Study Design and population
We conducted a cross-sectional study to describe the clinical and molecular characteristics of MJD/SCA3 patients with extended pedigrees, as well as to explore the allelic distribution of ATXN3-[CAG(n)] within a cohort of ataxic patients screened for MJD/SCA3 at a neurogenetics outpatient clinic in Lima, Peru. From January 1996 to February 2022, a total of 341 individuals from both genders with clinical diagnosis of hereditary ataxia followed up at a tertiary hospital were included in this study. The exclusion criteria were (a) the presence of any abnormality on the following tests at recruitment that explained the occurrence of ataxia or related neurological symptoms: basic blood biochemistry; vitamin B12, Vitamin E, VDRL (venereal disease research laboratory), chest X-ray; abdominal ultrasound; mammary ultrasound and mammography (in women); thyroid-stimulating hormone, lymphocyte and thrombocyte count, hemoglobin, erythrocyte mean corpuscular volume, sedimentation rate, v, antibodies (anti-HIV, Human Immunodeficiency Virus), qualitative urine test, anti-Yo and anti-Hu antibodies; (b) suggestive MRI for a vascular, autoimmune, or infectious process in the central nervous system. Informed consent was obtained from each participant. This study was approved by the Institutional Review Board from the Instituto Nacional de Ciencias Neurologicas, Lima, Peru.
ATXN3 Genotyping
Blood samples were collected, and then DNA was isolated from leukocytes at the Neurogenetics Lab in Lima using standard procedures [17]. DNA samples underwent ATXN3 genotyping based on the amplification of the CAG repeat within ATXN3 by Polymerase Chain Reaction (PCR) employing a modified procedure originally standardized by Kawaguchi et al., 1994 [18]. The modified PCR protocol had the following conditions: 1X PCR buffer, 10% DMSO, 1.5 mM MgCl2, 0.15 mM of each dNTPs, 1 µM of each primer (MJD52 5'-CCAGTGACTACTTTGATTCG-3' and MJD25 5'- TGGCCTTTCACATGGATGTGAA-3’), 0.5 U of Platinum Taq DNA Polymerase and 0.3 ng of DNA genomic in a final reaction volume of 10 µL. The amplification program had the following initial conditions: 2 min of initial denaturation at 94°C; 28 cycles of 30 sec denaturation at 94°C, 30 sec hybridization at 58.5°C and 1 min extension at 72°C; and 10 min of final extension at 72°C The amplicons were observed by 6% non-denaturing polyacrylamide gel electrophoresis. Allele sizing was performed using reference samples of known genotype analyzed by PCR and capillary electrophoresis through Rede Neurogenetica-Brazil.
Samples not displaying both alleles were further genotyped by Triple Repeat-PCR (TP-PCR) followed by 10% non-denaturing polyacrylamide gel electrophoresis. The TP-PCR was performed employing a modified procedure originally standardized by Melo et al., 2016 [19]. The modified TP-PCR protocol had the following conditions: 1X PCR buffer, 6% DMSO, 1.5 mM MgCl2, 0.2 mM of each dNTPs, 0.3 µM of primer MJD25R (5’-TGGCCTTTCACATGGATGTGA-3’), 0.06 µM of primer ForIntRep (5’-TACGCATCCCAGTTTGAGACG-3’) and 0.3 µM of primer ForTail (5’-TACGCATCCCAGTTTGAGACGCAGCAGCAGCAGCAG-3’) 0.4 U of Platinum Taq DNA Polymerase and 10 ng of DNA genomic in a final reaction volume of 10 µL. The amplification program had the following initial conditions: 2 min of initial denaturation at 94°C; 30 cycles of 30 sec denaturation at 94°C, 30 sec hybridization at 62°C and 45 sec extension at 72°C; and 10 min of final extension at 72°C.
Due to the lack of consensus to date, we classified ATXN3 alleles according to Saute & Jardim et al., 2015 [20]. 12 to 44 CAG repeat length alleles are considered normal and ≥51 CAG repeats as pathogenic. Within normal alleles, we identified a subgroup of large normal (LN) alleles with ≥27 CAG repeats [21].
MJD/SCA3 extended families identification and data collection
Among the 341 participants with ataxic symptoms, we identified seven non-related MJD/SCA3 probands. We extended the pedigrees of all seven probands and contacted most of the affected family members. We performed home visits to a total of 18 affected individuals by home visits in four different cities across the country to complete a standardized clinical questionnaire, neurological examination, SARA, MoCA and PHQ-9 rating scales assessment. Extended pedigrees were drawn using an online pedigree tool (https://www.progenygenetics.com). Age at onset was defined when the patient or a close relative noticed the first symptom of the disease, which was usually but not always gait ataxia. We collected information on the presence or absence of several neurological findings, as well as clinical and family history.
Statistical analysis
We estimated ATNX3 allelic frequencies and presented their distributions in vertical bars, grouped according to classification mentioned above. We calculated the mean, median and mode of the allelic frequency.