Christy L. Rhine

Substitutions that disrupt pre-mRNA splicing are a common cause of genetic disease. On average, 13.4% of all hereditary disease alleles are classified as splicing mutations mapping to the canonical 5' and 3' splice sites. However, splicing mutations present in exons and deeper intronic positions are vastly underreported. A recent re-analysis of coding mutations in exon 10 of the Lynch Syndrome gene, MLH1, revealed an extremely high rate (77%) of mutations that lead to defective splicing. This finding is confirmed by extending the sampling to five other exons in the MLH1 gene. Further analysis suggests a more general phenomenon of defective splicing driving Lynch Syndrome. Of the 36 mutations tested, 11 disrupted splicing. Furthermore, analyzing past reports suggest that MLH1 mutations in canonical splice sites also occupy a much higher fraction (36%) of total mutations than expected. When performing a comprehensive analysis of splicing mutations in human disease genes, we found that three main causal genes of Lynch Syndrome, MLH1, MSH2, and PMS2, belonged to a class of 86 disease genes which are enriched for splicing mutations. Other cancer genes were also enriched in the 86 susceptible genes. The enrichment of splicing mutations in hereditary cancers strongly argues for additional priority in interpreting clinical sequencing data in relation to cancer and splicing.

BACKGROUND: Critically ill patients with sepsis and acute respiratory distress syndrome have severely altered physiology and immune system modifications. RNA splicing is a basic molecular mechanism influenced by physiologic alterations. Immune checkpoint inhibitors, such as B and T Lymphocyte Attenuator (BTLA) have previously been shown to influence outcomes in critical illness. We hypothesize altered physiology in critical illness results in alternative RNA splicing of the immune checkpoint protein, BTLA, resulting in a soluble form with biologic and clinical significance. METHODS: Samples were collected from critically ill humans and mice. Levels soluble BTLA (sBTLA) were measured. Ex vivo experiments assessing for cellular proliferation and cytokine production were done using splenocytes from critically ill mice cultured with sBTLA. Deep RNA sequencing was done to look for alternative splicing of BTLA. sBTLA levels were fitted to models to predict sepsis diagnosis. RESULTS: sBTLA is increased in the blood of critically ill humans and mice and can predict a sepsis diagnosis on hospital day 0 in humans. Alternative RNA splicing results in a premature stop codon that results in the soluble form. sBTLA has a clinically relevant impact as splenocytes from mice with critical illness cultured with soluble BTLA have increased cellular proliferation. CONCLUSION: sBTLA is produced as a result of alternative RNA splicing. This isoform of BTLA has biological significance through changes in cellular proliferation and can predict the diagnosis of sepsis.