Genome-wide association study identifies APOE and ZMIZ1 variants as mitophagy modifiers in Lewy body disease.

The PINK1-PRKN pathway mediates a critical quality control to maintain mitochondrial health and function. Together the kinase-ligase pair identifies and decorate damaged mitochondria with phosphorylated ubiquitin (p-S65-Ub). This selective label serves as the mitophagy tag and facilitates their degradation via autophagy-lysosome system. While complete loss of PINK1 or PRKN function causes early-onset Parkinson disease, much broader mitophagy impairments are emerging across neurodegenerative disorders. We previously found age- and disease-dependent accumulation of p-S65-Ub signal in the hippocampus of autopsy brains with Lewy body disease (LBD). However, the contribution of genetic variation to mitochondrial damage and p-S65-Ub levels remains unknown in LBD cases. To identify novel regulators of PINK1-PRKN mitophagy in LBD, we performed an unbiased genome-wide association study of hippocampal p-S65-Ub level with 1,012 autopsy confirmed LBD samples. Using an established, mostly automated workflow, hippocampal sections were immunostained for p-S65-Ub, scanned, and quantified with unbiased algorithms. Functional validation of the significant hit was performed in animal model and human induced pluripotent stem cells (hiPSCs). We identified a strong association with p-S65-Ub for APOE4 (rs429358; ß : 0.50, 95% CI: 0.41 to 0.69; p =8.67x10 -25 ) and a genome-wide significant association for ZMIZ1 (rs6480922; ß : -0.33, 95% CI: -0.45 to -0.22; p =1.42x10 -8 ). The increased p-S65-Ub levels in APOE4 -carrier may be mediated by both co-pathology-dependent and -independent mechanisms, which was confirmed in Apoe-targeted replacement mice and hiPSC-derived astrocytes. Intriguingly, ZMIZ1 rs6480922 also significantly associated with increased brain weight and reduced neuropathological burden indicating a potential role as a resilience factor. Our findings nominate novel mitophagy regulators in LBD brain ( ZMIZ1 locus) and highlight a strong association of APOE4 with mitophagy alteration. With APOE4 being the strongest known risk factor for clinical Alzheimer's disease and dementia with Lewy bodies, our findings suggest a common mechanistic link underscoring the importance of mitochondrial quality control.

Lysosomal polygenic risk is associated with the severity of neuropathology in Lewy body disease.

Intraneuronal accumulation of misfolded α-synuclein is the pathological hallmark of Parkinson's disease and dementia with Lewy bodies, often co-occurring with variable degrees of Alzheimer's disease related neuropathology. Genetic association studies have successfully identified common variants associated with disease risk and phenotypic traits in Lewy body disease, yet little is known about the genetic contribution to neuropathological heterogeneity. Using summary statistics from Parkinson's disease and Alzheimer's disease genome-wide association studies, we calculated polygenic risk scores and investigated the relationship with Lewy, amyloid-ß and tau pathology. Associations were nominated in neuropathologically defined samples with Lewy body disease from the Netherlands Brain Bank (n = 217) and followed up in an independent sample series from the Mayo Clinic Brain Bank (n = 394). We also generated stratified polygenic risk scores based on single-nucleotide polymorphisms annotated to eight functional pathways or cell types previously implicated in Parkinson's disease and assessed for association with Lewy pathology in subgroups with and without significant Alzheimer's disease co-pathology. In an ordinal logistic regression model, the Alzheimer's disease polygenic risk score was associated with concomitant amyloid-ß and tau pathology in both cohorts. Moreover, both cohorts showed a significant association between lysosomal pathway polygenic risk and Lewy pathology, which was more consistent than the association with a general Parkinson's disease risk score and specific to the subset of samples without significant concomitant Alzheimer's disease related neuropathology. Our findings provide proof of principle that the specific risk alleles a patient carries for Parkinson's and Alzheimer's disease also influence key aspects of the underlying neuropathology in Lewy body disease. The interrelations between genetic architecture and neuropathology are complex, as our results implicate lysosomal risk loci specifically in the subset of samples without Alzheimer's disease co-pathology. Our findings hold promise that genetic profiling may help predict the vulnerability to specific neuropathologies in Lewy body disease, with potential relevance for the further development of precision medicine in these disorders.

Reconsidering the role of blood-brain barrier in Alzheimer's disease: From delivery to target.

The existence of a selective blood-brain barrier (BBB) and neurovascular coupling are two unique central nervous system vasculature features that result in an intimate relationship between neurons, glia, and blood vessels. This leads to a significant pathophysiological overlap between neurodegenerative and cerebrovascular diseases. Alzheimer's disease (AD) is the most prevalent neurodegenerative disease whose pathogenesis is still to be unveiled but has mostly been explored under the light of the amyloid-cascade hypothesis. Either as a trigger, bystander, or consequence of neurodegeneration, vascular dysfunction is an early component of the pathological conundrum of AD. The anatomical and functional substrate of this neurovascular degeneration is the BBB, a dynamic and semi-permeable interface between blood and the central nervous system that has consistently been shown to be defective. Several molecular and genetic changes have been demonstrated to mediate vascular dysfunction and BBB disruption in AD. The isoform ε4 of Apolipoprotein E is at the same time the strongest genetic risk factor for AD and a known promoter of BBB dysfunction. Low-density lipoprotein receptor-related protein 1 (LRP-1), P-glycoprotein, and receptor for advanced glycation end products (RAGE) are examples of BBB transporters implicated in its pathogenesis due to their role in the trafficking of amyloid-ß. This disease is currently devoid of strategies that change the natural course of this burdening illness. This unsuccess may partly be explained by our misunderstanding of the disease pathogenesis and our inability to develop drugs that are effectively delivered to the brain. BBB may represent a therapeutic opportunity as a target itself or as a therapeutic vehicle. In this review, we aim to explore the role of BBB in the pathogenesis of AD including the genetic background and detail how it can be targeted in future therapeutic research.

TREX1 p.A129fs and p.Y305C variants in a large multi-ethnic cohort of CADASIL-like unrelated patients.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and retinal vasculopathy with cerebral leukodystrophy and systemic manifestations (RVCL-S) are the most common forms of rare monogenic early-onset cerebral small vessel disease and share clinical, and, to different extents, neuroradiological and neuropathological features. However, whether CADASIL and RVCL-S overlapping phenotype may be explained by shared genetic risk or causative factors such as TREX1 coding variants remains poorly understood. To investigate this intriguing hypothesis, we used exome sequencing to screen TREX1 protein-coding variability in a large multi-ethnic cohort of 180 early-onset independent familial and apparently sporadic CADASIL-like Caucasian patients from the USA, Portugal, Finland, Serbia and Turkey. We report 2 very rare and likely pathogenic TREX1 mutations: a loss of function mutation (p.Ala129fs) clustering in the catalytic domain, in an apparently sporadic 46-year-old patient from the USA and a missense mutation (p.Tyr305Cys) in the well conserved C-terminal region, in a 57-year-old patient with positive family history from Serbia. In concert with recent findings, our study expands the clinical spectrum of diseases associated with TREX1 mutations.

Exome sequencing identifies rare damaging variants in ATP8B4 and ABCA1 as risk factors for Alzheimer's disease.

Alzheimer's disease (AD), the leading cause of dementia, has an estimated heritability of approximately 70%1. The genetic component of AD has been mainly assessed using genome-wide association studies, which do not capture the risk contributed by rare variants2. Here, we compared the gene-based burden of rare damaging variants in exome sequencing data from 32,558 individuals-16,036 AD cases and 16,522 controls. Next to variants in TREM2, SORL1 and ABCA7, we observed a significant association of rare, predicted damaging variants in ATP8B4 and ABCA1 with AD risk, and a suggestive signal in ADAM10. Additionally, the rare-variant burden in RIN3, CLU, ZCWPW1 and ACE highlighted these genes as potential drivers of respective AD-genome-wide association study loci. Variants associated with the strongest effect on AD risk, in particular loss-of-function variants, are enriched in early-onset AD cases. Our results provide additional evidence for a major role for amyloid-ß precursor protein processing, amyloid-ß aggregation, lipid metabolism and microglial function in AD.

Evaluation of a Proposed Approach for the Determination of the Bioequivalence Acceptance Range for Narrow Therapeutic Index Drugs in the European Union.

Bioequivalence (BE) of products containing narrow therapeutic index (NTI) drugs in the European Union is currently established by demonstrating that the 90% confidence interval for the ratio of the population geometric means of the test compared to the reference product's AUC, and in certain cases Cmax, is included within the tighter acceptance range of 90.00−111.11%. An alternative criterion, consisting of narrowed limits based on the within-subject variability of the reference product, was recently proposed. Its performance for a three-period partial replicate design was tested by simulation in terms of power to show BE, type I error (T1E) and sample size requirements. A new condition, a constraint on the test-to-reference geometric mean ratio (cGMR) to be contained within the range of 90.00−111.11%, was also tested. The probability of showing BE when the products differ more than 10% was increased, but only if the reference product's within-subject variability was moderate-to-high. The inclusion of the additional cGMR limited this. An increase in the T1E (<7%) was observed. The inclusion of the additional cGMR did not change the highest inflation of the T1E. Finally, a significant sample size reduction was observed and the inclusion of the cGMR usually did not increase the required sample size.

Independent phenotypic plasticity axes define distinct obesity sub-types.

Studies in genetically 'identical' individuals indicate that as much as 50% of complex trait variation cannot be traced to genetics or to the environment. The mechanisms that generate this 'unexplained' phenotypic variation (UPV) remain largely unknown. Here, we identify neuronatin (NNAT) as a conserved factor that buffers against UPV. We find that Nnat deficiency in isogenic mice triggers the emergence of a bi-stable polyphenism, where littermates emerge into adulthood either 'normal' or 'overgrown'. Mechanistically, this is mediated by an insulin-dependent overgrowth that arises from histone deacetylase (HDAC)-dependent ß-cell hyperproliferation. A multi-dimensional analysis of monozygotic twin discordance reveals the existence of two patterns of human UPV, one of which (Type B) phenocopies the NNAT-buffered polyphenism identified in mice. Specifically, Type-B monozygotic co-twins exhibit coordinated increases in fat and lean mass across the body; decreased NNAT expression; increased HDAC-responsive gene signatures; and clinical outcomes linked to insulinemia. Critically, the Type-B UPV signature stratifies both childhood and adult cohorts into four metabolic states, including two phenotypically and molecularly distinct types of obesity.

Whole-exome sequencing reveals PSEN1 and ATP7B combined variants as a possible cause of early-onset Lewy body dementia: a case study of genotype-phenotype correlation.

Dementia with Lewy bodies is a neurodegenerative disease, sharing features with Parkinson's and Alzheimer's diseases. We report a case of a patient dementia with Lewy bodies carrying combined PSEN1 and ATP7B mutations. A man developed dementia with Lewy bodies starting at the age of 60 years. CSF biomarkers were of Alzheimer's disease and DaTSCAN was abnormal. Whole-exome sequencing revealed a heterozygous p.Ile408Thr PSEN1 variant and a homozygous p.Arg616Trp ATP7B variant. This case reinstates the need of considering ATP7B mutations when evaluating a patient with parkinsonism and supports p.Ile408Thr as a pathogenic PSEN1 variant.

Exome Sequencing of a Portuguese Cohort of Frontotemporal Dementia Patients: Looking Into the ALS-FTD Continuum.

Introduction: Frontotemporal dementia (FTD) is considered to be part of a continuum with amyotrophic lateral sclerosis (ALS). Many genes are associated with both ALS and FTD. Yet, many genes associated with ALS have not been shown to cause FTD. We aimed to study a Portuguese cohort of FTD patients, searching for variants in genes associated with both FTD and/or ALS. Methods: We included 57 thoroughly characterized index FTD patients from our memory clinic, who were not carriers of pathogenic variants in GRN, MAPT or C9orf72. We performed exome sequencing and 1) prioritized potential FTD and ALS causing variants by using Exomiser to annotate and filter results; and 2) looked specifically at rare variability in genes associated with FTD (excluding GRN, MAPT and C9ORF72) and/or ALS. Results: We identified 13 rare missense variants in 10 patients (three patients had two variants) in the following genes: FUS, OPTN, CCNF, DCTN1, TREM2, ERBB4, ANG, CHRNA4, CHRNB4 and SETX. We found an additional frameshift variant on GLT8D1 in one patient. One variant (ERBB4 p.Arg1112His) gathered enough evidence to be classified as likely pathogenic by the ACMG criteria. Discussion: We report, for the first time, an expanded study of genes known to cause FTD-ALS, in the Portuguese population. Potentially pathogenic variants in ERBB4, FUS, SETX, ANG, CHRNA4 and CHRNB4 were identified in FTD patients. These findings provide additional evidence for the potential role of rare variability in ALS-associated genes in FTD, expanding the genetic spectrum between the two diseases.

MED27, SLC6A7, and MPPE1 Variants in a Complex Neurodevelopmental Disorder with Severe Dystonia.

BACKGROUND: Despite advances in next generation sequencing technologies, the identification of variants of uncertain significance (VUS) can often hinder definitive diagnosis in patients with complex neurodevelopmental disorders. OBJECTIVE: The objective of this study was to identify and characterize the underlying cause of disease in a family with two children with severe developmental delay associated with generalized dystonia and episodic status dystonicus, chorea, epilepsy, and cataracts. METHODS: Candidate genes identified by autozygosity mapping and whole-exome sequencing were characterized using cellular and vertebrate model systems. RESULTS: Homozygous variants were found in three candidate genes: MED27, SLC6A7, and MPPE1. Although the patients had features of MED27-related disorder, the SLC6A7 and MPPE1 variants were functionally investigated. SLC6A7 variant in vitro overexpression caused decreased proline transport as a result of reduced cell-surface expression, and zebrafish knockdown of slc6a7 exhibited developmental delay and fragile motor neuron morphology that could not be rescued by L-proline transporter-G396S RNA. Lastly, patient fibroblasts displayed reduced cell-surface expression of glycophosphatidylinositol-anchored proteins linked to MPPE1 dysfunction. CONCLUSIONS: We report a family harboring a homozygous MED27 variant with additional loss-of-function SLC6A7 and MPPE1 gene variants, which potentially contribute to a blended phenotype caused by multilocus pathogenic variants. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Genome-wide association of polygenic risk extremes for Alzheimer's disease in the UK Biobank.

In just over a decade, advances in genome-wide association studies (GWAS) have offered an approach to stratify individuals based on genetic risk for disease. Using recent Alzheimer's disease (AD) GWAS results as the base data, we determined each individual's polygenic risk score (PRS) in the UK Biobank dataset. Using individuals within the extreme risk distribution, we performed a GWAS that is agnostic of AD phenotype and is instead based on known genetic risk for disease. To interpret the functions of the new risk factors, we conducted phenotype analyses, including a phenome-wide association study. We identified 246 loci surpassing the significance threshold of which 229 were not reported in the base AD GWAS. These include loci that showed suggestive levels of association in the base GWAS and loci not previously suspected to be associated with AD. Among these, there are loci, such as IL34 and KANSL1, that have since been shown to be associated with AD in recent studies. We also show highly significant genetic correlations with multiple health-related outcomes that provide insights into prodromal symptoms and comorbidities. This is the first study to utilize PRS as a phenotype-agnostic group classification in AD genetic studies. We identify potential new loci for AD and detail phenotypic analysis of these PRS extremes.

Genetic analysis reveals novel variants for vascular cognitive impairment.

OBJECTIVES: The genetic background of vascular cognitive impairment (VCI) is poorly understood compared to other dementia disorders. The aim of the study was to investigate the genetic background of VCI in a well-characterized Finnish cohort. MATERIALS & METHODS: Whole-exome sequencing (WES) was applied in 45 Finnish VCI patients. Copy-number variant (CNV) analysis using a SNP array was performed in 80 VCI patients. This study also examined the prevalence of variants at the miR-29 binding site of COL4A1 in 73 Finnish VCI patients. RESULTS: In 40% (18/45) of the cases, WES detected possibly causative variants in genes associated with cerebral small vessel disease (CSVD) or other neurological or stroke-related disorders. These variants included HTRA1:c.847G>A p.(Gly283Arg), TREX1:c.1079A>G, p.(Tyr360Cys), COLGALT1:c.1411C>T, p.(Arg471Trp), PRNP: c.713C>T, p.(Pro238Leu), and MTHFR:c.1061G>C, p.(Gly354Ala). Additionally, screening of variants in the 3'UTR of COL4A1 gene in a sub-cohort of 73 VCI patients identified a novel variant c.*36T>A. CNV analysis showed that pathogenic CNVs are uncommon in VCI. CONCLUSIONS: These data support pathogenic roles of variants in HTRA1, TREX1 and in the 3'UTR of COL4A1 in CSVD and VCI, and suggest that vascular pathogenic mechanisms are linked to neurodegeneration, expanding the understanding of the genetic background of VCI.

Molecular Characterization of Portuguese Patients with Hereditary Cerebellar Ataxia.

Hereditary cerebellar ataxia (HCA) comprises a clinical and genetic heterogeneous group of neurodegenerative disorders characterized by incoordination of movement, speech, and unsteady gait. In this study, we performed whole-exome sequencing (WES) in 19 families with HCA and presumed autosomal recessive (AR) inheritance, to identify the causal genes. A phenotypic classification was performed, considering the main clinical syndromes: spastic ataxia, ataxia and neuropathy, ataxia and oculomotor apraxia (AOA), ataxia and dystonia, and ataxia with cognitive impairment. The most frequent causal genes were associated with spastic ataxia (SACS and KIF1C) and with ataxia and neuropathy or AOA (PNKP). We also identified three families with autosomal dominant (AD) forms arising from de novo variants in KIF1A, CACNA1A, or ATP1A3, reinforcing the importance of differential diagnosis (AR vs. AD forms) in families with only one affected member. Moreover, 10 novel causal-variants were identified, and the detrimental effect of two splice-site variants confirmed through functional assays. Finally, by reviewing the molecular mechanisms, we speculated that regulation of cytoskeleton function might be impaired in spastic ataxia, whereas DNA repair is clearly associated with AOA. In conclusion, our study provided a genetic diagnosis for HCA families and proposed common molecular pathways underlying cerebellar neurodegeneration.

Rare variants in TP73 in a frontotemporal dementia cohort link this gene with primary progressive aphasia phenotypes.

BACKGROUND AND PURPOSE: TP73 was recently reported to cause amyotrophic lateral sclerosis (ALS). ALS and frontotemporal dementia (FTD) are considered to form part of a continuum. We aimed to investigate whether TP73 variants may be associated with FTD. METHODS: We studied a thoroughly investigated cohort of 65 Portuguese patients with frontotemporal dementia using whole-exome sequencing. The patients had no other known genetic cause for their disease (C9orf72 expansion was also excluded). RESULTS: Of the 65 patients studied, two had rare variants in TP73 (p.Gly605Ser and p.Arg347Trp). Both variants had minor allele frequency <0.001 and were predicted to be pathogenic in silico. The two patients displayed a phenotype that included predominant language impairment, suggestive of non-fluent progressive aphasia. CONCLUSION: We show that two thoroughly studied patients without other known genetic changes harbored TP73 rare variants, which are pathogenic in silico. This adds evidence to support the role of TP73 in the ALS-FTD spectrum, especially in primary progressive aphasia cases.

In situ proximity labeling identifies Lewy pathology molecular interactions in the human brain.

The intracellular misfolding and accumulation of alpha-synuclein into structures collectively called Lewy pathology (LP) is a central phenomenon for the pathogenesis of synucleinopathies, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Understanding the molecular architecture of LP is crucial for understanding synucleinopathy disease origins and progression. Here we used a technique called biotinylation by antibody recognition (BAR) to label total (BAR-SYN1) and pathological alpha-synuclein (BAR-PSER129) in situ for subsequent mass spectrometry analysis. Results showed superior immunohistochemical detection of LP following the BAR-PSER129 protocol, particularly for fibers and punctate pathology within the striatum and cortex. Mass spectrometry analysis of BAR-PSER129-labeled LP identified 261 significantly enriched proteins in the synucleinopathy brain when compared to nonsynucleinopathy brains. In contrast, BAR-SYN1 did not differentiate between disease and nonsynucleinopathy brains. Pathway analysis of BAR-PSER129-enriched proteins revealed enrichment for 718 pathways; notably, the most significant KEGG pathway was PD, and Gene Ontology (GO) cellular compartments were the vesicle, extracellular vesicle, extracellular exosome, and extracellular organelle. Pathway clustering revealed several superpathways, including metabolism, mitochondria, lysosome, and intracellular vesicle transport. Validation of the BAR-PSER129-identified protein hemoglobin beta (HBB) by immunohistochemistry confirmed the interaction of HBB with PSER129 Lewy neurites and Lewy bodies. In summary, BAR can be used to enrich for LP from formalin-fixed human primary tissues, which allowed the determination of molecular signatures of LP. This technique has broad potential to help understand the phenomenon of LP in primary human tissue and animal models.

Prion-like α-synuclein pathology in the brain of infants with Krabbe disease.

Krabbe disease is an infantile neurodegenerative disorder resulting from pathogenic variants in the GALC gene that causes accumulation of the toxic sphingolipid psychosine. GALC variants are also associated with Lewy body diseases, an umbrella term for age-associated neurodegenerative diseases in which the protein α-synuclein aggregates into Lewy bodies. To explore whether α-synuclein in Krabbe disease has pathological similarities to that in Lewy body disease, we performed an observational post-mortem study of Krabbe disease brain tissue (n = 4) compared to infant controls (n = 4) and identified widespread accumulations of α-synuclein. To determine whether α-synuclein in Krabbe disease brain displayed disease-associated pathogenic properties we evaluated its seeding capacity using the real-time quaking-induced conversion assay in two cases for which frozen tissue was available and strikingly identified aggregation into fibrils similar to those observed in Lewy body disease, confirming the prion-like capacity of Krabbe disease-derived α-synuclein. These observations constitute the first report of prion-like α-synuclein in the brain tissue of infants and challenge the putative view that α-synuclein pathology is merely an age-associated phenomenon, instead suggesting it results from alterations to biological pathways, such as sphingolipid metabolism. Our findings have important implications for understanding the mechanisms underlying Lewy body formation in Lewy body disease.

Challenge accepted: uncovering the role of rare genetic variants in Alzheimer's disease.

The search for rare variants in Alzheimer's disease (AD) is usually deemed a high-risk - high-reward situation. The challenges associated with this endeavor are real. Still, the application of genome-wide technologies to large numbers of cases and controls or to small, well-characterized families has started to be fruitful.Rare variants associated with AD have been shown to increase risk or cause disease, but also to protect against the development of AD. All of these can potentially be targeted for the development of new drugs.Multiple independent studies have now shown associations of rare variants in NOTCH3, TREM2, SORL1, ABCA7, BIN1, CLU, NCK2, AKAP9, UNC5C, PLCG2, and ABI3 with AD and suggested that they may influence disease via multiple mechanisms. These genes have reported functions in the immune system, lipid metabolism, synaptic plasticity, and apoptosis. However, the main pathway emerging from the collective of genes harboring rare variants associated with AD is the Aß pathway. Associations of rare variants in dozens of other genes have also been proposed, but have not yet been replicated in independent studies. Replication of this type of findings is one of the challenges associated with studying rare variants in complex diseases, such as AD. In this review, we discuss some of these primary challenges as well as possible solutions.Integrative approaches, the availability of large datasets and databases, and the development of new analytical methodologies will continue to produce new genes harboring rare variability impacting AD. In the future, more extensive and more diverse genetic studies, as well as studies of deeply characterized families, will enhance our understanding of disease pathogenesis and put us on the correct path for the development of successful drugs.

A Proposed Approach for the Determination of the Bioequivalence Acceptance Range for Narrow Therapeutic Index Drugs in the European Union.

The current regulatory criterion for bioequivalence of narrow therapeutic index (NTI) drugs in the European Union requires that the 90% confidence interval for the ratio of the population geometric means of the test product compared with the reference for area under the plasma concentration-time curve (AUC), and in certain cases maximum plasma drug concentration (Cmax ), to be included within the tighter acceptance range of 90.00-111.11%. As a consequence, sponsors need to recruit a higher number of subjects to demonstrate bioequivalence and this may be seen as increasing the burden for the development of generics. This "one-size-fits-all" criterion is particularly questionable when the within-subject variability of the reference product is moderate to high. As an alternative, we propose a further refined statistical approach where the acceptance range is narrowed based on the within-subject variability of the reference product of the NTI drug, similar to the one used for widening the standard 80.00-125.00% acceptance range for highly variable drugs. The 80.00-125.00% acceptance range is narrowed, only if the within-subject variability is lower than 30%, down to the current NTI acceptance range of 90.00-111.11% when the within-subject variability is 13.93% or lower. Examples within the current European Medicines Agency list of NTI drugs show a considerable reduction in required sample size for drugs like tacrolimus and colchicine, where the predicted within-subject variability is 20-30%. In these cases, this approach is less sample size demanding without any expected increase in the therapeutic risks, since patients treated with reference products with moderate to high within-subject variability are frequently exposed to bioavailability differences larger than 10%.