Peter E. Thijssen

DNA Methylation Signatures Link Prenatal Famine Exposure to Growth and Metabolism

Periconceptional diet may persistently influence DNA methylation levels with phenotypic consequences. However, a comprehensive assessment of the characteristics of prenatal malnutrition-associated differentially methylated regions (P-DMRs) is lacking in humans. Here we report on a genome-scale analysis of differential DNA methylation in whole blood after periconceptional exposure to famine during the Dutch Hunger Winter. We show that P-DMRs preferentially occur at regulatory regions, are characterized by intermediate levels of DNA methylation and map to genes enriched for differential expression during early development. Validation and further exploratory analysis of six P-DMRs highlight the critical role of gestational timing. Interestingly, differential methylation of the P-DMRs extends along pathways related to growth and metabolism. P-DMRs located in INSR and CPT1A have enhancer activity in vitro and differential methylation is associated with birth weight and serum LDL cholesterol. Epigenetic modulation of pathways by prenatal malnutrition may promote an adverse metabolic phenotype in later life.

SOX Antibodies in Small-Cell Lung Cancer and Lambert-Eaton Myasthenic Syndrome: Frequency and Relation With Survival

PURPOSE SOX1 antibodies are common in small-cell lung carcinoma (SCLC) with and without paraneoplastic syndrome (PNS) and can serve as serological tumor marker. Addition of other antibodies might improve its diagnostic power. We validated an enzyme-linked immunosorbent assay (ELISA) to assess the diagnostic value of serum antibodies in SCLC and Lambert-Eaton myasthenic syndrome (LEMS). Clinical outcome with respect to SOX antibodies was evaluated, as the SOX-related antitumor immune response might help to control the tumor growth. PATIENTS AND METHODS We used recombinant SOX1, SOX2, SOX3, SOX21, HuC, HuD, or HelN1 proteins in an ELISA to titrate serum samples and validated the assay by western blot. We tested 136 consecutive SCLC patients, 86 LEMS patients (43 with SCLC), 14 patients with SCLC and PNS (paraneoplastic cerebellar degeneration or Hu syndrome), 62 polyneuropathy patients, and 18 healthy controls. RESULTS Our ELISA was equally reliable as western blot. Forty-three percent of SCLC patients and 67% of SCLC-LEMS patients had antibodies to one of the SOX or Hu proteins. SOX antibodies had a sensitivity of 67% and a specificity of 95% to discriminate between LEMS with SCLC and nontumor LEMS. No difference in survival was observed between SOX positive and SOX negative SCLC patients. CONCLUSION SOX antibodies are specific serological markers for SCLC. Our assay is suitable for high throughput screening, detecting 43% of SCLC. SOX antibodies have diagnostic value in discriminating SCLC-LEMS from nontumor LEMS, but have no relation to survival in patients with SCLC.

MuSK IgG4 autoantibodies cause myasthenia gravis by inhibiting binding between MuSK and Lrp4

Significance MuSK myasthenia gravis (MG) is a debilitating autoimmune disease: one-third of MuSK MG patients experience a life-threatening respiratory crisis, and long-term immunosuppression is the only current treatment option. Here we show that pathogenic human IgG4 MuSK antibodies bind to the first Ig-like domain in MuSK and prevent Lrp4 from binding MuSK, thereby inhibiting Agrin-stimulated MuSK phosphorylation. This inhibitory mechanism is likely responsible for disrupting the structure of the synapse, compromising synaptic transmission and causing disease. Our findings therefore suggest that therapeutic strategies designed to increase MuSK activity may prove effective in treating MuSK MG. Moreover, our studies provide mechanistic understanding of an IgG4-mediated autoimmune disease and may shed light on the mechanisms of other IgG4-mediated autoimmune diseases. Myasthenia gravis (MG) is a severely debilitating autoimmune disease that is due to a decrease in the efficiency of synaptic transmission at neuromuscular synapses. MG is caused by antibodies against postsynaptic proteins, including (i) acetylcholine receptors, the neurotransmitter receptor, (ii) muscle-specific kinase (MuSK), a receptor tyrosine kinase essential for the formation and maintenance of neuromuscular synapses, and (iii) low-density lipoprotein receptor-related protein 4 (Lrp4), which responds to neural Agrin by binding and stimulating MuSK. Passive transfer studies in mice have shown that IgG4 antibodies from MuSK MG patients cause disease without requiring complement or other immune components, suggesting that these MuSK antibodies cause disease by directly interfering with MuSK function. Here we show that pathogenic IgG4 antibodies to MuSK bind to a structural epitope in the first Ig-like domain of MuSK, prevent binding between MuSK and Lrp4, and inhibit Agrin-stimulated MuSK phosphorylation. In contrast, these IgG4 antibodies have no direct effect on MuSK dimerization or MuSK internalization. These results provide insight into the unique pathogenesis of MuSK MG and provide clues toward development of specific treatment options.

Gene set analysis of GWAS data for human longevity highlights the relevance of the insulin/IGF-1 signaling and telomere maintenance pathways

In genome-wide association studies (GWAS) of complex traits, single SNP analysis is still the most applied approach. However, the identified SNPs have small effects and provide limited biological insight. A more appropriate approach to interpret GWAS data of complex traits is to analyze the combined effect of a SNP set grouped per pathway or gene region. We used this approach to study the joint effect on human longevity of genetic variation in two candidate pathways, the insulin/insulin-like growth factor (IGF-1) signaling (IIS) pathway and the telomere maintenance (TM) pathway. For the analyses, we used genotyped GWAS data of 403 unrelated nonagenarians from long-lived sibships collected in the Leiden Longevity Study and 1,670 younger population controls. We analyzed 1,021 SNPs in 68 IIS pathway genes and 88 SNPs in 13 TM pathway genes using four self-contained pathway tests (PLINK set-based test, Global test, GRASS and SNP ratio test). Although we observed small differences between the results of the different pathway tests, they showed consistent significant association of the IIS and TM pathway SNP sets with longevity. Analysis of gene SNP sets from these pathways indicates that the association of the IIS pathway is scattered over several genes (AKT1, AKT3, FOXO4, IGF2, INS, PIK3CA, SGK, SGK2, and YWHAG), while the association of the TM pathway seems to be mainly determined by one gene (POT1). In conclusion, this study shows that genetic variation in genes involved in the IIS and TM pathways is associated with human longevity.

Intrinsic epigenetic regulation of the D4Z4 macrosatellite repeat in a transgenic mouse model for FSHD.

Facioscapulohumeral dystrophy (FSHD) is a progressive muscular dystrophy caused by decreased epigenetic repression of the D4Z4 macrosatellite repeats and ectopic expression of DUX4, a retrogene encoding a germline transcription factor encoded in each repeat. Unaffected individuals generally have more than 10 repeats arrayed in the subtelomeric region of chromosome 4, whereas the most common form of FSHD (FSHD1) is caused by a contraction of the array to fewer than 10 repeats, associated with decreased epigenetic repression and variegated expression of DUX4 in skeletal muscle. We have generated transgenic mice carrying D4Z4 arrays from an FSHD1 allele and from a control allele. These mice recapitulate important epigenetic and DUX4 expression attributes seen in patients and controls, respectively, including high DUX4 expression levels in the germline, (incomplete) epigenetic repression in somatic tissue, and FSHD–specific variegated DUX4 expression in sporadic muscle nuclei associated with D4Z4 chromatin relaxation. In addition we show that DUX4 is able to activate similar functional gene groups in mouse muscle cells as it does in human muscle cells. These transgenic mice therefore represent a valuable animal model for FSHD and will be a useful resource to study the molecular mechanisms underlying FSHD and to test new therapeutic intervention strategies.

Leukocyte telomere length associates with prospective mortality independent of immune-related parameters and known genetic markers

Background: Human leukocyte telomere length (LTL) decreases with age and shorter LTL has previously been associated with increased prospective mortality. However, it is not clear whether LTL merely marks the health status of an individual by its association with parameters of immune function, for example, or whether telomere shortening also contributes causally to lifespan variation in humans. Methods: We measured LTL in 870 nonagenarian siblings (mean age 93 years), 1580 of their offspring and 725 spouses thereof (mean age 59 years) from the Leiden Longevity Study (LLS). Results: We found that shorter LTL is associated with increased prospective mortality in middle (30–80 years; hazard ratio (HR) = 0.75, P = 0.001) and highly advanced age (≥90 years; HR = 0.92, P = 0.028), and show that this association cannot be explained by the association of LTL with the immune-related markers insulin-like growth factor 1 to insulin-like growth factor binding protein 3 molar ratio, C-reactive protein, interleukin 6, cytomegalovirus serostatus or white blood cell counts. We found no difference in LTL between the middle-aged LLS offspring and their spouses (β = 0.006, P = 0.932). Neither did we observe an association of LTL-associated genetic variants with mortality in a prospective meta-analysis of multiple cohorts (n = 8165). Conclusions: We confirm LTL to be a marker of prospective mortality in middle and highly advanced age and additionally show that this association could not be explained by the association of LTL with various immune-related markers. Furthermore, the approaches performed here do not further support the hypothesis that LTL variation contributes to the genetic propensity for longevity.

Heterogeneous clinical presentation in ICF syndrome: correlation with underlying gene defects

Immunodeficiency with centromeric instability and facial anomalies (ICF) syndrome is a primary immunodeficiency, predominantly characterized by agammaglobulinemia or hypoimmunoglobulinemia, centromere instability and facial anomalies. Mutations in two genes have been discovered to cause ICF syndrome: DNMT3B and ZBTB24. To characterize the clinical features of this syndrome, as well as genotype–phenotype correlations, we compared clinical and genetic data of 44 ICF patients. Of them, 23 had mutations in DNMT3B (ICF1), 13 patients had mutations in ZBTB24 (ICF2), whereas for 8 patients, the gene defect has not yet been identified (ICFX). While at first sight these patients share the same immunological, morphological and epigenetic hallmarks of the disease, systematic evaluation of all reported informative cases shows that: (1) the humoral immunodeficiency is generally more pronounced in ICF1 patients, (2) B- and T-cell compartments are both involved in ICF1 and ICF2, (3) ICF2 patients have a significantly higher incidence of intellectual disability and (4) congenital malformations can be observed in some ICF1 and ICF2 cases. It is expected that these observations on prevalence and clinical presentation will facilitate mutation-screening strategies and help in diagnostic counseling.

Correlation analysis of clinical parameters with epigenetic modifications in the DUX4 promoter in FSHD

The aim of our study was to identify relationships between epigenetic parameters correlating with a relaxed chromatin state of the DUX4 promoter region and clinical severity as measured by a clinical severity score or muscle pathologic changes in D4Z4 contraction-dependent (FSHD1) and –independent (FSHD2) facioscapulohumeral muscular dystrophy patients. Twenty primary fibroblast (5 control, 10 FSHD1 and 5 FSHD2) and 26 primary myoblast (9 control, 12 FSHD1 and 5 FSHD2) cultures originating from patients with FSHD and controls were analyzed. Histone modification levels were determined by chromatin immunoprecipitation. We examined correlations between the chromatin compaction score (ChCS) defined by the H3K9me3:H3K4me2 ratio and an age corrected clinical severity score (CSS) or muscle pathology score (MPS). Possible relationships were investigated using linear regression analysis and significance was tested by Pearson’s product-moment coefficient. We found a significant difference of the ChCS between controls and patients with FSHD1 and between controls and patients with FSHD2. Tissue specific differences in ChCS were also observed. We also found a near-significant relationship between ChCS and the age corrected CSS in fibroblasts but not in myoblasts. Surprisingly, we found a strong correlation between the MPS of the vastus lateralis and the CSS. Our results confirm the D4Z4 chromatin relaxation previously shown to be associated with FSHD in a small number of samples. A possible relationship between clinical and epigenetic parameters could be established in patient fibroblasts, but not in myoblasts. The strong correlation between the MPS of the vastus lateralis and the CSS suggests that this muscle can be used to study for surrogate markers of overall disease severity.

Mutations in CDCA7 and HELLS cause immunodeficiency-centromeric instability-facial anomalies syndrome.

The life-threatening Immunodeficiency, Centromeric Instability and Facial Anomalies (ICF) syndrome is a genetically heterogeneous autosomal recessive disorder. Twenty percent of patients cannot be explained by mutations in the known ICF genes DNA methyltransferase 3B or zinc-finger and BTB domain containing 24. Here we report mutations in the cell division cycle associated 7 and the helicase, lymphoid-specific genes in 10 unexplained ICF cases. Our data highlight the genetic heterogeneity of ICF syndrome; however, they provide evidence that all genes act in common or converging pathways leading to the ICF phenotype.

Remodeling and spacing factor 1 (RSF1) deposits centromere proteins at DNA double-strand breaks to promote non-homologous end-joining

The cellular response to ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) in native chromatin requires a tight coordination between the activities of DNA repair machineries and factors that modulate chromatin structure. SMARCA5 is an ATPase of the SNF2 family of chromatin remodeling factors that has recently been implicated in the DSB response. It forms distinct chromatin remodeling complexes with several non-canonical subunits, including the remodeling and spacing factor 1 (RSF1) protein. Despite the fact that RSF1 is often overexpressed in tumors and linked to tumorigenesis and genome instability, its role in the DSB response remains largely unclear. Here we show that RSF1 accumulates at DSB sites and protects human cells against IR-induced DSBs by promoting repair of these lesions through homologous recombination (HR) and non-homologous end-joining (NHEJ). Although SMARCA5 regulates the RNF168-dependent ubiquitin response that targets BRCA1 to DSBs, we found RSF1 to be dispensable for this process. Conversely, we found that RSF1 facilitates the assembly of centromere proteins CENP-S and CENP-X at sites of DNA damage, while SMARCA5 was not required for these events. Mechanistically, we uncovered that CENP-S and CENP-X, upon their incorporation by RSF1, promote assembly of the NHEJ factor XRCC4 at damaged chromatin. In contrast, CENP-S and CENP-X were dispensable for HR, suggesting that RSF1 regulates HR independently of these centromere proteins. Our findings reveal distinct functions of RSF1 in the 2 major pathways of DSB repair and explain how RSF1, through the loading of centromere proteins and XRCC4 at DSBs, promotes repair by non-homologous end-joining.