Aberrant Splicing in GJB1 and the Relevance of 5' UTR in CMTX1 Pathogenesis

Abstract

The second most common form of Charcot-Marie-Tooth disease (CMT) follows an X-linked dominant inheritance pattern (CMTX1), referring to mutations in the gap junction protein beta 1 gene (GJB1) that affect connexin 32 protein (Cx32) and its ability to form gap junctions in the myelin sheath of peripheral nerves. Despite the advances of next-generation sequencing (NGS), attention has only recently also focused on noncoding regions. We describe two unrelated families with a c.-17+1G>T transversion in the 5' untranslated region (UTR) of GJB1 that cosegregates with typical features of CMTX1. As suggested by in silico analysis, the mutation affects the regulatory sequence that controls the proper splicing of the intron in the corresponding mRNA. The retention of the intron is also associated with reduced levels of the transcript and the loss of immunofluorescent staining for Cx32 in the nerve biopsy, thus supporting the hypothesis of mRNA instability as a pathogenic mechanism in these families. Therefore, our report corroborates the role of 5' UTR of GJB1 in the pathogenesis of CMTX1 and emphasizes the need to include this region in routine GJB1 screening, as well as in NGS panels.

Keywords: 5′ UTR; CMT; Charcot-Marie-Tooth; Connexin 32; GJB1; X-linked Charcot-Marie-Tooth (CMTX1); noncoding; splicing.

Figure 1

Pedigree tree of both families. (A) First family; (B) Second family. Arrows indicate the probands. Black circle/square: female-/male-affected individuals; open circle/square: female/male healthy individuals; wt = wild-type. LOD (logarithm of the odds) score was 2.107, assuming an X-linked dominant model of inheritance.

Figure 3

Comparative transcriptional and retro-transcriptional analysis. (A–C): Real-time polymerase chain reaction (RT-PCR) of mRNA extracted from archived frozen sural nerve biopsies of a normal control (N) and patient III-8 (P), separated by 2% agarose. (D,E): Nucleotide sequence of GJB1 cDNA, as obtained from sural nerve biopsy of a normal control (D) and patient III-8 (E). ATG start codon is outlined by the blue rectangles. (A) Analysis of GJB1 RNA splicing using primers spanning exon 1B and the first 23 nucleotides of exon 2 (from g.13017 to g.13503): N shows an amplicon of 131 bp, as expected after regular mRNA splicing. In P, the same amplicon has an estimated length of 487 pb, which is consistent with the inclusion of intron 1B. (B) RT-PCR of GJB1 exon 2 (from g.13615 to g.13943) showed a decreased expression of the patient’s cDNA. A semiquantitative densitometric analysis demonstrated a 70% reduction of transcript level in P when compared to N. (C) Both PMP22 (438 bp, spanning from exon 1 to exon 4) and the housekeeping GAPDH (252 bp, spanning from exon 6 to exon 8) cDNAs in patient P had similar expression levels to control N. (D) Nucleotide sequence of a normal control: GJB1 exon 1B and exon 2 are joined together. A vertical line represents the boundary between exon 1B and exon 2. (E) Nucleotide sequence of patient III-8: an arrow points to the c.-17+1G>T mutation. The change of the canonical splice site sequence causes the retention of the whole (356 nt-long) intron 1B into the mRNA. Only the first and last 10 nucleotides of intron 1B are shown for a better output.

Figure 2

Sural nerve biopsies. Semithin sections stained with toluidin blue. (A) (20×): 17-year-old patient II-2, first family. (B) (10×): 18-year-old patient III-8, second family. Both biopsies show mild loss of large myelinated fibres, several clusters of regeneration (arrows) and few simple onion bulbs (asterisk).

Figure 4

Immunofluorescence study on nerve longitudinal sections, comparing patient III-8 (D–F) to a normal control (A–C). Original magnification 40×. (A,D) Green immunofluorescence following reaction with anti-MAG antibodies. (B,E) Red immunofluorescence using anti-Cx32 antibodies. (C,F) Merged images. In the control nerve sample, anti-MAG and anti-Cx32 antibodies colocalized at the paranodes (C), whereas in the patient, Cx32 signal was lacking (E) and the sole anti-MAG immunofluorescence was observed in merged images (F).