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REVIEW

Multiple sclerosis genetics is dead Christopher H. Hawkes Neuroscience Centre, Blizard Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, 4 Newark Street, London E1 2AT, UK Received 16 September 2012; received in revised form 17 December 2012; accepted 19 December 2012

KEYWORDS

Abstract

Multiple sclerosis; Genome wide association studies; Twin studies; Epigenetics; Epistasis; Vitamin D

This review addresses several areas of contention related to the genetic theory for multiple sclerosis (MS). It is argued (a) that the concept of MS as a ‘complex disease’ has little value, (b) just because a disorder is found in multiple families, it is not necessarily genetically based, (c) although twin studies are claimed to show that MS is ‘30% genetically based’ this concept derives from widely varying data, (d) although genome-wide association studies (GWAS) suggest the presence of several MS related genes this has yet to be proven, (e) monozygotic twins discordant for MS should have a different genetic sequence if the disorder has a genetic basis but data so far suggest this may not be correct and (f) epigenetics or epistasis are contentious topics and may not provide the answer. It is concluded that the role of genetics in MS etiology may be overstated and that attention should now be devoted to environmental causes. & 2012 Elsevier B.V. All rights reserved.

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.1. Multiple sclerosis is a complex disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.2. If a disorder is found in multiple families then it must be genetically based . . . . . . . . . . . . . . . . . . . . . 2 1.3. Twin studies show that MS is 30% genetic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.4. Genome-wide association studies (GWAS) suggest the presence of several MS related genes. . . . . . . . . . . . 4 1.5. Monozygotic twins discordant for MS should have a different genetic sequence . . . . . . . . . . . . . . . . . . . 4 1.6. Epigenetics or epistasis may provide the answer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2. Epistasis and MS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2.1. What is wrong with the environmental theory for MS? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 3. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

E-mail address: [email protected] 2211-0348/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msard.2012.12.006 Please cite this article as: Hawkes CH. Multiple sclerosis genetics is dead. Multiple Sclerosis and Related Disorders (2013), http://dx.doi.o rg/10.1016/j.msard.2012.12.006

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1.

Introduction

The words of the title are not mine actually; they were spoken informally by an eminent MS geneticist, much to the amazement of those within earshot. After some 30 years of intensive multi-national genetics research, what prompted such a defeatist remark? This paper reviews some facts and fallacies related to MS and why now an attack on environmental issues has become of prime importance.

1.1.

environment has substantial effect. It cannot be termed a ‘simple disease’; they probably do not exist. Multiple factors apply to every known disease in the animal kingdom. Conversely many disorders known to be infectious have susceptibility genes as described below. The terms ‘complex’ or ‘simple’ disease are now meaningless in scientific literature and should not be used.

1.2. If a disorder is found in multiple families then it must be genetically based

Multiple sclerosis is a complex disease

Many articles begin with this statement. Here is a recent sweeping remark: ‘complex genetic disorders are caused by polygenic inheritance of common variations, which are present within the normal population’ (Rose and Bell, 2012). It is not tenable. Others define complex disease (sometimes now called ‘common complex disease’) as a multifactorial condition due to a mixture of genetic and environmental factors. Single gene disorders are meant to be different. Not so; for example, the expression of Huntington’s Disease (HD) which is claimed to belong to this category is not entirely explained by a single gene nor the length of its trinucleotide repeats. As Wexler and colleagues conclude: ‘approximately 40% of the variance remaining in onset age is attributable to genes other than the HD gene and 60% is environmental’ (Wexler et al., 2004). This is a recognized single gene disorder yet the

Consider Fig. 1, without consulting the legend and then decide what patterns of disease are characterized. Many will consider they represent autosomal dominant conditions with partial penetrance in some pedigrees. In fact, the top three are families with tuberculosis, the middle three have leprosy and the lower three visceral leishmaniasis (Blackwell, 1998). Just because a disease is passed on with regularity from one generation to the next does not make it necessarily a genetic disorder. In one sense the pedigrees illustrated are compatible with some degree of heritability i.e. genetic susceptibility to infection. Alternatively those affected could have suffered unusually high exposure to infection and their genetic makeup would be almost irrelevant. In several disorders there is indeed evidence of mutations or linkage to genes that confer contagious disease susceptibility, for example, poliomyelitis (chromosome 19q13); measles (1q32);

Fig. 1 Pedigrees from three infectious diseases: top three families (P1) have tuberculosis; middle three families (P2) have leprosy and bottom three (P3) have visceral leishmaniasis. All families show a pattern compatible with autosomal dominant inheritance although some look atypical (Blackwell, 1998). Please cite this article as: Hawkes CH. Multiple sclerosis genetics is dead. Multiple Sclerosis and Related Disorders (2013), http://dx.doi.o rg/10.1016/j.msard.2012.12.006

Multiple sclerosis genetics is dead

3 important genetic component there should be significantly more concordant monozygotic twins (MZ) than dizygotic (DZ) thus creating a high MZ: DZ concordance ratio. If the condition is mainly environmentally based, the MZ: DZ concordance ratio should be approximately equal as the sibs’ different genetic make-up has little influence. Initial classic MS twin studies found a higher concordance ratio in MZ twins, around three times greater than DZ and this has led to the frequent statement that ‘MS is 30% genetic’. Rarely does one read that MS is ‘70% environmental’ although this would be equally simplistic. Unfortunately, there are multiple ascertainment problems with these classic-designed twin studies that explore a relatively rare disease such as MS. When the six largest population-based classical studies were re-appraised, an unclear picture emerged (Hawkes and Macgregor, 2009). The index of heritability (h2, which is the proportion of total phenotypic variance explained by genetic variance) was calculated; it was found to vary extensively (from 25% to 75%) and confidence intervals were extremely wide (Fig. 3). In essence, the classic twin approach is not able to make an accurate assessment of MS heritability, simply because there

AIDS (multiple loci); leprosy (10p13); tuberculosis (multiple loci). Conversely resistance to malaria is related to sickle hemoglobin, glucose-6-phosphate dehydrogenase deficiency, absence of the Duffy blood group and linkage to 1q32.2, 4q31.21 and many others. (Source: Online Mendelian Inheritance in Man). There is also evidence of resistance to HTLV-1 infection in Japanese who have the HLA-Cwn08 haplotype (Jeffery et al., 2000). Similar multicase families have been described in MS see Fig. 2 (Dyment et al., 2002) but rarely is it suggested they might demonstrate transmission of susceptibility to infection or even common environmental exposure as in Fig. 1. Thus, multiple instances of the same disease in the same family is not necessarily a primary genetic disorder; it could equally well be an infection or other environmental exposure.

1.3.

Twin studies show that MS is 30% genetic

In theory, the genetic contribution to MS may be dissected out by study of twin pairs. In general, if the disorder has an

Fig. 2 A multigenerational family with multiple sclerosis. Diamond = offspring, gender not specified; open circle = female, not affected with multiple sclerosis; open square = male, not affected with multiple sclerosis; filled circle = female, affected with multiple sclerosis; filled square = male, affected with multiple sclerosis; partially filled square or circle = possible multiple sclerosis; diagonal line = deceased; arrow = proband. Reproduced with permission from (Dyment et al., 2002).

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Fig. 3 To show the index of heritability (h2) on the vertical axis, with 95% confidence intervals. This is derived from six ‘populationbased’ MS twin studies. The greater the genetic contribution, the higher the index. It is clear that h2 varies widely from one country to another thus limiting meaningful interpretation (Hawkes and Macgregor, 2009) Please cite this article as: Hawkes CH. Multiple sclerosis genetics is dead. Multiple Sclerosis and Related Disorders (2013), http://dx.doi.o rg/10.1016/j.msard.2012.12.006

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are just not enough twin pairs with MS even worldwide to achieve a meaningful result. Such studies have not advanced our knowledge of MS etiology at all; they have only caused confusion. Conversely, between-pair twin studies can be extremely valuable as discussed below.

1.4. Genome-wide association studies (GWAS) suggest the presence of several MS related genes Numerous genetic variants such as hair or eye color, height, blood groups, etc. distinguish healthy people. These are known broadly as polymorphisms and when they affect a single nucleotide they are termed single nucleotide polymorphisms (SNPs). Many non-geneticists are unclear about what is a mutation. A common definition is the presence of an SNP that occurs in less than 1% of the population. In MS, numerous polymorphisms have been demonstrated and they clearly show a central role for the immune system. Skeptics might argue that we knew that long before any GWAS, and in any event, immunity is central to defense against the environment e.g. infection, toxins, etc. There are now over 50 non-HLA genetic risk factors associated with MS (Gourraud et al., 2012) and these authors state that there is still ‘a large proportion of the disease heritability still unaccounted for’. Despite this wishful prophesy, it is well known that over the past 30 years genetic studies have identified at least 10 single gene mutations in various forms of parkinsonism. Implementing exactly the same technique over a similar timeframe has revealed (sadly) not one mutation for MS. Thus, the likelihood of a single gene mutation for MS is remote. Furthermore it is probable that HLA polymorphisms are major genetic risk factors not just for MS, but for acknowledged infections such as leprosy (Zhang et al., 2009), visceral leishmaniasis (LeishGEN Consortium; WTCCC2, 2013) as well as putative autoimmune disease such as type 1 diabetes (Noble and Erlich, 2012) and rheumatoid arthritis (Yang et al., 2012). All this suggests there may be a major environmental factor for MS that just has not been identified. It also implies that so-called autoimmune disease might be an infection in susceptible people and perhaps a situation no different from, for example, the common cold where there is frequent susceptibility and rare resistance.

1.5. Monozygotic twins discordant for MS should have a different genetic sequence In a landmark study of twins published in Nature, genome sequences were examined in one MS-discordant MZ twin pair, and messenger RNA transcriptome and epigenome sequences of CD41 lymphocytes from three MS-discordant, MZ pairs (Baranzini et al., 2010). Clearly, if significant genetic differences were observed between MS twins, then this might reveal an ‘MS gene(s)’—if such exists. To quote from the paper which is heavy going for non-geneticists: ‘no differences were detected between co-twins among 3.6 million SNPs or 0.2 million insertion–deletion polymorphisms. Nor were any reproducible differences observed between siblings of the three twin pairs in HLA haplotypes, confirmed MS-susceptibility SNPs, copy number variations, mRNA and genomic SNP and insertion–deletion genotypes, or the expression of 19,000 genes in CD41 T cells’ It continues:

‘Only 2–176 differences in the methylation of 2 million CpG dinucleotides (CHH: sites where cytosine lies next to guanine) were detected between siblings of the three twin pairs, in contrast to 800 methylation differences between T cells of unrelated individuals and several thousand differences between tissues or between normal and cancerous tissues’. In other words, these MZ twins, all of whom carried known susceptibility genes were virtually identical and there was no evidence of any significant difference in their genes after fertilization i.e. no epigenetic effects (see below). So how do you interpret this? It could be a fluke, unrepresentative occurrence given that only four twin pairs were studied and we already know that the concordance in MS–MZ twins is not 100%. It could be that the discordance was simple bad luck; the affected twin was in the wrong place at the wrong time—although MZ twins share the same environment very closely throughout life, particularly until school leaving age. There is also the assumption that the discordant MZ twins will remain discordant. The mean lag time can vary from 7 to 8 years (Islam et al., 2006; Hansen et al., 2005). However all these explanations require an environmental influence upon twins who appear to be genetically identical. Assuming this highly detailed genomic analysis is correct and representative (and others may wish to confirm) then the most plausible explanation is that susceptibility genes have relatively weak effect and it is mainly environmental factors that cause MS.

1.6. Epigenetics or epistasis may provide the answer The word ‘epigenetics’ has several meanings and many clinicians do not have a clear understanding of the term. One definition is ‘the study of heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence’ (Wikipedia, 2012). Known mechanisms include DNA methylation, histone modification and changes in microRNAs (small noncoding RNAs that regulate gene expression) all of which may alter gene expression without changing the underlying DNA sequence. Usually, two copies of a gene are inherited, one from each parent, but if one copy is epigenetically inactivated and the remaining working gene is mutated, then significant disability may result. There is a good copy of the required gene but it cannot act because it has been turned off by an epigenetic mechanism. This is the ‘trans-generational mechanism of epigenetics’. Accepted examples of this are the Angleman and Praeder–Willi syndromes. More recently the definition of epigenetics has expanded to include potential effects of environmental agents on the expression of DNA but many authors do not make it clear which definition they are applying and the term is often used to convey almost anything that alters DNA expression. If epigenetic effects are important for MS then the discordant MZ twin study described above (Baranzini et al., 2010) should have demonstrated some differences between discordant twin pairs but they found virtually none in 2 million dinucleotides. A problem with this study is that it examined methylation status of lymphocytes rather than neurons, with the unproven implication that abnormalities in blood cells would be reflected in neural

Please cite this article as: Hawkes CH. Multiple sclerosis genetics is dead. Multiple Sclerosis and Related Disorders (2013), http://dx.doi.o rg/10.1016/j.msard.2012.12.006

Multiple sclerosis genetics is dead tissue. Epigenetic change, if relevant at all for MS may be cell specific. A further difficulty with the epigenetic theory for MS is that presumably there would be a specific event in utero or in early childhood/adult life that causes epigenetic change specific for MS i.e. secondary to environmental exposure. What agent in early life could do this? Some would maintain that vitamin D deficiency (whether related to diet or ultraviolet light exposure) is sufficient to cause epigenetic change resulting in MS and cite rare instances of familial Vitamin D resistant rickets associated with MS (Torkildsen et al., 2008; Ramagopalan et al., 2011). However, non-familial rickets is common where MS is infrequent (Dawodu et al., 1998; Guzel et al., 2001; Wondale et al., 2005) and although most Westerners with MS have low levels of vitamin D they rarely suffer rickets or osteomalacia. Defenders of the vitamin D theory would maintain that vitamin D receptor binding (VDR) is more likely to occur within suspected MS regions when compared to the rest of the genome and that more than 60% of MS regions are bound by the VDR. (Disanto et al., 2012). Emerging evidence suggests that vitamin D deficiency increases the risk of subsequent MS (along with smoking, latitude and EBV exposure). Whether this effect is mediated through an immune or epigenetic process is far from clear and such deficiency may just increase susceptibility to ‘autoimmune’ disease in general (Baranzini and Nickles, 2012).

2.

Epistasis and MS

If a mutation cannot be found, then apart from search for epigenetic effects, linkage or association studies, then the next hypothesis to force MS into the category of a primary genetic disorder revolves around gene-gene interaction or epistasis. For example, data from the massive MS Canadian database showed epistatic interactions between HLA-DRB1 haplotypes. The presence of HLA-DRB1n08 haplotype alone increased the risk of MS modestly but when it was present with HLA-DRB1n15 on the other parental haplotype, it doubled the risk associated with a single copy of HLADRB1n15 (Sadovnick, 2012). Other haplotypes conferred resistance to MS. A similar argument could be made for susceptibility and resistance to infection. This finding does not necessarily indicate that MS is a genetic disorder, only that it creates susceptibility to whatever causes the disease. Thus, the evidence so far, gives slender support to either epigenetic or epistatic mechanism; they are hypotheses awaiting further substantiation.

2.1. What is wrong with the environmental theory for MS? In a nutshell, nothing. Basically it has been overshadowed by the search for MS genes which some would say has failed to deliver. It is not planned to review all proposed environmental associations for MS but those generally agreed (which are admittedly not that startling) comprise latitude, gender, cigarette smoking, vitamin D deficiency and exposure to the Epstein Barr virus (Ascherio and Munger, 2007a, 2007b). It is very difficult to explain the widely accepted migration studies of MS, whether from high to low-risk regions or vice versa, by any mechanism other than

5 environmental, given that as far as known, moving from one country to another does not change an individual’s DNA. It could be argued that there might be some epigenetic effect as a result of migration. That remains a speculation, some would say a wild idea but a tantalizing area for neurogenetics research.

3.

Summary

We must stop asserting that ‘MS is a complex disease’ and that ‘MS is 30% genetic’. Just because the same disease is detected in several members of the same family does not necessarily mean the disorder is primarily genetically based. GWAS in MS have excluded significant DNA mutations although some would argue that there could still be rare mutations in several genes in multiple unrelated individuals. If genetics has a significant role in MS then that might relate to epigenetic effects or gene interactions. Despite this, epigenetics has far from universal support from international figures. One renowned neurogeneticist recently stated informally that ‘epigenetics is mostly nonsense’. This remark and that of this article title indicate rumblings of uncertainty emerging within the MS genetic community. Perhaps we are making the situation more complex than it really is; there may be a basic environmental agent or process that has been overlooked. So, is MS genetics dead? Not completely, but it is struggling to keep alive.

Conflict of interest The author had no conflict of interest and no relevant source of funding.

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Please cite this article as: Hawkes CH. Multiple sclerosis genetics is dead. Multiple Sclerosis and Related Disorders (2013), http://dx.doi.o rg/10.1016/j.msard.2012.12.006

Multiple sclerosis genetics is dead.

This review addresses several areas of contention related to the genetic theory for multiple sclerosis (MS). It is argued (a) that the concept of MS a...
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