Expanding the phenotype associated with the NEFL mutation: neuromuscular disease in a family with overlapping myopathic and neurogenic findings

Abstract

Importance: Newer sequencing technologies in combination with traditional gene mapping techniques, such as linkage analysis, can help identify the genetic basis of disease for patients with rare disorders of uncertain etiology. This approach may expand the phenotypic spectrum of disease associated with those genetic mutations.

Objective: To elucidate the molecular cause of a neuromuscular disease among a family in which 4 members, a mother and her 3 sons, were affected.

Design, setting, and participants: Two of 4 affected members manifested nemaline myopathy, a common subtype of congenital myopathy, while the other 2 had a nonspecific myopathy. Single-nucleotide polymorphism-based linkage analysis was performed on DNA samples from the 4 affected family members, and whole-genome sequencing was performed in the proband. Real-time quantitative reverse transcription-polymerase chain reaction, immunofluorescence, and Western blot analysis were performed on muscle biopsy specimens.

Main outcomes and measures: Whole-genome sequencing and linkage analysis identified a variant in a gene that explains the phenotype.

Results: We identified a novel neurofilament light polypeptide (NEFL) nonsense mutation in all affected members. NEFL mutations have been previously linked to Charcot-Marie-Tooth disease in humans. This led us to reevaluate the diagnosis, and we recognized that several of the findings, especially those related to the muscle biopsy specimens and electromyography, were consistent with a neurogenic disease.

Conclusions and relevance: NEFL mutations are known to cause Charcot-Marie-Tooth disease in humans and motor neuron disease in mice. We report the identification of an NEFL mutation in a family clinically manifesting congenital myopathy. We also describe potential overlap between myopathic and neurogenic findings in this family. These findings expand the phenotypic spectrum of diseases associated with NEFL mutations. This study is an example of the power of genomic approaches to identify potentially pathogenic mutations in unsuspected genes responsible for heterogeneous neuromuscular diseases.

Figure 1. Genetic Analysis and Confirmation of an NEFL Mutation in the Family With a Congenital Myopathy Diagnosis

A, Shown is the affected proband mother (20-1) and her 3 affected sons (20-2, 20-3, and 20-4). The square represents a male individual; a circle, a female individual; the arrow; the proband; and a slash mark, a deceased individual. B, Chromatograms from Sanger sequencing of genomic polymerase chain reaction products in the proband and her 3 affected sons show the heterozygous c.1261C>T change. C, Chromatograms of the sequenced real-time quantitative reverse transcription–polymerase chain reaction products derived from muscle biopsy samples in patient 20-4 and 2 controls (CO1 and CO2) show a mutant transcript peak of similar height to that of the wild type, suggesting no significant nonsense-mediated decay of the mutated NEFL messenger RNA. NEFL, neurofilament light polypeptide gene.

Figure 2. Histopathological Findings in Muscle Biopsy Specimens From Affected Members of the Family

A-C. Proband 20-1. Deltoid muscle biopsy was performed at age 42 years. Marked excess variability was observed in fiber size, with increased number of internalized nuclei and increased endomysial connective tissue (A). Occasional fibers contained nemaline bodies (arrowhead) (B), and occasional ring fibers (arrowhead) were also noted (C) (modified Gömöri trichrome, original magnification ×200 [A] and ×600 [B]; nicotinamide adenine dinucleotide– tetrazolium reductase, original magnification ×400 [C]). D-F. Patient 20-3. Quadriceps muscle biopsy was performed at age 32 years. The biopsy specimen revealed group atrophy (arrowheads) and marked adipose tissue replacement (D). Small group atrophy was present (arrowheads) even in more well-preserved areas (E). Some of the fibers contained nemaline bodies (arrowheads) (F) (hematoxylin-eosin, original magnification ×100 [D] and ×200 [E]; modified Gömöri trichrome, original magnification ×200 [F]). G-I. Patient 20-4. Quadriceps muscle biopsy was performed at age 11 years. Mild variability in fiber size was present and there was no significant increase in endomysial connective tissue (G and H). Nemaline bodies were not identified (I) (hematoxylin-eosin, original magnification ×100 [G] and ×200 [G]; modified Gömöri trichrome, original magnification ×200 [I]).

Figure 3. Normal Localization of NEFL at Motor End Plates in Affected Muscle

A and B, Indirect immunofluorescence shows similar localization of NEFL protein in transverse sections of control (A) and patient 20-2 (B) skeletal muscle biopsy specimens. C-E, Shown is double-label indirect immunofluorescence of a longitudinal section of myofiber from patient 20-2 stained for NEFL (green in C) and a-bungarotoxin (red in D). The merged image in E identifies NEFL-positive structures as motor end plates (scale bars are 50μm).