NGS-Based Genetic Analysis in a Cohort of Italian Patients with Suspected Inherited Myopathies and/or HyperCKemia

Abstract

Introduction/Aims HyperCKemia is considered a hallmark of neuromuscular diseases. It can be either isolated or associated with cramps, myalgia, weakness, myoglobinuria, or rhabdomyolysis, suggesting a metabolic myopathy. The aim of this work was to investigate possible genetic causes in order to help diagnose patients with recurrent hyperCKemia or clinical suspicion of inherited metabolic myopathy. Methods A cohort of 139 patients (90 adults and 49 children) was analyzed using a custom panel containing 54 genes associated with hyperCKemia. Results A definite genetic diagnosis was obtained in 15.1% of cases, while candidate variants or variants of uncertain significance were found in a further 39.5%. Similar percentages were obtained in patients with infantile or adult onset, with some different causative genes. RYR1 was the gene most frequently identified, either with single or compound heterozygous variants, while ETFDH variants were the most common cause for recessive cases. In one patient, mRNA analysis allowed identifying a large LPIN1 deletion missed by DNA sequencing, leading to a certain diagnosis. Conclusion These data confirm the high genetic heterogeneity of hyperCKemia and metabolic myopathies. The reduced diagnostic yield suggests the existence of additional genes associated with this condition but also allows speculation that a significant number of cases presenting with hyperCKemia or muscle symptoms are due to extrinsic, not genetic, factors.

Keywords: Next Generation Sequencing (NGS); creatine kinase; hyperCKemia; myoglobinuria; rhabdomyolysis; skeletal muscle damage.

Figure 1

Type of candidate variants and affected genes identified in this study. (A) Distribution of the identified variants according to the tiers defined by the ACMG criteria (P: pathogenic; LP: likely pathogenic; VUS: variant of uncertain significance; LB: likely benign; B: benign). (B) Number of variants (P: Pathogenic; LP: Like Pathogenic; VUS: variant of uncertain significance) found in different (38/54) genes analyzed by our NGS panel.

Figure 2

Distribution of the investigated patients in the selected groups according to their genetic diagnosis. Patients were classified as “solved” (with a definitive diagnosis), “uncertain” (with an inconclusive diagnosis), or “unsolved” (negative cases). Panel (A) reports infantile cases; panel (B) reports adult cases.

Figure 3

Deep DNA and RNA analysis of the subject with LPIN1 variants. (A) Pedigree of the patient 6/C (II-2), carrying two variants in LPIN1, and segregation analysis in the family. (B) Amplification of the LPIN1 transcript, which highlights two bands in the proband II-1, while control DNA (ct) has a single band. Sanger electropherograms show that the upper band corresponds to the wild-type transcript, and the lower one corresponds to a transcript missing exons 19–20. (C) Snapshots from Integrative Genomic Visualization showing NGS analysis of the genomic region containing LPIN1 exons 19–20. The lower coverage of exon 19 in II-2 compared to control (ct) is due to a heterozygous exon deletion; in II-2, some reads (arrow) map to the region upstream of the deletion. (D) Genomic DNA analysis by PCR using specific primers to define the deletion breakpoints (b = blank sample, ct = control sample, II-2 patient 6/C, I-1: father; I-2: mother). Sanger sequencing of the amplicon containing the deletion reveals it is c.2295–866_2410-30del.