A clinical report of the massive CAG repeat expansion in spinocerebellar ataxia type 2: Severe onset in a Mexican child and review previous cases

Abstract The spinocerebellar ataxia type 2 is a neurodegenerative disease with autosomal dominant inheritance; clinically characterized by progressive cerebellar ataxia, slow ocular saccades, nystagmus, ophthalmoplegia, dysarthria, dysphagia, cognitive deterioration, mild dementia, peripheral neuropathy. Infantile onset is a rare presentation that only has been reported in four instances in the literature. In the present work a boy aged 5 years 7 months was studied due to horizontal gaze-evoked nystagmus, without saccades, ataxic gait, dysarthria, dysphagia, dysmetria, generalized spasticity mainly pelvic, bilateral Babinsky. The mother aged 27 years-old presented progressive cerebellar ataxia, dysarthria, dysmetria, dysdiadochokinesis, limb ataxia and olivopontocerebellar atrophy. The molecular analysis was made by identifying the expansion repeats in tandem by long PCR to analyze the repeats in the ATXN2 gene. We found an extreme CAG expansion repeats of ~884 repeats in the child. We describe a Mexican child affected by SCA2 with an infantile onset, associated with a high number of CAG repeats previously no reported and anticipation phenomenon.

occurrence presentation with only nine reports in the literature and is associated with extremely large CAG expansions (range 100-200 repeats). On the other hand, there are severe infantile cases with expansions between 50-70 repeats, sometimes is related with interruption CTG or CAA as occurs in adults with SCA2 and amyotrophic lateral sclerosis, which also leads to instability of ATXN2 mRNA (Choudhry et al., 2001;Antenora et al., 2017), and meiotic instability a general feature of SCA2 without a familial history (Babovic et al., 1998;Mao et al., 2002;Moretti et al., 2004;Dirik et al., 2007;Abdel and Zaki, 2008;Paciorkowski et al., 2011;Di Fabio et al., 2012;Vinther-Jensen et al., 2013;Singh et al., 2014). However, cases with infantile SCA2 are explain by these features; there are childhood onset cases reported with mosaicism in germ cells such as spermatozoa with larger expanded alleles more than in peripheral blood cells (Moretti et al., 2004;Dirik et al., 2007;Abdel and Zaki, 2008;Vinther-Jensen et al., 2013).
The most frequent signs in infantile onset are developmental delay, visual impairment usually dependent on retinitis pigmentosa or optic atrophy, hypotonia, seizures with infantile spasms or myoclonic seizures, facial dysmorphism, dystonic features and early death (Tables 1 and 2) (Singh et al., 2014;Antenora et al., 2017). Brain MRI scans showed extreme cerebellar and brainstem atrophy, but also different degrees of supratentorial atrophy, ventricular enlargement, and white matter signal abnormalities probably attributable to dysmyelination and/or delayed myelination (Singh et al., 2014;Antenora et al., 2017). The aim of this report is to present the clinical findings and molecular studies in a Mexican child with familial SCA2 with extremely large CAG expansion repeats, and a literature review of clinical and molecular findings in early-onset cases with SCA2.
The clinical findings in the index case, a boy aged 5 yr 7 mo ( Figure 1A), was the product of the first pregnancy complicated by hyperemesis during the first trimester, delivery was by cesarean section due to macrosomia. Since birth the patient presented feeding difficulties due to dysphagia that persists at this time; at 3 months of age nystagmus was present. Psychomotor development, at 12 months he began to walk with evident ataxic gait. When he started speaking at 2-3 years of age, he presented dysarthria, at this moment he has progression of all the symptomatology, poor coordination, walking difficulty, incontinence (he never had sphincter control) and he continues with dysphagia. At physical examination he presented horizontal gaze-evoked nystagmus, without saccades, ataxic gait, dysarthria, dysphagia, dysmetria, generalized spasticity mainly pelvic, bilateral Babinsky. CT brain scan revealed mild pontine atrophy, moderate cerebellar atrophy (Table 2) ( Figure 1B,C).
Family data: Mother aged 27 years similarly affected, clinical symptomatology began at 7 years of age with walking and speech difficulties. At 10 years of age she started with seizures, controlled for 5 years. Currently presents ataxia, dysarthria, dysmetria, dysdiadochokinesia, ataxic gait with olivopontocerebellar atrophy. Father aged 30-year-old neurologically healthy. Two maternal aunts of 30 and 23 years and the maternal grandmother (age of onset 40 yr), were similarly affected. Mother with molecular analysis positive to SCA2 with a genotype 22/49 CAG expansion repeats and father with normal range or expansion repeats.
Genomic DNA was extracted from peripheral blood leukocytes using the Gene Catcher Kit (Invitrogen) of the index case. The analysis of the ataxin-2 gene was performed by long PCR using a pair of oligonucleotides flanking the repeated CAGs. The sequence of the oligonucleotides was as follows: FWP1 5'-GGGCCCCTCACCATGTCG-3'; and RWP 5'-CGGGCTTGCGGACATTGG-3' (Sigma Aldrich). PCR was carried out in a total volume of 10 ml containing 100 ng of human DNA, 0.4 mM of each oligonucleotide, 200 mM of each dNTP; 0.6 ml of the 10x reaction buffer (Roche Diagnostics GmbH), 2 mM MgCl2 (Roche Diagnostics), 0.5 U of the enzyme Taq DNA polymerase (Roche Diagnostics GmbH) and 10% DMSO (Sigma-Aldrich) (Magaña et al., 2008;Flores-Alvarado et al., 2016). The amplification program consisted of 28 cycles, including denaturation at 96°C for 60 s, hybridization at 59°C for 30 s and polymerization at 72°C for 60 s (Magaña et al., 2008;Flores-Alvarado et al., 2016).
The PCR product was mixed with deionized formamide and denatured in water bath for seven minutes. Then it was placed on ice frappe for five minutes, and subjected by electrophoresis on 10% polyacrylamide gels (PAGE, 29:1). From the PCR products, were made a dilution 1:5 to apply 5 mL in the respective lane. PAGE gel was run at 150 volts, during 6.25 hours (Magaña et al., 2008); and subsequently stained with a solution containing; 0.1 g silver nitrate, 0.5 mL of acetic acid and 10 mL of ethanol graduated at 100 mL (12). The development solution used in the PAGE gels contained 3% sodium hydroxide with 270 mL of 37% formaldehyde graduated to 100 ml (Flores-Alvarado et al., 2016).
The amplified fragment of 130 bp corresponds to a normal allele of 22 CAG repeats. The normal range is between 13-31 CAG repeats and in affected cases greater than 32 repeats (Guzmán-López et al., 2018). In the PCR-PAGE denaturing assay ( Figure 2) the first lane is a molecular weight marker of 100 bp, the second lane is the white control PCR which appears white, without amplified product, in lanes 4 and 6 is the sample 124 with a DNA dilution of 1:5, there is a band of 130 bp and another band at 2 kb that was calculated as 2,652 bp which generates the genotype 22/~884 repeats; in lanes 3 and 7 is the sample 125 with a DNA dilution of 1:5, a band of 130 bp is observed and in lane 5 there is a negative control sample for the trinucleotide expansion repeat. Thus, with these considerations, the index case presented a normal allele of 22 CAG repeats, and an allelic variant of~884 CAG expansion repeats (2,652 bp), being carrier of the genotype 22/~884. This result was corroborated by the RED (Repeat Expansion Detection) method in the IDIBELL Center of Molecular Diagnosis; showing a higher penetrance allele of 2600 bp.
The present case is the first report of an index case with autosomal dominant SCA2 of neonatal presentation due to Age of death Babovic et al, 1998 Child was born with neonatal hypotonia, at 2 wk progressive apnea, developmental delay, and dysphagia, Retinitis pigmentosa was noted at 10 mo. Next 7 mo, the child remained hypotonic with increase feeding difficulties, dysphagia, and more frequent apneas. The child continued to show slow progression of her neurological symptoms and died from respiratory complications. This infant girl had global developmental delay in infancy, and microcephaly. At 4 yr she was failing to thrive with poorly coordinated swallow, at the 10 mo later began to lose motor and cognitive milestones and had autonomic dysfunction, optic nerve atrophy and retinitis pigmentosa, brain MRI at 5 yr 10 mo showed diffuse T2 white matter signal abnormalities, with cavitations of the parietooccipital lobes and cortical and cerebellar volume loss.

mo 22/750
Father 22/40 NR massive expansion of more than 884 CAG repeats in exon 1 of ATXN2. Massive expansions in the SCA2 are considered above one hundred repeats, longer are associated with an early onset age, and also are related with severe clinical findings (Antenora et al., 2017). Certainly, the present case started with symptoms since birth and severe neurological deterioration and a genotype with a longer allele, compared with other neonatal cases (Babovic et al., 1998;Dirik et al., 2007). So, the range in 19 reported cases with SCA2 with age of onset in childhood was 0-48 months, which were carriers of the heterozygous genotype (22/X), in which X corresponds to an allele with abnormal expansion repeats with an average range of 62-841 repeats (Table 1) (Babovic et al., 1998;Mao et al., 2002;Moretti et al., 2004;Dirik et al., 2007;Abdel and Zaki, 2008;Paciorkowski et al., 2011;Di Fabio et al., 2012;Vinther-Jensen et al., 2013;Avelino et al., 2014;Singh et al., 2014).
Abnormal expansions in many trinucleotide expansion diseases are related with the anticipation phenomenon, resulting into longer mutant alleles of maternal origin. However, in infantile SCA2 with massive expansion, most of the reported cases with paternal origin contribute with a greater effect to the anticipation phenomenon (Table 1). On the other hand, there are three reported cases and the present one, with a maternal origin associated with a massive expansion (Mao et al., 2002;Paciorkowski et al., 2011;Trang et al., 2015).
There are two clinical prognostic severity markers in SCA2, which are saccades and dysphagia (Wadia et al., 1998). Dysphagia was reported in five cases with massive repeats, greater than 200 repeats (Babovic et al., 1998;Mao et al., 2002;Abdel and Zaki, 2008;Avelino et al., 2014). The present case presented dysphagia since birth.
Abnormalities in the electroencephalogram were frequent in infantile SCA2 cases reports, but the present case This infant girl presented at 2 mo with poor head control and lack of visual fixation, at 7 mo she had tonic seizures, EEG at 14 mo showed multifocal epileptiform discharges. At 12 mo with retinitis pigmentosa. Brain MRI at 12 months showed mild cerebellar atrophy. The patient died after a neurodegenerative course. Pathologic examination of the cerebellum showed profound loss of Purkinje and granular neurons with severe attenuation of the molecular layer. A 10-month male child was referred to regression of milestones since 6 mo of age.
Here was no obvious dysmorphology, decreased muscle tone, inability to roll in his bed, and decreased tendon reflexes in all limbs and retinitis pigmentosa. MRI (at age of 10 mo) showed a markedly small cerebellum and vermis with associated atrophy involving the brainstem and both cerebral hemispheres. There was massive enlargement of the ventricles and enlarged infratentorial subarachnoid spaces. Additionally, diffuse T2 hyperintensity was observed within the periventricular white matter. Arachnoid cysts within the posterior fossa and right anterior temporal region were incidental findings. A 1-year-old Brazilian girl presented the phenotype of very early-onset SCA2 (neonatal form) encephalopathy, with hypotonia, choreic movements, dystonia with dystonic jerks, seizures, and retinitis, motor developmental delay. During the neonatal period (first mo), she presented hypotonia, dysphagia, and frequent gastroesophageal reflux. Tonic seizures, hypotonic with increased feeding difficulties, and choreic movements and dystonia with dystonic jerks. Decreased tonus and global decreased tendon reflexes. Visual fixation was poor and erratic eye, ophthalmoplegia were observed. Fundoscopy disclosed abnormal retina, with white dots, suggesting retinitis punctata albescens. Brain MRI showed marked cerebellar and brainstem atrophy and mild delayed myelination.

Newborn 22/104
Father Non determinated NR NR -not recorded; mo-months, yr-years, wk-weeks. did not present abnormal patterns in the electroencephalogram or epilepsy with tonic seizures or myoclonic seizures (Mao et al., 2002;Paciorkowski et al., 2011;Vinther-Jensen et al., 2013;Singh et al., 2014). While cerebellar and brainstem atrophy were related with massive CAG expansions (> 200) in the ATXN2 gene (Babovic et al., 1998;Mao et al., 2002;Moretti et al., 2004;Dirik et al., 2007;Paciorkowski et al., 2011;Magaña et al., 2008;Vinther-Jensen et al., 2013;Singh et al., 2014;Avelino et al., 2014). A case whose brain at 5 years 10 months showed diffuse T2 white matter signal abnormalities, with cavitations of the parieto-occipital lobes and cortical and cerebellar volume loss, was found carrier of a genotype 22/~750 (Paciorkowski et al., 2011). In the present case the CT brain scan revealed mild pontine atrophy, moderate cerebellar atrophy, with longer repeats 22/~841. This indicates that the severity of the damage to the brain structures not only depend of the ATXN2 VNTR expansion, but may influence other genes in which ATXN2 forms a network, that corresponds to a new frontier of research in SCA2. The present case presented gaze-evoked nystagmus without retinitis pigmentosa common in childhood SCA2 due to long CAG repeats in ATXN2 gene (see Table 2), greater than one hundred repeats (Babovic et al., 1998;Mao et al., 2002;Paciorkowski et al., 2011;Di Fabio et al., 2012;Vinther-Jensen et al., 2013;Avelino et al., 2014;Singh et al., 2014). In the literature has been reported massive expansion repeats (more than 750 repeats) in SCA2 with autonomic dysfunction, retinitis pigmentosa, and infantile spasms; the difference in the present case without retinitis pigmentosa, not only be explained by the age of onset, but also by the same molecular and allelic heterogeneity of retinitis, which is polygenic with different inheritance patterns (Fahim et al., 2018).
In patients with infantile SCA2, some craniofacial alterations were found, such as microcephaly in four cases and one case showed facial dysmorphism (Paciorkowski et al., Severe onset spinocerebellar ataxia type 2 5 Notes. Abnormalities in the electroencephalogram are considered hypsarrhythmia, repetive discharges of sharp and slow wave activit; pronounced slowing, infantile spasms with hypsarrhythmia, highamplitude bursts of slow waves with polyspikes reminiscent of hypsarrhythmia, multifocal epileptiform discharges, bilateral spike foci in the frontal and parietal regions and seizures. MRI = magnetic resonance imaging; CT = computed tomography. 2011). These findings were not relevant in the present case, but it would be worthwhile to analyze them clinically in detail, because there is a possibility that they are not syndromic but may be related to an ATXN2 effect on cranial morphogenesis.
Peripheral nerve affection, polyphagia and obesity were striking manifestations in the middle stage of the disease, as well as generalized edema and proteinuria, and minimal change glomerulonephritis that were found in an isolated patient. This might support a previously suggested relationship between the ataxin-2 gene and body weight and insulin resistance, which is modulated by ataxin-2 by the pathway of insulin by GRB2 and SRC (Flores-Alvarado et al., 2016;Guzmán-López et al., 2018;Ramirez-Garcia et al., 2019). The two carriers of these phenotypic findings had an expanded alleles range between 90 and less than 130 CAG repeats, which corroborates the participation of ataxin-2 gene in the development of metabolic syndrome traits (Flores-Alvarado et al., 2016;Ramirez-Garcia et al., 2019). However, our tester has the highest repeating range in the literature, and has not developed any of these findings, which suggests that there are other genes that can modify the SCA2 phenotype, especially in the case of children (Ramírez-Garcia et al., 2011;Trang et al., 2015).
In conclusion, the present report describes a child with infantile SCA2 with a massive expansion of CAG trinucleotides of the ATXN2 gene reported up to date in the literature, which resulted in a very early onset of clinical findings, since birth, and whose phenotype mainly corresponds to the severe affection of the central and peripheral nervous system. Also, this is the first childhood case in a Mexican and Latin-American population. The review of the literature of 18 cases of infantile SCA2 and the comparison with the present case shows a great clinical and molecular heterogeneity, which does not always correlate with the greater number of CAG repeats of the ATXN2 gene. The most frequent clinical signs were hypsarrhythmia, hypotonia, pigmentosa retinitis, cerebellar atrophy, as well as middle brain and lengthening of the ventricular system.
The range of CAG repeats ranged between 69-884 repeats; however, longer repeats have more severe phenotypes. 6 Sánchez-Corona et al.