Identification and Characterization of Novel Founder Mutations in NDRG1: Refining the Genetic Landscape of Charcot-Marie-Tooth Disease Type 4D in Bulgaria

Abstract

Charcot-Marie-Tooth neuropathy type 4D (CMT4D) is a rare genetic disorder of the peripheral nervous system caused by biallelic mutations in the N-Myc Downstream Regulated 1 gene (NDRG1). Patients present with an early onset demyelinating peripheral neuropathy causing severe distal muscle weakness and sensory loss, leading to loss of ambulation and progressive sensorineural hearing loss. The disorder was initially described in the Roma community due to a common founder mutation, and only a handful of disease-causing variants have been described in this gene so far. Here, we present genetic and clinical findings from a large Bulgarian cohort of demyelinating CMT patients harboring recurrent and novel variants in the NDRG1 gene. Notably, two splice-site variants are exclusive to Bulgarian Muslims and reside in ancestral haplotypes, suggesting a founder effect. Functional characterization of these novel variants implicates a loss-of-function mechanism due to shorter gene products. Our findings contribute to a deeper understanding of the genetic and clinical heterogeneity of CMT4D and highlight novel founder mutations in the ethnic minority of Bulgarian Muslims.

Keywords: Charcot–Marie–Tooth neuropathy type 4D; NDRG1; demyelinating neuropathy; founder mutation; non-Roma ethnicity.

Figure 1

Haplotype schematics in families harboring the splice-site variants. The c.538-1G>A variant resides on a shared haplotype block of 2.4 Mb between the three families (I, II, and IV), shown in light green, and the c.327-2G variant resides on a 9.6 Mb haplotype block shared between families III and IV, shown in dark green. The individual alleles can be followed by additional colors in each pedigree. The splice site variants are denoted in red letters. Circles represent females, squares represent males, filled in shapes represent affected individuals.

Figure 2

Splicing analysis of the c.327-2A>G and c.538-1G>A alleles. Pathogenic variants are denoted with red letters. (A) An amount of 2.5% agarose gel ran for 3 h at 100V, showing amplification of cDNA with primers flanking exon 6 of the NDRG1 gene; (B) graphical overview and interpretation of (A); (C) 2.5% agarose gel ran for 3 h at 100V, showing amplification of cDNA with primers flanking exon 9 of the NDRG1 gene; (D) graphical overview and interpretation of (C). Cyan boxes represent exons, arrows represent the primer positions used for the analyses.