Novel Genomic Approach to the Arrhytmogenic Sudden Cardiac Death (Cardiac Arrhythmias and Genetics) Part 1

Introduction

Unfortunately, most of the common diseases in cardiology do not show traditional Mendelian genetics, they usually are complex genetic diseases resulting from the combination of multiple heritable and environmental factors. However, one of the cardiology dysfunction that can affect apparently healthy young adults or with any previous heart disease, such as sudden cardiac death (SCD), could be the first symptom of a Mendelian disease such as cardiomyopathies or channelopathies.

In many of the SCD cases, especially in case of young people, the cause of death cannot be explained neither after autopsy nor after laboratory tests. Inherited heart diseases such as hypertrophic cardiomyopathy (HCM), arrhythmogenic right ventricular cardiomyopathy (ARVC) and primary electrical diseases such as long QT syndrome (LQTS), Brugada syndrome (BrS) or catecholaminergic polymorphic ventricular tachycardia (CPVT), are the main cause of death in young adults with no previous clinical history. Most often these inherited cardiac disorders give rise to lethal ventricular arrhythmias and show an autosomal dominant mode of inheritance.

Genetic screening of the genes described as implicated in the different pathologies may help to determine the cause of death and to evaluate the potential risk of the relatives. Today we know which are the main causes of sudden cardiac death in young adults and we also know which are the genes responsible of these diseases in a high percentage of cases. The aim of this article is to present to the reader the estate of the art of the use of the new next generation sequencing technologies for the study of arrhythmogenic sudden cardiac deaths. We will discuss the different available technologies, and the different applications: Candidate gene resequencing. We will describe the interesting genes to be studied and the different strategies available for their enrichment and sequencing Whole exome resequencing. We will describe the application of this approach to those cases were we need to look for new genes.


Inherited arrhythmogenic diseases

There are various arrhythmogenic disorders, with different electrocardiographic patterns, which are not always present or are not evident in carriers of mutations implicated in the pathology. In general these are diseases with low penetrance so the genetic study is of great importance in patients with personal or family history of sudden cardiac death. The term inherited arrhythmogenic diseases typically includes cardiac diseases caused by mutations in ion channels and ion channel-controlling proteins such as the long-QT syndrome (LQTS), the short QT syndrome (SQTS), the Brugada syndrome (BrS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). Ion channels are integral membrane proteins that regulate the flow of ions across the cell membrane. They consist of multimeric units generally encoded by different genes. The a subunit forms the pore and mediates ion current, while the p subunits are regulatory. Defects in these channels due to mutations in genes that encode proteins, or mutations in proteins associated with these channels may lead to an electrical disturbance in the heart linked to the development of channelopathies.

Long QT syndrome

The Long QT Syndrome (LQTS) is characterized by prolongation of the QT interval on the electrocardiogram, which indicates ventricular repolarisation unusually long, due either to a decrease in the K+ flow of repolarisation or to a delayed entry of Na+ into the myocyte. The estimated incidence is between 1:2000 -1:5000 people (Hedley et al., 2009), and its penetrance is variable, ranging from sub clinical phenotypes with a QT interval at the limit, without arrhythmias or syncope (Napolitano et al., 2005; Priori et al., 1999; Vicent et al., 1992) to sudden cardiac death, being a major cause of sudden death in young people. To determine wheter the QT interval is prolonged the corrected QT interval (QTc) is used, which is calculated using the Bazzet formula QTc = QT / V RR (Bazzet et al., 1920) Are considered abnormally prolonged QTc values when exceeding 440 ms in men and those over 460 ms in women (values corrected for heart rate). It is estimated that a patient with a QTc interval of 550 ms has a 63% higher risk of suffering a cardiac event that an individual with a value of QTc interval of 450 ms. (Zareba et al., 2008).

The LQTS shows a congenital form of the disease in about 85% of cases and a sporadic form which corresponds to 15% [85]. There is also an acquired form of QT prolongation caused mainly by drugs, both cardiac (e.g. antiarrhythmics) and other medications (e.g. antidepressants) or derived from an electrolyte imbalance. The congenital form shows two basic patterns of inheritance, one autosomal dominant called Romano-Ward syndrome and another autosomal recessive known as Jervell Lange-Nielsen syndrome, which presents with congenital deafness. To date 12 genes have been described in the pathology. The most common are genes that encode K + channels, KCNQ1, KCNH2, which represent about 4055% and 35-45% of cases respectively, and the SCN5A gene coding for Na + channels which represents a 2 – 8% of cases (Hedley et al., 2009). The involvement of each of these genes leads to defined clinical phenotypes, so KCNQ1 gene leads to LQTS type 1 (LQT1), KCNH2 causes LQTS type 2 (LQT2) and SCN5A leads to LQTS3 (LQT3).

Short QT syndrome

The short QT syndrome (SQTS) has been recognized as a clinical entity characterized recently by a shortened QT interval that can lead to arrhythmias and sudden cardiac death (Gussak et al., 2000). Although there is no established consensus, it is accepted that a value between 320ms and 340 ms are considered short (although it could be considered normal 340ms) if there is a history of cardiac symptoms such as syncope or aborted sudden death. Nevertheless, as in LQTS, the transition zone of normal QT intervals to pathological intervals is difficult to establish (Schimpf et al., 2007).

In SQTS, mutations in genes encoding K + channels: KCNH2, KCNJ2, and KCNQ1, cause a gain of function of these channels and give rise to SQTS1, SQTS2and SQTS3 respectively. However, mutations in genes encoding a and p2 subunits of the Cav1.2 channel, the CACNB2 and CACNA1C genes, cause loss of function of these channels and give rise to SQTS4 and SQTS5 phenotype respectively (Hedley et al., 2009).

Brugada syndrome

Brugada Syndrome (BrS) is characterized by ST segment elevation in right precordial leads (V1-V3) of the electrocardiogram and can also be associated with an increase in PR interval and bundle branch block. Penetrance is also variable, and can trigger polymorphic ventricular tachycardia and / or ventricular fibrillation and sudden death. The distribution and incidence of this disease is difficult to determine because it is a syndrome recently described and because electrocardiographic manifestations are not always present. Campuzano et al. (2010) estimate a prevalence of approximately 35/100000 persons / year and they say that, although symptoms usually develop around the age of 40, sudden death can affect individuals of any age. They estimate sudden death affects 75% of the affected males, and between 20 and 50% of the affected people have a family history of sudden death.

The dominant pattern of inheritance is autosomal dominant with expression probably age dependent and incomplete penetrance. Today we have described more than 100 mutations in 7 different genes that give rise to the 7 types of BrS. These genes encode proteins of both Na + channels and other ion channels. The mutations affect the proper development of phases 1 and 2 of the cardiac action potential (Hedley et al., 2009b). It is estimated that 2025% of BrS patients show mutations in the SCN5A gene (Schott et al., 1999), which also represents 5-10% of mutations found in cases of sudden cardiac death in adults and children (Hedley et al., 2009b) .

Catecholaminergic polymorphic ventricular tachycardia

Catecolaminergic polymorphic ventricular tachycardia (CPVT) occurs with a characteristic pattern of bidirectional or polymorphic tachycardia related to stress without structural cardiac abnormalities (Coumel et al., 1978). These clinical manifestations often occur during childhood and adolescence.

The CPVT has two modes of inheritance: autosomal dominant and recessive. The autosomal dominant form is caused by mutations in the gene encoding the ryanodine receptor RyR2, which is a large protein that forms the calcium release channel in sarcoplasmic reticulum. The recessive form of the disease is caused by mutations in the cardiac isoform calciquestrina gene (CASQ2), which binds to the ryanodine receptor and participates in the control of excitation-contraction (Ylanen et al., 2010). The steps of the molecular pathogenesis of CPVT are not entirely clear, but Mutations of the two interacting proteins, RyR2 and CASQ2, seem to result in inadequately controlled Ca2+ bursts into the sarcoplasm, with concomitant risk of delayed afterdepolarizations and triggered arrhythmia.

Genetic screening of arrhythmogenic diseases

The importance of knowing the molecular substrate in patients with inherited cardiac channelopathies is recognized and highlighted in the guidelines for the prevention of SCD developed by the American Heart Association, the American College of Cardiology, and the European Society of Cardiology (Zipes et al., 2006). Screening for mutations in genes that encode cardiac ion channels associated with LQTS, SQTS, BrS, and CPVT is primarily sought in clinically affected patients to tailor risk stratification and management and to further identify family members (Priori et al., 2002a, b, 2003). However, genetic analysis is not yet available at most clinical centres and it is still mainly performed in finite research laboratories.

Our present understanding of human inherited arrhythmia diseases has become increasingly complex. Several clinical syndromes have been identified as human inherited arrhythmia diseases and at least 21 genes are known to cause these diseases. These genes and the associated syndromes are given in Table 1. Mutations associated with inherited arrhythmia syndromes occur in ion channel pore-forming proteins, associating subunit proteins and channel interacting proteins ,Ca2+ handling proteins, components of the ion cannel macromolecular complex, and regulatory pathways. Although most inherited arrhythmia syndromes are rare clinical findings, sometimes with just a single family described.

Several studies have been published trying to determine the effectiveness of genetic screening (Bai et al., 2009; Kapplinget et al., 2009) in terms of efficiency and cost. Bay et al. (2009) showed that the current cost of genetic testing for inherited cardiac channelopathies is reasonable for those who have a conclusive diagnosis and that these patients should have priority access to genetic screening (Fuster et al., 2008) However, until now these studies were limited by two main drawbacks, the reduced effectiveness of the techniques of genetic determination employed and the high cost of the same.

Gen

Symbol

Locus

CPVT LQTS

SQTS

BrS

A kinase anchor protein (yotiao) 9

AKAP9

7q21-q22

x


ankyrin 2

ANK2

4q25-q27

x

calcium channel, voltage-dependent, L type, alpha 1C subunit

CACNA1C

12p13.3

x

x

x

calcium channel, voltage-dependent, beta 2 subunit

CACNB2

10p12

x

x

calsequestrin 2

CASQ2

1p13.3-p11

x

caveolin 3

CAV3

3p25

x

glycerol-3-phosphate dehydrogenase 1-like

GPD1L

3p22.3

x

hyperpolarization activated cyclic nucleotide-gated potassium channel 4

HCN4

15q24.1

x

potassium voltage-gated channel, Isk-related family, member 1

KCNE1

21q22.12

x

potassium voltage-gated channel, Isk-related family, member 2

KCNE2

21q22.12

x

potassium voltage-gated channel, Isk-related family, member 3

KCNE3

11q13.4

x

potassium voltage-gated channel, subfamily H, member 2

KCNH2

7q36.1

x

x

potassium inwardly-rectifying channel, subfamily J, member 2

KCNJ2

17q24.3

x

x

potassium inwardly-rectifying channel, subfamily J, member 5

KCNJ5

11q24

x

potassium voltage-gated channel, KQT-like subfamily, member 1

KCNQ1

11p15.5

x

x

ryanodine receptor 2

RYR2

1q43

x

sodium channel, voltage-gated, type I, beta

SCN1B

19q13,1

x

sodium channel, voltage-gated, type III, beta

SCN3B

11q23,3

x

sodium channel, voltage-gated, type IV, beta

SCN4B

11q23.3

x

sodium channel, voltage-gated, type V, alpha subunit

SCN5A

3p21

x

x

syntrophin, alpha 1

SNTA1

20q11.2

x

Table 1. Genes related to arrhythmogenic sudden cardiac death

Today, with the development of the next generation sequencing strategies, these two problems are being overcome, so that on one hand, we managed to sequence as many genes as we want, detecting both, genetic variants already described and new variants not yet known; and on the other hand, we have significantly reduced the cost of each genetic screening and we hope that this reduction will still see increased in the future days. The new next generation sequencing technologies are allowing us sequencing large number of DNA fragments or genes, using target resequencing strategies, in a fast, reliable and effective way. The selection of the genes will depend on the researcher’s own interests, so that in our case, we could focus on those genes previously described as involved in arrhythmogenic heart diseases or we can make the sequencing of all genes and search exome mutations also in genes that have not previously been associated with the pathology. If we consider the aforementioned 21 genes as candidate genes to be sequenced, It would involve the sequencing of approximately 400 exons, accounting around 120.000 base pairs of coding DNA. This work, in terms of time and cost of each analysis represents a major handicap for the routine work of many small laboratories dedicated to genetic diagnosis of these pathologies. An indicative example of this type of analysis is the Familion test for Long QT syndrome (Kapplinger et al., 2009), a bidirectional DNA sequencing-based assay that comprises analysis of 73 polymerase chain reaction (PCR) amplicons to analyse the 3 major LQTS-susceptibility genes (KCNQ1 [LQT1], KCNH2 [LQT2], SCN5A [LQT3]) along with 2 minor genes (KCNE1 [LQT5] and KCNE2 [LQT6]). Kapplinger et al (2009) evaluated the Familion Test in 2500 unrelated LQTS cases and they found 903 positive genetic tests describing 562 putative mutations absent in 2600 reference alleles. They reported that despite the passage of 14 years since the first LQTS-causative mutations were discovered, still one-third of the mutations being discovered today are novel; therefore, this study is further evidence of the need for genetic screening strategies that allow us to detect both known mutations and new genetic variants, such as the sequencing. In addition, the study highlights the need for functional studies providing evidence on the possible pathogenicity for new genetic variants that are being described. Here we describe the implementation of a new research strategy using next generation sequencing, that allows the simultaneous study of the sequence of all the genes described in relation to arrhythmogenic disorders at risk of sudden cardiac death (candidate gene approach), or the study of the complete sequence of the human exome (whole exome approach), searching for genetic variants both in genes previously associated with sudden cardiac death and in new genes whose involvement in the fatal event is currently unknown

Next generation sequencing

Capillary electrophoresis based in Sanger sequencing is the technology widely used for analyzing genes involves in different pathologies. However, over the past five years, Next Generation Sequencing (NGS) technologies have became a reliable tool for massive parallel sequencing, overcoming the limitations in throughput and speed of capillary electrophoresis (Shendure & Ji, 2008; Metzer, 2010; Glenn , 2011) .

On this topic we will focus on commercially available platforms: 454 (Roche), Illumina Genome Analyzer (Illumina Inc.), SOLiD and Ion Torrent (Life Technologies) (Table 2). The 454 Genome Sequencer (Roche) was the first NGS platform available (Margulies et al., 2005). Small fragments of DNA are attached onto the surface of beads and amplified via emulsion PCR. Millions of beads are deposited onto a picotitre plate. Sequencing is performed in parallel by pyrosequencing, where the incorporation of a nucleotide by a DNA polymerase results in the release of a pyrophosphate, which initiates a series of downstream reactions that ultimately produce light by a luciferase. The light can be correlated with the nucleotide incorporated, because the nucleotides are added following a sequential order. The Illumina Genome Analyzer (Illumina Inc.) relies on bridge PCR on a glass slide to amplify small fragments of DNA. In this approach, forward and reverse PCR primers are attached to a solid surface, and as a consequence, amplification products originating from any single template molecule remain immobilized and clustered to a physical position on the array. Sequencing chemistry is based on sequencing by synthesis with reversible terminators (Fedurco et al., 2006; Turcatti et al., 2008), where all fluorescently labeled four nucleotides are added simultaneously to the flow cell channels, along with the polymerase, for incorporation into the oligo-primed cluster fragments obtained after bridge PCR. The SOLiD system (Life Technologies) is based on sequencing by ligation and the use of two-base encoded probes (Valouev et al., 2008). A universal sequencing primer is hybridized to templates and a pool of fluorescently labelled octamer probes containing all possible combination of A, C, G and T at positions 1-5, interrogates the sequence of the unknown template on each bead. Only the probe homologous to the first five bases of the template will be ligated to the universal sequencing primer. Up to ten cycles of ligation, detection and cleavage record the colour at every fifth position. Templates for sequencing are prepared via emulsion PCR.

In the case of the Ion PGM Sequencer (Life Technologies), sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all natural nucleotides on the ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in 1.2 million wells, which allow parallel and simultaneous detection of independent sequencing reactions (Rothberg et al., 2011). As 454 and SOLiD, template preparation is performed by emulsion PCR. Unlike the other technologies where the throughput is determined by the equipment, the Ion PGM throughput is determined by the chip used for sequencing (Table 2)

Very promising NGS approaches are the ones based on single molecule sequencing like Helicos Biosciences (Harris et al., 2008) and Pacific Biosciences (Eid et al., 2009), where sequencing is performing directly on the DNA, avoiding any amplification step. However, these platforms are not commercially available so they are only mentioned.

Instrument

Read length (bp)

Maximum Throughput

Run time

454-GS Junior

400

50 Mb

10 h

454-FLX+

700

900 Mb

23 h

Illumina-MiSeq

150+150

> 1 Gb

27 h

Illumina-GAII

150+150

95 Gb

14 days

Illumina-HiScanSQ

100+100

150 Gb

11 days

Illumina-HiSeq1000

100+100

300 Gb

11 days

Illumina-HiSeq2000

100+100

600 Gb

11 days

SOLiD-5500

75+35

90 Gb

7 days

SOLiD-5500xl

75+35

180 Gb

7 days

Ion PGM – 314 chip

200

>10 Mb

2 h

Ion PGM – 316 chip

200

>100 Mb

2 h

Table 2. Comparison of NGS platforms.

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