Objective. To determine whether the neuropsychological profiles of ALS patients with (ALSC9+) and without (ALSC9-) C9orf72 expansion are different we administered a battery of neuropsychological tests to 741 ALS patients (68 ALSC9+ and 673 ALSC9-) and 129 controls.

Methods. The study population includes 741 ALS patients who were consecutively diagnosed at the Turin ALS expert center in the period 2010-2018 and who underwent both cognitive/behavioral and genetic testing. Patients’ neuropsychological patterns were compared (a) at the same degree of cognitive and behavioral deficit according to the revised ALS-FTD Consensus Criteria; and (b) at the same level of motor impairment according to the King’s staging system.

Results. Despite being about 7 years younger, ALSC9+ patients had significant lower scores in tests exploring executive functions, and verbal memory both when classified as cognitively normal and when diagnosed in the intermediate cognitive categories. Considering the clinical perspective, ALSC9+ patients showed significantly lower scores compared to ALSC9- patients at King’s stage 1 and 3 in almost all the examined neuropsychological domains, while at King’s stage 2 ALSC9+ patients were more severely affected only in the verbal memory domain. Behavioral function was comparably impaired in the two cohorts.

Conclusions. ALSC9+ patients show a different neuropsychological profile compared to ALSC9- ones, being more impaired in executive functions and verbal memory domains at all King’s stages. Verbal memory emerged as a particularly vulnerable function in ALSC9+, with worse performances even when patients were still classified as cognitively normal.

The study population includes all ALS patients who were consecutively seen at the Turin ALS expert center in the period 2010-2018 and who underwent both cognitive/behavioral and genetic testing. Patients were diagnosed according to El Escorial revised criteria.¹⁰All patients were evaluated with the ALS Functional Rating Scale revised (ALSFRS-R) at time of cognitive testing.

Patients with history of neurological disorders affecting cognition (major stroke, severe head injuries, mental retardation), alcohol-dependence and drug-dependence, severe mental illness, and use of high-dose psychoactive medications were tested but not included in data analysis. Patients who were not of Italian mother tongue were assessed only through an unstructured interview and therefore were excluded from the analysis. A total of 129 population-based controls were also tested with the same battery.

Neuropsychological battery. ALS patients underwent a battery of neuropsychological tests encompassing executive function, memory, visuospatial function, and language, selected according to the Diagnostic Criteria for the Behavioural variant of Frontotemporal Dementia,¹¹ and ALS-FTD Consensus Criteria.⁴ Patients underwent the following neuropsychological battery: Letter Fluency test (FAS); Category Fluency Test (CAT); Frontal Assessment Battery (FAB); Trail Making Test (TMT) A, B and B-A; Rey-Osterrieth Complex Figure Test (ROCF), immediate (IR) and differed recall (DR); Rey Auditory Verbal Learning Test (RAVL), immediate (IR) and differed recall (DR); Babcock Story Recall Test (BSRT), immediate (IR) and differed recall (DR); Digit Span Forward and Backward; Raven’s Colored Progressive Matrices (CPM47); Mini Mental State Examination (MMSE). The raw scores of all tests were age-, sex-, and education-corrected using the more recent Italian normative (for reference see Supplementary Table 1).

In selected cases, according to the judgment of the neuropsychologist, supplementary tasks were administered for a comprehensive evaluation of language; the following tests were used: semantic systems tests (7 and 8) of the Battery for the Analysis of Aphasic Deficits and the Token test. However, since these test were not performed in all patients, they results are not reported.

Neurobehavioral dysfunction was determined with the Frontal Systems Behaviour Scale (FrSBe), using the Family-form evaluated by a close relative/caregiver (scores: normal ≤59, borderline 60-64; pathological ≥65). For the purpose of this study, we considered the change in points for each of the 3 domains of FrSBe (apathy, disinhibition, executive) from the before disease to the disease scores. If a subject had scores reflecting a frontal systems abnormality both in the premorbid and in the post-illness forms, he/she was considered pathological only if there was an increase of ≥10 points at the T-score between the two forms.³

Anxiety and depression were assessed with the Hospital Anxiety and Depression Scale (HADS); the item “I feel slowed down” was discussed with patients in order to have him/her not to refer to physical disability.³

Domain classification. Tests were classified according the main neuropsychological domain they assess (Table 1).^4,12-14 A domain was considered impaired if at least one of the tests exploring that domain had a score under the normative cut-off, with the exception of executive functions and verbal memory, which were considered impaired if at least two not overlapping tests had a score under the cut-off.⁴

Classification of cognitive phenotypes. Patients were classified according to the consensus criteria for the diagnosis of frontotemporal cognitive and behavioral syndromes in ALS.⁴ We considered five categories: ALS-CN,  ALSci, ALSbi, ALScbi and ALS co-morbid with frontotemporal dementia (ALS-FTD). For the aims of this study, intermediate cognitive categories (ALSbi, ALSci and ALScbi) were merged.

ALS Staging. Patients’ motor impairment was classified according to the King’s staging system. King’s staging is based on the spreading of motor symptoms in three different body regions (bulbar, upper limbs, and lower limbs), and on the use of non-invasive ventilation (NIV) and enteral nutrition.⁹ The five stages of the King’s staging are: 1, one region involved; 2, two regions involved; 3 three regions involved; 4A, patient needs gastrostomy; 4B, patient needs non-invasive ventilation. The stage can be derived from the direct observation of the patients and also from the ALSFRS-R scale.¹⁵ For the purpose of this study, we considered only stages 1 to 3, since stages 4A and 4B are not anatomical but functional and thus do not necessarily correspond to the spreading of the anatomical lesions.

Genetic testing. All the coding exons and 50 bp of the flanking intron-exon boundaries of SOD1, of exon 6 of TARDBP, and of exons 14 and 15 of FUS have been PCR amplified, sequenced using the Big-Dye Terminator v3.1 sequencing kit (Applied Biosystems Inc.), and run on an ABIPrism 3130 genetic analyzer. These exons were selected as the vast majority of known pathogenic variants are known to lie within these mutational hotspots. A repeat-primed PCR assay was used to screen for the presence of the GGGGCC hexanucleotide expansion in the first intron of C9orf72. A cut-off of ≥30 repeats was considered pathological.¹⁶

Statistical analysis. Comparisons were performed on age-, sex-, and education-corrected scores. Since all cognitive tests had not a normal distribution, the Mann-Whitney U test was used for comparisons. Data were analyzed in subsequent steps. First, we compared the results of cognitive tests of ALSC9+ vs ALSC9- patients for each cognitive and behavioral level, merging the intermediate cognitive categories (ALS-CN; ALSci/ALSbi/ALScbi; ALS-FTD). Second, we compared the results of cognitive tests of ALSC9+ vs ALSC9- patients for each King’s stage, independently from their level of cognitive impairment. Third, we grouped the tests according to their domain and compared the number of ALSC9+ vs. ALSC9- cases who showed an impairment in each domain.

Two-tailed p-values are reported; Holmes correction for multiple testing was used. Effect sizes were also calculated (Supplementary Table 2).¹⁷ All analyses were performed with SPSS 26.0 statistical package (SPSS, Chicago, IL, USA).