Leber Hereditary Optic Neuropathy in a Family of Carriers of MT-ND5 m.13042G>T (A236S) Novel Variant.

BACKGROUND: A Slovenian three-generation family with 3 individuals with bilateral optic neuropathy and 2 unaffected relatives with a novel homoplasmic missense variant m.13042G > T (A236S) in the ND5 gene is described. A detailed phenotype at initial diagnosis and a follow-up of bilateral optic neuropathy progression is presented for 2 affected individuals. METHODS: A detailed phenotype analysis with clinical examination in the early and chronic phase with electrophysiology and OCT segmentation is presented. Genotype analysis with full mitochondrial genome sequencing was performed. RESULTS: Two affected male individuals (maternal cousins) had a profound visual loss at an early age (11 and 20 years) with no recovery. The maternal grandmother exhibited bilateral optic atrophy with a history of visual loss at the age 58 years. The visual loss of both affected male individuals was characterized by centrocecal scotoma, abnormal color vision, abnormal PERG N95, and VEP. Later with disease progression, retinal nerve fiber layer thinning was observed on OCT. We observed no other extraocular clinical features. Mitochondrial sequencing identified a homoplasmic novel variant m.13042G > T (A236S) in the MT-ND5 gene, belonging to a haplogroup K1a. CONCLUSIONS: Novel homoplasmic variant m.13042G > T (A236S) in the ND5 gene in our family was associated with Leber hereditary optic neuropathy-like phenotype. However, predicting the pathogenicity of a novel ultra-rare missense variant in the mitochondrial ND5 gene is challenging. Genetic counseling should consider genotypic and phenotypic heterogeneity, incomplete penetrance, haplogroup type, and tissue-specific thresholds.

The Potential Connection between Molecular Changes and Biomarkers Related to ALS and the Development and Regeneration of CNS.

Neurodegenerative diseases are one of the greatest medical burdens of the modern age, being mostly incurable and with limited prognostic and diagnostic tools. Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease characterized by the loss of motoneurons, with a complex etiology, combining genetic, epigenetic, and environmental causes. The neuroprotective therapeutic approaches are very limited, while the diagnostics rely on clinical examination and the exclusion of other diseases. The recent advancement in the discovery of molecular pathways and gene mutations involved in ALS has deepened the understanding of the disease pathology and opened the possibility for new treatments and diagnostic procedures. Recently, 15 risk loci with distinct genetic architectures and neuron-specific biology were identified as linked to ALS through common and rare variant association analyses. Interestingly, the quantity of related proteins to these genes has been found to change during early postnatal development in mammalian spinal cord tissue (opossum Monodelphis domestica) at the particular time when neuroregeneration stops being possible. Here, we discuss the possibility that the ALS-related genes/proteins could be connected to neuroregeneration and development. Moreover, since the regulation of gene expression in developmental checkpoints is frequently regulated by non-coding RNAs, we propose that studying the changes in the composition and quantity of non-coding RNA molecules, both in ALS patients and in the developing central nervous (CNS) system of the opossum at the time when neuroregeneration ceases, could reveal potential biomarkers useful in ALS prognosis and diagnosis.

Genetic prediction profile for adalimumab response in Slovenian Crohn's disease patients.

INTRODUCTION: Response to anti-TNF therapy is crucial for life expectancy and life quality in patients with severe Crohn's disease. We investigated if a previously reported gene expression profile predictive for infliximab response could be also applied to adalimumab response in an independent cohort. METHODS: Forty-seven Slovene Crohn's disease patients indicated for adalimumab therapy were enrolled in the study. Inflamed and non-inflamed colon biopsy samples were obtained during routine colonoscopy prior to adalimumab treatment. Response to adalimumab was measured with IBDQ. Gene expression in inflamed and non-inflamed colon biopsy samples was measured with RT-qPCR. Genotypes were extracted from previously available genotype data. Statistical analysis was performed with SPSS software. The R package e1071 was used to train bootstrap aggregated support vector machines (SVM). RESULTS: SVM prediction model analysis was used to analyze pooled, non-inflamed, and inflamed colon tissue datasets using IBDQ response after 4, 12, 20 and 30 weeks of adalimumab treatment. The bagging approach was used in an endeavor to obtain 100 % accuracy using 10 × 100 or 100 × 100 iterations. Average adalimumab response prediction accuracy is 75.5 % for pooled samples, 90.5 % for inflamed samples, and 100 % for non-inflamed samples. Moreover, models trained on selected SNPs from analyzed genes had an average accuracy of 92.8 %, confirming the involvement of genetic regions mapping the reported genes. Finally, using combined gene expression and SNP data we observed 100 % adalimumab response prediction accuracy for pooled, inflamed, and non-inflamed datasets. DISCUSSION: Our study supports the reported genetic anti-TNF response profile and extends it for adalimumab prediction.

Single-Cell Transcriptomic and Targeted Genomic Profiling Adjusted for Inflammation and Therapy Bias Reveal CRTAM and PLCB1 as Novel Hub Genes for Anti-Tumor Necrosis Factor Alpha Therapy Response in Crohn's Disease.

BACKGROUND: The lack of reliable biomarkers in response to anti-TNFα biologicals hinders personalized therapy for Crohn's disease (CD) patients. The motivation behind our study is to shift the paradigm of anti-TNFα biomarker discovery toward specific immune cell sub-populations using single-cell RNA sequencing and an innovative approach designed to uncover PBMCs gene expression signals, which may be masked due to the treatment or ongoing inflammation; Methods: The single-cell RNA sequencing was performed on PBMC samples from CD patients either naïve to biological therapy, in remission while on adalimumab, or while on ustekinumab but previously non-responsive to adalimumab. Sieves for stringent downstream gene selection consisted of gene ontology and independent cohort genomic profiling. Replication and meta-analyses were performed using publicly available raw RNA sequencing files of sorted immune cells and an association analysis summary. Machine learning, Mendelian randomization, and oligogenic risk score methods were deployed to validate DEGs highly relevant to anti-TNFα therapy response; Results: This study found PLCB1 in CD4+ T cells and CRTAM in double-negative T cells, which met the stringent statistical thresholds throughout the analyses. An additional assessment proved causal inference of both genes in response to anti-TNFα therapy; Conclusions: This study, jointly with an innovative design, uncovered novel candidate genes in the anti-TNFα response landscape of CD, potentially obscured by therapy or inflammation.

MIF Variant rs755622 Is Associated with Severe Crohn's Disease and Better Response to Anti-TNF Adalimumab Therapy.

Crohn's disease (CD), rheumatoid arthritis, psoriatic arthritis and other inflammatory diseases comprise a group of chronic diseases with immune-mediated pathogenesis which share common pathological pathways, as well as treatment strategies including anti-TNF biologic therapy. However, the response rate to anti-TNF therapy among those diseases varies, and approximately one third of patients do not respond. Since pharmacogenetic studies for anti-TNF therapy have been more frequent for other related diseases and are rare in CD, the aim of our study was to further explore markers associated with anti-TNF response in other inflammatory diseases in Slovenian CD patients treated with the anti-TNF drug adalimumab (ADA). We enrolled 102 CD patients on ADA, for which the response was defined after 4, 12, 20 and 30 weeks of treatment, using an IBDQ questionnaire and blood CRP value. We genotyped 41 SNPs significantly associated with response to anti-TNF treatment in other diseases. We found novel pharmacogenetic association between SNP rs755622 in the gene MIF (macrophage migration inhibitory factor) and SNP rs3740691 in the gene ARFGAP2 in CD patients treated with ADA. The strongest and most consistent association with treatment response was found for the variant rs2275913 in gene IL17A (p = 9.73 × 10-3).

Gene Ontology Analysis Highlights Biological Processes Influencing Responsiveness to Biological Therapy in Psoriasis.

Psoriasis is a chronic, immune-mediated and inflammatory skin disease. Although various biological drugs are available for psoriasis treatment, some patients have poor responses or do not respond to treatment. The aim of the present study was to highlight the molecular mechanism of responsiveness to current biological drugs for psoriasis treatment. To this end, we reviewed previously published articles that reported genes associated with treatment response to biological drugs in psoriasis, and gene ontology analysis was subsequently performed using the Cytoscape platform. Herein, we revealed a statistically significant association between NF-kappaB signaling (p value = 3.37 × 10-9), regulation of granulocyte macrophage colony-stimulating factor production (p value = 6.20 × 10-6), glial cell proliferation (p value = 2.41 × 10-5) and treatment response in psoriatic patients. To the best of our knowledge, we are the first to directly associate glial cells with treatment response. Taken together, our study revealed gene ontology (GO) terms, some of which were previously shown to be implicated in the molecular pathway of psoriasis, as novel GO terms involved in responsiveness in psoriatic disease patients.

The expression IL1B correlates negatively with the clinical response to adalimumab in Crohn's disease patients: An ex vivo approach using peripheral blood mononuclear cells.

AIMS: Understanding of the molecular mechanisms of anti-TNFα therapy non-response and reliable biomarkers are essential for personalized medicine in Crohn's disease (CD) patients. Using RNA-seq data adjusted for deconvoluted fractions of peripheral blood cells, we recently described MMD gene, coding for a monocyte to macrophage differentiation factor, as a biomarker of adalimumab (anti-TNFα) therapy response in CD. The results also suggest that cell subtype-specific biomarkers may be superior to those measured in bulk peripheral blood. Here, we used functional cell model to further investigate the role of the monocyte to macrophage differentiation in adalimumab treatment response and evaluate monocyte/macrophage specific expression of the inflammatory cytokines as potential biomarkers for (non)response to adalimumab in CD patients. MAIN METHODS: The peripheral monocytes of CD patients responsive and non-responsive to adalimumab were isolated, differentiated into macrophages, and exposed to inflammation and concurrent adalimumab therapy in vitro. The results were correlated to the clinical response of the donor patients. KEY FINDINGS: Correlation is shown of the expression of two macrophage differentiation related genes- CD68 and MMD, with the expression of the inflammatory cytokines TNF, IL1B, IL6 and CXCL8. Monocytes and in vitro differentiated macrophages of adalimumab non-responders express more inflammatory cytokines than those of responders. The biggest difference was in the IL1B expression. Additionally, IL1B expression in the in vitro differentiated macrophages of CD patients correlates negatively with their clinical response to adalimumab. SIGNIFICANCE: We propose the IL1B expression in the macrophages as a possible biomarker for adalimumab response in CD patients.

Gene Ontology Analysis Highlights Biological Processes Influencing Non-Response to Anti-TNF Therapy in Rheumatoid Arthritis.

Anti-TNF therapy has significantly improved disease control in rheumatoid arthritis, but a fraction of rheumatoid arthritis patients do not respond to anti-TNF therapy or lose response over time. Moreover, the mechanisms underlying non-response to anti-TNF therapy remain largely unknown. To date, many single biomarkers of response to anti-TNF therapy have been published but they have not yet been analyzed as a system of interacting nodes. The aim of our study is to systematically elucidate the biological processes underlying non-response to anti-TNF therapy in rheumatoid arthritis using the gene ontologies of previously published predictive biomarkers. Gene networks were constructed based on published biomarkers and then enriched gene ontology terms were elucidated in subgroups using gene ontology software tools. Our results highlight the novel role of proteasome-mediated protein catabolic processes (p = 2.91 × 10-15) and plasma lipoproteins (p = 4.55 × 10-11) in anti-TNF therapy response. The results of our gene ontology analysis help elucidate the biological processes underlying non-response to anti-TNF therapy in rheumatoid arthritis and encourage further study of the highlighted processes.

Meta-Analytic Comparison of Global RNA Transcriptomes of Acute and Chronic Myeloid Leukemia Cells Reveals Novel Gene Candidates Governing Myeloid Malignancies.

BACKGROUND: Acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) represent a group of hematological malignancies characterized by the pathogenic clonal expansion of leukemic myeloid cells. The diagnosis and clinical outcome of AML and CML are complicated by genetic heterogeneity of disease; therefore, the identification of novel molecular biomarkers and pharmacological targets is of paramount importance. METHODS: RNA-seq-based transcriptome data from a total of five studies were extracted from NCBI GEO repository and subjected to an in-depth bioinformatics analysis to identify differentially expressed genes (DEGs) between AML and CML. A systemic literature survey and functional gene ontology (GO) enrichment analysis were performed for the top 100 DEGs to identify novel candidate genes and biological processes associated with AML and CML. RESULTS: LINC01554, PTMAP12, LOC644936, RPS27AP20 and FAM133CP were identified as novel risk genes for AML and CML. GO enrichment analysis showed that DEGs were significantly associated with pre-RNA splicing, reactive oxygen species and glycoprotein metabolism, the cellular endomembrane system, neutrophil migration and antimicrobial immune response. CONCLUSIONS: Our study revealed novel biomarkers and specific biological processes associated with AML and CML. Further studies are required to evaluate their value as molecular targets for managing and treating the myeloid malignancies.

Identification of Novel Loci Involved in Adalimumab Response in Crohn's Disease Patients Using Integration of Genome Profiling and Isoform-Level Immune-Cell Deconvoluted Transcriptome Profiling of Colon Tissue.

Crohn's disease is a consequence of dysregulated inflammatory response to the host's microbiota. Although anti-TNF treatment improves the quality of the patient's life, a large proportion of patients lose response to the treatment. The past decade of research has led to a continuum of studies showcasing the heterogeneity of anti-TNF response; thus, the aim of the present study was to dissect transcriptome-wide findings to transcript isoform specific levels and combine the analyses with refined information of immune cell landscapes in colon tissue, and subsequently select promising candidates using gene ontology and genomic integration. We enrolled Slovenian Crohn's disease patients who were naïve with respect to adalimumab treatment. We performed colon tissue RNA sequencing and peripheral blood mononuclear cell DNA genotyping with a subsequent contemporary integrative approach to combine immune cell deconvoluted isoform transcript specific transcriptome analysis, gene ontology layering and genomic data. We identified nine genes (MACF1, CTSE, HDLBP, HSPA9, HLA-DMB, TAP2, LGMN, ANAPC11, ACP5) with 15 transcripts and 16 variants involved in the adalimumab response. Our study identified loci, some of which were previously shown to contribute to inflammatory bowel disease susceptibility, as novel loci involved in adalimumab response in Crohn's disease patients.

Omics data integration identifies ELOVL7 and MMD gene regions as novel loci for adalimumab response in patients with Crohn's disease.

Response to anti-TNF therapy is of pivotal importance in patients with Crohn's disease (CD). Here we integrated our and previously reported PBMC derived transcriptomic and genomic data for identification of biomarkers for discrimination between responders and non-responders to anti-TNF therapy. CD patients, who were naïve with respect to the treatment with biologicals, were enrolled in the study. DNA and RNA were extracted from peripheral blood mononuclear cells. RNA-seq was performed using BGISEQ-500. Genotyping was performed using Infinium Global Screening Array. Association regressions were carried out with 12 week response to adalimumab as an outcome variable. RNA-seq analysis confirmed 7 out of 65 previously suggested genes involved in anti-TNF response. Subsequently, analysis of single nucleotide variants in regions of confirmed genes identified 5 variants near MMD and two in ELOVL7 intronic regions associated with treatment response to anti-TNF. Functional analysis has shown that rs1465352, rs4422035 and rs78620886 are listed at H3K9ac_Pro histone modification epigenetic mark. The present study confirmed MMD and ELOVL7 involvement in anti-TNF response and revealed that the regulation of MMD and ELOVL7 gene regions in ADA response may be a part of a complex interplay extending from genetic to epigenetic and to transcriptomic level.

MFUM-BrTNBC-1, a Newly Established Patient-Derived Triple-Negative Breast Cancer Cell Line: Molecular Characterisation, Genetic Stability, and Comprehensive Comparison with Commercial Breast Cancer Cell Lines.

Triple-negative breast cancer (TNBC) is a breast cancer (BC) subtype that accounts for approximately 15-20% of all BC cases. Cancer cell lines (CLs) provide an efficient way to model the disease. We have recently isolated a patient-derived triple-negative BC CL MFUM-BrTNBC-1 and performed a detailed morphological and molecular characterisation and a comprehensive comparison with three commercial BC CLs (MCF-7, MDA-MB-231, MDA-MB-453). Light and fluorescence microscopy were used for morphological studies; immunocytochemical staining for hormone receptor, p53 and Ki67 status; RNA sequencing, qRT-PCR and STR analysis for molecular characterisation; and biomedical image analysis for comparative phenotypical analysis. The patient tissue-derived MFUM-BrTNBC-1 maintained the primary triple-negative receptor status. STR analysis showed a stable and unique STR profile up to the 6th passage. MFUM-BrTNBC-1 expressed EMT transition markers and displayed changes in several cancer-related pathways (MAPK, Wnt and PI3K signalling; nucleotide excision repair; and SWI/SNF chromatin remodelling). Morphologically, MFUM-BrTNBC-1 differed from the commercial TNBC CL MDA-MB-231. The advantages of MFUM-BrTNBC-1 are its isolation from a primary tumour, rather than a metastatic site; good growth characteristics; phenotype identical to primary tissue; complete records of origin; a unique identifier; complete, unique STR profile; quantifiable morphological properties; and genetic stability up to (at least) the 6th passage.

Molecular Genetic Architecture of Monogenic Pediatric IBD Differs from Complex Pediatric and Adult IBD.

Inflammatory bowel disease (IBD) manifests as a complex disease resulting from gene-environment interactions or as a monogenic disease resulting from deleterious mutations. While monogenic IBD is predominantly pediatric, only one-quarter of complex IBD is pediatric. In this study, we were the first to systematically compare genetic architecture between monogenic and complex pediatric and adult IBD on genetic and molecular pathway levels. Genes reported as causal for monogenic pediatric IBD and related syndromes and as risk factors for pediatric and adult complex IBD were analyzed using CytoScape and ClueGO software tools to elucidate significantly enriched Gene Ontology (GO) terms. Despite the small overlap (seven genes) between monogenic IBD genes (85) and complex IBD loci (240), GO analysis revealed several enriched GO terms shared between subgroups (13.9%). Terms Th17 cell differentiation and Jak/STAT signaling were enriched in both monogenic and complex IBD subgroups. However, primary immunodeficiency and B-cell receptor signaling pathway were specifically enriched only for pediatric subgroups, confirming existing clinical observations and experimental evidence of primary immunodeficiency in pediatric IBD patients. In addition, comparative analysis identified patients below 6 years of age to significantly differ from complex pediatric and adult IBD and could be considered a separate entity.

Comprehensive genetic study of fatty acids helps explain the role of noncoding inflammatory bowel disease associated SNPs and fatty acid metabolism in disease pathogenesis.

Fatty acids and their derivatives play an important role in inflammation. Diet and genetics influence fatty acid profiles. Abnormalities of fatty acid profiles have been observed in inflammatory bowel diseases (IBD), a group of complex diseases defined by chronic gastrointestinal inflammation. IBD associated fatty acid profile abnormalities were observed independently of nutritional status or disease activity, suggesting a common genetic background. However, no study so far has attempted to look for overlap between IBD loci and fatty acid associated loci or investigate the genetics of fatty acid profiles in IBD. To this end, we conducted a comprehensive genetic study of fatty acid profiles in IBD using iCHIP, a custom microarray platform designed for deep sequencing of immune-mediated disease associated loci. This study identifies 10 loci associated with fatty acid profiles in IBD. The most significant associations were a locus near CBS (p = 7.62 × 10-8) and a locus in LRRK2 (p = 1.4 × 10-7). Of note, this study replicates the FADS gene cluster locus, previously associated with both fatty acid profiles and IBD pathogenesis. Furthermore, we identify 18 carbon chain trans-fatty acids (p = 1.12 × 10-3), total trans-fatty acids (p = 4.49 × 10-3), palmitic acid (p = 5.85 × 10-3) and arachidonic acid (p = 8.58 × 10-3) as significantly associated with IBD pathogenesis.