Guidelines for genetic testing of healthy adults who deposit samples and related data in bioresource collections and biobanks

Currently, a significant part of research in the fields of human and medical genetics is carried out using tissue samples, genealogical, population, medical and personal data. Their use is of particular relevance in the “genome era”, since only joint analysis of genomic data and health status of the population is crucial for understanding how genes are associated with health and disease. Genetic studies of adults without symptoms of diseases are carried out to obtain data on a possible predisposition to multifactorial diseases, to establish the carrier status of autosomal recessive mutations as part of preconception care and to assess individual sensitivity to drugs. In addition, healthy individuals can be tested to detect an inherited disease at presymptomatic stage. This situation increasingly emphasizes the importance of storing data on genome sequencing or any other patient tests for subsequent data reanalysis, as well as their safety, including biosamples from an individual and one’s family. The review article, based on international experience, summarizes guidelines for genetic testing of healthy individuals. The options for storing biological samples and related data are considered.

Impaired Retromer Function in Niemann-Pick Type C Disease Is Dependent on Intracellular Cholesterol Accumulation

Niemann-Pick type C disease (NPC) is a rare inherited neurodegenerative disorder characterized by an accumulation of intracellular cholesterol within late endosomes and lysosomes due to NPC1 or NPC2 dysfunction. In this work, we tested the hypothesis that retromer impairment may be involved in the pathogenesis of NPC and may contribute to increased amyloidogenic processing of APP and enhanced BACE1-mediated proteolysis observed in NPC disease. Using NPC1-null cells, primary mouse NPC1-deficient neurons and NPC1-deficient mice (BALB/cNctr-Npc1m1N), we show that retromer function is impaired in NPC. This is manifested by altered transport of the retromer core components Vps26, Vps35 and/or retromer receptor sorLA and by retromer accumulation in neuronal processes, such as within axonal swellings. Changes in retromer distribution in NPC1 mouse brains were observed already at the presymptomatic stage (at 4-weeks of age), indicating that the retromer defect occurs early in the course of NPC disease and may contribute to downstream pathological processes. Furthermore, we show that cholesterol depletion in NPC1-null cells and in NPC1 mouse brains reverts retromer dysfunction, suggesting that retromer impairment in NPC is mechanistically dependent on cholesterol accumulation. Thus, we characterized retromer dysfunction in NPC and propose that the rescue of retromer impairment may represent a novel therapeutic approach against NPC.

Nucleolar stress controls mutant Huntington toxicity and monitors Huntington’s disease progression

Transcriptional and cellular-stress surveillance deficits are hallmarks of Huntington’s disease (HD), a fatal autosomal-dominant neurodegenerative disorder caused by a pathological expansion of CAG repeats in the Huntingtin (HTT) gene. The nucleolus, a dynamic nuclear biomolecular condensate and the site of ribosomal RNA (rRNA) transcription, is implicated in the cellular stress response and in protein quality control. While the exact pathomechanisms of HD are still unclear, the impact of nucleolar dysfunction on HD pathophysiology in vivo remains elusive. Here we identified aberrant maturation of rRNA and decreased translational rate in association with human mutant Huntingtin (mHTT) expression. The protein nucleophosmin 1 (NPM1), important for nucleolar integrity and rRNA maturation, loses its prominent nucleolar localization. Genetic disruption of nucleolar integrity in vulnerable striatal neurons of the R6/2 HD mouse model decreases the distribution of mHTT in a disperse state in the nucleus, exacerbating motor deficits. We confirmed NPM1 delocalization in the gradually progressing zQ175 knock-in HD mouse model: in the striatum at a presymptomatic stage and in the skeletal muscle at an early symptomatic stage. In Huntington’s patient skeletal muscle biopsies, we found a selective redistribution of NPM1, similar to that in the zQ175 model. Taken together, our study demonstrates that nucleolar integrity regulates the formation of mHTT inclusions in vivo, and identifies NPM1 as a novel, readily detectable peripheral histopathological marker of HD progression.

The circadian rest-activity pattern predicts cognitive decline among mild-moderate Alzheimer’s disease patients

Background Alterations in circadian rhythms are present in the presymptomatic stage of Alzheimer’s disease (AD), possibly contributing to its pathogenesis. However, it is unknown whether such alterations are associated with worse outcomes once individuals are diagnosed with symptomatic disease. We aimed to evaluate the association between the circadian rest-activity pattern and AD-related features in patients with mild-moderate AD. Methods We assessed the circadian rest-activity pattern of consecutive patients with mild-moderate AD through actigraphy for 14 days. Cerebrospinal fluid was obtained to determine the levels of important pathological markers including amyloid-beta protein (Aβ42), phosphorylated tau (P-tau), total tau (T-tau), and neurofilament light (NF-L). Neuropsychological evaluation was conducted at the beginning of the study and after 12 months of follow-up. Linear regression models were performed considering the global population and Aβ42+ patients only. Results The cohort included 100 patients with mild-moderate AD. The median age [p25;p75] was 76.0 [73.0;80.0] years and 63.0% were female. Older age (effect size [SE] of 0.324 [0.096]; p = 0.001) and male sex (0.780 [0.193]; p = 0.001) were associated with increased fragmentation and decreased synchronization of the rhythm, respectively. After adjusting for age, sex, and season of the year, increased levels of T-tau (effect size [95% CI] of 0.343 [0.139 to 0.547]; p = 0.001) and NF-L (0.444 [0.212 to 0.676]; p = 0.001) were associated with a higher amplitude of the rest-activity rhythm. Increased fragmentation of the rhythm at baseline was associated with greater cognitive decline after one year of follow-up independent of age, sex, T-tau/Aβ42 ratio, educational level, and season of the year (− 0.715 [− 1.272 to − 0.157]; p = 0.013). Similar findings were obtained considering only the Aβ42+ patients. Conclusions Our results suggest a potential role of the circadian rest-activity pattern in predicting the cognitive decline of patients with mild-moderate AD. Further studies are warranted to confirm these findings and to elucidate whether there is causality among the observed associations.

Cerebellar hemorrhages in patients with Dutch-type hereditary cerebral amyloid angiopathy

Background Recent studies suggest that superficially located cerebellar intracerebral hemorrhage (ICH) and microbleeds might point towards sporadic cerebral amyloid angiopathy (CAA). Aims We investigated the proportion of cerebellar ICH and asymptomatic macro- and microbleeds in Dutch-type hereditary CAA (D-CAA), a severe and essentially pure form of CAA. Methods Symptomatic patients with D-CAA (defined as ≥1 symptomatic ICH) and presymptomatic D-CAA mutation-carriers were included. We assessed magnetic resonance imaging scans for symptomatic (cerebellar) ICH and asymptomatic cerebellar macro- and microbleeds according to the STRIVE-criteria. Location was assessed as superficial-cerebellar (cortex, vermis or juxta-cortical) or deep-cerebellar (white matter, pedunculi cerebelli and gray nuclei). Results We included 63 participants (mean age 58 years, 60% women, 42 symptomatic). In total, the 42 symptomatic patients with D-CAA had 107 symptomatic ICH (range 1–7). None of these ICH were located in the cerebellum. Six of 42 (14%, 95%CI 4–25%) symptomatic patients and none of the 21 (0%, 95%CI 0–0%) presymptomatic carriers had ≥ 1 asymptomatic cerebellar macrobleed(s). All macrobleeds were superficially located. Cerebellar microbleeds were found in 40 of 63 (64%, 95%CI 52–76) participants (median 1.0, range 0–159), 81% in symptomatic patients and 29% in presymptomatic carriers. All microbleeds were strictly or predominantly superficially (ratio superficial versus deep 15:1) located. Conclusions Superficially located asymptomatic cerebellar macrobleeds and microbleeds are common in D-CAA. Cerebellar microbleeds are already present in the presymptomatic stage. Despite the high frequency of cerebellar micro and macrobleeds, CAA pathology did not result in symptomatic cerebellar ICH in patients with D-CAA.

Oxaloacetate treatment preserves motor function in SOD1G93A mice and normalizes select neuroinflammation-related parameters in the spinal cord

Amyotrophic lateral sclerosis (ALS) remains a devastating motor neuron disease with limited treatment options. Oxaloacetate treatment has a neuroprotective effect in rodent models of seizure and neurodegeneration. Therefore, we treated the ALS model superoxide dismutase 1 (SOD1) G93A mice with oxaloacetate and evaluated their neuromuscular function and lifespan. Treatment with oxaloacetate beginning in the presymptomatic stage significantly improved neuromuscular strength measured during the symptomatic stage in the injected mice compared to the non-treated group. Oxaloacetate treatment starting in the symptomatic stage significantly delayed limb paralysis compared with the non-treated group. For lifespan analysis, oxaloacetate treatment did not show a statistically significant positive effect, but the treatment did not shorten the lifespan. Mechanistically, SOD1G93A mice showed increased levels of tumor necrosis factor-α (TNFα) and peroxisome proliferative activated receptor gamma coactivator 1α (PGC-1α) mRNAs in the spinal cord. However, oxaloacetate treatment reverted these abnormal levels to that of wild-type mice. Similarly, the altered expression level of total NF-κB protein returned to that of wild-type mice with oxaloacetate treatment. These results suggest that the beneficial effects of oxaloacetate treatment in SOD1G93A mice may reflect the effects on neuroinflammation or bioenergetic stress.

Presymptomatic treatment of classic late-infantile neuronal ceroid lipofuscinosis with cerliponase alfa

Background Neuronal ceroid lipofuscinosis type 2 (CLN2 disease) is a rare rapidly progressive neurodegenerative disorder, resulting in early death. Intracerebroventricular enzyme replacement therapy (ERT) with cerliponase alfa is now available and has shown to delay disease progression in symptomatic patients. It is yet unknown if cerliponase alfa can prevent disease onset in presymptomatic patients. Results We evaluated the effect of 2 years of intracerebroventricular ERT in two siblings with CLN2 disease, one symptomatic (age 47 months) and one presymptomatic (age 23 months) at treatment start, using the CLN2 Clinical Rating Scale (CLN2 CRS), Gross Motor Function Measure-66 (GMFM-66) for motor function, Bayley Scales of Infant and Toddler Development, 3rd Edition, Dutch (BSID-III-NL) for neurocognitive development, brain MRI, and visual evoked potentials (VEP), electroretinogram (ERG) and retinoscopy for visual function. On the CLN2 CRS patient 1 showed a decline from 3 to 2 in the combined motor and language score due to regression in language use (CLN2 CRS total score after 2 years of treatment: 8), whereas a decline of 2 or more points in the combined motor and language score would be expected without treatment. Patient 2 retained the maximum score of 3 in all 4 subdomains (CLN2 CRS total score after 2 years of treatment: 12). The GMFM-66 total score declined from 46 to 39 in patient 1 and showed an age-appropriate increase from 66 to 84 in patient 2. Cognitive-developmental age decreased from 24 to 11 months in patient 1, whereas an increase in cognitive-developmental age from 21 to 39 months was seen in patient 2. Cerebral and cerebellar atrophy observed on MRI in patient 1 at age 42 months (before treatment) was not observed in patient 2 at age 48 months (after 2 years of treatment). Conclusion We show that cerliponase alfa is able to delay the onset of symptoms when treatment is started in a presymptomatic stage of CLN2 disease. Our results advocate the start of treatment at an early age before symptom onset, but should be confirmed in a larger cohort study.

Natural history of patients with Leber hereditary optic neuropathy—results from the REALITY study

Background/objectives REALITY is an international observational retrospective registry of LHON patients evaluating the visual course and outcome in Leber hereditary optic neuropathy (LHON). Subjects/methods Demographics and visual function data were collected from medical charts of LHON patients with visual loss. The study was conducted in 11 study centres in the United States of America and Europe. The collection period extended from the presymptomatic stage to at least more than one year after onset of vision loss (chronic stage). A Locally Weighted Scatterplot Smoothing (LOWESS) local regression model was used to analyse the evolution of best-corrected visual acuity (BCVA) over time. Results 44 LHON patients were included; 27 (61%) carried the m.11778G>A ND4 mutation, 8 (18%) carried the m.3460G>A ND1 mutation, and 9 (20%) carried the m.14484T>C ND6 mutation. Fourteen (32%) patients were under 18 years old at onset of vision loss and 5 (11%) were below the age of 12. The average duration of follow-up was 32.5 months after onset of symptoms. At the last observed measure, mean BCVA was 1.46 LogMAR in ND4 patients, 1.52 LogMAR in ND1 patients, and 0.97 LogMAR in ND6 patients. The worst visual outcomes were reported in ND4 patients aged at least 15 years old at onset, with a mean BCVA of 1.55 LogMAR and no tendency for spontaneous recovery. The LOESS modelling curve depicted a severe and permanent deterioration of BCVA. Conclusions Amongst LHON patients with the three primary mtDNA mutations, adult patients with the m.11778G>A ND4 mutation had the worst visual outcomes, consistent with prior reports.

Preclinical Detection of Alpha-Synuclein Seeding Activity in the Colon of a Transgenic Mouse Model of Synucleinopathy by RT-QuIC

In synucleinopathies such as Parkinson’s disease (PD) and dementia with Lewy body (DLB), pathological alpha-synuclein (α-syn) aggregates are found in the gastrointestinal (GI) tract as well as in the brain. In this study, using real-time quaking-induced conversion (RT-QuIC), we investigated the presence of α-syn seeding activity in the brain and colon tissue of G2-3 transgenic mice expressing human A53T α-syn. Here we show that pathological α-syn aggregates with seeding activity were present in the colon of G2-3 mice as early as 3 months old, which is in the presymptomatic stage prior to the observation of any neurological abnormalities. In contrast, α-syn seeding activity was not detectable in 3 month-old mouse brains and only identified at 6 months of age in one of three mice. In the symptomatic stage of 12 months of age, RT-QuIC seeding activity was consistently detectable in both the brain and colon of G2-3 mice. Our results indicate that the RT-QuIC assay can presymptomatically detect pathological α-syn aggregates in the colon of G2-3 mice several months prior to their detection in brain tissue.

Routine saliva testing for the identification of silent coronavirus disease 2019 (COVID-19) in healthcare workers

Objective: Current COVID-19 guidelines recommend symptom-based screening and regular nasopharyngeal (NP) testing for healthcare personnel in high-risk settings. We sought to estimate case detection percentages with various routine NP and saliva testing frequencies. Design: Simulation modeling study. Methods: We constructed a sensitivity function based on the average infectiousness profile of symptomatic coronavirus disease 2019 (COVID-19) cases to determine the probability of being identified at the time of testing. This function was fitted to reported data on the percent positivity of symptomatic COVID-19 patients using NP testing. We then simulated a routine testing program with different NP and saliva testing frequencies to determine case detection percentages during the infectious period, as well as the presymptomatic stage. Results: Routine biweekly NP testing, once every 2 weeks, identified an average of 90.7% (SD, 0.18) of cases during the infectious period and 19.7% (SD, 0.98) during the presymptomatic stage. With a weekly NP testing frequency, the corresponding case detection percentages were 95.9% (SD, 0.18) and 32.9% (SD, 1.23), respectively. A 5-day saliva testing schedule had a similar case detection percentage as weekly NP testing during the infectious period, but identified ~10% more cases (mean, 42.5%; SD, 1.10) during the presymptomatic stage. Conclusion: Our findings highlight the utility of routine noninvasive saliva testing for frontline healthcare workers to protect vulnerable patient populations. A 5-day saliva testing schedule should be considered to help identify silent infections and prevent outbreaks in nursing homes and healthcare facilities.