated nearby biologically plausible candidate genes for future functional investigation including LARS1, a gene implicated in osteosarcoma and skeletal muscle dysgenesis, at the 5q32 locus and COX5BP6, a gene implicated in age at menarche and testosterone levels, at 13q32.3.Our results add to evidence supporting a strong inherited genetic component to EwS risk and particularly a genetic architecture harboring a substantial number of common variants with moderate effect. Ongoing work is focusing on enrichment analyses of GGAA microsatellite repeat sequences in identified risk loci to search for evidence for germline-somatic interactions with EWSR1::FLI1. Citation Format: Aubrey K. Hubbard, Weiyin Zhou, Carrie Boyce, Javed Khan, Melissa M. Hudson, Kirsten K. Ness, Zhaoming Wang, Katherine A. Janeway, Philip J. Lupo, Lindsay M. Morton, Stephen J. Chanock, Thomas G. Grünewald, Olivier Delattre, Mitchell J. Machiela, Ewing Sarcoma Germline Genetics Group. Multiple new Ewing sarcoma susceptibility loci expand knowledge of germline genetic etiology and nominate mechanisms of risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6148.

A capture-based method of prenatal cell-free DNA screening for autosomal recessive non-syndromic hearing loss.

This study aimed to develop and validate a prenatal cell-free DNA (cfDNA) screening method that uses capture-based enrichment to genotype fetal autosomal recessive disorders. This method was applied in pregnancies at high risk of autosomal recessive non-syndromic hearing loss (ARNSHL) to assess its accuracy and effectiveness. This assay measured the allele counts in both white blood cell DNA and cfDNA from the blood samples of pregnant women using a capture-based next-generation sequencing method. It then applied a binomial model to infer the fetal genotypes with the maximum likelihood. Ninety-four pregnant couples that were carriers of variants of ARNSHL in GJB2 or SLC26A4 were enrolled. The fetal genotypes deduced using this screening method were compared with the results of genetic diagnosis using amniocentesis. Of the 94 couples, 65 carried more than one variant, resulting in 170 single-nucleotide polymorphism (SNP) loci to be inferred in the fetuses. Of the 170 fetal SNP genotypes, 150 (88.2%) had high confidence calls and 139 (92.7%) of these matched the genotypes obtained by amniocentesis result. Out of the remaining 20 (11.8%) cases with low-confidence calls, only 14 (70.0%) were concordant with genetic diagnosis using amniocentesis. The concordance rate was 100% for sites where the maternal genotype was wild-type homozygous. The discordance was site-biased, with each locus showing a consistent direction of discordance. Genetic diagnosis identified a total of 19 wild-type homozygotes, 46 heterozygotes, 19 compound heterozygotes, and 10 pathogenic homozygotes. This screening method correctly genotyped 81.9% (77/94) of fetuses and demonstrated a sensitivity of 89.7% and a specificity of 89.2% for correctly identifying ARNSHL. This capture-based method of prenatal screening by cfDNA demonstrated strong potential for fetal genotyping of autosomal recessive disorders.

A targeted amplicon next-generation sequencing assay for tryptase genotyping to support personalized therapy in mast cell-related disorders.

Tryptase, the most abundant mast cell granule protein, is elevated in severe asthma patients independent of type 2 inflammation status. Higher active β tryptase allele counts are associated with higher levels of peripheral tryptase and lower clinical benefit from anti-IgE therapies. Tryptase is a therapeutic target of interest in severe asthma and chronic spontaneous urticaria. Active and inactive allele counts may enable stratification to assess response to therapies in asthmatic patient subpopulations. Tryptase gene loci TPSAB1 and TPSB2 have high levels of sequence identity, which makes genotyping a challenging task. Here, we report a targeted next-generation sequencing (NGS) assay and downstream bioinformatics analysis for determining polymorphisms at tryptase TPSAB1 and TPSB2 loci. Machine learning modeling using multiple polymorphisms in the tryptase loci was used to improve the accuracy of genotyping calls. The assay was tested and qualified on DNA extracted from whole blood of healthy donors and asthma patients, achieving accuracy of 96%, 96% and 94% for estimation of inactive α and βΙΙΙFS tryptase alleles and α duplication on TPSAB1, respectively. The reported NGS assay is a cost-effective method that is more efficient than Sanger sequencing and provides coverage to evaluate known as well as unreported tryptase polymorphisms.

Improved detection of low-frequency within-host variants from deep sequencing: A case study with human papillomavirus.

High-coverage sequencing allows the study of variants occurring at low frequencies within samples, but is susceptible to false-positives caused by sequencing error. Ion Torrent has a very low single nucleotide variant (SNV) error rate and has been employed for the majority of human papillomavirus (HPV) whole genome sequences. However, benchmarking of intrahost SNVs (iSNVs) has been challenging, partly due to limitations imposed by the HPV life cycle. We address this problem by deep sequencing three replicates for each of 31 samples of HPV type 18 (HPV18). Errors, defined as iSNVs observed in only one of three replicates, are dominated by C→T (G→A) changes, independently of trinucleotide context. True iSNVs, defined as those observed in all three replicates, instead show a more diverse SNV type distribution, with particularly elevated C→T rates in CCG context (CCG→CTG; CGG→CAG) and C→A rates in ACG context (ACG→AAG; CGT→CTT). Characterization of true iSNVs allowed us to develop two methods for detecting true variants: (1) VCFgenie, a dynamic binomial filtering tool which uses each variant's allele count and coverage instead of fixed frequency cut-offs; and (2) a machine learning binary classifier which trains eXtreme Gradient Boosting models on variant features such as quality and trinucleotide context. Each approach outperforms fixed-cut-off filtering of iSNVs, and performance is enhanced when both are used together. Our results provide improved methods for identifying true iSNVs in within-host applications across sequencing platforms, specifically using HPV18 as a case study.

A massive alteration of gene expression in undescended testicles of dogs and the association of KAT6A variants with cryptorchidism

Cryptorchidism is the most common form of disorder of sex development in male dogs, but its hereditary predisposition is poorly elucidated. The gonadal transcriptome of nine unilaterally cryptorchid dogs and seven control dogs was analyzed using RNA-seq. Comparison between the scrotal and inguinal gonads of unilateral cryptorchid dogs revealed 8,028 differentially expressed genes (DEGs) (3,377 up-regulated and 4,651 down-regulated). A similar number of DEGs (7,619) was found by comparing the undescended testicles with the descended testicles of the control dogs. The methylation status of the selected DEGs was also analyzed, with three out of nine studied DEGs showing altered patterns. Bioinformatic analysis of the cDNA sequences revealed 20,366 SNP variants, six of which showed significant differences in allelic counts between cryptorchid and control dogs. Validation studies in larger cohorts of cryptorchid (n = 122) and control (n = 173) dogs showed that the TT genotype (rs850666472, p.Ala1230Val) and the AA genotype in 3'UTR (16:23716202G>A) in KATA6, responsible for acetylation of lysine 9 in histone H3, are associated with cryptorchidism (P = 0.0383). Both the transcript level of KAT6A and H3K9 acetylation were lower in undescended testes, and additionally, the acetylation depended on the genotypes in exon 17 and the 3'UTR. Our study showed that the massive alteration of the transcriptome in undescended testicles is not caused by germinal DNA variants in DEG regulatory sequences but is partly associated with an aberrant DNA methylation and H3K9 acetylation patterns. Moreover, variants of KAT6A can be considered markers associated with the risk of this disorder.