Single Variants Research Articles

Unravelling the effect of blood group on FVIII:C levels and response to DDAVP in 20 males with a single genotype (Twillingate Variant) causing Haemophilia A.

The genetic variant responsible for haemophilia A (HA) significantly impacts endogenous coagulant factor VIII (FVIII:C) level, thus impacting DDAVP responsiveness. Blood group (BG) also impacts FVIII:C levels, but this is difficult to evaluate in a genetically heterogeneous population. Canada has a large cohort of mild-moderate HA due to a single point variant: c.6104T>C, p.Val2035Ala-the Twillingate variant. To evaluate the impact of BG on endogenous FVIII:C levels and DDAVP responsiveness in a single genotype of mild-moderate HA. This was a retrospective, single-centre study. BG and FVIII:C levels were obtained for males with the Twillingate variant. One-hour absolute and fold increases in FVIII:C post-DDAVP were calculated. T-tests and Mann-Whitney U tests were used to compare FVIII:C levels and DDAVP challenge variables between individuals according to BGs (O vs. non-O). Twenty males were included. There were significant differences between BGs (O vs. non-O) in their lowest FVIII:C level at age <12years (medians: 0.05vs. 0.08 IU/mL; P=.05). Fifteen subjects underwent DDAVP challenges. Mean 1-h FVIII:C were 0.29 (O BG) versus 0.41 IU/mL (non-O BG); P=.04. There were no significant differences between BGs (O vs. non-O) in mean absolute FVIII:C increase (0.20vs. 0.27 IU/mL; P=.10) and FVIII:C fold increase (3.3-fold vs. 3.8-fold; P=.51). In HA subjects with an identical genotype, BG significantly impacts baseline FVIII:C levels and FVIII:C levels post-DDAVP, but does not impact absolute and fold increases in FVIII:C with DDAVP.

Benchmarking of bioinformatics tools for NGS-based microRNA profiling with RT-qPCR method

MicroRNAs are vital gene expression regulators, extensively studied worldwide. The large-scale characterization of miRNAomes is possible using next-generation sequencing (NGS). This technology offers great opportunities, but these cannot be fully exploited without proper and comprehensive bioinformatics analysis. This may be achieved by the use of reliable dedicated software; however, different programs may generate divergent results, leading to additional discrepancies. Thus, the aim of this study was to compare three bioinformatic algorithms dedicated to NGS-based microRNA profiling and validate them using an alternative method, namely RT-qPCR. The comparison analysis revealed differences in the number and sets of identified miRNAs. The qPCR confirmed the expression of the investigated microRNAs. The correlation analysis of NGS and qPCR measurements showed strong and significant coefficients for a subset of the tested miRNAs, including those detected by all three algorithms. Single miRNA variants (isomiRs) showed different levels of correlation with the qPCR data. The obtained results revealed the good performance of all tested programs, despite the observed differences. Moreover, they implied that some specific miRNAs may be differentially estimated using NGS technology and the qPCR method, regardless of the used bioinformatics software. These discrepancies may stem from many factors, including the composition of the isomiR profile, their abundance, length, and investigated species. In conclusion, in this study, we shed light on the bioinformatics aspects of miRNAome profiling, elucidating its complexity and pinpointing potential features influencing validation. Thus, qPCR validation results should be open to interpretation when not fully concordant with NGS results until further, additional analyses are conducted.

A missense GDF5 variant causes brachydactyly type A1 and multiple-synostoses syndrome 2.

This study aimed to identify the molecular defects and clinical manifestations in a Chinese family with brachydactyly (BD) type A1 (BDA1) and multiple-synostoses syndrome 2 (SYNS2). A Chinese family with BDA1 and SYNS2 was enrolled in this study. Whole-exome sequencing was used to analyze the gene variants in the proband. The sequences of the candidate pathogenic variant in GDF5 was validated via Sanger sequencing. I-TASSER and PyMOL were used to analyze the functional domains of the corresponding mutant proteins. The family was found to have an autosomal-dominantly inherited combination of BDA1 and SYNS2 caused by the S475N variant in the GDF5 gene. The variant was located within the functional region, and the mutated residue was found to be highly conserved among species. Via bioinformatic analyses, we predicted this variant to be deleterious, which perturb the protein function. The substitution of the negatively charged amino acid S475 with the neutral N475 was predicted to disrupt the formation of salt bridges with Y487 and impair the structure, stability, and function of the protein, consequently, the abnormalities in cartilage and bone development ensue. A single genetic variant (S475N) which disrupt the formation of salt bridges with Y487, in the interface of the antagonist- and receptor-binding sites of GDF5 concurrently causes two pathological mechanisms. This is the first report of this variant, identified in a Chinese family with BDA1 and SYNS2.

EBSD study of variant reorientation, texture, and twin formation in a martensitic NiTi alloy deformed in compression

Martensitic crystallography plays a vital role in the texture evolution and mechanical properties in Nickel-Titanium (NiTi) shape memory alloys when subjected to deformation. However, their microstructural changes during deformation are not well known and understood. In this study, we systematically investigate the stress-induced change of the microstructure of NiTi shape memory alloy under compression using the electron backscattered diffraction (EBSD) technique, and more specifically the variant reorientation, the evolution of textures, and the formation of twins. The EBSD maps reveal that upon strain the martensite morphologies shift from needle to block and their orientations are strongly reinforced along the loading axis. To quantify these microstructural changes, we used the Interaction Work (IW) associated with the lattice distortion of single variant, which provides a good fit with the experimental observation. A strong dependence of reorientation on the pre-textures of the parent B2 grains was also put in evidence. Other plasticity sources were confirmed such as dislocations and deformation twins. The calculations indicate that, for some specific orientations of parent grains, the active deformation twins could be easier for {011}M twin than that of (201¯)M deformation twin, whereas other orientations only favor the (201¯)M deformation twin.

Variant Classification Discordance: Contributing Factors and Predictive Models

An ever-growing catalog of human variants is hosted in the ClinVar database. In this database, submissions on a variant are combined into a multi-submitter record and in the case of discordance in variant classification between submitters, the record is labelled as conflicting. We used ClinVar data to identify characteristics that would make variants more likely to be associated with the conflict class of variants. Further, we used the Extreme Gradient Boosting (XGBoost) algorithm to train classifier models to provide prediction of classification discordance for single submission variants in ClinVar database.We showed that population allele frequency, the gene harboring the variant, variant type, consequence on protein, variant deleteriousness score, first submitter identity and submission count are associated with conflict in variant classification. Using such features, the optimized classifier showed accuracy on the test set of 88% with the weighted average of precision, recall and f1-score of 0.84, 0.88 and 0.85, respectively.There are pronounced associations between variant classification discordance and allele frequency, gene type, and the identity of the first submitter. We provide the predicted discordance status for single-submitter variants deposited in ClinVar. Our approach can be used to assess whether single-submitter variants are likely to be supported, or in conflict with, future entries; this knowledge might help laboratories with clinical variant assessment.

On the co-nucleation of adjoining twin pairs at grain boundaries in hexagonal close-packed materials

This work addresses the formation of adjoining twin pairs (ATPs) at grain boundaries (GBs) in hexagonal close-packed (hcp) metals from the perspective of the co-nucleation (CN) of pairs of deformation twins. A continuum defect mechanics model is proposed to investigate the energetic feasibility of the CN of ATPs resulting from the dissociation of GB dislocations. Application of the model to a set of GBs in Mg reveals that CN is largely preferred over the nucleation of a single twin variant for low misorientation angle GBs, which is consistent with previous experimental analyses. The CN events are subsequently analyzed considering the GB character, and the twin system alignment (m′) and Schmid factors to obtain better insights into the conditions governing the formation of ATPs via CN. The likelihood of CN even at low m′ values reveals that CN events could be responsible for the formation of ATP pairs in these scenarios.

Penetrance and expressivity of mitochondrial variants in a large clinically unselected population.

Whole genome sequencing (WGS) from large clinically unselected cohorts provides a unique opportunity to assess the penetrance and expressivity of rare and/or known pathogenic mitochondrial variants in population. Using WGS from 179 862 clinically unselected individuals from the UK Biobank, we performed extensive single and rare variant aggregation association analyses of 15 881 mtDNA variants and 73 known pathogenic variants with 15 mitochondrial disease-relevant phenotypes. We identified 12 homoplasmic and one heteroplasmic variant (m.3243A>G) with genome-wide significant associations in our clinically unselected cohort. Heteroplasmic m.3243A>G (MAF = 0.0002, a known pathogenic variant) was associated with diabetes, deafness and heart failure and 12 homoplasmic variants increased aspartate aminotransferase levels including three low-frequency variants (MAF ~0.002 and beta~0.3 SD). Most pathogenic mitochondrial disease variants (n = 66/74) were rare in the population (<1:9000). Aggregated or single variant analysis of pathogenic variants showed low penetrance in unselected settings for the relevant phenotypes, except m.3243A>G. Multi-system disease risk and penetrance of diabetes, deafness and heart failure greatly increased with m.3243A>G level ≥ 10%. The odds ratio of these traits increased from 5.61, 12.3 and 10.1 to 25.1, 55.0 and 39.5, respectively. Diabetes risk with m.3243A>G was further influenced by type 2 diabetes genetic risk. Our study of mitochondrial variation in a large-unselected population identified novel associations and demonstrated that pathogenic mitochondrial variants have lower penetrance in clinically unselected settings. m.3243A>G was an exception at higher heteroplasmy showing a significant impact on health making it a good candidate for incidental reporting.

A new compact, self-compressing, vertical one and two-stage gas gun at the University of Kent

A new gas gun configuration has been developed at the Centre for Astrophysics and Planetary Science, University of Kent, to produce vertical impacts at up to 2 km s−1. The vertical arrangement allows impact into non-cohesive target materials such as sand (representing loose regolith on the surface of solar system bodies) and water. Three key constraints had to be met: (1) the vertical gun had to fit in a laboratory with a ceiling of 3.5 m height, (2) the gun had to operate without a chemical propellant (i.e. gunpowder) and (3) it had to fire into the same target chamber as the existing horizontal Kent gun. In addition, the use of standard pipeline elements was adopted. The result was a self-compressing right angle shaped gun design, with single and two-stage variants for low and high speeds respectively. The gun assembly and its operation are described, together with ancillary components. The self-compressing first stage of the gun (using pressure generated by boiling liquid nitrogen) is horizontal leading into a vertical second stage. The gun has performed as expected, with shots in the speed range 0.3 to 2 km s−1. Results from the first study using the gun are presented for impacts of 1 mm diameter stainless steel spheres into sand over the full speed range, and are found to be compatible with previous work at low and high speed.

Crystallography of stress-induced martensitic transformation and giant elastocaloric effect in a A textured Ni27Cu21Mn46Sn6 shape memory alloy

Shape memory alloys exploit the latent heat yielded by stress-induced martensitic transformation to achieve elastocaloric effect, being suited for solid-state cooling technology as an alternative to conventional vapor-compression refrigeration. In this work, the crystallography of stress-induced martensitic transformation and elastocaloric effect in a polycrystalline Ni27Cu21Mn46Sn6 shape memory alloy with <001>A preferential orientation produced by means of directional solidification were studied. By performing ex-situ electron backscatter diffraction (EBSD) measurements, the transition from austenite to a single variant of 6 M martensite induced by compressive loading was evidenced, following a specific transformation orientation relationship of {1 0 1}A//{1 –2 –6}M and <1 0 − 1>A//<–6 –6 1>M between two phases. Moreover, owing to the synergistic effect of large transformation entropy change tailored through manipulating the lattice volume change across the phase transformation and microstructure texturing promoted by temperature gradient effect during solidification process, the present alloy can demonstrate very remarkable elastocaloric effect, with an extremely high value of adiabatic temperature variation up to –31.8 K upon releasing a moderate compressive loading of 460 MPa. This work is expected to give insight into stress-induced martensitic transformation crystallography and offer some inspirations for designing high-performance elastocaloric materials.

Spastic Paraplegia Type 7-Associated Optic Neuropathy: A Case Series.

Hereditary optic neuropathies comprise a group of clinically and genetically heterogeneous disorders. Optic neuropathy has been previously reported in families with spastic paraplegia type 7 (SPG7) gene mutations. However, the typical time course and clinical presentation of SPG7-associated optic neuropathy is poorly understood. We report a series of 5 patients harboring pathogenic SPG7 mutations who originally presented to a neuro-ophthalmology clinic with symptoms of optic neuropathy. Retrospective case series of 5 patients with pathogenic SPG7 mutations and optic atrophy from 3 neuro-ophthalmology clinics. Demographic, clinical, diagnostic, and treatment data were collected and reported by the clinician authors. Five patients ranging in age from 8 to 48 years were evaluated in the neuro-ophthalmology clinic. Although there were variable clinical presentations for each subject, all noted progressive vision loss, typically bilateral, and several also had previous diagnoses of peripheral neuropathy (e.g., Guillain-Barré Syndrome). Patients underwent neuro-ophthalmic examinations and testing with visual fields and optic coherence tomography of the retinal nerve fiber layer. Genetic testing revealed pathogenic variants in the SPG7 gene. Five patients presented to the neuro-ophthalmology clinic with progressive vision loss and were diagnosed with optic atrophy. Although each patient harbored an SPG7 mutation, this cohort was phenotypically and genotypically heterogeneous. Three patients carried the Ala510Val variant. The patients demonstrated varying degrees of visual acuity and visual field loss, although evaluations were completed during different stages of disease progression. Four patients had a previous diagnosis of peripheral neuropathy. This raises the prospect that a single pathogenic variant of SPG7 may be associated with peripheral neuropathy in addition to optic neuropathy. These results support the consideration of SPG7 testing in patients with high suspicion for genetic optic neuropathy, as manifested by symmetric papillomacular bundle damage without clear etiology on initial workup. Applied judiciously, genetic testing, including for SPG7, may help clarify the cause of unexplained progressive optic neuropathies.

The VUS Challenge in Cystic Kidney Disease: A Case-Based Review.

Genetic testing in nephrology is becoming increasingly important to diagnose patients and to provide appropriate care. This is especially true for autosomal dominant polycystic kidney disease (ADPKD) because this is a common cause of kidney failure and genetically complex. In addition to the major genes, PKD1 and PKD2 , there are at least six minor loci, and phenotypic, and in some cases, genetic overlap with other cystic disorders. Targeted next-generation sequencing, a low-cost, high-throughput technique, has made routine genetic testing viable in nephrology clinics. Appropriate pre- and post-testing genetic counseling is essential to the testing process. Carefully assessing variants is also critical, with the genetic report classifying variants in accordance with American College of Medical Genetics and Genomics guidelines. However, variant of uncertain significance (VUSs) may pose a significant challenge for the ordering clinician. In ADPKD, and particularly within PKD1 , there is high allelic heterogeneity; no single variant is present in more than 2% of families. The Mayo/Polycystic Kidney Disease Foundation variant database, a research tool, is the best current database of PKD1 and PKD2 variants containing over 2300 variants identified in individuals with polycystic kidney disease, but novel variants are often identified. In patients with a high pretest probability of ADPKD on the basis of clinical criteria, but no finding of a pathogenic (P) or likely pathogenic (LP) variant in a cystic kidney gene, additional evaluation of cystic gene VUS can be helpful. In this case-based review, we propose an algorithm for the assessment of such variants in a clinical setting and show how some can be reassigned to a diagnostic grouping. When assessing the relevance of a VUS, we consider both patient/family-specific and allele-related factors using population and variant databases and available prediction tools, as well as genetic expertise. This analysis plus further family studies can aid in making a genetic diagnosis.

BIOM-09. OPPOSING EFFECTS OF LINKED CX3CR1 GERMLINE VARIANTS ON SURVIVAL AND IMMUNE ACTIVATION IN GLIOMA

Abstract Linkage-disequilibrium connects CX3CR1 V249I and T280M single-nucleotide polymorphisms; V249I occurs as a single-variant (SV) and T280M exclusively as a double-variant (DV) concurrent with V249I. In low-grade gliomas (LGG), we found improved median overall-survival (mOS) in SV (n = 14, mOS 21.03yrs), compared to WT (n = 45, mOS 9.41yrs) and DV (n = 31, mOS 10.49yrs; p = 0.037 Log-rank). In TCGA glioblastoma (GBM), SV (n = 69, mOS 15.65mo) improved survival over DV (n = 106, mOS 11.74mo; p = 0.044 Log-rank). In a separate GBM cohort, bulk RNA-sequencing revealed MMP9 over-expression in DV (n = 15) compared to SV (n = 10; log2FC 2.87, p-adj 0.079). On a single-gene basis, MMP9 expression was decreased in SV compared to DV and WT (n = 33; p = 0.044, Kruskal-Wallis). MMP9 promotes tumor angiogenesis and invasion. SV showed SPP1 over-expression compared to DV (log2FC -1.75, p-adj 0.026), with decreased SPP1 expression in both WT and DV compared to SV (p = 0.004, Kruskal-Wallis). SPP1 is a pro-inflammatory cytokine enriched in glioma-associated macrophages. Gene Ontology revealed enrichment of biological processes: neutrophil migration, granulocyte chemotaxis, and neutrophil chemotaxis in SV (p&amp;lt; 0.05). We single-cell RNA-sequenced IDH mutant gliomas (51777 cells). We subset and re-clustered myeloid-derived cells (microglia/macrophages, 8944 cells), identifying thirteen clusters. Myeloid-derived cells clustered by CX3CR1 status, with 50.8% of SV cells in cluster five and 85.3% of DV cells in clusters six or seven. Cluster five over-expressed IL1B (average-log2FC 3.23), a tumor-suppressive M1 macrophage marker. Clusters six and seven were characterized by IFIT genes – immunomodulatory in myeloid-derived cells (IFIT3 average-log2FC 2.65 and 2.06). Assessing CX3CR1 status in normal population – 74.2% are WT, 10.0% SV, and 15.7% DV. In glioma patients, we identify 59.9% WT, 14.9% SV, and 25.2% DV – an increase of 49% in SV and 61% in DV (p = 1.95e-6, Chi-square). Our findings demonstrate opposing effects of linked CX3CR1 germline variants on glioma survival and indicate enhanced immune activation in SV.

An Analysis of the Neutralizing Antibodies against the Main SARS-CoV-2 Variants in Healthcare Workers (HCWs) Vaccinated against or Infected by SARS-CoV-2.

Although the anti-COVID-19 vaccination has proved to be an effective preventive tool, "breakthrough infections" have been documented in patients with complete primary vaccination courses. Most of the SARS-CoV-2 neutralizing antibodies produced after SARS-CoV-2 infection target the spike protein receptor-binding domain which has an important role in facilitating viral entry and the infection of the host cells. SARS-CoV-2 has demonstrated the ability to evolve by accumulating mutations in the spike protein to escape the humoral response of a host. The aim of this study was to compare the titers of neutralizing antibodies (NtAbs) against the variants of SARS-CoV-2 by analyzing the sera of recovered and vaccinated healthcare workers (HCWs). A total of 293 HCWs were enrolled and divided into three cohorts as follows: 91 who had recovered from SARS-CoV-2 infection (nVP); 102 that were vaccinated and became positive after the primary cycle (VP); and 100 that were vaccinated with complete primary cycles and concluded the follow-up period without becoming positive (VN). Higher neutralization titers were observed in the vaccinated subjects' arms compared to the nVP subjects' arms. Differences in neutralization titers between arms for single variants were statistically highly significant (p < 0.001), except for the differences between titers against the Alpha variant in the nVP and in VP groups, which were also statistically significant (p < 0.05). Within the nVP group, the number of subjects with an absence of neutralizing antibodies was high. The presence of higher titers in patients with a complete primary cycle compared to patients who had recovered from infection suggested the better efficacy of artificial immunization compared to natural immunization, and this further encourages the promotion of vaccination even in subjects with previous infections.

Plasma Neuronal Growth Regulator 1 May Link Physical Activity to Reduced Risk of Type 2 Diabetes: A Proteome-Wide Study of ARIC Participants.

Habitual physical activity (PA) impacts the plasma proteome and reduces the risk of developing type 2 diabetes (T2D). Using a large-scale proteome-wide approach in Atherosclerosis Risk in Communities Study participants, we aimed to identify plasma proteins associated with PA and determine which of these may be causally related to lower T2D risk. PA was associated with 92 plasma proteins in discovery (p<1.01x10-5), and 40 remained significant in replication (p<5.43x10-4). Eighteen of these proteins were independently associated with incident T2D (p<1.25x10-3) including neuronal growth regulator 1 (NeGR1) (HR per SD: 0.85; p-value=7.5x10-11). Two-sample Mendelian randomization inverse variance weighted analysis indicated that higher NeGR1 reduces T2D risk (OR per SD: 0.92; p=0.03) and was consistent with MR-Egger, weighted median, and weighted mode sensitivity analyses. A stronger association was observed for the single cis-acting NeGR1 genetic variant (OR per SD: 0.80; p=6.3x10-5). Coupled with previous evidence that low circulating NeGR1 levels promote adiposity, its association with PA and potential causal role in T2D shown here suggest that NeGR1 may link PA exposure with metabolic outcomes. Further research is warranted to confirm our findings and examine the interplay of PA, NeGR1, adiposity, and metabolic health.

Modulation of warm temperature-sensitive growth using a phytochrome B dark reversion variant, phyB[G515E], in Arabidopsis and rice

IntroductionAmbient temperature-induced hypocotyl elongation in Arabidopsis seedlings is sensed by the epidermis-localized phytochrome B (phyB) and transduced into auxin biosynthesis via a basic helix-loop-helix transcription factor, phytochrome-interacting factor 4 (PIF4). Once synthesized, auxin travels down from the cotyledons to the hypocotyl, triggering hypocotyl cell elongation. Thus, the phyB–PIF4 module involved in thermosensing and signal transduction is a potential genetic target for engineering warm temperature-insensitive plants. ObjectivesThis study aims to manipulate warm temperature-induced elongation of plants at the post-translational level using phyB variants with dark reversion, the expression of which is subjected to heat stress. MethodsThe thermosensitive growth response of Arabidopsis was manipulated by expressing the single amino acid substitution variant of phyB (phyB[G515E]), which exhibited a lower dark reversion rate than wild-type phyB. Other variants with slow (phyB[G564E]) or rapid (phyB[S584F]) dark reversion or light insensitivity (phyB[G767R]) were also included in this study for comparison. Warming-induced transient expression of phyB variants was achieved using heat shock-inducible promoters. Arabidopsis PHYB[G515E] and PHYB[G564E] were also constitutively expressed in rice in an attempt to manipulate the heat sensitivity of a monocotyledonous plant species. ResultsAt an elevated temperature, Arabidopsis seedlings transiently expressing PHYB[G515E] under the control of a heat shock-inducible promoter exhibited shorter hypocotyls than those expressing PHYB and other PHYB variant genes. This warm temperature-insensitive growth was related to the lowered PIF4 and auxin responses. In addition, transgenic rice seedlings expressing Arabidopsis PHYB[G515E] and PHYB[G564E] showed warm temperature-insensitive shoot growth. ConclusionTransient expression of phyB variants with altered dark reversion rates could serve as an effective optogenetic technique for manipulating PIF4–auxin-mediated thermomorphogenic responses in plants.

Exploring Mitochondrial DNA Heteroplasmy in a Mouse Model of Sickle Cell Disease

BACKGROUND: Mitochondria are important in the pathology of sickle cell disease (SCD). They are abnormally retained in sickle red blood cells (RBCs) and the likely source of cell-free mitochondrial DNA (mtDNA), an erythrocytic-DAMP that triggered formation of neutrophil extracellular traps (Tumburu et al, 2022). Acquired mutations in the mitochondrial genome, referred to mtDNA heteroplasmy, are emerging markers of aging and inflammation. Using whole genome sequence data from peripheral blood DNA, we previously established that patients with SCD not only have increased mtDNA heteroplasmy, but the burden also varied with the sickle genotype [Ahmad et. al, 2021]. The burden of mtDNA heteroplasmy is known to vary across different organs and tissues. Here, we utilized the Townes mouse model of SCD to explore differential mtDNA heteroplasmy burden across the different tissues and sickle genotypes in SCD. METHODS: Genomic DNA was extracted from 9 different organs (heart, brain, muscle, kidney, blood, bone marrow, liver, lungs, spleen) of littermate mice (3 male and 3 female) of three genotypes (HbAA, HbAS, HbSS) in three replica sets (total number of mice = 18, 9 different tissue samples per mouse, total sample number = 162). Mitochondria DNA was enriched from genomic DNA by long range PCR in 2 amplicons of 9kb and 7kb sizes (Misa Hirose et al, 2018). The enriched mtDNA PCR amplicons were mixed together and subjected to library preparation for deep sequencing using Illumina platform. Sequence reads aligned to mtDNA were mapped and called for variants using LoFreq variant calling tool. RESULTS: Of the 162 samples, 1 sample (HbAA, lung) was excluded from analysis due to low coverage. Using LoFreq variant caller tool (VAF cut-off 4% &amp; Coverage Filter 3500X), and after removing the repeating variants, 11 unique variants spanning 16kb mouse mitochondrial genome, were found in 106 of the 161 (65.8%) samples. The variants were differentially distributed across the tissues and genotypes with the variant at mito-position 9820 (MT: 9820) being most frequent, present in 19, 16 and 18 samples in AA, AS and SS mice, respectively (Fig. 1). Two variants at MT: 11866 and MT: 6881 were shared between AA and AS mice. Four variants were unique to SS mice: MT: 6753 in COX1 gene causing Phe476-Leu substitution, MT: 6794 in intergenic region upstream to COX2 gene, MT: 1220 in intergenic region upstream to ND1 gene, and MT425 in regulatory/D-Loop region (Table -1). The SS-unique variants were present in all tissues except for Cox1 Phe476-Leu (MT: 6753), a single variant that was found only in the spleen. Overall, HbSS had the highest concentration of variants distributed among 41/54 (75.9%) followed by AS 38 /54 (70.3 %) and AA 27/53 (50.9%). Female mice had higher mtDNA heteroplasmy burden compared to male mice in all genotypes. Across all genotypes and tissues, spleen had the highest number of variants (16.03%) and liver, the lowest (9.4%). CONCLUSION: In keeping with data in patients with SCD, HbSS mice had the highest mtDNA heteroplasmic burden followed by HbAS and HbAA genotypes. Four mito-variants were unique to HbSS. We also found differentially higher mtDNA heteroplasmic burden in splenic tissues and speculate that this could arise from metabolic stress conditions in the spleen.

Spectrum of ASXL1 Mutations Show Similar Impact on Outcomes in Patients with Myelofibrosis Undergoing Transplantation

Background The discovery of prognostically informative mutations in patients with primary myelofibrosis (PMF) prompted the development of mutation-enhanced risk scores. Among these mutations, those in ASXL1 are invariably associated with adverse outcome, either as single variants or as part of a high-mutation-risk category (HMR) including SRSF2, EZH2, and IDH1/2. Furthermore, it was suggested that ASXL1 mutations show distinct outcomes in PMF and post polycythemia vera / essential thrombocythemia myelofibrosis (PPV/PET MF). Particularly, the ASXL1G646fs* variant was reported to have a strong adverse impact on survival, specifically in patients with PMF. For patients undergoing curative treatment with hematopoietic cell transplantation (HCT), ASXL1 mutations were associated with increased risk for post-transplant mortality and therefore included in transplant-specific risk stratification. However, results on outcomes of different variants have not been reported so far. Patients and Methods We here report characteristics and outcomes of 273 patients with PMF (n=169, 62%) and PPV/PET MF (n=104; 38%) who underwent first HCT. Patients were homogenously treated with reduced-intensity conditioning using busulfan-fludarabine and anti-T-lymphocyte globulin for graft-versus-host disease prophylaxis. Patient samples were collected prior to start of conditioning and analyzed using next-generation sequencing. Primary endpoint was post-transplant overall survival, secondary endpoints were non-relapse mortality and incidence of relapse. Results The median age at the time of HCT was 59 years (range, 29-75). 57% of patients were male and 43% were female, 62% tested positive for cytomegalovirus (CMV). DIPSS at time of HCT was low (1%), intermediate-1 (22%), intermediate-2 (55%), and high (22%). Transfusion-dependency at time of HCT was present in 54% of patients, 50% received Ruxolitinib prior to HCT. Fibrosis grade 2 or higher was detected in 85% of patients. In terms of driver mutation genotype, 10% of patients were triple negative 63% of patients tested positive for JAK2, 22% for CALR, and 6% for MPL. In the total cohort, 24% of patients had a detectable ASXL1 mutation (7% had ASXL1G646fs* and 3% ASXL1R634fs*). There was a significant difference in distribution of mutations according to type of MF, with 83% and 71% of patients with ASXL1G646fs* or ASXL1R634fs* having PMF, while 75% of patients with other ASXL1 mutations had PMF compared with 25% having PPV/PET MF (P=0.04) . ASXL1G646fs* was associated with a tendency towards higher absolute leukocyte counts and lower platelet counts at time of HCT compared with wild-type or other ASXL1 mutations (P=0.10 and 0.07). DIPSS categories did not differ significantly between ASXL1G646fs*, other ASXL1 mutations and wild-type (P=0.66). No difference in co-occurring driver mutations between the groups was detected (P=0.38). Notably, more patients with ASXL1G646fs* had co-occurring mutations in EZH2 (P=0.004) and RUNX1 (P=0.003). In terms of outcomes, ASXL1 mutations showed worse overall survival (P=0.01), with no difference between ASXL1G646fs* and other ASXL1 mutations (P=0.51). 5-year survival was 54% for ASXL1G646fs* 53% for ASXL1 and 74% for the wild-type group. Poorer survival was seen in both PMF and PPV/PET MF (P=0.002 and P=0.03, respectively). Patients with ASXL1G646fs* showed significantly higher rates of early relapse 1 year after HCT, with incidence of 22% compared with 17% for other ASXL1 mutations and 10% for the wild-type group (P&amp;lt;0.001; Figure). Multivariable modelling (including donor type, leukocyte and platelet counts, Karnofsky performance status, and age) confirmed similar outcomes for the ASXL1 groups but higher risk for death compared with wild-type configuration, with hazard ratios of 1.50 for ASXL1G646fs* and 1.62 for other ASXL1 mutations. ASXL1G646fs* was associated with significantly higher risk for relapse (hazard ratio, 1.95). Conclusion ASXL1 mutations showed significantly inferior post-transplant outcomes in both PMF and PPV/PET MF. While the newly identified mutation ASXL1G646fs* was associated with worse outcomes (especially for post-transplant relapse), HCT seems to abrogate the recently suggested onset poorer outcomes for this mutation compared with other ASXL1 mutations.

Congenital Dyserythropoietic Anemia Type II: An Update from the Congenital Dyseryhtropoietic Anemia Registry of North America (CDAR)

Congenital Dyserythropoietic Anemias (CDAs) are a heterogeneous group of rare anemias characterized by ineffective erythropoiesis and hemolysis. The CDA registry in North America (CDAR; NCT02964494) was established to facilitate natural history studies. Participants who do not have a genetic diagnosis are offered family-trio whole genome sequencing (WGS) and elect to provide samples to CDAR biorepository to support collaborative mechanistic studies. CDA type II (CDA-II), the most common CDA type, is an autosomal recessive disease caused by biallelic SEC23Bvariants. CDA-II presents with hemolytic anemia with suboptimal reticulocytosis, iron overload, splenomegaly, and characteristic binucleation in 10-30% of the bone marrow (BM) erythroblasts. Although CDA-II is a recessive disorder, only one SEC23B variant is found in some patients. Here, we present the genetic and clinical data of CDA-II patients enrolled in CDAR so far. As of July 2023, 169 individuals (85 patients; 84 unaffected family members) enrolled in CDAR. Nine have a confirmed CDA-II diagnosis. Six patients have biallelic SEC23B pathogenic variants and 3 have only one variant identified. WGS for the 3 patients with a single variant revealed rare deep-intronic candidate variants. Since SEC23Bis ubiquitously expressed, we studied SEC23B protein levels in patient-derived lymphoblastoid cell lines generated for the CDAR biorepository. Patients with a single SEC23B variant exhibited an expression level comparable to those with biallelic mutations (Figure). The candidate deep intronic variants are currently being investigated for pathogenicity. Seven patients presented with hemolytic anemia during infancy, but the age of diagnosis was delayed by a mean of 12.5 yrs (0.1 to 55 y.o). Blood smears showed mild macrocytosis, poikilocytosis, and several spherocytes congruent with an osmotic gradient ektacytometry resembling mild hereditary spherocytosis. BM examination, when performed, exhibited erythroid hyperplasia, prominent dyserythropoiesis, and binucleation in ≥ 10% of erythroblasts. Anemia severity was variable: 6 patients were transfusion-dependent, 1 received intermittent transfusions, and 2 did not need blood transfusions. Two became transfusion-independent after splenectomy at ages 6 and 7 y.o, and two underwent successful matched-sibling BMT at age 10 and 26 y.o, after adequate chelation (liver iron concentration [LIC]&amp;lt;7 mg/g) Iron overload, disproportionate to transfusions, was present in 6 patients; 5 received iron chelation. All 5 patients with available MRI results had hepatic hemosiderosis (LIC range 3.4 to 21.8 mg/g) and evidence of myocardial hemosiderosis (T2* range 6.3 to 20 ms). Ferritin values were variable (range 58 to 1631.5 ng/ml) and discordant from LIC and T2* measurements. Notably, ferritin underestimated iron overload burden: 2 patients with normal ferritin (58 and 160 ng/mL) had hepatic hemosiderosis (LIC 6.1 and 3.4 mg/g respectively), and patients with severe hepatic and myocardial hemosiderosis (LIC 16.6-21.8 mg/g; T2* 6.3-7.8 ms) had modest ferritin values 1475-1631 ng/mL. Iron overload was present even in a non-transfusion-dependent patient and worsened in a patient post-splenectomy, suggesting a role for ineffective erythropoiesis. To further investigate this possibility, we measured erythroferrone (ERFE), GDF-15, and hepcidin levels in 2 patients so far. ERFE and GDF-15 levels were remarkably higher than controls, and hepcidin-to-ferritin ratios were significantly lower, suggesting that high ERFE, caused by ineffective erythropoiesis, inappropriately suppressed hepcidin for the body's iron levels. In summary, CDA-II diagnosis is often delayed despite early onset of anemia. Genetic testing may expedite the diagnosis. The severity of anemia is variable and there are several patients requiring transfusions. The risk of iron overload, including myocardial overload, is high and appears to be related to ineffective erythropoiesis. While frequency of transfusions may improve post-splenectomy, iron overload is not determined by blood transfusions alone and does not resolve post-splenectomy. Serum ferritin underestimated the burden of tissue iron in CDA-II, suggesting that MRI scans should be employed early. Future studies may identify a role for SEC23B deep intronic variants in CDA-II patients with heterozygous variants.

First-Hit SETBP1 Mutations Cause a Myeloproliferative Disorder with Bone Marrow Fibrosis

Somatic SETBP1 mutations are found in various myeloid disorders covering both myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS). To characterize the early steps of SETBP1-mediated leukemogenesis, we generated a conditional mouse model expressing SETBP1 G870S mutant in the entire hematopoietic tissue through Cre-mediated recombination driven by the Vav1 promoter. In all mice signs of a hematological disease appeared between 30 and 90 days: longitudinal analysis revealed accumulation of white blood cells (WBC) in virtually all heterozygous SETBP1 G870S mice, with a marked imbalance between the lymphoid and myeloid lineages in favor of the latter, and an increase in mature myeloid cells in absence of circulating blasts or non-segmented myeloid precursors. Kaplan-Meier analysis revealed a dramatic decrease in event-free survival in SETBP1 G870S mice. BM histology showed overt myeloid hyperplasia with fibrosis and no evidence of dysplasia except for the megakaryocytic lineage. Exploration of visceral organs highlighted the presence of severe hepatosplenomegaly with massive infiltration by myeloid elements, disruption of normal tissue architecture, and signs of extramedullary hematopoiesis. Single-cell RNA-sequencing (scRNA) on BM Lin - cells identified Mecom, Setbp1 and Hoxa9 among the top upregulated genes in SETBP1 G870S precursors. Pseudotime analysis of scRNA data revealed the presence of 261 spatially autocorrelated genes. Of them 158 were also differentially expressed in SETBP1 G870Svs control mice. Spi1, encoding for the master regulator of hematopoietic differentiation PU.1, was significantly upregulated in SETBP1 G870S precursors. PU.1 promotes the maturation of bone marrow early precursors towards the granulocytic/monocytic lineages by directly impairing the transcription of Gata2 and Gata1. In line with these data, Gata2 and Gata1 expression was profoundly suppressed in the early myeloid precursors of SETBP1 G870S mice, which associated with the down-modulation of markers of the erythroid lineage, such as the Carbonic Anhydrase 1, in the MEP differentiation branch. Ourmouse model recapitulates many clinical features of primary myelofibrosis (PMF). As up to 10% of PMF are triple-negative for the classical JAK2, CALR, and MPL mutations (TN-PMF), we set out to assess SETBP1 mutations in this context. We analyzed 36 TN-PMF patients by exome sequencing. In 29 we did not find any evidence of somatic mutations; in the remaining 7 (7/36; 19.4%) high VAF SETBP1 degron mutations were identified. In two patients SETBP1 was found as a single somatic variant, while in the others it coexisted with ASXL1, NRAS TET2 SRSF2, RIT1 CBL or CSF3R mutations. Notably, SETBP1 mutations were the only shared alterations among all seven patients. A markedly reduced overall survival was observed for SETBP1 positive patients, with a median survival time of 24 months (median survival not reached at 60 months for SETBP1 negative patients). To dissect the clonal architecture of SETBP1 positive TN-PMF at single-cell resolution, we applied single-cell targeted DNA sequencing on 3 SETBP1-mutated TN-PMF samples using the Tapestri technology, showing that, as opposite to MDS/MPN, in all TN-PMF cases SETBP1 is a very early clonal event. Therefore, our study suggests a clear partition of TN-PMF into two groups, the first one characterized by oncogenic, high VAF SETBP1 mutations accompanied by other oncogenic variants and poor prognosis and the other one characterized by no evidence of an active clonal process or of driver oncogenic events and much lower aggressiveness. SETBP1 positive TN-PMF disorders lie in a gray zone comprised between the MPN and MPN/MDS boundary. In MDS/MPN, SETBP1 mutations are often found as mid or late events. In contrast, we show here that in TN-PMF SETBP1 appears to be one of the earliest hits, therefore highlighting a potentially relevant biological difference occurring in the two subsets. In this context the presence of early SETBP1 mutations seems to promote the occurrence of a myeloid disorder characterized by the triad: leukocytosis without differentiation block or dysplasia, BM fibrosis and progressive splenomegaly, hence recapitulating many clinical features of overt or prefibrotic/early PMF and resembling the SETBP1 mouse model.

Ponatinib Versus Imatinib in Patients with Newly Diagnosed Ph+ ALL: Subgroup Analysis of the Phase 3 Phallcon Study

Background: BCR::ABL1 tyrosine kinase inhibitors (TKIs) in combination with chemotherapy or steroids remain the standard of care in patients with newly diagnosed Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). Most patients treated with first- or second-generation TKIs eventually experience disease progression due to treatment resistance. Ponatinib is the only currently approved pan-BCR::ABL1 inhibitory TKI that potently inhibits wild-type and single resistance mutation variants of BCR::ABL1, including T315I. Multiple studies have shown promising minimal residual disease (MRD) negativity (neg) rates and survival outcomes with ponatinib in combination with chemotherapy or chemotherapy-free regimens. PhALLCON (NCT03589326) is the first randomized study comparing frontline TKIs in adults with Ph+ ALL. The primary endpoint for PhALLCON was met, with a clinically significantly higher MRD-neg complete remission (CR) rate at end of induction (EOI) with ponatinib vs imatinib (34.4% vs 16.7%; risk difference: 0.18 [95% confidence interval (CI): 0.06‒0.29]; P=0.0021) and a manageable safety profile comparable with imatinib. Here we report subgroup efficacy analyses from PhALLCON. Methods: This global, registrational, phase 3, open-label study randomized newly diagnosed adult patients with Ph+ ALL 2:1 to receive ponatinib (30 mg once daily [QD]) or imatinib (600 mg QD) plus reduced-intensity chemotherapy through EOI (Cycles 1-3), consolidation (Cycles 4-9), and post-consolidation (Cycles 10-20). Following post-consolidation, patients continued to receive single-agent ponatinib or imatinib until disease progression or unacceptable toxicity. The composite primary endpoint was MRD-neg ( BCR:: ABL1 ≤0.01% [MR4])CR for 4 weeks at EOI. Event-free survival (EFS; any-cause death, failure to achieve CR by EOI, relapse from CR) was a key secondary endpoint. A post-hoc analysis of progression-free survival (PFS; EFS-defined events, failure to achieve MRD-neg by the end of treatment, and loss of MRD-neg) was conducted. Subgroup analyses were performed relative to baseline demographic and disease characteristics. Results: Of 245 randomized patients, 232 (ponatinib, n=154; imatinib, n=78) had baseline BCR::ABL1 p190/p210 variants verified by central lab (efficacy-evaluable population); median age was 54 years (37.1% ≥60 years). As of Aug 2022, 78 patients (ponatinib/imatinib: 68 [41.5%]/10 [12.3%]) remained on study treatment; the top 3 reasons for discontinuation were hematopoietic stem cell transplantation (HSCT; 30.5%/37.0%), adverse events (12.2%/12.3%), and lack of efficacy (7.3%/25.9%). Median follow-up in the ponatinib and imatinib arms was 20 months and 18 months, respectively. Benefit for ponatinib was observed across all subgroups analyzed ( Table). All age subgroups had higher rates of MRD-neg CR with ponatinib vs imatinib, with the greatest benefit observed in patients ≥60 years (40.0% ponatinib vs 10.3% imatinib; P=0.0005). MRD-neg CR rate was higher for ponatinib vs imatinib among those with the BCR::ABL1 p190 variant (38.6%/17.0%; P=0.0017); it was higher for the p210 variant as well, but not significantly (22.5% vs 16.0%; P=0.51). Median PFS was longer with ponatinib vs imatinib regardless of age, with the greatest difference observed in the subgroup of patients ≥60 years (22.5 months for ponatinib vs 8.3 months for imatinib; hazard ratio: 0.594 [95% CI: 0.332-1.063]). Conclusions: Ponatinib was superior to imatinib in combination with reduced-intensity chemotherapy in the front-line setting for patients with Ph+ ALL, with a clinically significantly higher MRD-neg CR rate at EOI. Benefit was observed across all subgroups, particularly for patients ≥60 years and for those with the BCR::ABL1 p190 variant.