Brain Advance Access originally published online on October 26, 2005
Brain 2006 129(1):235-242; doi:10.1093/brain/awh651
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SCA28, a novel form of autosomal dominant cerebellar ataxia on chromosome 18p11.22–q11.2
1 Dipartimento di Genetica Biologia e Biochimica, Università degli Studi di Torino and S.C. Genetica Medica, Ospedale San Giovanni Battista di Torino, Torino, 2 UO Biochimica e Genetica and 3 UO Neuroradiologia, Istituto Nazionale Neurologico Carlo Besta, Milano and 4 UO Genetica Medica, Università degli Studi di Bologna, Bologna, Italy
Correspondence to: Stefano Di Donato, MD, UO Biochimica e Genetica, Istituto Nazionale Neurologico Carlo Besta, via Celoria, 11-20133 Milano, Italy E-mail: didonato{at}istituto-besta.it
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Summary |
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We describe a four-generation Italian family with a novel form of juvenile-onset, slowly progressive, autosomal dominant cerebellar ataxia. Eleven affected family members have been evaluated. The mean age at onset was 19.5 years with no evidence of anticipation. The first symptoms were invariably unbalanced standing and mild gait incoordination. Gaze-evoked nystagmus was prominent at onset, while patients with longer disease duration developed slow saccades, ophthalmoparesis and, often, ptosis. Deep tendon reflexes in lower limbs were increased in 80% of the cases. Genetic analysis excluded the presence of pathological repeat expansions in spinocerebellar ataxia (SCA) types 1–3, 6–8, 10, 12 and 17, and DRPLA genes. Linkage exclusion tests showed no evidence of association with other known SCA loci. A genome-wide screen analysis identified linkage with chromosome 18 markers. A maximum two-point limit of determination score of 4.20 was found for marker D18S53. Haplotype analysis refined a critical region of 7.9 Mb between markers D18S1418 and D18S1104. This new SCA locus on 18p11.22–q11.2 has been designated SCA28. Candidate genes within the critical interval are currently screened for mutations.
Key Words: spinocerebellar ataxia; SCA; oculomotor function; autosomal dominant cerebellar ataxia; linkage analysis
Abbreviations: ADCA = autosomal dominant cerebellar ataxia; DRPLA = dentato-rubral-pallido-luysian atrophy; LOD = log of the odds; SCA = spinocerebellar ataxia; SCA1 = spinocerebellar ataxia type 1
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Received July 22, 2005. Revised August 29, 2005. Accepted August 31, 2005.
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Introduction |
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TopSummaryIntroductionPatients and methodsResultsDiscussionReferences |
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Autosomal dominant cerebellar ataxias (ADCAs) are a clinically and genetically heterogeneous group of neurodegenerative disorders primarily characterized by imbalance, progressive gait and limb ataxia, and dysarthria (Harding, 1982
). The clinical phenotype appears often complicated by the presence of additional neurological signs, which are highly variable among and within families. Since the identification of the first gene responsible for spinocerebellar ataxia type 1 (SCA1) in 1993 (Orr et al., 1993), an increasing number of genes and chromosomal loci have been characterized, demonstrating large genetic heterogeneity in these hereditary disorders (Schöls et al., 2004; Taroni and Di Donato, 2004).
At present, 25 distinct genetic forms of spinocerebellar ataxias (SCAs) are known: SCA1–8, SCA10–23, SCA25–26 and SCA27/FGF14 (Knight et al., 2004; Schöls et al., 2004; Taroni and Di Donato, 2004; Ishikawa et al., 2005; Yu et al., 2005; www.gene.ucl.ac.uk/hugo) (Table 1). The genetic form designated as dentato-rubral-pallido-luysian atrophy (DRPLA) is also commonly classified within this group of disorders. The disease gene has been identified in 12 SCAs and in DRPLA. In SCAs 1, 2, 3, 6, 7 and 17 subtypes and in DRPLA the molecular mutation is a trinucleotide CAG repeat expansion within the coding region of the corresponding gene (Schöls et al., 2004; Taroni and Di Donato, 2004).
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The prevalence of SCAs has been estimated to be
3 in 100 000, being the relative frequency of specific genotypes variable in different geographical areas and in populations of different ethnic origins (Schöls et al., 2004; Brusco et al., 2004). In addition, population screening for the currently known SCA gene mutations demonstrates that 30–60% of the clinically identified ADCA families remain genetically unassigned, indicating further genetic heterogeneity (Schöls et al., 2004). We recently demonstrated that 45% of the Italian families are associated with SCA1 and SCA2 genotypes, a small percentage of cases are caused by expansions in SCA3, SCA6, SCA7 and SCA17 and DRPLA genes, whereas 40% of the families are negative for mutations in the known SCA genes (Brusco et al., 2004). From this group, we have characterized a four-generation Italian family with an inherited form of slowly progressive cerebellar ataxia. Genome-wide linkage studies allowed mapping of the disease locus on chromosome 18p11.22–q11.2. This new locus has been assigned the SCA28 symbol by the Human Genome Nomenclature Committee (www.gene.ucl.ac.uk/hugo). We report here the clinical features of the affected subjects and the results of genetic studies.
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Patients and methods |
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Patients
The pedigree of the family (MI-A091) is shown in Fig. 1. In the past years, we have collected clinical information and blood samples from the proband III-1 and other family members in generation II. More recently, all the available affected subjects (11 patients) have been clinically evaluated. Four affected patients (III-1; III-7; III-14 and IV-4) were admitted at the Istituto Neurologico Carlo Besta for more extensive clinical evaluation including brain MRI, electrophysiology, electro-oculographic examination and muscle biopsy. Clinical information and blood samples from additional subjects of generation III and IV were collected for linkage studies. A total of 26 subjects (both affected and healthy individuals, and spouses) have been included in the study. Clinical and genetic investigations have been performed after obtaining informed consent from all participants or the parents of participating minors.
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Analysis of known SCA genes and repeat expansion detection (RED) analysis
PCR analysis of the SCA1–3, 6–8, 10, 12 and 17, and DRPLA gene expansions, and the RED analysis were performed as described previously (Brusco et al., 2004
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Genotyping and linkage analysis
Linkage exclusion was carried out using an affected-only approach on nine patients (II-2, II-5, II-6, II-7, III-1, III-7, III-8, III-14 and III-16) and three spouses (II-1, II-4 and II-8) (Fig. 1). Genotyping was performed using 3–13 microsatellite markers for each of the following SCA loci (primer sequences are available on the Genome Database website, www.gdb.org): SCA4, SCA5/20, SCA11, SCA13-16, SCA18, SCA19/22, SCA21, SCA25 and SCA27.
To localize the disease gene, we performed a genome-wide scan using 383 fluorescent-labelled microsatellite markers from the ABI-PRISM Linkage Mapping Set MD-10 version 2.5 (Applied Biosystems, Foster City, CA) spaced at intervals of 10 cM on autosomes. Chromosome X was excluded because a male-to-male transmission was present. In addition to the subjects used for linkage exclusion, three patients (III-10, IV-1 and IV-4), one spouse (III-15) and one healthy relative (III-6) were included in the analysis. Polymerase chain reactions (PCRs) were performed as recommended by the supplier on GeneAmp 9700 or 2700 PCR machines (Applied Biosystems). Amplified fragments labelled with VIC, FAM or NED were pooled in 27 different panels, added to a mix of highly deionized formamide (10 µl) and LIZ-500 fluorescent marker (0.05 µl), denatured for 1 min at 95°C and loaded on an ABI-Prism 3730 automatic sequencer (Applied Biosystems). Data were collected and analysed using the Genemapper software (ver3.0; Applied Biosystems). Eleven additional polymorphic markers (D18S843, D18S1418, D18S1150, D18S453, D18S1104, D18S1107, D18S66, D18S1143, D18S1126, D18S1127 and D18S69, indicated in bold in Table 4) were analysed to narrow the positive candidate region on chromosome 18. Relative positions were derived from the human genome draft sequence and the Marshfield map (www.ncbi.nlm.nih.gov).
Pairwise and multipoint log of the odds (LOD) scores were calculated using the MLINK and SIMWALK2 programs (Lathrop et al., 1984; Sobel et al., 1996). The disease was considered to be autosomal dominant with a frequency of 0.001%. Recombination fractions were assumed equal for men and women, and were converted to map distances using the Kosambi mapping function. Multipoint analysis was performed with the sex-averaged recombination fractions and the order of loci as calculated on Marshfield map. Final linkage data were calculated using 12 patients, 4 spouses and 10 healthy relatives (Fig. 1). Age-dependent penetrance was taken into account by assigning healthy subjects to one of two liability classes determined from the age at onset curve of the family [25–35 years (80%), 36–50 years (90%)]. Allele frequencies at each microsatellite marker were assumed to be equal.
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Results |
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Clinical features
As shown in Fig. 1 the disease was probably transmitted through individual I-1, originating from Southern Italy, who was reported to have gait and speech difficulties. This subject died at the age of 74 of heart failure. The disease was inherited by four of his five children, three of whom are still alive and have been examined by us. The original index case was subject III-1, who was first evaluated at our Institute at the age of 39 years. He suffered from disequilibrium and gait difficulties since the age of 20 years. Neurological examination showed: nystagmus in lateral and vertical gaze, mild ophthalmoparesis in the horizontal plane, dysarthria, mild gait and limb ataxia, increased deep tendon reflexes at the four limbs, bilateral ankle clonus and Babinski sign. Muscle tone, sensation and mental status were normal. IQ was 83. Routine blood tests and cerebrospinal fluid (CSF) examination were normal. The patient was subsequently re-evaluated at 42 and 54 years of age. At the last examination, after 34 years of disease, the patient showed a moderate progression of the gait ataxia and was still able to walk independently. Neurological examination revealed severe ophthalmoparesis in the horizontal and vertical planes, very slow saccades and bilateral palpebral ptosis, wide-based ataxic gait and moderate limb dysmetria. Tendon reflexes were still hyperactive but there was no spasticity.
The other affected members of the family had similar clinical histories and phenotypes. Two patients had died: subject II-5 deceased at age 73 of gastric tumour, while subject III-3 died of acute myocardial infarction at the age of 45. The disease course was slowly progressive with most of the patients remaining ambulant in their late sixties. Patients II-2, II-7 and II-5 required a walking support at the age of 72, 75 and 70, respectively, whereas patient II-6 was confined to a wheelchair at 73 years of age. The clinical features of the 11 affected subjects are summarized in Table 2. The mean age at onset was 19.5 years (range 12–36). Ascertained ages at onset in generation II were 20 and 36 years; ages at onset in generation III ranged from 16 to 20 years, whereas in the two affected subjects of generation IV the onset was at 12 and 16 years. These data do not suggest overt anticipation.
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The first symptoms were invariably imbalance in standing and mild gait and limb incoordination. Disease duration ranged from 1 to 58 years. No cognitive impairment was observed at clinical examinations. Increased patellar tendon reflexes were found in 9 out of 10 patients, increased muscle tone in lower limbs in 3 out of 9 and Babinski sign in 4 out of 8. There was no clinical evidence of sensory involvement. In 5 out of 11 patients a mild-to-severe horizontal gaze-evoked nystagmus was present, 6 out of 10 had slow saccades and 8 out of 11 had mild-to-severe ophthalmoparesis. In 6 out of 10 subjects palpebral ptosis was observed.
Patients III-1, III-7, III-14 and IV-4 underwent electrophysiological, electro-oculographic and brain imaging studies. Nerve conduction studies, visual, auditory, somatosensory and motor evoked potentials were normal in all cases. Brain MRI showed cerebellar atrophy (Fig. 2).
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At electro-oculographic evaluation, hypometric saccades were found in patients III-1, III-7 and III-14, and hypermetric saccades in patient IV-4. Reduced gain in smooth horizontal pursuit was detected in the three patients (range 0.5–0.6; frequency 0.2 Hz), and saccadic break-down of pursuit was present in patients III-14 and IV-4. Optokinetic gain was reduced in patients III-14 and IV-4 (0.3 and 0.7, at drum velocity 20°/s), while it was abolished in patient III-1 and III-7.
Muscle biopsies, performed in patients III-1, III-7, III-14 and IV-4, showed no ragged-red fibres. Biochemical assays on muscle homogenates demonstrated normal activities of the respiratory chain enzymes (complexes I-V), and Southern blot analysis excluded the presence of mitochondrial DNA deletions (data not shown).
Genetic tests and linkage analysis
SCA1–3, SCA6–8, SCA10, SCA12, SCA17 and DRPLA gene repeat expansions were excluded, and RED analysis did not reveal CAG/CTG expansions >40 repeats. In addition, there was no evidence of linkage of this family to most of known mapped SCA loci (SCA4, SCA5/20, SCA11, SCA13-16, SCA18, SCA19/22, SCA21, SCA25 and SCA27) (Table 3).
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We used a genome-wide scan to map the new locus. The most suggestive evidence of linkage was obtained at two distinct non-consecutive markers on chromosome 18, D18S53 and D18S474. To confirm linkage in one of the two regions and for fine mapping, we tested 11 additional polymorphic markers. Pairwise and multipoint linkage analyses confirmed the presence of two regions, one on the short arm and one on the long arm of chromosome 18 (Fig. 3 and Table 4). The highest LOD scores were found in the first region between markers D18S1418 and D18S1104 with a maximum of Z = 4.77 at marker D18S453 in multipoint analysis. LOD scores in the second region, spanning markers D18S1102 and D18S69, were below the threshold of Z = 3.3 both in two-point and multipoint tests (Table 4).
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Haplotype reconstruction (Fig. 1) showed that a single haplotype segregated with the disease in the family. Two key recombination events, centromeric to D18S1418 in patient II-6 and to D18S1104 in patient III-8, defined the minimal in-linkage interval between D18S1418 and D18S1104 as a 7.9 Mb tract. The region is located on cytogenetic bands 18p11.22–q11.2. This new locus has been assigned the SCA28 symbol by the Human Nomenclature Committee (www.gene.ucl.ac.uk/hugo).
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Discussion |
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TopSummaryIntroductionPatients and methodsResultsDiscussionReferences |
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We studied an Italian family with hereditary cerebellar ataxia characterized by juvenile onset, slow disease progression, eye movement abnormalities and, in some cases, pyramidal signs. The transmission of the trait was compatible with autosomal dominant inheritance. The phenotype observed in the affected family members was homogeneous as regards neurological symptoms and disease progression. The clinical features of this family are not fully distinctive in comparison with other SCA subtypes. However, the association of very slowly progressive cerebellar ataxia, ophthalmoparesis, increased deep tendon reflexes and Babinski sign is peculiar. The neurological condition observed in this family combines a few clinical features characteristic of ADCA type I as described by Harding (1982)
, with an unusually slow disease course. ADCA type I is characterized by the association of cerebellar ataxia, supranuclear ophthalmoplegia, pyramidal or extrapyramidal signs, mild dementia and peripheral neuropathy. In addition, it has been estimated that the mean time to wheelchair confinement in patients with ADCA type I is 17 years of disease duration (Klockgether et al., 1998). In our patients, the disease course before overt disability was much longer.
An interesting and consistent finding in our family was the presence of eye movement abnormalities. Two distinct clinical patterns were observed: in patients with short disease duration, persistent gaze-evoked nystagmus was the prevalent finding, while in patients with more than 20 years of disease duration, slow saccades, ophthalmoparesis and ptosis were also observed. A similar progression of oculomotor alterations has been described in other SCA genotypes; for example, in SCA1 patients, possibly as the result of brainstem degeneration (Schöls L et al., 1995; Klostermann et al., 1997; Bürk et al., 1999). However, in SCA1 and SCA2 patients, brainstem involvement is usually supported by the MRI finding of pontine atrophy, whereas in our affected patients neuroimaging did not clearly demonstrate brainstem abnormalities. We have also considered the possibility that the neurological phenotype in this family might be due to one of the nuclear-encoded disease genes that affects mitochondrial DNA stability and causes ophthalmoparesis and ataxia transmitted in an autosomal dominant fashion (Zeviani and Di Donato, 2004). This hypothesis was ruled out by normal morphological, biochemical and molecular findings in muscle biopsies from four patients.
Genetic analysis excluded the presence of known SCA gene mutations, and linkage exclusion tests showed no evidence of linkage to most of the known SCA loci, suggesting a genetically distinct form of SCA. A genome-wide linkage analysis allowed us to map a new locus on chromosome region 18p11.22–q11.2, designated SCA28, thus confirming that the disorder observed in this family represents a novel form of autosomal dominant SCA. The disease locus spans a 7.9 Mb region on chromosome 18, a chromosome that has never been associated with hereditary cerebellar ataxias.
Since anticipation was not present in this family and RED analysis did not reveal expanded CTG/CAG 
40 repeats, we speculate that repeat expansions are an unlikely cause of the disease. A common disease haplotype was found in all affected members but also in a 47-year-old woman (III-11) who had no symptoms of cerebellar ataxia. This subject was neurologically evaluated and found to present horizontal gaze-evoked nystagmus with no gait or limb ataxia. These findings can be explained by reduced penetrance or expressivity, or a late onset of the disease in this subject.
Approximately 65 known genes map in the critical interval (Human Genome Assembly NCBI35.1) including one disease-gene (MC2R) that encodes the melanocortin-2 receptor and is mutated in the autosomal recessive glucocorticoid deficiency type 1 (GCCD1, OMIM 202200
[OMIM]
). Since none of the genes in the critical region exhibits obvious similarity with any of the currently known ADCA-causing genes, a candidate gene approach based on expression in disease-affected tissues is being used (Tiffin et al., 2005). According to the GNF Affy HG U95 expression dataset (Hide et al., 2003; Ensembl EnsMart Genome Browser, http://www.ensembl.org/Homo_sapiens/martview), 12 genes in the region are significantly expressed in the CNS including cerebellum and their sequence analysis is currently underway.
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Notes |
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* These authors contributed equally to this work
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Acknowledgements |
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The authors are grateful to the family members for participating in this study. This study was promoted by the European Integrated Project on Spinocerebellar Ataxias (EUROSCA) awarded to S.D.D. The work was partially supported by grants from the Italian Ministry of Health (‘Malattie Neurodegenerative’ ex art 56 to S.D.D. and RF2002/160 to F.T.), Telethon-Italia (GGP04254 to A.B.), "National Organization for Rare Disorders" NORD (to A.B.), ‘Associazione Emma & Ernesto Rulfo per la Genetica Medica’ and ‘Associazione Gli Amici di Valentina’ (to A.B.). We are indebted to Mr Franco Carrara, and Dr Federica Invernizzi for the analysis of mtDNA and respiratory chain complexes, and Dr Barbara Castellotti for preliminary molecular studies. We also wish to thank Drs Antonella Antonelli, Ivano Dones and Massimo Pandolfo for early clinical studies on this family.
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