Gene expression profiling of epithelium-associated FcRL4+ B cells in primary Sjögren's syndrome reveals a pathogenic signature.

In primary Sjögren's syndrome (pSS), FcRL4+ B cells are present in inflamed salivary gland tissue, within or in close proximity to ductal epithelium. FcRL4 is also expressed by nearly all pSS-related mucosa-associated lymphoid tissue (MALT) B cell lymphomas, linking FcRL4 expression to lymphomagenesis. Whether glandular FcRL4+ B cells are pathogenic, how these cells originate, and how they functionally differ from FcRL4- B cells in pSS is unclear. This study aimed to investigate the phenotype and function of FcRL4+ B cells in the periphery and parotid gland tissue of patients with pSS. First, circulating FcRL4+ B cells from 44 pSS and 54 non-SS-sicca patients were analyzed by flow cytometry. Additionally, RNA sequencing of FcRL4+ B cells sorted from parotid gland cell suspensions of 6 pSS patients was performed. B cells were sorted from cell suspensions as mini bulk (5 cells/well) based on the following definitions: CD19+CD27-FcRL4- ('naive'), CD19+CD27+FcRL4- ('memory'), and CD19+FcRL4+ B cells. We found that, although FcRL4+ B cells were not enriched in blood in pSS compared with non-SS sicca patients, these cells generally exhibited a pro-inflammatory phenotype. Genes coding for CD11c (ITGAX), T-bet (TBX21), TACI (TNFRSF13B), Src tyrosine kinases and NF-κB pathway-related genes were, among others, significantly upregulated in glandular FcRL4+ B cells versus FcRL4- B cells. Pathway analysis showed upregulation of B cell activation, cell cycle and metabolic pathways. Thus, FcRL4+ B cells in pSS exhibit many characteristics of chronically activated, pro-inflammatory B cells and their gene expression profile suggests increased risk of lymphomagenesis. We postulate that these cells contribute significantly to the epithelial damage seen in the glandular tissue and that FcRL4+ B cells are an important treatment target in pSS.

Exome sequencing and network analysis identifies shared mechanisms underlying spinocerebellar ataxia.

The autosomal dominant cerebellar ataxias, referred to as spinocerebellar ataxias in genetic nomenclature, are a rare group of progressive neurodegenerative disorders characterized by loss of balance and coordination. Despite the identification of numerous disease genes, a substantial number of cases still remain without a genetic diagnosis. Here, we report five novel spinocerebellar ataxia genes, FAT2, PLD3, KIF26B, EP300, and FAT1, identified through a combination of exome sequencing in genetically undiagnosed families and targeted resequencing of exome candidates in a cohort of singletons. We validated almost all genes genetically, assessed damaging effects of the gene variants in cell models and further consolidated a role for several of these genes in the aetiology of spinocerebellar ataxia through network analysis. Our work links spinocerebellar ataxia to alterations in synaptic transmission and transcription regulation, and identifies these as the main shared mechanisms underlying the genetically diverse spinocerebellar ataxia types.

An interconnected data infrastructure to support large-scale rare disease research.

The Solve-RD project brings together clinicians, scientists, and patient representatives from 51 institutes spanning 15 countries to collaborate on genetically diagnosing ("solving") rare diseases (RDs). The project aims to significantly increase the diagnostic success rate by co-analyzing data from thousands of RD cases, including phenotypes, pedigrees, exome/genome sequencing, and multiomics data. Here we report on the data infrastructure devised and created to support this co-analysis. This infrastructure enables users to store, find, connect, and analyze data and metadata in a collaborative manner. Pseudonymized phenotypic and raw experimental data are submitted to the RD-Connect Genome-Phenome Analysis Platform and processed through standardized pipelines. Resulting files and novel produced omics data are sent to the European Genome-Phenome Archive, which adds unique file identifiers and provides long-term storage and controlled access services. MOLGENIS "RD3" and Café Variome "Discovery Nexus" connect data and metadata and offer discovery services, and secure cloud-based "Sandboxes" support multiparty data analysis. This successfully deployed and useful infrastructure design provides a blueprint for other projects that need to analyze large amounts of heterogeneous data.

Practical Barriers and Facilitators Experienced by Patients, Pharmacists and Physicians to the Implementation of Pharmacogenomic Screening in Dutch Outpatient Hospital Care-An Explorative Pilot Study.

Pharmacogenomics (PGx) can provide optimized treatment to individual patients while potentially reducing healthcare costs. However, widespread implementation remains absent. We performed a pilot study of PGx screening in Dutch outpatient hospital care to identify the barriers and facilitators to implementation experienced by patients (n = 165), pharmacists (n = 58) and physicians (n = 21). Our results indeed suggest that the current practical experience of healthcare practitioners with PGx is limited, that proper education is necessary, that patients want to know the exact implications of the results, that healthcare practitioners heavily rely on their computer systems, that healthcare practitioners encounter practical problems in the systems used, and a new barrier was identified, namely that there is an unclear allocation of responsibilities between healthcare practitioners about who should discuss PGx with patients and apply PGx results in healthcare. We observed a positive attitude toward PGx among all the stakeholders in our study, and among patients, this was independent of the occurrence of drug-gene interactions during their treatment. Facilitators included the availability of and adherence to Dutch Pharmacogenetics Working Group guidelines. While clinical decision support (CDS) is available and valued in our medical center, the lack of availability of CDS may be an important barrier within Dutch healthcare in general.

Transcriptome analysis suggests a compensatory role of the cofactors coenzyme A and NAD+ in medium-chain acyl-CoA dehydrogenase knockout mice.

During fasting, mitochondrial fatty-acid ß-oxidation (mFAO) is essential for the generation of glucose by the liver. Children with a loss-of-function deficiency in the mFAO enzyme medium-chain acyl-Coenzyme A dehydrogenase (MCAD) are at serious risk of life-threatening low blood glucose levels during fasting in combination with intercurrent disease. However, a subset of these children remains asymptomatic throughout life. In MCAD-deficient (MCAD-KO) mice, glucose levels are similar to those of wild-type (WT) mice, even during fasting. We investigated if metabolic adaptations in the liver may underlie the robustness of this KO mouse. WT and KO mice were given a high- or low-fat diet and subsequently fasted. We analyzed histology, mitochondrial function, targeted mitochondrial proteomics, and transcriptome in liver tissue. Loss of MCAD led to a decreased capacity to oxidize octanoyl-CoA. This was not compensated for by altered protein levels of the short- and long-chain isoenzymes SCAD and LCAD. In the transcriptome, we identified subtle adaptations in the expression of genes encoding enzymes catalyzing CoA- and NAD(P)(H)-involving reactions and of genes involved in detoxification mechanisms. We discuss how these processes may contribute to robustness in MCAD-KO mice and potentially also in asymptomatic human subjects with a complete loss of MCAD activity.

Running-wheel activity delays mitochondrial respiratory flux decline in aging mouse muscle via a post-transcriptional mechanism.

Loss of mitochondrial respiratory flux is a hallmark of skeletal muscle aging, contributing to a progressive decline of muscle strength. Endurance exercise alleviates the decrease in respiratory flux, both in humans and in rodents. Here, we dissect the underlying mechanism of mitochondrial flux decline by integrated analysis of the molecular network. Mice were given a lifelong ad libitum low-fat or high-fat sucrose diet and were further divided into sedentary and running-wheel groups. At 6, 12, 18 and 24 months, muscle weight, triglyceride content and mitochondrial respiratory flux were analysed. Subsequently, transcriptome was measured by RNA-Seq and proteome by targeted LC-MS/MS analysis with 13 C-labelled standards. In the sedentary groups, mitochondrial respiratory flux declined with age. Voluntary running protected the mitochondrial respiratory flux until 18 months of age. Beyond this time point, all groups converged. Regulation Analysis of flux, proteome and transcriptome showed that the decline of flux was equally regulated at the proteomic and at the metabolic level, while regulation at the transcriptional level was marginal. Proteomic regulation was most prominent at the beginning and at the end of the pathway, namely at the pyruvate dehydrogenase complex and at the synthesis and transport of ATP. Further proteomic regulation was scattered across the entire pathway, revealing an effective multisite regulation. Finally, reactions regulated at the protein level were highly overlapping between the four experimental groups, suggesting a common, post-transcriptional mechanism of muscle aging.

Practical guidelines for interpreting copy number gains detected by high-resolution array in routine diagnostics.

The correct interpretation of copy number gains in patients with developmental delay and multiple congenital anomalies is hampered by the large number of copy number variations (CNVs) encountered in healthy individuals. The variable phenotype associated with copy number gains makes interpretation even more difficult. Literature shows that inheritence, size and presence in healthy individuals are commonly used to decide whether a certain copy number gain is pathogenic, but no general consensus has been established. We aimed to develop guidelines for interpreting gains detected by array analysis using array CGH data of 300 patients analysed with the 105K Agilent oligo array in a diagnostic setting. We evaluated the guidelines in a second, independent, cohort of 300 patients. In the first 300 patients 797 gains of four or more adjacent oligonucleotides were observed. Of these, 45.4% were de novo and 54.6% were familial. In total, 94.8% of all de novo gains and 87.1% of all familial gains were concluded to be benign CNVs. Clinically relevant gains ranged from 288 to 7912 kb in size, and were significantly larger than benign gains and gains of unknown clinical relevance (P < 0.001). Our study showed that a threshold of 200 kb is acceptable in a clinical setting, whereas heritability does not exclude a pathogenic nature of a gain. Evaluation of the guidelines in the second cohort of 300 patients revealed that the interpretation guidelines were clear, easy to follow and efficient.

A chronic obstructive pulmonary disease related signature in squamous cell lung cancer.

The epidemiological relationship between squamous cell lung cancer (SCC) and chronic obstructive pulmonary disease (COPD), both smoking-related diseases, suggests that they have also a genetic basis. We compared 35 SCC patients with and without COPD with whole-genome gene expression profiles of laser microdissected tissue. Validation of differential expression was performed for 25 genes using quantitative (q)RT-PCR. Subsequently, we performed array-based CGH on the same tumor samples. We found that 374 probes were differentially expressed in SCC from patients with and without COPD. Forty-four probes were derived from genes with mitochondrial functions and 34 probes were located on 5q. All these probes showed a lower expression level in SCC from non-COPD patients. For a random selection of 25 mitochondrial and 5q genes, we confirmed the differential expression by qRT-PCR. Loss of 3p, 5q and 9p was observed, via array-CGH, to be more frequent in SCC from non-COPD patients as compared to SCC from COPD patients. Combination of chromosomal aberrations and the location of the differentially expressed genes showed significant association for loss of the 5q31.2-31.3 region and reduced expression of the 5q genes. In conclusion, a more frequent loss of 5q and a low expression of genes located on 5q in SCC tumors of non-COPD patients compared to tumors from COPD patients was identified suggesting that different oncogenetic pathways are operational in patients with and without COPD.

Genomic aberrations in squamous cell lung carcinoma related to lymph node or distant metastasis.

About 50% of patients presenting with resectable lung cancer develop distant metastases within 5 years. Genomic markers predicting metastatic behaviour of squamous cell lung carcinoma (SCC) are currently underexposed. We analyzed a cohort of patients with primary SCC using array-based comparative genomic hybridization (aCGH) to identify which genomic aberrations are related to metastatic behaviour. The cohort consisted of 34 patients with a follow-up of at least 5 years, 8 with metastases in regional lymph nodes only and 26 patients without any metastases at the time of surgery. Eleven of the latter 26 developed metastases in distant organs within 3 years after surgery. Copy number changes observed in at least 40% of all SCC included gains at chromosomal arms 3q, 5p, 8q, 19q, 20p, 22q and losses at 3p, 4p, 4q, 5q, 8p and 9p. High copy number amplifications were observed at 2p15-p16, 3q24-q29, 8p11-p12, 8q23-q24, and 12p12, containing candidate oncogenes such as BCL11A, REL, ECT2, PIK3CA, ADAM9, MYC and KRAS. Amplification of 2p15-p16 is a novel finding in SCC. Another novel finding is the homozygous deletion observed at 4q33-34.1 in 15% of the SCC cases. Gains at 7q36, 8p12, 10q22, 12p12, loss at 4p14 and the homozygous deletions at 4q occurred significantly more frequent in SCC from patients with lymph node metastases only. SCC from patients with distant metastases showed a significantly higher gain frequency at 8q22-q24 and loss at 8p23 and 13q21, and a significantly lower gain frequency at 2p12 and 2p16 and loss at 11q25 compared with SCC from patients without metastases. Of these, gains at 7q, 8p and 10q were restricted to SCC with lymph node metastasis and gain at 8q was restricted to patients with distant metastasis. Two genomic aberrations, i.e. loss of 4p and gain of 19q12 were observed more frequently in SCC with only lymph node metastases as compared to SCC with distant metastases. In conclusion, we identified genomic aberrations in primary SCC that were related to lymph node or distant metastases.