Innate frequency-discrimination hyperacuity in Williams-Beuren syndrome mice.

Williams-Beuren syndrome (WBS) is a rare disorder caused by hemizygous microdeletion of ∼27 contiguous genes. Despite neurodevelopmental and cognitive deficits, individuals with WBS have spared or enhanced musical and auditory abilities, potentially offering an insight into the genetic basis of auditory perception. Here, we report that the mouse models of WBS have innately enhanced frequency-discrimination acuity and improved frequency coding in the auditory cortex (ACx). Chemogenetic rescue showed frequency-discrimination hyperacuity is caused by hyperexcitable interneurons in the ACx. Haploinsufficiency of one WBS gene, Gtf2ird1, replicated WBS phenotypes by downregulating the neuropeptide receptor VIPR1. VIPR1 is reduced in the ACx of individuals with WBS and in the cerebral organoids derived from human induced pluripotent stem cells with the WBS microdeletion. Vipr1 deletion or overexpression in ACx interneurons mimicked or reversed, respectively, the cellular and behavioral phenotypes of WBS mice. Thus, the Gtf2ird1-Vipr1 mechanism in ACx interneurons may underlie the superior auditory acuity in WBS.

Blinatumomab for MRD-Negative Acute Lymphoblastic Leukemia in Adults.

BACKGROUND: Many older adults with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) have a relapse despite having a measurable residual disease (MRD)-negative complete remission with combination chemotherapy. The addition of blinatumomab, a bispecific T-cell engager molecule that is approved for the treatment of relapsed, refractory, and MRD-positive BCP-ALL, may have efficacy in patients with MRD-negative remission. METHODS: In a phase 3 trial, we randomly assigned patients 30 to 70 years of age with BCR::ABL1-negative BCP-ALL (with :: indicating fusion) who had MRD-negative remission (defined as <0.01% leukemic cells in bone marrow as assessed on flow cytometry) after induction and intensification chemotherapy to receive four cycles of blinatumomab in addition to four cycles of consolidation chemotherapy or to receive four cycles of consolidation chemotherapy alone. The primary end point was overall survival, and relapse-free survival was a secondary end point. RESULTS: The data and safety monitoring committee reviewed the results from the third efficacy interim analysis and recommended that they be reported. Complete remission with or without full count recovery was observed in 395 of 488 enrolled patients (81%). Of the 224 patients with MRD-negative status, 112 were assigned to each group. The characteristics of the patients were balanced between the groups. At a median follow-up of 43 months, an advantage was observed in the blinatumomab group as compared with the chemotherapy-only group with regard to overall survival (at 3 years: 85% vs. 68%; hazard ratio for death, 0.41; 95% confidence interval [CI], 0.23 to 0.73; P = 0.002), and the 3-year relapse-free survival was 80% with blinatumomab and 64% with chemotherapy alone (hazard ratio for relapse or death, 0.53; 95% CI, 0.32 to 0.87). A higher incidence of neuropsychiatric events was reported in the blinatumomab group than in the chemotherapy-only group. CONCLUSIONS: The addition of blinatumomab to consolidation chemotherapy in adult patients in MRD-negative remission from BCP-ALL significantly improved overall survival. (Funded by the National Institutes of Health and others; E1910 ClinicalTrials.gov number, NCT02003222.).

SYK-CARD9 Signaling Axis Promotes Gut Fungi-Mediated Inflammasome Activation to Restrict Colitis and Colon Cancer.

Fungi represent a significant proportion of the gut microbiota. Aberrant immune responses to fungi are frequently observed in inflammatory bowel diseases (IBD) and colorectal cancer (CRC), and mutations in the fungal-sensing pathways are associated with the pathogenesis of IBD. Fungal recognition receptors trigger downstream signaling via the common adaptor protein CARD9 and the kinase SYK. Here we found that commensal gut fungi promoted inflammasome activation during AOM-DSS-induced colitis. Myeloid cell-specific deletion of Card9 or Syk reduced inflammasome activation and interleukin (IL)-18 maturation and increased susceptibility to colitis and CRC. IL-18 promoted epithelial barrier restitution and interferon-γ production by intestinal CD8+ T cells. Supplementation of IL-18 or transfer of wild-type myeloid cells reduced tumor burden in AOM-DSS-treated Card9-/- and Sykfl/flLysMCre/+ mice, whereas treatment with anti-fungal agents exacerbated colitis and CRC. CARD9 deletion changes the gut microbial landscape, suggesting that SYK-CARD9 signaling maintains a microbial ecology that promotes inflammasome activation and thereby restrains colitis and colon tumorigenesis.

Genomic determinants of response and resistance to inotuzumab ozogamicin in B-cell ALL.

ABSTRACT: Inotuzumab ozogamicin (InO) is an antibody-drug conjugate that delivers calicheamicin to CD22-expressing cells. In a retrospective cohort of InO-treated patients with B-cell acute lymphoblastic leukemia, we sought to understand the genomic determinants of the response and resistance to InO. Pre- and post-InO-treated patient samples were analyzed by whole genome, exome, and/or transcriptome sequencing. Acquired CD22 mutations were observed in 11% (3/27) of post-InO-relapsed tumor samples, but not in refractory samples (0/16). There were multiple CD22 mutations per sample and the mechanisms of CD22 escape included epitope loss (protein truncation and destabilization) and epitope alteration. Two CD22 mutant cases were post-InO hyper-mutators resulting from error-prone DNA damage repair (nonhomologous/alternative end-joining repair, or mismatch repair deficiency), suggesting that hypermutation drove escape from CD22-directed therapy. CD22-mutant relapses occurred after InO and subsequent hematopoietic stem cell transplantation (HSCT), suggesting that InO eliminated the predominant clones, leaving subclones with acquired CD22 mutations that conferred resistance to InO and subsequently expanded. Acquired loss-of-function mutations in TP53, ATM, and CDKN2A were observed, consistent with a compromise of the G1/S DNA damage checkpoint as a mechanism for evading InO-induced apoptosis. Genome-wide CRISPR/Cas9 screening of cell lines identified DNTT (terminal deoxynucleotidyl transferase) loss as a marker of InO resistance. In conclusion, genetic alterations modulating CD22 expression and DNA damage response influence InO efficacy. Our findings highlight the importance of defining the basis of CD22 escape and eradication of residual disease before HSCT. The identified mechanisms of escape from CD22-targeted therapy extend beyond antigen loss and provide opportunities to improve therapeutic approaches and overcome resistance. These trials were registered at www.ClinicalTrials.gov as NCT01134575, NCT01371630, and NCT03441061.

Acute myeloid leukemias with UBTF tandem duplications are sensitive to menin inhibitors.

ABSTRACT: UBTF tandem duplications (UBTF-TDs) have recently emerged as a recurrent alteration in pediatric and adult acute myeloid leukemia (AML). UBTF-TD leukemias are characterized by a poor response to conventional chemotherapy and a transcriptional signature that mirrors NUP98-rearranged and NPM1-mutant AMLs, including HOX-gene dysregulation. However, the mechanism by which UBTF-TD drives leukemogenesis remains unknown. In this study, we investigated the genomic occupancy of UBTF-TD in transformed cord blood CD34+ cells and patient-derived xenograft models. We found that UBTF-TD protein maintained genomic occupancy at ribosomal DNA loci while also occupying genomic targets commonly dysregulated in UBTF-TD myeloid malignancies, such as the HOXA/HOXB gene clusters and MEIS1. These data suggest that UBTF-TD is a gain-of-function alteration that results in mislocalization to genomic loci dysregulated in UBTF-TD leukemias. UBTF-TD also co-occupies key genomic loci with KMT2A and menin, which are known to be key partners involved in HOX-dysregulated leukemias. Using a protein degradation system, we showed that stemness, proliferation, and transcriptional signatures are dependent on sustained UBTF-TD localization to chromatin. Finally, we demonstrate that primary cells from UBTF-TD leukemias are sensitive to the menin inhibitor SNDX-5613, resulting in markedly reduced in vitro and in vivo tumor growth, myeloid differentiation, and abrogation of the UBTF-TD leukemic expression signature. These findings provide a viable therapeutic strategy for patients with this high-risk AML subtype.

Genomic landscape of Down syndrome-associated acute lymphoblastic leukemia.

Trisomy 21, the genetic cause of Down syndrome (DS), is the most common congenital chromosomal anomaly. It is associated with a 20-fold increased risk of acute lymphoblastic leukemia (ALL) during childhood and results in distinctive leukemia biology. To comprehensively define the genomic landscape of DS-ALL, we performed whole-genome sequencing and whole-transcriptome sequencing (RNA-Seq) on 295 cases. Our integrated genomic analyses identified 15 molecular subtypes of DS-ALL, with marked enrichment of CRLF2-r, IGH::IGF2BP1, and C/EBP altered (C/EBPalt) subtypes compared with 2257 non-DS-ALL cases. We observed abnormal activation of the CEBPD, CEBPA, and CEBPE genes in 10.5% of DS-ALL cases via a variety of genomic mechanisms, including chromosomal rearrangements and noncoding mutations leading to enhancer hijacking. A total of 42.3% of C/EBP-activated DS-ALL also have concomitant FLT3 point mutations or insertions/deletions, compared with 4.1% in other subtypes. CEBPD overexpression enhanced the differentiation of mouse hematopoietic progenitor cells into pro-B cells in vitro, particularly in a DS genetic background. Notably, recombination-activating gene-mediated somatic genomic abnormalities were common in DS-ALL, accounting for a median of 27.5% of structural alterations, compared with 7.7% in non-DS-ALL. Unsupervised hierarchical clustering analyses of CRLF2-rearranged DS-ALL identified substantial heterogeneity within this group, with the BCR::ABL1-like subset linked to an inferior event-free survival, even after adjusting for known clinical risk factors. These results provide important insights into the biology of DS-ALL and point to opportunities for targeted therapy and treatment individualization.

Discovery of novel predisposing coding and noncoding variants in familial Hodgkin lymphoma.

Familial aggregation of Hodgkin lymphoma (HL) has been demonstrated in large population studies, pointing to genetic predisposition to this hematological malignancy. To understand the genetic variants associated with the development of HL, we performed whole genome sequencing on 234 individuals with and without HL from 36 pedigrees that had 2 or more first-degree relatives with HL. Our pedigree selection criteria also required at least 1 affected individual aged <21 years, with the median age at diagnosis of 21.98 years (3-55 years). Family-based segregation analysis was performed for the identification of coding and noncoding variants using linkage and filtering approaches. Using our tiered variant prioritization algorithm, we identified 44 HL-risk variants in 28 pedigrees, of which 33 are coding and 11 are noncoding. The top 4 recurrent risk variants are a coding variant in KDR (rs56302315), a 5' untranslated region variant in KLHDC8B (rs387906223), a noncoding variant in an intron of PAX5 (rs147081110), and another noncoding variant in an intron of GATA3 (rs3824666). A newly identified splice variant in KDR (c.3849-2A>C) was observed for 1 pedigree, and high-confidence stop-gain variants affecting IRF7 (p.W238∗) and EEF2KMT (p.K116∗) were also observed. Multiple truncating variants in POLR1E were found in 3 independent pedigrees as well. Whereas KDR and KLHDC8B have previously been reported, PAX5, GATA3, IRF7, EEF2KMT, and POLR1E represent novel observations. Although there may be environmental factors influencing lymphomagenesis, we observed segregation of candidate germline variants likely to predispose HL in most of the pedigrees studied.

The genomic landscape of acute lymphoblastic leukemia with intrachromosomal amplification of chromosome 21.

Intrachromosomal amplification of chromosome 21 defines a subtype of high-risk childhood acute lymphoblastic leukemia (iAMP21-ALL) characterized by copy number changes and complex rearrangements of chromosome 21. The genomic basis of iAMP21-ALL and the pathogenic role of the region of amplification of chromosome 21 to leukemogenesis remains incompletely understood. In this study, using integrated whole genome and transcriptome sequencing of 124 patients with iAMP21-ALL, including rare cases arising in the context of constitutional chromosomal aberrations, we identified subgroups of iAMP21-ALL based on the patterns of copy number alteration and structural variation. This large data set enabled formal delineation of a 7.8 Mb common region of amplification harboring 71 genes, 43 of which were differentially expressed compared with non-iAMP21-ALL ones, including multiple genes implicated in the pathogenesis of acute leukemia (CHAF1B, DYRK1A, ERG, HMGN1, and RUNX1). Using multimodal single-cell genomic profiling, including single-cell whole genome sequencing of 2 cases, we documented clonal heterogeneity and genomic evolution, demonstrating that the acquisition of the iAMP21 chromosome is an early event that may undergo progressive amplification during disease ontogeny. We show that UV-mutational signatures and high mutation load are characteristic secondary genetic features. Although the genomic alterations of chromosome 21 are variable, these integrated genomic analyses and demonstration of an extended common minimal region of amplification broaden the definition of iAMP21-ALL for more precise diagnosis using cytogenetic or genomic methods to inform clinical management.

Gain-of-function mutations in RPA1 cause a syndrome with short telomeres and somatic genetic rescue.

Human telomere biology disorders (TBD)/short telomere syndromes (STS) are heterogeneous disorders caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify 3 germline heterozygous missense variants in the RPA1 gene in 4 unrelated probands presenting with short telomeres and varying clinical features of TBD/STS, including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, or skin manifestations. All variants cluster to DNA-binding domain A of RPA1 protein. RPA1 is a single-strand DNA-binding protein required for DNA replication and repair and involved in telomere maintenance. We showed that RPA1E240K and RPA1V227A proteins exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function, whereas RPA1T270A has binding properties similar to wild-type protein. To study the mutational effect in a cellular system, CRISPR/Cas9 was used to knock-in the RPA1E240K mutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patients with RPA1E240K, we discovered somatic genetic rescue in hematopoietic cells due to an acquired truncating cis RPA1 mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the 2 somatic genetic rescue events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1 variants in individuals with TBD/STS.

Somatic and Germline Variant Calling from Next-Generation Sequencing Data.

Re-sequencing of the human genome by next-generation sequencing (NGS) has been widely applied to discover pathogenic genetic variants and/or causative genes accounting for various types of diseases including cancers. The advances in NGS have allowed the sequencing of the entire genome of patients and identification of disease-associated variants in a reasonable timeframe and cost. The core of the variant identification relies on accurate variant calling and annotation. Numerous algorithms have been developed to elucidate the repertoire of somatic and germline variants. Each algorithm has its own distinct strengths, weaknesses, and limitations due to the difference in the statistical modeling approach adopted and read information utilized. Accurate variant calling remains challenging due to the presence of sequencing artifacts and read misalignments. All of these can lead to the discordance of the variant calling results and even misinterpretation of the discovery. For somatic variant detection, multiple factors including chromosomal abnormalities, tumor heterogeneity, tumor-normal cross contaminations, unbalanced tumor/normal sample coverage, and variants with low allele frequencies add even more layers of complexity to accurate variant identification. Given the discordances and difficulties, ensemble approaches have emerged by harmonizing information from different algorithms to improve variant calling performance. In this chapter, we first introduce the general scheme of variant calling algorithms and potential challenges at distinct stages. We next review the existing workflows of variant calling and annotation, and finally explore the strategies deployed by different callers as well as their strengths and caveats. Overall, NGS-based variant identification with careful consideration allows reliable detection of pathogenic variant and candidate variant selection for precision medicine.

Estimated number of adult survivors of childhood cancer in United States with cancer-predisposing germline variants.

PURPOSE: To estimate the absolute number of adult survivors of childhood cancer in the U.S. population who carry a pathogenic or likely pathogenic variant in a cancer predisposition gene. METHODS: Using the Surveillance, Epidemiology, and End Results (SEER) Program, we estimated the number of childhood cancer survivors on December 31, 2016 for each childhood cancer diagnosis, multiplied this by the proportion of carriers of pathogenic/likely pathogenic variants in the St. Jude Lifetime Cohort (SJLIFE) study, and projected the resulting number onto the U.S. RESULTS: Based on genome sequence data, 11.8% of 2450 SJLIFE participants carry a pathogenic/likely pathogenic variant in one of 156 cancer predisposition genes. Given this information, we estimate that 21 800 adult survivors of childhood cancer in the United States carry a pathogenic/likely pathogenic variant in one of these genes. The highest estimated absolute number of variant carriers are among survivors of central nervous system tumors (n = 4300), particularly astrocytoma (n = 1800) and other gliomas (n = 1700), acute lymphoblastic leukemia (n = 4300), and retinoblastoma (n = 3500). The most frequently mutated genes are RB1 (n = 3000), NF1 (n = 2300), and BRCA2 (n = 800). CONCLUSION: Given the increasing number of childhood cancer survivors in the United States, clinicians should counsel survivors regarding their potential genetic risk, consider referral for genetic counseling and testing, and, as appropriate, implement syndrome-specific cancer surveillance or risk-reducing measures.

Structure and evolution of double minutes in diagnosis and relapse brain tumors.

Double minute chromosomes are extrachromosomal circular DNA fragments frequently found in brain tumors. To understand their evolution, we characterized the double minutes in paired diagnosis and relapse tumors from a pediatric high-grade glioma and four adult glioblastoma patients. We determined the full structures of the major double minutes using a novel approach combining multiple types of supporting genomic evidence. Among the double minutes identified in the pediatric patient, only one carrying EGFR was maintained at high abundance in both samples, whereas two others were present in only trace amounts at diagnosis but abundant at relapse, and the rest were found either in the relapse sample only or in the diagnosis sample only. For the EGFR-carrying double minutes, we found a secondary somatic deletion in all copies at relapse, after erlotinib treatment. However, the somatic mutation was present at very low frequency at diagnosis, suggesting potential resistance to the EGFR inhibitor. This mutation caused an in-frame RNA transcript to skip exon 16, a novel transcript isoform absent in EST database, as well as about 700 RNA-seq of normal brains that we reviewed. We observed similar patterns involving longitudinal copy number shift of double minutes in another four pairs (diagnosis/relapse) of adult glioblastoma. Overall, in three of five paired tumor samples, we found that although the same oncogenes were amplified at diagnosis and relapse, they were amplified on different double minutes. Our results suggest that double minutes readily evolve, increasing tumor heterogeneity rapidly. Understanding patterns of double minute evolution can shed light on future therapeutic solutions to brain tumors carrying such variants.

Comparative Genomics of the Sigatoka Disease Complex on Banana Suggests a Link between Parallel Evolutionary Changes in Pseudocercospora fijiensis and Pseudocercospora eumusae and Increased Virulence on the Banana Host.

The Sigatoka disease complex, caused by the closely-related Dothideomycete fungi Pseudocercospora musae (yellow sigatoka), Pseudocercospora eumusae (eumusae leaf spot), and Pseudocercospora fijiensis (black sigatoka), is currently the most devastating disease on banana worldwide. The three species emerged on bananas from a recent common ancestor and show clear differences in virulence, with P. eumusae and P. fijiensis considered the most aggressive. In order to understand the genomic modifications associated with shifts in the species virulence spectra after speciation, and to identify their pathogenic core that can be exploited in disease management programs, we have sequenced and analyzed the genomes of P. eumusae and P. musae and compared them with the available genome sequence of P. fijiensis. Comparative analysis of genome architectures revealed significant differences in genome size, mainly due to different rates of LTR retrotransposon proliferation. Still, gene counts remained relatively equal and in the range of other Dothideomycetes. Phylogenetic reconstruction based on a set of 46 conserved single-copy genes strongly supported an earlier evolutionary radiation of P. fijiensis from P. musae and P. eumusae. However, pairwise analyses of gene content indicated that the more virulent P. eumusae and P. fijiensis share complementary patterns of expansions and contractions in core gene families related to metabolism and enzymatic degradation of plant cell walls, suggesting that the evolution of virulence in these two pathogens has, to some extent, been facilitated by convergent changes in metabolic pathways associated with nutrient acquisition and assimilation. In spite of their common ancestry and shared host-specificity, the three species retain fairly dissimilar repertoires of effector proteins, suggesting that they likely evolved different strategies for manipulating the host immune system. Finally, 234 gene families, including seven putative effectors, were exclusively present in the three Sigatoka species, and could thus be related to adaptation to the banana host.

Gut Microbiome Composition Predicts Infection Risk During Chemotherapy in Children With Acute Lymphoblastic Leukemia.

Background: Myelosuppression-related infections remain important causes of morbidity and mortality in children with acute lymphoblastic leukemia (ALL). Methods: By analyzing fecal samples collected at diagnosis and after each of the initial 3 phases of chemotherapy, we evaluated the role of gut microbiota in predicting infections in 199 children with newly diagnosed ALL. The bacterial 16S rRNA gene was analyzed by high-depth sequencing to determine the diversity and composition of the microbiome. Results: After the induction and reinduction I phases of chemotherapy, microbial diversity decreased significantly relative to the prechemotherapy value. After chemotherapy, the relative abundance of certain bacterial taxa (eg, Bacteroidetes) decreased significantly, whereas that of other taxa (eg, Clostridiaceae and Streptococcaceae) increased. A baseline gut microbiome characterized by Proteobacteria predicted febrile neutropenia. Adjusting for the chemotherapy phase and ALL risk level, Enterococcaceae dominance (relative abundance ≥30%) predicted significantly greater risk of subsequent febrile neutropenia and diarrheal illness, whereas Streptococcaceae dominance predicted significantly greater risk of subsequent diarrheal illness. Conclusions: In children undergoing therapy for newly diagnosed ALL, the relative abundance of Proteobacteria before chemotherapy initiation predicts development of febrile neutropenia, and domination of the gut microbiota by Enterococcaceae or Streptococcaceae at any time during chemotherapy predicts infection in subsequent phases of chemotherapy. Clinical Trial Registration: NCT00549848.

Pyruvate Oxidase as a Critical Link between Metabolism and Capsule Biosynthesis in Streptococcus pneumoniae.

The pneumococcus is one of the most prodigious producers of hydrogen peroxide amongst bacterial pathogens. Hydrogen peroxide production by the pneumococcus has been implicated in antibiotic synergism, competition between other bacterial colonizers of the nasopharynx, and damage to epithelial cells. However, the role during invasive disease has been less clear with mutants defective in hydrogen peroxide production demonstrating both attenuation and heightened invasive disease capacity depending upon strain and serotype background. This work resolves these conflicting observations by demonstrating that the main hydrogen peroxide producing enzyme of the pneumococcus, SpxB, is required for capsule formation in a strain dependent manner. Capsule production by strains harboring capsules with acetylated sugars was dependent upon the presence of spxB while capsule production in serotypes lacking such linkages were not. The spxB mutant had significantly lower steady-state cellular levels of acetyl-CoA, suggesting that loss of capsule arises from dysregulation of this intermediary metabolite. This conclusion is corroborated by deletion of pdhC, which also resulted in lower steady-state acetyl-CoA levels and phenocopied the capsule expression profile of the spxB mutant. Capsule and acetyl-CoA levels were restored in the spxB and lctO (lactate oxidase) double mutant, supporting the connection between central metabolism and capsule formation. Taken together, these data show that the defect in pathogenesis in the spxB mutant is due to a metabolic imbalance that attenuates capsule formation and not to reduced hydrogen peroxide formation.

Male-specific region of the bovine Y chromosome is gene rich with a high transcriptomic activity in testis development.

The male-specific region of the mammalian Y chromosome (MSY) contains clusters of genes essential for male reproduction. The highly repetitive and degenerative nature of the Y chromosome impedes genomic and transcriptomic characterization. Although the Y chromosome sequence is available for the human, chimpanzee, and macaque, little is known about the annotation and transcriptome of nonprimate MSY. Here, we investigated the transcriptome of the MSY in cattle by direct testis cDNA selection and RNA-seq approaches. The bovine MSY differs radically from the primate Y chromosomes with respect to its structure, gene content, and density. Among the 28 protein-coding genes/families identified on the bovine MSY (12 single- and 16 multicopy genes), 16 are bovid specific. The 1,274 genes identified in this study made the bovine MSY gene density the highest in the genome; in comparison, primate MSYs have only 31-78 genes. Our results, along with the highly transcriptional activities observed from these Y-chromosome genes and 375 additional noncoding RNAs, challenge the widely accepted hypothesis that the MSY is gene poor and transcriptionally inert. The bovine MSY genes are predominantly expressed and are differentially regulated during the testicular development. Synonymous substitution rate analyses of the multicopy MSY genes indicated that two major periods of expansion occurred during the Miocene and Pliocene, contributing to the adaptive radiation of bovids. The massive amplification and vigorous transcription suggest that the MSY serves as a genomic niche regulating male reproduction during bovid expansion.

Copy number variations of the extensively amplified Y-linked genes, HSFY and ZNF280BY, in cattle and their association with male reproductive traits in Holstein bulls.

BACKGROUND: Recent transcriptomic analysis of the bovine Y chromosome revealed at least six multi-copy protein coding gene families, including TSPY, HSFY and ZNF280BY, on the male-specific region (MSY). Previous studies indicated that the copy number variations (CNVs) of the human and bovine TSPY were associated with male fertility in men and cattle. However, the relationship between CNVs of the bovine Y-linked HSFY and ZNF280BY gene families and bull fertility has not been investigated. RESULTS: We investigated the copy number (CN) of the bovine HSFY and ZNF280BY in a total of 460 bulls from 15 breeds using a quantitative PCR approach. We observed CNVs for both gene families within and between cattle breeds. The median copy number (MCN) of HSFY among all bulls was 197, ranging from 21 to 308. The MCN of ZNF280BY was 236, varying from 28 to 380. Furthermore, bulls in the Bos taurus (BTA) lineage had a significantly higher MCN (202) of HSFY than bulls in the Bos indicus (BIN) lineage (178), while taurine bulls had a significantly lower MCN (231) of ZNF280BY than indicine bulls (284). In addition, the CN of ZNF280BY was positively correlated to that of HSFY on the BTAY. Association analysis revealed that the CNVs of both HSFY and ZNF280BY were correlated negatively with testis size, while positively with sire conception rate. CONCLUSION: The bovine HSFY and ZNF280BY gene families have extensively expanded on the Y chromosome during evolution. The CN of both gene families varies significantly among individuals and cattle breeds. These variations were associated with testis size and bull fertility in Holstein, suggesting that the CNVs of HSFY and ZNF280BY may serve as valuable makers for male fertility selection in cattle.

Machine learning to optimize automated RH genotyping using whole-exome sequencing data.

ABSTRACT: Rh phenotype matching reduces but does not eliminate alloimmunization in patients with sickle cell disease (SCD) due to RH genetic diversity that is not distinguishable by serological typing. RH genotype matching can potentially mitigate Rh alloimmunization but comprehensive and accessible genotyping methods are needed. We developed RHtyper as an automated algorithm to predict RH genotypes using whole-genome sequencing (WGS) data with high accuracy. Here, we adapted RHtyper for whole-exome sequencing (WES) data, which are more affordable but challenged by uneven sequencing coverage and exacerbated sequencing read misalignment, resulting in uncertain predictions for (1) RHD zygosity and hybrid alleles, (2) RHCE∗C vs. RHCE∗c alleles, (3) RHD c.1136C>T zygosity, and (4) RHCE c.48G>C zygosity. We optimized RHtyper to accurately predict RHD and RHCE genotypes using WES data by leveraging machine learning models and improved the concordance of WES with WGS predictions from 90.8% to 97.2% for RHD and 96.3% to 98.2% for RHCE among 396 patients in the Sickle Cell Clinical Research and Intervention Program. In a second validation cohort of 3030 cancer survivors (15.2% Black or African Americans) from the St. Jude Lifetime Cohort Study, the optimized RHtyper reached concordance rates between WES and WGS predications to 96.3% for RHD and 94.6% for RHCE. Machine learning improved the accuracy of RH predication using WES data. RHtyper has the potential, once implemented, to provide a precision medicine-based approach to facilitate RH genotype-matched transfusion and improve transfusion safety for patients with SCD. This study used data from clinical trials registered at ClinicalTrials.gov as #NCT02098863 and NCT00760656.

Metabolic reprogramming of cancer cells by JMJD6-mediated pre-mRNA splicing associated with therapeutic response to splicing inhibitor.

Dysregulated pre-mRNA splicing and metabolism are two hallmarks of MYC-driven cancers. Pharmacological inhibition of both processes has been extensively investigated as potential therapeutic avenues in preclinical and clinical studies. However, how pre-mRNA splicing and metabolism are orchestrated in response to oncogenic stress and therapies is poorly understood. Here, we demonstrate that jumonji domain containing 6, arginine demethylase, and lysine hydroxylase, JMJD6, acts as a hub connecting splicing and metabolism in MYC-driven human neuroblastoma. JMJD6 cooperates with MYC in cellular transformation of murine neural crest cells by physically interacting with RNA binding proteins involved in pre-mRNA splicing and protein homeostasis. Notably, JMJD6 controls the alternative splicing of two isoforms of glutaminase (GLS), namely kidney-type glutaminase (KGA) and glutaminase C (GAC), which are rate-limiting enzymes of glutaminolysis in the central carbon metabolism in neuroblastoma. Further, we show that JMJD6 is correlated with the anti-cancer activity of indisulam, a 'molecular glue' that degrades splicing factor RBM39, which complexes with JMJD6. The indisulam-mediated cancer cell killing is at least partly dependent on the glutamine-related metabolic pathway mediated by JMJD6. Our findings reveal a cancer-promoting metabolic program is associated with alternative pre-mRNA splicing through JMJD6, providing a rationale to target JMJD6 as a therapeutic avenue for treating MYC-driven cancers.

Somatic mitochondrial DNA mutations are a source of heterogeneity among primary leukemic cells.

Somatic mitochondrial DNA (mtDNA) mutations are prevalent in tumors, yet defining their biological significance remains challenging due to the intricate interplay between selective pressure, heteroplasmy, and cell state. Utilizing bulk whole-genome sequencing data from matched tumor and normal samples from two cohorts of pediatric cancer patients, we uncover differences in the accumulation of synonymous and nonsynonymous mtDNA mutations in pediatric leukemias, indicating distinct selective pressures. By integrating single-cell sequencing (SCS) with mathematical modeling and network-based systems biology approaches, we identify a correlation between the extent of cell-state changes associated with tumor-enriched mtDNA mutations and the selective pressures shaping their distribution among individual leukemic cells. Our findings also reveal an association between specific heteroplasmic mtDNA mutations and cellular responses that may contribute to functional heterogeneity among leukemic cells and influence their fitness. This study highlights the potential of SCS strategies for distinguishing between pathogenic and passenger somatic mtDNA mutations in cancer.