Injury distance limits the transcriptional response to spinal injury.

The ability of neurons to sense and respond to damage is fundamental to homeostasis and nervous system repair. For some cell types, notably dorsal root ganglia (DRG) and retinal ganglion cells (RGCs), extensive profiling has revealed a large transcriptional response to axon injury that determines survival and regenerative outcomes. In contrast, the injury response of most supraspinal cell types, whose limited regeneration constrains recovery from spinal injury, is mostly unknown. Here we employed single-nuclei sequencing in mice to profile the transcriptional responses of diverse supraspinal cell types to spinal injury. Surprisingly, thoracic spinal injury triggered only modest changes in gene expression across all populations, including corticospinal tract (CST) neurons. Moreover, CST neurons also responded minimally to cervical injury but much more strongly to intracortical axotomy, including upregulation of numerous regeneration and apoptosis-related transcripts shared with injured DRG and RGC neurons. Thus, the muted response of CST neuron to spinal injury is linked to the injury's distal location, rather than intrinsic cellular characteristics. More broadly, these findings indicate that a central challenge for enhancing regeneration after a spinal injury is the limited sensing of distant injuries and the subsequent modest baseline neuronal response.

eyeVarP: A computational framework for the identification of pathogenic variants specific to eye disease.

PURPOSE: Disease-specific pathogenic variant prediction tools that differentiate pathogenic variants from benign have been improved through disease specificity recently. However, they have not been evaluated on disease-specific pathogenic variants compared with other diseases, which would help to prioritize disease-specific variants from several genes or novel genes. Thus, we hypothesize that features of pathogenic variants alone would provide a better model. METHODS: We developed an eye disease-specific variant prioritization tool (eyeVarP), which applied the random forest algorithm to the data set of pathogenic variants of eye diseases and other diseases. We also developed the VarP tool and generalized pipeline to filter missense and insertion-deletion variants and predict their pathogenicity from exome or genome sequencing data, thus we provide a complete computational procedure. RESULTS: eyeVarP outperformed pan disease-specific tools in identifying eye disease-specific pathogenic variants under the top 10. VarP outperformed 12 pathogenicity prediction tools with an accuracy of 95% in correctly identifying the pathogenicity of missense and insertion-deletion variants. The complete pipeline would help to develop disease-specific tools for other genetic disorders. CONCLUSION: eyeVarP performs better in identifying eye disease-specific pathogenic variants using pathogenic variant features and gene features. Implementing such complete computational procedure would significantly improve the clinical variant interpretation for specific diseases.

Clinical reassessments and whole-exome sequencing uncover novel BEST1 mutation associated with bestrophinopathy phenotype.

BACKGROUND: The diagnosis of retinal dystrophies can be challenging due to the spectrum of protean phenotypic manifestations. This study employed trio-whole-exome sequencing (trio-WES) to unveil the genetic cause of an inherited retinal disorder in a south Indian family. MATERIALS AND METHODS: Proband's initial ophthalmic examinations was performed in the year 2016. WES was performed on a proband-parent trio to identify causative mutation followed by Sanger validation, segregation analysis, sequence and structure-based computational analysis to assess its pathogenicity. Based on the genetic findings, detailed clinical reassessments were performed in year 2020 for the proband and available family members. RESULTS: WES revealed a novel homozygous BEST1 mutation c.G310A (p.D104N) in the proband and heterozygous for the parents, indicating autosomal recessive inheritance. Segregation analysis showed heterozygous mutation in maternal grandfather and normal genotype for younger brother and maternal grandmother. Moreover, the structure-based analysis revealed the mutation p.D104N in the cytoplasmic domain, causing structural hindrance by altering hydrogen bonds and destabilizing the BEST1 protein structure. Proband's clinical assessments were consistent with autosomal recessive bestrophinopathy (ARB) phenotype. Additionally, characteristic absent light rise and decreased light peak-to-dark trough ratio (LP:DT) was observed bilaterally in EOG. CONCLUSIONS: Our study demonstrates the utility of WES and clinical re-evaluations in establishing the precise diagnosis of autosomal recessive bestrophinopathy associated with a novel mutation, thus expanding the BEST1-related mutation spectrum.

Whole-exome sequencing identifies multiple pathogenic variants in a large South Indian family with primary open-angle glaucoma.

PURPOSE: To identify the pathogenic variants associated with primary open-angle glaucoma (POAG) using whole-exome sequencing (WES) data of a large South Indian family. METHODS: We recruited a large five-generation South Indian family (n = 84) with a positive family history of POAG (n = 19). All study participants had a comprehensive ocular evaluation. We performed WES for 16 samples (nine POAG and seven unaffected controls) since Sanger sequencing of the POAG candidate genes (MYOC, OPTN, and TBK1) showed no genetic variation. We used an in-house pipeline for prioritizing the pathogenic variants based on their segregation among the POAG individual. RESULTS: We identified one novel and five low-frequency pathogenic variants with consistent co-segregation in all affected individuals. The variant c.G3719A in RPGR-interacting domain of RPGRIP1 that segregated heterozygously with the six POAG cases is distinct from variants causing photoreceptor dystrophies, reported affecting the RPGR protein complex signaling in primary cilia. The cilia in trabecular meshwork (TM) cells has been reported to mediate the intraocular pressure (IOP) sensation. Furthermore, we identified a novel c.A1295G variant in Rho guanine nucleotide exchange factors Gene 40 (ARHGEF40) and a likely pathogenic variant in the RPGR gene, suggesting that they may alter the RhoA activity essential for IOP regulation. CONCLUSION: Our study supports that low-frequency pathogenic variants in multiple genes and pathways probably affect Primary Open Angle Glaucoma's pathogenesis in the large South Indian family. Furthermore, it requires larger case-controls to perform family-based association tests and to strengthen our analysis.

Genetic characterization of Stargardt clinical phenotype in South Indian patients using sanger and targeted sequencing.

BACKGROUND: Stargardt disease 1 (STGD1; MIM 248200) is a monogenic form of autosomal recessive genetic disease caused by mutation in ABCA4. This gene has a major role in hydrolyzing N-retinylidene-phosphatidylethanolamine to all-trans-retinal and phosphatidylethanolamine. The purpose of this study is to identify the frequency of putative disease-causing mutations associated with Stargardt disease in a South Indian population. METHODS: A total of 28 clinically diagnosed Stargardt-like phenotype patients were recruited from south India. Ophthalmic examination of all patients was carefully carried out by a retina specialist based on the stages of fundus imaging and ERG grouping. Genetic analysis of ABCA4 was performed for all patients using Sanger sequencing and clinical exome sequencing. RESULTS: This study identified disease-causing mutations in ABCA4 in 75% (21/28) of patients, 7% (2/28) exhibited benign variants and 18% (5/28) were negative for the disease-causing mutation. CONCLUSION: This is the first study describing the genetic association of ABCA4 disease-causing mutation in South Indian Stargardt 1 patients (STGD1). Our findings highlighted the presence of two novel missense mutations and an (in/del, single base pair deletion & splice variant) in ABCA4. However, genetic heterogeneity in ABCA4 mutants requires a larger sample size to establish a true correlation with clinical phenotype.

Performance assessment of variant calling pipelines using human whole exome sequencing and simulated data.

BACKGROUND: Whole exome sequencing (WES) is a cost-effective method that identifies clinical variants but it demands accurate variant caller tools. Currently available tools have variable accuracy in predicting specific clinical variants. But it may be possible to find the best combination of aligner-variant caller tools for detecting accurate single nucleotide variants (SNVs) and small insertion and deletion (InDels) separately. Moreover, many important aspects of InDel detection are overlooked while comparing the performance of tools, particularly its base pair length. RESULTS: We assessed the performance of variant calling pipelines using the combinations of four variant callers and five aligners on human NA12878 and simulated exome data. We used high confidence variant calls from Genome in a Bottle (GiaB) consortium for validation, and GRCh37 and GRCh38 as the human reference genome. Based on the performance metrics, both BWA and Novoalign aligners performed better with DeepVariant and SAMtools callers for detecting SNVs, and with DeepVariant and GATK for InDels. Furthermore, we obtained similar results on human NA24385 and NA24631 exome data from GiaB. CONCLUSION: In this study, DeepVariant with BWA and Novoalign performed best for detecting accurate SNVs and InDels. The accuracy of variant calling was improved by merging the top performing pipelines. The results of our study provide useful recommendations for analysis of WES data in clinical genomics.