Homozygous defects in LMNA, encoding lamin A/C nuclear-envelope proteins, cause autosomal recessive axonal neuropathy in human (Charcot-Marie-Tooth disorder type 2) and mouse

Abstract

The Charcot-Marie-Tooth (CMT) disorders comprise a group of clinically and genetically heterogeneous hereditary motor and sensory neuropathies, which are mainly characterized by muscle weakness and wasting, foot deformities, and electrophysiological, as well as histological, changes. A subtype, CMT2, is defined by a slight or absent reduction of nerve-conduction velocities together with the loss of large myelinated fibers and axonal degeneration. CMT2 phenotypes are also characterized by a large genetic heterogeneity, although only two genes---NF-L and KIF1Bbeta---have been identified to date. Homozygosity mapping in inbred Algerian families with autosomal recessive CMT2 (AR-CMT2) provided evidence of linkage to chromosome 1q21.2-q21.3 in two families (Zmax=4.14). All patients shared a common homozygous ancestral haplotype that was suggestive of a founder mutation as the cause of the phenotype. A unique homozygous mutation in LMNA (which encodes lamin A/C, a component of the nuclear envelope) was identified in all affected members and in additional patients with CMT2 from a third, unrelated family. Ultrastructural exploration of sciatic nerves of LMNA null (i.e., -/-) mice was performed and revealed a strong reduction of axon density, axonal enlargement, and the presence of nonmyelinated axons, all of which were highly similar to the phenotypes of human peripheral axonopathies. The finding of site-specific amino acid substitutions in limb-girdle muscular dystrophy type 1B, autosomal dominant Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy type 1A, autosomal dominant partial lipodystrophy, and, now, AR-CMT2 suggests the existence of distinct functional domains in lamin A/C that are essential for the maintenance and integrity of different cell lineages. To our knowledge, this report constitutes the first evidence of the recessive inheritance of a mutation that causes CMT2; additionally, we suggest that mutations in LMNA may also be the cause of the genetically overlapping disorder CMT2B1.

Figure 1

Ultrastructural micrograph of peripheral nerve from an individual from family ALG.16-301 who is affected with CMT2. A severe rarefaction of myelinated and nonmyelinated fibers can be observed. Large myelinated fibers are almost totally lacking. Neither onion bulbs (proliferations of Schwann cells) nor regenerating clusters are present. Nerve biopsies were performed, and the samples were prepared for electron microscopy as described elsewhere (Hahn et al. 2001).

Figure 2

Pedigree of largest Algerian family with AR-CMT2, mutant haplotypes, and integrated map at the LMNA locus. A, Pedigrees and genotypes of Algerian families ALG.16-301 and ALG.16-315, both with AR-CMT2. Blackened symbols represent subjects with clinical, electrophysiological, and histological diagnosis of CMT2. The homozygosity interval in each affected individual is boxed and shaded. B, Genetic, physical, and partial transcriptional map of 1q21.2-q21.3 region. The common haplotype for D1S303, D1S2777, and D1S2721 that is shared by all affected individuals from the three Algerian families is shown (see “Results” section). Position of genetic markers is indicated with two-point LOD-score values. The positions of A1U, SEMB, and LMNA are indicated.

Figure 3

Lamin A/C R298C mutation. A, Sequence analysis and identification of R298C mutation in LMNA. DNA sequences from normal control (+/+), unaffected heterozygote (+/−), and homozygote (−/−) are presented. The R→C amino acid substitution at position 298 of human lamin A/C peptidic sequence is indicated. B, Segregation of the nucleotidic 892C→T mutation in a branch of family ALG.16-301 assayed by digestion with AciI. The 306-bp PCR product encompassing exon 5 includes one invariant AciI restriction site in wild-type alleles, which disappears when transition C→T is present, thereby leading to a 306-bp digestion fragment compared with the 126-bp and 180-bp bands observed for the wild-type allele. Normal (n) and mutant (m) fragments, as well as the mutation status (+/+, +/−, and −/−), are indicated for each individual. Undigested and digested fragment sizes, respectively, are 40 and 20 bp larger than described, owing to the M13 universal and reverse primers' sequencing tags (see “Subjects and Methods” section).

Figure 4

Alignment of amino acid sequence of human lamin A/C with its orthologs from various species and human lamin B1, showing conservation of arginine at position 298. Fully conserved amino acids are shown atop a shaded background. The conservation of arginine at position 298 and its substitution in patients with AR-CMT2 are shown in boldface. The DNA sequences encoding the normal and mutant proteins are shown above and below the amino acid sequences, respectively.

Figure 5

Electron micrographs from transverse sections of sciatic nerves of Lmna knockout and control mice at age 7 wks. A, Sciatic nerve of Lmna +/+ control. B, Sciatic nerve of Lmna +/− mouse. No abnormality was detected, although a slight accumulation of neurofilaments was observed. Axon density, size, and myelination were preserved. C, Sciatic nerve of Lmna −/− mouse. Axon density was reduced, and several pathological axons are indicated by asterisks (*). The axons' diameters are enlarged, and no myelin sheath is visible; additionally, they present focal accumulations of neurofilaments, as observed in nerves of Lmna +/− mouse (bars correspond to 1 μm).