Circuit-specific gene therapy reverses core symptoms in a primate Parkinson's disease model.

Parkinson's disease (PD) is a debilitating neurodegenerative disorder. Its symptoms are typically treated with levodopa or dopamine receptor agonists, but its action lacks specificity due to the wide distribution of dopamine receptors in the central nervous system and periphery. Here, we report the development of a gene therapy strategy to selectively manipulate PD-affected circuitry. Targeting striatal D1 medium spiny neurons (MSNs), whose activity is chronically suppressed in PD, we engineered a therapeutic strategy comprised of a highly efficient retrograde adeno-associated virus (AAV), promoter elements with strong D1-MSN activity, and a chemogenetic effector to enable precise D1-MSN activation after systemic ligand administration. Application of this therapeutic approach rescues locomotion, tremor, and motor skill defects in both mouse and primate models of PD, supporting the feasibility of targeted circuit modulation tools for the treatment of PD in humans.

Efficient embryonic homozygous gene conversion via RAD51-enhanced interhomolog repair.

Searching for factors to improve knockin efficiency for therapeutic applications, biotechnology, and generation of non-human primate models of disease, we found that the strand exchange protein RAD51 can significantly increase Cas9-mediated homozygous knockin in mouse embryos through an interhomolog repair (IHR) mechanism. IHR is a hallmark of meiosis but only occurs at low frequencies in somatic cells, and its occurrence in zygotes is controversial. Using multiple approaches, we provide evidence for an endogenous IHR mechanism in the early embryo that can be enhanced by RAD51. This process can be harnessed to generate homozygotes from wild-type zygotes using exogenous donors and to convert heterozygous alleles into homozygous alleles without exogenous templates. Furthermore, we identify additional IHR-promoting factors and describe features of IHR events. Together, our findings show conclusive evidence for IHR in mouse embryos and describe an efficient method for enhanced gene conversion.

Genomic analyses in Cornelia de Lange Syndrome and related diagnoses: Novel candidate genes, genotype-phenotype correlations and common mechanisms.

Cornelia de Lange Syndrome (CdLS) is a rare, dominantly inherited multisystem developmental disorder characterized by highly variable manifestations of growth and developmental delays, upper limb involvement, hypertrichosis, cardiac, gastrointestinal, craniofacial, and other systemic features. Pathogenic variants in genes encoding cohesin complex structural subunits and regulatory proteins (NIPBL, SMC1A, SMC3, HDAC8, and RAD21) are the major pathogenic contributors to CdLS. Heterozygous or hemizygous variants in the genes encoding these five proteins have been found to be contributory to CdLS, with variants in NIPBL accounting for the majority (>60%) of cases, and the only gene identified to date that results in the severe or classic form of CdLS when mutated. Pathogenic variants in cohesin genes other than NIPBL tend to result in a less severe phenotype. Causative variants in additional genes, such as ANKRD11, EP300, AFF4, TAF1, and BRD4, can cause a CdLS-like phenotype. The common role that these genes, and others, play as critical regulators of developmental transcriptional control has led to the conditions they cause being referred to as disorders of transcriptional regulation (or "DTRs"). Here, we report the results of a comprehensive molecular analysis in a cohort of 716 probands with typical and atypical CdLS in order to delineate the genetic contribution of causative variants in cohesin complex genes as well as novel candidate genes, genotype-phenotype correlations, and the utility of genome sequencing in understanding the mutational landscape in this population.

Genetic, epigenetic, and environmental contributions to neural tube closure.

The formation of the embryonic brain and spinal cord begins as the neural plate bends to form the neural folds, which meet and adhere to close the neural tube. The neural ectoderm and surrounding tissues also coordinate proliferation, differentiation, and patterning. This highly orchestrated process is susceptible to disruption, leading to neural tube defects (NTDs), a common birth defect. Here, we highlight genetic and epigenetic contributions to neural tube closure. We describe an online database we created as a resource for researchers, geneticists, and clinicians. Neural tube closure is sensitive to environmental influences, and we discuss disruptive causes, preventative measures, and possible mechanisms. New technologies will move beyond candidate genes in small cohort studies toward unbiased discoveries in sporadic NTD cases. This will uncover the genetic complexity of NTDs and critical gene-gene interactions. Animal models can reveal the causative nature of genetic variants, the genetic interrelationships, and the mechanisms underlying environmental influences.

The Interplay between Drivers of Erythropoiesis and Iron Homeostasis in Rare Hereditary Anemias: Tipping the Balance.

Rare hereditary anemias (RHA) represent a group of disorders characterized by either impaired production of erythrocytes or decreased survival (i.e., hemolysis). In RHA, the regulation of iron metabolism and erythropoiesis is often disturbed, leading to iron overload or worsening of chronic anemia due to unavailability of iron for erythropoiesis. Whereas iron overload generally is a well-recognized complication in patients requiring regular blood transfusions, it is also a significant problem in a large proportion of patients with RHA that are not transfusion dependent. This indicates that RHA share disease-specific defects in erythroid development that are linked to intrinsic defects in iron metabolism. In this review, we discuss the key regulators involved in the interplay between iron and erythropoiesis and their importance in the spectrum of RHA.

Diencephalic Size Is Restricted by a Novel Interplay Between GCN5 Acetyltransferase Activity and Retinoic Acid Signaling.

Diencephalic defects underlie an array of neurological diseases. Previous studies have suggested that retinoic acid (RA) signaling is involved in diencephalic development at late stages of embryonic development, but its roles and mechanisms of action during early neural development are still unclear. Here we demonstrate that mice lacking enzymatic activity of the acetyltransferase GCN5 ((Gcn5hat/hat )), which were previously characterized with respect to their exencephalic phenotype, exhibit significant diencephalic expansion, decreased diencephalic RA signaling, and increased diencephalic WNT and SHH signaling. Using a variety of molecular biology techniques in both cultured neuroepithelial cells treated with a GCN5 inhibitor and forebrain tissue from (Gcn5hat/hat ) embryos, we demonstrate that GCN5, RARα/γ, and the poorly characterized protein TACC1 form a complex in the nucleus that binds specific retinoic acid response elements in the absence of RA. Furthermore, RA triggers GCN5-mediated acetylation of TACC1, which results in dissociation of TACC1 from retinoic acid response elements and leads to transcriptional activation of RA target genes. Intriguingly, RA signaling defects caused by in vitro inhibition of GCN5 can be rescued through RA-dependent mechanisms that require RARß. Last, we demonstrate that the diencephalic expansion and transcriptional defects seen in (Gcn5hat/hat ) mutants can be rescued with gestational RA supplementation, supporting a direct link between GCN5, TACC1, and RA signaling in the developing diencephalon. Together, our studies identify a novel, nonhistone substrate for GCN5 whose modification regulates a previously undescribed, tissue-specific mechanism of RA signaling that is required to restrict diencephalic size during early forebrain development.SIGNIFICANCE STATEMENT Changes in diencephalic size and shape, as well as SNPs associated with retinoic acid (RA) signaling-associated genes, have been linked to neuropsychiatric disorders. However, the mechanisms that regulate diencephalic morphogenesis and the involvement of RA signaling in this process are poorly understood. Here we demonstrate a novel role of the acetyltransferase GCN5 in a previously undescribed mechanism of RA signaling in the developing forebrain that is required to maintain the appropriate size of the diencephalon. Together, our experiments identify a novel nonhistone substrate of GCN5, highlight an essential role for both GCN5 and RA signaling in early diencephalic development, and elucidate a novel molecular regulatory mechanism for RA signaling that is specific to the developing forebrain.

HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle.

Cornelia de Lange syndrome (CdLS) is a dominantly inherited congenital malformation disorder, caused by mutations in the cohesin-loading protein NIPBL for nearly 60% of individuals with classical CdLS, and by mutations in the core cohesin components SMC1A (~5%) and SMC3 (<1%) for a smaller fraction of probands. In humans, the multisubunit complex cohesin is made up of SMC1, SMC3, RAD21 and a STAG protein. These form a ring structure that is proposed to encircle sister chromatids to mediate sister chromatid cohesion and also has key roles in gene regulation. SMC3 is acetylated during S-phase to establish cohesiveness of chromatin-loaded cohesin, and in yeast, the class I histone deacetylase Hos1 deacetylates SMC3 during anaphase. Here we identify HDAC8 as the vertebrate SMC3 deacetylase, as well as loss-of-function HDAC8 mutations in six CdLS probands. Loss of HDAC8 activity results in increased SMC3 acetylation and inefficient dissolution of the 'used' cohesin complex released from chromatin in both prophase and anaphase. SMC3 with retained acetylation is loaded onto chromatin, and chromatin immunoprecipitation sequencing analysis demonstrates decreased occupancy of cohesin localization sites that results in a consistent pattern of altered transcription seen in CdLS cell lines with either NIPBL or HDAC8 mutations.

Loss-of-function HDAC8 mutations cause a phenotypic spectrum of Cornelia de Lange syndrome-like features, ocular hypertelorism, large fontanelle and X-linked inheritance.

Cornelia de Lange syndrome (CdLS) is a multisystem genetic disorder with distinct facies, growth failure, intellectual disability, distal limb anomalies, gastrointestinal and neurological disease. Mutations in NIPBL, encoding a cohesin regulatory protein, account for >80% of cases with typical facies. Mutations in the core cohesin complex proteins, encoded by the SMC1A, SMC3 and RAD21 genes, together account for ∼5% of subjects, often with atypical CdLS features. Recently, we identified mutations in the X-linked gene HDAC8 as the cause of a small number of CdLS cases. Here, we report a cohort of 38 individuals with an emerging spectrum of features caused by HDAC8 mutations. For several individuals, the diagnosis of CdLS was not considered prior to genomic testing. Most mutations identified are missense and de novo. Many cases are heterozygous females, each with marked skewing of X-inactivation in peripheral blood DNA. We also identified eight hemizygous males who are more severely affected. The craniofacial appearance caused by HDAC8 mutations overlaps that of typical CdLS but often displays delayed anterior fontanelle closure, ocular hypertelorism, hooding of the eyelids, a broader nose and dental anomalies, which may be useful discriminating features. HDAC8 encodes the lysine deacetylase for the cohesin subunit SMC3 and analysis of the functional consequences of the missense mutations indicates that all cause a loss of enzymatic function. These data demonstrate that loss-of-function mutations in HDAC8 cause a range of overlapping human developmental phenotypes, including a phenotypically distinct subgroup of CdLS.

Whole exome sequencing identifies genetic variants in inherited thrombocytopenia with secondary qualitative function defects.

Inherited thrombocytopenias are a heterogeneous group of disorders characterized by abnormally low platelet counts which can be associated with abnormal bleeding. Next-generation sequencing has previously been employed in these disorders for the confirmation of suspected genetic abnormalities, and more recently in the discovery of novel disease-causing genes. However its full potential has not yet been exploited. Over the past 6 years we have sequenced the exomes from 55 patients, including 37 index cases and 18 additional family members, all of whom were recruited to the UK Genotyping and Phenotyping of Platelets study. All patients had inherited or sustained thrombocytopenia of unknown etiology with platelet counts varying from 11×109/L to 186×109/L. Of the 51 patients phenotypically tested, 37 (73%), had an additional secondary qualitative platelet defect. Using whole exome sequencing analysis we have identified "pathogenic" or "likely pathogenic" variants in 46% (17/37) of our index patients with thrombocytopenia. In addition, we report variants of uncertain significance in 12 index cases, including novel candidate genetic variants in previously unreported genes in four index cases. These results demonstrate that whole exome sequencing is an efficient method for elucidating potential pathogenic genetic variants in inherited thrombocytopenia. Whole exome sequencing also has the added benefit of discovering potentially pathogenic genetic variants for further study in novel genes not previously implicated in inherited thrombocytopenia.

A genome-wide association study of resistance to HIV infection in highly exposed uninfected individuals with hemophilia A.

Human genetic variation contributes to differences in susceptibility to HIV-1 infection. To search for novel host resistance factors, we performed a genome-wide association study (GWAS) in hemophilia patients highly exposed to potentially contaminated factor VIII infusions. Individuals with hemophilia A and a documented history of factor VIII infusions before the introduction of viral inactivation procedures (1979-1984) were recruited from 36 hemophilia treatment centers (HTCs), and their genome-wide genetic variants were compared with those from matched HIV-infected individuals. Homozygous carriers of known CCR5 resistance mutations were excluded. Single nucleotide polymorphisms (SNPs) and inferred copy number variants (CNVs) were tested using logistic regression. In addition, we performed a pathway enrichment analysis, a heritability analysis, and a search for epistatic interactions with CCR5 Δ32 heterozygosity. A total of 560 HIV-uninfected cases were recruited: 36 (6.4%) were homozygous for CCR5 Δ32 or m303. After quality control and SNP imputation, we tested 1 081 435 SNPs and 3686 CNVs for association with HIV-1 serostatus in 431 cases and 765 HIV-infected controls. No SNP or CNV reached genome-wide significance. The additional analyses did not reveal any strong genetic effect. Highly exposed, yet uninfected hemophiliacs form an ideal study group to investigate host resistance factors. Using a genome-wide approach, we did not detect any significant associations between SNPs and HIV-1 susceptibility, indicating that common genetic variants of major effect are unlikely to explain the observed resistance phenotype in this population.

RAD21 mutations cause a human cohesinopathy.

The evolutionarily conserved cohesin complex was originally described for its role in regulating sister-chromatid cohesion during mitosis and meiosis. Cohesin and its regulatory proteins have been implicated in several human developmental disorders, including Cornelia de Lange (CdLS) and Roberts syndromes. Here we show that human mutations in the integral cohesin structural protein RAD21 result in a congenital phenotype consistent with a "cohesinopathy." Children with RAD21 mutations display growth retardation, minor skeletal anomalies, and facial features that overlap findings in individuals with CdLS. Notably, unlike children with mutations in NIPBL, SMC1A, or SMC3, these individuals have much milder cognitive impairment than those with classical CdLS. Mechanistically, these mutations act at the RAD21 interface with the other cohesin proteins STAG2 and SMC1A, impair cellular DNA damage response, and disrupt transcription in a zebrafish model. Our data suggest that, compared to loss-of-function mutations, dominant missense mutations result in more severe functional defects and cause worse structural and cognitive clinical findings. These results underscore the essential role of RAD21 in eukaryotes and emphasize the need for further understanding of the role of cohesin in human development.

Preoperative diagnosis of benign thyroid nodules with indeterminate cytology.

BACKGROUND: Approximately 15 to 30% of thyroid nodules evaluated by means of fine-needle aspiration are not clearly benign or malignant. Patients with cytologically indeterminate nodules are often referred for diagnostic surgery, though most of these nodules prove to be benign. A novel diagnostic test that measures the expression of 167 genes has shown promise in improving preoperative risk assessment. METHODS: We performed a 19-month, prospective, multicenter validation study involving 49 clinical sites, 3789 patients, and 4812 fine-needle aspirates from thyroid nodules 1 cm or larger that required evaluation. We obtained 577 cytologically indeterminate aspirates, 413 of which had corresponding histopathological specimens from excised lesions. Results of a central, blinded histopathological review served as the reference standard. After inclusion criteria were met, a gene-expression classifier was used to test 265 indeterminate nodules in this analysis, and its performance was assessed. RESULTS: Of the 265 indeterminate nodules, 85 were malignant. The gene-expression classifier correctly identified 78 of the 85 nodules as suspicious (92% sensitivity; 95% confidence interval [CI], 84 to 97), with a specificity of 52% (95% CI, 44 to 59). The negative predictive values for "atypia (or follicular lesion) of undetermined clinical significance," "follicular neoplasm or lesion suspicious for follicular neoplasm," or "suspicious cytologic findings" were 95%, 94%, and 85%, respectively. Analysis of 7 aspirates with false negative results revealed that 6 had a paucity of thyroid follicular cells, suggesting insufficient sampling of the nodule. CONCLUSIONS: These data suggest consideration of a more conservative approach for most patients with thyroid nodules that are cytologically indeterminate on fine-needle aspiration and benign according to gene-expression classifier results. (Funded by Veracyte.).

Enrichment of FLI1 and RUNX1 mutations in families with excessive bleeding and platelet dense granule secretion defects.

We analyzed candidate platelet function disorder genes in 13 index cases with a history of excessive bleeding in association with a significant reduction in dense granule secretion and impaired aggregation to a panel of platelet agonists. Five of the index cases also had mild thrombocytopenia. Heterozygous alterations in FLI1 and RUNX1, encoding Friend leukemia integration 1 and RUNT-related transcription factor 1, respectively, which have a fundamental role in megakaryocytopoeisis, were identified in 6 patients, 4 of whom had mild thrombocytopenia. Two FLI1 alterations predicting p.Arg337Trp and p.Tyr343Cys substitutions in the FLI1 DNA-binding domain abolished transcriptional activity of FLI1. A 4-bp deletion in FLI1, and 2 splicing alterations and a nonsense variation in RUNX1, which were predicted to cause haploinsufficiency of either FLI1 or RUNX1, were also identified. Our findings suggest that alterations in FLI1 and RUNX1 may be common in patients with platelet dense granule secretion defects and mild thrombocytopenia.

Scribble is required for normal epithelial cell-cell contacts and lumen morphogenesis in the mammalian lung.

During lung development, proper epithelial cell arrangements are critical for the formation of an arborized network of tubes. Each tube requires a lumen, the diameter of which must be tightly regulated to enable optimal lung function. Lung branching and lumen morphogenesis require close epithelial cell-cell contacts that are maintained as a result of adherens junctions, tight junctions and by intact apical-basal (A/B) polarity. However, the molecular mechanisms that maintain epithelial cohesion and lumen diameter in the mammalian lung are unknown. Here we show that Scribble, a protein implicated in planar cell polarity (PCP) signalling, is necessary for normal lung morphogenesis. Lungs of the Scrib mouse mutant Circletail (Crc) are abnormally shaped with fewer airways, and these airways often lack a visible, 'open' lumen. Mechanistically we show that Scrib genetically interacts with the core PCP gene Vangl2 in the developing lung and that the distribution of PCP pathway proteins and Rho mediated cytoskeletal modification is perturbed in Scrib(Crc/Crc) lungs. However A/B polarity, which is disrupted in Drosophila Scrib mutants, is largely unaffected. Notably, we find that Scrib mediates functions not attributed to other PCP proteins in the lung. Specifically, Scrib localises to both adherens and tight junctions of lung epithelia and knockdown of Scrib in lung explants and organotypic cultures leads to reduced cohesion of lung epithelial cells. Live imaging of Scrib knockdown lungs shows that Scrib does not affect bud bifurcation, as previously shown for the PCP protein Celsr1, but is required to maintain epithelial cohesion. To understand the mechanism leading to reduced cell-cell association, we show that Scrib associates with ß-catenin in embryonic lung and the sub-cellular distribution of adherens and tight junction proteins is perturbed in mutant lung epithelia. Our data reveal that Scrib is required for normal lung epithelial organisation and lumen morphogenesis by maintaining cell-cell contacts. Thus we reveal novel and important roles for Scrib in lung development operating via the PCP pathway, and in regulating junctional complexes and cell cohesion.

Evaluation of participants with suspected heritable platelet function disorders including recommendation and validation of a streamlined agonist panel.

Light transmission aggregometry (LTA) is used worldwide for the investigation of heritable platelet function disorders (PFDs), but interpretation of results is complicated by the feedback effects of ADP and thromboxane A(2) (TxA(2)) and by the overlap with the response of healthy volunteers. Over 5 years, we have performed lumi-aggregometry on 9 platelet agonists in 111 unrelated research participants with suspected PFDs and in 70 healthy volunteers. Abnormal LTA or ATP secretion test results were identified in 58% of participants. In 84% of these, the patterns of response were consistent with defects in Gi receptor signaling, the TxA(2) pathway, and dense granule secretion. Participants with defects in signaling to Gq-coupled receptor agonists and to collagen were also identified. Targeted genotyping identified 3 participants with function-disrupting mutations in the P2Y(12) ADP and TxA(2) receptors. The results of the present study illustrate that detailed phenotypic analysis using LTA and ATP secretion is a powerful tool for the diagnosis of PFDs. Our data also enable subdivision at the level of platelet-signaling pathways and in some cases to individual receptors. We further demonstrate that most PFDs can be reliably diagnosed using a streamlined panel of key platelet agonists and specified concentrations suitable for testing in most clinical diagnostic laboratories.

Identifying and testing candidate genetic polymorphisms in the irritable bowel syndrome (IBS): association with TNFSF15 and TNFα.

OBJECTIVES: The postinfectious irritable bowel syndrome (PI-IBS) suggests that impaired resolution of inflammation could cause IBS symptoms. The authors hypothesised that polymorphisms in genes whose expression were altered by gastroenteritis might be linked to IBS with diarrhoea (IBS-D) which closely resembles PI-IBS. DESIGN: Part 1: 25 healthy volunteers (HVs), 21 patients 6 months after Campylobacter jejuni infection, 37 IBS-D and 19 IBS with constipation (IBS-C) underwent rectal biopsy for gene expression analysis and peripheral blood mononuclear cell cytokine production assessment. Part 2: Polymorphisms in genes whose expression was altered in Part 1 were assessed in 179 HV, 179 IBS-D, 122 IBS-C and 41 PI-IBS. RESULTS: Part 1: Mucosal expression of seven genes was altered in IBS: CCL11, CCL13, Calpain 8 and TNFSF15 increased while NR1D1, GPR161 and GABRE decreased with similar patterns after infection with C jejuni. Part 2: The authors assessed 21 known single nucleotide polymorphisms (SNPs) in these seven genes and one SNP in each of the TNFα and IL-10 genes. Three out of five TNFSF15 SNPs (rs6478108, rs6478109 and rs7848647) showed reduced minor allele frequency (MAF) (0.28, 0.27 and 0.27) in subjects with IBS-D compared with HV (0.38, 0.36 and 0.37; p=0.007, 0.015 and 0.007, respectively) confirming others recent findings. The authors also replicated the previously reported association of the TNFα SNP rs1800629 with PI-IBS which showed an increase in the MAF at 0.30 versus 0.19 for HV (p=0.04). CONCLUSION: IBS-D and PI-IBS patients are associated with TNFSF15 and TNFα genetic polymorphisms which also predispose to Crohn's disease suggesting possible common underlying pathogenesis.

Enhancement of mediodorsal thalamus rescues aberrant belief dynamics in a mouse model with schizophrenia-associated mutation.

Optimizing behavioral strategy requires belief updating based on new evidence, a process that engages higher cognition. In schizophrenia, aberrant belief dynamics may lead to psychosis, but the mechanisms underlying this process are unknown, in part, due to lack of appropriate animal models and behavior readouts. Here, we address this challenge by taking two synergistic approaches. First, we generate a mouse model bearing patient-derived point mutation in Grin2a (Grin2aY700X+/-), a gene that confers high-risk for schizophrenia and recently identified by large-scale exome sequencing. Second, we develop a computationally trackable foraging task, in which mice form and update belief-driven strategies in a dynamic environment. We found that Grin2aY700X+/- mice perform less optimally than their wild-type (WT) littermates, showing unstable behavioral states and a slower belief update rate. Using functional ultrasound imaging, we identified the mediodorsal (MD) thalamus as hypofunctional in Grin2aY700X+/- mice, and in vivo task recordings showed that MD neurons encoded dynamic values and behavioral states in WT mice. Optogenetic inhibition of MD neurons in WT mice phenocopied Grin2aY700X+/- mice, and enhancing MD activity rescued task deficits in Grin2aY700X+/- mice. Together, our study identifies the MD thalamus as a key node for schizophrenia-relevant cognitive dysfunction, and a potential target for future therapeutics.

A novel composition of endogenous metabolic modulators improves red blood cell properties in sickle cell disease.

The most common forms of sickle cell disease (SCD) are sickle cell anemia (SCA; HbSS) and HbSC disease. In both, especially the more dense, dehydrated and adherent red blood cells (RBCs) with reduced deformability are prone to hemolysis and sickling, and thereby vaso-occlusion. Based on plasma amino acid profiling in SCD, a composition of 10 amino acids and derivatives (RCitNacQCarLKHVS; Axcella Therapeutics, USA), referred to as endogenous metabolic modulators (EMMs), was designed to target RBC metabolism. The effects of ex vivo treatment with the EMM composition on different RBC properties were studied in SCD (n = 9 SCA, n = 5 HbSC disease). Dose-dependent improvements were observed in RBC hydration assessed by hemocytometry (MCV, MCHC, dense RBCs) and osmotic gradient ektacytometry (Ohyper). Median (interquartile range [IQR]) increase in Ohyper compared to vehicle was 4.9% (4.0%-5.5%), 7.5% (6.9%-9.4%), and 12.8% (11.5%-14.0%) with increasing 20×, 40×, and 80X concentrations, respectively (all p < 0.0001). RBC deformability (EImax using oxygen gradient ektacytometry) increased by 8.1% (2.2%-12.1%; p = 0.0012), 9.6% (2.9%-15.1%; p = 0.0013), and 13.3% (5.7%-25.5%; p = 0.0007), respectively. Besides, RBC adhesion to subendothelial laminin decreased by 43% (6%-68%; p = 0.4324), 58% (48%-72%; p = 0.0185), and 71% (49%-82%; p = 0.0016), respectively. Together, these results provide a rationale for further studies with the EMM composition targeting multiple RBC properties in SCD.

Activation of pyruvate kinase as therapeutic option for rare hemolytic anemias: Shedding new light on an old enzyme.

Novel developments in therapies for various hereditary hemolytic anemias reflect the pivotal role of pyruvate kinase (PK), a key enzyme of glycolysis, in red blood cell (RBC) health. Without PK catalyzing one of the final steps of the Embden-Meyerhof pathway, there is no net yield of adenosine triphosphate (ATP) during glycolysis, the sole source of energy production required for proper RBC function and survival. In hereditary hemolytic anemias, RBC health is compromised and therefore lifespan is shortened. Although our knowledge on glycolysis in general and PK function in particular is solid, recent advances in genetic, molecular, biochemical, and metabolic aspects of hereditary anemias have improved our understanding of these diseases. These advances provide a rationale for targeting PK as therapeutic option in hereditary hemolytic anemias other than PK deficiency. This review summarizes the knowledge, rationale, (pre)clinical trials, and future advances of PK activators for this important group of rare diseases.

Maximizing Small Biopsy Patient Samples: Unified RNA-Seq Platform Assessment of over 120,000 Patient Biopsies.

Despite its wide-ranging benefits, whole-transcriptome or RNA exome profiling is challenging to implement in a clinical diagnostic setting. The Unified Assay is a comprehensive workflow wherein exome-enriched RNA-sequencing (RNA-Seq) assays are performed on clinical samples and analyzed by a series of advanced machine learning-based classifiers. Gene expression signatures and rare and/or novel genomic events, including fusions, mitochondrial variants, and loss of heterozygosity were assessed using RNA-Seq data generated from 120,313 clinical samples across three clinical indications (thyroid cancer, lung cancer, and interstitial lung disease). Since its implementation, the data derived from the Unified Assay have allowed significantly more patients to avoid unnecessary diagnostic surgery and have played an important role in guiding follow-up decisions regarding treatment. Collectively, data from the Unified Assay show the utility of RNA-Seq and RNA expression signatures in the clinical laboratory, and their importance to the future of precision medicine.