Spinocerebellar Ataxia Type 1 in China: Title and subTitle BreakMolecular Analysis and Genotype-Phenotype Correlation in 5 Families FREE

THE DOMINANTLY inherited spinocerebellar ataxias (SCAs) represent a group of genetically diverse neurological conditions that are characterized by progressive deterioration of balance due to degeneration of the cerebellum and its afferent and efferent pathways.^{1 - 2} Extracerebellar symptoms include nuclear or supranuclear ophthalmoparesis, slow saccades, pyramidal and extrapyramidal signs, and axonal neuropathy. Several genetically distinct types of autosomal dominant ataxia have been mapped: SCA type 1 (SCA1) to chromosome 6p,³ SCA2 to 12q,⁴ SCA3/Machado-Joseph disease (MJD) to 14q,^{5 - 6} SCA4 to 16q,⁷ SCA5 to 11cen,⁸ SCA6 to 19p,⁹ SCA7 to 3p,^{10 - 12} SCA8 to 10q24,¹³ SCA10 to 22q13,¹⁴ SCA11 to 15q14-21.3¹⁵ and SCA12 to 5q31-33.¹⁶ In 6 types, SCA1,¹⁷ SCA2,^{18 - 20} SCA3/MJD,²¹ SCA6,⁹ SCA7,²² and SCA12,¹⁶ the disease-causing gene has been cloned and the mutation identified as an expansion of a CAG trinucleotide repeat in the gene coding region. CAG repeat expansion is the mutational mechanism in a large group of neurodegenerative diseases that includes, in addition to ataxias, spinobulbar muscular atrophy,²³ Huntington disease,²⁴ and dentatorubral pallidoluysian atrophy (DRPLA).^{25 - 26}

Spinocerebellar ataxia type 1 is an autosomal dominant neurodegenerative disorder characterized by cerebellar ataxia, progressive motor deterioration, and loss of cerebellar Purkinje cells and brainstem neurons. This type of disease was first reported by Schut²⁷ in a large US family of Russian extraction. The SCA1 locus was initially linked to the vicinity of the HLA locus on the short arm of chromosome 6 in a small Japanese family,³ and subsequently the SCA1 gene was mapped to the 6p22-p23 chromosomal region.^{28 - 32} The SCA1 gene was subsequently cloned and the mutation identified as an unstable trinucleotide CAG repeat.¹⁷

This study was part of a larger effort to characterize 75 Chinese families diagnosed as having autosomal dominant SCA. The results of molecular analysis allowed the identification of SCA3/MJD in 26 of these families,³³ SCA2 in 9,³⁴ SCA6 in 2 (Y.-X.Z., et al, unpublished data, 2000), and SCA7 in 2 (W.-H.G., et al, unpublished data, 2000), and we provide herein the first documentation of molecularly confirmed SCA1 in Mainland China.

One hundred nine individuals from 75 kindreds with autosomal dominant SCA, 16 patients with sporadic SCA or spastic paraplegia, 280 control chromosomes of the Chinese population, and 120 control chromosomes of the Sakha population were selected for this study. The affected families originated in Beijing, Shanghai, Shenyang, Hunan, Hubei, Jiangxi, Zhenjiang, Jiangsu, Hebei, and Inner Mongolia, representing equally the southern and northern parts of China. Table 1 shows the distribution of patients according to geographic origin. All subjects were examined by the same 3 neurologists (G.-X.W., Y.-X.Z., and B.-S.T.). Studies of families with SCA2 and SCA3/MJD have previously been reported.^{33 - 42}

Blood samples were collected from 125 individuals affected with autosomal dominant SCA and 16 patients with sporadic SCA or spastic paraplegia, 140 healthy Chinese controls, and 60 healthy Sakha controls after obtaining informed consent. High-molecular-weight genomic DNA was extracted from either peripheral leukocytes or lymphoblastoid cell lines transformed by Epstein-Barr virus following standard procedures.⁴³

The number of CAG repeat units in the SCA1 gene was determined by polyacrylamide gel electrophoresis of polymerase chain reaction (PCR) fragments produced by amplification with primer pair Rep-1 and Rep-2 as outlined by Orr et al¹⁷ and Goldfarb et al.⁴⁴ SCA2 and SCA3/MJD1 alleles were amplified and analyzed on agarose and acrylamide gels using previously described methods.^{18 ,43} DRPLA alleles were amplified using primers and conditions described by Koide et al.²⁵ SCA6, SCA7, SCA8, and SCA12 alleles were amplified using gene-specific primers.^{9 ,16 ,22 ,45} The antisense primer was fluorescently labeled. For PCR amplification, genomic DNA was denatured at 95°C for 2 minutes and the reaction carried out for 32 cycles consisting of 1-minute denaturation at 95°C, 1-minute annealing at 60°C, and 1-minute elongation at 72°C. This was followed by a final elongation at 72°C for 7 minutes, in a total volume of 20 µL containing 200 ng of genomic DNA; 20 pmol/mL of each primer; 200 mmol/L each of deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxythymidine triphosphate, and deoxycytidine triphosphate; 50-mmol/L potassium chloride; 1.5-mmol/L magnesium chloride; 10-mmol/L tromethamine, pH 8.8; and 5 U of Taq polymerase. The PCR products were electrophoresed in a 5% denaturing polyacrylamide gel on an automated ABI 373A sequencer, and analysis was performed by using the GeneScan 672 program (ABI-Perkin Elmer, Foster City, Calif).^{18 ,44} With some samples, PCR was performed in a total volume of 20 µL containing 200 ng of genomic DNA; 20 pmol/mL of each primer; 200 mmol/L each of deoxyadenosine triphosphate, deoxyguanosine triphosphate, and deoxythymidine triphosphate, 200 mmol/L for each; 30-µmol/L deoxycytidine triphosphate; 370 KBq of [α-³²P]–deoxycytidine triphosphate (370 MBq/mL); 50-mmol/L potassium chloride; 1.5-µmol/L magnesium chloride; 10-µmol/L tromethamine, pH 8.8; and 5 U of Taq polymerase.⁴⁶ The PCR products were electrophoresed in 5% denaturing polyacrylamide gel together with PCR products from cloned templates of normal and expanded SCA1 alleles as size standards and subjected to autoradiography. A molecular weight standard was included to accurately determine the number of CAG repeats.

The PCR products amplified from normal and SCA1 chromosomes were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. DNA was recovered from agarose gel plugs using the GeneClean kit (Bio101), subcloned into pGEM-T easy vector (Promega, Madison, Wis), and transfected into DH5a-competent cells. Insert-containing clones were sequenced using the Sequenase version 2.0 DNA sequencing kit. The numbers of CAG repeats and CAT interruptions were determined from sequence analysis.

Correlation of the age of disease onset with the number of CAG repeat units was determined by Pearson correlation coefficient test. Other statistical analyses were performed using the t test or χ² test.

Most of our patients originated in the midnorthern, northeastern, mideastern, and midsouthern regions of China, such as Beijing, Shanghai, Shenyang, Hunan, Hubei, Jiangxi, Zhejiang, Jiangsu, Hebei, and Inner Mongolia. Table 1 shows the distribution of patients according to geographic origin. The SCA1 mutation was detected in 5 (7%) of 75 Chinese families with autosomal dominant SCA. All 12 patients with the SCA1 phenotype were heterozygous for alleles with CAG repeat numbers within a range of 51 to 64. The number of trinucleotide repeats in the normal alleles of the Chinese control subjects ranged from 26 to 35. As in all other disorders with CAG repeat expansion, we observed a statistically significant negative correlation between the age of disease onset and the number of CAG repeat units in the expanded alleles, with a Pearson correlation coefficient of r = −0.914.

Clinical features of the studied patients with SCA1 are summarized in Table 2. The mean ± SD age at last examination was 37.3 ± 4.3 years. Patients with SCA1 in our series shared classic clinical features of gait and limb ataxia, dysarthria, pyramidal tract signs (spasticity, hyperreflexia, and extensor plantar responses), and variable degree of oculomotor dysfunction, which includes 1 or more of the following: nystagmus, slow saccades, and ophthalmoparesis. Saccades were slowed in 50%. Gaze paralysis was most frequent in the vertical plane, usually upward (33%). Nystagmus on lateral gaze was rare. Motor weakness, amyotrophy, and mild sensory deficits manifested as proprioceptive loss were seen in some patients. Dementia was evident in 4 (33%) of 12 patients with SCA1. Although ataxia, dysarthria, and cranial nerve dysfunction were consistently present in every SCA1-affected individual, considerable intrafamilial variability was noted with regard to all of the other clinical features. Several of the kindreds that did not have an expanded SCA1 CAG repeat displayed the same clinical findings as were observed in SCA1 kindreds, reflecting the inherent difficulty of clinical classification. The frequencies of specific clinical signs were analyzed by the Mann-Whitney U test in an effort to determine whether any correlation existed with the number of CAG repeats, but none were found in our data set.

The combined frequency of SCA1, SCA2, and SCA3/MJD in the Chinese population was 53%. None of the 16 patients with sporadic SCA or spastic paraplegia tested positive for SCA1, SCA2, SCA3, SCA6, or SCA7, nor did any of our patients with inherited or sporadic ataxia test positive for SCA8, SCA12, or DRPLA, including a large family with myoclonus epilepsy, tremor, and ataxia (data not shown). Clinical finding in 12 patients from 5 SCA1 families were compared with 16 patients from 9 SCA2 families and 72 patients from 26 SCA3/MJD families in Table 2. Onset age was not statistically different. Hyperactive reflexes and spasticity were more frequent in SCA1, whereas hypoactive reflexes and slow saccades were more frequent in SCA2; facial myokymia and horizontal nystagmus were seen more frequently in SCA3/MJD. Of patients with SCA3/MJD, only 12 (46%) were correctly diagnosed on clinical grounds. These patients manifested with typical adult-onset (type II-III MJD) phenotype that included ophthalmoparesis with eyelid retraction, facial myokymia, ataxia, spasticity, and amyotrophy. The remaining 54% of the patients with SCA3/MJD had clinical features indistinguishable from patients with other types of ataxia, such as SCA1 or SCA2. Of SCA2 families, only 5 (56%) and of SCA1 families only 2 (40%) were diagnosed on clinical grounds alone. Molecular diagnosis was helpful in identifying or confirming the type of SCA in each of these cases.

The results of sequence analysis showed that 65% of the Siberian Sakha and only 8% of Chinese normal alleles had a single CAT interruption. Based on historic and ethnographic studies,⁴⁷ the Sakha (Ilakut) population, currently numbering 350 000, is a relatively recent arrival in eastern Siberia. The prevalence of SCA1 is extremely high in this small population,⁴⁸ whereas the prevalence of SCA1 in the Chinese population is significantly lower.

In 1992, at an MJD international workshop, families with a dominantly inherited ataxia were discussed in detail, and several MJD families from China were described as well as other SCA-type families.⁴⁹ The discussion and agreement that dominantly inherited ataxias were present in China led us to do a more detailed clinical-molecular analysis. Subsequently, we have had the opportunity to evaluate members representing 75 Chinese kindreds with dominantly inherited ataxia. The cloning of genes and identification of the causative mutations in several types of SCA have provided a powerful tool for establishing a definitive diagnosis by genetic testing. Our data indicate that 5 (7%) of the studied families with hereditary ataxia originating from various regions of China, including Beijing, Shanghai, Jiangsu, Zhejiang, Hebei, Liaoning, Hunan, Hubei, Jiangxi, and Inner Mongolia, had SCA1. This frequency is similar to the frequencies recently reported in other population groups.^{50 - 52} The prevalence of SCA2 and SCA3/MJD in China is also within the range observed in other large populations.^{50 - 55} The combined frequency of SCA1, SCA2, and SCA3/MJD among the Chinese families with autosomal dominant ataxia is 53%. The prevalence of SCA6 and SCA7 is 4% and 3%, respectively (Y.-X.Z. and W.-H.G., unpublished data, 2000). None of our patients had DRPLA, including a large family whose disease manifested as myoclonus epilepsy, tremor, and ataxia, suggesting that DRPLA is rare in non-Japanese populations. Analysis of 280 normal chromosomes from 140 individuals demonstrated that the number of CAG repeats in the coding region ranges in size from 26 to 35 repeats. The number of CAG repeats in normal and SCA1 Chinese chromosomes is similar to those described in other reports.^{50 - 52} We found a strong inverse correlation between the age at disease onset and the CAG repeat number in the expanded alleles, similar to features previously described in Huntington disease and other studied SCAs.

We observed a wide spectrum of clinical features in patients with SCA1, significantly overlapping with SCA2 and SCA3/MJD, but there was no statistically significant correlation between the number of CAG repeat units in the SCA1 gene and clinical manifestations. Such correlations were previously reported in patients with SCA3/MJD⁵⁶ and SCA2. This discrepancy may be due to the small size of our data set. The comparison of the clinical features of our patients with 3 reported series^{57 - 59} showed an overall clinical similarity (Table 3). Some statistical differences among series may be explained by differences in evaluation criteria and examiners. Meanwhile, the fact that low occurrence of slow saccades and sphincter disturbances and high occurrence of horizontal nystagmus, lower limb weakness, lower limb amyotrophy, and knee reflexes occurred in our patients with autosomal dominant cerebellar ataxia type 1 compared with 3 other large groups^{60 - 62} could be explained by occurrence of different mutations in our population (Table 4). The existence of different clinical subtypes within autosomal dominant cerebellar ataxia type 1 has been addressed.²

Of the patients with SCA1 whom we studied, only 2 could be reliably diagnosed without genetic testing. A wide variety of phenotypes seen in SCA1 and a significant degree of clinical overlap with SCA2 and SCA3/MJD highlight the difficulty of making the diagnosis on the basis of clinical information alone. Of the patients with SCA2 we studied, only 5 (56%) and of the patients with SCA3/MJD only 12 (46%) could be reliably diagnosed without genetic testing. We found that SCA3/MJD, more often than other SCA types, manifests with specific features such as ophthalmoparesis and characteristic eyelid retraction, facial myokymia, ataxia, spasticity, and amyotrophy. Patients with SCA3/MJD who merely have ataxia with slowed saccades were clinically indistinguishable from patients with SCA1 or SCA2. The PCR-based testing makes it possible to diagnose the type of ataxia with great certainty. This underscores the importance of genetic testing for diagnostic accuracy in patients with autosomal dominant ataxia.

In the present study, we found a close association between the prevalence of SCA1 in Chinese and Sakha populations and the frequency of CAT interruptions in the SCA1 gene. A similar association between the relative prevalence of SCA1 (15%)⁶³ in the North American population and the frequency of CAT interruptions (11%) was reported.⁶⁴ If a substitution of CAT for CAG was the initial event contributing to generation of expanded alleles, the Sakha population must have been predisposed to such a transition by simply having more individuals carrying a single CAT interruption.

Accepted for publication October 27, 2000.

We thank the families for their active involvement in this project. We also thank Roger N. Rosenberg, MD, for his constructive and critical comments. We thank S. G. Brodie, PhD, for valuable comments.

We gratefully acknowledge research support from the National Natural Science Foundation of China and Research Foundation of the Ministry of Public Health of China (Drs Y.-X. Zhou and Wang).

Corresponding author and reprints: Yong-Xing Zhou, MD, PhD, Genetics of Development and Disease Branch, Bldg 10/9N104, NIDDK, NIH, 10 Center Dr Bethesda, MD 20892.