Abstract
Evaluation of: Baranzini SE, Mudge J, van Velkinburgh JC et al. Genome, epigenome and RNA sequences of monozygotic twins discordant for multiple sclerosis. Nature 464, 1351–1356 (2010).
Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS. Genetically identical (monozygotic) twins have a concordance rate for MS of approximately 30%, lending support to the notion that the disease has a complex etiology, developing as a result of genetic and environmental factors and their interactions. However, recent studies have highlighted the fact that monozygotic twins might not actually be genetically identical. In an effort to see if this can explain MS twin discordance, Baranzini and colleagues sequenced the genome from a pair of monozygotic twins discordant for MS, and also examined DNA methylation and gene expression across the genome in this twin pair and an additional two more twin pairs. No consistent differences in DNA sequence, DNA methylation or gene expression were found. Here we put these findings into context and discuss their significance.
Classical twin research provides overwhelming evidence that both genetic and environmental factors are important in the etiology of multiple sclerosis (MS) Citation[1]. Disease discordance in monozygotic (MZ) or identical twins is often attributed to disparity in environmental exposures based on the assumption that there are no genetic or epigenetic differences between twins Citation[2]. However, several lines of research provide compelling evidence that such differences exist. Mosaicism denotes the presence of two populations of cells with different genotypes that have developed from a single fertilized egg as a result of postzygotic alterations of the genome Citation[3]. If the event leading to mosaicism occurs early during development, it is possible that both somatic and germline cells will become mosaic. Conversely, if the triggering event occurs later in life, it may only affect a certain somatic cell population Citation[3]. Differences in the genetic sequence of MZ twins can be thought of as an extreme form of mosaicism, and differences in the DNA sequence of MZ twins have indeed been reported Citation[4].
Epigenetics refers to DNA and chromatin modifications that regulate genomic functions, and includes DNA methylation of C–G dinucleotides and histone modifications Citation[5]. Nearly all cells in the human body have the same genotype, but cells can have hugely different phenotypes and this, to some extent, arises from epigenetics Citation[5]. Recent studies have highlighted the fact that MZ twins differ in terms of DNA methylation across the genome Citation[6,7]. It is plausible that mosaicism or epigenetic differences can explain twin phenotypic discordance. Indeed, for neurofibromatosis type I and the rare epigenetic disorder, Beckwith–Wiedemann syndrome, MZ twin discordance has been shown to be due to differences in DNA sequence and methylation, respectively Citation[8,9]. The availability of next-generation sequencing technologies has the potential to revolutionize genomic research. We are now able to study genetic variation on a genome-wide scale and characterize gene regulatory processes at unprecedented resolution Citation[10]. Using these new technologies, Baranzini and colleagues have attempted to determine whether twin discordance in MS can be explained by genetic, epigenetic or transcriptomic differences Citation[11]. This is the first study of its kind, and the authors should be commended for their efforts.
Methods & results
Baranzini et al. performed whole-genome sequencing of genomic DNA from CD4+ T cells taken from a pair of MZ twins discordant for MS Citation[11]. Genome-wide mRNA sequencing, reduced representation bisulfite sequencing (RRBS) of DNA and genome-wide single nucleotide polymorphism (SNP) typing by arrays was also performed on CD4+ cells from this twin pair and from another two MZ twin pairs discordant for MS. Approximately 3.6 million SNPs and 0.2 million insertions and deletions (indels) were detected in the genome of the twin pair that was sequenced. A small fraction of SNP and indel differences were found between twin pairs by each of the methods used (genomic DNA sequencing, mRNA sequencing or by SNP arrays) but no differences inferred by one approach were recapitulated by a second method, suggesting that this variation was due to sequencing/genotyping errors.
More than 18,000 genes were expressed, as assessed by mRNA sequencing, but the transcript abundance was not significantly different between twins. Thus, robust gene-expression differences were not observed between MS-affected and unaffected twins in CD4+ lymphocytes. Finally, methylation at greater than 1.5 million C–G dinucleotides was assessed in the twin pairs by RRBS, and the twins differed at 176 sites at the most. However, these differences were not consistent between twin pairs. In summary, Baranzini and colleagues did not find any convincing evidence for genetic, epigenetic or transcriptome differences that could explain MS discordance in MZ twins.
Discussion & significance
While this study would lend support to a predominant role of the environment in determining twin concordance for MS, there are a number of factors with regard to the study design that are worthy of consideration. The fact that Baranzini et al. found no reproducible differences in SNPs or indels between twins is surprising. It is suggested that somatic mutations occur at a rate of 8.4 × 10-9 to 4.6 × 10-10 per nucleotide per generation Citation[11]. Given the enormous number of cell divisions that would have taken place between the twinning event and sampling of the CD4+ cells that were analyzed, one would have expected at least a handful of differences. The analysis of next-generation sequencing data is challenging, particularly because most sequencing platforms provide short reads, which are difficult to align and assemble Citation[10]. It should be remembered that there were regions of low sequencing coverage or repetitive genomic sequences that were not fully addressed. Furthermore, the power of the Baranzini study to detect somatic mutations was not optimal. Previous studies have calculated that in order to identify true somatic mutations, a sequencing depth of 30-fold offers high sensitivity, ruling out both sequencing errors and germline mutations Citation[12]. This study falls short, with the average depths of coverage being approximately 22-fold for each twin. It is also important to note that these results only represent CD4+ T cells. Although CD4+ lymphocytes are relevant to MS, this is by no means the only cell type known to be involved in disease pathogenesis Citation[13,14] and, thus, further investigation is needed in other cell types. Therefore, this study cannot conclusively rule out the possible involvement of somatic changes in twins discordant for MS.
The DNA methylation analysis was also not perfect. RRBS (where genomic DNA is cleaved by a restriction enzyme to sequences enriched with CpG dinucleotides and resulting fragments bisulfite converted and sequenced) offers poor coverage of the genome (only 3% of the human reference sequence) compared with bisulfite conversion or affinity-based enrichment of the methylated portion of the genome and subsequent sequencing (whole-genome bis-seq or MeDIP-seq) Citation[15]. Therefore, much of the genome remains to be interrogated for differences in DNA methylation. Furthermore, DNA methylation is not the entire field of epigenetics. For example, a recently discovered epigenetic change is hydroxymethylation of DNA Citation[16]. Thus, other epigenetic changes may contribute to twin discordance. These caveats aside, we were surprised to find that Baranzini et al. only examined differences in methylation of 20% or less in one twin, compared with 80% or more in the other or vice versa, given that previous studies have demonstrated the existence of smaller differences between MZ twins Citation[8] and the importance of such differences to autoimmune disease Citation[17] and autism Citation[18]. This approach would miss perhaps the most common type of epigenetic mutation, namely loss of imprinting Citation[19].
Baranzini and coworkers also found an absence of significant mRNA expression differences between discordant twins. However, important regions, such as the expression of T-cell receptors, were not examined. Furthermore, unlike the hard-wired genome, expression and methylation patterns are dynamic in nature and influenced by the environment, and thus key causative differences may only have been present before disease onset. Finally, there is also the assumption that the discordant twins will remain so. There is a well-known delay between diagnosis of concordant twin pairs Citation[20]. In this case, one of the twin pairs was very young (19 years old), and thus it remains to be seen whether these twin pairs later become concordant. Even if the twin pairs do remain discordant for a clinical diagnosis of MS – the apparently unaffected twin may have ‘subclinical’ MS – unaffected MS twins have been shown to have demyelinating pathology on MRI Citation[21] and immunological changes Citation[22] similar to their affected sibling, and thus no genetic differences may be expected.
Expert commentary & five-year view
In conclusion, the study provides support for a major role for the environment in determining concordance for MS in MZ twin pairs. This is in accordance with epidemiological data highlighting the fact that twin concordance is dependent on latitude of birth/residence Citation[20]. This study also highlights the very exciting potential applications of sequencing technology. Next-generation sequencing platforms allow us to survey multiple levels of natural variation at unprecedented resolution and depth. As sequencing costs continue to decrease, and both laboratory and computational protocols improve, we will see ever increasing use of this technology, enabling us to focus on a large number of outstanding questions that previously could not be addressed effectively. However, in this instance, the experimental design was not equipped to comprehensively test any of the primary aims. As a consequence, Baranzini et al. cannot conclusively exclude possible contributions of genetic, epigenetic or transcriptomic differences to disease discordance in MS MZ twins, and further studies are warranted. Studies targeting larger cohorts of MZ twins discordant for MS should ideally be performed to address the limitations of the Baranzini study.
Key issues
• Multiple sclerosis is a complex neurological disease, with a monozygotic twin concordance rate of approximately 30%.
• Recent studies have highlighted the fact that monozygotic twins might not actually be genetically or epigenetically identical.
• Baranzini and colleagues sequenced the genome from a pair of monozygotic twins discordant for multiple sclerosis, and also examined DNA methylation and gene expression across the genome.
• No consistent difference in DNA sequence, DNA methylation or gene expression was found between affected and unaffected twins.
• This result suggests that the environment is the key driver of twin discordance.
• However, a number of limitations of the study mean that DNA sequence, DNA methylation or gene expression differences that influence twin discordance cannot be ruled out.
Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
References
- Handel AE, Giovannoni G, Ebers GC, Ramagopalan SV. Environmental factors and their timing in adult-onset multiple sclerosis. Nat. Rev. Neurol.6, 156–166 (2010).
- Ramagopalan SV, Dyment DA, Ebers GC. Genetic epidemiology: the use of old and new tools for multiple sclerosis. Trends Neurosci.31, 645–652 (2008).
- Youssoufian H, Pyeritz RE. Mechanisms and consequences of somatic mosaicism in humans. Nat. Rev. Genet.3, 748–758 (2002).
- Bruder CE, Piotrowski A, Gijsbers AA et al. Phenotypically concordant and discordant monozygotic twins display different DNA copy-number-variation profiles. Am. J. Hum. Genet.82, 763–771 (2008).
- Feinberg AP. Epigenetics at the epicenter of modern medicine. JAMA299, 1345–1350 (2008).
- Fraga MF, Ballestar E, Paz MF et al. Epigenetic differences arise during the lifetime of monozygotic twins. Proc. Natl Acad. Sci. USA102, 10604–10609 (2005).
- Kaminsky ZA, Tang T, Wang SC et al. DNA methylation profiles in monozygotic and dizygotic twins. Nat. Genet.41, 240–245 (2009).
- Kaplan L, Foster R, Shen Y et al. Monozygotic twins discordant for neurofibromatosis 1. Am. J. Med. Genet. A152A, 601–606 (2010).
- Weksberg R, Shuman C, Caluseriu O et al. Discordant KCNQ1OT1 imprinting in sets of monozygotic twins discordant for Beckwith–Wiedemann syndrome. Hum. Mol. Genet.11, 1317–1325 (2002).
- Gilad Y, Pritchard JK, Thornton K. Characterizing natural variation using next-generation sequencing technologies. Trends Genet.25, 463–471 (2009).
- Baranzini SE, Mudge J, van Velkinburgh JC et al. Genome, epigenome and RNA sequences of monozygotic twins discordant for multiple sclerosis. Nature464, 1351–1356 (2010).
- Bentley DR, Balasubramanian S, Swerdlow HP et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature456, 53–59 (2008).
- De Jager PL, Rossin E, Pyne S et al. Cytometric profiling in multiple sclerosis uncovers patient population structure and a reduction of CD8low cells. Brain131, 1701–1711 (2008).
- Friese MA, Fugger L. Pathogenic CD8+ T cells in multiple sclerosis. Ann. Neurol.66, 132–141 (2009).
- Laird PW. Principles and challenges of genome-wide DNA methylation analysis. Nat. Rev. Genet.11, 191–203 (2010).
- Kriaucionis S, Heintz N. The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science324, 929–930 (2009).
- Javierre BM, Fernandez AF, Richter J et al. Changes in the pattern of DNA methylation associate with twin discordance in systemic lupus erythematosus. Genome Res.20, 170–179 (2010).
- Nguyen A, Rauch TA, Pfeifer GP, Hu VW. Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contributions to autism spectrum disorders and a novel autism candidate gene, RORA, whose protein product is reduced in autistic brain. FASEB J. DOI: 10.1096/fj.10-154484 (2010) (Epub ahead of print).
- Kaneda A, Feinberg AP. Loss of imprinting of IGF2: a common epigenetic modifier of intestinal tumor risk. Cancer Res.65, 11236–11240 (2005).
- Islam T, Gauderman WJ, Cozen W et al. Differential twin concordance for multiple sclerosis by latitude of birthplace. Ann. Neurol.60, 56–64 (2006).
- Okuda DT. Unanticipated demyelinating pathology of the CNS. Nat. Rev. Neurol.5, 591–597 (2009).
- Somma P, Ristori G, Battistini L et al. Characterization of CD8+ T cell repertoire in identical twins discordant and concordant for multiple sclerosis. J. Leukoc. Biol.81, 696–710 (2007).