Cohort Analysis of 67 Charcot-Marie-Tooth Italian Patients: Identification of New Mutations and Broadening of Phenotype Expression Produced by Rare Variants

Rosangela Ferese, Rosa Campopiano, Simona Scala, Carmelo D'Alessio, Marianna Storto, Fabio Buttari, Diego Centonze, Giancarlo Logroscino, Chiara Zecca, Stefania Zampatti, Francesco Fornai, Vittoria Cianci, Elisabetta Manfroi, Emiliano Giardina, Mauro Magnani, Antonio Suppa, Giuseppe Novelli, Stefano Gambardella, Rosangela Ferese, Rosa Campopiano, Simona Scala, Carmelo D'Alessio, Marianna Storto, Fabio Buttari, Diego Centonze, Giancarlo Logroscino, Chiara Zecca, Stefania Zampatti, Francesco Fornai, Vittoria Cianci, Elisabetta Manfroi, Emiliano Giardina, Mauro Magnani, Antonio Suppa, Giuseppe Novelli, Stefano Gambardella

Abstract

Charcot-Marie-Tooth (CMT) disease is the most prevalent inherited motor sensory neuropathy, which clusters a clinically and genetically heterogeneous group of disorders with more than 90 genes associated with different phenotypes. The goal of this study is to identify the genetic features in the recruited cohort of patients, highlighting the role of rare variants in the genotype-phenotype correlation. We enrolled 67 patients and applied a diagnostic protocol including multiple ligation-dependent probe amplification for copy number variation (CNV) detection of PMP22 locus, and next-generation sequencing (NGS) for sequencing of 47 genes known to be associated with CMT and routinely screened in medical genetics. This approach allowed the identification of 26 patients carrying a whole gene CNV of PMP22. In the remaining 41 patients, NGS identified the causative variants in eight patients in the genes HSPB1, MFN2, KIF1A, GDAP1, MTMR2, SH3TC2, KIF5A, and MPZ (five new vs. three previously reported variants; three sporadic vs. five familial variants). Familial segregation analysis allowed to correctly interpret two variants, initially reported as "variants of uncertain significance" but re-classified as pathological. In this cohort is reported a patient carrying a novel familial mutation in the tail domain of KIF5A [a protein domain previously associated with familial amyotrophic lateral sclerosis (ALS)], and a CMT patient carrying a HSPB1 mutation, previously reported in ALS. These data indicate that combined tools for gene association in medical genetics allow dissecting unexpected phenotypes associated with previously known or unknown genotypes, thus broadening the phenotype expression produced by either pathogenic or undefined variants. Clinical trial registration: ClinicalTrials.gov (NCT03084224).

Keywords: Charcot-Marie-Tooth disease; diagnosis; multiple ligation dependent probe amplification; neurogenetics; next-generation sequencing.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2021 Ferese, Campopiano, Scala, D’Alessio, Storto, Buttari, Centonze, Logroscino, Zecca, Zampatti, Fornai, Cianci, Manfroi, Giardina, Magnani, Suppa, Novelli and Gambardella.

Figures

Figure 1
Figure 1
Pedigree and electropherograms of class 4 and 5 mutations identified by next-generation sequencing (NGS). (A) Family ID: 564, MPLA analysis and identified a mosaic duplication of 1.5-Mb (17p11.2–12); (B) Family ID: 882, heterozygous stop mutation in mitofusin-2 (MFN2) gene: NM_001127660.1:c.[2258dupT], NP_001121132.1:p.(Gln754AlafsTer9; rs773371488); (C) Family ID: 896, heterozygous stop mutation in MPZ gene: NM_000530.8: c.[306delA], NP_000521: p.(Asp104ThrfsTer14), (rs281865125); (D) Family ID: 402, heterozygous missense mutation in HSPB1 gene: NM_001540.3:[c.570G > C], NP_001531.1:p.(Gln190His); (E) Family ID: 184, heterozygous missense mutation in KIF1A gene: NM_001244008.2:c.[5332C > T], NP_ p.(Arg1778Trp), (rs765668490); (F) Family ID: 1196, heterozygous stop mutation in GADP1 gene: NM_018972.2: c.[140delA], NP_061845.2: p.(Lys47ArgfsTer3); (G) Family ID: 1251, homozygous splicing mutations in SH3TC2 gene: NM_024577.3: c.[805 + 2T > C], (rs139052887); (H) Family ID: 1141, heterozygous deletion mutation in KIF5A gene: NM_004984.2:c.[2868_2870delTCT], NP_004975.2:p.(Leu957del), (rs575223790); and (I) Family ID: 580, homozygous missense mutation in MTMR2 gene: NM_016156.5:c.[463T > C], NP_057240.3:p.(Cys155Arg).
Figure 2
Figure 2
Genotype and phenotype of Charcot-Marie-Tooth (CMT) patients from this study (A), and CMT patients reviewed from the literature (B). (A) Variants identified in 67 CMT patients (blue: copy number variation 38.8%; orange: MPZ mutation 1.5%; gray: HSPB1 mutation 1.5%; light yellow: MFN2 mutation 1.5%; light blue: KIF1A mutation 1.5%; green: GAPD1 mutation 1.5%; dark blue: MTMR2 mutation 1.5%; brown: KIF5A mutation 1.5%; dark gray: SH3TC2 mutation 1.5%; yellow: no cause detected 41.8%; and dark green: uncertain pathogenetic 7.4%). (B) This graphic compares detection yield in CMT phenotypes obtained from the literature. (light yellow: CMT1; orange: CMT2; gray: CMT4; light blue: CMTX; green: HMSN; and blue: undiagnosed). Bar 1: data obtained in this study; Bar 2: data obtained from three Italian studies; and Bar 3: data obtained from 20 European studies (see Supplementary Material).
Figure 3
Figure 3
Pedigree and electropherograms of variants of uncertain significance identified by NGS. (A) Family ID: 721, two missense variants in (a) AGRN gene NM_198576.4:c.[5851C > T], NP_940978.2:p.(Arg1951Cys), (rs746117937) and (b) SCP2 gene NM_002979.5 c.[886C > T], p.(Pro296Ser); (B) Family ID: 856, missense variant in DNM2 gene NM_001005360.2:c.[890G > A], NP_001005360.1:p.(Arg297His); (rs763894364); (C) Family ID: 608, missense variant in MED25 gene NM_030973.3:c.[949G > T], NP_112235.2: p.(Gly317Cys); (rs1280659782); (D) Family ID: 962, N missense variant in DYNC1H1 gene NM_001376.4:c.[9919G > T], NP_001367.2:p.(Val3307Leu); (E) Family ID: 731, missense variant in DYNC1H1 gene NM_001376.4:c.[6743A > G], NP_001367.2:p.(Glu2248Gly); and (F) Family ID: 1261, missense variant in BSCL2 gene NM_001122955.3:c.[124C > T], NP_001116427.1:p.(Arg42Cys); (rs201493373).

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