Deletions Causing Spinal Muscular Atrophy Do Not Predispose to Amyotrophic Lateral Sclerosis

ORIGINAL CONTRIBUTION Deletions Causing Spinal Muscular Atrophy Do Not Predispose to Amyotrophic Lateral Sclerosis

Jillian S. Parboosingh, MSc; Vincent Meininger, MD; Diane McKenna-Yasek; Robert H. Brown, Jr, MD; Guy A. Rouleau, MD

Background: Amyotrophic lateral sclerosis (ALS) is a ing neurodegeneration in spinal muscular atrophy are rapidly progressive, invariably lethal disease resulting present in patients with ALS in whom the copper/zinc from the premature death of motor neurons of the superoxide dismutase gene is not mutated. motor cortex, brainstem, and spinal cord. In approximately 15% of familial ALS cases, the copper/zinc super- Patients and Methods: Patients in whom ALS oxide dismutase gene is mutated; a juvenile form of was diagnosed were screened for mutations in the familial ALS has been linked to chromosome 2. No SMN and NAIP genes by single strand conformation cause has been identified in the remaining familial ALS analysis. cases or in sporadic cases and the selective neurodegen- erative mechanism remains unknown. Deletions in 2 Results: We found 1 patient with an exon 7 deletion in genes on chromosome 5q, SMN (survival motor neuron the SMN gene; review of clinical status confirmed the mo- gene) and NAIP (neuronal apoptosis inhibitory protein lecular diagnosis of spinal muscular atrophy. No muta- gene), have been identified in spinal muscular atrophy, a tions were found in the remaining patients. disease also characterized by the loss of motor neurons. These genes are implicated in the regulation of apopto- Conclusion: The SMN and NAIP gene mutations are spe- sis, a mechanism that may explain the cell loss found in cific for spinal muscular atrophy and do not predispose the brains and spinal cords of patients with ALS. individuals to ALS.

Objective: To determine whether the mutations caus- Arch Neurol. 1999;56:710-712

OTOR NEURON loss is progression of muscular weakness to death characteristic of sev- typically 2 to 5 years after disease onset eral neurodegenera- in the fourth or fifth decade of life. tive diseases, includ- Spinal muscular atrophy was mapped ing spinal muscular to chromosome 5q11.2-13.3,4,5 and par- Matrophy (SMA) and amyotrophic lateral tial deletions of genes in this region are re- sclerosis (ALS). Spinal muscular atrophy ported. In 67% of type 1 and 42% of types is a relatively common childhood disor- II and III SMA chromosomes, deletions der resulting from the loss of spinal mo- were identified in the neuronal apoptosis tor neurons. Childhood SMA is subdi- inhibitory protein (NAIP) gene,6 while ex- vided into 3 groups according to clinical ons 7 and 8 of the telomeric survival mo- From the Centre for Research in severity and age at onset; all forms are in- tor neuron (SMN) gene are mutated in 1 Neuroscience, McGill herited recessively. An adult form of SMA 98.6% of patients with autosomal reces- 7 University and the Montreal exists with an age of onset of 17 to 55 years sive childhood-onset SMA and in some General Hospital Research and either recessive or dominant modes patients with adult-onset SMA.8,9 SMN is Institute, Montreal, Quebec of inheritance.2,3 Amyotrophic lateral scle- proposed to be the disease-determining (Ms Parboosingh and rosis is characterized pathologically by the gene. Clinical severity and age at onset Dr Rouleau); Centre S. L. A., premature death of cortical, spinal, and appear to be correlated with SMN gene Hôtel Dieu de Paris, Paris, brainstem motor neurons. In approxi- expression.10 SMN protein is detectable in France (Dr Meininger); and mately 10% of ALS cases, an autosomal patients with deletions in the telomeric Day Neuromuscular Laboratory, Massachusetts dominant mode of inheritance is ob- SMN gene, indicating the presence of General Hospital, Harvard served; familial ALS (FALS) cases cannot protein encoded by the highly homolo- c Medical School, Boston be distinguished clinically or pathologi- gous centromeric copy ( BCD541). The (Ms McKenna-Yasek and cally from sporadic ALS (SALS) cases. loss of the NAIP gene and other as yet Dr Brown). Clinically, the neuronal loss results in rapid unknown genes in the duplicated SMA

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©1999 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/23/2021 Summary of Single Strand Conformation Analysis PATIENTS AND METHODS of SMN Gene and cBCD541 Exons 7 and 8*

c PATIENT DATA Exon SMN BCD541 No. of Cases 7 − + 1/262 Two hundred sixty-two unrelated patients with ALS + + 221/262 were analyzed for the SMA deletion. Sixty-eight of +/p + 22/262 the patients had a family history of ALS. The diag- + − 18/262 nosis of ALS was established by at least one neurolo- 8 + + 84/93 gist subspecialized in the assessment and treatment +/SSCP variants + 3/93 of ALS based on the El Escorial World Federation of + − 6/93 Neurology criteria.14 All cases studied were negative for mutations in the SOD1 gene. *Plus sign indicates present; minus sign, absent; p, C/T intron 7 polymorphism; SMN, survival motor neuron; and cBCD541, protein encoded by the highly homologous centromeric copy. MUTATION ANALYSIS

DNA was extracted from whole blood specimens and 1234567 used to amplify exon 6 of the NAIP gene by the poly- merase chain reaction (PCR); coamplification of exon 13 was used as a control. Multiplex PCR was performed using the conditions and primers previously described.6 Electrophoresis was performed on the PCR ∗ products using a 1% agarose gel for 11⁄2 hours at 100 V; the PCR products were then stained with ethidium † bromide and visualized under UV light. Exons 7 and 8 of the SMN gene were amplified via PCR sulfur 35–adenosine triphosphate incorpo- ration.7 Electrophoresis was performed on the PCR products using a ϫ0.5 mutation detection enhance- † ment acrylamide gel overnight at 4 W and 4°C. The gel was dried and exposed to x-ray film. ∗

region may also play a role in the variable clinical Single strand conformation analysis of exon 7 of the survival motor neuron expression of the disease. ( SMN) gene (asterisk) and protein encoded by the highly homologous The mechanism of neuronal death in ALS is un- centromeric copy (cBCD541) (dagger). Lanes 2, 3, 5, and 6 show the 4-band pattern of normal chromosomes; lane 1, deletion of exon 7 of cBCD541; lane known, but abnormal apoptosis is considered a possi- 4, deletion of exon 7 of the SMN gene in a patient with spinal muscular bility. Mutations in the copper/zinc superoxide dismu- atrophy; and lane 7, polymorphism in exon 7 (exon 7 of the SMN gene and tase (SOD1) gene have been identified in approximately cBCD541 are also present). 15% of FALS cases, although the mechanism by which these mutations lead to motor neuron death is un- light revealed 2 bands of 434 base pairs (bp) (exon 6) known.11 Although mutations in the SOD1 gene have been and 242 bp (exon 13), indicating the absence of homo- identified in some patients with ALS, in most cases the zygous deletions seen in some patients with SMA. cause, genetic or otherwise, remains a mystery. Patients were also screened for deletions or other Reports of the coexistence of SMA and ALS within mutations in exons 7 and 8 of the SMN gene by single families suggest the possibility of a common genetic ba- strand conformation analysis; the Table summarizes these sis.12,13 Therefore, we elected to explore a possible role results. In controls, 4 bands are apparent after PCR for the NAIP and SMN genes in the pathogenesis of FALS amplification and single strand conformation analysis, and SALS. Genomic DNA specimens from 69 patients with revealing the presence of the SMN gene and cBCD541 non–SOD1-mutated FALS and 194 patients with SALS (Figure). Ninety-three percent of the patients in whom were analyzed for the absence of exon 6 in the NAIP gene SMA is diagnosed are reported to have a deletion of exon and for deletions or aberrant single strand conforma- 7intheSMN gene.7 One patient, in whom ALS was clini- tional polymorphism bands in exons 7 and 8 of the SMN cally diagnosed, was identified to have an exon 7 dele- gene. tion in the SMN gene as shown in lane 4 of the Figure; the centromeric copy remains. Review of this patient’s RESULTS clinical history confirmed that symptoms were consistent with a diagnosis of SMA. A polymorphism, C to T We analyzed 262 patients with ALS for the NAIP and SMN transition in intron 7, was observed in 22 patients (9 with gene deletions found in patients with SMA. Exon 6 of the FALS and 13 with SALS; Figure, lane 7); however, in each NAIP gene was successfully amplified via PCR, and elec- case, the intact SMN gene and cBCD541 genes were also trophoresis was performed using a 1% agarose gel (coam- observed. Eighteen patients (2 with FALS and 16 with plification of exon 13 provided an internal control). In SALS) lack the centromeric cBCD541 copy as shown in all cases, ethidium bromide staining visualized under UV the Figure, lane 1. Ninety-three patients were screened

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©1999 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/23/2021 for mutations in exon 8. Deletions in exon 8 of cBCD541 patients with ALS, without whose support these studies could were observed in 6 cases. In 3 more cases, 2 cases of FALS not be done. We also acknowledge support from the Mus- and 1 of SALS, aberrant migration patterns were ob- cular Dystrophy Association, Tucson, Ariz, and from the served; however, in each case the SMN gene remained Medical Research Council of Canada. intact. These variants were due to sequence variations Corresponding author: Guy A. Rouleau, MD, Centre in the 3Ј untranslated region: a 3-bp deletion,aCtoT for Research in Neuroscience, Montreal General Hospital substitution in cBCD541, and an A to T substitution. Research Institute, 1650 Cedar Ave, Montreal, Quebec, Canada H3G 1A4. COMMENT REFERENCES We found a deletion in exon 7 of the SMN gene in 1 patient with a clinical diagnosis of ALS. Symptoms exhib- ited by this patient were consistent with a diagnosis of SMA. 1. Dubowitz V. Muscle Disorders in Childhood. Philadelphia, Pa: WB Saunders Co; No evidence was found of homozygous deletions in exon 1978. 2. Pearn JH, Hudgson P, Walton JN. A clinical and genetic study of spinal muscu- 6oftheNAIP gene or in exons 7 or 8 of the telomeric SMN lar atrophy of adult onset: the autosomal recessive form as a discrete disease gene in the remaining 261 patients with FALS, confirm- entity. Brain. 1978;101:591-606. ing a previous report of the absence of deletions in 10 uni- 3. Pearn J. Autosomal dominant spinal muscular atrophy: a clinical and genetic study. generational patients with FALS and 54 patients with J Neurol Sci. 1978;38:263-275. 15 4. Brzustowicz LM, Lehner T, Castilla LH, et al. Genetic mapping of chronic childhood- SALS. Furthermore, microdeletions or missense muta- onset spinal muscular atrophy to chromosome 5q11.2-13.3. 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These data confirm the absence of a com- muscular atrophy to motor neuron disease: investigation of SMN and NAIP gene mon genetic basis between ALS and SMA, strongly sug- deletions in sporadic and familial ALS. J Neurol Sci. 1997;145:55-61. 16. Devriendt K, Lammens M, Schollen E, et al. Clinical and molecular genetic fea- gesting that the pathogenic molecular and cellular pro- tures of congenital spinal muscular atrophy. Ann Neurol. 1996;40:731-738. cesses of ALS and SMA differ. This study provides evidence 17. Korinthenberg R, Sauer M, Ketelsen U-P, et al. Congenital axonal neuropathy caused that mutations in the SMN gene are restricted to SMA. by deletions in the spinal muscular atrophy region. Ann Neurol. 1997;42: Thus, while molecular detection of the SMN deletion will 364-368. 18. Velasco E, Valero C, Valero A, Moreno F, Hernandez-Chico C. Molecular analysis be useful and accurate in diagnosing SMA, in more than of the SMN and NAIP genes in Spanish spinal muscular atrophy (SMA) families 95% of ALS cases (non-SOD1), the diagnosis will con- and correlation between number of copies of cBCD541 and SMA phenotype. Hum tinue to be based on clinical criteria and the exclusion Mol Genet. 1996;5:257-263. of other motor neuron disorders. 19. Ferlini A, Patrosso MC, Guidetti D, et al. Androgen receptor gene (CAG)n repeat analysis in the differential diagnosis between Kennedy disease and other motor neuron disorders. Am J Med Genet. 1995;55:105-111. Accepted for publication September 17, 1998. 20. Parboosingh JS, Figlewicz DA, Krizus A, et al. Spinobulbar muscular atrophy can We thank Alexander MacKenzie, MD, PhD, for pro- mimic ALS: the importance of genetic testing on male patients with atypical ALS. viding us with the NAIP primers. We also thank the many Neurology. 1997;49:568-572.

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