A mother and her daughter carrying a pathogenic expansion of the HTT gene with a phenotype encompassing motor neuron disease and Huntington's disease

Recently, pathogenic expansions (range 40–64 CAG repeats) in the HTT gene have been found in patients diagnosed with pure frontotemporal dementia/amyotrophic lateral sclerosis (FTD/ALS). We report a mother with Huntington's disease (HD) associated with motor neuron disease (MND) signs and her daughter suffering from ALS with subtle signs of HD, both carrying a pathogenic allele of the HTT gene (i.e., >39 repeats). The co‐occurrence of MND and chorea has been reported in previous cases. Subjects showing both ALS and HD signs and carrying HTT pathogenic expansions in two generations of the same kindred have never been reported so far. The study of the overlap of disease mechanisms at the cellular level between TDP‐43 and Huntingtin is relevant in an era offering promising strategies of targeted treatments in neurodegenerative disorders.


| INTRODUCTION
Motor neuron diseases (MND) are a group of degenerative diseases affecting upper and lower motor neurons to a variable extent, thus leading to considerable phenotypic heterogeneity.Amyotrophic lateral sclerosis (ALS) is the most frequent MND, causing death within 3-5 years from respiratory failure.Approximately 50% of ALS cases show some degree of cognitive and/or behavioural impairment in the spectrum of Frontotemporal Dementia (FTD).Currently, up to 20% of ALS cases are attributed to genetic causes. 1 The remaining cases are likely due to the interaction of multiple genetic and environmental factors. 2 Among genetic factors involved in ALS pathogenesis, short tandem repeats (STR) have been implicated in determining ALS risk.STR are tracts of repetitive DNA units that are prone to germline and somatic length variation.The intronic STR expansion in C9ORF72 gene is the only one which has been already demonstrated to cause ALS and FTD. 3 Nevertheless, intermediate STR expansions in genes associated with other neurodegenerative disorders have been called into question as risk factors for ALS.They include ATXN1 (spinocerebellar ataxia type 1, SCA1), ATXN2 (SCA2), ATXN8 (SCA8), Salvatore Gallone and Adriano Chiò contributed equally to this work.TBP (SCA17), HTT (Huntington's disease, HD), DMPK (myotonic dystrophy type 1, DM1), CNBP (DM2), and FMR1 (fragile-X disorders). 4 regards the HTT gene, wild-type alleles have 6 to 26 CAG repeats.Alleles with ≥27 repeats are unstable and have a tendency to expand in future generations, particularly with paternal transmission.
Intermediate expansions with 27 to 35 repeats may rarely cause disease.Nevertheless, the generally accepted threshold for developing HD is 36 repeats, with a variable penetrance of the HD phenotype in the range 36 to 39. Full penetrance occurs with ≥40 repeats. 5The CAG repeats are translated as poly-glutamine tract (Poly-Q), with neurotoxic effects. 6Noteworthy, the co-occurrence of ALS and chorea has been reported in association with HTT expansions. 7Recently, pathogenic expansions (range 40-64 CAG repeats) in the HTT gene have been found even in patients diagnosed with pure FTD/ALS. 8re we report a mother and her daughter both showing an overlap of MND and HD signs.

| MATERIALS AND METHODS
The proband was evaluated at the ALS Centre of Turin, Italy.She underwent a repeat-primed PCR assay screen for the presence of the GGGGCC hexanucleotide expansion in the first intron of C9ORF72.A cut-off of <23 repeats was considered normal.
Next-Generation Sequencing was performed on the proband including the panel of genes reported in Appendix A.
Finally, the proband underwent the analysis of the HTT gene through the amplification with fluorescent oligonucleotides of the region of exon 1 including the CAG repeats and the determination of the expansion length with capillary electrophoresis.
Clinical and genetic data of the proband's mother were obtained from medical records.

| CASE PRESENTATION
The proband developed progressive spastic paraparesis at the age of 60.One year after the onset, bilateral weakness and muscle wasting of the hands appeared.She was referred to our Centre 18 months after the onset.The neurological examination showed head tremor; motor impersistence of the tongue; mild dysarthria; cough weakness;

| DISCUSSION
We report a mother and her daughter, the former suffering from HD complicated by MND signs and the latter affected by ALS with mild signs ascribable to HD, both carrying a pathogenic allele of the HTT gene with >39 repeats.
The co-occurrence of MND and chorea has been reported in previous cases.Approximately 50% of cases have a positive family history for ALS, chorea or both.Almost half of patients show cognitive impairment.The most frequent underlying genetic cause are HTT gene mutations, but single cases with FUS and SETX mutations have been reported, in addition to one patient carrying both VCP and HTT gene mutations. 7However, to the best of our knowledge, a clinical picture including both ALS and HD signs and associated with HTT pathogenic expansions in two generations of the same kindred has never been reported so far.Noteworthy, in cases with the co-occurrence of MND and HD the atrophy of caudate nucleus and putamen was also found, 7 similarly to our cases.Moreover, in the largest autopsy series of HD/ALS published so far, 9 striatal atrophy and corticospinal tract degeneration have been demonstrated in selected cases, together with the occurrence of an HD/ALS patient with a positive HD family history.
The proband and her mother differed by some characteristics.First, we observed a pathogenic HTT allele with 43 repeats in the mother and 41 in the daughter.This discrepancy may be due to the 20-year difference between the mother and daughter's tests and the technical limitations of the testing procedure.
Phenotype also varied between the two patients.Phenotypic heterogeneity could arise from the different somatic instability of CAG-repeats across brain regions 10 and blood samples.Moreover, environmental factors and gene modifiers should be considered, although the very extensive genetic screening performed on the proband resulted negative.Finally, it is possible that the proband has developed ALS before showing the full picture of HD.Indeed, mild signs ascribable to HD (head tremor, tongue motor impersistence, upper limb rigidity) were already present at the time of ALS diagnosis, while the rapid worsening of limb weakness due to MND might have masked choreic movements.
Based on literature data and our cases, we suggest performing HTT genetic test in ALS patients with positive personal or family history for any of the following conditions: MND, chorea, parkinsonism, dementia, or psychiatric disorders.Since also C9ORF72 can be associated with these features, eventual HTTspecific characteristics should be investigated in the context of larger studies.
Recently, pathogenic HTT repeat expansions in patients diagnosed with FTD/ALS were found.Autopsy from these patients showed TDP-43 pathology along with polyglutamine inclusions in the frontal cortex but without the striatal degeneration typically seen in HD. 8 However, the pathogenic link between ALS and HD at the cellular level remains partially unclear.Interestingly, the colocalisation of phosphorylated TDP-43, which is the main component of intraneuronal inclusions across the ALS-FTD continuum, and mutant huntingtin has been observed in the brains of HD patients. 11Moreover, in cell models TDP-43 has been shown to be sequestered into polyglutamine aggregates via the binding to a glutamine/asparagine (Q/N)-rich region in the C-terminal domain of TDP-43.As a result, TDP-43 is cleared from the nucleus and becomes detergent insoluble, with a consequent loss of TDP-43-mediated splicing in the nucleus.Strikingly, in cellular models polyQ toxicity can be partially rescued by increasing the expression of TDP-43. 12The study of the overlap of disease mechanisms at the cellular level between TDP-43 and Huntingtin is relevant in the light of recent clinical trials with antisense oligonucleotides (ASO) targeting HTT. 13 This perspective has been already realised in an experimental setting for Ataxin 2 (ATXN2), a polyglutamine (polyQ) protein mutated in SCA2.It has been shown to be a potent modifier of TDP-43 toxicity in animal and cellular models 14 and a clinical trial with ASO targeting ATXN2 is currently ongoing for ALS patients with an without intermediate expansions of ATXN2 (please, see at https://clinicaltrials.gov/ using 'Amyotrophic Lateral Sclerosis' and 'ATXN2' as search terms).
Our report is in agreement with the finding that clinical and pathological pleiotropy of neurodegenerative disease genes carrying STR can have a relevant contribution in the ALS-FTD spectrum.In this context, in addition to the already known role of C9ORF72, ATXN2, and HTT, further genes have been called into question, including those causing SCA1, SCA8, SCA17, DM1, DM2, and fragile-X disorders.Beyond the importance for the design of novel experimental therapeutic strategies, the study of pleiotropy associated with other neurodegenerative disease genes would be relevant for appropriate genetic counselling and prognostic prediction. 4 conclusion, our report underlines the importance of the study of the possible link between ALS and Huntingtin mutations in an era offering promising strategies of targeted treatments in neurodegenerative disorders.
hypophonia; brisk jaw jerk reflex; weakness of neck muscles; spasticity, muscle weakness and wasting, and hyperreflexia at upper and lower limbs; extrapyramidal rigidity at upper limbs; bilateral Hoffmann and Babinski signs; bilateral ankle clonus; bilateral palmomental reflex.Brain MRI showed bilateral caudate nuclei and putamen atrophy.The electromyography (EMG) found active denervation at the upper and lower limbs.A diagnosis of ALS was made despite some findings, namely head tremor, motor impersistence of the tongue, and upper limb rigidity, did not fit with the typical ALS clinical presentation.Two years after the onset she started non-invasive ventilation and cough mechanical assistance.Six months later she underwent percutaneous radiologic gastrostomy because of severe dysphagia.She died 42 months after the onset due to the worsening of respiratory failure, since she refused invasive ventilation.During the family history collection the proband reported that her mother was diagnosed with HD and MND.From the mother's medical records, we found that she resulted positive for the genetic test for HD (19/43 CAG).Neurological symptoms began at the age of 69.Her clinical picture was characterised by a cognitive impairment documented by a full neuropsychological assessment, showing deficits in letter and category fluency, long-term spatial memory, and abstract reasoning.The neurological examination revealed fatuous attitude, upward gaze impairment, pursuit eye movements fragmentation, slow saccades, ocular convergence deficit, orofacial apraxia, dysarthria, dysphagia, cough weakness, bilateral tongue and masseter wasting, brisk jaw jerk reflex, diffuse muscle hypotonia, and wasting, diffuse choreic involuntary movements, wide-gait ataxia, hyperreflexia of the four limbs, bilateral Babinski sign.Brain MRI showed diffuse cortical atrophy and reduced volume of the caudate nuclei and midbrain.A needle EMG revealed active denervation in the bulbar and bilateral hand muscles.A diagnosis of HD associated with MND was made.Based on the family history and the clinical picture, the proband underwent genetic analysis for the search of C9ORF72 repeat expansion and NGS for MND, hereditary spastic paraplegias, motor neuropathies, distal amyotrophies, and frontotemporal dementia, which resulted normal, and the screening for the expansion of the HTT gene, showing a positive result (17/41 CAG).