Copy Of Allele Research Articles

Epistatic modifiers influence the expression of continual flowering in strawberry

Society Impact StatementUntil the 1970s, the majority of commercial strawberry varieties produced a single bloom of flowers. However, continuously flowering, everbearing strawberries are now routinely cultivated and use is increasing. Indeed, introgression of the everbearing flowering trait can lead to economic benefits for growers through the production of a continual crop from the same plant. Genetically guided improvement has the power to streamline everbearing generation. As such, the genetic markers reported here can help to identify everbearing individuals at an early time point in the breeding process. Furthermore, these markers can help to improve the predictions of progeny segregation ratios.Summary Previous work within the community led to the identification of a single dominant allele that controls the everbearing trait. However, frequent observations have indicated that crosses do not segregate in a Mendelian fashion, as would be expected for a trait controlled by a single dominant gene. Therefore, it was hypothesised that one or more unidentified epistatic alleles interact with the major gene. A genome‐wide association study (GWAS) was conducted on 587 June bearers and 207 everbearers to assess the genetic components associated with flowering habit. The segregation ratios of parental strawberry lines with known phenotypes were used to validate the identified alleles. Three loci including the known major FaPFRU locus and two epistatic modifiers were identified. These modifiers function as enhancers of the everbearing trait in individuals containing a single copy of the FaPFRU everbearing allele and appear to be functionally redundant. Principally, heterozygous individuals required the presence of two modifying alleles in order to allow expression of the everbearing trait. Inclusion of the epistatic alleles improved the prediction of everbearing segregation ratios; beyond that of a single allele model, however, a large proportion of the variation remained unexplained. Future work should identify the additional repressor and enhancer modifiers not identified here. Discovering the genetic components controlling the everbearing trait can enable genetic informed strawberry improvement.

COVID-19 Survivor Patients Carrying the Rs35705950 Risk Allele in MUC5B Have Higher Plasma Levels of Mucin 5B.

Background: Genetic susceptibility to infectious diseases is partly due to the variation in the human genome, and COVID-19 is not the exception. This study aimed to identify whether risk alleles of known genes linked with emphysema (SERPINA1) and pulmonary fibrosis (MUC5B) are associated with severe COVID-19, and whether plasma mucin 5B differs according to patients’ outcomes. Materials and methods: We included 1258 Mexican subjects diagnosed with COVID-19. We genotyped rs2892474 and rs17580 of the SERPINA1 gene and rs35705950 of MUC5B. Based on the rs35705950 genotypes, mucin 5B plasma protein levels were quantified. Results: Homozygous for the risk alleles of the three polymorphisms were found in less than 5% of the study population, but no statistically significant difference in the genotype or allele association analysis. At the protein level, non-survivors carrying one or two copies of the risk allele rs35705950 in MUC5B (GT + TT) had lower levels of mucin 5B compared to the survivors (0.0 vs. 0.17 ng/mL, p = 0.0013). Conclusion: The polymorphisms rs28929474 and rs17580 of SERPINA1 and rs35705950 of MUC5B are not associated with the risk of severe COVID-19 in the Mexican population. COVID-19 survivor patients bearing one or two copies of the rs35705950 risk allele have higher plasma levels of mucin 5B.

Association of germline TYK2 variation with lung cancer and non-Hodgkin lymphoma risk.

Deucravacitinib, a novel, selective inhibitor of TYK2 is currently under review at the FDA and EMA for treatment of moderate-to-severe plaque psoriasis. It is unclear whether recent safety concerns (ie, elevated rates of lung cancer and lymphoma) related to similar medications (ie, other JAK inhibitors) are shared with this novel TYK2 inhibitor. We used a partial loss-of-function variant in TYK2 (rs34536443), previously shown to protect against psoriasis and other autoimmune diseases, to evaluate the potential effect of therapeutic TYK2 inhibition on risk of lung cancer and non-Hodgkin lymphoma. Summary genetic association data on lung cancer risk were obtained from a GWAS meta-analysis of 29 266 cases and 56 450 controls in the Integrative Analysis of Lung Cancer Risk and Aetiology (INTEGRAL) consortium. Summary genetic association data on non-Hodgkin lymphoma risk were obtained from a GWAS meta-analysis of 8489 cases and 374 506 controls in the UK Biobank and InterLymph consortium. In the primary analysis, each copy of the minor allele of rs34536443, representing partial TYK2 inhibition, was associated with an increased risk of lung cancer (OR 1.15, 95% CI 1.09-1.23, P=2.29 × 10-6 ) and non-Hodgkin lymphoma (OR 1.18, 95% CI 1.05-1.33, P=5.25 × 10-3 ). Our analyses using an established partial loss-of-function mutation to mimic TYK2 inhibition provide genetic evidence that therapeutic TYK2 inhibition may increase risk of lung cancer and non-Hodgkin lymphoma. These findings, consistent with recent reports from postmarketing trials of similar JAK inhibitors, could have important implications for future safety assessment of deucravacitinib and other TYK2 inhibitors in development.

Small Peptide Derivatives Within the Carbohydrate Recognition Domain of SP-A2 Modulate Asthma Outcomes in Mouse Models and Human Cells.

Surfactant Protein-A (SP-A) is an innate immune modulator that regulates a variety of pulmonary host defense functions. We have shown that SP-A is dysfunctional in asthma, which could be partly due to genetic heterogeneity. In mouse models and primary bronchial epithelial cells from asthmatic participants, we evaluated the functional significance of a particular single nucleotide polymorphism of SP-A2, which results in an amino acid substitution at position 223 from glutamine (Q) to lysine (K) within the carbohydrate recognition domain (CRD). We found that SP-A 223Q humanized mice had greater protection from inflammation and mucin production after IL-13 exposure as compared to SP-A-2 223K mice. Likewise, asthmatic participants with two copies the major 223Q allele demonstrated better lung function and asthma control as compared to asthmatic participants with two copies of the minor SP-A 223K allele. In primary bronchial epithelial cells from asthmatic participants, full-length recombinant SP-A 223Q was more effective at reducing IL-13-induced MUC5AC gene expression compared to SP-A 223K. Given this activity, we developed 10 and 20 amino acid peptides of SP-A2 spanning position 223Q. We show that the SP-A 223Q peptides reduce eosinophilic inflammation, mucin production and airways hyperresponsiveness in a house dust mite model of asthma, protect from lung function decline during an IL-13 challenge model in mice, and decrease IL-13-induced MUC5AC gene expression in primary airway epithelial cells from asthmatic participants. These results suggest that position 223 within the CRD of SP-A2 may modulate several outcomes relevant to asthma, and that short peptides of SP-A2 retain anti-inflammatory properties similar to that of the endogenous protein.

Monogenic TCRβ Assembly and Expression Are Paramount for Uniform Antigen Receptor Specificity of Individual αβ T Lymphocytes.

The ability of individual T and B cells to display Ag receptors of unique uniform specificity is the molecular basis of adaptive immunity. Most αβ T cells achieve uniform specificity by assembling in-frame genes on only one allelic copy of TCRβ and TCRα loci, while others prevent incorporation of TCRα protein from both alleles into TCRs. Analysis of mice expressing TCR proteins from a restricted combination of transgenes showed that TCR protein pairing restrictions achieve uniform specificity of cells expressing two types of TCRβ protein. However, whether this mechanism operates in the physiological context where each dual-TCRβ cell expresses one set of a vast number of different TCRβ proteins remains an open question, largely because there is a low, but significant, portion of cells carrying two in-frame TCRβ genes. To resolve this issue, we inactivated one allelic copy of the TCRα locus in a new mouse strain that assembles two in-frame TCRβ genes in an elevated fraction of cells. This genetic manipulation has no effect on the frequency of cells that display multiple types of αβ TCR, yet increases the representation of cells displaying TCRβ proteins that generate more highly expressed TCRs. Our data demonstrate that some TCRβ proteins exhibit differential functional pairing with TCRα proteins, but these restrictions have negligible contribution for ensuring uniform specificity of cells that express two types of TCRβ protein. Therefore, we conclude that mechanisms governing monogenic assembly and expression of TCRβ genes in individual cells are paramount for uniform specificity of αβ T lymphocytes.

Genetic LGALS1 Variants Are Associated with Heterogeneity in Galectin-1 Serum Levels in Patients with Early Arthritis.

Galectin 1 (Gal1) exerts immunomodulatory effects leading to therapeutic effects in autoimmune animal models. Patients with rheumatoid arthritis have been reported to show higher Gal1 serum levels than the healthy population. Our study aimed to find genetic variants on the Gal1 gene (LGALS1) modulating its expression and/or clinical features in patients with early arthritis (EA). LGALS1 was sequenced in 53 EA patients to characterize all genetic variants. Then, we genotyped rs9622682, rs929039, and rs4820293, which covered the main genetic variation in LGALS1, in 532 EA patients. Gal1 and IL-6 serum levels were measured by ELISA and Gal1 also by western blot (WB) in lymphocytes from patients with specific genotypes. Once disease activity improved with treatment, patients with at least one copy of the minor allele in rs9622682 and rs929039 or those with GG genotype in rs4820293 showed significantly higher Gal1 serum levels (p < 0.05). These genotypic combinations were also associated with higher Gal1 expression in lymphocytes by WB and lower IL-6 serum levels in EA patients. In summary, our study suggests that genetic variants studied in LGALS1 can explain heterogeneity in Gal1 serum levels showing that patients with higher Gal1 levels have lower serum IL-6 levels.

A multiplexed electrochemical quantitative polymerase chain reaction platform for single-base mutation analysis

Detection of single-based mutation (SbM), which is of ultra-low abundance against wild-type alleles, are typically constrained by the level of multiplexing, sensitivity for single-base resolution and quantification accuracy. In this work, an electrochemical quantitative polymerase chain reaction (E-PCR) platform was developed for multiplexed and quantitative SbM analysis in limited and precious samples with single-nucleotide discrimination. A locked nucleic acid (LNA)-mediated multiplexed PCR system in a single, closed tube setup was firstly constructed to selectively amplify the SbM genes while suppressing the wild-type alleles. The amplicons were detected simultaneously through hybridization with the sequence-specific hairpin probes anchored on a reduced graphene oxide-gold nanoparticles functionalized electrode surface. With the inclusion of an LNA-mediated PCR step upstream of the electrochemical detection, we improved the limit of detection (LOD) by 2 orders of magnitude, down to an ultralow-level of 5 copies μL−1. The platform achieved an ultra-sensitive and specific detection with 0.05% against a background of 10, 000 copies of wild-type alleles. It is highly adaptive and has the potential to enable expanded multiplexed detection in parallel, thus providing a universal tool for multiplexed SbM identification.

3′UTR-HLA-G polymorphisms and circulating sHLA-G are associated with breast cancer: Evidence from a meta-analysis

BackgroundHuman leukocyte antigen-G (HLA-G) gene polymorphisms and circulating sHLA-G have often been linked to the risk of breast cancer (BC). However, the results remain controversial. To resolve this issue, we performed a meta-analysis of HLA-G gene polymorphisms and sHLA-G levels in BC. MethodsWe performed a meta-analysis on the association of HLA-G 14-bp Insertion/Deletion (Ins/Del) and HLA-G +3142 C/G polymorphisms with BC as well as the relationship between sHLA-G and the disease outcome. ResultsPooled analysis showed a statistically significant association between the HLA-G 14-bp Ins/Del polymorphism and BC susceptibility for the overall population and for Caucasians. The Del allele and genotypes with at least one copy of the Del allele presented significant risks for BC. For HLA-G +3142 C/G polymorphism, the G allele significantly decreased the risk of BC for the overall population and for Caucasians, indicating that the G allele was a protective factor against BC and that the C allele was a significant risk factor for BC. The meta-analysis revealed a significantly increased level of sHLA-G patients with BC compared to the control group for the overall population, Caucasians and Asians. ConclusionThe present meta-analysis showed a major association of both HLA-G 14-bp Ins/Del and +3142 C/G polymorphisms with BC susceptibility, suggesting Del and C variants as highly significant risk factors for BC. The present study also showed significantly higher sHLA-G levels in patients with BC compared to healthy controls. Our pooled results suggested a critical role of HLA-G in BC, thereby providing evidence to use HLA-G as a biomarker and a therapeutic tool.

Abstract 5725: HOXA9 demonstrates allelic imbalance in KMT2A-rearranged infant acute lymphoblastic leukemia

Abstract Background: KMT2A-rearrangement is a characteristic finding in approximately 70% of infant acute lymphoblastic leukemia (ALL) cases. We previously utilized a computational screen to find that KMT2A-r infant ALL demonstrated somatic allele specific expression for 431 genes when compared to non-KMT2A-r samples (Abstract Number 3649, AACR 2019). The gene that exhibited the most differential allele specific expression (ASE) pattern was HOXA9. Here, we follow up that analysis with manual examination of additional infant ALL samples. Methods: We performed whole genome sequencing (WGS) and RNA sequencing (RNAseq) on peripheral blood or bone marrow samples from 48 infants with ALL (33 KMT2A-r cases and 15 non-KMT2A-r cases) treated on Children’s Oncology Group protocol AALL0631, at diagnosis (DX), at remission (MD), and at relapse (RL), if applicable. WGS data at MD was phased using a 1000 Genomes reference panel. HOXA9 expression was quantified in transcripts per million (TPM). Biallelic expression was manually calculated on the phased genome for samples with at least 1 germline single nucleotide polymorphism (SNP) within HOXA9 and at least 5 aligned RNAseq reads over the HOXA9 transcript. ASE was defined as minor allele fraction (MAF; allele fraction for the less prevalent allele) of ≤ 0.35. Whole genome bisulfite sequencing data and sequencing-derived copy number data were also analyzed to investigate possible explanations for HOXA9 ASE. Results: We identified diagnostic samples from 21 patients, 9 KMT2A-AFF1, 9 KMT2A-MLLT1, and 3 non-KMT2A-r, that passed manual filtering. Data included 5 patients with paired DX and RL samples. Of the 15 KMT2A-r samples unable to be analyzed for ASE, 13 lacked a SNP for phasing and 2 had &amp;lt; 5 reads. For the 12 non-KMT2A-r samples unanalyzable for ASE, 6 lacked a SNP and 6 had &amp;lt; 5 reads. Average HOXA9 expression was 15.17 TPM (range 0.01 to 44.34) in KMT2A-r samples and 2.91 TPM (range 0 to 29.29) in non-KMT2A-r samples. ASE of HOXA9 (MAF ≤ 0.35) was observed in 14 samples, 72% of KMT2A-r (13 of 18 samples) and 33% of non-KMT2A-r (1 of 3 samples). RL samples were found to have HOXA9 ASE in all cases with ASE of HOXA9 in the DX sample. Of the 18 KMT2A-r samples, 6 of 9 with KMT2A-AFF1 and 7 of 9 with KMT2A-MLLT1 demonstrated HOXA9 ASE. Frequency of HOXA9 ASE at diagnosis was similar between patients known to later relapse versus those known to remain in remission. Allele specific methylation, mono-allelic deletion, or copy-neutral loss of heterozygosity (cn-LOH) for HOXA9 were not observed in KMT2A-r nor non-KMT2A-r samples. Conclusion: Our expanded cohort supports the ASE of HOXA9 in KMT2A-r infant ALL. This phenomenon did not appear to segregate by KMT2A-r partner gene, nor could it be explained by allele specific methylation or copy number variation (e.g., deletion of one HOXA9 allele or cn-LOH). Overall, further investigation into a causal mechanism is needed. Citation Format: Sarah E. Mc Dermott, Byunggil Yoo, Warren A. Cheung, Emily Farrow, Bing Ge, Margaret Gibson, Patrick A. Brown, Erin Guest, Tomi Pastinen, Midhat S. Farooqi. HOXA9 demonstrates allelic imbalance in KMT2A-rearranged infant acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5725.

Abstract 3818: Infrequent chromosomal loss and recurrent gains lead to imbalanced expression of HLA genes in melanoma

Abstract Introduction: Loss-of-heterozygosity (LOH) events in chromosome 6p, comprising the human leukocyte antigen (HLA) genes, have been reported in about 10% of cutaneous melanoma (compared to 20-40% of squamous cell carcinomas), while copy number gains in this region have been observed in over 50% of melanoma. Recent studies focused in HLA allelic loss have been restricted to DNA-based approaches, and have not been validated orthogonally at the RNA or protein levels. Here, using clinical melanoma biopsies and patient-derived melanoma cell lines, we show that genetic alterations in HLA genes results in imbalanced allele expression, subsequently skewing antigen presentation by melanoma cells. Methods: Whole exome and RNA sequencing (WES, RNAseq) analyses were performed on 760 melanoma biopsies and 60 patient-derived melanoma cell lines. Patient-matched normal WES was used to perform HLA haplotyping across Class I and II HLA genes. Tumor WES was analyzed for copy number alterations in chromosome 6, identifying which alleles were lost or gained. Differential expression of HLA alleles was quantified in tumor RNAseq, correlating the allelic imbalance at the DNA and RNA levels. Melanoma cell lines heterozygous for HLA-A*02, A*03, and A*24 were analyzed by flow cytometry for surface-level HLA protein expression using allele-specific antibodies to quantify allelic densities and compare the imbalance of HLA-A alleles at the DNA, RNA, and protein levels. Results: Across 760 melanoma biopsies, copy number alterations in chromosome 6p were identified in 76% of tumors; 12% had LOH, and 54% had copy number gains that resulted in imbalanced copies of alleles. In paired tumor WES and RNAseq (N=682), genetic imbalance was correlated with imbalanced expression of HLA alleles in the classical Class I HLA genes (Spearman rho=0.64-0.7; p=2.2e-16); this association was strengthened in tumors with high tumor cellularity, and was not associated with the total expression of the HLA genes.These patterns were explored in a 60 patient-derived melanoma cell lines with matched tumor WES and RNAseq, confirming that alleles gained at the genetic level were also expressed at higher levels than alleles that were not gained or lost. In 10 cell lines heterozygous for either HLA-A*02, A*03, or A*24, allelic imbalance at the DNA and RNA level resulted in correlative imbalanced surface presentation of alleles at the protein level. Conclusions: Evaluation of paired tumor WES and RNAseq revealed orthogonal validation of HLA allelic imbalance, and analysis in cell lines suggested that these patterns were likely tumor intrinsic. Experimental validation of these findings at the protein level suggests that antigen presentation density can be modulated by chromosomal gains, and not just allelic loss, in HLA genes. This knowledge is important for the design of cancer vaccines or T cell therapies targeting neoantigens presented by HLA class I complexes. Citation Format: Katie M. Campbell, Justin Saco, Egmidio Medina, Meelad Amouzgar, Shannon M. Pfeiffer, Cynthia R. Gonzalez, Gabriela Steiner, Ameya Champhekar, Cristina Puig Saus, Jesse Zaretsky, Gabriel Abril Rodriguez, Agustin Vega-Crespo, Ignacio Baselga Carretero, Mito Tariveranmoshabad, Anusha Kalbasi, Christine Spencer, Zachary L. Skidmore, Malachi Griffith, Obi L. Griffith, Daniel K. Wells, Antoni Ribas. Infrequent chromosomal loss and recurrent gains lead to imbalanced expression of HLA genes in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3818.

Additive genetic effect of GCKR, G6PC2, and SLC30A8 variants on fasting glucose levels and risk of type 2 diabetes.

Impaired glucose tolerance is a major risk factor for type 2 diabetes (T2D) and several cardiometabolic disorders. To identify genetic loci underlying fasting glucose levels, we conducted an analysis of 9,232 individuals of European ancestry who at enrollment were either nondiabetic or had untreated type 2 diabetes. Multivariable linear mixed models were used to test for associations between fasting glucose and 7.9 million SNPs, with adjustment for age, body mass index (BMI), sex, significant principal components of the genotypes, and cryptic relatedness. Three previously discovered loci were genome-wide significant, with the lead SNPs being rs1260326, a missense variant in GCKR (p = 1.06×10-8); rs560887, an intronic variant in G6PC2 (p = 3.39×10-11); and rs13266634, a missense variant in SLC30A8 (p = 4.28×10-10). Fine mapping, genome-wide conditional analysis, and functional annotation indicated that the three loci were independently associated with fasting glucose. Each copy of an alternate allele at any of these three SNPs was associated with a reduction of 0.012 mmol/L in fasting glucose levels (p = 8.0×10-28), and this association was replicated in trans-ethnic analysis of 14,303 individuals (p = 2.2×10-16). The three SNPs were jointly associated with significantly reduced T2D risk, with an odds ratio (95% CI) of 0.93 (0.88, 0.98) per protective allele. Our findings implicate additive effects across pathophysiological pathways involved in type 2 diabetes, including glycolysis, gluconeogenesis, and insulin secretion. Since none of the individuals homozygous for the alternate alleles at all three loci has T2D, it might be possible to use a genetic predictor of fasting glucose levels to identify individuals at low vs. high risk of developing type 2 diabetes.

Genetic Determination of the Amount of White Spotting: A Case Study in Siberian Cats.

The current hypothesis, along with the opinion of the breeders, is that a cat with two copies of the white spotting allele (SS) has white on more than half of its body, while a cat with only one copy (Ss) has white on less than half of its body. The present study was based on the analysis of two large pedigree databases of Siberian cats (23,905 individuals in PawPeds and 21,650 individuals in Felis Polonia database). The distribution of the amount of white spotting in the offspring of cats with different amounts of white was investigated. Significant differences compared to expected distributions were observed. In many cases the amount of white in cats that were supposed to be homozygous was less than 50% of the body, while in many supposedly heterozygous cats a very large amount of white (over 50%) was observed. This phenomenon was also presented on the verified examples of the specific families excluding possible errors in determining the amount of white by the breeder. The collected evidence suggests that there are other factors involved in the inheritance of the amount of white in cats and the current hypothesis should be revised.

Allelic dosage of RB1 drives CDK4/6 inhibitor treatment resistance in metastatic breast cancer.

1010 Background: We recently reported inferior outcomes to CDK4/6 inhibitors and endocrine therapy (CDK4/6i-ET) associated with germline BRCA2 (g BRCA2) in a cohort of estrogen receptor (ER) positive breast cancers. Co-occurrence of gBRCA2 with loss of heterozygosity (LOH) of neighboring RB1 was found to portend particularly poor outcomes. Here, we sought to define the effects of pre-treatment RB1 allelic copy number status on outcomes of CDK4/6i-ET and the likelihood of developing RB1 loss-of-function (LOF) mutations on CDK4/6i through the analysis of an expanded cohort of metastatic ER+ breast cancer patients. Methods: Patients who underwent sequencing on MSK-IMPACT from April 2014 to May 2021 were included. For every sample preceding CDK4/6i-ET, we performed FACETS to infer RB1 allele specific copy number, ploidy, tumor purity and fraction genome altered (FGA). Patients were categorized based on RB1 allelic status: HetLoss (total of one allelic copy), copy neutral LOH (CNLOH), other allelic imbalance including all other aneuploidy states, and diploid. Progression free survival (PFS) was assessed using univariate and multivariate Cox proportional hazard models adjusted for ET partner and FGA. Firth penalized logistic regression was used to study association of pre-treatment RB1 status with acquired RB1 LOF variants in paired post-CDK4/6i samples. Results: Of 2,630 potentially eligible patients, 279 patients had genomic sequencing performed prior to 1st line CDK4/6i-ET. Of these, 75 (26.8%) exhibited RB1 HetLoss, 39 (14.0%) had CNLOH of RB1, 111 (39.7%) exhibited diploid RB1 state, while 54 (19.4%) had other patterns of RB1 allelic imbalance. All non-diploid RB1 states were associated with significantly shortened PFS relative to diploid (univariate HetLoss HR: 2.05, 95% CI: 1.42, 2.97; CNLOH HR: 2.08, 95% CI: 1.32, 3.25; other imbalance HR: 1.70, 95% CI: 1.11, 2.58). Only HetLoss remained significant when adjusted for FGA (HR 1.61, 95% CI: 1.09, 2.38, p = 0.017). RB1 LOF was rare in pre-CDK4/6i tumors (&lt; 1%); excluding these cases did not change our results. Of the 176 patients with paired pre- and post-CDK4/6i samples, only RB1 HetLoss in pre-CDK4/6i sample was significantly associated with development of RB1 LOF mutations in post-CDK4/6i sample (18.4%) as compared to diploid (4.2%, OR 4.25, 95% CI 1.02, 17.7, p = 0.047). These results indicate that tumors with one functional copy of RB1 are more likely to acquire RB1 LOF on CDK4/6i to achieve biallelic RB1 loss as a mechanism of CDK4/6i resistance. Conclusions: We demonstrate that LOH and allelic imbalance of RB1 are associated with shorter PFS on CDK4/6-ET. We postulate this may occur partly as a result of more frequent acquired RB1 LOF mutations under selective pressure of CDK4/6i. These data supports the implementation of more refined allele-specific copy number methods and identifies a high-risk population for escalated monitoring and treatment approaches.

In vivo impact of JAK3 A573V mutation revealed using zebrafish

BackgroundJanus kinase 3 (JAK3) acts downstream of the interleukin-2 (IL-2) receptor family to play a pivotal role in the regulation of lymphoid cell development. Activating JAK3 mutations are associated with a number of lymphoid and other malignancies, with mutations within the regulatory pseudokinase domain common.MethodsThe pseudokinase domain mutations A572V and A573V were separately introduced into the highly conserved zebrafish Jak3 and transiently expressed in cell lines and zebrafish embryos to examine their activity and impact on early T cells. Genome editing was subsequently used to introduce the A573V mutation into the zebrafish genome to study the effects of JAK3 activation on lymphoid cells in a physiologically relevant context throughout the life-course.ResultsZebrafish Jak3 A573V produced the strongest activation of downstream STAT5 in vitro and elicited a significant increase in T cells in zebrafish embryos. Zebrafish carrying just a single copy of the Jak3 A573V allele displayed elevated embryonic T cells, which continued into adulthood. Hematopoietic precursors and NK cells were also increased, but not B cells. The lymphoproliferative effects of Jak3 A573V in embryos was shown to be dependent on zebrafish IL-2Rγc, JAK1 and STAT5B equivalents, and could be suppressed with the JAK3 inhibitor Tofacitinib.ConclusionsThis study demonstrates that a single JAK3 A573V allele expressed from the endogenous locus was able to enhance lymphopoiesis throughout the life-course, which was mediated via an IL-2Rγc/JAK1/JAK3/STAT5 signaling pathway and was sensitive to Tofacitinib. This extends our understanding of oncogenic JAK3 mutations and creates a novel model to underpin further translational investigations.

Influence of CYP2D6 genetic variation on adverse events with propafenone in the pediatric and young adult population.

Propafenone is an antiarrhythmic drug metabolized primarily by cytochrome P450 2D6 (CYP2D6). In adults, propafenone adverse events (AEs) are associated with CYP2D6 poor metabolizer status; however, pediatric data are lacking. Subjects were tested for 10 CYP2D6 allelic variants and copy number status, and activity scores assigned to each genotype. Seventy‐six individuals (median 0.3 [range 0–26] years old) were included. Propafenone AEs occurred in 29 (38%); 14 (18%) required drug discontinuation due to AE. The most common AEs were QRS (n = 10) and QTc (n = 6) prolongation. Those with AEs were older at the time of propafenone initiation (1.58 [0.13–9.92] vs. 0.20 [0.08–2.01] years old; p = 0.042). CYP2D6 activity scores were not associated with presence of an AE (odds ratio [OR] 0.48 [0.22–1.03]; p = 0.055) but with the total number of AE (β 1 = −0.31 [−0.60, −0.03]; p = 0.029), systemic AEs (OR 0.33 [0.13–0.88]; p = 0.022), and drug discontinuation for systemic AEs (OR 0.28 [0.09–0.83]; p = 0.017). Awareness of CYP2D6 activity score and patient age may aid in determining an individual's risk for an AE with propafenone administration.

Spectrum of germline AIRE mutations causing APS-1 and familial hypoparathyroidism.

ObjectiveThe autoimmune polyendocrine syndrome type 1 (APS-1) is an autosomal recessive disorder characterised by immune dysregulation and autoimmune endocrine gland destruction. APS-1 is caused by biallelic mutations affecting the autoimmune regulator (AIRE) gene on chromosome 21q22.3, which facilitates immunological self-tolerance. The objective was to investigate >300 probands with suspected APS-1 or isolated hypoparathyroidism for AIRE abnormalities.MethodsProbands were assessed by DNA sequence analysis. Novel variants were characterised using 3D modelling of the AIRE protein. Restriction enzyme and microsatellite analysis were used to investigate for uniparental isodisomy.ResultsBiallelic AIRE mutations were identified in 35 probands with APS-1 and 5 probands with isolated hypoparathyroidism. These included a novel homozygous p.(His14Pro) mutation, predicted to disrupt the N-terminal caspase activation recruitment domain of the AIRE protein. Furthermore, an apparently homozygous AIRE mutation, p.Leu323fs, was identified in an APS-1 proband, who is the child of non-consanguineous asymptomatic parents. Microsatellite analysis revealed that the proband inherited two copies of the paternal mutant AIRE allele due to uniparental isodisomy. Hypoparathyroidism was the most common endocrine manifestation in AIRE mutation-positive probands and >45% of those harbouring AIRE mutations had at least two diseases out of the triad of candidiasis, hypoparathyroidism, and hypoadrenalism. In contrast, type 1 diabetes and hypothyroidism occurred more frequently in AIRE mutation-negative probands with suspected APS-1. Around 30% of AIRE mutation-negative probands with isolated hypoparathyroidism harboured mutations in other hypoparathyroid genes.ConclusionsThis study of a large cohort referred for AIRE mutational analysis expands the spectrum of genetic abnormalities causing APS-1.

GSTM1 Copy Number and Kidney Disease in People With HIV

GSTM1 Copy Number and Kidney Disease in People With HIV

Accounting for population structure in genetic studies of cystic fibrosis.

SummaryCFTR F508del (c.1521_1523delCTT, p.Phe508delPhe) is the most common pathogenic allele underlying cystic fibrosis (CF), and its frequency varies in a geographic cline across Europe. We hypothesized that genetic variation associated with this cline is overrepresented in a large cohort (N > 5,000) of persons with CF who underwent whole-genome sequencing and that this pattern could result in spurious associations between variants correlated with both the F508del genotype and CF-related outcomes. Using principal-component (PC) analyses, we showed that variation in the CFTR region disproportionately contributes to a PC explaining a relatively high proportion of genetic variance. Variation near CFTR was correlated with population structure among persons with CF, and this correlation was driven by a subset of the sample inferred to have European ancestry. We performed genome-wide association studies comparing persons with CF with one versus two copies of the F508del allele; this allowed us to identify genetic variation associated with the F508del allele and to determine that standard PC-adjustment strategies eliminated the significant association signals. Our results suggest that PC adjustment can adequately prevent spurious associations between genetic variants and CF-related traits and are therefore effective tools to control for population structure even when population structure is confounded with disease severity and a common pathogenic variant.

A nickase Cas9 gene-drive system promotes super-Mendelian inheritance in Drosophila.

SUMMARYCRISPR-based gene-drives have been proposed for managing insect populations, including disease-transmitting mosquitoes, due to their ability to bias their inheritance toward super-Mendelian rates (>50%). Current technologies use a Cas9 that introduces DNA double-strand breaks into the opposing wild-type allele to replace it with a copy of the gene-drive allele via DNA homology-directed repair. However, the use of different Cas9 versions is unexplored, and alternative approaches could increase the available toolkit for gene-drive designs. Here, we report a gene-drive that relies on Cas9 nickases that generate staggered paired nicks in DNA to propagate the engineered gene-drive cassette. We show that generating 5′ overhangs in the system yields efficient allelic conversion. The nickase gene-drive arrangement produces large, stereotyped deletions that are advantageous to eliminate viable animals carrying small mutations when targeting essential genes. Our nickase approach should expand the repertoire for gene-drive arrangements aimed at applications in mosquitoes and beyond.

Germinal center expansion but not plasmablast differentiation is proportional to peptide-MHCII density via CD40-CD40L signaling strength.

SUMMARYT follicular helper (TFH) cells promote expansion of germinal center (GC) B cells and plasma cell differentiation. Whether cognate peptide-MHCII (pMHCII) density instructs selection and cell fate decisions in a quantitative manner remains unclear. Using αDEC205-OVA to differentially deliver OVA peptides to GC B cells on the basis of DEC205 allelic copy number, we find DEC205+/+ B cells take up 2-fold more antigen than DEC205+/− cells, leading to proportional TFH cell help and B cell expansion. To validate these results, we establish a caged OVA peptide, which is readily detected by OVA-specific TFH cells after photo-uncaging. In situ uncaging of peptides leads to multiple serial B-T contacts and cell activation. Differential CD40 signaling, is both necessary and sufficient to mediate 2-fold differences in B cell expansion. While plasmablast numbers are increased, pMHCII density does not directly control the output or quality of plasma cells. Thus, we distinguish the roles TFH cells play in expansion versus differentiation.