Genetic Analysis of Arrhythmogenic Diseases in the Era of NGS: The Complexity of Clinical Decision-Making in Brugada Syndrome

Abstract

Background: The use of next-generation sequencing enables a rapid analysis of many genes associated with sudden cardiac death in diseases like Brugada Syndrome. Genetic variation is identified and associated with 30-35% of cases of Brugada Syndrome, with nearly 20-25% attributable to variants in SCN5A, meaning many cases remain undiagnosed genetically. To evaluate the role of genetic variants in arrhythmogenic diseases and the utility of next-generation sequencing, we applied this technology to resequence 28 main genes associated with arrhythmogenic disorders.

Materials and methods: A cohort of 45 clinically diagnosed Brugada Syndrome patients classified as SCN5A-negative was analyzed using next generation sequencing. Twenty-eight genes were resequenced: AKAP9, ANK2, CACNA1C, CACNB2, CASQ2, CAV3, DSC2, DSG2, DSP, GPD1L, HCN4, JUP, KCNE1, KCNE2, KCNE3, KCNH2, KCNJ2, KCNJ5, KCNQ1, NOS1AP, PKP2, RYR2, SCN1B, SCN3B, SCN4B, SCN5A, SNTA1, and TMEM43. A total of 85 clinically evaluated relatives were also genetically analyzed to ascertain familial segregation.

Results and discussion: Twenty-two patients carried 30 rare genetic variants in 12 genes, only 4 of which were previously associated with Brugada Syndrome. Neither insertion/deletion nor copy number variation were detected. We identified genetic variants in novel candidate genes potentially associated to Brugada Syndrome. These include: 4 genetic variations in AKAP9 including a de novo genetic variation in 3 positive cases; 5 genetic variations in ANK2 detected in 4 cases; variations in KCNJ2 together with CASQ2 in 1 case; genetic variations in RYR2, including a de novo genetic variation and desmosomal proteins encoding genes including DSG2, DSP and JUP, detected in 3 of the cases. Larger gene panels or whole exome sequencing should be considered to identify novel genes associated to Brugada Syndrome. However, application of approaches such as whole exome sequencing would difficult the interpretation for clinical purposes due to the large amount of data generated. The identification of these genetic variants opens new perspectives on the implications of genetic background in the arrhythmogenic substrate for research purposes.

Conclusions: As a paradigm for other arrhythmogenic diseases and for unexplained sudden death, our data show that clinical genetic diagnosis is justified in a family perspective for confirmation of genetic causality. In the era of personalized medicine using high-throughput tools, clinical decision-making is increasingly complex.

Fig 1. Case-Family 1.

Segregation of the ANK2 c.8843C>G (p.(Ala2948Gly)) (rs138438183) and lack of segregation on PKP2 c.1577C>T (p.(Thr526Met)) (rs146882581_CM113820[11]), rare variant also detected in case 3. Case 1 (II.1), 47-year-old woman. Individuals (II.2 and II.3) were also diagnosed with BrS both with previous syncope and one of them (II.2) showed a positive ajmaline test.

Fig 2. Case-Family 2.

Positive segregation of ANK2 c.7334A>G (p.Asp2445Gly) and lack of segregation on ANK2 c.7132G>A (p.(Glu2378Lys)) (rs141191319) and AKAP9 c.5246T>C (p.(Ile1749Thr)) (rs150016098). The family history includes two brothers diagnosed at an age of 36 and 34 (III.2 and III.3, respectively), his asymptomatic sister (III.4), and his mother (II.2) affected by BrS. Individual III.2 showed a positive ECG pattern and a positive EPS although remaining asymptomatic. An ICD was implanted. Individual III.3 had a positive ajmaline test. Their previously asymptomatic mother (II.2) showed a positive EPS and an ICD was implanted.

Fig 3. Case-Family 3.

CASQ2 c.1148A>G (p.(Asp383Gly)) and KCNJ2 c.532G>A (p.(Ala178Thr)) segregation is shown. Also the lack of segregation of CACNB2 c.1511C>T (p.(Thr504Ile)) (rs143326262) and PKP2 c.1577C>T (p.(Thr526Met)) (rs146882581 also detected in Case 1). Individual I.1 died at age 65 because of chronic cardiovascular disease. Individual I.2 died at age 76 because of congestive heart failure. The second generation of this family includes 9 siblings (II.1-II.9), 5 of them-all male- died suddenly at ages between 42 and 53 years (II.1-II.5) and also a brother who died stillborn (II.9). Two siblings (II.7 and II.8), both female, died from other causes. The last sibling, a 91-year-old woman (II.6), is still alive and was diagnosed with atrial fibrillation (AF) at a young age. The third generation includes our index case (III.6), a 66-year-old man who was diagnosed with AF at 18 years of age. Notably, one SIDS case (III.4) was recorded in this generation. Clinical history and evaluation of the index case includes syncope at age 54, BrS pattern at ECG, and positive EPS, leading to an ICD implantation. His offspring are a 39-year-old asymptomatic daughter (IV.1) with negative EPS and procainamide test, a 20-year-old man (IV.2) who died suddenly, and a 31-year-old man (IV.3) diagnosed with AF, with negative procainamide test, positive EPS, and previously asymptomatic.

Fig 4. Case-Family 4.

Positive segregation of HCN4 c.3577G>C (p.(Glu1193Gln)) (rs200507617) with the pathology in the family. Individual II.1 is a symptomatic 70-year-old man (II.1) who showed a pathologic BrS ECG pattern and positive EPS, and had an ICD implanted. The genetic variant, predicted as deleterious, was absent in his healthy brother (II.2), with a non-pathological ECG.

Fig 5. Case-Family 5.

Positive segregation of JUP c.475G>T (p.(Val159Leu)) with the pathology in the family. This variant was previously considered as a pathogenic variant (CM1010258). Case II.1 is a 30-year-old man with a positive basal BrS ECG, syncope, and positive EPS. His brother (II.2), a 26-year-old man diagnosed with a positive ECG after flecainide test, and a positive EPS was also a carrier of the detected variation. Both relatives carry an ICD. Family history includes BrS diagnosed in their father (I.1).

Fig 6. Case-Family 6.

Positive segregation of the de novo detected variant RYR2 c.3803T>C (p.(Ile1268Thr)) with the pathology in the family. The index case (II.1) is a 48-year-old woman, previously asymptomatic, with a positive flecainide test and negative EPS study. The variant was also detected in one of her two daughters (III.1), clinically affected of BrS showing a positive ajmaline test. The second daughter (III.2) was clinically unaffected.

Fig 7. Case-Family 7.

Incomplete penetrance pattern of both variations in AKAP9 c.3827G>A (p.(Arg1276Gln)) (rs146797353) and the de novo detected variant AKAP9 c.8573A>G (p.(Tyr 2858Cys)) in the family. The index case, II.1, is a 44-year-old man with a positive basal ECG, previously asymptomatic, and positive EPS and flecainide test. No family history of SD was reported. The variant AKAP9 c.3827G>A (p.Arg1276Gln) was also detected in 4 more relatives: his asymptomatic mother (I.2); one clinically-affected sister with a positive flecainide test (II.3); an asymptomatic brother with negative ajmaline test (II.4); and an asymptomatic nephew (III.4). Familial segregation showed that the variant was absent in four clinically unaffected relatives: I.1, III.1, III.2, and III.3. Additionally, the index case showed another de novo variant in AKAP9 c.8573A>G (p.(Tyr2858Cys)), also identified in his two asymptomatic children (III.1 and III.2). This last variant, absent in the remaining available relatives, is also absent in the international databases.

Fig 8. Case Family 8.

Incomplete penetrance pattern of AKAP9 c.8656A>G (p.(Ile2886Val)) (rs143283097) in the family. Family history of sudden death was related. Individual III.3 is a 43-year-old woman previously asymptomatic, with a negative basal ECG but positive procainamide test and EPS. The variant was detected in her asymptomatic daughter (IV.4) and also in (III.2), clinically affected. Her asymptomatic brother (III.1) did carry the variation. The asymptomatic nephew (IV.3) did not carry the variant.

Fig 9. Case-Family 9.

Incomplete penetrance pattern of ANK2 c.3914G>A (p.(Arg1305Gln)) and the lack of penetrance of PKP2 c.1781T>C (p.(Ile594Thr)) in the family. Individual I.1 is a 74-year-old symptomatic man with both a positive ajmaline test and EPS. His 51-year-old daughter (II.1) was also diagnosed with BrS, showing a positive ajmaline test. Her 21-year-old granddaughter (III.1) is asymptomatic and had a negative ajmaline test. p.Arg1305Gln was not previously described and is predicted deleterious. A second variant considered deleterious, PKP2 (p.(Ile594Thr)), was detected in the index case (I.1), but was absent in II.1 and III.1.

Fig 10. Case-Family 10.

Incomplete penetrance of DSP c.1150G>C (p.(Glu384Gln)). Individual II.3 is a 48-year-old woman clinically affected with BrS. It was also confirmed in two more relatives: both asymptomatic sister and daughter (II.1 and III.3). The variant was absent in another sister and two daughters and a son (II.2, III.1, III.4 and III.2). Family history includes SCD in a 12-year-old daughter (III.5).

Fig 11. Case-Family 11.

Incomplete penetrance of DSP c.5218G>A (p.(Glu1740Lys)) (rs142885240). Individual II.2 is a 68-year-old woman, previously asymptomatic but with positive basal ECG and procainamide test. An ICD was implanted. Her affected son (III.1) and two clinically asymptomatic sons (III.2 and III.4) also carry the same genetic variant. Her husband (II.1) and children (III.3 and III.6), also asymptomatic, did not carry the variant.

Fig 12. Case-family 12.

Incomplete penetrance of PKP2 c.2504A>G (p.(Lys835Arg)) (rs372729739) in the family. Individual II.1 is a 38-year-old woman who suffered several syncopes and showed positive ECG-Type I after ajmaline test and had no family history of sudden death. Her son and daughter (III.1 and III.2) and two brothers (II.3 and II.4), all asymptomatic, did not carry the genetic variant. The variant was identified in an asymptomatic brother (II.2), and an affected sister (II.6) and child (III.5).