Categorized Genetic Analysis in Childhood-Onset Cardiomyopathy

Two-hundred five unrelated families with childhood-onset cardiomyopathy, who were referred from different regions of Saudi Arabia, were consecutively recruited over a period of 7 years. The vast majority of the families (n=182; 88.8%) were consanguineous. There were a family history of cardiomyopathy in 105 (51.2%) families. The median age at presentation was 10 months (range:1 day–17 years, average 2.2 years). Cardiological evaluation revealed that 117 (57.1%) children were affected with dilated cardiomyopathy (DCM), 82 (40.0%) with hypertrophic cardiomyopathy (HCM), 7 (3.4%) with isolated or associated left ventricular noncompaction, and 3 (1.5%) with restrictive cardiomyopathy (Figure 1).

For genes known to be associated with cardiomyopathy, the yield was 53.7% (110 out of 205 cases). In 62 (30.2%) families, the genetic alteration was identified through Targeted Genetic Test which had a yield of 82.7%. In the second category, 143 cases underwent WES (n=118) or WGS (n=25), which had a yield of 37.3% and 16.0%, respectively (Figure 2).

In multiplex families (n=105), the genetic test was positive in 61.0%, whereas in singleton families it was 46.0% (Figure 3). The yield of genetic test in DCM families was 41.9%; 69.4% of which were uncovered by WES or WGS, whereas in HCM families, the yield was much higher at 72.0%; however, only 22.0% were uncovered by WES or WGS (Table III in the Data Supplement).

In 8 families in which samples from affected children were not available, heterozygous pathogenic mutations were identified in the parents in 5 (Tables III and IV in the Data Supplement).

Strikingly, 96.4% (106/110) of the families were segregating homozygous variants, whereas only 2.7% (3/110) had de novo heterozygous and 0.9% (1/110) hemizygous variants. Two founder alleles in ELAC2 and ACADVL represented 15.1% and 8.3% of cases, respectively (Table IV in the Data Supplement). However, a tremendous genetic and allelic heterogeneity was observed with 50 different alleles detected in 27 cardiomyopathy-associated genes (Figure 4A, Table III in the Data Supplement). Out of the 50 alleles, 29 (58.0%) were truncating, and 19 (38.0%) were missense (Figure 4B). Half of the variants were novel (Table III in the Data Supplement). No plausible copy number variants or deep intronic variants were detected through WGS.

In this largest published series of children with cardiomyopathy that underwent WES/WGS, we report the value, yield, and implications of a categorized genetic approach. As a consequence of the common practice of consanguineous marriage in our population, a remarkable difference in the frequency of homozygous mutations was observed. In this study, which had a yield of 53.7%, homozygous variants were detected in 96.4% of the probands. Although not unexpected, this finding is extremely different from what has been known in childhood-onset cardiomyopathy where genetic analysis typically reveals a heterogeneous patterns of inheritance.^6–8

The allelic and genetic heterogeneity observed in this study reflects the heterogeneous tribal structure of the Saudi population. With the exception of the 2 prevalent founder mutations in ELAC2 and ACADVL, no single allele represented the vast majority of the genetic causes of cardiomyopathy.

Two other factors had clear impact on the diagnostic yield: positive family history and type of cardiomyopathy. In multiplex families the detection rate was higher compared with singleton families (61% versus 46%) as it was for HCM compared with DCM (72% versus 42%); a finding that is similar to previously published reports.^22,23

Sarcomere genes, which are well known to cause a large proportion of cases with HCM,²⁴ were mutated in only 11.8% of cases with positive result. The spectrum of sarcomere genes detected in our cases was also different with NRAP being the most common. Mutated NRAP, recently reported in 2 families,^8,14 was found in 7 probands in our cohort which represented 14.3% of DCM cases with positive result, suggesting that it is not an uncommon cause of recessively inherited form of childhood cardiomyopathy.

Several genes are known to cause cardiomyopathy in a dominant and recessive pattern of inheritance.^25–27 In our cohort, 9 (4.4%) families segregated homozygous variants in genes (FLNC, MYLK3, NEXN, VCL) that have only been reported to cause a dominant form of cardiomyopathy. The affected children were homozygous, whereas their parents were unaffected heterozygous carriers. The lack of clinical manifestations in the parents suggests that the variants are either nonpenetrant or potentially causative of cardiac involvement at an older age in heterozygous state. The high rate of consanguinity has apparently uncovered these variants that would otherwise be missed.

The majority (73%) of children that had positive result presented with only, or predominantly, cardiac involvement without a clinical clue to the gene defect which underscores the importance of devising an appropriate approach to the genetic diagnosis. Knowing that detecting a founder mutation is very likely in our highly consanguineous population, we adopted a categorized approach which would give an opportunity for rapid diagnosis at a lower cost compared with multigene panel, WES, or WGS. By adopting this approach, Targeted Genetic Test detected almost one-third of the cases (30.2%) and had a high yield (82.7%). In view of the tribal structure of our population, we predicted at the time of devising our approach that some of the uncovered variants would be in genes not included in commercially available multigene panels or would be found in novel candidate genes. Hence, in cases where clinical clues to a specific genetic defect were not observed or a founder mutation was unlikely or negative, we shifted to the most comprehensive genetic analysis by WES/WGS instead of using a multigene cardiomyopathy panel. In our cases, using WES and WGS detected variants in 13 families (11.8% of positive cases) in 4 genes (KLHL24, MYLK3, NRAP, PPP1R13L) that have been recently implicated in cardiomyopathy and are normally not included in multigene panels.

The clinical use of WGS has been recently investigated as a diagnostic tool in cardiomyopathy. The added advantage of this approach is that it provides a platform for detecting variants (eg, in deep intronic or regulatory regions) that would not be detected by WES or multigene panel. Bagnall et al²⁸ reported the result of performing WGS in adult-onset HCM as a first-line genetic test. A pathogenic variant was detected in 5 of 12 families (42%) that had never received prior genetic testing. We have performed WGS in 25 cases which revealed a yield of 16.0% for variants in cardiomyopathy-associated genes, none of which were outside the coding regions. However, if we had adopted WGS as a first-line test in all our cases it would be expected that we would have a similar yield (53.4%) if not higher should other variants (eg, deep intronic and Copy Number Variants) be detected.

Furthermore, and as indicated by Cirino et al,²⁹ WGS offers the advantage of reanalysis over time to incorporate advances in knowledge. In our cohort, 74 families have more than one child with cardiomyopathy, which is highly suggestive of a genetic cause, and yet 41 (55%) of them had negative WES or WGS. The availability of a comprehensive genetic data in these families provides a continuing opportunity for reanalysis that would hopefully facilitate the identification of new genes for cardiomyopathy.

With the rapid decline in the cost of WGS and the improvement of available tools of filtering and interpreting genetic variants, WGS will likely be a first-line genetic test in many genetically heterogeneous conditions. It is needless to say, however, that the risk of uncovering incidental findings when WES or WGS are used should be taken into account at the time of consenting for the genetic test.³⁰ The genetic analysis in our cases was conducted on a research basis; we opted not to report the incidental findings.

Early detection of the genetic cause of cardiomyopathy is critical for the initiation of treatment in treatable conditions; notably, fatty acid oxidation defect and Pompe disease. Furthermore, in cases with severe cardiomyopathy that may require heart transplantation, identifying the genetic cause helps in prioritizing candidacy of transplantation. Cases with mutations in genes implicated in potentially treatable conditions (SLC22A5, ACADVL, GAA), or in multisystem diseases (eg, mitochondrial genes) are not normally considered as appropriate candidates for transplantation.

In addition to providing the appropriate medical intervention to affected cases, identifying the genetic cause in families with cardiomyopathy would serve several other clinical purposes. Establishing the diagnosis helps in providing the proper genetic counseling to patients and their families. Recurrence of the disease in next pregnancy can be prevented either by prenatal diagnosis or preimplantation genetic diagnosis. Also, testing at-risk family members for carrier status and targeted premarital/carrier screening would also be possible to prevent the recurrence of a potentially fatal disease.

The identification of recessive inheritance of dominant genes highlights the importance of long-term follow-up of asymptomatic parents, should they be presymptomatic rather than nonpenetrant. In contrast to classic recessive disease, preventive intervention utilizing preimplantation genetic diagnosis with selecting embryos that are wild type only would provide a prudent approach to avoid the uncertainty of clinical affection in individuals who are heterozygous carriers.

Our work and previously published data clearly indicate that other genes implicated in cardiomyopathy are yet to be discovered. We report variants in 7 genes (ACACB, AASDH, CASZ1, FLII, RHBDF1, RPL3L, ULK1) that were filtered utilizing several methods. All the detected variants were either novel or have a minor allele frequency of <0.001, predicted to be damaging, segregated with the phenotype in all the tested families, and lay within a region of homozygosity. All the candidate genes encode for proteins that are highly expressed in heart muscles.

Yet, implicating these genes in cardiomyopathy is still very preliminary; biological and functional studies on the encoded proteins and animal models may either confirm or exclude a causal effect. However, we elected to report these variants should other cases be identified to have other alleles in the same genes which would strengthen the association with cardiomyopathy.

The cohort of our study was mostly from consanguineous families which had enriched the results with recessively inherited homozygous variants on the expense of other modes of inheritance. The categorized approach of genetic testing adopted in the study may therefore have limited value in nonconsanguineous population.

In such a highly consanguineous cohort, the risk of bias towards homozygous variants cannot be entirely excluded. Tendency to refer a case for genetic evaluation might have been higher in the presence of consanguinity compared with singleton nonconsanguineous cases which could have resulted in missing de novo mutations. In addition, filtering WES/WGS for homozygous variants in consanguineous cases is relatively simpler than for dominant and de novo variants.

The utilization of a comprehensive genetic analysis with WES/WGS has provided insight on its value and yield; however, a larger multiethnic sample size, especially for WGS, is required to better understand the clinical utility of adopting this approach in pediatric cardiomyopathy.

Our work expands the diversity of genetic cause in childhood cardiomyopathy. It demonstrates the value, yield, and implications of adopting a categorized genetic approach that is population-sensitive. It also illustrates the impact of consanguinity on the prevalence of homozygous variants and on the recessive inheritance of dominant genes. The lack of a genetic diagnosis in a large proportion of cases with childhood cardiomyopathy suggests that adopting WES/WGS as a first-line test may be considered.