Objective To estimate the frequency of SOD1 mutations in a large referral cohort of familial amyotrophic lateral sclerosis (FALS) and sporadic amyotrophic lateral sclerosis (SALS) patients from The Netherlands and to compare this frequency with that of other developed countries.
Methods A total of 451 sporadic and 55 FALS patients were screened for SOD1 mutations. The authors performed PCR amplification of all five coding exons of SOD1 followed by direct DNA sequencing using forward and reverse primers.
Results One novel mutation (p.I99V) and a homozygous p.D90A mutation were identified in SALS patients. In a pedigree with Mendelian dominant FALS, one patient was found to be heterozygous for the p.D90A mutation. SOD1 mutation frequency was found to be significantly lower in The Netherlands compared with other countries with p=0.0004 for FALS (21.9% vs 2.5%) and p=0.005 for SALS (2.5% vs 0.44%).
Conclusions The authors demonstrate that SOD1 mutations are rare in The Netherlands in familial and SALS. This observation suggests that the genetic background of amyotrophic lateral sclerosis differs between different populations, countries and regions. This may have consequences for the interpretation of association studies and explain why replication of association studies has proven difficult in amyotrophic lateral sclerosis.
- association study
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Amyotrophic lateral sclerosis (ALS) is an adult-onset, fatal neurodegenerative syndrome characterised mainly by the progressive loss of upper and lower motor neurons and their axons resulting in wasting, paresis and death from respiratory failure within a few years on average. The incidence (around 1–2 per 100 000 person-years) and prevalence (4–13 per 100 000) of ALS are remarkably similar across European and North American populations.1 To date, the only effective therapeutic strategy is treatment with riluzole, which prolongs survival by about 3 months after 18 months' treatment.
In retrospective, epidemiological studies, 1–13% of ALS patients are claimed to have a familial disposition for the syndrome (FALS), most commonly with a Mendelian dominant mode of inheritance. In the remaining 90% of ALS patients where there is no family history, the disease is considered to be sporadic (denoted as SALS).
Linkage analysis in 18 familial ALS pedigrees associated the gene encoding Cu/Zn superoxide dismutase (SOD1) on chromosome 21 to the syndrome. Mutational analysis revealed point-mutations in the SOD1 gene to cosegregate with the disease in these pedigrees.2 Subsequent studies have identified over 140 different SOD1 mutations in ALS patients. Mutations in SOD1 have been reported in approximately 12–23% of the patients diagnosed as having FALS and in 1–4% of patients diagnosed as having SALS.3
Although SOD1 mutations have been identified in many western populations at comparable frequencies, screening in clinical practice suggests that these mutations are rare in The Netherlands. This observation indicates that the genetic background of ALS may differ between countries and regions. This could have consequences for the interpretation of association studies and may explain why replication of associations has proven difficult in ALS. We report the frequency of SOD1 mutations in a large Dutch referral population of sporadic and familial ALS patients, and a comparison is made with other countries by reviewing studies on SOD1 mutation frequencies in populations from Europe, USA and Japan.
The University Medical Center Utrecht is part of the National Center for ALS in The Netherlands, to which an estimated 60% of ALS patients in the country are referred. We collected DNA samples from all patients who were diagnosed as having ALS from February 2006 to February 2008 at our outpatient clinic. All patients were diagnosed according to the revised El Escorial criteria and fulfilled the criteria for probable or definite ALS.4 Additionally, we included DNA samples from all FALS patients who were referred to our clinic from July 2000 to July 2006. Patients with one or more affected relatives were diagnosed as having familial ALS. We included one individual per family, and detailed family histories were taken to exclude the possibility of distant kinship. In total, 451 sporadic cases and 55 familial index cases were screened for SOD1 gene mutations. Baseline characteristics for the study population are provided in table 1. The study was performed according to the tenants of the Declaration of Helsinki (WMA, 1964) and was approved by the local medical ethical committee, with all participants providing written informed consent.
DNA isolation and mutation analysis
Venous blood samples were drawn using 10 ml EDTA tubes, and genomic DNA was extracted from leucocytes using a standard salting out procedure. PCR amplification of all five coding exons of the SOD1 gene and at least 50 bp of flanking intronic sequence was performed. Direct DNA sequencing was performed on the PCR products with an Applied Biosystems 3730xL automated fluorescent DNA sequencer (Applied Biosystems, Foster City, California). Each product was sequenced using forward and reverse primers.
To increase the likelihood of finding patients with mutations, we also measured the SOD1 enzymatic activity in erythrocytes from 201 of the patients, as described previously.5 6 The assay was performed directly on haemolysates without prior precipitation of haemoglobin, and the SOD1 enzyme activity was expressed as units per milligram of haemoglobin (U/mg Hb).
Review of SOD1 mutation studies
We searched Pubmed (http://www.ncbi.nlm.nih.gov/sites/entrez/) and EMBASE (http://www.embase.com) for studies on SOD1 mutation frequency using the following search terms: Cu/Zn-SOD, SOD1, Amyotrophic Lateral Sclerosis, ALS, SLA, Motor Neuron Disease and Familial ALS. We also cross-checked references in the obtained studies to assess completeness of the search, which did not result in additional studies.
Comparison of mutation frequencies was made using the Fisher Exact test in the statistical analysis program R (CRAN; http://www.R-project.org). We compared the mutation frequency in all populations combined in FALS and SALS versus mutation frequency in FALS and SALS in The Netherlands.
Three (0.6%) of the 506 patients were found to carry exonic missense mutations in SOD1. No patients were found to carry non-sense mutations. We identified a novel mutation p.I99V and a homozygous p.D90A mutation in two patients with a diagnosis of SALS. A heterozygous p.D90A mutation was detected in a patient with a diagnosis of FALS. The mutations are shown in figure 1.
A novel mutation was found in exon 4 of the gene at position 298 where a G to A (c.298G→A) substitution was identified, resulting in an isoleucine to valine amino-acid exchange, or p.I99V.
The sporadic patient carrying the p.I99V mutation presented to our outpatient clinic at 60 years of age with a speech impairment and difficulty swallowing, followed by weakness of the legs within a year. On neurological examination, he had pseudobulbar dysarthria, signs of atrophy and fasciculations of the tongue. Fasciculations were also seen in the arms and legs, and pathological hyper-reflexia was seen in the arms and legs. Although the initial development of symptoms was quite rapid, the patient has survived over 4 years and is still alive.
The apparently sporadic patient homozygous for the p.D90A mutation presented at 55 years of age with problems maintaining balance and frequent falling. A year prior to his presentation, he had experienced passing tingling sensations in his lower limbs, for which he had not sought medical attention. On neurological examination, there were no sensory abnormalities. Fasciculations were seen in both legs and arms. There was weakness of the quadriceps and iliopsoas muscles in both legs. Weakness was, however, more pronounced on the left side. Initially, reflexes were symmetrically brisk in arms and legs but were not considered pathological. Plantar responses were seen on both sides. Electromyography revealed signs of deinnervation and reinnervation in the legs and arms, which fulfilled the El Escorial criteria for ALS. Over time, the symptoms were progressive, and the patient developed marked atrophy in the legs as well as clearly pathological hyper-reflexia, including bilateral Babinski signs. Disease progression is relatively slow, and the patient has survived over 86.5 months. The patient's parents had died at an old age of causes not related to motor neuron disease. DNA was not available from these individuals.
Analysis of the pedigree of the heterozygous p.D90A FALS patient revealed a Mendelian dominant pattern of inheritance. An anonymous version of this large Dutch pedigree is shown in figure 2. Clinical data were available for only two patients.
Unfortunately, DNA were not available from additionally affected family members to show cosegregation of the p.D90A SOD1 allele with disease. Individual III:1 (index patient) presented at the age of 51 with bilateral weakness of the hands and cramps in arms and calf muscles. On neurological examination, there were fasciculations in the arms and legs. Generalised amyotrophy was observed, which was most prominent in the hands. There was severe weakness of the hands and mild weakness in the proximal arm muscles. No involvement of the bulbar or lumbar regions was initially seen. Cognition was intact. Disease progression is relatively slow, and the patient is alive after a disease duration of more than 6 years. Limited clinical information was available for III:2. This patient suffered from bulbar-onset ALS and died within 3 years as a result of respiratory failure.
SOD1 mutations were identified in 0.44% of sporadic cases and 1.8% of familial cases in our study population. Our Pubmed search identified 18 studies, which analysed patients from 15 different countries (Ireland,7 Scotland,8 Canada,9 10 Japan,11 UK,12–14 USA,15 Italy,16–19 Spain,20 France,21 Denmark,22 Norway,22 Finland,22 Sweden,22 Germany23 and Belgium24) on SOD1 mutation frequencies. In all studies combined, a total of 187 SOD1 mutations were identified in 854 FALS patients with an overall frequency of 21.9%. The combined studies identified 45 mutations in 1792 SALS patients with an overall frequency of 2.5%. Comparison of frequencies in FALS between the Dutch population and the other studies (1.8% vs 21.9%) yielded a Fisher Exact p value of 0.0004. A similar result was observed for SALS with SOD1 mutations in 0.4% in The Netherlands and in 2.5% in the other studies with a Fisher Exact p value of 0.005. The results are summarised in table 2.
The SOD1 dismutation acitivities in erytrocytes from 201 patients were no different from controls (52.9±5.8 vs 54.1±5.7 U/mg Hb, controls n=66). The two patients with the D90A mutation had activities of 55.55 and 56.54 U/mg Hb, respectively. Unfortunately, erytrocytes were not available for dismutation analysis from the patient with the p.I99V SOD1 mutation.
This study identified one novel mutation (p.I99V) in a patient with SALS. The mutation has been added to the online database (http://www.alsod.org).26 We also identified patients homozygous and heterozygous for the p.D90A mutation. This mutation is of particular interest, because in the majority of cases, the p.D90A mutation is recessive.27 However, nine pedigrees with ALS patients carrying the p.D90A SOD1 allele in heterozygous form have also been reported. The majority of these heterozygous patients were diagnosed as having SALS, but a few FALS patients have also been reported.24 The heterozygous case with a p.D90A mutation found in this study was identified as FALS, and analysis of the pedigree demonstrated that the disease is transmitted as a dominant Mendelian trait. The patient homozygous for the p.D90A mutation was found in an apparently sporadic ALS patient. With our finding of the p.D90A allele in The Netherlands, this allele has now been found in 20 countries and in almost all countries where a large number of samples have been screened for SOD1 mutations (the reported exceptions being Ireland, Denmark, China and Japan). This makes the SOD1 p.D90A allele the most common identified cause of ALS.
Studies have revealed that the p.D90A SOD1 allele probably arose from a single founder at least 18 000 years ago (or 865 generations), and all carriers of the p.D90A mutation share the same basic haplotype of this common ancestor.28
Clinically, it has been observed that the p.D90A mutation can cause ALS either as a dominant trait or as a recessive trait. In these recessive families, no p.D90A heterozygous carriers ever develop clinical symptoms of ALS. It has been hypothesised that a second, protective mutation occurred on the p.D90A haplotype, causing the recessive pattern of inheritance. Studies indicate that this second putative mutation occurred approximately 15 000 years ago in the founding population of Finno-Scandinavia and Russia, and is quite common in the northern Scandinavian populations (5% of the population carries the p.D90A SOD1 allele in the large Torne Valley in northern Sweden).
All studied homozygous p.D90A patients around the world appear to be of Russian-Finno-Scandinavian descent, and share a common haplotype and thus the potential to carry this second, protective DNA mutation.
All reported homozygous carriers of the recessive p.D90A mutation have a unique homogenous phenotype, with insidious onset of an initial preparetic phase followed by hyper-reflexia in the lower extremities. Muscle paresis and wasting always first appear in the lower limbs with slowly ascending paresis to the upper limbs and bulbar muscles. The mean survival time is about 14 years, with some patients surviving more than 25 years. In contrast, the reported ALS patients heterozygous for the p.D90A allele have presented with diverse phenotypes (sites of onset, survival times) even within families, as is typical for FALS associated with heterozygous mutations in the SOD1 gene.19 22 27 29
The homozygous p.D90A patient identified in this study presented with symptoms as seen in the Scandinavian subtype and indeed carries the Scandinavian haplotype. As expected, the disease phenotype was variable in the autosomal dominant p.D90A family. The identification of new pedigrees carrying the recessive and dominant p.D90A haplotypes may add value to the ongoing search to identify the cis-acting protecting modifier on the recessive haplotype.
Although mutations were identified in three patients, the results of this study support the clinical observation that SOD1 mutations are rare in The Netherlands. A review of 18 studies from different countries demonstrated mutations in 187 out of 854 familial index cases (21.9%) and in 45 out of 1792 sporadic ALS cases (2.5%). In contrast, we found mutation in one out of 55 familial cases (1.8%) and in two out of 451 sporadic cases (0.44%). The Fisher exact test shows that this difference is significant with p=0.0004 for FALS and p=0.005 for SALS. These estimates are almost certainly subject to various statistical biases. Many of the studies were performed on selected populations with a focus on FALS and younger patients (which are more likely to carry a gene defect and to come into contact with an ALS research centre). Only one of the previous studies was performed prospectively and was designed with the specific aim to determine the frequency of SOD1 mutations. With these limitations in mind, statistical analyses revealed that the SOD1 mutation frequency in The Netherlands is lower than in the studies from European countries, North America and Japan.
A recent population-based study from Italy suggests that the frequency of SOD1 mutations in SALS is likely overestimated in these series due to referral bias.19 This study, however, did report similar figures for FALS (13.6%). It is possible that our study finds a lower mutation frequency, because the referral rate is high in The Netherlands. However, the frequency of SOD1 mutations in our referral population is also lower than in the Italian population-based study by a factor 7.60 for FALS and 1.49 for SALS, suggesting that our finding is not solely due to referral bias but indicates that the frequency of genetic risk factor(s) for ALS differs between countries and regions.
Similar observations were made in association studies in SALS. For instance, polymorphisms in the promoter region of the vascular endothelial growth factor gene (VEGF) are a susceptibility factor in Belgium,30 but not in The Netherlands.31 An SNP in the gene encoding the inositol 1,4,5-trisphosphate receptor type 2 (ITPR2) has been demonstrated to be in association with SALS in two independent Dutch populations and in Sweden, but not in Belgium.32
FALS is a genetically heterogeneous disease with known mutations in eight genes (SOD1,2 ALSin,33 VAPB,34 SETX,35 ANG,36 TARDBP,37 DCTN138 and FUS39 40), and significant linkage has been reported for eight other loci. Mutations in SOD1, ANG, DCTN1, CHMP2B and TDP-43 have also been reported in apparently SALS patients. This finding suggests that the genetics underlying SALS may also be just as heterogenic.
If SALS is indeed a genetically heterogeneous syndrome, and the frequency of these risk factors varies between ethnic populations, this could be a possible explanation why replication of genetic association studies in SALS has often proven difficult. Although a false-positive association must remain a serious consideration in association studies, it seems that population-specific genetic risk factors (like in the D90A-homozygous cases in Finno-Scandinavia) exist for ALS.
We thank the patients and their families for their participation.
Leonard H van den Berg and Peter M Andersen contributed equally to this paper
Funding This work was supported by Netherlands Organization for Scientific Research (NWO) and the Prinses Beatrix Foundation (PBF). We would also like to thank H Kersten and M Kersten, for their generous support, as well as JR van Dijk and the Adessium foundation and the VSB fonds (LHvdB), The Brain Foundation of The Netherlands (JHV), the Kempe Foundation, the Swedish Brain Research Foundation and Bertil Hallsten, the Bjorklund Foundation for ALS Research, the Swedish Brain Power Foundation and the Swedish Association for the Neurologically Disabled (PMA).
Competing interests None.
Patient consent Obtained.
Ethics approval Ethics approval was provided by the regional medical school ethics committee.
Provenance and peer review Not commissioned; externally peer reviewed.