Cerebellar Ataxia Phenotype

In summary, out of 33 patients with cerebellar ataxia with TGM6 variants, 18 (54%) were male and the rest were female. Around half of the patients (48%) had onset in the fifth decade, while 12% of patients each had onset in the third, fourth, and sixth decades. Only two (6%) and three (9%) patients had onset of the illness in the first and second decades. The patients were diagnosed after a lag period of median 15 years of illness (range: 3–30 years). All patients had signs of persistent cerebellar ataxia, apart from the one with EA2 phenotype. He had episodes of ataxia lasting for hours together, with spontaneous resolution from 54 years of age and the episodes did not respond to acetazolamide. He also had nystagmus in between the episodes, but no residual truncal ataxia, migraine, or seizures. The demographic profile and clinical presentation of patients with spinocerebellar/EA have been described in Table 1.

Around 87, 81, and 54% of patients had dysarthria, hand tremor, and nystagmus. The average SARA score was 17.56 ± 8.37. Oculomotor apraxia was present in eight (24%) of cases, in the form of saccadic/pursuit aberrations. Other less frequent clinical features include spasmodic torticollis in eight (24%), impaired proprioception in six (18%), myoclonus in three (9%), epilepsy in one (3%), extrapyramidal features in three (9%), parkinsonism in two (6%), and dystonia in four (12%) patients. However, on clinical examination, 31 out of 33 patients had brisk deep tendon reflexes suggestive of pyramidal involvement. One patient presented at 55 years of age with isolated dystonic tremors of the right upper limb for 20 years, which subsequently involved voice and contralateral upper limb. He was tried with multiple medications without any sustainable response including the following drugs, that is, primidone, propranolol, trihexyphenidyl, gabapentin, levodopa, and topiramate. He did not show response to even deep brain stimulation of the ventral intermediate nucleus of the thalamus.

Parkinsonism Phenotype

Among the 15 patients, 9 (60%) were males, who presented with parkinsonism caused by TGM6 variants. A family history of PD was present in eight (53%) patients, although genetic testing was not available for the affected family members in any of the cases. The average age at onset of PD symptoms was 48.3 ± 13.9 years, whereas the average age at diagnosis was 52.7 ± 14.2 years, with an average disease duration of 5.5 ± 3.7 years. The clinical course of parkinsonism in these patients more or less resembled that of cases with idiopathic PD, but with milder symptom severity at onset and more insidious disease progression, over a longer duration. Clinical presentation and demographic profile of patients with parkinsonism phenotype have been described in Table 2.

Resting tremor and bradykinesia were the initial presenting symptoms in eight (53%) and seven (47%) patients, respectively. However, at the time of determination of genetic etiology, most of them had all classical features of parkinsonism, including bradykinesia in all patients, resting tremors in 14 (93.3%) patients, and rigidity in 12(80%) patients. Other less prevalent clinical features described in these patients were as follows: postural instability in five (33.3%), olfactory problems in seven (47%), rapid eye movement sleep-related behavior disorder in six (40%), depression in six (40%), constipation in nine (60%), sexual impotence in two (13.3%), sleep benefit in symptoms in five (33.3%), and motor fluctuation in three(20%) patients. The average Beck depression inventory score of PD patients was 9.8 ± 7.0, average Sniffing Stick's test of olfactory function score was 5.4 ± 2.3, Mini-Mental State Examination (MMSE) score was 26.0 ± 5.2, Epworth sleepiness scale score was 6.6 ± 5.3, unified PD rating scale score was 23.3 ± 9.1, and stage of parkinsonism according to Hoehn and Yahr's scale was 2.3 ± 0.7. Only one case had a significantly abnormal MMSE score. All patients had a favorable response to the levodopa/carbidopa combination. Pramipexole and trihexyphenidyl were also tried successfully in four and one patient, respectively.

Polyglutamine Aggregation Deposition

Apart from TG6, some of the other TG proteins have also been suggested in the pathogenesis of various other neurodegenerative diseases. TG6 and TG2 proteins convert soluble forms of poly-Q into an insoluble form, thereby promoting poly-Q aggregate formation. Apart from SCAs that are well known to be poly-Q diseases, other neurodegenerative diseases like Alzheimer's disease, Huntington disease, and idiopathic parkinsonism have also been postulated to be caused by aberrant or reduced expression of TGs.3 7 12

Disruption of Autophagy and α-Synuclein Deposition

TGM6 variants cause parkinsonism by disrupting the normal autophagy process and reducing metabolism of α-synuclein. TG2 has been shown to catalyze α-synuclein crosslink formation, which is one of the key initial steps in the pathogenesis of PD. The in vitro studies on A53T-SH-SY5Y cells prepared as PD models showed consistent results. While the cells overexpressing wild-type (WT) TG protected the cells by enhancing autophagy and reducing α-synuclein, this protective effect was found to be attenuated in cells with mutant TG6. Functional TG activity grossly reduces in cells with mutated TGM6. These recently revealed novel pathogenetic mechanisms delineate the correlation between TGM6 and parkinsonism and provide a potential therapeutic target for PD.4

Resemblances with Gluten Ataxia

In patients with celiac disease, anti-TG6 antibodies are associated with gluten ataxia; and ataxic symptoms could be reproduced, when anti-TG2/3/6 cross-reactive antibodies or single-chain variable fragments are injected intraventricularly in mice. None of the patients with TGM6 variants have been demonstrated to have anti-TG6 antibodies. In celiac disease, gluten-specific CD4 T cells drive autoantibody production, hence removing gluten is an effective treatment through preventing T cell help to B cells. However, in genetic TG6 deficiency this most probably has no role to play.6 Larger studies with longer follow-up periods are required to demonstrate whether any true resemblance exists between these two illnesses.

Unfolded Protein Response

Tripathy et al9 in 2017 analyzed in vitro pathogenicity of 12 reported TGM6 variants, proposed to be linked with SCA-35, including seven novels and five previously reported mutations, and found another novel mechanism to explain the molecular basis behind neurotoxicity caused by mutant TG6. Predominantly in the nucleus and perinuclear area, the WT-TG6 could be localized. In cells with mutated TG6, there was loss of enzymatic function and insolubility of TG6 mutants, leading to nuclear depletion of TG6 and aberrant accumulation in the perinuclear area. An unfolded protein response gets activated by the increased and abnormal accumulation of mutated TG6 proteins in the perinuclear area. This endoplasmic reticulum stress response activated by mutated, nonfunctional TG6 proteins with abnormal conformation is the leading cause behind neuronal death and this has been shown in animal models of SCA-35 in drosophila melanogaster.9

Sensitization of Neurons to Apoptosis

Guan et al18 have demonstrated that overexpression of the TG6 mutants increases the activity of caspases and sensitizes cells to staurosporine-induced apoptosis, apart from exhibiting reduced TG activity and stability. The pathogenesis of SCA-35 might be related to the proapoptotic role of TG6 mutants.

Characteristics of Patients with SCA-35 Phenotype

In patients with SCA, presenting symptoms included insidiously progressive gait ataxia, pursuit and saccadic aberrations, dysarthria, action tremor, and pyramidal/extrapyramidal signs in a few cases. The exact description for the neuroimaging discrepancy between both phenotypes remains inconclusive. However, most patients with idiopathic parkinsonism also demonstrate grossly normal neuroimaging in the early stages, whereas a significant proportion of patients with other types of SCA also have cerebellar atrophy.3 4 5 6 7 8 9 10 11 12

SCA-35 was first reported by Wang et al3 in 2010 in a four-generation Han Chinese family, caused by mutations in the TGM6 gene, encoding for TG6. They utilized both whole-exome sequencing and linkage analyses together in four patients to identify this novel gene probably responsible for ataxia in human beings. They identified a missense variation, c.1550T-G transition (L517W), involving exon 10 of the TGM6 gene. They proposed that this mutation had a significant functional impact in bioinformatics study, although it was later contradicted by Thomas et al17). This mutation was, however, at a highly conserved position and completely cosegregated with the SCA phenotype in the fourth-generation family. The mutation was also located in 20p13–12.2, the same region identified by linkage analysis. They found further evidence that TGM6 mutations can cause SCA in human beings when they found another pathogenic missense variant cosegregating with the phenotype in an additional Chinese family with SCA.21 This second single-nucleotide substitution mutation, D327G, was found to interfere with Ca²⁺ binding at site 3 in structural remodeling and therefore may have compromised enzyme activation. But the mechanism by which the first mutation caused the deleterious effect remained obscure. Haploinsufficiency has been proposed as one possible mechanism by Thomas et al,17 by which mutation of a single allele can produce the dominant ataxia phenotype, but they also proposed that difficulties in the folding of mutant proteins leading to neurotoxicity could be another possible mechanism.

Subsequently, two additional Chinese families with SCA-35 were reported, harboring TGM6 pathogenic variants. However, the search for TGM6 mutations in 118 Caucasian cases of sporadic SCA did not yield any positive results. But similar search in 109 molecularly unassigned probands from Taiwanese SCA families, in whom underlying etiology remained unexplained, yielded three distinct TGM6 mutations, out of which two were novel mutations, like our case. Another study estimated that SCA-35 constituted 0.6% of patients with SCA phenotype from the Han-Chinese population of Taiwanese descent.5 7

Initially, SCA-35 was considered to be a disease of elderly adults universally, with onset from the fifth decade of life onwards. Later, cases with much younger-onset and even two adolescents affected with SCA-35 were reported. The case we presented is atypical not only because of the first reported case from India with a novel missense mutation but also due to the younger age of onset in the first decade of life. Despite all these previous cases, it is notable to mention that the role of TGM6 mutations accompanying potential early-onset SCA-35 is not well supported in a recent article, as the presence of this mutation poorly correlates with what is seen in cases where linkage has been more formally established and also in segregation analysis in affected family members in a report by Chen et al.22 Moreover, the mutation reported in this case report was the same L517W variant, reported initially by Wang et al.3 Although the affected family members in this report had CAG expansions in the ATN1 gene, the possible interaction between ATN1 and TGM6 cannot be ruled out completely.

As we have previously described, early manifestations are nonspecific and the same as for other SCAs including gait difficulties, dysarthria, and hand tremors. But spasmodic torticollis, impaired proprioception, and slow ocular pursuits are some of the relatively specific features, which may help clinicians in suspecting SCA-35 in patients with SCA phenotype. However, these findings have been reported only in a minority of affected patients. Another peculiar feature found in SCA-35 patients with onset in the first decade or adolescence is cognitive impairment (as found in our case), also seen in some other SCAs (SCA-2, -8, -13, -17, -19/22, -21, -36, and -44).1 Future studies are likely to report more distinct features and broaden the clinical spectrum of this illness.

Characteristics of Patients with Parkinson's Disease Phenotype

Compared with PD patients without any definite monogenetic abnormality, early onset PD patients with TGM6 variants exhibited milder symptoms for a longer duration, with slower disease progression. PET imaging findings in patients with PD caused by TGM6 variants resembled other PD patients. Although these two diverse, apparently mutually exclusive phenotypes caused by TGM6 variants were attributed to genetic heterogeneity, this might not be the true cause, as there are four cases of SCA-35, as described above, who also demonstrated some extrapyramidal features, like dystonia and parkinsonism. However, the reverse clinical picture has not been reported until now, as all described cases of PD did not have any evidence of significant cerebellar involvement.4

At the same time, it is noteworthy to mention that few families having molecularly confirmed patients with other SCA mutations (SCA-2 and -3) demonstrated pure parkinsonism or parkinsonism predominant phenotype. SCA-2 family with six affected individuals showing pure parkinsonism phenotype was described by Sun et al21 along with two other individuals with cerebellar ataxia.

Furtado et al23 have described another SCA-2 family in 2004 with PET brain imaging showing abnormalities similar to results found in idiopathic PD patients. Bettencourt et al24 and Buhmann et al25 have described SCA-3 patients initially presenting with parkinsonism and responding favorably to levodopa. However, they manifested cerebellar signs too after several years of the disease progression. Further prolong monitoring in the reported parkinsonism patients with TGM6 variants might show the development of cerebellar signs.