Cognitive impairment in stable Wilson disease across phenotype

In Wilson disease (WD), mutations in the gene encoding the ATP7B copper transport protein causes accumulation of copper especially in liver and brain. WD typically presents with hepatic and/or neuropsychiatric symptoms. Impaired cognition is a well-described feature in patients with neurological WD, while the reports on cognition in hepatic WD patients are fewer and less conclusive. We examined cognition in a cohort of WD patients with both phenotypes. In this cross-sectional pilot study, we investigated cognition in 28 stable Danish WD patients by the PortoSystemic Encephalopathy (PSE) and the Continuous Reaction Time (CRT) tests. Half of the patients were female, and their median age was 35.5 years (IQR 24.5). Their phenotype was hepatic in 14 (50%), neurologic in 10 (36%) and mixed in 4 (14%). The duration of treatment was > 2 year in all patients, and their condition was stable as judged by urinary copper excretion, liver enzymes, and clinical assessment. The hepatic patients did not show signs of liver failure. In total, 16 (57%) patients performed worse than normal in the PSE and/or the CRT tests. The two tests were correlated (rho = 0.60, p = 0.0007), but neither correlated with phenotype, MELD-, Child–Pugh score, 24 h-U-Cu, or treatment type. Measurable cognitive impairment was present in more than half of the stable WD patients independent of phenotype. Thus, our data questions the existence of a purely hepatic phenotype.


Background
In Wilson disease (WD) copper accumulates in body tissues particularly the liver and brain (European Association for the Study of the Liver 2012). The disease typically presents with either hepatic or neuropsychiatric symptoms and in some with a combination. The hepatic and neurological symptoms most often improve with treatment, but patients still hold an increased risk of psychiatric and somatic comorbidity including depression, anxiety, cardiac myopathy and decreased liver function (European Association for the Study of the Liver 2012 ;Schaefer 2016;Litwin 2018;Grandis 2017;Björklund 2018).
Cognitive impairment in WD patients with neuropsychiatric presentation is well described (Frota 2013;Hegde 2010;Seniów 2002;Wenisch 2013) and probably related to the cerebral lesions detected by MRI (Frota 2013;Dezortova 2020;Yu 2019). However, recent studies suggest subtle cerebral structural changes even in hepatic WD patients (Tinaz 2020). Few papers assessing cognition in hepatic WD are available (Seniów 2002;Wenisch 2013;Iwański 2015). While most standard cognitive tests were normal in the hepatic WD patients, more sensitive tests of the sustained attention were also affected in hepatic patients according to one study (Iwański 2015). In order to evaluate cognition across phenotype we studied a stable WD cohort and employed two psychometric tests developed to detect subtle cognitive disturbances with relation to sustained attention: the portosystemic encephalopathy (PSE) and the continuous reaction time (CRT) tests (Elsass 1986;Nabi and Bajaj 2014).

Methods
In this cross-sectional pilot study, all stable Danish WD patients at the Danish Wilson Center, Aarhus University Hospital over the age of 18 with at least 2 years of treatment duration were examined over a time span of 9 months. Baseline characteristics and inflammatory data were recently published (Björklund 2018). The patients' phenotype was defined by the incident patient's dominating debut symptoms.
Stability was defined as stable urinary copper and liver enzymes over a 1-year period, and absence of clinically manifest neurological or hepatic decay. Patients were excluded if liver transplanted or unable to collect urine. No participant had known concomitant non-Wilsonian neurological disease.
After informed consent was obtained, the patients were evaluated by the PSE and CRT tests. The PSE test is a validated and widely used pen and paper test battery most established for identification of minimal hepatic encephalopathy (Nabi and Bajaj 2014;Weissenborn 2001). Complex cognitive functions such as attention, accuracy, working speed, and visual orientation are assessed through five subtests. A score of ≤ − 5 is abnormal, and lower than 2 SD below the median in a healthy German age-adjusted population (Weissenborn 2001). A Danish norm is under establishment but does not differ significantly from the German one [Lauridsen MM, Personal Communication]. The CRT is also validated as a diagnostic tool for minimal HE. It consists of 100 serial and randomly delivered auditory stimuli and gives a measure of sustained attention, vigilance, and inhibitory control (Elsass 1986;Lauridsen 2013). The key test outcome is the reaction time stability quantified by the CRTindex, the ratio reaction time 50 percentile/(90-10 percentile). An index below 1.9 is abnormal (Elsass 1985). The CRTindex is not affected by age, gender, or educational level (Lauridsen 2012). The PSE and CRT tests were chosen as they are commonly used in the field of hepatology, used widely in local practice with especially CRT having a large Danish experience base. These choices also allowed us to determine dyscognition in Wilson patients by the test we use in daily clinical practice. Fibroscan and blood test results were secured within 1 month of the test day (Table 1).
The study was approved by The Central Denmark Region Committees on Health Research Ethics (50611), and the Danish Data Protection Agency (1-16-02-614-15), registered at clinicaltrials.gov (NCT02702765), and conducted in accordance with The Helsinki declaration.

Statistical analysis
Histograms and Q-Q plots were used to check normality. Results are presented as median and IQR. Wilcoxon Mann-Whitney test was used to compare groups and Spearman's correlation was used for analysis of correlations within the dataset. A p value < 0.05 was considered statistically significant. We used STATA version 16.1 (StataCorp LP, College Station, TX) for data analysis.

Results
Of 38 screened patients, 9 could not be included (4 < 18 years of age; 1 newly diagnosed; 1 too ill; 3 without hospital contact during the study period), 1 refused and twenty-eight patients were included in the study. Figure 1 displays the results of PSE and CRT related to the WD disease phenotype. Table 1 presents patient characteristics, PSE subtest scores and CRT percentiles comparing those with and without cognitive impairment.
Five (18%-2 hepatic and 3 neurological phenotype) patients presented with a pathological PSE score (score median − 6, IQR − 2). These patients scored lowest in the digit symbol and line tracing A subtests (attention, visuospatial construction, working speed, accuracy, and visual orientation).
In total, sixteen (57%) patients performed worse than normal in cognitive function by either one or both the PSE and CRT tests, and four performed worse than normal in both tests. In these, patient characteristics were similar to those with normal PSE and CRT tests (Table 1). Thus, there was no correlation between PSE or CRTindex and phenotype, Model for End-stage Liver Disease (MELD)-, CP-score, treatment, or 24-h urine copper excretion.

Discussion
We showed that more than half of our stable WD cohort performed below normal in cognitive function tests regardless of liver or brain disease involvement. More patients subperformed in the CRT test than in the PSE test. This suggests that these patients suffered from impaired motor reaction speed, sustained attention ability and inhibitory control (Elsass 1986;Lauridsen 2013). In the PSE test, patients performed worse in the digit symbol and line tracing A subtests suggesting impaired attention, as well as working speed, accuracy, visual orientation and visuo-spatial construction (Weissenborn 2001). Our findings are in line with the known impact of neurological WD on cognition (Frota 2013;Hegde 2010;Seniów 2002). In the few available reports on cognition in WD patients without neurological symptoms, cognition was found normal (Seniów 2002;Wenisch 2013;Iwański 2015) except for tests of sustained attention (Iwański 2015). Both the PSE and CRT tests are widely used to detect subclinical cognitive dysfunction in cirrhosis patients with so-called minimal hepatic encephalopathy and because they are very sensitive to purpose and familiar to our staff, we chose them for initial cognitive screening of Wilson patients. A general trait seems to be loss of sustained attention and our results are thus in agreement with those of Iwański (2015) and Favre (2017) where tests of sustained attention were also impaired in both phenotypes.
Taken together, our data suggest that mild cognitive disturbances are common in both phenotypes. The problem may be related to copper-dependent structural changes in the liver, even in stable patients and after long periods of treatment (Frota 2013;Scheinberg 1987), although we found that neither urinary copper or treatment modality correlated with the PSE or CRT measurements. Free copper was not measurable at the study time but is presumably under control in stable patients. Recent advanced MRI reports suggest subtle changes in brain domains even in hepatic WD patients, compatible with weakened attention, as we found in both phenotypes (Tinaz 2020). Further studies are awaited.
Alternatively, Wilsonian liver disease might in itself be associated with the attention deficit, as the same is seen in other liver diseases, e.g., non-alcoholic fatty liver disease and -steatohepatitis, hepatitis C, and alcoholic cirrhosis (Colognesi et al. 2020;Kjaergaard, et al. 2021;Perry et al. 2008;San Martín-Valenzuela, et al. 2020;Yohannes 2017). The liver is also involved in most neurologic WD patients, and if cirrhosis emerges this may contribute towards cognitive impairment (European Association for the Study of the Liver 2012). However, our patients presented with normal or only mildly affected liver function tests (Table 1) and neither their MELD nor CP-scores were related to the cognition test results, also not when only analyzing results from WD patients with hepatic presentation. Minimal HE is thus unlikely to be causative of the measured cognitive impairment.
Our findings thus indicate that the occurrence of subclinical cognitive dysfunction in stable WD is much more common than previously thought and may be present regardless of phenotype. While current treatment often is highly effective in controlling the major neurological and hepatic manifestations of WD, it may still be inadequate to treat the cognitive manifestations as well. This is not a trivial matter because such mild dyscognition is closely related to impaired quality-of-life, not recorded here but widely documented in other liver patients. Our study and the discussion above suggest that WD patients without apparent neurological involvement should also be offered thorough neurological examination including brain MRI and neuropsychiatric examinations.
Our small study population (n = 28) remains a limitation of this study, somewhat counterbalanced by the wellcharacterized cohort of stable Danish patients and the high rate of disease population participation. Studies in larger populations as well as introduction of neurological tests of cognition are clearly motivated needed. These studies should also explore if and how these subtle cognitive disturbances affect the daily living of the patients.
In conclusion, our cohort of stable well treated WD patients performed worse than normal in cognitive function tests regardless of neurological or hepatic phenotype despite long-term successful therapy. Authors' contributions FK wrote the manuscript, analyzed the data and acquired parts of the data. DM, TL, MML, TS assisted in writing the manuscript. TL acquired data and assisted in data analysis. DM and TS also assisted in data analysis. HV, PO and HG helped form the project and gave inputs to the manuscript. All authors read and approved the final manuscript.
Funding This study was supported with a grant from "The memorial foundation of manufacturer Vilhelm Pedersen and wife-and the Aarhus Wilson consortium". Design of the study and collection, analysis and interpretation of data as well as writing of the manuscript was independently performed by the research group. Research grants were obtained and accounted for in the Competing interest's section.

Data availability
The datasets used in the current study are available from the corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate The study was approved by The Central Denmark Region Committees on Health Research Ethics (50611) and approved by the Danish Data Protection Agency (1-16-02-614-15). The study was conducted in accordance with the Helsinki II declaration.
Consent for publication Not applicable.