Abstract
Objective: To characterize a distinct form of autosomal dominant cerebellar ataxia (ADCA) clinically and genetically.
Background: ADCAs are a clinically, pathologically, and genetically heterogeneous group of neurodegenerative disorders. Nine responsible genes have been identified for SCA-1, -2, -3, -6, -7, -8, -10, and -12 and dentatorubral-pallidoluysian atrophy (DRPLA). Loci for SCA-4, -5, -11, -13, and -14 have been mapped.
Methods: The authors studied a four-generation Japanese family with ADCA. The 19 members were enrolled in this study. The authors performed the mutation analysis by PCR and a genome-wide linkage analysis.
Results: Nine members (five men and four women) were affected. The ages at onset ranged from 20 to 66 years. All affected members showed pure cerebellar ataxia, and three patients also had head tremor. Head MRI demonstrated cerebellar atrophy without brain stem involvement. The mutation analysis by PCR excluded diagnoses of SCA-1, -2, -3, -6, -7, -8, and -12 and DRPLA. The linkage analysis suggested linkage to a locus on chromosome 8q22.1-24.1, with the highest two-point lod score at D8S1804 (Z = 3.06 at θ = 0.0). The flanking markers D8S270 and D8S1720 defined a candidate region of an approximately 37.6-cM interval. This candidate region was different from the loci for SCA-4, -5, -10, -11, -13, and -14.
Conclusion: The family studied had a genetically novel type of SCA (SCA-16).
The autosomal dominant cerebellar ataxias (ADCAs) are a genetically heterogeneous group of neurodegenerative disorders affecting the cerebellum and other components of the nervous system. Classification of ADCA based on clinical and neuropathologic features is useful to make a clinical diagnosis. Harding1 classified ADCAs into three different groups based on the associated signs: ADCA I includes optic atrophy, ophthalmoplegia, pyramidal signs, extrapyramidal signs, peripheral neuropathy, or dementia; ADCA II, retinopathy; and ADCA III, absence of associated signs. However, the clinical and pathologic features are variable even in the same family, and recent genetic studies have revealed a great heterogeneity in these subgroups. Thus, classification based on the responsible genes or genetic loci is becoming prevalent.
Several distinct SCA genes and genetic loci have been identified. In the relationship between the genetic and clinical classifications, SCA-1, -2, -3, -4, -8, -12, -13, and -14 belong to ADCA I, SCA-7 to ADCA II, and SCA-5, -6, -10, and -11 to ADCA III. The genes or loci have not been identified in one-third of ADCA families worldwide and about one-fifth of Japanese families,2 implying the presence of other unidentified responsible genes. We report the clinical and genetic studies on a Japanese ADCA family and provide evidence for a genetically novel SCA.
Methods and patients.
Clinical studies.
We examined a four-generation Japanese family that had cerebellar ataxia. All the members in the first generation, whose medical histories were taken, were dead, but 19 members in the second to fourth generations were enrolled in this study. They were all neurologically examined by the same neurologist (Y.M.). Those who presented overt cerebellar signs such as scanning speech and limb and truncal ataxia were regarded as affected. An affected mother and her affected son were hospitalized for extensive clinical evaluation including neuroradiologic and electrophysiologic studies and ophthalmologic examinations.
Genetic studies and linkage analysis.
After informed consent was obtained, blood samples were drawn from each family member. DNA was extracted from the peripheral whole blood using standard techniques. CAG or CTG expansions were examined in the SCA-1,3 -2,4-6⇓⇓ -3,7 -6,8 -7,9 -8,10 -12,11 and dentatorubral-pallidoluysian atrophy (DRPLA)12,13⇓ genes by PCR.
A genome-wide genotyping was performed using the ABI Prism™ Linkage Mapping Set version 2 (PE Applied Biosystems, Foster City, CA) comprising 400 highly polymorphic markers to cover the entire genome, with an average interval of 10 cM. Then 13 flanking markers in chromosome 8q (Généthon chromosome 8 linkage map) were analyzed to refine the candidate region. The reaction mixture was prepared in a final volume of 16 μL GeneAmp PCR Buffer II containing 20 ng of genomic DNA, 250 μM deoxynucleoside triphosphate (dNTP), 2.5 mM MgCl2, 300 nM each primer, and 0.7 units of AmpliTaq Gold DNA polymerase (PE Applied Biosystems, Foster City, CA). PCR was performed at 95 °C for 12 minutes to activate AmpliTaq Gold DNA polymerase; for 10 cycles of denaturation at 94 °C for 15 seconds, annealing at 55 °C for 15 seconds and elongation at 72 °C for 30 seconds; for 25 cycles of denaturation at 89 °C for 15 seconds, annealing at 55 °C for 15 seconds and elongation at 72 °C for 30 seconds. The pooled reaction products were supplemented with the internal-size standards and analyzed with a model 377 automated DNA sequencer (PE Applied Biosystems) and GENESCAN (version 2.0) (PE Applied Biosystems) peak-calling software. The genotypes were defined and edited using the GENOTYPER (version 1.1) program (PE Applied Biosystems). Haplotypes were constructed manually based on the family structure to minimize the number of recombinations.
Two-point linkage analysis was performed with the LINKAGE program version 5.10 (Dr. Ott, Rockefeller University, New York, NY),14 under the assumption of autosomal dominant inheritance and a disease frequency of one in 100,000. The allele frequencies were assumed to be equal for each marker. Because the penetrance of the disorder is a function of age, the penetrance in each individual was assigned to the following five liability classes determined from the age at onset in nine informative patients: 0.133 at age 10 to 20 years, 0.329 at age 20 to 30, 0.525 at age 30 to 40, 0.721 at age 40 to 50, and 0.977 at age 50 years or more.
Results.
Clinical phenotype.
An autosomal dominant transmission was the most likely, although a male-to-male transmission was absent in the pedigree (figure 1). Among 19 family members, nine (five men and four women) were affected. In the affected individuals, the mean age at onset was 39.6 ± 15.5 (SD) years and the range was 20 to 66 years (table 1). Anticipation was not apparent in this family. The mean age at examination was 55.0 ± 17.9 (SD) years and the range was 26 to 80 years, and the disease duration was 1 to 40 years. The initial symptom was gait disturbance in six members, ataxic speech in two, and head trembling in one. The progression was slow but varied. It took several as long as 20 years to require assistive devices or wheelchair-bound status. The cardinal clinical features were relatively pure cerebellar signs including truncal and limb ataxia, dysarthria of a scanning type, horizontal gaze-evoked nystagmus, and impaired smooth pursuit of the eyes. Three of the nine affected individuals (33%) also showed an involuntary movement of the head, whose regular and rotatory character indicated tremor and distinguished it from titubation. None had other extracerebellar signs.
Figure 1. Pedigree of the current family. Squares = men; circles = women. Affected patients are indicated by black symbols, unaffected individuals by white symbols, and a possible carrier by a symbol with a dot. Deceased individuals are indicated by a diagonal line. Haplotypes for chromosome 8 markers are presented below symbols. Haplotypes cosegregating with the trait are boxed. Informative recombination events place the SCA-16 disease locus between markers D8S270 and D8S1720.
Clinical characteristics of patients affected with SCA-16
Patient II-3.
A 75-year-old woman had noted difficulty in climbing stairs without a handrail when she was about 35 years old. The ability gradually deteriorated, and she noticed clumsiness in walking on even ground in her 40s and dysarthria in her 50s. She could not walk without aid at 65 years old. On neurologic examination, she showed horizontal gaze-evoked nystagmus, severe dysarthria, and severe limb and truncal ataxia. Muscle strength, sensation, deep tendon reflexes, and mentation were normal. Nerve conduction studies, motor evoked potentials, and somatosensory evoked potentials of all four limbs were normal. Head MRI demonstrated cerebellar atrophy without brain stem involvement (figure 2, A and B). The ophthalmologic examination revealed bilateral drusen.
Figure 2. T1-weighted head MRI shows atrophy of the cerebellum without brain stem involvement. Repetition time = 400, echo time = 12. (A and B) Patient II-3. (C and D) Patient III-8.
Patient III-8.
A 42-year-old man, the youngest son of patient II-4, had noted clumsiness in walking when he was about 31 years old. He was apt to fall down and noticed dysarthria when he was about 38 years old. He developed difficulty in climbing stairs without a handrail when he was about 40 years old. On neurologic examination, he had horizontal gaze-evoked nystagmus, moderate dysarthria, and moderate limb and truncal ataxia. Muscle strength, sensation, deep tendon reflexes, and mentation were normal. Nerve conduction studies, motor evoked potentials, and somatosensory evoked potentials were normal. Head MRI demonstrated cerebellar atrophy without brain stem involvement (see figure 2, C and D). 18F-fluoro-2-deoxyglucose PET demonstrated no abnormalities. Ophthalmologic findings were normal.
Genetic studies.
Polymerase chain reaction analyses did not identify CAG or CTG expansion in the SCA-1, -2, -3, -6, -7, -8, -12, or DRPLA genes.
Of the 400 markers, 389 were available for this study due to a failure of PCR. Markers with two-point lod scores of ≥1.0 were found on chromosomes 1, 2, 4, 8, 9, 11, 13, and 19. Haplotype analyses of markers on these chromosomes suggested a possible linkage of the disease locus to the region flanking D8S514 and D8S284 markers (data not shown).
Subsequently, further genotyping was performed using 13 markers more closely flanking D8S514 and D8S284. Results of two-point analyses are shown in table 2. The highest two-point lod score was at D8S1804 (Z = 3.06 at θ = 0.0). Recombination events were identified in the second generation for D8S270, III-1 for D8S270 and D8S1801, and IV-5 for D8S1720 (see figure 1). Affected members shared identical haplotypes of markers from D8S1784 to D8S1774, and unaffected ones did not. IV-4 had the same haplotypes as the affected members but did not present with cerebellar ataxia yet. She was 19 years old and could be an asymptomatic carrier. Thus, the markers D8S270 and D8S1720 defined a candidate interval of approximately 37.6 cM on chromosome 8q22.1-24.1.
Two-point lod scores for SCA-16 vs markers in chromosome 8q22.1-24
Discussion.
We have identified an SCA locus on human chromosome 8q22.1-24.1. To date, the genetic loci of SCA have been mapped as follows: SCA-1 on chromosome 6p,15 -2 on 12q,16 -3 on 14q,17 -4 on 16q,18 -5 on 11,19 -6 on 19p,8 -7 on 3p,20-22⇓⇓ -8 on 13q,10 -10 on 22q,23 -11 on 15q,24 -12 on 5q,11 -13 on 19q,25 -14 on 19q,26 and DRPLA on 12p.13 Thus, the presently identified locus of SCA is novel, and this form of SCA has been designated SCA-16 (with approval from the HUGO Genome Nomenclature Committee).
The clinical feature of SCA-16 is pure cerebellar ataxia with head tremor. Head tremor may be a rare symptom for all SCA types. It was described in SCA-2,27 -7,28 and -1211 but not in ADCA III. Head tremor is most commonly associated with essential tremor. PET studies have identified overactivity of the cerebellar connection in essential tremor.29 Thus, head tremor is not necessarily an extracerebellar feature in the present family, which may be considered among ADCA III.
The relative prevalences of ADCAs vary in different ethnic backgrounds. Before assigning SCA-7, -8, -10, and -12 loci, the relative prevalences in Japanese were estimated as follows: SCA-1 (3%), -2 (5%), -3 (43%), -6 (11%), and DRPLA (20%). Relative prevalences in whites were estimated as follows: SCA-1 (15%), -2 (14%), -3 (30%), -6 (5%), and DRPLA (0%).2 SCA-6 and DRPLA are more frequent in Japanese. Linkage studies on unassigned pedigrees of ADCA with markers on 8q22.1-24.1 in various races will determine whether SCA-16 is predominant in Japanese ADCA or not.
In SCA-1, -2, -3, -6, -7, -8, -10, -12, and DRPLA, the responsible genes have been identified. All of them present anticipation, and their genetic mutation involves an expansion of the trinucleotide CAG/CTG repeat or the pentanucleotide repeat ATTCT.30 Anticipation was not apparent in SCA-16, suggesting that this mutation is different from the expansion of the trinucleotide repeat. The neuronal potassium channel α subunit gene (Kv8.1)31 has been mapped to 8q22.3-24.1 and the KQT-like potassium channel gene (KCNQ3)31 has been mapped to 8q24. These ion channel genes may be the candidate genes for SCA-16. Kv8.1 is widely distributed in the brain, including the granule cell and Purkinje cell layers of the cerebellum, and may be involved in cerebellar function. The dysfunction of channel molecules often causes episodic symptoms. KCNQ3 is related to benign familial neonatal convulsion,32 shaker-related potassium voltage-gated channel (KCNA1) to episodic ataxia type 133, and α1A-voltage-dependent calcium channel (CACNA1A) to familial hemiplegic migraine and episodic ataxia type 2.34 Episodic symptoms are absent in SCA-16. Because SCA-6, which lacks apparent episodic symptoms, is caused by a small expansion of the CAG repeat in the CACNA1A gene, the possibility of a channel gene mutation cannot be excluded in SCA-16. Identification of another SCA gene is helpful to the investigation, diagnosis, and genetic counseling of patients with ADCAs.
Acknowledgments
Acknowledgment
The authors thank all members of this family for participation in this study and Mrs. N. Shinnoh, Mrs. T. Tokuyasu, Miss K. Fukuyama, and Miss M. Obo for technical assistance.
- Received January 12, 2001.
- Accepted March 10, 2001.
References
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