Whole-exome Sequencing Identifies SLC52A1 and ZNF106 Variants as Novel Genetic Risk Factors for (Early) Multiple-organ Failure in Acute Pancreatitis.

OBJECTIVE: The aim of this study was to identify genetic variants associated with early multiple organ failure (MOF) in acute pancreatitis. SUMMARY BACKGROUND DATA: MOF is a life-threatening complication of acute pancreatitis, and risk factors are largely unknown, especially in early persistent MOF. Genetic risk factors are thought to enhance severity in complex diseases such as acute pancreatitis. METHODS: A 2-phase study design was conducted. First, we exome sequenced 9 acute pancreatitis patients with early persistent MOF and 9 case-matched patients with mild edematous pancreatitis (phenotypic extremes) from our initial Dutch cohort of 387 patients. Secondly, 48 candidate variants that were overrepresented in MOF patients and 10 additional variants known from literature were genotyped in a replication cohort of 286 Dutch and German patients. RESULTS: Exome sequencing resulted in 161,696 genetic variants, of which the 38,333 non-synonymous variants were selected for downstream analyses. Of these, 153 variants were overrepresented in patients with multiple-organ failure, as compared with patients with mild acute pancreatitis. In total, 58 candidate variants were genotyped in the joined Dutch and German replication cohort. We found the rs12440118 variant of ZNF106 to be overrepresented in patients with MOF (minor allele frequency 20.4% vs 11.6%, Padj=0.026). Additionally, SLC52A1 rs346821 was found to be overrepresented (minor allele frequency 48.0% vs 42.4%, Padj= 0.003) in early MOF. None of the variants known from literature were associated.Conclusions: This study indicates that SLC52A1, a riboflavin plasma membrane transporter, and ZNF106, a zinc finger protein, may be involved in disease progression toward (early) MOF in acute pancreatitis.

Cis D4Z4 repeat duplications associated with facioscapulohumeral muscular dystrophy type 2.

Facioscapulohumeral muscular dystrophy, known in genetic forms FSHD1 and FSHD2, is associated with D4Z4 repeat array chromatin relaxation and somatic derepression of DUX4 located in D4Z4. A complete copy of DUX4 is present on 4qA chromosomes, but not on the D4Z4-like repeats of chromosomes 4qB or 10. Normally, the D4Z4 repeat varies between 8 and 100 units, while in FSHD1 it is only 1-10 units. In the rare genetic form FSHD2, a combination of a 4qA allele with a D4Z4 repeat size of 8-20 units and heterozygous pathogenic variants in the chromatin modifier SMCHD1 causes DUX4 derepression and disease. In this study, we identified 11/79 (14%) FSHD2 patients with unusually large 4qA alleles of 21-70 D4Z4 units. By a combination of Southern blotting and molecular combing, we show that 8/11 (73%) of these unusually large 4qA alleles represent duplication alleles in which the long D4Z4 repeat arrays are followed by a small FSHD-sized D4Z4 repeat array duplication. We also show that these duplication alleles are associated with DUX4 expression. This duplication allele frequency is significantly higher than in controls (2.9%), FSHD1 patients (1.4%) and in FSHD2 patients with typical 4qA alleles of 8-20 D4Z4 units (1.5%). Segregation analysis shows that, similar to typical 8-20 units FSHD2 alleles, duplication alleles only cause FSHD in combination with a pathogenic variant in SMCHD1. We conclude that cis duplications of D4Z4 repeats explain DUX4 expression and disease presentation in FSHD2 families with unusual long D4Z4 repeats on 4qA chromosomes.

Diurnal rhythms in the white adipose tissue transcriptome are disturbed in obese individuals with type 2 diabetes compared with lean control individuals.

AIMS/HYPOTHESIS: Animal studies have indicated that disturbed diurnal rhythms of clock gene expression in adipose tissue can induce obesity and type 2 diabetes. The importance of the circadian timing system for energy metabolism is well established, but little is known about the diurnal regulation of (clock) gene expression in obese individuals with type 2 diabetes. In this study we aimed to identify key disturbances in the diurnal rhythms of the white adipose tissue transcriptome in obese individuals with type 2 diabetes. METHODS: In a case-control design, we included six obese individuals with type 2 diabetes and six healthy, lean control individuals. All participants were provided with three identical meals per day for 3 days at zeitgeber time (ZT, with ZT 0:00 representing the time of lights on) 0:30, 6:00 and 11:30. Four sequential subcutaneous abdominal adipose tissue samples were obtained, on day 2 at ZT 15:30, and on day 3 at ZT 0:15, ZT 5:45 and ZT 11:15. Gene expression was measured using RNA sequencing. RESULTS: The core clock genes showed reduced amplitude oscillations in the individuals with type 2 diabetes compared with the healthy control individuals. Moreover, in individuals with type 2 diabetes, only 1.8% (303 genes) of 16,818 expressed genes showed significant diurnal rhythmicity, compared with 8.4% (1421 genes) in healthy control individuals. Enrichment analysis revealed a loss of rhythm in individuals with type 2 diabetes of canonical metabolic pathways involved in the regulation of lipolysis. Enrichment analysis of genes with an altered mesor in individuals with type 2 diabetes showed decreased activity of the translation initiating pathway 'EIF2 signaling'. Individuals with type 2 diabetes showed a reduced diurnal rhythm in postprandial glucose concentrations. CONCLUSIONS/INTERPRETATION: Diurnal clock and metabolic gene expression rhythms are decreased in subcutaneous adipose tissue of obese individuals with type 2 diabetes compared with lean control participants. Future investigation is needed to explore potential treatment targets as identified by our study, including clock enhancement and induction of EIF2 signalling. DATA AVAILABILITY: The raw sequencing data and supplementary files for rhythmic expression analysis and Ingenuity Pathway Analysis have been deposited in NCBI Gene Expression Omnibus (GEO series accession number GSE104674).

Systemic inhibition of the membrane attack complex impedes neuroinflammation in chronic relapsing experimental autoimmune encephalomyelitis.

The complement system is a key driver of neuroinflammation. Activation of complement by all pathways, results in the formation of the anaphylatoxin C5a and the membrane attack complex (MAC). Both initiate pro-inflammatory responses which can contribute to neurological disease. In this study, we delineate the specific roles of C5a receptor signaling and MAC formation during the progression of experimental autoimmune encephalomyelitis (EAE)-mediated neuroinflammation. MAC inhibition was achieved by subcutaneous administration of an antisense oligonucleotide specifically targeting murine C6 mRNA (5 mg/kg). The C5a receptor 1 (C5aR1) was inhibited with the C5a receptor antagonist PMX205 (1.5 mg/kg). Both treatments were administered systemically and started after disease onset, at the symptomatic phase when lymphocytes are activated. We found that antisense-mediated knockdown of C6 expression outside the central nervous system prevented relapse of disease by impeding the activation of parenchymal neuroinflammatory responses, including the Nod-like receptor protein 3 (NLRP3) inflammasome. Furthermore, C6 antisense-mediated MAC inhibition protected from relapse-induced axonal and synaptic damage. In contrast, inhibition of C5aR1-mediated inflammation diminished expression of major pro-inflammatory mediators, but unlike C6 inhibition, it did not stop progression of neurological disability completely. Our study suggests that MAC is a key driver of neuroinflammation in this model, thereby MAC inhibition might be a relevant treatment for chronic neuroinflammatory diseases.

Expression of complement components in the peripheral nervous system.

We have generated a SAGE (serial analysis of gene expression) library of normal sciatic nerve and found tags encoding for mRNAs of the complement system highly represented. RNA (RT-PCR and northern blot hybridization) and protein (western blot analysis and immunohistochemistry) studies confirmed these findings. High expression of classical pathway components, alternative pathway components and inhibitory components was observed in specific regions of the sciatic nerve. The first components of complement were found in axons, whereas the inhibitory components were detected in the perineurium, thereby protecting the nerve from a complement attack. Immunoreactivity towards activated complement factors was noted in post traumatic neuromas and after acute crush injury, which exemplify nerve regeneration and degeneration. We propose that local production of complement in the peripheral nervous system participates in the protection of healthy nerve and is needed for efficient clearance of myelin after injury: a prerequisite for normal regeneration and remyelination of the peripheral nerve.

Transcriptional profile of the human peripheral nervous system by serial analysis of gene expression.

The peripheral nerve contains both nonmyelinating and myelinating Schwann cells. The interactions between axons, surrounding myelin, and Schwann cells are thought to be important for the correct functioning of the nervous system. To get insight into the genes involved in human myelination and maintenance of the myelin sheath and nerve, we performed a serial analysis of gene expression of human sciatic nerve and cultured Schwann cells. In the sciatic nerve library, we found high expression of genes encoding proteins related to lipid metabolism, the complement system, and the cell cycle, while cultured Schwann cells showed mainly high expression of genes encoding extracellular matrix proteins. The results of our study will assist in the identification of genes involved in maintenance of myelin and peripheral nerve and of genes involved in inherited peripheral neuropathies.