Genetics 101 for Cardiologists: Rare Genetic Variants and Monogenic Cardiovascular Disease

Canadian Journal of Cardiology 29 (2013) 18 –22

Special Article

Genetics 101 for Cardiologists: Rare Genetic Variants and Monogenic Cardiovascular Disease Sali M.K. Farhan, BSc, and Robert A. Hegele, MD, FRCPC Departments of Medicine and Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada

ABSTRACT

RÉSUMÉ

Monogenic diseases have a distinctive familial inheritance that follows Mendel’s laws, showing patterns like dominant, recessive, or X-linked. There are ⬎ 7000 monogenic diseases curated in databases, and together they account for up to 10% of all illnesses encountered in the emergency room or clinic. Despite the rarity of individual monogenic conditions, mapping their causative genes and mutations is important for several reasons. First, knowing the causative gene and mutation could provide actionable information for genetic counselling. Sometimes, knowing the gene and mutation allows for early diagnosis in affected families, which is important if there is an evidence-based intervention. Second, the implication of a mutant gene as being causative for a clinical phenotype provides strong evidence of the importance of the gene product in a cellular or biochemical pathway. Discovery of new molecular pathways in families with rare diseases can serve as the first step toward developing rational therapies to help not only affected families, but also patients with less extreme, nongenetic forms of the same condition. For instance, the study of rare patients with familial hypercholesterolemia helped in developing statin drugs, initially as a treatment for familial hypercholesterolemia but now a widely used therapy to reduce low-density lipoprotein cholesterol and cardiovascular disease risk.

Les maladies monogéniques proviennent d’une hérédité familiale distincte qui suit les lois de Mendel, montrant des modèles dominants, récessifs ou liés à l’X. On traite de ⬎7000 maladies monogéniques dans les bases de données qui comptent dans leur ensemble jusqu’à 10 % de toutes les maladies rencontrées en salle des urgences ou en clinique. En dépit de la rareté d’affections monogéniques individuelles, la cartographie de leurs gènes et de leurs mutations causales est importante pour plusieurs raisons. Premièrement, la connaissance du gène et de la mutation causale pourrait fournir des informations concrètes au counseling génétique. Parfois, la connaissance du gène et de la mutation permet un diagnostic précoce chez les familles atteintes, lequel est important s’il y a une intervention fondée sur des preuves. Deuxièmement, l’implication d’un gène mutant à titre causal d’un phénotype clinique fournit des données convaincantes sur l’importance du produit génétique dans une voie cellulaire ou biochimique. La découverte de nouvelles voies moléculaires dans les familles ayant des maladies rares peut servir de première étape au développement de traitements rationnels pour aider non seulement les familles atteintes, mais aussi les patients ayant des formes non génétiques, moins extrêmes, de la même maladie. Par exemple, l’étude de patients ayant une hypercholestérolémie familiale rare a aidé au développement de médicaments à base de statines, initialement pour traiter l’hypercholestérolémie familiale, mais maintenant grandement utilisés pour réduire le cholestérol à lipoprotéines de faible densité et le risque de maladies cardiovasculaires.

Most common cardiovascular diseases (CVDs) are complex, multifactorial conditions that result from interactions between genetic and nongenetic factors.1 Because of their economic and societal impact, CVDs such as ischemic heart disease, heart failure, and arrhythmias have been the focus of genome research efforts over the past decade.2 This research has shown that the genetic contribution to most “garden variety” or common adult-onset CVDs comes largely from multiple inherited

DNA variants, each of which has a small effect on disease risk, but which cumulatively can play a substantial role.2 However, much remains to be learned by studying monogenic forms of CVD that result from large effects of rare mutations.3-5

Received for publication September 6, 2012. Accepted October 10, 2012. Corresponding author: Dr Robert A. Hegele, Blackburn Cardiovascular Genetics Laboratory, 4288A-100 Perth Dr, Robarts Research Institute, Western University, London, Ontario N6A 5K8, Canada. Tel.: ⫹1-519-931-5271; fax: ⫹1-519-931-5218. E-mail: [email protected] See page 22 for disclosure information.

What Are Monogenic Diseases? Monogenic diseases are caused by a mutation in a single gene that cripples or perturbs a protein involved in a major mechanism or cellular pathway.4,5 Monogenic diseases are individually rare, but collectively involve a substantial proportion—perhaps up to 10%— of all patients in the health care system.4 They are also called Mendelian diseases, because the inheritance of the clinical phenotypes in families follows classic patterns, such as dominant, recessive, or X-linked.6

0828-282X/$ – see front matter © 2013 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cjca.2012.10.010

Farhan and Hegele Rare Genetic Variants and Monogenic CVD

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Table 1. Examples of monogenic disorders that affect LDL-C Disorder Elevated LDL-C Familial hypercholesterolemia Familial defective apolipoprotein B Autosomal dominant hypercholesterolemia Autosomal recessive hypercholesterolemia Depressed LDL-C Abetalipoproteinemia Hypobetalipoproteinemia PCSK9 deficiency with low LDL Familial combined hypolipidemia

Gene symbol

Chromosomal localization

Affected protein

MIM reference numbers

Inheritance

LDLR APOB PCSK9

19p13.3 2p24-p23 1p32.3

LDL receptor Apolipoprotein B PCSK9

143890, 606945 144010 603776, 607786

Autosomal codominant Autosomal dominant Autosomal dominant

LDLRAP1 (ARH)

1p36-p35

LDL receptor-associated protein 1

603813, 605747

Autosomal recessive

Microsomal triglyceride transfer protein Apolipoprotein B PCSK9 Angiopoietin-like protein 3

200100, 157147

Autosomal recessive

144010, 605019 607786; 613589 605019, 604774

Autosomal codominant Autosomal dominant Autosomal recessive

MTP APOB PCSK9 ANGPTL3

4q24 2p24-p23 1p32.3 1p31.1-p22.3

C, cholesterol; LDL, low-density lipoprotein; MIM, Mendelian Inheritance in Man; PCSK9, proprotein convertase subtilisin/kexin type 9.

Approximately half of known Mendelian disorders have been solved at the molecular genetic level: the Online Mendelian Inheritance in Man database (OMIM) (http://omim.org/ statistics/entry; downloaded August 18, 2012) recently reported that, of 7220 human diseases either known or suspected to have a single-gene basis, 3551 had their causative gene defined with at least 1 well-characterized human gene mutation. Of the rare diseases reported in OMIM, ⬎ 2000 involve the cardiovascular system directly or indirectly, and approximately 200 primarily involve the heart or blood vessels. Some examples of monogenic disorders affecting plasma lipids5,7—specifically low-density lipoprotein (LDL) cholesterol levels—are shown in Table 1. Monogenic CVDs often present early in life and, if not lethal in childhood, can persist into adulthood, typically leading to complications as the disease progresses.5,8 Furthermore, affected individuals often have poor reproductive fitness, either being infertile or unable to survive to sexual maturity. Thus, a disease-causing mutation is often not passed on to the next generation from an affected individual. However, a causative allele for a recessive or X-linked disease can persist and be passed on through asymptomatic carriers. In contrast, in autosomal dominant conditions, carrying 1 copy of a mutant gene is enough to result in the expression of the disease. DNA Variation: Many Types and Sizes A wide range of DNA variant types can cause human diseases.8 Human genomic variation spans the spectrum ranging from large-scale chromosomal changes to small-scale single nucleotide changes.8 Single DNA base pair changes range from common and typically benign neutral variants known as single nucleotide polymorphisms to rare and deleterious genetic variants that are referred to as “mutations.”8 By convention, rare mutations occur within the population with a minor allele frequency of ⬍ 1%. Though rare mutations would appear to be more likely responsible for dysfunction of the protein product of the gene, there are many examples of missense mutations in coding regions, variants in DNA regions of unknown function, and silent changes in coding regions for which pathogenicity is uncertain. Indeed, recent massive sequencing efforts in normal populations reveal that each human genome contains tens of thousands of these rare variants— often unique to the individual—and that the vast majority of these have no obvious clinical consequences.9,10

A DNA variant is more likely to be dysfunctional and disease-causing if it alters a sequence that encodes an evolutionarily conserved amino acid residue, thus increasing the likelihood that it alters the encoded protein’s function.11 Other criteria that implicate a mutation as disease-causing include: (1) statistical association of the mutation’s presence in affected members of a family or population together with its absence from unaffected subjects; (2) computer modelling to predict the consequences of the mutation on normal protein structure or function; (3) test tube or petri dish studies showing that the mutant protein functions abnormally in a controlled experimental situation; or (4) genetic engineering experiments in living organisms proving that the mutated protein causes an abnormal phenotype resembling the human disease.12 Why Study Monogenic Diseases? Because complex pathologies like atherosclerosis leading to ischemic heart disease or stroke extract such a burden on the health care system, why should resources continue to be directed toward deciphering the DNA basis of various rare CVDs? First and foremost, we have much still to learn from monogenic diseases.4 By understanding the gene, and thus the protein whose function has gone awry, one can hope to develop specific treatments— genetically based or otherwise—for patients with rare genetic conditions,7 but treatments arising from studying monogenic illnesses could have even broader impact in cardiovascular medicine. For instance, old-time clinicians say “when you hear hoof beats, think of horses, not zebras.” Though this maxim holds true under the stress of making a diagnosis in the emergency room, genetics researchers have long recognized that studying zebras often reveals something about the horse.13 In other words, because monogenic disorders are mechanistically more straightforward than complex traits, finding the causative mutation in a new gene might uncover an important new pathway or rate-limiting step in a common disease process. If genetically perturbing the protein causes a disease, it implies that the pathway must be important. Deriving Treatments From Studying Rare Diseases An illustration of how this paradigm was successfully applied (see Fig. 1) was the discovery of the LDL receptor (encoded by the LDLR gene), which is defective in familial hyper-

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Canadian Journal of Cardiology Volume 29 2013

Learning from monogenic diseases PROBLEM: LDL-C MECHANISTIC RESEARCH: clinical, biochemical, cellular, genetic

MECHANISTIC INSIGHT: LDL receptor clears LDL particles

CLINICAL TRIALS: statin effects on biochemistry, angiography, CVD endpoints, mortality

MONOGENIC MODEL: Familial hypercholesterolemia

FUNDAMENTAL DISCOVERY: LDL receptor and receptorNobel mediated endocytosis

DRUG TARGET:

prize

statins to upregulate LDL receptor

IMPACT ON PRACTICE: statins = standard of care

Figure 1. Learning from monogenic diseases. The example of familial hypercholesterolemia (FH) illustrates how studying a rare monogenic disease yielded broad clinical benefits. The clinical issue was the well-known epidemiologic relationship between elevated plasma levels of low-density lipoprotein (LDL) cholesterol (LDL-C) and increased atherosclerosis risk. A rare monogenic condition defined by heritable high LDL cholesterol is FH, which became the model system for many of the experiments of Goldstein and Brown.15 Study of FH patients was central to discovering the cell-surface LDL receptor and its gene LDLR, which was mutated in FH patients. Describing the process of receptor-mediated endocytosis of LDL particles through the LDL receptor won Brown and Goldstein the Nobel Prize in Medicine and Physiology in 1985. This discovery suggested that suppressing intracellular cholesterol biosynthesis using 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitors (statins) would force upregulation of the nonmutated LDLR allele in heterozygous FH patients. This in turn enhanced removal of LDL particles from plasma. Because non-FH patients have 2 functioning copies of the LDLR gene, statins very effectively lower plasma LDL cholesterol in the general CVD population, as demonstrated by clinical trials. Statins are now prescribed to millions of Canadians at risk of major CVD end points.

cholesterolemia (FH), a disease that affects approximately 1:500 Canadians.14 The LDL receptor is central to receptormediated endocytosis of plasma LDL cholesterol; this insight suggested that the nonmutated LDLR allele in heterozygous FH patients could be upregulated if intracellular cholesterol biosynthesis is suppressed by a statin drug.15 Enhanced removal of LDL particles from plasma and reductions in LDL cholesterol were subsequently proven in both FH patients and in the general population. Statins are now widely used to lower plasma LDL cholesterol and delay the onset of CVD.16 The LDLR-FH discovery has become the poster child showing how studying a rare monogenic condition can yield farreaching benefits for general cardiology and public health. A more recent example is the study of the relatively uncommon genetic condition known as proprotein convertase subtilisin/ kexin 9 (PCSK9) deficiency, not really a disease but rather a protected state, in which carriers of a rare variant in the PCSK9 gene have reduced PCSK9 activity, preventing LDL receptor degradation, ultimately reducing both LDL cholesterol and CVD risk.17 At least 9 pharmaceutical firms are now developing inhibitors of PCSK9 to pharmacologically mimic the human deficiency state. Early phase trials suggest that targeting this pathway dramatically lowers LDL cholesterol18 and, assuming that there are no safety issues, these drugs could find future utility for at-risk patients who either fail to respond to statins or have adverse reactions to them. Many drug development efforts for various forms of CVD are following this approach, exploiting the understanding of

rare human diseases gained through genetics to define new drug targets. The patients in monogenic disease studies are in effect “probes” for discovery of new disease mechanisms; some of these mechanisms might turn out to be so important that treating them will have direct relevance not just for rare patients, but also for those with common forms of CVD and its risk factors.

Diagnostic Implications of Studying Monogenic Diseases In addition to development of new therapies, studying rare monogenic diseases might help to develop diagnostic tools.2 For instance, in families with a rare illness, knowing the precise causative mutation could enable presymptomatic diagnosis and early evidence-based interventions. Examples include rare electrophysiological conditions associated with sudden onset of life-threatening arrhythmias or sudden cardiac death, as reviewed in this issue of the Canadian Journal of Cardiology. Furthermore, insights from studying Mendelian CVDs might help to identify susceptibility alleles that can be used to study or predict complex nongenetic CVD phenotypes or perhaps response to therapies.19 However, most recent progress to define susceptibility to “garden variety” CVD has emerged as a result of studying single nucleotide polymorphisms detected by microarrays rather than rare mutations detected by sequencing.20

Farhan and Hegele Rare Genetic Variants and Monogenic CVD

Are Monogenic Diseases Really So Simple? While monogenic diseases are simpler than common diseases, there are still numerous complexities. For instance, in some families, variable penetrance of an autosomal dominant disease occurs when carriers of the identical mutation vary in the severity of the disease phenotype. Reasons for this include: (1) interactions with other genetic variants that modulate the severity of disease expression (gene-gene interactions or “genetic modifiers”); and (2) interactions with environmental factors, such as diet, exercise, or drugs that similarly modulate severity of gene expression (gene-environment interactions). Some environmental factors are thought to induce “epigenetic” changes, that is changes to DNA that are not initially inherited but then can affect gene function and can even be passed on to the next generation, as reviewed in this issue of the Canadian Journal of Cardiology. Other types of complexity include instances in which mutations in different genes can result in an identical disease phenotype, a phenomenon referred to as “locus heterogeneity.”21 The Effect of Next-Generation DNA Sequencing on Monogenic Diseases Next-generation sequencing (NGS) of DNA refers to a range of technologies that all have the goal of deciphering astronomical quantities of genetic information at microscopic cost. For instance, in 2001, it had taken approximately 14 years and more than 2 billion dollars to assemble the 3 billion DNA base pairs of a single human genome. In 2007, a project of this scale required approximately 1 year and approximately 2 million dollars. Currently, a human genome can be sequenced within a week at a cost of less than 10 thousand dollars. The

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cost of reporting only coding sequences (called “exome sequencing”) is rapidly approaching 1 thousand dollars or less. Readers interested in the technological aspects are referred to several excellent reviews of these methods.9-11 The widespread application of NGS since 2010 in the human genetics field has been staggeringly successful, particularly for monogenic diseases. An NGS experiment produces a mountain of data from numerous small reads that are spliced together to yield the patient’s intact genome, a computational challenge that is like trying to assemble the Encyclopedia Britannica (or Wikipedia) using millions of brief Twitter feeds. Fortunately, high capacity computers and bioinformatic software are up to the task, so that within a short time it is now possible to obtain all DNA variants present in a patient’s genome. Although these experiments are revealing that each of us carries a tremendous load of genetic variants— often new rare variants never previously reported—the number of variants is finite. For a researcher or health care provider seeking the molecular cause of a monogenic disease, the fact that the smoking gun lies within a finite list of suspect mutations makes the entire process manageable, although still challenging. The list of possible mutations still needs to be whittled down using prioritization criteria; clues and circumstantial evidence from other types of experiments. Despite these challenges, NGS has already helped to identify the causative genes and mutations for approximately 200 monogenic diseases since 2010, with dozens of new reports monthly. Important issues going forward include determining how NGS methods can be applied clinically and also extended to study “garden variety” complex forms of CVD.7-9

Table 2. Glossary of terms Term Autosomal codominant Autosomal dominant Autosomal recessive Complex traits Environment Epigenetics Exome Exon Haplotype Heritability Intron Locus heterogeneity Minor allele frequency (MAF) Next-generation DNA sequencing Variable penetrance X-linked

Explanation An additive pattern of inheritance, in which each mutated copy of the gene inherited produces a further clinically more extreme deviation of the phenotype. A pattern of inheritance in which 1 mutated copy of the gene is sufficient to cause the expression of disease. A pattern of inheritance in which 2 mutated copies of the gene, 1 inherited from each parent, is required for the disease to be expressed. Carriers with a single mutated copy are clinically normal. Traits that are influenced by the environment and/or through a combination of variants in at least several genes, each of which has a small effect. Any nongenetic factors such as nutrition, physical activity, and exposure to pathogens or toxins. The environment can play a role in certain diseases. “Above genetics.” Heritable chemical groups involved in DNA modification that are not encoded in the DNA sequence but still contribute to gene function by influencing gene expression. The subset of a genome that is protein-coding. In addition to the exome, commercially available capture probes target noncoding exons, sequences flanking exons, and microRNAs. DNA sequences within a gene transcript that are translated into proteins. A combination of alleles along a single chromosome. The proportion of the total phenotypic variation in a given characteristic that can be attributed to additive genetic effects. DNA sequences transcribed into the gene transcript, but not translated into protein. These are typically removed before translation by splicing mechanisms. A condition in which mutations in 1 of 2 or more different genes can result in the same clinical phenotype (eg, mutations in LDLR or APOB or PCSK9 can each cause a phenotype that resembles familial hypercholesterolemia) (see Table 1). A numeric value derived from large powered population studies assessing the prevalence and distribution of an allele. MAF acts a great tool by providing an appropriate estimate of the disease prevalence. Highly parallelized DNA sequencing technologies that produce many hundreds of thousands or millions of short reads (25-500 base pair) for a low cost and in a short time. A phenomenon often observed in autosomal dominant diseases in which the severity of disease expression can vary among carriers of the same mutation. A pattern of inheritance in which a mutated gene on the X-chromosome causes expression of a disease when a Ychromosome is present (ie, males are affected), but is phenotypically silent when another X-chromosome is present (ie, females are carriers).

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How Does This Apply to My Practice? At present, clinical utility of testing patients at risk for a rare monogenic CVD is highly variable. For instance, the diagnosis of FH is most often made on clinical and biochemical grounds; screening families to find additional affected individuals can be performed with a simple lipid profile, which in affected members would typically show LDL cholesterol ⬎ 5 mmol/L. DNA testing is often unnecessary based on our current understanding of the illness. In contrast, knowing and being able to test for the precise DNA mutation in certain long QT syndrome or dilated cardiomyopathy families might allow very early presymptomatic diagnosis, which could in turn be life-saving in some instances. Furthermore, a substantial amount of emerging genetic research has a translational or clinical orientation, and will undoubtedly provide new evidence for how knowledge of the patient’s precise mutation can affect the course of disease or choice of treatment.

2. O’Donnell CJ, Nabel EG. Genomics of cardiovascular disease. N Engl J Med 2011;365:2098-109.

Conclusion Thus, rare monogenic diseases that are classically inherited in families, as compared with complex diseases which might cluster in families but show no distinct pattern of inheritance, comprise a minor, but clinically relevant portion of CVD. A glossary of terms related to the topics discussed here is provided in Table 2. Identifying causative genes has clinical implications for diagnosis and development of new therapies. This is also academically important because each new characterized gene helps fill in the complex biological jigsaw puzzle of human pathways and mechanisms. The rate of monogenic disease gene discovery will increase with the recent developments in NGS technology.

10. Majewski J, Schwartzentruber J, Lalonde E, et al. What can exome sequencing do for you? J Med Genet 2011;48:580-9.

Funding Sources S.M.K.F. is supported by the CIHR Strategic Training Program in Vascular Research. R.A.H. is supported by the Jacob J. Wolfe Distinguished Medical Research Chair, the Edith Schulich Vinet Canada Research Chair in Human Genetics, the Martha G. Blackburn Chair in Cardiovascular Research, and operating grants from the CIHR (MOP-13430, MOP-79523, CTP-79853), the Heart and Stroke Foundation of Ontario (NA-6059, T-6018, PRG-4854), and Genome Canada through the Ontario Genomics Institute.

15. Goldstein JL, Brown MS. The LDL receptor. Arterioscler Thromb Vasc Biol 2009;29:431-8.

Disclosures The authors have no conflicts of interest to disclose. References 1. Lanktree MB, Hegele RA. Gene-gene and gene-environment interactions: new insights into the prevention, detection and management of coronary artery disease. Genome Med 2009;1:28.

3. Nabel EG. Cardiovascular disease. N Engl J Med 2003;349:60-72. 4. Antonarakis SE, Beckmann JS. Mendelian disorders deserve more attention. Nat Rev Genet 2006;4:277-82. 5. Rahalkar AR, Hegele RA. Monogenic pediatric dyslipidemias: classification, genetics and clinical spectrum. Mol Genet Metab 2008;93:282-94. 6. Nehring WM, Faux SA. Clinical genetics: an overview. J Cardiovasc Nurs 1999;4:19-33. 7. Hegele RA. Plasma lipoproteins: genetic influences and clinical implications. Nat Rev Genet 2009;10:109-21. 8. Pollex RL, Hegele RA. Copy number variation in the human genome and its implications for cardiovascular disease. Circulation 2007;115:3130-8. 9. Bick D, Dimmock D. Whole exome and whole genome sequencing. Curr Opin Pediatr 2011;23:594-600.

11. Glissen C, Hoischen A, Brunner HG, et al. Unlocking Mendelian disease using exome sequencing. Genome Biol 2011;12:228-39. 12. Lahiry P, Wang J, Robinson JF, et al. A multiplex human syndrome implicates a key role for intestinal cell kinase in development of central nervous, skeletal, and endocrine systems. Am J Hum Genet 2009;84:134-47. 13. Hegele RA, Reue K. Hoofbeats, zebras, and insights into insulin resistance. J Clin Invest 2009;119:249-51. 14. Yuan G, Wang J, Hegele RA. Heterozygous familial hypercholesterolemia: an under-recognized cause of early cardiovascular disease. CMAJ 2006;174:1124-9.

16. Cholesterol Treatment Trialists’ (CTT) Collaborators, Mihaylova B, Emberson J, et al. The effects of lowering LDL cholesterol with statin therapy in people at low risk of vascular disease: meta-analysis of individual data from 27 randomised trials. Lancet 2012;380:581-90. 17. Cohen JC, Boerwinkle E, Mosley TH Jr, et al. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N Engl J Med 2006;354:1264-72. 18. Stein EA, Mellis S, Yancopoulos GD, et al. Effect of a monoclonal antibody to PCSK9 on LDL cholesterol. N Engl J Med 2012;366:1108-18. 19. Torkamani A, Scott-Van Zeeland AA, Topol EJ, et al. Annotating individual human genomes. Genomics 2011;98:233-41. 20. Musunuru K, Kathiresan S. Genetics of coronary artery disease. Annu Rev Genomics Hum Genet 2010;11:91-108. 21. Scriver CR. Science, medicine and phenylketonuria. Acta Paediatr Suppl 1994;407:11-8.