DNA/RNA helicase gene mutations in a form of juvenile amyotrophic lateral sclerosis (ALS4)

Abstract

Juvenile amyotrophic lateral sclerosis (ALS4) is a rare autosomal dominant form of juvenile amyotrophic lateral sclerosis (ALS) characterized by distal muscle weakness and atrophy, normal sensation, and pyramidal signs. Individuals affected with ALS4 usually have an onset of symptoms at age <25 years, a slow rate of progression, and a normal life span. The ALS4 locus maps to a 1.7-Mb interval on chromosome 9q34 flanked by D9S64 and D9S1198. To identify the molecular basis of ALS4, we tested 19 genes within the ALS4 interval and detected missense mutations (T3I, L389S, and R2136H) in the Senataxin gene (SETX). The SETX gene encodes a novel 302.8-kD protein. Although its function remains unknown, SETX contains a DNA/RNA helicase domain with strong homology to human RENT1 and IGHMBP2, two genes encoding proteins known to have roles in RNA processing. These observations of ALS4 suggest that mutations in SETX may cause neuronal degeneration through dysfunction of the helicase activity or other steps in RNA processing.

Figure A1

Human multiple-tissue northern-blot (Clontech) analysis. a, Northern-blot analysis of SETX mRNA. We performed northern-blot analysis on human 12-lane MTN blot (Clontech), with a SETX 3′ IMAGE clone insert (number 4136196) labeled with dCTP-[α-³²P], and we identified two transcripts in all tissues at 11.5 kb and 9.0 kb. The lower panel represents the same blots hybridized with human β-actin. b, Northern-blot analysis of 10 μg of total RNA isolated from normal control lymphoblast cell lines. Probe derived from exon 10 of SETX was labeled with dCTP-[α-³²P], which identified two transcripts of 11.5 kb and 9.0 kb.

Figure 1

Identification of the ALS4 gene. a, ALS4 gene region on 9q34. Markers D9S64 and D9S1198 define a 1.7-Mb interval containing 19 genes. Sequencing of 340 exons and flanking regions in probands with ALS4 revealed mutations in SETX or KIAA0625, a gene encoding a novel member of the RNA/DNA helicase family. Corresponding human, mouse, and rat cDNA sequences are shown. The SETX gene consists of 26 exons. An asterisk (*) indicates the locations of mutations in three unrelated pedigrees with ALS4. b, Mutations in SETX in pedigrees with ALS4 (K7000, CMT-106, and CMT-61). As shown, mutations are heterozygous, except for the mutant hybrid cell line (prepared from an affected individual from K7000). Protein alignment showed that mutations of human SETX are well conserved with rat and murine orthologs. c, Restriction pattern of the NdeI digest of the 441-bp exon 3 fragment, with the c.8C→T mutation seen in CMT-106 giving rise to two bands of 284 bp and 157 bp. BsaHI exon 19 restriction digestion pattern of a 284-bp fragment and 157-bp fragment, with the c.6407G→A mutation seen in CMT-61, resulting in an undigested band of 433 bp.

Figure 2

Alignment of the helicase domain regions of the human SETX, IGHMBP2, RENT1, and yeast SEN1 proteins. Identities of the SETX helicase domain with that of IGHMBP2, RENT1, and yeast SEN1 are 42%, 46%, and 42%, respectively. SETX has an ATP/GTP–binding site motif A (P-loop), indicated by a bar. An asterisk (*) indicates the position of the mutation in CMT-61.