C A R D I O M Y O P AT H Y

Tackling the Achilles’ Heel of Genetic Testing Hugh Watkins

CREDIT (ILLUSTRATION): V. ALTOUNIAN/SCIENCE TRANSLATIONAL MEDICINE

Assigning pathogenicity to rare genetic variants is at its hardest with the enormous titin gene, but comprehensive genomic analysis makes the task more tractable (Roberts et al., this issue).

Detection of human mutations has been made much easier by advances in sequencing technology, but determination of the pathogenicity of variants has turned out to be far harder than envisaged. In part this refects the increasing awareness of the wide diversity of disease genes underlying a given condition. But even more problematic has been the realization of the remarkably high prevalence of rare, potentially deleterious DNA variants. Now, in a large human genetic medicine study, Roberts et al. assess mutations in the gene (TTN) encoding the giant protein titin and seek to separate those that cause cardiomyopathy and those that do not (1). Deciphering of the molecular basis of dilated cardiomyopathy that results from TTN mutations and understanding the apparent low penetrance of TTN alleles should improve the utility of these commonly encountered variants in clinical management of this important disease. Te cumulative burden of even truly rare coding variants in proven disease genes (for example, even singletons among the 61,000 exomes in the recently released Exome Aggregator data) (2) is far in excess of the prevalence of relevant diseases (3). Tus, most cannot be disease causing. Past frequency thresholds for evaluating a variant have not been sufciently stringent. In the era of linkage and candidate gene analysis, absence of a variant from as few as 200 control chromosomes was considered sufcient to exclude the variant as a “neutral polymorphism.” More recently, variants with frequency of 0.1% or even 1% have been retained in fltering strategies designed to identify pathogenic variants in rare monogenic diseases. Simple inspection of disease frequency and allele frequency indicates that this cannot be right! Te ensuing confusion has many undesirable consequences that impede the clinical adoption of genetic testing. Genes with a high burden of rare variants will tend not to Radclife Department of Medicine, University of Oxford, Oxford OX3 9DU, U.K. Corresponding author. E-mail: hugh.watkins@rdm. ox.ac.uk

be adopted for clinical use. Erroneous inclusion of nonpathogenic variants in surveys of diferent diseases will generate apparent overlap that confuses true gene-disease relationships (4). (A characteristic of nonpathogenic rare polymorphisms is that they get erroneously associated with multiple, mechanistically unrelated diseases.) Importantly, inclusion of “false positive” DNA variants will cause errors in family-based genetic testing and cascade screening—the

sequential testing of frst-degree relatives of each newly ascertained case in a family. Tis problem will be particularly extreme for “incidental fndings” for which the pretest likelihood of a pathogenic mutation is lower than in afected cases and the confrmation provided by co-segregation within a family is ofen not available. Tese challenges—encountered in genetic testing in all areas of Mendelian disease—are particularly well illustrated by inherited cardiac conditions, such as cardiomyopathies and cardiac channelopathies, as these autosomal dominant disorders are common, serious, and, because they can be treated, actionable. At best, genetic testing with reliably implicated variants has transformed diagnosis and family management. Genetic analysis for cascade testing is now a Class I indication (which means that a test is recommended and should be

Sarcomere I-band

Z-disc

Titin N2B isoform

Titin N2BA isoform

A-band

I-band

Thin flament (actin)

Proximal Ig domains

N2-Bus

N2-Bus

Middle Ig domains

Truncation mutations in elastic I-band region, typically in low-usage exons, have low pathogenicity; common in controls.

PEVK

PEVK

Distal Ig domains

Distal Ig domains

Truncation mutations in A-band region, which afect both N2B and N2BA isoforms, have relatively high pathogenicity; common in DCM.

Thick flament (myosin)

M-band

TK

TK

Mutations in M-band region underrepresented in DCM; instead associated with recessive skeletal myopathies.

Fig. 1. Anatomy of a giant. The giant myofilament protein titin spans half the sarcomere of striated muscle, from the Z-disc that anchors the actin thin filament and many associated proteins to the M-band signaling complex of the myosin-containing thick filament. Two isoforms predominate in the heart, N2B (stiff ) and N2BA (compliant), each with complex alternative splicing. Roberts et al. (1) show that the impact of truncation alleles is determined by exon usage (low in the I-band, where most exons are symmetrical and can be spliced out without frameshift), occurrence in the different isoforms, and localization in terms of the sarcomeric region affected. This will aid, though not completely resolve, discrimination between individually rare benign and pathogenic truncation variants. Putative pathogenic variants are enriched in the A-band region but even here appear to be of low penetrance. N2-Bus, N2B unique sequence (a site of phosphorylation); PEVK, titin region rich in proline, glutamate, valine, and lysine (contributes to spring function); TK, titin kinase domain (important signaling roles). Titin schematic is adapted from W. A. Linke and N. Hamdani, Circulation Res. 2014 (10.1161/ CIRCRESAHA). http://circres.ahajournals.org/content/114/6/1052.figures-only. Electron micrograph courtesy of Pradeep Luther (www.sarcomere.org). www.ScienceTranslationalMedicine.org 14 January 2015 Vol 7 Issue 270 270fs1

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performed and remunerated) in European guidelines for hypertrophic cardiomyopathy—the most common inherited cardiac disorder (5). But genetic testing for dilated cardiomyopathy (DCM), the commonest cause of heart failure in younger patients, is hindered by marked genetic heterogeneity. Until 2012, genetic testing for DCM was rather unproductive, as no gene accounted for more than a few percent of cases. Tis changed with the demonstration by Christine and Jon Seidman and colleagues that truncating variants of the giant muscle flament protein titin can be found in up to 25% of cases with severe and familial DCM (6). Tis was a welcome surprise. Titin is the largest human protein. Pairs of titin molecules are arranged head to head, spanning the whole sarcomere—the contractile unit of heart and skeletal muscle (Fig. 1). Titin is highly modular, and it was the repeated sequences with high homology as much as the vast size that had precluded efective sequencing until the advent of next-generation sequencing (NGS) technologies. With this discovery came the realization that titin exemplifes the challenge of assigning pathogenicity to individual mutations on a grand scale. Truncating titin variants were shown to occur in 2% to 3% of apparently normal individuals—a radically diferent situation from the more familiar cardiomyopathy-causing missense and truncation variants in other myoflament proteins (7). Te new work by Stuart Cook and colleagues (1) presents a sophisticated and comprehensive analysis of the giant TTN gene and its transcripts in an adequately powered set of DCM cases and controls and substantially addresses, yet cannot completely resolve, this challenge. In simple monogenic traits with penetrant mutations, robust evidence of cosegregation within an extended pedigree is key to assigning pathogenicity. Tis generates reliable information for clinical and research use whenever the same variant is seen again; aggregation of such data into a curated database (for example, ClinVar; www.ncbi.nlm.nih.gov/clinvar) is a key responsibility for all scientists and clinicians. But in cases in which data are available only for probands or nuclear families, other data provide a probabilistic framework for ranking the likely pathogenicity of an individual mutation. Bioinformatic prediction of impact on RNA and protein has a place in diagnostics but unsurprisingly does not correlate with pathogenicity well enough for

clinical reliance. Functional assays work if the readout is specifc, but they can mislead if the assay is crude (clinically silent variants may alter, even radically, protein structure and function but not trigger disease). In any case, functional assays have not displayed a high enough throughput to cope with a large series of private (that is, unique to a given family) alleles. Tus, the challenge faced by Roberts et al. was to move from statistically robust evidence by burden testing that truncating alleles of titin are overrepresented in patients with DCM to a usable classifcation of the myriad individual truncation alleles. Teir substantial progress is a testament to the rigor and breadth of the approaches adopted. A frst requirement was to defne a metatranscript to ensure that all variants could be cataloged in an unambiguous manner; the remarkable size and complex splicing of titin had meant that no such reference was previously available. Next, a systematic assessment of NGS revealed the pitfalls of mapping short reads against such a repetitive sequence. Tis is being made easier as longer read lengths are achieved. Perhaps most informative was detailed exon usage data from RNA sequencing of transcripts in human myocardium. As Roberts et al. showed [in their Figure 1 (1)], I-band titin contained a high proportion of symmetrical exons with very low “percentage spliced-in” in expressed transcripts; indeed, some exons ascribed to the canonical N2BA and N2B titin isoforms (Fig. 1) were not actually included in the transcripts. Once this was known, it was clear why many truncation alleles seen in normal controls do not cause pathology. Similarly, certain titin isoforms have very diferent functions, such as the cardiacspecifc Novex3 isoform, which does not span the sarcomere; mutations unique to these isoforms can also be set aside. In contrast, mutations that truncate exons consistently expressed in both of the dominant N2BA and N2B isoforms have a higher likelihood of pathogenicity (Fig. 1). Tus, truncations in the C-terminal portion of the protein, encoding A-band titin (thought to function as a “molecular ruler”), are the predominant class of pathogenic variants. Te A-band predominance appears to be more than just the consequence of exon and isoform usage (and nor is it explained by uneven distribution of sites susceptible to truncating mutations). Te most distal M-band region of titin—with similar exon usage and isoform inclusion—was signifcantly under-

represented in DCM alleles. In contrast, truncation and other loss-of-function alleles in this domain have been well characterised in skeletal muscle titinopathies, ofen with autosomal recessive inheritance. A number of important questions now arise. Foremost is whether identifcation of a TTN truncation allele in DCM is now clinically useful. In keeping with other genetic cardiomyopathies, the main gain should be for preclinical diagnosis and screening in families. Potential genotype:phenotype correlations were also explored by the authors, and some convincing trends were shown, but as usual, there was sufcient overlap that the quantitative diferences do not look likely to drive clinical decisionmaking. About half of the truncation variants identifed in population cohorts can now be set aside with reasonable confdence that they are not disease causing. However, the other half are indistinguishable and, indeed, some were seen in both cases and controls. Overall, truncations that ft the criteria for pathogenicity were seen in ~1% of the normal population and 15% of all DCM cases. Te consequence is that in typical patients with DCM, putative pathogenic truncating variants still only have an estimated 93% probability of pathogenicity (a likelihood ratio of ~14); when such a variant is identifed in a proband, there is still a 7% likelihood that it is a chance fnding. Tis suggests that such variants are not yet reliable for cascade screening, not least because this requires that it be reasonable to discharge a relative found not to carry the variant. As an incidental fnding there appears to be no actionable signifcance, as most people with putative pathogenic TTN truncating variants do not have DCM: In a random sample of 1000, such variants will be found in around 10 individuals, but there will only be ~2 DCM, cases very likely with neither carrying such a mutation. What do the fndings tell us about pathogenesis, and will this aid further refnement to allow clinical testing in the future? Te authors found no evidence of signifcant nonsense-mediated decay or marked allelic imbalance, and total levels of TTN transcript were similar in DCM and normal hearts; thus, it can be predicted that the truncated proteins would be expressed. Total levels of N2BA and N2B protein were also similar in cases and controls. Tis, and the observation that distal truncations were more pathogenic than proximal ones, favors a dominant-negative mechanism. A-band

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domains of titin would be needed to anchor the truncated peptides in the sarcomere, perhaps explaining why distal truncations, but not the more proximal I-band truncations, tend to be pathogenic. However, it has not been possible to document even low levels of mutant titin protein in human heart samples, a situation that contrasts with the dominant-negative proteins typical of other myoflament cardiomyopathies. A key question is why the truncation variants are relatively weak predictors of disease. An optimistic interpretation is that some specifc truncations, while ftting the new criteria, are nevertheless clinically silent while others cause DCM and that lumping together gives the modest likelihood ratio of 14. Tis is compatible with a dominantnegative mechanism and would imply potential for more clear-cut discrimination; but it is not easy to explain how apparently similar truncations would behave so differently. More likely—and more problematically—is that each A-band truncation has pathogenic potential but consistently low penetrance. Tis would ft the late-onset phenotype (mean age at diagnosis was ~50) and the scarcity of extended autosomal dominant families. If titin truncations are tolerated in some patients throughout life and in all patients at least until adulthood, this implies signifcant bufering of the phenotype, perhaps requiring a second genetic “hit” or an environmental stress to reveal a phenotype. One such factor has been postulated: that a TTN truncation allele may just unmask a pathogenic missense mutation on the other chromosome. Tere are clear precedents for recessive titin mutations—indeed, this is a frequent basis of skeletal muscle titinopathies—but substantial obstacles have made it hard to prove or refute this hypothesis. First, most individuals carry unique TTN missense mutations; clearly the large majority cannot be pathogenic on their own and are likely not to be pathogenic even in a hemizygous state, but some certainly can be (8, 9). Te way to resolve this would be

through extended family studies to formally document inheritance patterns with the truncation variants. In the case series studied by Roberts et al. (1), the prevalence of familial disease was low and no extended families were reported. Tis is perhaps a weakness of the study design but may also re!ect the natural history of titin-truncation DCM. Either way, it is a problem: the preponderance of families with just one or two afected relatives is compatible with both a low-penetrance late-onset, dominant model or with compound heterozygous inheritance of truncation and deleterious missense alleles. It does not seem likely that true recessive inheritance is the primary explanation, but the impact of diferent missense alleles in trans is at least a plausible explanation of why some instances of titin truncation, but not others, manifest with DCM (8, 9). Tis would of course confound attempts at genetic diagnosis in families. Investigation of titin missense alleles remains an important task. Simple burden tests that compare cumulative mutation frequencies in cases and controls are unlikely to be helpful. A focus on rare or unique variants may reveal enrichment, but it is likely to require a focus on missense alleles in well-characterized titin domains together with careful documentation of inheritance patterns in families to fully understand both the pathogenicity of truncating variants and the possibility of interplay with missense alleles. A further area of interest will be an exploration of the splicing machinery, as this too may be a factor that determines the extent to which truncations can be bufered. Te recent demonstration that deleterious alleles of the titin-splicing factor RBM20 cause DCM (10) is an interesting point of convergence. REFERENCES AND NOTES 1. A. M. Roberts, J. S. Ware, D. S. Herman, S. Schafer, J. Baksi, A. G. Bick, R. J. Buchan, R. Walsh, S. John, S. Wilkinson, F. Mazzarotto, L. E. Felkin, S. Gong, J. A. L. MacArthur, F. Cunningham, J. Flannick, S. B. Gabriel, D. M. Altshuler, P. S. Macdonald, M. Heinig, A. M. Keogh, C. S. Hayward,

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10.1126/scitranslmed.aaa4276 Citation: H. Watkins, Tackling the Achilles’ heel of genetic testing. Sci. Transl. Med. 7, 270fs1 (2015).

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