Physicians' Academy for Cardiovascular Education

The role of next-generation DNA sequencing in identifying cardiovascular conditions

Mogensen J et al., Eur Heart J 2015

The current role of next-generation DNA sequencing in routine care of patients with hereditary cardiovascular conditions: a viewpoint paper of the European Society of Cardiology working group on myocardial and pericardial diseases and members of the European Society of Human Genetics

Mogensen J, Van Tintelen JP, Fokstuen S, et al.
Eur Heart J published 5 April 2015, 10.1093/eurheartj/ehv122


Various guidelines recommend genetic testing to screen for certain relatively frequent cardiovascular (CV) conditions with Mendelian inheritance. Via cascade screening, pre-symptomatic testing of relatives ensures adequate clinical surveillance of mutation carriers and allows non-carriers to be discharged from clinical follow-up.
Recent advances in DNA sequencing technologies allow to investigate large numbers of disease genes simultaneously, known as Next-Generation Sequencing (NGS). A consequence of this approach is that sequence variants of uncertain clinical significance (VUS) may also be identified. Interpreting the not always straightforward results of NGS analysis is therefore challenging. This current opinion paper reviews the implications of NGS for cardio-genetic services.

Methods for identification of disease-causing mutations

In the context of hereditary CV conditions for which a number of underlying causative mutations is considerable, the parallelized sequencing process in NGS is very useful. NGS can be performed at higher speed and at lower cost than the previously commonly used Sanger sequencing. NGS also allows analysis of very large disease genes.
NGS can be performed by focussing on ‘targeted gene panels’, or as whole exome sequencing (WES) or whole-genome sequencing (WGS). Targeted gene panels yield results with identical analytical quality to Sanger sequencing, and are faster and cheaper with better coverage and sensitivity than WES and WGS. WES and WGS allow an unbiased search for mutations. Currently it is, however, not considered the most appropriate for routine diagnostic purposes, since not every part of the coding sequences is sufficiently covered, which may lead to false-negative results. Management of the huge amounts of data generated by WES and WGS is also a practical challenge that needs to be solved before it can be used routinely.

Interpretation of sequencing results

With the vastly growing numbers of genes identified by NGS, comes the need for classification of variants as either pathogenic, benign or VUS. Complementary investigations and rigorous interpretation are crucial to avoid misclassification.
  1. Frequency of variants in healthy controls
    After identification of a sequence variant in a patient, it needs to be determined whether this variant is present or absent in one of several public databases about the frequency of variants within the coding sequence of the human genome in apparently healthy controls. The frequency within controls will indicate whether a variant represents normal variation of a potential disease-associated mutation.
  2. Published data
    It should be checked whether a variant has been reported as disease causing, keeping in mind that data generated in the pre-NGS era involved a limited number of controls. A significant number of variants previously reported as pathogenic now appear to be benign based on their frequency in the normal population.
  3. Co-segregation in families
    Co-segregation of a variant with the condition in a large family with many affected individuals strongly suggests causation. This is, however, not a common clinical scenario. Many novel variants identified by NGS will often first be classified as VUS.
  4. Likely effect on the transcribed protein and evolutionary conservation
    The type of mutation (nonsense, missense, splice site), the level of conservation through evolution (compare with other species) and use of in silico prediction tools can give a hint at the impact on the protein structure that it may have (pathogenic or not).

Informed consent and ethical considerations

Prior to WES or WGS it is essential to discuss with patients wither genes unrelated to their conditions should be interpreted and whether they want to be informed about potentially relevant findings in genes unrelated to the condition for which they are being tested. Another challenge of NGS is the possibility of reclassification of genetic variants from benign to disease causing, or vice versa at a later stage.
WES and WGS are therefore currently considered to be a diagnostic method in development. The authors suggest that only recognized disease genes with substantial evidence of causality be investigated when offering routine genetic testing. In case of less evidence of causality genes should be classified as candidate genes, and primarily e investigated for research purposes.

Sharing information in public databases

In order to take full advantage of NGS, dedicated databases need to be developed that combine sequencing data and clinical information about patients. Sharing, comparing, and continuously updating this knowledge will facilitate correct interpretation of identified sequence variants, ensure clinical efficiency and maintain on-going evaluation of reported sequencing data.

Complexities in diagnosis and organisation of cardio-genetic services

The development of NGS makes is feasible and maybe tempting to apply less stringent indications for genetic testing. However, the diagnostic yield of genetic investigations is highest in patients with familial disease who meet diagnostic criteria for the condition under investigation. In case of an ambiguous clinical diagnosis, it is generally difficult to establish whether a sequence variant is causative.
It is therefore essential that cardiologists, molecular biologists, bio-informaticians and clinical geneticists work closely together to ensure accurate clinical diagnosis, provide optimal counselling and management of families with hereditary CV conditions. Pooling the experience of multidisciplinary teams in public databases will allow translating all data generated by NGS into usable knowledge for the benefit of patients and their families.

Find this article at the European Heart Journal