Targeted next-generation sequencing panels in the diagnosis of Charcot-Marie-Tooth disease

Abstract

Objective: To investigate the effectiveness of targeted next-generation sequencing (NGS) panels in achieving a molecular diagnosis in Charcot-Marie-Tooth disease (CMT) and related disorders in a clinical setting.

Methods: We prospectively enrolled 220 patients from 2 tertiary referral centers, one in London, United Kingdom (n = 120), and one in Iowa (n = 100), in whom a targeted CMT NGS panel had been requested as a diagnostic test. PMP22 duplication/deletion was previously excluded in demyelinating cases. We reviewed the genetic and clinical data upon completion of the diagnostic process.

Results: After targeted NGS sequencing, a definite molecular diagnosis, defined as a pathogenic or likely pathogenic variant, was reached in 30% of cases (n = 67). The diagnostic rate was similar in London (32%) and Iowa (29%). Variants of unknown significance were found in an additional 33% of cases. Mutations in GJB1, MFN2, and MPZ accounted for 39% of cases that received genetic confirmation, while the remainder of positive cases had mutations in diverse genes, including SH3TC2, GDAP1, IGHMBP2, LRSAM1, FDG4, and GARS, and another 12 less common genes. Copy number changes in PMP22, MPZ, MFN2, SH3TC2, and FDG4 were also accurately detected. A definite genetic diagnosis was more likely in cases with an early onset, a positive family history of neuropathy or consanguinity, and a demyelinating neuropathy.

Conclusions: NGS panels are effective tools in the diagnosis of CMT, leading to genetic confirmation in one-third of cases negative for PMP22 duplication/deletion, thus highlighting how rarer and previously undiagnosed subtypes represent a relevant part of the genetic landscape of CMT.

Figure 1. Distribution of patients receiving genetic diagnosis according to Charcot-Marie-Tooth disease (CMT) subtype

CMTR = Charcot-Marie-Tooth disease and related disorders; dHMN = distal hereditary motor neuropathy; HSN = hereditary sensory neuropathy.

Figure 2. Representative examples of ancillary testing to next-generation sequencing (NGS) performed in selected cases

(A) Case 122 presented with early onset of demyelinating neuropathy associated with scoliosis and cranial nerve involvement. He had a sister with a similar condition. NGS for genes associated with Charcot-Marie-Tooth disease type 1 (CMT1) and intermediate was performed and identified a single c.386-2A>C mutation in SH3TC2. Relative read-depth analysis of NGS was performed (A.b) looking for copy number variant in SH3TC2 and identified a deletion of exon 7 (indicated by a red * on the read depth plots), which was confirmed by long PCR in both siblings (A.a), in compound heterozygous state with the c.386-2A>C. (B) Patient 139 was diagnosed in the first decade of life with CMT1. A targeted NGS panel was performed at age 72, which identified 2 variants in FDG4 1304_1305delinsAA p.(Arg435Gln) and FDG4:c.1192-48_1233del. Long-range PCR was performed followed by Sanger sequencing of the gel band-extracted PCR product (red square box) identifying the breakpoints of 90–base pair FGD4 deletion. (C) Patient 164 presented with early-onset CMT1. NGS targeted panel for CMT1 genes was performed at age 35 and identified a homozygous 892-1 G>T variant in NDRG1, bearing potential to disrupt splicing of the flanking exons. RNA was extracted from peripheral blood and retrotranscribed into cDNA showing that the splicing mutation leads to a 9–base pair deletion of NDRG1 transcript (c.892_900delCCGGCCAAG) resulting in an in-frame deletion of 3 amino acids (red box). As opposed to typical CMT4D cases due to stop mutations in NDRG1, patient 164 presented a relatively mild neuropathy without clinical evidence of hearing loss, suggesting that the splicing mutation leading to in-frame deletion of 3 amino acidic residues may not abolish NDRG1 function.