The dynamic mutation investigation and whole exome sequencing in a cohort of Chinese autosomal dominant cerebellar ataxia patients

Background Spinocerebellar ataxias (SCAs) are the autosomal dominant cerebellar ataxia (ADCA) with great clinical and genetic heterogeneity. Genetic testing will contribute to the final diagnosis. Methods A total of 204 Chinese ADCA patients were recruited and 190 had genetic testing. Dynamic mutations of SCA1, 2, 3, 6, 7, 8, 10, 12, 17 and dentatorubral-pallidoluysian atrophy (DRPLA) were screened firstly. For the patients with negative results, the dynamic mutations of HTT of Huntington Disease (HD), SCA31, 36 and even the whole exome sequencing (WES) were further performed. We investigated the genetic results and clinical characteristics retrospectively. Results Among these 190 index cases, 177(93.16%) were identified SCA dynamic mutations. SCA3 was the commonest, accounting for 70.06%, followed by SCA1 (9.6%), 2 (9.05%), 12 (3.39%), 6 (2.26%), DRPLA (2.26%), 7(1.13%), 8 (1.13%) and 17(0.56%). One patient carried a compound dynamic mutation of SCA6 and SCA17 (SCA6/17). No SCA10 or SCA36 was found. Among the remaining 13 patients, three were diagnosed with HD (1.58%) and one with Episodic Ataxia 2 (EA2). WES did reveal several variants with uncertain significance (VUS) in the remaining nine patients, but failed to detect causative mutations. Conclusion We illustrated the approach and challenge of genetic testing in Chinese ADCA patients. Dynamic mutations of SCAs should be screened firstly. When the results were negative, dynamic mutation of HTT would better be screened consequently. In early-onset ADCA patients, WES might be effective to identify causative mutations, but in adult-onset cases, WES might be less effective.


Introduction
Spinocerebellar ataxias (SCAs) are the autosomal dominant cerebellar ataxia (ADCA) with progressive cerebellar ataxia combined with other manifestations such as peripheral neuropathy, ophthalmoplegia, pigmentary retinopathy, pyramidal signs, extrapyramidal symptoms and cognitive impairment [1] . Up to date, more than 40 SCA subtypes including dentatorubral-pallidoluysian atrophy (DRPLA) and DNMT1 [2] have been reported. Since the diverse clinical heterogeneity of SCAs, it is difficult to distinguish different subtypes on the basis of clinical features. Therefore, genetic testing will contribute to the final diagnosis.
Non-repeat mutations, especially missense mutations are another important cause of ADCA. Whole exome sequencing (WES) has been adopted as a cost-effective and highyield way to discover the non-repeat mutations related to ataxia, which might help to confirm the diagnoses of ADCA patients with negative SCA or HD dynamic mutations [19,20] .
Here we reported the genetic and clinical characteristics of 190 Chinese ADCA patients.
We illustrated the approach of genetic testing and discussed the significance and challenge of performing WES, which would help clinicians prioritize genetic testing.
The WES was carried out by Sure Select Human All Exon Kit V5 (Agilent, Santa Clara, CA) and high throughput sequencing by Illumina novasEq. The quality control and the variant screening were as previously reported [22] .
The flowchart of genetic testing was shown in Fig. 1 SCA: spinocerebellar ataxia; SD: standard deviation. *The CAG repeat numbers of five SCA1 patients were less than 45 but the CAG triplet repeats were proven without CAT repeat interruption by Sanger sequencing and were ascribed pathogenic.

Patients presenting with positional tremor
Positional tremor was the most noticeable symptom of SCA12, which could serve as a clue to confirm SCA12. In our cohort, 80% of SCA12 patients had positional tremor in the head or hands. 19% of SCA1 and 33% of SCA2 patients also complained about positional tremor.

Patients presenting with saccadic movement disorders
Saccadic movement disorders include saccadic starting slowly, sudden setback in the process and dysmetria. 73.3% of SCA2, 50% of SCA1 and 33.7% of SCA3 patients developed saccadic movement disorders.

Patients presenting with visual impairment
Both SCA7 patients presented with severe visual impairment. One patient could hardly read and the other was diagnosed with retinal pigmentosa by optical coherence tomography (OCT). 11.8% of SCA1 and 6.9% of SCA3 patients showed blurred vision.

Patients presenting with psychiatric symptoms
Three patients (11.1%) in four DRPLA pedigrees showed irritability. SCA17 could not cooperate well with the physical examination because of emotional swings and temper tantrum.

Patients presenting with Parkinsonism
SCA3 has been classified into several subgroups previously [23] . Our patients fell into three subgroups of levodopa responsive Parkinsonism, ataxia combined with pyramidal signs and peripheral neuropathy. Five SCA3 patients showed Parkinsonism, accounting for 5%, manifesting as bradykinesia, rigidity, and postural instability. One male SCA8 patient developed a typical Progressive Supranuclear Palsy (PSP) phenotype with bradykinesia, rigidity, festination and freezing gait.

Patients presenting with epilepsy
Nine patients (33.3%) in our four DRPLA pedigrees (Fig. 3) presented with epilepsy. The detailed clinical manifestations were concluded in Table 2.

Clinical characteristics of HD patients
Three HD patients were detected in our study. The average onset age was 45.67 ± 7.37.
Their repeat numbers of HTT were 46, 46 and 43 respectively. All of them presented with unstable walking and blurred speech at early stage. One patient developed chorea and cognitive decline later and the other two were still dominated by cerebellar ataxia. These atypical clinical symptoms would often lead to the initial misdiagnosis.

The results of WES
One patient was diagnosed with EA2 after performing WES. A heterozygous frameshift mutation of c.2764-2765insc (p.Arg922ProfsTer145, NM_023035) in CACNA1A [24] was revealed, which has not been reported and according to the American College of Medical Genetics and Genomics (ACMG) guideline [25] , it was rated as likely pathogenic (PM2 + PM4 + PP3 + PP4). But in the remaining nine patients, except for some VUS, no causative mutations were found. So the detection rate of WES was 10%. We concluded the clinical manifestations and predicted the pathogenicity of these VUS in Table 3.

Discussion
As a single-center study, we recruited cerebellar ataxia patients with a dominant inheritance consecutively. To investigate the relationship between genotypes and clinical phenotypes, we developed a genetic testing program. Dynamic mutations of SCA1, 2, 3, 6, 7, 8, 10, 12, 17 and DRPLA were screened firstly. Then the dynamic mutations of HD, SCA31, 36 and even the WES were further performed.
In this research, we found that SCAs were the overwhelming majority. Perhaps the limitation of dominant inheritance increased the frequency of SCAs. On the other hand, the incidence of HD in our cohort was 1.58%, no less than some SCA subtypes, suggesting that HD should be considered when patients manifested as cerebellar ataxia. When dynamic mutations of SCAs were negative, the dynamic mutation of HTT would better be detected subsequently.
As for clinical manifestations, some typical signs could help discriminate different subtypes [2] , such as saccadic movement disorders in SCA2, vision impairment in SCA7, positional tremor in SCA12, chorea, epilepsy and dementia in DRPLA, psychiatric symptoms and dementia in SCA17. Parkinsonism could occur in SCA3, but in SCA8 it has been reported as a rare phenotype [26,27] , suggesting SCA8 should not be ignored when dealing with parkinsonism. SCA31 and SCA36 were really rare in Chinese population, no SCA37 reported. Therefore, we detected SCA31 and SCA36 in the remaining ten patients.
SCA37 was characterized by ataxia with distinctive abnormal ocular movements, including dysmetric vertical and horizontal saccades and pursuit, which were apparent in the early stage, even as a pre-symptomatic feature [29] . By reviewing all the clinical characteristics of the remaining patients, no patient was consisted with the phenotypes of SCA31 and SCA37.
WES has been recognized as effective to identify the pathogenic missense mutations in cerebellar ataxia patients, with the overall detection rate 18%-41% [20,30] . And in earlyonset (≤ 20 years old) patients, the detection rate was much higher with 39%-46% [31,32] , especially in familial cases, the rate was up to 69%-75% [30,32] . These data suggested WES was effective in early-onset patients. While in adult-onset cases, the detection rate was much lower, only accounting for 8.3% [30] and 15% [19] . In 183 Chinese cases excluded for dynamic mutations, the diagnostic rate was only 9.83%, suggesting the frequencies of non-polyglutamine SCA cases might be originally low in China [13] . In our study, the detection rate was 10% and the remaining nine patients were all adult-onset patients (> 40 years old), which might not be the preferred population for WES.
These remaining patients might be caused by novel genetic abnormalities, including new dynamic mutations or non-repeat mutations, or the mitochondrial mutations which had not been screened. Therefore, in cerebellar ataxia patients, especially in adult-onset ADCA patients, WES might not play such a huge role in the final diagnosis. There still be a high possibility to discover novel genetic mutations, which would broaden the spectrums of genotypes and phenotypes related to ataxia.

Conclusion
In this study, we illustrate the approach and challenge of genetic testing in Chinese ADCA

Consent for publication
Written informed consents for publication were obtained from both patients and their relatives.

Availability of data and material
Data sharing neo applicable to this article as no datasets were generated or analyzed during the current study.

Funding
This work has no funding to support.
conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

Figure 2
Results of the genetic testing and frequencies of SCA subtypes.