SUMMARY Exome sequencing is an effective strategy for identifying human disease genes. However, this methodology is difficult in late-onset diseases where limited availability of DNA from informative family members prohibits comprehensive segregation analysis. To overcome this limitation, we performed an exome-wide rare variant burden analysis of 363 index cases with familial ALS (FALS). The results revealed an excess of patient variants within TUBA4A, the gene encoding the Tubulin, Alpha 4A protein. Analysis of a further 272 FALS cases and 5,510 internal controls confirmed the overrepresentation as statistically significant and replicable. Functional analyses revealed that TUBA4A mutants destabilize the microtubule network, diminishing its repolymerization capability. These results further emphasize the role of cytoskeletal defects in ALS and demonstrate the power of gene-based rare variant analyses in situations where causal genes cannot be identified through traditional segregation analysis.

Tsetse flies (Glossina spp.) can harbor up to three distinct species of endosymbiotic bacteria that exhibit unique modes of transmission and evolutionary histories with their host. Two mutualist enterics, Wigglesworthia and Sodalis, are transmitted maternally to tsetse flies' intrauterine larvae. The third symbiont, from the genus Wolbachia, parasitizes developing oocytes. In this study, we determined that Sodalis isolates from several tsetse fly species are virtually identical based on a phylogenetic analysis of their ftsZ gene sequences. Furthermore, restriction fragment-length polymorphism analysis revealed little variation in the genomes of Sodalis isolates from tsetse fly species within different subgenera (Glossina fuscipes fuscipes and Glossina morsitans morsitans). We also examined the impact on host fitness of transinfecting G. fuscipes fuscipes and G. morsitans morsitans flies with reciprocal Sodalis strains. Tsetse flies cleared of their native Sodalis symbionts were successfully repopulated with the Sodalis species isolated from a different tsetse fly species. These transinfected flies effectively transmitted the novel symbionts to their offspring and experienced no detrimental fitness effects compared to their wild-type counterparts, as measured by longevity and fecundity. Quantitative PCR analysis revealed that transinfected flies maintained their Sodalis populations at densities comparable to those in flies harboring native symbionts. Our ability to transinfect tsetse flies is indicative of Sodalis ' recent evolutionary history with its tsetse fly host and demonstrates that this procedure may be used as a means of streamlining future paratransgenesis experiments.

IMPORTANCE Juvenile amyotrophic lateral sclerosis (ALS) is a rare form of ALS characterized by age of symptom onset less than 25 years and a variable presentation.OBJECTIVE To identify the genetic variants associated with juvenile ALS. DESIGN, SETTING, AND PARTICIPANTSIn this multicenter family-based genetic study, trio whole-exome sequencing was performed to identify the disease-associated gene in a case series of unrelated patients diagnosed with juvenile ALS and severe growth retardation. The patients and their family members were enrolled at academic hospitals and a government research facility between March 1, 2016, and March 13, 2020, and were observed until October 1, 2020. Whole-exome sequencing was also performed in a series of patients with juvenile ALS. A total of 66 patients with juvenile ALS and 6258 adult patients with ALS participated in the study. Patients were selected for the study based on their diagnosis, and all eligible participants were enrolled in the study. None of the participants had a family history of neurological disorders, suggesting de novo variants as the underlying genetic mechanism. MAIN OUTCOMES AND MEASURESDe novo variants present only in the index case and not in unaffected family members. RESULTSTrio whole-exome sequencing was performed in 3 patients diagnosed with juvenile ALS and their parents. An additional 63 patients with juvenile ALS and 6258 adult patients with ALS were subsequently screened for variants in the SPTLC1 gene. De novo variants in SPTLC1 (p.Ala20Ser in 2 patients and p.Ser331Tyr in 1 patient) were identified in 3 unrelated patients diagnosed with juvenile ALS and failure to thrive. A fourth variant (p.Leu39del) was identified in a patient with juvenile ALS where parental DNA was unavailable. Variants in this gene have been previously shown to be associated with autosomal-dominant hereditary sensory autonomic neuropathy, type 1A, by disrupting an essential enzyme complex in the sphingolipid synthesis pathway.CONCLUSIONS AND RELEVANCE These data broaden the phenotype associated with SPTLC1 and suggest that patients presenting with juvenile ALS should be screened for variants in this gene.

Background Patients with repaired tetralogy of Fallot (TOF) account for a substantial proportion of cases with late-onset right ventricular (RV) failure. The current surgical approach, which includes pulmonary valve replacement/insertion (PVR), has yielded mixed results. Therefore, it may be clinically useful to identify parameters that can potentially be used to predict RV function response to PVR. Methods and Results Cardiac magnetic resonance (CMR) data before and 6-month after PVR were obtained from 16 patients with repaired TOF (8 m, 8 f, median age 42.75). RV ejection fraction (EF) change from pre- to post-PVR was used as the outcome. The patients were divided into Group 1 (n=8, better outcome) and Group 2 (n=8, worst outcome). CMR-based patient-specific computational RV/LV models were constructed and RV mechanical stress and strain, wall thickness (WT), curvature, and volumes were obtained for analysis. Our results indicated that RV wall stress was the best single predictor for post-PRV outcome with an area under the receiver operating characteristic curve of 0.819. Mean values of stress, strain, WT, and longitudinal curvature differed significantly between the two groups with RV wall stress showing the largest difference. Mean RV stress from Group 2 was 103% higher than that from Group 1. Conclusion Computational modeling and RV stress may be used as a potential tool to identify RV function response to PVR. Large-scale clinical studies are needed to validate these preliminary findings.

The identification of several hundred genomic regions affecting disease risk has proven the ability of genome-wide association studies have proven their ability to identify genetic contributors to disease. Currently, single-nucleotide polymorphism (SNP) association analysis is the most widely used method of genome-wide association data, but recent research shows that multi-marker tests of association may provide greater power, especially when more than one mutation is present within a gene and the mutations are in low linkage disequilibrium with each other. Here we use a multi-marker association test based on regression to SNPs located within known genes to obtain a gene-level score of association. We then perform pathway analysis using this score as a measure of gene importance. We use two tests of pathway enrichment - a binomial test and a random set method. By utilizing publicly available gene and pathway information, we identify B cell, cytokine and inflammation response, and antigen presentation pathways as being associated with rheumatoid arthritis. These results confirm known biological mechanisms for auto-immunity disorders, of which rheumatoid arthritis is one.