Ciliary neurotrophic factor genotype does not influence clinical phenotype in amyotrophic lateral sclerosis.код для вставкиСкачать
5. Globus A, Scheibel AB. Synaptic locie on parietal cortical neurons: termination of corpus callosum fibers. Science 1967; 156:1127–1129. 6. Jacobson S, Trojanowski JQ. The cells of origin of the corpus callosum in rat, cat and rhesus monkey. Brain Res 1974;74: 149 –155. 7. Bogen JE, Schultz DH, Vogel PJ. Completeness of callosotomy shown by magnetic resonance imaging in the long term. Arch Neurol 1988;45:1203–1205. 8. Zaidel E. Stereognosis in the chronic split brain: hemispheric differences, ipsilateral control, and sensory integration across the midline. Neurophychologia 1998;36:1033–1047. 9. Millhouse E. The Golgi methods. In: Heimer L, Robards M, eds. Neuroanatomical tract-tracing methods. New York: Plenum, 1982:311–344. 10. Jacobs B, Driscoll L, Schall M. Life-span dendritic and spine changes in areas 10 and 18 of human cortex: a quantitative Golgi study. J Comp Neurol 1997;386:661– 680. 11. Riley JN. A reliable Golgi-Kopsch modification. Brain Res Bull 1979;4:127–129. 12. Swann JW, Al-Noori S, Jiang M. Lee CL. Spine loss and other dendritic abnormalities in epilepsy. Hippocampus 2000;10: 617– 625. 13. Scheibel ME, Scheibel AB. The dendritic structure of the human Betz cell. In: Brazier MAB, Petsche H, eds. Architectonics of the cerebral cortex. New York: Raven, 1978:43–57. 14. Belichenko PV, Sourander P, Malmgren K, et al. Dendritic morphology in epileptogenic cortex from TRPE patients, revealed by intracellular Lucifer Yellow microinjection and confocal laser scanning microscopy. Epilepsy Res 1994;18: 233–247. 15. DeFelipe J, Sola RG, Marco P, et al. Selective changes in the microorganization of the human epileptogenic neocortex revealed by parvalbumin immunoreactivity. Cereb Cortex 1993; 3:39 – 48. 16. Innocenti GM. General organization of callosal connections in the cerebral cortex. In: Jones EG, Peters A, eds. Cerebral cortex. Vol 5. New York: Plenum, 1986:291–345. 17. Meyer G. Forms and spatial arrangement of neurons in the primary motor cortex of man. J Comp Neurol 1987;262: 402– 428. 18. Diamond MC, Rosenzweig MR, Bennett, EL, et al. Effects of environmental enrichment and impoverishment on rat cerebral cortex. J Neurobiol 1972;3:47— 64. 19. Adkins DL, Bury SD, Jones TA. Laminar-dependent dendritic spine alterations in the motor cortex of adult rats following callosal transection and forced forelimb use. Neurobiol Learn Mem 2002;78:35–52. 20. Gorden HW, Bogen JE, Sperry RW. Absence of deconnexion syndrome in two patients with partial section of the neocommissures. Brain 1971;94:327–336. Ciliary Neurotrophic Factor Genotype Does Not Influence Clinical Phenotype in Amyotrophic Lateral Sclerosis Ammar Al-Chalabi, PhD, MRCP,1,2 Margaret D. Scheffler, BA,2 Bradley N. Smith, BSc,1 Matthew J. Parton, PhD, MRCP,1 Merit E. Cudkowicz, MD,3 Peter M. Andersen, DMSc,4 Douglas L. Hayden, MA,5 Valerie K. Hansen, BSc,1 Martin R. Turner, MRCP,1 Christopher E. Shaw, MD, FRCP,6 P. Nigel Leigh, PhD, FRCP,1 and Robert H. Brown, Jr, MD, DPhil2 Ciliary neurotrophic factor (CNTF) maintains survival of adult motor neurons. Mice lacking the CNTF gene develop mild, progressive motor neuron loss. In the normal human population, 1 to 2.3% are homozygous for a null allele, and reports suggest this mutant is associated with a younger onset of amyotrophic lateral sclerosis (ALS). We have tested this hypothesis in a study of 400 subjects with ALS and 236 controls. There was no difference in age of onset, clinical presentation, rate of progression, or disease duration for those with one or two copies of the null allele, excluding CNTF as a major disease modifier in ALS. Ann Neurol 2003;54:130 –134 Amyotrophic lateral sclerosis (ALS) is a disease in which there is progressive degeneration of motor neurons resulting in paralysis and death, usually over approximately 3 years. Approximately 10% of cases show autosomal dominant inheritance. Mutations in the gene for Cu/Zn superoxide dismutase (SOD1) account From the 1Department of Neurology, Academic Neuroscience Centre, Institute of Psychiatry, King’s College London, London, United Kingdom; 2Day Neuromuscular Research Laboratory, MGH East, Charlestown, MA; 3Neurology Clinical Trials Unit, Massachusetts General Hospital, Boston, MA; 4Department of Neurology, Umeå University, Umeå, Sweden; 5General Clinical Research Center, Massachusetts General Hospital, Boston, MA; and 6Departments of Neurology and Medical and Molecular Genetics, Guy’s King’s and St. Thomas’ School of Medicine, and Institute of Psychiatry, King’s College London, United Kingdom. Received Jul 15, 2002, and in revised form Apr 7, 2003. Accepted for publication Apr 7, 2003. Address correspondence to Dr Al-Chalabi, Department of Neurology, PO41, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK. E-mail: email@example.com 130 © 2003 American Neurological Association Published by Wiley-Liss, Inc., through Wiley Subscription Services for 2 to 7% of all cases, but no other genes clearly associated with typical disease have yet been identified.1,2 There is considerable phenotypic heterogeneity in ALS, even within families, but no definite phenotypic modifier genes have been found. The ciliary neurotrophic factor (CNTF) gene codes for a 200 –amino acid protein that plays an important role in maintenance and regeneration of the adult nervous system.3 It prolongs neuronal survival of various culture systems4 and also improves survival in the pmn and wobbler mutant mouse models of motor neuron disease,5,6 even though these mice do not have an obvious defect in CNTF or its receptor. Conversely, mice with disruption of CNTF or its receptor do develop motor neuron degeneration.7 These observations led to the unsuccessful use of CNTF in therapeutic clinical trials in ALS on the basis that increased levels improve survival regardless of the underlying cause of disease.8 –10 In humans, a null allele of CNTF is generated by a G to A intronic point mutation that results in a new splice site, a reduction in polypeptide length from 200 to 62 (only the first 39 are part of the normal protein), and prevention of expression of normal protein.11 Between 1 and 2.3% of the population may be homozygous for this null mutant but are not at increased risk for neurological disease, including ALS.11–14 Some studies have shown differences in CNTF protein and CNTF receptor mRNA expression in spinal cord from patients with ALS compared with normal controls,15 suggesting that this pathway may be involved in ALS pathogenesis. It is possible that possession of the null allele influences ALS once the disease process has started because motor neurons cannot be protected or rescued from the disease-causing insult. In support of such an idea, anecdotal reports suggest that the null allele may be associated with a younger onset and more rapid disease.16,17 CNTF genotype therefore is an interesting candidate as a phenotypic modifier, particularly for disease onset and duration. We have tested this hypothesis in two populations of patients with sporadic ALS and a second group with familial ALS, homozygous for the D90A mutation of SOD1, using for comparison a set of neurologically normal controls. Subjects and Methods Clinical Resource Subjects were seen in tertiary referral clinics in the United States, United Kingdom, and Scandinavia. The diagnosis of ALS was made by a consultant neurologist after exclusion of other conditions. Demographic data, age of symptom onset, site of first weakness, time to referral, and disease duration from first symptom were recorded. In a subset of patients from the United States, rate of progression also was recorded, as assessed by repeated ALS functional rating scale and forced vital capacity assessments. Controls were anonymous blood donors from the United Kingdom. Genotyping Genotypes were determined as previously described.12 Statistics The 2 test was used to test the association of ALS with the null allele of CNTF. Analysis of variance (ANOVA) was used to compare means of continuous variables. Kaplan– Meier analysis (K-M) and Cox regression were used to test potential continuous and categorical predictors of age of onset and survival. SPSS statistical software (version 10.0.7; SPSS, Chicago, IL) and SAS version 8.1 (SAS Institute, Cary, NC) were used for all statistical analysis. Results CNTF genotypes were obtained for 636 subjects; 217 were neurologically normal controls, 167 were from the United Kingdom, and 50 were from Scandinavia. There were also 400 subjects with ALS, of whom 351 had sporadic ALS and 49 had familial ALS, homozygous for the D90A mutation of SOD1, which is prevalent in Scandinavia. The remaining 19 people were homozygous for the D90A mutation but were unaffected by ALS as of January 1, 2000. For the subjects with sporadic disease, the age of onset distribution, gender ratio, and site of onset were similar to those reported in other retrospective studies. Mean age of onset was 53.8 years, 64% were men, and the first symptoms were in the bulbar region in 31%. CNTF genotype frequencies were in Hardy–Weinberg equilibrium for controls, sporadic ALS, and familial ALS (Table), and comparison of the sporadic ALS genotypes and controls confirmed there was no association of the null allele with ALS (2 ⫽ 0.11, p ⫽ 0.95). In contrast with the expected findings, there was no significant difference in mean or median age of onset for different CNTF genotypes (ANOVA p ⫽ 0.26; K-M log rank 4.0, p ⫽ 0.14), and homozygosity for CNTF null mutant tended to delay disease onset (Fig 1). In addition, there was no significant difference in survival for different CNTF genotypes (K-M log rank 1.5, p ⫽ 0.47; Fig 2) or when controlling for known prognostic factors using multivariate regression analysis with available data (Cox model p ⫽ 0.44). For 47 subjects with sporadic ALS, there was no significant difference in disease progression for different CNTF genotypes ( p ⫽ 0.38). For the Scandinavian D90A homozygous subjects with symptoms, genotype frequencies were in agreement with Hardy–Weinberg proportions (2 1.56, p ⫽ 0.21), and there was no association between the null CNTF allele and ALS (2 1.19, p ⫽ 0.55). Mean age of onset of paresis was 47.5 years, and mean age of the unaffected was 49 years. There was no significant difference in mean or median age of onset for different CNTF genotypes (ANOVA p ⫽ 0.73; K-M log rank 0.49, p ⫽ 0.78). Median survival could not be calcu- Al-Chalabi et al: CNTF and ALS 131 Table.1 Sporadic and Homozygous D90A ALS Test Values for Each Model (P) Genotypes Sporadic ALS SALS (n) Controls (n) NN NM MM Total 260 84 7 351 122 42 3 167 Bulbar onset (n) 63 25 1 89 Limb onset (n) Male (n) 145 168 45 52 5 5 195 225 91 54 32 53 2 64 125 55 56 68 223 36 74 41 5 31 302 173 53 5 231 34 15 0 49 36 8 13 10 1 1 50 19 Female (n) Mean age of onset (yr) Median age of onset (yr) No. of subjects Median survival (mo) No. of subjects Homozygous D90A ALS D90A ALS (n) Controls (n) D90A disease-free subjects (n) Mean age of onset (yr) Mean age of disease-free D90A subjects at January 1, 2000 (yr) Median age of onset (yr) 47 48 N/A 54 46 40 45 50 N/A Test Dominant Recessive 1. No association of cases with CNTF null mutant 2. Case alleles in HardyWeinberg equilibrium 3. Random assortment of alleles by site of onset 2 0.805 0.59 (one tailed) 2 0.52 0.67 (two tailed) 4. Random assortment of alleles by gender 2 0.70 1.00 (two tailed) Null Hypothesis 5. No differences in mean ages of onset 6. No differences in median ages of onset 2 Test Values (P) 0.92 ANOVA 0.26 Log rank 0.14 7. No differences in survival Log rank 0.47 1. No association of cases with CNTF null mutant 2 0.77 0.51 (one tailed) 0.21 2. Case alleles in Hardy– Weinberg equilibrium 3. No differences in mean ages of onset 4. No differences in mean ages of disease-free D90A subjects 5. No differences in median ages of onset 2 0.21 ANOVA 0.73 ANOVA 0.11 Log rank 0.78 The wild-type allele is coded as N, the null mutant as M. Numbers for each genotype and phenotypic category are shown with the statistical test used and p value obtained. Dominant model refers to possession of at least one M allele; recessive model refers to possession of two. For MM genotype, there are low numbers, and the Fisher exact test was used. In all D90A cases included, subjects were homozygous for the D90A mutation of SOD1. There was no evidence for an effect of the CNTF null mutant allele on ALS phenotype in sporadic or D90A ALS. ALS ⫽ amyotrophic lateral sclerosis; CNTF ⫽ ciliary neurotrophic factor; SALS ⫽ sporadic ALS; ANOVA ⫽ analysis of variance; N/A ⫽ not applicable. lated because of the long survival of people with this mutation. There was no difference in survival between homozygous wild-type and heterozygous subjects (K-M log rank 0.2, p ⫽ 0.66). Discussion Studies using recombinant mice suggest that CNTF is not required for motor neuron development but has a role in maintenance of motor neuron survival in adults.18 Mice null for CNTF develop normally but in adulthood experience progressive loss of motor neurons accompanied by mild weakness. In contrast, mice null for the CNTF receptor gene either die in the perinatal period or have severe motor neuron loss.7 Mice transgenic for the G93A SOD1 mutation develop motor neuron degeneration significantly earlier if they are also 132 Annals of Neurology Vol 54 No 1 July 2003 CNTF deficient,17 suggesting that CNTF can act as a disease modifier. Direct evidence that CNTF might act as a diseasemodifying gene in ALS comes from two anecdotal reports. In one study of 154 subjects, the 3 homozygous for the null mutant CNTF allele had a younger onset of motor neuron diseases.16 In the other, subjects from a family with SOD1-mediated ALS had an age of onset and disease duration related to CNTF genotype.17 A man with a V148G SOD1 mutation had a young onset of ALS at 25 years and a rapid disease course with death by 11 months. He was homozygous for the CNTF null mutant allele. His sister and mother also carried the SOD1 mutation but were not homozygous for the null mutant CNTF allele. They were disease free at 35 and 54 years of age, respectively. If CNTF is acting as a disease modifier in ALS, various dominance relationships between the null mutant and wild-type alleles are possible. The most likely in a loss of function mutation is that the mutant is recessive to wild type, but a dose effect or even overdominance, in which the heterozygote has a phenotype not seen in the homozygotes, is possible. Levels of CNTF postmortem in human sciatic nerve are, in fact, determined mainly by gene dose such that heterozygotes have approximately half the level of CNTF as wild-type homozygotes.19 This would suggest that heterozygotes should have a phenotype intermediate between the two homozygotes. It is possible that any effect might be masked by the natural heterogeneity of ALS or because sporadic ALS could be different in some way from familial ALS. We therefore included a population of 49 subjects with ALS caused by homozygosity for the D90A mutation of SOD1.20 This SOD1 mutation is important because it has arisen on a genetically homogeneous background, and subjects with ALS related to this mutation have a uniform phenotype, with very slowly progressive ascending paresis and a mean survival time of 14 years. This makes it ideal for the study of phenotypic modifiers, because deviation would be more obvious. In the two sporadic ALS groups we studied, there was no significant difference in age of onset for the three CNTF genotypes, and, for the five null homozygotes with data, there was actually a later age of onset than for the other two genotypes, although this was not statistically significant. The possibility that the null allele only has an effect which is mild and recessive Fig 2. Kaplan–Meier curve of survival for the three ciliary neurotrophic factor (CNTF) genotypes. There is no significant difference in survival between any groups. N is the wild-type and M the null mutant allele. Cross marks represent subjects who were alive at the censor date and therefore do not reduce the proportion surviving. Numbers of subjects: NN 173, NM 53, MM 5. cannot be excluded by this or previous studies but seems unlikely given the later age of onset for homozygotes that we have observed. Because subjects homozygous for the null mutant constitute at most 2.3% of the population, to have sufficient power to detect a subtle effect would require a study of thousands. These findings suggest that the null allele of CNTF does not have a major modifying effect on ALS onset, rate of progression, or duration. This work was supported by a Medical Research Council Clinician Scientist Fellowship (A.A.-C.); the Medical Research Council (V.K.H.); a Wellcome Trust Fellowship (M.R.T.); the MNDA, ALSA, MDA, Project ALS and the Angel Fund; and the National Institutes of Health (NINDS, NIA; R.B.). References Fig 1. Kaplan–Meier curve of age of onset for the three different ciliary neurotrophic factor (CNTF) genotypes. There is no significant difference in age of onset between any groups. N is the wild-type and M the null mutant allele. Numbers of subjects: NN 223, NM 74, MM 5. The marked step-like shape for the MM genotype survival curve reflects the low number of subjects in this group. 1. Hadano S, Hand CK, Osuga H, et al. A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2. Nat Genet 2001;29:166 –173. 2. Yang Y, Hentati A, Deng HX, et al. 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