Clinical and Molecular Correlates of NLRC5 Expression in Patients With Melanoma

NLRC5 is an important regulator in antigen presentation and inflammation, and its dysregulation promotes tumor progression. In melanoma, the impact of NLRC5 expression on molecular phenotype, clinical characteristics, and tumor features is largely unknown. In the present study, public datasets from the Cancer Cell Line Encyclopedia (CCLE), Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), and cBioPortal were used to address these issues. We identify that NLRC5 is expressed in both immune cells and melanoma cells in melanoma samples and its expression is regulated by SPI1 and DNA methylation. NLRC5 expression is closely associated with Breslow thickness, Clark level, recurrence, pathologic T stage, and ulceration status in melanoma. Truncating/splice mutations rather than missense mutations in NLRC5 could compromise the expression of downstream genes. Low expression of NLRC5 is associated with poor prognosis, low activity of immune-related signatures, low infiltrating level of immune cells, and low cytotoxic score in melanoma. Additionally, NLRC5 expression correlates with immunotherapy efficacy in melanoma. In summary, these findings suggest that NLRC5 acts as a tumor suppressor in melanoma via modulating the tumor immune microenvironment. Targeting the NLRC5 related pathway might improve efficacy of immunotherapy for melanoma patients.

Identification of Deep-Intronic Splice Mutations in a Large Cohort of Patients With Inherited Retinal Diseases

High throughput sequencing technologies have revolutionized the identification of mutations responsible for a diverse set of Mendelian disorders, including inherited retinal disorders (IRDs). However, the causal mutations remain elusive for a significant proportion of patients. This may be partially due to pathogenic mutations located in non-coding regions, which are largely missed by capture sequencing targeting the coding regions. The advent of whole-genome sequencing (WGS) allows us to systematically detect non-coding variations. However, the interpretation of these variations remains a significant bottleneck. In this study, we investigated the contribution of deep-intronic splice variants to IRDs. WGS was performed for a cohort of 571 IRD patients who lack a confident molecular diagnosis, and potential deep intronic variants that affect proper splicing were identified using SpliceAI. A total of six deleterious deep intronic variants were identified in eight patients. An in vitro minigene system was applied to further validate the effect of these variants on the splicing pattern of the associated genes. The prediction scores assigned to splice-site disruption positively correlated with the impact of mutations on splicing, as those with lower prediction scores demonstrated partial splicing. Through this study, we estimated the contribution of deep-intronic splice mutations to unassigned IRD patients and leveraged in silico and in vitro methods to establish a framework for prioritizing deep intronic variant candidates for mechanistic and functional analyses.

Bacillus Calmette-Guérin (BCG) Infections at High Frequency in Both AR-CGD and X-CGD Patients Following BCG Vaccination

Background Patients with chronic granulomatous disease (CGD) develop severe infections, including Bacillus Calmette-Guérin (BCG). Although the autosomal recessive CGD (AR-CGD) patients should hypothetically develop relatively fewer infections compared to the X-linked CGD (X-CGD) patients due to more residual reactive oxygen intermediates, the impacts of BCG vaccination on AR-CGD and X-CGD patients are unclear. Herein, we demonstrated the clinical features of BCG infections, treatments, and genetic factors in CGD patients after BCG vaccination under the Japanese immunization program. Methods We collected data retrospectively from 43 patients with CGD and assessed their history of initial infection, age at diagnosis of CGD, BCG vaccination history, clinical course, treatment for BCG infections, and genetic mutations associated with CGD. Results Fourteen CGD patients avoided BCG vaccination because of other preceding infections and family history. Of 29 patients with CGD who received BCG vaccination, 20 patients developed BCG infections. Although the age at onset of initial infection in X-CGD patients was significantly younger than that in AR-CGD patients (P < .01), the onset and frequency of BCG infections were similar in X-CGD and AR-CGD patients. In X-CGD patients, BCG infections equally developed in the patients carrying missense, insertion, deletion, nonsense, and splice mutations of CYBB. All CGD patients with BCG infections were successfully treated with anti-tuberculous drugs. Conclusions Although X-CGD patients develop severe infections at a younger age than AR-CGD patients, our data suggested that BCG infections develop at high frequency in both AR-CGD and X-CGD patients, regardless of genotype and mutant forms.

Molecular and Clinical Investigations on Portuguese Patients with Multiple acyl-CoA Dehydrogenase Deficiency

Background: Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) is a congenital rare metabolic disease with broad clinical phenotypes and variable evolution. This inborn error of metabolism is caused by mutations in the ETFA, ETFB or ETFDH genes, which encode for the mitochondrial ETF and ETF:QO proteins. A considerable group of patients has been described to respond positively to riboflavin oral supplementation, which constitutes the prototypic treatment for the pathology. Objectives: To report mutations in ETFA, ETFB and ETFDH genes identified in Portuguese patients, correlating, whenever possible, biochemical and clinical outcomes with the effects of mutations on the structure and stability of the affected proteins, to better understand MADD pathogenesis at the molecular level. Methods: MADD patients were identified based on the characteristic urinary profile of organic acids and/or acylcarnitine profiles in blood spots during newborn screening. Genotypic, clinical and biochemical data were collected for all patients. In silico structural analysis was employed using bioinformatic tools carried out in an ETF:QO molecular model for the identified missense mutations. Results: A survey describing clinical and biochemical features of eight Portuguese MADD patients was made. Genotype analysis identified five ETFDH mutations, including one extension (p.X618QextX*14), two splice mutations (c.34+5G>C and c.405+3A>T) and two missense mutations (ETF:QO-p.Arg155Gly and ETF:QO-p.Pro534Leu), and one ETFB mutation (ETFβ- p.Arg191Cys). Homozygous patients containing the ETFDH mutations p.X618QextX*14, c.34+5G>C and ETF:QO-p.Arg155Gly, all presented severe (lethal) MADD phenotypes. However, when any of these mutations are in heterozygosity with the known ETF:QO-p.Pro534Leu mild variant, the severe clinical effects are partly and temporarily attenuated. Indeed, the latter destabilizes an ETF-interacting loop, with no major functional consequences. However, the position 155 in ETF:QO is localized at the ubiquinone binding and membrane interacting domain, and is thus expected to perturb protein structure and membrane insertion, with severe functional effects. Structural analysis of molecular models is therefore demonstrated to be a valuable tool to rationalize the effects of mutations in the context of the clinical phenotype severity. Conclusion: Advanced molecular diagnosis, structural analysis and clinical correlations reveal that MADD patients harboring a severe prognosis mutation in one allele can actually revert to a milder phenotype by complementation with a milder mutation in the other allele. However, such patients are nevertheless in a precarious metabolic balance which can revert to severe fatal outcomes during catabolic stress or secondary pathology, thus requiring strict clinical follow-up.

Evaluation of circulating tumor DNA (ctDNA) with respect to germline alterations in metastatic castrate resistant prostate cancer patients.

254 Background: Circulating tumor-derived DNA (ctDNA) is an accessible method for characterizing tumoral alterations. We report ctDNA screenings of mCRPC patients (pts) who have had germline testing. Methods: Guardant360 (Guardant Health, Inc.) assesses ctDNA using sequencing to identify genomic alterations in 73 cancer-related genes. Alterations were categorized by type which included amplifications, deletions, frameshift mutations, insertions, missense mutations, splice mutations, truncations, and other. A total of 186 PCa pts in various stages of therapy had both ctDNA and germline DNA tested. Results: Of the 186 pts tested for germline mutations, 26 (14%) were germline positive. The most common germline mutation was BRCA2 with 12 (46%) pts, followed by ATM with 3 (11%). Of the total gene alterations were detected on ctDNA analysis of germline positive pts, with the most common genes being TP53 (n = 14/73, 19%), NF1 (n = 6/73, 8%), PIK3CA (n = 6/73, 8%), and BRCA2 (n = 5/73, 7%). Of the total gene alteration were detected on ctDNA analysis of germline negative pts, with the most common genes being TP53 (n = 94/588, 16%), AR (n = 90/588, 15%), EGFR (n = 31/588, 5%), and BRAF (n = 29/588, 5%). Germline negative pts showed had more amplifications (p = 0.008) while germline positive patients had more frameshift mutations (p = 0.025). Other alterations (deletion, missense, insertion, other, splicing, and truncating) were not significantly different. Missense mutations were the most prevalent type of gene alteration in germline negative (n = 306/609, 44%) and germline positive (n = 45/77, 48%), followed by amplifications (n = 210/609, 25% germline negative and n = 15/45, 18% germline positive). The median percent ctDNA values for missense mutations in germline negative and positive patients were 0.5% and 0.3% respectively. Of the germline positive pts, BRCA2 mutation was associated with the highest number of genes with alterations (n = 39), followed by RECQL4 (n = 8), ATM (n = 5), and MSH2 (n = 5). Conclusions: Germline positive pts had a higher number of frameshift mutations compared to germline negative pts. Additionally, pts with BRCA2 had the highest number of genes altered in ctDNA.