Early onset of severe familial amyotrophic lateral sclerosis with a SOD-1 mutation: potential impact of CNTF as a candidate modifier gene

Abstract

Mutations in the copper/zinc superoxide dismutase 1 (SOD-1) gene are found in approximately 20% of patients with familial amyotrophic lateral sclerosis (FALS), or amyotrophic lateral sclerosis 1. Here we describe a 25-year-old male patient who died from FALS after a rapid disease course of 11 mo. Sequencing of the SOD-1 gene revealed a heterozygous T-->G exchange at position 1513 within exon 5, coding for a V-->G substitution at position 148 of the mature protein. Genetic analysis of this family revealed the same mutation in both his healthy 35-year-old sister and his mother, who did not develop the disease before age 54 years. Screening for candidate modifier genes that might be responsible for the early onset and severe course of the disease in the 25-year-old patient revealed an additional homozygous mutation of the CNTF gene not found in his yet unaffected sister. hSOD-1G93A mice were crossbred with CNTF(-/-) mice and were investigated with respect to disease onset and duration, to test the hypothesis that CNTF acts as a candidate modifier gene in FALS with mutations in the SOD-1 gene. Such hSOD-1G93A/CNTF-deficient mice develop motoneuron disease at a significantly earlier stage than hSOD-1G93A/CNTF-wild-type mice. Linkage analysis revealed that the SOD-1 gene was solely responsible for the disease. However, disease onset as a quantitative trait was regulated by the allelic constitution at the CNTF locus. In addition, patients with sporadic amyotrophic lateral sclerosis who had a homozygous CNTF gene defect showed significantly earlier disease onset but did not show a significant difference in disease duration. Thus, we conclude that CNTF acts as a modifier gene that leads to early onset of disease in patients with FALS who have SOD-1 mutations, in patients with sporadic amyotrophic lateral sclerosis, and in the hSOD-1G93A mouse model.

Figure 1

Characterization of mutations in the genes that encode SOD-1 and CNTF in a family with autosomal dominant ALS. A, Family pedigree with FALS. Squares denote male family members, circles denote female family members, black symbols denote affected family members, gray symbols denote affected members who are possibly affected on the basis on family history, and slash marks (/) through symbols denote deceased family members. The arrow indicates the 25-year-old male index patient who carried both a V148G mutation in SOD-1 and a null mutation in CNTF. B, Heterozygous T→G transition at position 1513 within exon 5 of the SOD-1 gene in genotyped family members. Arrows indicate the T→G transition in family members III.4, IV.3, and IV.4, as revealed by automated DNA sequencing of the PCR product of exon 5 of the SOD-1 gene. Family members III.3, III.5, IV.1, and IV.2 do not show the T→G transition. The large arrow indicates index patient IV.4. C, CNTF genotyping in family members. Demonstration of the G→A transition within the intron-exon boundary of the second exon of the CNTF gene, leading to a truncated, biologically inactive CNTF protein. CNTF^+/+ (i.e., no mutation) was found in subjects IV.2 and IV.3; CNTF^+/− (i.e., a heterozygous G→A transition) was found in subjects III.3, III.4, III.5, and IV.1; and CNTF^−/− (i.e., a homozygous G→A transition) was found in index patient IV.4. D, Identification of the CNTF null mutation by HaeIII-restriction-polymorphism analysis of PCR-amplified genomic DNA. The three different genotypes were identified by characteristic digestion patterns: CNTF^+/+ showed two bands of 94 and 40 bp (in subjects IV.2 and IV.3); CNTF^+/− showed three bands of 134, 94, and 40 bp (in subjects III.3, III.4, III.5, and IV.1); and CNTF^−/− showed one band of 134 bp (in index patient IV.4). “M” denotes the Phi-x-174/HaeIII marker.

Figure 2

Motoneuron disease in mice with combined CNTF and SOD-1 gene defects. A, CNTF genotyping of mice. Ethidium bromide–stained 2% agarose gel reveals bands of 97 bp for the wild-type allele (CNTF^+/+) and 1.197 bp for the mutated allele (CNTF^−/−). Heterozygous mice (CNTF^+/−) show both bands. “M” denotes the Phi-x-174/HaeIII marker. B, Automated DNA sequencing of PCR products of exon 4 of the human SOD-1 gene, showing either the wild-type sequence of the human SOD-1 gene in transgenic hSOD-1wt mice (arrow) or the guanine→cytosine transition at position 1087 (arrow) that leads to an amino acid exchange from glycine to alanine at position 93 of the SOD-1 protein in transgenic hSOD-1 G93A mice. C, Kaplan-Meier curves, showing survival of hSOD-1G93A/CNTF^+/+(n=25), hSOD-1G93A/CNTF^+/− (n=24), and hSOD-1G93A/CNTF^−/− mice (n=32) (P<.001 by log-rank test). D and E, Motoneuron numbers in lumbar spinal cord and facial nuclei. Values shown are mean ± SD of motoneuron numbers in lumbar spinal cord (L1–L5; D) and facial nuclei (E) corrected for split nuclei as described (Oppenheim et al. 2001). The differences between the four groups of mice were tested by one-way ANOVA; at the levels for which P<.0001, differences were significant. D, Motoneuron counts of the lumbar spinal cord. Comparison of individual groups by Bonferroni's multiple-comparison test gave the following results: P<.01 (**) and P<.001 (***). E, Motoneuron counts of the facial nucleus. Comparison of individual groups by Bonferroni's multiple comparison test gave the following results: P<.05 (*), P<.01 (**), and P<.001 (***). F–I, Morphology of degenerating motoneurons in the lumbar spinal cord of mice with combined SOD-1 and CNTF gene mutations. In hSOD-1wt/CNTF^+/+ mice (F), the spinal cord appeared to be histologically normal. In hSOD-1wt/CNTF^−/− (G) and hSOD-1G93A/CNTF^+/+ (H) mice, evident reduction in motoneuron number is detectable. In mice carrying the double mutation, hSOD-1G93A/CNTF^−/− (I), the spinal cord appeared to be nearly void of motoneurons. The more severe reduction in motoneuron number of the spinal cord compared to the facial nucleus and the obvious gliosis suggests a caudorostral progression of the disorder, confirming the clinical phenotype. Bar = 50 μm.