The chromosome 9 ALS and FTD locus is probably derived from a single founder

doi:10.1016/j.neurobiolaging.2011.08.005

Neurobiol Aging. Author manuscript; available in PMC 2013 January 1.

Published in final edited form as:

Neurobiol Aging. 2012 January; 33(1): 209.e3–209.e8.

Published online 2011 September 16. doi: 10.1016/j.neurobiolaging.2011.08.005

PMCID: PMC3312749

NIHMSID: NIHMS364198

The chromosome 9 ALS and FTD locus is probably derived from a single founder

Kin Mok,¹ Bryan J. Traynor,² Jennifer Schymick,² Pentti J. Tienari,³ Hannu Laaksovirta,^2,³ Terhi Peuralinna,³ Liisa Myllykangas,⁴ Adriano Chiò,⁵ Aleksey Shatunov,⁶ Bradley F. Boeve,⁷ Adam L. Boxer,⁸ Mariely DeJesus-Hernandez,⁹ Ian R. Mackenzie,¹⁰ Adrian Waite,¹¹ Nigel Williams,¹¹ Huw R. Morris,¹¹ Javier Simón-Sánchez,¹² John C. van Swieten,^12,¹³ Peter Heutink,¹² Gabriella Restagno,¹⁴ Gabriele Mora,¹⁵ Karen E. Morrison,¹⁶ Pamela J. Shaw,¹⁷ Pamela Sara Rollinson,¹⁸ Ammar Al-Chalabi,¹¹ Rosa Rademakers,⁹ Stuart Pickering-Brown,¹⁸ Richard W. Orrell,¹ Michael A. Nalls,¹⁹ and John Hardy¹⁺

¹Reta Lila Weston Research Laboratories, Departments of Molecular Neuroscience and of Clinical Neuroscience, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK

²Molecular Genetics Section and Neuromuscular Diseases Research Group, Laboratory of Neurogenetics, NIA, NIH, Bethesda, MD 20892, USA

³Helsinki University Central Hospital, Department of Neurology, Molecular Neurology Research Program, Biomedicum, University of Helsinki, Haartmaninkatu 8, FIN-00290, Helsinki, Finland

⁴Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, and Folkhalsan Institute of Genetics, POB 21, 00014 Helsinki, Finland

⁵Department of Neuroscience, University of Turin, and Azienda Ospedaliera Universitaria San Giovanni Battista, Turin, Italy

⁶MRC Centre for Neurodegeneration Research, King's College London, Institute of Psychiatry, London SE5 8AF, UK

⁷Department of Neurology, Mayo Clinic, Rochester, MN, USA

⁸Memory and Aging Center, Department of Neurology, University of California, San Francisco, San Francisco, CA, USA

⁹Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224, USA

¹⁰Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada

¹¹MRC Centre for Neuropsychiatric Genetics and Genomics, Cardiff University School of Medicine, Cardiff, CF14 4XN, UK

¹²Department of Clinical Genetics, Section of Medical Genomics, VU University Medical Centre, Amsterdam, The Netherlands

¹³Department of Neurology, Erasmus Medical Center, Rotterdam, The Netherlands

¹⁴Molecular Genetics Laboratory, Azienda Ospedaliera OIRM-Sant’Anna, Turin, Italy

¹⁵Fondazione Salvatore Mangeri, IRCCS Scientific Institute of Milan, Milan, Italy

¹⁶School of Clinical and Experimental Medicine, University of Birmingham, Birmingham B15 2TT and Queen Elizabeth Hospital, University Hospitals Birmingham NHS Foundation Trust, Birmingham B15 2TH, UK

¹⁷The Sheffield Institute for Translational Neuroscience (SITraN, Department of Neuroscience, University of Sheffield, 385A Glossop Road, S10 2HQ, UK

¹⁸Neurodegeneration and Mental Health Research Group, Faculty of Human and Medical Sciences, University of Manchester, AV Hill Building, Oxford Road, Manchester, M13 9PT, UK

¹⁹Molecular Genetics Section, Laboratory of Neurogenetics, NIA, NIH, Bethesda, MD20892, USA

⁺Correspondence to ; Email: j.hardy/at/ion.ucl.ac.uk

The publisher's final edited version of this article is available at Neurobiol Aging

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Abstract

We and others have recently reported an association between ALS and single nucleotide polymorphisms on chromosome 9p21 in several populations. Here we show that the associated haplotype is the same in all populations and that several families previously shown to have genetic linkage to this region also share this haplotype. The most parsimonious explanation of these data is that there is a single founder for this form of disease.

Keywords: Genetics, amyotrophic lateral sclerosis, frontotemporal dementia, Finland

Introduction

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons characterized by rapidly progressive weakness and ultimately death from respiratory failure typically within three years of symptom onset. Understanding the genetic etiology of the disease has been a focus for the ALS research community, as each new gene provides fundamental insights into the pathogenesis of motor neuron degeneration, as well as accelerating disease modeling and the design and testing of targeted therapeutics.

Using a genome-wide association (GWAs) approach, we recently reported that a locus on chromosome 9p21 accounted for >40% of familial ALS and nearly one fourth of all ALS cases in a sample of 405 Finnish patients (Laaksovirta et al.2010). This association signal had previously been reported by van Es and colleagues as showing association with ALS (2009) and a meta-analysis amongst many studies showed that this was indeed the major signal for this disease (Shatunov et al. 2010). Similarly, a recent GWAs for Frontotemporal Dementia (FTD) with TDP-43 pathology had also identified this locus (van Deerlin et al. 2010)

Linkage analysis of kindreds affected with multiple cases of ALS, FTD and FTD-ALS with type 2 TDP-43 pathology had suggested there was an important locus for the disease on chromosome 9p (Vance et al. 2006, Morita et al. 2006, Boxer et al. 2010, Pearson et al. 2011) but it had not been clear whether the linkage and association signals related to a single locus or whether the different studies were reporting the same alleles at that locus.

The analysis in the Finnish population had narrowed the association signal to a 232kb block of linkage disequilibrium, and allowed the identification of a haplotype that increased risk of disease by over twenty-fold. Despite considerable efforts in our laboratories and others the underlying causative variant and deleterious mutation has not yet been identified.

Here, we examine the prevalence of the Finnish risk haplotype in other European populations to determine its geographical distribution and to analyze the possibility that it represents a founder mutation. We then tested this haplotype in ALS and FTD families with evidence of linkage to this region to determine if the same haplotype is responsible for both ALS and FTD. In the 4 families for which we had access to primary genetic data, the haplotype was consistent with the Finnish one.

Methods

We analyzed GWAs data obtained for ALS patients and neurologically-normal controls in five populations in which we have access to the raw genotype data. These are the Finnish dataset (Laaksovirta et al. 2010), the Irish dataset (Cronin et al. 2008), the UK dataset (Shatunov et al. 2010), the US dataset (Schymick et al. 2007) and the Italian dataset (Chio et al. 2009). All cohorts had been genotyped using Illumina SNP arrays. Standard quality control procedures were applied to each dataset prior to combining summary statistics for meta-analysis. In brief, samples were excluded if they had call rates less than 95%, phenotype-genotype gender discordance, demonstrated cryptic relatedness (defined as pi_hat greater than 12.5%, effectively removing all first or second degree relatives) or outliers from the populations with European ancestry (defined as > 3 standard deviations away from the combined CEU/TSI (Hapmap3 release 3 2010) population mean in components vectors 1 and 2, using PLINK Multi-dimensional scaling plot). SNPs were excluded if they had a minor allele frequency (MAF) < 0.01, Hardy–Weinberg equilibrium p-value < 10⁻⁶ in controls, missing by haplotype p-value < 10⁻⁴, or evidence of non-random missingness in cases versus controls (p-value < 10⁻⁴). Meta-analyses were performed with METAL (Abecasis et al. 2007) for fixed-effect and PLINK (Purcell et al. 2007) for random-effects model. Haplotype analysis was performed using Haploview 4.2 to evaluate the possibility of population-based differences (Barrett et al. 2005). The meta-analysis of the haploytpe was performed under random effect model. Additional statistical analyses including the meta-analysis of haplotype were performed using R (version 2.11.1, R Development Core Team 2010). Subsequently, we tested families where phased genotype data generated on various SNP chips was available to establish the relationship between the 9p21 susceptibility region and the Mendelian linkage regions.

Results

We performed a meta-analysis of five ALS genome-wide association studies involving a total of 2017 cases and 3639 controls drawn from the five datasets. As expected, meta-analysis confirmed the presence of previously identified locus on chromosome 9p21 (most significantly with imputed SNP = rs2477521, p-value = 4.51×10⁻¹¹ based on fixed-effect model with heterogeneity p value of 1.5×10⁻⁴, and an overall p-value = 0.00876 based on more conservative random-effects model). Heterogeneity estimates suggested significant variation in the effect size from different populations, with the Italian population being a frequent outlier. Secondary analysis without the Italian cohort yielded a markedly more robust p-value for the same SNP (p-value for rs2477521 under the fixed effect model = 1.24×10⁻¹³; rs10967973 with p-value under the random effects model =1.55× 10⁻¹⁰). This suggests that the effect at this SNP differs markedly when comparing between populations of Northern and Southern European ancestry.

The original risk haplotype identified within the Finnish ALS population consisted of 42-SNPs stretching over a 232kb region of chromosome 9p21 (Laaksovirta et al, 2010). This block of linkage disequilibrium was shorter in the Caucasian CEU HapMap data (24-SNPs over a 140kb region), as would be expected in an outbred European population compared to the genetically homogeneous Finnish population (Shifman et al, 2001). Of these 24 SNPs, only 21 had been genotyped in all 5 populations. Furthermore, the most centromeric SNP of these 21 (rs1444533) did not show convincing association with disease in either the UK or Irish population. This SNP was therefore dropped from subsequent analysis, leaving a 20 SNP risk haplotype common to all Northern European ancestry groups in this meta-analysis. Thus, we restricted subsequent analyses to the region chr9:27467874-27579657 (NCBI36/hg18) between SNP rs1444533 and rs696826.

This 20 SNP haplotype was associated with disease in Finland, was less significantly associated in the UK and US populations (Table 1, ,3);3); this haplotype had only a trend towards association in the Irish population (p = 0.17) and showed no evidence of association at all in the Italian population. The odds ratio meta-analysis showed a combined odd ratio 1.44 (95% CI 1.01–2.06) based on random effects model. The odd ratio was 1.64 (95% CI 1.16–2.32) if Italian population was excluded. Heterogeneity X² distribution P value also dropped from 0.0018 in all cohorts to 10 fold less (0.019) if Italian was excluded (Figure 1). The 20 SNP haplotype is consistent with the association recently reported for both FTD (Van Deerlin et al. 2010, Rollinson et al. 2011: see Table 2) and for ALS in a Dutch study although the incompleteness of the published data in these studies precludes a formal comparison.

Table 1

20 SNP haplotype frequencies in cases and controls

Table 3

Metaanalysis of the 20-SNPs Haplotypes using Odd ratio

Fig 1

Forest Plot of the haplotype Odd ratio

Table 2

The 24-SNP Finnish haplotype compared with 20 SNP haplotype and data from other populations, families and publications

Analysis of SNP chip data in four families with FTD or FTD-ALS (Selaar et al. 2010, Pearson et al, 2011, Boxer et al. 2011, Traynor and Hardy unpublished) in which was have generated linkage data using SNP chips (Table 2) revealed that a similar disease haplotype was found in all patients with the exception of the most distal SNP (rs2477518) in the family reported by Seelaar et al (2010). These data suggest that the same conserved chromosome 9p21 20 SNP risk haplotype underlies both ALS and FTD in multiple populations and that a proportion at least of the families showing genetic linkage to the region also share this rather short haplotypic region.

Discussion

These results are consistent with a single haplotype being associated with ALS, FTD and FTD-ALS in most of the populations studied with the strength of the association being strongest in populations from Northern Europe that exhibit some estimated degree of Scandinavian ancestry and progressively weaker as one moves south and the contribution of this ancestral background is reduced. This interpretation is also consistent with the data from Van Es and colleagues (2009) who first identified this association and showed a stronger association in a Swedish population than in the others included in their analysis (note that this analysis partially overlaps with our analysis reported here). This haplotype has the structure shown in Table 2 and extends over 140kb and three genes MOBKL2B, C9orf72 and IFNK.

The observations described above have several implications. First, if only a single founding haplotype bears the mutation this suggests that all, or at least the majority of individuals, with the disease possess the same pathogenic variant. Second, the lack of pathogenic coding mutations in the known genes within this locus suggests that the mutation(s) is of an unusual type involving something other than a simple missense or nonsense change. Possibilities would include inversions similar to the MAPT H2 haplotype, or the inclusion of cryptic exons or the exclusion of exons caused by variants distant from splice sites. Third, it seems that the same associated haplotype is found in both FTD and ALS. In this latter regard, it is interesting that, whereas a founder mutation of the MAPT gene largely explains the Manchester focus of FTD (Pickering Brown et al. 2002) the well documented Lund focus of FTD in Sweden remains unexplained. Fourth, our data is consistent with the same haplotype being responsible for the disease in families showing linkage to this region suggesting they harbor the same pathogenic mutation: certainly this is the case in those families that we have acces to. By explicitly publishing this haplotype, our data will enable those who have acess to other families toassess whether this same haplotype is present in their families. It remains unclear as to why the apparent penetrance of the haplotype appears to be so variable. It could be that this reflects ascertainment bias, or that there have been subsequent additional variants accrued onto this ancient haplotype, or it could be that there is another epistatic locus elsewhere in the genome which influences penetrance as Gijselinck and colleagues have suggested (2009)

Clearly, the identification of this locus remains a major goal of ALS and FTD research. Our data suggest that this will be a difficult task and will require complete sequencing of the locus and of all the transcripts emanating from it.

Supplementary Material

supplementary

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Acknowledgments

Funding

This work was supported in part by the Intramural Research Programs of the NIH, the National Institute on Aging (Z01-AG000949-02), and the National Institute on Neurological Disorders and Stroke. Extramural NIH grants R01AG031278, R01AG038791 supported some family assessments: NIH/NIA grant R01 AG26251 (RR) funded some analytical work. We thank the NIHR specialist Biomedical Research Centre for Mental Health at the South London and Maudsley NHS Foundation Trust (SLaM) and the Institute of Psychiatry, King's College London as well as the NIHR funded UCL/UCLH Comprehensive Biomedical Research Centre. The research leading to these results has received funding from the European Community's Health Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 259867. We thank the Motor Neurone Disease Association of Great Britain for several grants relating to this work (RWO, AAC, PJS, HM), the ALS Association, The Angel Fund, the ALS Therapy Alliance and the Wellcome Trust (PJS) for support. This work was also funded by the Reta Lila Weston Foundation, and by an MRC returning scientist (JH) and fellowship (SPB) awards, by Microsoft Research Foundation, by the ALS Association, Helsinki University Central Hospital, and the Finnish Academy. This work was also funded by Ministero della Salute, Progetti Finalizzati 2007, Fondazione Vialli e Mauro for ALS, and Federazione Italiana Giuoco Calcio. We also want to thank the Hersenstichting Nederland (http://www.hersenstichting.nl) for supporting this work. The research leading to these results has additionally received funding from the European Community's Health Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 259867.

Footnotes

conflict of interest statement

The funding organizations had no part in the study design or the writing up of this work, and the authors report no conflicts of interest.

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