SMN1 Duplications Are Associated With Progressive Muscular Atrophy, but Not With Multifocal Motor Neuropathy and Primary Lateral Sclerosis.

OBJECTIVE: To assess the association between copy number (CN) variation in the survival motor neuron (SMN) locus and multifocal motor neuropathy (MMN), progressive muscular atrophy (PMA), and primary lateral sclerosis (PLS) susceptibility and to determine the association of SMN1 and SMN2 CN with MMN, PMA, and PLS disease course. METHODS: In this monocenter study, we used multiplex ligation-dependent probe amplification to determine SMN1 and SMN2 CN in Dutch patients with MMN, PMA, and PLS and controls. We stratified clinical parameters for SMN1 and SMN2 CN. We analyzed SMN1 and SMN2 exons 1-6, intron 6, and exon 8 CN to study the genetic architecture of SMN1 duplications. RESULTS: SMN1 and SMN2 CN were determined in 132 patients with MMN, 150 patients with PMA, 104 patients with PLS, and 956 control subjects. MMN and PLS were not associated with CN variation in SMN1 or SMN2. By contrast, patients with PMA more often than controls carried SMN1 duplications (≥3 SMN1 copies, 12.0% vs 5.0%, odds ratio 2.69 (1.43-4.91), p 0.0020). SMN1 and SMN2 CN status was not associated with MMN, PLS, or PMA disease course. In case of SMN1 exon 7 duplications, exons 1-6, exon 8, and introns 6 and 7 were also duplicated, suggesting full SMN1 duplications. CONCLUSIONS: SMN1 duplications are associated with PMA, but not with PLS and MMN. SMN1 duplications in PMA are balanced duplications. The results of this study highlight the primary effect of altered SMN CN on lower motor neurons.

Whole blood transcriptome analysis in amyotrophic lateral sclerosis: A biomarker study.

The biological pathways involved in amyotrophic lateral sclerosis (ALS) remain elusive and diagnostic decision-making can be challenging. Gene expression studies are valuable in overcoming such challenges since they can shed light on differentially regulated pathways and may ultimately identify valuable biomarkers. This two-stage transcriptome-wide study, including 397 ALS patients and 645 control subjects, identified 2,943 differentially expressed transcripts predominantly involved in RNA binding and intracellular transport. When batch effects between the two stages were overcome, three different models (support vector machines, nearest shrunken centroids, and LASSO) discriminated ALS patients from control subjects in the validation stage with high accuracy. The models' accuracy reduced considerably when discriminating ALS from diseases that mimic ALS clinically (N = 75), nor could it predict survival. We here show that whole blood transcriptome profiles are able to reveal biological processes involved in ALS. Also, this study shows that using these profiles to differentiate between ALS and mimic syndromes will be challenging, even when taking batch effects in transcriptome data into account.

Analysis of the KIFAP3 gene in amyotrophic lateral sclerosis: a multicenter survival study.

Sporadic amyotrophic lateral sclerosis is a multifactorial disease of environmental and genetic origin. In a previous large multicenter genome wide study, common genetic variation in the Kinesin-Associated Protein 3 (KIFAP3) gene (rs1541160) was reported to have a significant effect on survival in amyotrophic lateral sclerosis patients. However, this could not be replicated in 3 smaller independent cohorts. We conducted a large multicenter multivariate survival analysis (n = 2362) on the effect of genetic variation in rs1541160. The previously reported beneficial genotype did not show a significant improvement in survival in this patient group.

Gene expression profile of SOD1-G93A mouse spinal cord, blood and muscle.

The exact pathway leading to neuron death and muscle atrophy in amyotrophic lateral sclerosis (ALS) has not yet been elucidated. Gene expression profile of spinal cord, blood and muscle could provide signalling pathways and systemic alterations useful for future biomarker development. In our study we compared whole genome expression profiles of lumbar spinal cord with peripheral blood and tibialis anterior muscle in 16 mutant SOD1-G93A mice and 15 wild-type littermates. In SOD1-G93A mice, 11 genes were significantly differentially expressed in spinal cord, and 16 genes in blood, while much larger transcriptional changes were noted in muscle (1745 genes significant; six overlapping with spinal cord (0.3%)) probably due to muscle atrophy. Overlap with spinal cord was enriched for significant genes in blood (six of 16 overlapping with spinal cord (37.5%)). Three genes were significantly down-regulated in all three tissues, and were closely related to mitochondrial function. Furthermore, clustering the significant genes in spinal cord and in blood, but not in muscle, could identify the SOD1-G93A mice. We conclude that blood gene expression profile overlapped with profile of spinal cord, allowing differentiation of SOD1-G93A mice from wild-type littermates. Blood gene expression profiling may be a promising biomarker for ALS patients.

H63D polymorphism in HFE is not associated with amyotrophic lateral sclerosis.

The H63D polymorphism in HFE has frequently been associated with susceptibility to amyotrophic lateral sclerosis (ALS). Regarding the role of HFE in iron homeostasis, iron accumulation is considered an important process in ALS. Furthermore, novel therapeutic strategies are being developed targeting this process. Evidence for this genetic association is, however, limited to several small studies. For this reason we studied the H63D polymorphism in a large European cohort including 3962 ALS patients and 5072 control subjects from 7 countries. After meta-analysis of previous studies and current findings we conclude that the H63D polymorphism in HFE is not associated with susceptibility to ALS, age at disease onset, or survival.

Screening for rare variants in the coding region of ALS-associated genes at 9p21.2 and 19p13.3.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease that causes progressive muscle weakness, eventually resulting in death because of respiratory failure. Genetic variants are thought to predispose to the disease. A recent, large, genome-wide association study identified 2 loci that increase susceptibility to ALS. These 2 loci on chromosomes 9 and 19 consist of 4 genes: UNC13a, IFNK, MOBKL2b, and C9ORF72. A hexanucleotide repeat expansion in the noncoding region of C9ORF72 was recently identified as the cause of chromosome 9-linked ALS-FTD (frontotemporal dementia). In this study, our aim was to determine whether the coding regions of these genes harbor rare, nonsynonymous variants that play a role in ALS pathogenesis. In DNA from 1019 sporadic ALS patients and 1103 control subjects of Dutch descent, we performed a mutation screening analysis in the coding region of these 4 genes by resequencing the exons. A total of 16 amino acid-changing rare variations were identified, 11 in UNC13a and 5 on chromosome 9. Some of these were unique to ALS, but were detected in a single patient. None of the genes showed significant enrichment of rare variants in the coding sequence. Rare variants in the coding region of UNC13a, IFNK, MOBKL2b, and C9ORF72 are unlikely to be a genetic cause of ALS.

Mapping of gene expression reveals CYP27A1 as a susceptibility gene for sporadic ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disease characterized by loss of upper and lower motor neurons. ALS is considered to be a complex trait and genome-wide association studies (GWAS) have implicated a few susceptibility loci. However, many more causal loci remain to be discovered. Since it has been shown that genetic variants associated with complex traits are more likely to be eQTLs than frequency-matched variants from GWAS platforms, we conducted a two-stage genome-wide screening for eQTLs associated with ALS. In addition, we applied an eQTL analysis to finemap association loci. Expression profiles using peripheral blood of 323 sporadic ALS patients and 413 controls were mapped to genome-wide genotyping data. Subsequently, data from a two-stage GWAS (3,568 patients and 10,163 controls) were used to prioritize eQTLs identified in the first stage (162 ALS, 207 controls). These prioritized eQTLs were carried forward to the second sample with both gene-expression and genotyping data (161 ALS, 206 controls). Replicated eQTL SNPs were then tested for association in the second-stage GWAS data to find SNPs associated with disease, that survived correction for multiple testing. We thus identified twelve cis eQTLs with nominally significant associations in the second-stage GWAS data. Eight SNP-transcript pairs of highest significance (lowest p = 1.27 × 10(-51)) withstood multiple-testing correction in the second stage and modulated CYP27A1 gene expression. Additionally, we show that C9orf72 appears to be the only gene in the 9p21.2 locus that is regulated in cis, showing the potential of this approach in identifying causative genes in association loci in ALS. This study has identified candidate genes for sporadic ALS, most notably CYP27A1. Mutations in CYP27A1 are causal to cerebrotendinous xanthomatosis which can present as a clinical mimic of ALS with progressive upper motor neuron loss, making it a plausible susceptibility gene for ALS.

NIPA1 polyalanine repeat expansions are associated with amyotrophic lateral sclerosis.

Mutations in NIPA1 cause Hereditary Spastic Paraplegia type 6, a neurodegenerative disease characterized by an (upper) motor neuron phenotype. Deletions of NIPA1 have been associated with a higher susceptibility to amyotrophic lateral sclerosis (ALS). The exact role of genetic variation in NIPA1 in ALS susceptibility and disease course is, however, not known. We sequenced the entire coding sequence of NIPA1 and genotyped a polyalanine repeat located in the first exon of NIPA1. A total of 2292 ALS patients and 2777 controls from three independent European populations were included. We identified two sequence variants that have a potentially damaging effect on NIPA1 protein function. Both variants were identified in ALS patients; no damaging variants were found in controls. Secondly, we found a significant effect of 'long' polyalanine repeat alleles on disease susceptibility: odds ratio = 1.71, P = 1.6 × 10(-4). Our analyses also revealed a significant effect of 'long' alleles on patient survival [hazard ratio (HR) = 1.60, P = 4.2 × 10(-4)] and on the age at onset of symptoms (HR = 1.37, P = 4.6 × 10(-3)). In patients carrying 'long' alleles, median survival was 3 months shorter than patients with 'normal' genotypes and onset of symptoms occurred 3.6 years earlier. Our data show that NIPA1 polyalanine repeat expansions are a common risk factor for ALS and modulate disease course.

Rare and common paraoxonase gene variants in amyotrophic lateral sclerosis patients.

Polymorphisms in the paraoxonase family (PON) have been reported in patients with amyotrophic lateral sclerosis (ALS), but a recent meta-analysis did not show a clear association. Recently, PON mutations have also been identified in ALS patients. In this study, we assessed the frequency of PON variants in 1118 sporadic ALS patients, 93 familial ALS patients, and 1240 control subjects of Dutch descent. We identified PON mutations in 1.4% of sporadic ALS patients, 2.1% of familial ALS patients, and 2.5% of control subjects. There were no significant differences in mutational burden for rare variants or in allele frequencies of polymorphisms between patients and control subjects. Thus, this study does not support the premise that mutations or polymorphisms in PON contribute to ALS susceptibility.

UNC13A is a modifier of survival in amyotrophic lateral sclerosis.

A large genome-wide screen in patients with sporadic amyotrophic lateral sclerosis (ALS) showed that the common variant rs12608932 in gene UNC13A was associated with disease susceptibility. UNC13A regulates the release of neurotransmitters, including glutamate. Genetic risk factors that, in addition, modify survival, provide promising therapeutic targets in ALS, a disease whose etiology remains largely elusive. We examined whether UNC13A was associated with survival of ALS patients in a cohort of 450 sporadic ALS patients and 524 unaffected controls from a population-based study of ALS in The Netherlands. Additionally, survival data were collected from individuals of Dutch, Belgian, or Swedish descent (1767 cases, 1817 controls) who had participated in a previously published genome-wide association study of ALS. We related survival to rs12608932 genotype. In both cohorts, the minor allele of rs12608932 in UNC13A was not only associated with susceptibility but also with shorter survival of ALS patients. Our results further corroborate the role of UNC13A in ALS pathogenesis.

Novel optineurin mutations in sporadic amyotrophic lateral sclerosis patients.

Optineurin (OPTN) mutations have been reported in a cohort of Japanese patients with familial (FALS) and sporadic (SALS) amyotrophic lateral sclerosis. In Caucasian patients, OPTN mutations have been identified in FALS patients, but were not detected in a cohort of 95 SALS patients. Moreover, single nucleotide polymorphisms (SNPs) in OPTN that could raise amyotrophic lateral sclerosis (ALS) susceptibility have not been investigated. Therefore, we screened a large Dutch cohort of 1191 patients with SALS, 94 patients with FALS, and 1415 control subjects for mutations and SNPs in OPTN. We identified 1 novel nonsense mutation (Q165X) and 1 unreported missense mutation (Q454E) in individual SALS patients. These patients demonstrated rapid disease progression with an average survival of 24.5 months. No heterozygous or homozygous OPTN mutations were identified in our cohort of FALS patients. SNP analysis did not reveal significant differences between ALS patients and control subjects. Therefore, variations in OPTN appear to be a rare cause of rapidly progressive SALS in the Netherlands.

VCP mutations in familial and sporadic amyotrophic lateral sclerosis.

Mutations in the valosin-containing protein (VCP) gene were recently reported to be the cause of 1%-2% of familial amyotrophic lateral sclerosis (ALS) cases. VCP mutations are known to cause inclusion body myopathy (IBM) with Paget's disease (PDB) and frontotemporal dementia (FTD). The presence of VCP mutations in patients with sporadic ALS, sporadic ALS-FTD, and progressive muscular atrophy (PMA), a known clinical mimic of inclusion body myopathy, is not known. To determine the identity and frequency of VCP mutations we screened a cohort of 93 familial ALS, 754 sporadic ALS, 58 sporadic ALS-FTD, and 264 progressive muscular atrophy patients for mutations in the VCP gene. Two nonsynonymous mutations were detected; 1 known mutation (p.R159H) in a patient with familial ALS with several family members suffering from FTD, and 1 mutation (p.I114V) in a patient with sporadic ALS. Conservation analysis and protein prediction software indicate the p.I114V mutation to be a rare benign polymorphism. VCP mutations are a rare cause of familial ALS. The role of VCP mutations in sporadic ALS, if present, appears limited.

Angiogenin variants in Parkinson disease and amyotrophic lateral sclerosis.

OBJECTIVE: Several studies have suggested an increased frequency of variants in the gene encoding angiogenin (ANG) in patients with amyotrophic lateral sclerosis (ALS). Interestingly, a few ALS patients carrying ANG variants also showed signs of Parkinson disease (PD). Furthermore, relatives of ALS patients have an increased risk to develop PD, and the prevalence of concomitant motor neuron disease in PD is higher than expected based on chance occurrence. We therefore investigated whether ANG variants could predispose to both ALS and PD. METHODS: We reviewed all previous studies on ANG in ALS and performed sequence experiments on additional samples, which allowed us to analyze data from 6,471 ALS patients and 7,668 controls from 15 centers (13 from Europe and 2 from the USA). We sequenced DNA samples from 3,146 PD patients from 6 centers (5 from Europe and 1 from the USA). Statistical analysis was performed using the variable threshold test, and the Mantel-Haenszel procedure was used to estimate odds ratios. RESULTS: Analysis of sequence data from 17,258 individuals demonstrated a significantly higher frequency of ANG variants in both ALS and PD patients compared to control subjects (p = 9.3 × 10(-6) for ALS and p = 4.3 × 10(-5) for PD). The odds ratio for any ANG variant in patients versus controls was 9.2 for ALS and 6.7 for PD. INTERPRETATION: The data from this multicenter study demonstrate that there is a strong association between PD, ALS, and ANG variants. ANG is a genetic link between ALS and PD.

Multifocal motor neuropathy is not associated with genetic variation in PTPN22, BANK1, Blk, FCGR2B, CD1A/E, and TAG-1 genes.

The contribution of genetic heterogeneity to the pathogenesis of multifocal motor neuropathy (MMN) has not been elucidated. We investigated frequencies of single nucleotide polymorphisms (SNPs) in the candidate genes protein tyrosine phosphatase, non-receptor type 22 (PTPN22), B-cell scaffold protein with ankyrin repeats (BANK1), B lymphocyte kinase (Blk), and Fc gamma receptor class IIB (FCGR2B), which have been found to be associated with other autoimmune diseases, CD1A and CD1E, important for antigen presentation of glycolipids, and transient axonal glycoprotein 1 (TAG-1), which is associated with responsiveness to intravenous immunoglobulin in patients with chronic inflammatory demyelinating polyneuropathy. SNP frequencies were determined by means of TaqMan SNP genotyping assay and direct sequencing of candidate genes in 92 Dutch patients with MMN and 1152 healthy controls. SNP frequencies did not differ between patients and controls (all p-values >0.15) and disease characteristics were not associated with SNP genotypes. Our results suggest that allelic variation in these genes does not play a major role in determining MMN susceptibility.

A large genome scan for rare CNVs in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease selectively affecting motor neurons in the brain and spinal cord. Recent genome-wide association studies (GWASs) have identified several common variants which increase disease susceptibility. In contrast, rare copy-number variants (CNVs), which have been associated with several neuropsychiatric traits, have not been studied for ALS in well-powered study populations. To examine the role of rare CNVs in ALS susceptibility, we conducted a CNV association study including over 19,000 individuals. In a genome-wide screen of 1875 cases and 8731 controls, we did not find evidence for a difference in global CNV burden between cases and controls. In our association analyses, we identified two loci that met our criteria for follow-up: the DPP6 locus (OR = 3.59, P = 6.6 × 10(-3)), which has already been implicated in ALS pathogenesis, and the 15q11.2 locus, containing NIPA1 (OR = 12.46, P = 9.3 × 10(-5)), the gene causing hereditary spastic paraparesis type 6 (HSP 6). We tested these loci in a replication cohort of 2559 cases and 5887 controls. Again, results were suggestive of association, but did not meet our criteria for independent replication: DPP6 locus: OR = 1.92, P = 0.097, pooled results: OR = 2.64, P = 1.4 × 10(-3); NIPA1: OR = 3.23, P = 0.041, pooled results: OR = 6.20, P = 2.2 × 10(-5)). Our results highlight DPP6 and NIPA1 as candidates for more in-depth studies. Unlike other complex neurological and psychiatric traits, rare CNVs with high effect size do not play a major role in ALS pathogenesis.

FUS mutations in familial amyotrophic lateral sclerosis in the Netherlands.

OBJECTIVES: To assess the frequency of FUS mutations in 52 probands with familial amyotrophic lateral sclerosis (FALS) and to provide careful documentation of clinical characteristics. DESIGN: FUS mutation analysis was performed using capillary sequencing on all coding regions of the gene in a cohort of patients with FALS. The clinical characteristics of patients carrying FUS mutations were described in detail. SETTING: Three university hospitals in the Netherlands (referral centers for neuromuscular diseases). PATIENTS: Fifty-two probands from unrelated pedigrees with FALS. MAIN OUTCOME MEASURE: FUS mutations. RESULTS: We identified 3 mutations in 4 of 52 probands. We observed 2 previously identified mutations (p.Arg521Cys and p.Arg521His) and 1 novel mutation (p.Ser462Phe). In addition, a p.Gln210His polymorphism was identified in 1 proband and 3 healthy control subjects. Phenotypic analysis demonstrated that patients may lack upper motor neuron signs, which was confirmed at autopsy, and disease survival was short (<36 months for 8 of 10 patients). CONCLUSIONS: We discovered FUS mutations in Dutch patients with FALS and the occurrence of benign variations in the gene. Therefore, caution is warranted when interpreting results in a clinical setting. Although the phenotype associated with FUS mutations is variable, most patients predominantly demonstrate loss of lower motor neurons and have short disease survival.

Analysis of FGGY as a risk factor for sporadic amyotrophic lateral sclerosis.

A genome-wide association study (GWAS) using pooled DNA samples from 386 sporadic ALS patients and 542 controls from the USA, identified genetic variation in FGGY (FLJ10986) as a risk factor, as well as 66 additional candidate SNPs. Considering the large number of hypotheses that are tested in GWAS, independent replication of associations is crucial for identifying true-positive genetic risk factors for disease. The primary aim of this study was to study the association between FGGY and sporadic ALS in large, homogeneous populations from northern Europe. Genotyping experiments were performed using Illumina Beadchips, Sequenom iPLEX assays and Taqman technology on large case-control series from The Netherlands, Belgium, Sweden and Ireland (total: 1883 sporadic ALS patients and 2063 controls). No significant association between sporadic ALS and the six previously reported associated SNPs in FGGY was observed: rs6700125 (p =0.56), rs6690993 (p =0.30), rs10493256 (p =0.68), rs6587852 (p =0.64), rs1470407 (p =0.28) and rs333662 (p =0.44). Screening of the additional candidate loci did not yield significant associations either, with the lowest p-value in joint analysis for rs7772593 (p =0.14). We concluded that common genetic variation in FGGY is not associated with susceptibility to sporadic ALS in genetically homogeneous populations from northern Europe.

Genome-wide association study identifies 19p13.3 (UNC13A) and 9p21.2 as susceptibility loci for sporadic amyotrophic lateral sclerosis.

We conducted a genome-wide association study among 2,323 individuals with sporadic amyotrophic lateral sclerosis (ALS) and 9,013 control subjects and evaluated all SNPs with P < 1.0 x 10(-4) in a second, independent cohort of 2,532 affected individuals and 5,940 controls. Analysis of the genome-wide data revealed genome-wide significance for one SNP, rs12608932, with P = 1.30 x 10(-9). This SNP showed robust replication in the second cohort (P = 1.86 x 10(-6)), and a combined analysis over the two stages yielded P = 2.53 x 10(-14). The rs12608932 SNP is located at 19p13.3 and maps to a haplotype block within the boundaries of UNC13A, which regulates the release of neurotransmitters such as glutamate at neuromuscular synapses. Follow-up of additional SNPs showed genome-wide significance for two further SNPs (rs2814707, with P = 7.45 x 10(-9), and rs3849942, with P = 1.01 x 10(-8)) in the combined analysis of both stages. These SNPs are located at chromosome 9p21.2, in a linkage region for familial ALS with frontotemporal dementia found previously in several large pedigrees.