Introduction and objectives
Spinocerebellar ataxia autosomal recessive (SCAR) represents a heterogeneous chronic and progressive neurological diseases group. They usually occur at an early age in a progressive manner. Diagnosis is complex due to phenotypic overlap. SCARs account for more than 50% of all ataxia cases of genetic origin, with a prevalence of 3–4/100 000. According to international published series, Friedreich’s ataxia (FA) is the most common. In Mexico, more than 90% of patients with suspected SCAR remain without etiologic diagnosis after ruling out FA and acquired causes of ataxia. Our main goal was to reach a diagnosis using genomic tools in this group of patients.
Materials and methods
At the National Institute of Genomic Medicine, we used next-generation sequencing as a diagnostic tool in 4 patients with a clinical diagnosis of SCAR to identify and classify etiologic variants responsible for this group of disorders.
Two novel pathogenic variants were identified in the SACS gene, and the diagnosis of autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) was established.
This is the first report of spastic ataxia of Charlevoix-Saguenay cases in Mexico.
Introducción y objetivos
Las ataxias espinocerebelosas autosómico recesivas (SCAR) representan un grupo heterogéneo de enfermedades crónicas progresivas. Este grupo de enfermedades inician a una edad temprana y suelen ser progresivas. Su diagnóstico es complejo debido a superposición fenotípica. Las SCAR representan más del 50% de los casos de ataxias de origen genético con una prevalencia de 3–4/100,000, siendo la ataxia de Friedreich (FA) la más común de acuerdo con series internacionales publicadas. En México, después de descartar FA, más del 90% de pacientes con sospecha de SCAR quedsn sin diagnóstico etiológico. Nuestro principal objetivo es el de establecer un diagnóstico etiológico mediante el uso de herramientas genómicas en este grupo de pacientes.
Material y métodos
En el Instituto Nacional de Medicina Genómica, utilizamos secuenciación de siguiente generación como herramienta diagnóstica en 4 pacientes con SCAR para identificar y clasificar las variantes patogénicas responsables del cuadro clínico.
Identificamos dos variantes nuevas en el gen SACS y establecimos el diagnóstico de ataxia espástica recesiva de Charlevoix-Saguenay (ARSACS).
Este es el primer reporte de ataxia espástica recesiva de Charlevoix-Saguenay en México.
Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) was first reported by JP Bouchard in 1978 among the population from the Charlevoix-Saguenay-Lac-Saint-Jean (CSLSJ) region of Quebec, Canada, where the estimated carrier prevalence of the original mutation (c.8844delT) is 1/22 due to a founder effect in the French-Canadian population.
ARSACS is considered a rare recessive disease occurring worldwide. , Cases have been reported in European, Asian, Middle Eastern, North America, and African countries. For the Latin American region, cases have been reported only from Brazil and Chile. , , ,
ARSACS is classically characterized by a triad of early-onset, slowly progressive cerebellar ataxia, pyramidal spasticity, and axonal-demyelinating sensorimotor peripheral neuropathy. However, significant clinical variability has been described between patients from different countries and between affected members of the same family. , Clinical phenotypes include disease onset in early adult years, spasticity, pyramidal involvement, dysarthria, distal muscle wasting, foot deformities, truncal ataxia, absence of sensory evoked potentials in the lower limbs, retinal striation, and mitral valve prolapse in some cases. Biochemically, hyperbilirubinemia and impaired pyruvate oxidation have been reported.
The disease is caused by homozygous or compound heterozygous pathogenic variants in the SACS gene. More than 200 pathogenic or likely pathogenic SACS variants have been identified, which mainly affect exon 10 (as of January 2022, clinvarminer.genetics.utah.edu/ ). The gene is located on chromosome 13q12.12 and encodes the sacsin protein. Sacsin is a large 520-kDa 5-domain protein; individual domains act as a giant hub functioning at multiple levels of neurofilament biology. Sacsin regulates intermediate filament assembly and dynamics in many neuronal populations, including Purkinje and cortical motor neurons. Sacsin is involved in chaperon activities, microtubule balance, and cell migration. Several studies suggest that sacsin may also play an essential role in mitochondrial dynamics related to neurodegeneration. ,
Historically, the clinical diagnosis of ARSACS has been based on the presence of 3 main clinical signs: ataxia, pyramidal involvement, and axonal neuropathy. However, it is often difficult to establish a diagnosis due to the lack of typical clinical manifestations or the presence of other signs and symptoms such as deafness, intellectual disability, or seizures. Differential diagnoses include other spinocerebellar recessive ataxias, such as FA, abetalipoproteinemia, ataxia-telangiectasia, Troyer syndrome, spastic paraplegia 7, autosomal recessive ataxia with vitamin E deficiency, and spastic paraplegia 30. Genetic testing is required for the specific diagnosis of ARSACS.
Over the past years, massively parallel sequencing for whole-genome, whole-exome, or clinical exome sequencing and the development and application of powerful bioinformatic tools have promoted significant advancements in research and diagnosis of common and rare inherited diseases. However, in low- and middle-income countries in Latin America, the use of these methodologies in routine diagnosis is limited by cost. This situation contributes to significant delays in establishing the etiological diagnosis of patients with rare diseases, such as SCAR, and the lack of epidemiological data on the mutational spectrum of most genetic diseases.
In collaboration with other public health institutions, the Genomic Diagnostic Laboratory from the National Institute of Genomic Medicine has been performing exome sequencing in patients with clinical suspicion of SCAR to identify its genetic cause. This approach allowed us to report the first cases of spastic ataxia of Charlevoix-Saguenay in Mexico and to describe 2 new mutations in the SACS gene. This study expands on the mutational spectrum and geographic location of the disease.
Material and methods
Study design and participants
Four Mexican mestizo patients of 2 unrelated families ( Fig. 1 ) with clinical suspicion of SCAR were evaluated by clinical geneticists and neurologists. The evaluation confirmed that patients had chronic and progressive gait or limb ataxia. All of them tested negative for FA by PCR analysis. Acquired causes of ataxia were also ruled out according to standard protocols.
Written informed consent for clinical evaluation, exome sequencing, and data report was obtained from all studied participants. A clinical geneticist provided pre- and post-test counseling to patients and their families. A clinical report with the genetic findings was given to each patient. The National Institute of Genomic Medicine in Mexico provided financial support to conduct this research study.
DNA quality and quantity
Genomic DNA (gDNA) was extracted from peripheral blood samples using Maxwell® 16 Blood DNA Purification Kit (Promega, Madison, WI, USA). DNA purity and concentration were determined using NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). A total of 50 ng determined by Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) was used for clinical exome sequencing.
Clinical exome sequencing
Library preparation was performed according to the manufacturer’s protocol using the reagents provided in the Clinical Exome Solution panel kit v2 (Sophia Genetics SA, Saint-Sulpice, Switzerland). Sequencing (2 x 150 bp paired-end reads) was performed on NextSeq Instrument (Illumina, San Diego, CA, USA). Sequencing data analysis and variant annotation were performed using Sophia DDM® (Sophia Genetics SA, Saint-Sulpice, Switzerland), Varsome ( varsome.com/ ), ClinVar ( www.ncbi.nlm.nih.gov/clinvar/ ), and Franklin by Genoox ( franklin.genoox.com/clinical-db/home ) platforms. Scientific literature was also consulted for variant annotation and classification. Variants were classified following the criteria published by the American College of Medical Genetics and Genomics.
FASTQ files were aligned against the reference sequence of the human genome of the National Center for Biotechnology Information of the National Institutes of Health version 37/hg19. Bioinformatic filters were applied, including genes previously reported to be related to SCARs and phenotypes such as ataxia (HP:0001251), peripheral neuropathy (HP:0009830), cerebellar atrophy (HP:0007360), spinal atrophy (HP:0007344), and spasticity (HP:0001257). The coverage depth for variants analyzed ranged from 96 to 135x, with a mean of 115x. The transcript NM_014363.6 was used for SACS gene variant annotation.
Case 1 is a 50-year-old male born from a non-consanguineous marriage in Mexico City ( Fig. 1 A). At age 5, he developed progressive ataxia, dysmetria, cerebellar tremor, and slurred speech. The initial neurological examination revealed bilateral gaze-evoked nystagmus, preserved strength 5/5 except for distal lower limbs (2/5), absent reflexes in all extremities, increased muscle tone in lower limbs, absent Babinski sign, and decreased vibration sensation in lower extremities. Clinical findings are summarized in Table 1 . Pes cavus and hammertoes were evident on the last examination at age 47 ( Fig. 2 ); gait was ataxic and only possible with crutches. Brain MRI revealed severe cerebellar and cervical spine atrophy ( Fig. 3 ).
|Gender||Age||AAO a||Clinical manifestations||MRI findings|
|Patient 1 (Family 1)||M||47||5||Ataxia, slurred speech, nystagmus, spasticity and decreased vibration sensation in lower extremities, pes cavus and hammertoes||Severe cerebellar and cervical spine atrophy Fig. 3|
|Patient 2 (Family 2)||M||41||30||Ataxia, tremor, dysarthria, spasticity, pes cavus, mild cognitive impairment, nystagmus, complete pyramidal syndrome, decreased sensitivity in distal extremities||Cerebellar and cortical atrophy, hypointense pontine striae, retrovermian arachnoid cyst Fig. 4 and Fig. 5|
|Patient 3 (Family 2)||M||45||10||Ataxia, mild cognitive impairment, spasticity, slurred speech, pyramidal syndrome, decreased sensitivity in distal extremities||Cerebellar and cortical atrophy, retrovermian arachnoid cyst similar to brother Fig. 6|
|Patient 4 (Family 2)||F||50||15||Ataxia, slurred speech, mild cognitive impairment, urinary incontinence||Decreased subcortical and supratentorial cortical volume, superior cerebellar vermis atrophy, hypointense pontine striae|
Sequencing results showed the patient was compound heterozygous for the following variants: SACS (NM_014363.6):c.1201C > T(p.Arg401*) and SACS (NM_014363.6):c.11624G > A(p.Arg3875His) ( Table 2 ). Variant c.1201C > T (p.Arg401*) is located in exon 8, which creates a premature translational stop at codon 401. This mutation is expected to result in an absent protein product since 4179 amino acids are lost. According to the Genome Aggregation Database (gnomAD), its frequency is very low; it has only been identified in 1 individual of European ethnicity (as of January 2022). The variant was not found in a local database of 480 Mexican mestizo exomes. Loss-of-function variants in SACS are known to be pathogenic. Additionally, the variant is enlisted in the ClinVar database (Allele ID 840614) and classified as pathogenic (as of January 2022) and is also classified as pathogenic according to the ACMG criteria.