Genetic testing now enables diagnoses that could not be made in the past. However, it depends on the method, which significance is behind the results.
Hereditary cardiovascular diseases, such as Marfan syndrome (MFS), are among the monogenic diseases and are considered rare, with a frequency (prevalence) of <1:2000. However, because there are many different hereditary cardiovascular diseases, the total number of affected individuals is relatively large and requires appropriate attention. Thanks to the steady progress in genetics, diagnostically and therapeutically relevant clarifications can be made today. More and more people can be diagnosed, confirmed or ruled out by means of genetic testing for medical purposes (genetic testing) of the genetic material (DNA) – not to be confused with (lifestyle) genetic testing for non-medical purposes from the Internet or the pharmacy ( Fig. 1). Moreover, medical-genetic clarifications of hereditary diseases do not represent a snapshot of the state of health, but are valid for life and have consequences both for the patient and his family (blood relatives). Appropriate genetic counseling by medical geneticists is therefore required by law (GUMG Art. 14). Genetic clarification of late manifesting diseases and carrier status of recessive diseases is reserved for adults.
Increasing importance of genetic diagnostics
The influence of genetic causes must be considered in the diagnosis as well as in the therapy of diseases. Familiarity may be indicative, but hereditary diseases also occur sporadically. In common multifactorial diseases, such as hypertension, the genetic effects are usually multiple and only strong in the aggregate, whereas monogenic diseases are caused by the mutation of a single gene. Often, the clinical picture in such diseases is not clearly pronounced, and genetics with new methods and findings is crucial to the correct diagnosis. This is the basis for prognosis and targeted disease management and, if possible, for successful therapy and prevention [1]. Without diagnosis, no conclusive statement can be made about therapy or change of therapy. Unfortunately, this aspect is often not taken into account in our healthcare system. Even when a clinical diagnosis is apparently certain, identification of the disease-causing gene defect (mutation) may be essential, as disease management and therapies may be gene- or even mutation-specific.
In contrast to rapid routine tests of blood parameters, medical genetic tests can only be automated and performed in a practice laboratory to a limited extent. They are mainly used in situations where clinical investigations do not allow a conclusive diagnosis. This is particularly important in diseases with overlapping or nonspecific clinical phenotype and in the early phase of a disease. It can also be clarified pre-symptomatically whether or not there is a genetic predisposition to the familial disease.
High-throughput DNA sequencing
The most important method for the targeted investigation of genes is DNA sequencing, which can determine the sequence of nucleotide bases of the genetic material (A, T, G, C) and thus precisely detect gene mutations. Such genetic analyses are performed with unprecedented efficiency using high-throughput sequencing (“Next Generation Sequencing”, NGS ). NGS is more efficient than classical single-gene analysis using Sanger sequencing and is particularly successful in detecting the causes of disease as well as analyzing cell-free DNA circulating in the blood and single-cell examination of minute biospecimens.
In NGS, either a selected combination (so-called panel) of genes (“Targeted Sequencing”, TS), the whole genome (“Whole Genome Sequencing”, WGS ; ~3 billion nucleotide bases) or its coding region (“Whole Exome Sequencing”, WES ; ~25’000 genes) are investigated. Due to these differences alone, not all NGS is the same (Fig. 2). In addition, there is the difference in performance and quality between the NGS methods, which is relevant for genetic diagnostics [2].
TS can analyze certain gene regions particularly intensively by capturing the sequence of more than 1000 DNA copies (“sequencing reads”) and thus detecting small amounts (<1:100) of non-reference alleles, which are present as a so-called mosaic. This is necessary, for example, in the genetic testing of somatic cancers. TS is also inexpensive and is therefore often used as the first step in mutation screening. However, if the disease-causing mutation is not found with TS, the disease remains undiagnosed and testing must be repeated with WES or, better yet, WGS. Caveat: A negative finding (i.e., no known/unambiguous pathogenic mutation) in the tested genes does not exclude a genetic cause of the present disease.
Challenges of genome analyses
With NGS, there are limitations relevant to genetic diagnostics. First, the sequence read length of Illumina’s market-leading NGS technology is too short (~150 nucleotide bases) to assign longer repetitive/homologous regions to the reference genome with unique location [2]. The latest sequencing technologies (e.g. from Pacific Biosciences or Oxford Nanopore Technologies), which can read DNA fragments from several thousand nucleotide bases, promise to remedy the situation. Second, NGS of GC-rich DNA regions is more difficult because the nucleotide base pair G and C has stronger binding than the pair A and T. Especially in TS and WES, GC-rich gene regions are not adequately detected, as is often the case at the beginning of a gene (i.e., with sequencing reads covered), which is why the quality requirements of genetic diagnostics are often not met. With WGS, this problem occurs much less, so WGS not only has the advantage of covering the non-coding region (98.5%) of the genome, but also covers the clinically most important coding region (1.5%) better than WES, especially GC-rich regions. WGS thus allows the best possible genetic diagnosis of hereditary diseases whose causes are unknown or which are caused by mutations in large and/or complex genes, such as the cardiomyopathy-causing genes DMD (~2.3 million nucleotide bases) and TTN (364 exons), which are our largest and exon-richest genes, respectively.
The American College of Medical Genetics and Genomics (ACMG) working group published a list of 59 genes whose mutations greatly increase the risk of aortic diseases, arrhythmias, and cardiomyopathies, among others, so that preventive measures are possible and indicated [3]. The ACMG working group recommends that all these risk genes should be examined as a standard part of comprehensive genetic examinations such as WES or WGS and that patients should be informed of pathogenic sequence deviations (mutations) if they so wish, so that any preventive measures can be initiated at an early stage (Table 1).
The large amount of data generated in the context of a WGS can be reduced to the level of TS or WES using virtual (in silico) gene panels and focused on the clinical question [4, 5]. In addition, quality control and interpretation of the volume of data generated by NGS is an elaborate challenge that is both costly and intellectually challenging, but this is not yet adequately represented by the current analysis list item for NGS (Fig. 2). Especially the acquisition and interpretation of those NGS data that are important for diagnostics requires a lot of human genetic expertise and can be particularly time-consuming. This is the case, for example, when sequence deviations are found in genes whose significance or function is not yet (fully) known, which is the case for about half of all human genes today. Although today’s interpretation software takes into account many different parameters and provides important bases for assessment, the interpretation of such sequence deviations (so-called “variants of unknown significance”, VUS) requires at least a segregation analysis in the family (if possible) and/or a corresponding partly complex functional characterization (Fig. 2).
To detect the disease-causing sequence deviation, extensive databases with linked genotype and phenotype information are required, as well as the generation and curation of gene-disease associations and their classification. For example, the ClinGen Working Group has categorized 53 genes that, when mutated, can cause syndromic or nonsyndromic hereditary thoracic aortic aneurysms and dissections, ranging from “definite/strong” to “no evidence” [6]. Such categories or gene panels may facilitate the targeted search for the cause of disease, but require an accurate clinical diagnosis of suspicion and regular updating [5].
Digenic Inheritance
It is little known in clinical practice that multiple monogenic diseases or mutations in multiple genes may underlie the clinical picture, which may segregate in the family and therefore be important in the genetic workup of family members. As an example, consider two families whose members have a mutation in the FBN1 gene(associated with MFS) and/or the FBN2 gene(associated with congenital contractural arachnodactyly, CCA) [7]. In those family members in whom both diseases co-occur, there is not only a mixing but also an intensification of clinical symptoms, with either the clinical signs of MFS or of CCA predominating. For example, this is true in a 20-year-old patient clinically diagnosed with classic MFS (aortic dilation with Z-score >2, severe myopia, kyphoscoliosis) and compared to his parents with mild signs of CCA (father with FBN2-mutation) or MFS (mother with FBN1-mutation) shows a more pronounced phenotype of MFS (Fig. 3). Equally clinically relevant is an attenuation or modification of symptoms, as this may cause a disease to be overlooked or misdiagnosed, but the causative mutation may still be inherited. Digenic inheritance highlights the importance and complexity of genetic clarification.
From diagnosis to therapy
Based on early correct diagnosis, in cardiovascular diseases such as aortopathies (e.g., MFS or vascular Ehlers-Danlos syndrome, vEDS), adequate lifestyle adjustments, check-ups, and/or prophylactic treatments can contribute to effective prevention. The first choice of drug treatment is usually antihypertensive drugs such as beta-blockers and/or angiotensin II type 1 receptor antagonists (sartans). However, their properties and effects are not without controversy for both certain beta-blockers (e.g., atenolol) and sartans (e.g., losartan) despite promising (pre)clinical studies [8,9]. This leads to the use of different preparations of these drug classes depending on the preferences of the treating physician.
While losartan is considered the drug therapy of choice for preventing and stabilizing aortic dilatation in MFS, its medication in vEDS has been uncertain, despite a suggestive Lancet study [10]. Certainty is provided by a recent paper demonstrating a clear positive effect of the beta-blocker celiprolol (Selectol), but not losartan, on aortic mechanical stability in an experimental mouse vEDS model [11]. This demonstrates that the treatment success of antihypertensive drugs can vary greatly depending on the aortopathy.
Pharmacogenetics – The Future of Drug Therapy
Drugs are often prescribed according to the “one fits all” principle, but they are not always or in all patients (equally) effective. Not only the disease-causing mutation plays a role, but also pharmacogenetics, i.e. the genetic predisposition that influences the effect of a drug through the underlying pharmacokinetics and dynamics. The field of pharmacogenetics is still relatively young and, despite its unrecognized importance, is only gradually being implemented into clinical practice. Pharmacogenetic profiles for patients already exist in Holland and within the EU project Ubiquitous Pharmacogenomics (U-PGx). These profiles include information on the major known genetic predispositions that influence drug effects. Together with the findings, the patient receives a card in credit card format with QR code (Fig. 4), which contains his genetic predispositions and the dosage of medications based on them according to international guidelines. In the U-PGx project, these are currently 45 sequence variations in 12 genes and their influence on the effect of 76 drugs. For example, for individuals who are CYP2D6 slow metabolizers (~5-10% of Europeans), a dose reduction of the beta-blocker metoprolol to 25% of the normal dose is recommended, whereas for ultrafast metabolizers (~3% of Europeans), a dose increase to 250% is recommended [12]. New findings are continuously integrated and the latest data can be retrieved using the card’s QR code. The card can be presented when visiting a doctor or pharmacy and thus the choice or dosage of a medication can be individually adjusted (personalized or precision medicine). This not only saves lengthy adjustment of the correct dosage and possible (fatal) side effects or ineffectiveness of drugs in the patient, but also associated healthcare costs [13].
Outlook
In the future, genetic diagnostics will be crucial not only for the diagnosis of the disease-causing mutation and thus the adequate (drug) therapy, but also for the pharmacogenetic analysis of genes that determine the choice and dosage of the drugs used. Increasingly, (epi-)genetic changes that do not directly affect the DNA sequence of genes, but rather their regulation, are also being studied and understood. Increasingly, genetic effects can also be identified in common multifactorial cardiovascular diseases.
New methods such as CRISPR/Cas open up possibilities, but also dangers, in the treatment of genetic diseases. Despite promising results from studies to date, this technology is still a long way from finding its way into everyday medical practice. On the one hand, because the method is not yet fully developed, on the other hand, because ethical and legal issues need to be regulated in order to limit any potential for abuse. Nevertheless, it is obvious that genetics will determine the future of medicine and that medicine will be guided by genetics in prevention, diagnosis and therapy.
Take-Home Messages
- Genetic testing can make accurate diagnoses that would not otherwise be possible. However, not all genetic tests are the same: The method used decisively determines the respective significance.
- Familial and/or early or syndromal occurrence of a disease may indicate a genetic cause. However, with current knowledge, the genetic cause cannot be identified in every case.
- Genetic workup has importance not only for diagnosis, prognosis, prevention and family counseling, but also for causal or pharmacological treatment.
- For vascular Ehlers-Danlos syndrome, celiprolol (Selectol) rather than losartan is the drug therapy of choice.
Literature:
- Attenhofer Jost CH, Greutmann M, et al: Medical treatment of aortic aneurysms in Marfan syndrome and other heritable conditions. Curr Cardiol Rev 2014; 10(2): 161-171.
- Caspar SM, Dubacher N, et al: Clinical sequencing: From raw data to diagnosis with lifetime value. Clin Genet 2018; 93(3): 508-519.
- Kalia SS, et al: Recommendations for reporting of secondary findings in clinical exome and genome sequencing, 2016 update (ACMG SF v2.0): a policy statement of the American College of Medical Genetics and Genomics. Genet Med 2017; 19(2): 249-255.
- Plüss M, Kopps A, et al: Need for speed in accurate whole-genome data analysis: GENALICE MAP challenges BWA/GATK more than PEMapper/PECaller and Isaac. Proc Natl Acad Sci U S A 2017; 114(40): E8320-E8322.
- Caspar SM, et al: More genes for thoracic aortic aneurysms and dissections. J Am Coll Cardiol 2019; 73(4): 528-529.
- Renard M, et al: Clincal Validity of Genes for Heritable Thoracic Aortic Aneurysms and Dissections. J Am Coll Cardiol 2018; 72(6): 605-615.
- Najafi A & Caspar SM, et al: Variant filtering, digenic inheritance, and other challenges in the current genomics era: a lesson from fibrillinopathies (submitted).
- Carlberg B, et al: Atenolol in hypertension: is it a wise choice? Lancet 2004; 364(9446): 1684-1689.
- Gao L & Chen L, et al: The effect of losartan on progressive aortic dilatation in patients with Marfan’s syndrome: a meta-analysis of prospective randomized clinical trials. Int J Cardiol 2018; 217: 190-194.
- Ong KT, et al: Effect of celiprolol on prevention of cardiovascular events in vascular Ehlers-Danlos syndrome: a prospective randomised, open, blinded-endpoints trial. Lancet 2010; 376(9751): 1476-1484.
- Dubacher N & Münger J, et al: Celiprolol but not losartan improves the biomechanical integrity of the aorta in a mouse model of vascular Ehlers-Danlos syndrome. Cardiovasc Res 2019 (Epub ahead of print).
- Swen JJ, et al: Pharmacogenetics: from bench to byte – an update of guidelines. Clin Pharmacol Ther 2011; 89(5): 662-673.
- Verbelen M, et al: Cost-effectiveness of pharmacogenetic-guided treatment: are we there yet? Pharmacogenomics J 2017; 17(5): 395-402.
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