FUS-P525L Juvenile Amyotrophic Lateral Sclerosis and Intellectual Disability: Evidence for Association and Oligogenic Inheritance.

Background and Objectives: Amyotrophic lateral sclerosis (ALS) is characterized by upper and lower motor neuron degeneration, with juvenile ALS (jALS) defined as disease with age at onset (AAO) before 25 years. We aimed to identify the genetic basis of 2 unrelated patients with jALS with very rapid deterioration and early age intellectual disability (ID) and to assess association of genetic findings with both phenotypes in a large cohort of patients with ALS and controls, and in the literature. Methods: Exome sequencing was performed in 2 unrelated probands and their parents. Trio analyses included de novo, rare homozygosity, and compound heterozygosity analyses. A TaqMan genotyping assay was used to genotype ALS cohorts. A systematic literature review was conducted and additional information from authors obtained to assess prevalence of fused in sarcoma (FUS)-ALS associated with ID. Results: A de novo mutation FUS-P525L was identified in both patients. Additional variations were identified in other genes related to intellectual disabilities. Among 8 additional unrelated juvenile patients, one carried the same FUS mutation and had a similar medical history of mild ID and fulminant ALS, whereas the others did not carry any FUS coding mutations and had no reported learning or intellectual disabilities (p = 0.0083). In addition, 486 patients with ALS with AAO ≥25 years were negative for this mutation. An extensive literature review showed that among all patients with FUS-related ALS with full phenotype reports, 10.3% exhibited additional learning/intellectual disabilities. Discussion: FUS-P525L mutation was identified in 3 among 10 patients with jALS (30%) in our clinical cohort, all with a very aggressive disease course and ID. Together with literature reports, these results support a novel association between mutations in FUS and early life ID. Additional variations identified in genes related to ID and brain development in our patients (GPT2, DNAH10, and SCUBE2) may suggest a complex oligogenic inheritance for this phenotype. We propose that this mutation should be screened in patients with ALS with very early AAO, aggressive disease course, and sporadic occurrence, especially when ALS is accompanied by ID.

Genetic markers of Restless Legs Syndrome in Parkinson disease.

INTRODUCTION: Several studies proposed that Restless Legs Syndrome (RLS) and Parkinson disease (PD) may be clinically and/or etiologically related. To examine this hypothesis, we aimed to determine whether the known RLS genetic markers may be associated with PD risk, as well as with PD subtype. METHODS: Two case-control cohorts from Tel-Aviv and New-York, including 1133 PD patients and 867 controls were genotyped for four RLS-related SNPs in the genes MEIS1, BTBD9, PTPRD and MAP2K5/SKOR1. The association between genotype, PD risk and phenotype was tested using multivariate regression models. RESULTS: None of the tested SNPs was significantly associated with PD risk, neither in any individual cohort nor in the combined analysis after correction for multiple comparisons. The MAP2K5/SKOR1 marker rs12593813 was associated with higher frequency of tremor in the Tel-Aviv cohort (61.0% vs. 46.5%, p = 0.001, dominant model). However, the risk allele for tremor in this gene has been associated with reduced RLS risk. Moreover, this association did not replicate in Tremor-dominant PD patients from New-York. CONCLUSION: RLS genetic risk markers are not associated with increased PD risk or subtype in the current study. Together with previous genetic, neuropathological and epidemiologic studies, our results further strengthen the notion that RLS and PD are likely to be distinct entities.

CHRNB3 c.-57A>G functional promoter change affects Parkinson's disease and smoking.

Cigarette smoking is protective in Parkinson's disease (PD), possibly because of nicotine action on brain nicotinic-acetylcholine receptors. The ß3 nicotinic-acetylcholine receptor subunit (encoded by CHRNB3) is depleted in the striatum of PD patients and associated with nicotine dependence. Herein, the CHRNB3 gene was sequenced, and the c.-57G allele frequency was 0.31 and 0.26 among patients (n = 596) and controls (n = 369), respectively (p = 0.02, odds ratio = 1.33, 95% confidence interval = 1.03-1.73). The c.-57G allele was strongly associated with smoking in patients, as 48.4% of c.-57G carriers compared with 32.6% of noncarriers reported smoking history (p < 0.0001). The transcription factor Oct-1 binding was almost eliminated in lymphoblasts with the c.-57G/G genotype, to only 6.5% percent, and the CHRNB3 promoter activity was reduced in cells with the c.-57G/G genotype by 96%-70%. These findings suggest that the CHRNB3 c.-57A>G alteration affects the promoter activity and is associated with PD and smoking in PD patients. It is therefore possible that nicotine may be valuable for patients who carry this alteration and beneficial in PD only for patients with specific genotypes.

Tnfα, Cox2 and AdipoQ adipokine gene expression levels are modulated in murine adipose tissues by both nicotine and nACh receptors containing the ß2 subunit.

Studies have provided evidences for the effects of nicotine on adipose tissues, as well as in inflammatory response. We hypothesized that nicotine affects adipokine gene expression in adipose tissues via specific neuronal nicotinic acetylcholine receptors (nAChRs). First, we described the expression of multiple nAChR subunit genes in mouse white and brown adipose tissues (WAT and BAT), and detected differential expression in WAT and BAT (α2>α5>ß2 and α2>ß2>ß4, respectively). Additionally, when nicotine was administered to wild-type mice, it significantly affected the expression of adipokine genes, such as Tnfα, AdipoQ, Haptoglobin and Mcp1 in WAT. Next, we demonstrated that in mice deficient for the ß2 nAChR subunit (ß2-/- mice), the expression levels of Cox2 and Ngfß genes in WAT, and Leptin, Cox2, AdipoQ and Haptoglobin in BAT, were significantly altered. Furthermore, interactions between mouse ß2 subunit and nicotine treatment affected the expression levels of the adipokine genes Tnfα, Cox2 and AdipoQ in WAT and of AdipoQ in BAT. Finally, analysis of a cellular model of cultured adipocytes demonstrated that application of nicotine after silencing of the ß2 nAChR subunit significantly elevated the expression level of Cox2 gene. Together, our data suggest a molecular link between the ß2 nACh receptor subunit and the expression levels of specific adipokines, which is also affected by nicotine.

Requirement of ATM-dependent monoubiquitylation of histone H2B for timely repair of DNA double-strand breaks.

The cellular response to DNA double-strand breaks (DSBs) is mobilized by the protein kinase ATM, which phosphorylates key players in the DNA damage response (DDR) network. A major question is how ATM controls DSB repair. Optimal repair requires chromatin relaxation at damaged sites. Chromatin reorganization is coupled to dynamic alterations in histone posttranslational modifications. Here, we show that in human cells, DSBs induce monoubiquitylation of histone H2B, a modification that is associated in undamaged cells with transcription elongation. We find that this process relies on recruitment to DSB sites and ATM-dependent phosphorylation of the responsible E3 ubiquitin ligase: the RNF20-RNF40 heterodimer. H2B monoubiquitylation is required for timely recruitment of players in the two major DSB repair pathways-nonhomologous end-joining and homologous recombination repair-and optimal repair via both pathways. Our data and previous data suggest a two-stage model for chromatin decondensation that facilitates DSB repair.

The histone H2B-specific ubiquitin ligase RNF20/hBRE1 acts as a putative tumor suppressor through selective regulation of gene expression.

Histone monoubiquitylation is implicated in critical regulatory processes. We explored the roles of histone H2B ubiquitylation in human cells by reducing the expression of hBRE1/RNF20, the major H2B-specific E3 ubiquitin ligase. While H2B ubiquitylation is broadly associated with transcribed genes, only a subset of genes was transcriptionally affected by RNF20 depletion and abrogation of H2B ubiquitylation. Gene expression dependent on RNF20 includes histones H2A and H2B and the p53 tumor suppressor. In contrast, RNF20 suppresses the expression of several proto-oncogenes, which reside preferentially in closed chromatin and are modestly transcribed despite bearing marks usually associated with high transcription rates. Remarkably, RNF20 depletion augmented the transcriptional effects of epidermal growth factor (EGF), increased cell migration, and elicited transformation and tumorigenesis. Furthermore, frequent RNF20 promoter hypermethylation was observed in tumors. RNF20 may thus be a putative tumor suppressor, acting through selective regulation of a distinct subset of genes.

Imatinib binding and cKIT inhibition is abrogated by the cKIT kinase domain I missense mutation Val654Ala.

Several activating mutations in the cKIT receptor tyrosine kinase are associated with the development and progression of gastrointestinal stromal tumors (GIST). Treatment of GIST with the tyrosine kinase inhibitor imatinib (Gleevec, STI571; Novartis, Basel, Switzerland) increases patient survival. However, many patients develop resistance to imatinib following initial responses. We sequenced cKIT exons from two patients with GIST after the development of imatinib resistance, revealing a point mutation in kinase domain I (exon 13), Val654Ala, which has been associated previously with relapse and resistance. Molecular modeling of cKIT-imatinib complexes shows that this residue is located in the drug-binding site and that the Val654Ala mutation disrupts drug binding by removing hydrophobic contacts with the central diaminophenyl ring of imatinib. Loss of these contacts results in a destabilizing effect on two key hydrogen bonds between imatinib and Asp310 and Thr670 of cKIT. Calculations based on published crystallography data show an estimated destabilization energy of 2.25 kcal/mol in the Val654Ala cKIT compared with wild type. When present on the same cKIT allele as an oncogenic mutation, the Val654Ala mutation abolishes imatinib-mediated inhibition of cKIT phosphoactivation in vitro. These results highlight some of the structural and functional consequences of the Val654Ala mutation in relapsing imatinib-resistant GIST and emphasize the importance of tumor genetics in drug development and patient-specific cancer treatment regimens.

The Ras inhibitor S-trans,trans-farnesylthiosalicylic acid chemosensitizes human tumor cells without causing resistance.

Ras transformation requires Ras membrane anchorage, which is promoted by a farnesylcysteine carboxymethyl ester and by additional sequences specific to each Ras isoform. We showed previously that S-trans,trans-farnesylthiosalicylic acid (FTS) disrupts Ras membrane anchorage and that this disturbance contributes to inhibition of cell transformation and tumor growth. Most tumor cells develop resistance to anticancer agents. Here we examined whether tumor cells develop resistance to FTS and evaluated the therapeutic potential of FTS combined with cytotoxic drugs, because oncogenic Ras promotes antiapoptotic signals in tumors of epithelial origin. We showed that Panc-1 pancreatic cancer cells, SW480 colon cancer cells, and H-ras (EJ)-transformed Rat-1 fibroblasts exposed to FTS for prolonged periods (>6 months) do not escape FTS-induced growth inhibition and do not develop drug resistance. These cells continued to express reduced amounts of Ras, exhibit a reversed phenotype, and show an altered response to the cytotoxic drugs doxorubicin and gemcitabine. FTS-treated Panc-1 or SW480 cells acquired sensitivity to the cytotoxic drugs, whereas FTS-treated EJ cells lost sensitivity to doxorubicin, reflecting the opposite effects of oncogenic Ras on the survival of epithelial cells and fibroblasts. Treatment with FTS led to a marked increase in sensitivity to gemcitabine of the formerly resistant SW480 cells and a 100-fold increase in sensitivity to gemcitabine of Panc-1 cells. Such treatment in mice with preexisting Panc-1 tumors provided a synergistic effect of FTS and gemcitabine, leading to enhanced inhibition of tumor growth and a 65% increase in survival rate.