The SATB1-MIR22-GBA axis mediates glucocerebroside accumulation inducing a cellular senescence-like phenotype in dopaminergic neurons.

Idiopathic Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, which is associated with neuroinflammation and reactive gliosis. The underlying cause of PD and the concurrent neuroinflammation are not well understood. In this study, we utilize human and murine neuronal lines, stem cell-derived dopaminergic neurons, and mice to demonstrate that three previously identified genetic risk factors for PD, namely SATB1, MIR22HG, and GBA, are components of a single gene regulatory pathway. Our findings indicate that dysregulation of this pathway leads to the upregulation of glucocerebrosides (GluCer), which triggers a cellular senescence-like phenotype in dopaminergic neurons. Specifically, we discovered that downregulation of the transcriptional repressor SATB1 results in the derepression of the microRNA miR-22-3p, leading to decreased GBA expression and subsequent accumulation of GluCer. Furthermore, our results demonstrate that an increase in GluCer alone is sufficient to impair lysosomal and mitochondrial function, thereby inducing cellular senescence. Dysregulation of the SATB1-MIR22-GBA pathway, observed in both PD patients and normal aging, leads to lysosomal and mitochondrial dysfunction due to the GluCer accumulation, ultimately resulting in a cellular senescence-like phenotype in dopaminergic neurons. Therefore, our study highlights a novel pathway involving three genetic risk factors for PD and provides a potential mechanism for the senescence-induced neuroinflammation and reactive gliosis observed in both PD and normal aging.

Drug driving: a secondary analysis of factors associated with driving under the influence of cannabis in Jamaica.

OBJECTIVES: To determine cannabis use patterns, the predictive sociodemographic correlates of driving under the influence of cannabis (DUIC) and the association between risk perception and cannabis dependence among vehicle drivers in Jamaica. DESIGN: Secondary data analysis. SETTING: Used the Jamaica National Drug Prevalence Survey 2016 dataset. PARTICIPANTS: 1060 vehicle drivers extracted from the population sample of 4623. PRIMARY AND SECONDARY OUTCOME MEASURES: Analysis used Pearson's χ2 test and logistic regression. ORs and 95% CIs were recorded. A p<0.05 was considered statistically significant. RESULTS: More than 10% of Jamaican drivers admitted to DUIC in the past year. Approximately 43.3% of drivers who currently use cannabis reported DUIC only. Evidently, 86.8% of drivers who DUIC were heavy cannabis users. Approximately 30% of drivers with moderate to high-risk perception of smoking cannabis sometimes or often were dependent on cannabis. Notwithstanding, drivers with no to low-risk perception of smoking cannabis sometimes or often were significantly likelier to be dependent (p<0.001 and p<0.001, respectively). Logistic regression highlighted male drivers (OR 4.14, 95% CI 1.59 to 14.20, p=0.009) that were 34 years and under (OR 2.97, 95% CI 1.71 to 5.29, p<0.001) and were the head of the household (OR 2.22, 95% CI 1.10 to 4.75, p=0.031) and operated a machine as part of their job (OR 1.87, 95% CI 1.09 to 3.24, p=0.023) were more likely to DUIC, while those who were married (OR 0.42, 95% CI 0.22 to 0.74, p=0.004) and had achieved a tertiary-level education (OR 0.26, 95% CI 0.06 to 0.76, p=0.031) were less likely. CONCLUSIONS: Two in five Jamaican drivers, who currently smoke cannabis, drive under its influence, with over 85% engaging in heavy use. Public health implications necessitate policy-makers consider mobile roadside drug testing and amending drug-driving laws to meet international standards.

TCF4 Mutations Disrupt Synaptic Function Through Dysregulation of RIMBP2 in Patient-Derived Cortical Neurons.

BACKGROUND: Genetic variation in the TCF4 (transcription factor 4) gene is associated with risk for a variety of developmental and psychiatric conditions, which includes a syndromic form of autism spectrum disorder called Pitt-Hopkins syndrome (PTHS). TCF4 encodes an activity-dependent transcription factor that is highly expressed during cortical development and in animal models has been shown to regulate various aspects of neuronal development and function. However, our understanding of how disease-causing mutations in TCF4 confer pathophysiology in a human context is lacking. METHODS: To model PTHS, we differentiated human cortical neurons from human induced pluripotent stem cells that were derived from patients with PTHS and neurotypical individuals. To identify pathophysiology and disease mechanisms, we assayed cortical neurons with whole-cell electrophysiology, Ca2+ imaging, multielectrode arrays, immunocytochemistry, and RNA sequencing. RESULTS: Cortical neurons derived from patients with TCF4 mutations showed deficits in spontaneous synaptic transmission, network excitability, and homeostatic plasticity. Transcriptomic analysis indicated that these phenotypes resulted in part from altered expression of genes involved in presynaptic neurotransmission and identified the presynaptic binding protein RIMBP2 as the most differentially expressed gene in PTHS neurons. Remarkably, TCF4-dependent deficits in spontaneous synaptic transmission and network excitability were rescued by increasing RIMBP2 expression in presynaptic neurons. CONCLUSIONS: Taken together, these results identify TCF4 as a critical transcriptional regulator of human synaptic development and plasticity and specifically identifies dysregulation of presynaptic function as an early pathophysiology in PTHS.

The SATB1-MIR22-GBA axis mediates glucocerebroside accumulation inducing a cellular senescence-like phenotype in dopaminergic neurons.

Idiopathic Parkinson's Disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, which is associated with neuroinflammation and reactive gliosis. The underlying cause of PD and the concurrent neuroinflammation are not well understood. In this study, we utilized human and murine neuronal lines, stem cell-derived dopaminergic neurons, and mice to demonstrate that three previously identified genetic risk factors for PD, namely SATB1, MIR22HG, and GBA, are components of a single gene regulatory pathway. Our findings indicate that dysregulation of this pathway leads to the upregulation of glucocerebrosides (GluCer), which triggers a cellular senescence-like phenotype in dopaminergic neurons. Specifically, we discovered that downregulation of the transcriptional repressor SATB1 results in the derepression of the microRNA miR-22-3p, leading to decreased GBA expression and subsequent accumulation of GluCer. Furthermore, our results demonstrate that an increase in GluCer alone is sufficient to impair lysosomal and mitochondrial function, thereby inducing cellular senescence dependent on S100A9 and stress factors. Dysregulation of the SATB1-MIR22-GBA pathway, observed in both PD patients and normal aging, leads to lysosomal and mitochondrial dysfunction due to the GluCer accumulation, ultimately resulting in a cellular senescence-like phenotype in dopaminergic neurons. Therefore, our study highlights a novel pathway involving three genetic risk factors for PD and provides a potential mechanism for the senescence-induced neuroinflammation and reactive gliosis observed in both PD and normal aging.

Using brain cell-type-specific protein interactomes to interpret neurodevelopmental genetic signals in schizophrenia.

Genetics have nominated many schizophrenia risk genes and identified convergent signals between schizophrenia and neurodevelopmental disorders. However, functional interpretation of the nominated genes in the relevant brain cell types is often lacking. We executed interaction proteomics for six schizophrenia risk genes that have also been implicated in neurodevelopment in human induced cortical neurons. The resulting protein network is enriched for common variant risk of schizophrenia in Europeans and East Asians, is down-regulated in layer 5/6 cortical neurons of individuals affected by schizophrenia, and can complement fine-mapping and eQTL data to prioritize additional genes in GWAS loci. A sub-network centered on HCN1 is enriched for common variant risk and contains proteins (HCN4 and AKAP11) enriched for rare protein-truncating mutations in individuals with schizophrenia and bipolar disorder. Our findings showcase brain cell-type-specific interactomes as an organizing framework to facilitate interpretation of genetic and transcriptomic data in schizophrenia and its related disorders.

Protein interaction studies in human induced neurons indicate convergent biology underlying autism spectrum disorders.

Autism spectrum disorders (ASDs) have been linked to genes with enriched expression in the brain, but it is unclear how these genes converge into cell-type-specific networks. We built a protein-protein interaction network for 13 ASD-associated genes in human excitatory neurons derived from induced pluripotent stem cells (iPSCs). The network contains newly reported interactions and is enriched for genetic and transcriptional perturbations observed in individuals with ASDs. We leveraged the network data to show that the ASD-linked brain-specific isoform of ANK2 is important for its interactions with synaptic proteins and to characterize a PTEN-AKAP8L interaction that influences neuronal growth. The IGF2BP1-3 complex emerged as a convergent point in the network that may regulate a transcriptional circuit of ASD-associated genes. Our findings showcase cell-type-specific interactomes as a framework to complement genetic and transcriptomic data and illustrate how both individual and convergent interactions can lead to biological insights into ASDs.

TCF4 mutations disrupt synaptic function through dysregulation of RIMBP2 in patient-derived cortical neurons.

Genetic variation in the transcription factor 4 ( TCF4) gene is associated with risk for a variety of developmental and psychiatric conditions, which includes a syndromic form of ASD called Pitt Hopkins Syndrome (PTHS). TCF4 encodes an activity-dependent transcription factor that is highly expressed during cortical development and in animal models is shown to regulate various aspects of neuronal development and function. However, our understanding of how disease-causing mutations in TCF4 confer pathophysiology in a human context is lacking. Here we show that cortical neurons derived from patients with TCF4 mutations have deficits in spontaneous synaptic transmission, network excitability and homeostatic plasticity. Transcriptomic analysis indicates these phenotypes result from altered expression of genes involved in presynaptic neurotransmission and identifies the presynaptic binding protein, RIMBP2 as the most differentially expressed gene in PTHS neurons. Remarkably, TCF4-dependent deficits in spontaneous synaptic transmission and network excitability were rescued by increasing RIMBP2 expression in presynaptic neurons. Together, these results identify TCF4 as a critical transcriptional regulator of human synaptic development and plasticity and specifically identifies dysregulation of presynaptic function as an early pathophysiology in PTHS.

Directed mechanisms for apical dendrite development during neuronal polarization.

The development of the dendrite and the axon during neuronal polarization underlies the directed flow of information in the brain. Seminal studies on axon development have dominated the mechanistic analysis of neuronal polarization. These studies, many originating from examinations in cultured hippocampal and cortical neurons in vitro, have established a prevalent view that axon formation precedes and is necessary for neuronal polarization. There is also in vivo evidence supporting this view. Nevertheless, the establishment of bipolar polarity, the leading edge, and apical dendrite development in pyramidal neurons in vivo occur when axon formation is prevented. Furthermore, recent mounting evidence suggest that directed mechanisms might mediate bipolar polarity/leading process and subsequent apical dendrite development. In the presence of spatially directed extracellular cues in the developing brain, these events may operate independently of axon forming events. In this perspective we summarize evidence in support of these evolving views in neuronal polarization and highlight recent findings on dedicated mechanisms acting in apical dendrite development.

Semaphorin3A/PlexinA3 association with the Scribble scaffold for cGMP increase is required for apical dendrite development.

The development of the apical dendrite from the leading process of the bipolar pyramidal neuron might be directed by spatially organized extrinsic cues acting on localized intrinsic determinants. The extracellular cues regulating apical dendrite polarization remain elusive. We show that leading process and apical dendrite development are directed by class III Semaphorins and mediated by a localized cGMP-synthesizing complex. The scaffolding protein Scribble that associates with the cGMP-synthesizing enzyme soluble guanylate cyclase (sGC) also associates with the Semaphorin3A (Sema3A) co-receptor PlexinA3. Deletion or knockdown of PlexinA3 and Sema3A or disruption of PlexinA3-Scribble association prevents Sema3A-mediated cGMP increase and causes defects in apical dendrite development. These manipulations also impair bipolar polarity and leading process establishment. Local cGMP elevation or sGC expression rescues the effects of PlexinA3 knockdown or PlexinA3-Scribble complex disruption. During neuronal polarization, leading process and apical dendrite development are directed by a scaffold that links Semaphorin cue to cGMP increase.

Genoppi is an open-source software for robust and standardized integration of proteomic and genetic data.

Combining genetic and cell-type-specific proteomic datasets can generate biological insights and therapeutic hypotheses, but a technical and statistical framework for such analyses is lacking. Here, we present an open-source computational tool called Genoppi (lagelab.org/genoppi) that enables robust, standardized, and intuitive integration of quantitative proteomic results with genetic data. We use Genoppi to analyze 16 cell-type-specific protein interaction datasets of four proteins (BCL2, TDP-43, MDM2, PTEN) involved in cancer and neurological disease. Through systematic quality control of the data and integration with published protein interactions, we show a general pattern of both cell-type-independent and cell-type-specific interactions across three cancer cell types and one human iPSC-derived neuronal cell type. Furthermore, through the integration of proteomic and genetic datasets in Genoppi, our results suggest that the neuron-specific interactions of these proteins are mediating their genetic involvement in neurodegenerative diseases. Importantly, our analyses suggest that human iPSC-derived neurons are a relevant model system for studying the involvement of BCL2 and TDP-43 in amyotrophic lateral sclerosis.

Choice of Intensive Lifestyle Change and/or Metformin after Shared Decision Making for Diabetes Prevention: Results from the Prediabetes Informed Decisions and Education (PRIDE) Study.

INTRODUCTION: While the Diabetes Prevention Program Study demonstrated that intensive lifestyle change and metformin both reduce type 2 diabetes incidence, there are little data on patient preferences in real-world, clinical settings. METHODS: The Prediabetes Informed Decisions and Education (PRIDE) study was a cluster-randomized trial of shared decision making (SDM) for diabetes prevention. In PRIDE, pharmacists engaged patients with prediabetes in SDM using a decision aid with information about both evidence-based options. We recorded which diabetes prevention option(s) participants chose after the SDM visit. We also evaluated logistic regression models examining predictors of choosing intensive lifestyle change ± metformin, compared to metformin or usual care, and predictors of choosing metformin ± intensive lifestyle change, compared to intensive lifestyle change or usual care. RESULTS: Among PRIDE participants (n = 515), 55% chose intensive lifestyle change, 8.5% chose metformin, 15% chose both options, and 21.6% declined both options. Women (odds ratio [OR] = 1.60, P = 0.023) had higher odds than men of choosing intensive lifestyle change. Patients >60 years old (OR = 0.50, P = 0.028) had lower odds than patients <50 years old of choosing metformin. Participants with higher body mass index (BMI) had higher odds of choosing intensive lifestyle change (OR = 1.07 per BMI unit increase, P = 0.005) v. other options and choosing metformin (OR = 1.06 per BMI unit increase, P = 0.008) v. other options. CONCLUSIONS: Patients with prediabetes are making choices for diabetes prevention that generally align with recommendations and expected benefits from the published literature. Our results are important for policy makers and clinicians, as well as program planners developing systemwide approaches for diabetes prevention.

Granulocyte macrophage colony-stimulating factor has come of age: From a vaccine adjuvant to antiviral immunotherapy.

GM-CSF acts as a pro-inflammatory cytokine and a key growth factor produced by several immune cells such as macrophages and activated T cells. In this review, we discuss recent studies that point to the crucial role of GM-CSF in the immune response against infections. Upon induction, GM-CSF activates four main signalling networks including the JAK/STAT, PI3K, MAPK, and NFκB pathways. Many of these transduction pathways such as JAK/STAT signal via proteins commonly activated with other antiviral signalling cascades, such as those induced by IFNs. GM-CSF also helps defend against respiratory infections by regulating alveolar macrophage differentiation and enhancing innate immunity in the lungs. Here, we also summarize the numerous clinical trials that have taken advantage of GM-CSF's mechanistic attributes in immunotherapy. Moreover, we discuss how GM-CSF is used as an adjuvant in vaccines and how its activity is interfered with to reduce inflammation such as in the case of COVID-19. This review brings forth the current knowledge on the antiviral actions of GM-CSF, the associated signalling cascades, and its application in immunotherapy.

Mind the translational gap: using iPS cell models to bridge from genetic discoveries to perturbed pathways and therapeutic targets.

Autism spectrum disorder (ASD) comprises a group of neurodevelopmental disorders characterized by impaired social interactions as well as the presentation of restrictive and repetitive behaviors. ASD is highly heritable but genetically heterogenous with both common and rare genetic variants collaborating to predispose individuals to the disorder. In this review, we synthesize recent efforts to develop human induced pluripotent stem cell (iPSC)-derived models of ASD-related phenotypes. We firstly address concerns regarding the relevance and validity of available neuronal iPSC-derived models. We then critically evaluate the robustness of various differentiation and cell culture protocols used for producing cell types of relevance to ASD. By exploring iPSC models of ASD reported thus far, we examine to what extent cellular and neuronal phenotypes with potential relevance to ASD can be linked to genetic variants found to underlie it. Lastly, we outline promising strategies by which iPSC technology can both enhance the power of genetic studies to identify ASD risk factors and nominate pathways that are disrupted across groups of ASD patients that might serve as common points for therapeutic intervention.

Case Report: Application of hepatitis B virus (HBV) deep sequencing to distinguish between acute and chronic infection.

Deep sequencing of the full-length hepatitis B virus (HBV) genome provides the opportunity to determine the extent to which viral diversity, genotype, polymorphisms, insertions and deletions may influence presentation and outcomes of disease. Increasing experience with analysis of HBV genomic data opens up the potential for using these data to inform insights into pathophysiology of infection and to underpin decision making in clinical practice. We here set out to undertake whole genome HBV sequencing from an adult who presented acutely unwell with a new diagnosis of HBV infection, and tested positive for both HBV anti-core IgM and IgG, possibly representing either acute hepatitis B infection (AHB) or chronic hepatitis B with an acute reactivation (CHB-AR). The distinction between these two scenarios may be important in predicting prognosis and underpinning treatment decisions, but can be challenging based on routine laboratory tests. Through application of deep whole-genome sequencing we typed the isolate as genotype-D1, and identified several minority variants including G1764A and G1986A substitutions in the pre-core promoter and pre-core regions, which support CHB-AR rather than AHB. In the longer term, enhanced deep sequencing data for HBV may provide improved evidence to distinguish between acute and chronic infection, to predict outcomes and to stratify treatment.

Effectiveness of Shared Decision-making for Diabetes Prevention: 12-Month Results from the Prediabetes Informed Decision and Education (PRIDE) Trial.

IMPORTANCE: Intensive lifestyle change (e.g., the Diabetes Prevention Program) and metformin reduce type 2 diabetes risk among patients with prediabetes. However, real-world uptake remains low. Shared decision-making (SDM) may increase awareness and help patients select and follow through with informed options for diabetes prevention that are aligned with their preferences. OBJECTIVE: To test the effectiveness of a prediabetes SDM intervention. DESIGN: Cluster randomized controlled trial. SETTING: Twenty primary care clinics within a large regional health system. PARTICIPANTS: Overweight/obese adults with prediabetes (BMI ≥ 24 kg/m2 and HbA1c 5.7-6.4%) were enrolled from 10 SDM intervention clinics. Propensity score matching was used to identify control patients from 10 usual care clinics. INTERVENTION: Intervention clinic patients were invited to participate in a face-to-face SDM visit with a pharmacist who used a decision aid (DA) to describe prediabetes and four possible options for diabetes prevention: DPP, DPP ± metformin, metformin only, or usual care. MAIN OUTCOMES AND MEASURES: Primary endpoint was uptake of DPP (≥ 9 sessions), metformin, or both strategies at 4 months. Secondary endpoint was weight change (lbs.) at 12 months. RESULTS: Uptake of DPP and/or metformin was higher among SDM participants (n = 351) than controls receiving usual care (n = 1028; 38% vs. 2%, p < .001). At 12-month follow-up, adjusted weight loss (lbs.) was greater among SDM participants than controls (- 5.3 vs. - 0.2, p < .001). LIMITATIONS: Absence of DPP supplier participation data for matched patients in usual care clinics. CONCLUSIONS AND RELEVANCE: A prediabetes SDM intervention led by pharmacists increased patient engagement in evidence-based options for diabetes prevention and was associated with significantly greater uptake of DPP and/or metformin at 4 months and weight loss at 12 months. Prediabetes SDM may be a promising approach to enhance prevention efforts among patients at increased risk. TRIAL REGISTRATION: This study was registered at clinicaltrails.gov (NCT02384109)).

Illumina and Nanopore methods for whole genome sequencing of hepatitis B virus (HBV).

Advancing interventions to tackle the huge global burden of hepatitis B virus (HBV) infection depends on improved insights into virus epidemiology, transmission, within-host diversity, drug resistance and pathogenesis, all of which can be advanced through the large-scale generation of full-length virus genome data. Here we describe advances to a protocol that exploits the circular HBV genome structure, using isothermal rolling-circle amplification to enrich HBV DNA, generating concatemeric amplicons containing multiple successive copies of the same genome. We show that this product is suitable for Nanopore sequencing as single reads, as well as for generating short-read Illumina sequences. Nanopore reads can be used to implement a straightforward method for error correction that reduces the per-read error rate, by comparing multiple genome copies combined into a single concatemer and by analysing reads generated from plus and minus strands. With this approach, we can achieve an improved consensus sequencing accuracy of 99.7% and resolve intra-sample sequence variants to form whole-genome haplotypes. Thus while Illumina sequencing may still be the most accurate way to capture within-sample diversity, Nanopore data can contribute to an understanding of linkage between polymorphisms within individual virions. The combination of isothermal amplification and Nanopore sequencing also offers appealing potential to develop point-of-care tests for HBV, and for other viruses.

Building resilient families: Developing family interventions for preventing adolescent depression and HIV in low resource settings.

Depression contributes significantly to the global burden of disease in low- and middle-income countries. In South Africa, individuals may be at elevated risk for depression due to HIV and AIDS, violence, and poverty. For adolescents, resilience-focused prevention strategies have the potential to reduce onset of depression. Involving families in promoting adolescent mental health is developmentally appropriate, but few existing interventions take a family approach to prevention of adolescent depression. We conducted a qualitative investigation from 2013-2015 to inform the development of a family intervention to prevent adolescent depression in South Africa among families infected or at risk for HIV. Using focus groups with adolescents and parents (eight groups, n = 57), and interviews (n = 25) with clinicians, researchers, and others providing mental health and related services, we identified context-specific factors related to risk for family depression, and explored family interactions around mental health more broadly as well as depression specifically. Findings indicate that HIV and poverty are important risk factors for depression. Future interventions must address linguistic complexities in describing and discussing depression, and engage with the social interpretations and meanings placed upon depression in the South African context, including bewitchment and deviations from prescribed social roles. Participants identified family meetings as a context-appropriate prevention strategy. Family meetings offer opportunities to practice family problem solving, involve other family members in communal parenting during periods of parental depression, and serve as forums for building Xhosa-specific interpretations of resilience. This study will guide the development of Our Family Our Future, a resilience-focused family intervention to prevent adolescent depression (ClinicalTrials.gov #NCT02432352).

Risperidone Provides Better Improvement of Sleep Disturbances Than Haloperidol Therapy in Schizophrenia Patients With Cannabis-Positive Urinalysis.

A high percentage of persons with Schizophrenia also uses Cannabis and this may potentially alter the therapeutic benefits of the antipsychotic medications prescribed. The aim of this study was to assess the impact of Cannabis usage on antipsychotic therapy of sleep disturbances in schizophrenia subjects. Male subjects, ≥18 years, admitted to the University Hospital of the West Indies psychiatric ward between October 2015 and October 2016, and diagnosed with schizophrenia were recruited for the study. Following written informed consent to the study, subjects were prescribed either risperidone monotherapy or haloperidol monotherapy orally for 14 days and classified as Cannabis users (CU) or non-users (non-CU), with presence/absence of Cannabis metabolite in urine samples. After 1 week of admission, subjects wore the Actiwatch-2 device, to record sleep data for 7 consecutive nights. Inferential statistical analysis involved non-parametric tests, expressed as median and inter-quartile ranges (IQR), with p<0.05 considered statistically significant. Fifty subjects were assessed, with a median (IQR) age of 28 (14) years. Majority (30; 60%) were CU, displaying longer sleep latency (SL) than non-CU when receiving haloperidol; but equivalent SL when receiving risperidone. In comparison to non-CU, the CU also displayed longer time in bed, but shorter durations asleep, awoke more frequently during the nights and for longer durations, whether receiving haloperidol or risperidone. This resulted in lower sleep efficiency for the CU (<85%) compared to the non-CU (≥85%). Over the study period, sleep efficiency was significantly improved for non-CU receiving either risperidone (p = 0.032) or haloperidol (p = 0.010); but was only significantly improved with risperidone for the CU (p = 0.045). It is apparent that the presence of Cannabis may be impacting the therapeutic benefits of antipsychotic drugs on sleep. In schizophrenia subjects with concomitant Cannabis use, risperidone is more beneficial than haloperidol in improving sleep efficiency.

Targeted exome sequencing defines novel and rare variants in complex blood group serology cases for a red blood cell reference laboratory setting.

BACKGROUND: We previously demonstrated that targeted exome sequencing accurately defined blood group genotypes for reference panel samples characterized by serology and single-nucleotide polymorphism (SNP) genotyping. Here we investigate the application of this approach to resolve problematic serology and SNP-typing cases. STUDY DESIGN AND METHODS: The TruSight One sequencing panel and MiSeq platform was used for sequencing. CLC Genomics Workbench software was used for data analysis of the blood group genes implicated in the serology and SNP-typing problem. Sequence variants were compared to public databases listing blood group alleles. The effect of predicted amino acid changes on protein function for novel alleles was assessed using SIFT and PolyPhen-2. RESULTS: Among 29 unresolved samples, sequencing defined SNPs in blood group genes consistent with serologic observation: 22 samples exhibited SNPs associated with varied but known blood group alleles and one sample exhibited a chimeric RH genotype. Three samples showed novel variants in the CROM, LAN, and RH systems, respectively, predicting respective amino acid changes with possible deleterious impact. Two samples harbored rare variants in the RH and FY systems, respectively, not previously associated with a blood group allele or phenotype. A final sample comprised a rare variant within the KLF1 transcription factor gene that may modulate DNA-binding activity. CONCLUSION: Targeted exome sequencing resolved complex serology problems and defined both novel blood group alleles (CD55:c.203G>A, ABCB6:c.1118_1124delCGGATCG, ABCB6:c.1656-1G>A, and RHD:c.452G>A) and rare variants on blood group alleles associated with altered phenotypes. This study illustrates the utility of exome sequencing, in conjunction with serology, as an alternative approach to resolve complex cases.