Autosomal dominant amyotrophic lateral sclerosis: a novel mutation in the Cu/Zn superoxide dismutase-1 gene. Markus Kostrzewa, Uta Burck-Lehmann1 and ...
©1994 Oxford University Press
Human Molecular Genetics, 1994, Vol. 3, No. 12
2261-2262
Autosomal dominant amyotrophic lateral sclerosis: a novel mutation in the Cu/Zn superoxide dismutase-1 gene Markus Kostrzewa, Uta Burck-Lehmann1 and Ulrlch Muller* Institut fur Humangenetjk, Justus-Liebig-Universitat Gie6en, Schlangenzahl 14, D-35392 GieBen and 'Getreideweg 20, D-50933 KOIn, Gemiany Received September 30, 1994; Accepted October 4, 1994
Amyotrophic lateral sclerosis (ALS) is a severe degenerative disorder of the motor neurons. While most cases are sporadic, approximately 10% are familial (1). Most familial cases are inherited as autosomal dominant traits with high penetrance. A mutation in the Cu/Zn superoxide dismutase-1 (SOD-1) gene was identified as the underlying cause of some familial ALS (FALS) cases (2,3). Presently, mutations have been identified in four of the five exons of the SOD-1 gene (2-6). Here we report on a previously not recognized mutation in an evolutionarily highly conserved region of exon 4 in a FALS family. The family investigated is given in Figure la. n/2 and H/3 had died from ALS at the ages of 68 and 51 years within 2 and 3 years of manifestation of the first symptoms. Similarly, their father (I/I) has died from ALS. Their presently asymptomatic offspring (HI/1, 30 years, m/2, 36 years, and m/3, 32 years of age) were tested for mutations in SOD-1. Exons 1, 2, 3, 4, and 5 of SOD-1 were amplified in the polymerase chain reaction (PCR) according to published procedures (2,3). For SSCP analysis (7), the amplification products were denatured by heat and separated on a 0.5 xMDE gel (AT Biochem) at 4°C/5 W. A typical band shift of the amplification products of both exons 3 (not shown) and 4 (Fig. lb) was found in individuals HI/2 and HI/3 but not in individual HI/1. No band shift of amplification products of exon 4 was found in a total of 90 unrelated controls (not shown). Both exons 3 and 4 were further analysed in the three individuals by direct cycle sequencing of PCR products using sequencing grade Taq polymerase (Promega). A previously reported A/C polymorphism at position 34 in the flanking intron 3 (6) was found to have caused the SSCP findings with exon 3 primers. Sequencing of exon 4 of the three probands revealed a C - G transversion at codon 115 in IQ/2 and m/3 but not in TW1. This base change results in the replacement of an arginine by a glycine in SOD-1. Several findings strongly suggest that this C—G transversion is the underlying cause of ALS in this family, (i) There is no indication that the mutation observed is a neutral polymorphism since it was detected neither in 90 controls of the present study nor in 212 normal individuals in previous investigations (3,6). (ii) The mutated exon 4 in the individuals with the mutation was shown to be of maternal origin. Although the mothers are dead, SSCP analysis of their fathers' exons 4 did not reveal any band shift (not shown), (iii) Alignment of exon 4 sequences of different species using the computer program Clustal V (8) reveals high evolutionary conservation of arginine at position 115 in vertebrates, invertebrates, higher plants and yeast (Fig. 2). This indicates an important function of Arg at this position of the enzyme, (iv) Arg 115 was found to be located at a dimer contact site of the SOD-1 molecule (2) and thus appears to be relevant for structural integrity of the enzyme. This notion is further supported by the finding that • To whom correspondence should be addressed
EMBL accession no. X81859
arginine, a positively charged amino acid, was replaced by the neutral amino acid glycine. Significantly, an adjacent amino acid (Del 13) in this dimer contact region of SOD-1 was previously shown to be mutated in FALS (3). Presently, it is not known how mutations in the SOD-1 gene cause ALS. Findings in mice transgenic for mutated SOD-1, however, suggest that the mutations result in an abnormal gain of function of SOD-1 (9). ACKNOWLEDGEMENT We thank Dr Teepu Siddique for D N A samples and primer information.
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6
Cl C2 C3
Ill/l 111/2 M/3 C4
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Figure 1. (a) Pedigree of the ALS family investigated, (b) SSCP analysis of four normal controls ( Q and members m/1 - I D / 3 of the ALS family, (c) sequence of a part of exon 4 of individual m/1 (left) and ID/2 (right). The site of mutation is marked by arrows.
2262 Human Molecular Genetics, 1994, Vol. 3, No. 12 FALS human
a HVGDLGHv^ADKDaVADVSIEDSVISLSGDHCIIGKrLV
COW
HVGDIXHTVTADKNOVAIVDIVDPLISLSGKYSIiaRTMV
pig
HV(H>LGNVTAaKD0VATVYIEDSVIAL3(roHSII(H«TMV
mouse
HVGDUJNVTAOKDOVAHVSIEDKVISLSGEHSIiaKrMV
swordfish
HVGDLGNVTMDAHOTWUDITDK-ISLTQI'YSIIGRTMV
DfOSOphila HLOTHXaJIKATQDCPTKVNITDSKITLFaADSIICaCTVV tomato
EAGDI/WIWGEDGTASlTriTDKQIPLTGPQSIIGHAVV
spinach
HAfflDLCTJIVANTDGVAEATIVDNQIPLTOPKSWORALV
rice
HArHJIXaJVTAOEDGVANIHWDSQIPLTGPHSIIGBAW
N.crassa
HVGDLGNIETDAQGHAKGTVTDNLVKLIOVESVIORTW
S.cervisiae
HV(H3MONVKTDENGVAKGSFKDSLIKLIOPTSWGRSW
Figure 2. Alignment of human SOD1 exon 4 encoded amino acid sequence with sequences from different organisms (human, refs 10,11, mouse, rcf. 12, swordfish, ref. 13, Drosophila melanogaster, ref. 14, tomato, ref. 15, rice, 16, Neurospora crassa, ref. 17, Saccharomyces cerevisiae, ref. 18, and others, ref. 11). Amino acids identical in all proteins aligned are marked by asterisks, conserved residues are marked by dots. The residue mutated in the present family is highlighted in the uppermost row.
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