Disorders of fatty acid homeostasis.

Humans derive fatty acids (FA) from exogenous dietary sources and/or endogenous synthesis from acetyl-CoA, although some FA are solely derived from exogenous sources ("essential FA"). Once inside cells, FA may undergo a wide variety of different modifications, which include their activation to their corresponding CoA ester, the introduction of double bonds, the 2- and ω-hydroxylation and chain elongation, thereby generating a cellular FA pool which can be used for the synthesis of more complex lipids. The biological properties of complex lipids are very much determined by their molecular composition in terms of the FA incorporated into these lipid species. This immediately explains the existence of a range of genetic diseases in man, often with severe clinical consequences caused by variants in one of the many genes coding for enzymes responsible for these FA modifications. It is the purpose of this review to describe the current state of knowledge about FA homeostasis and the genetic diseases involved. This includes the disorders of FA activation, desaturation, 2- and ω-hydroxylation, and chain elongation, but also the disorders of FA breakdown, including disorders of peroxisomal and mitochondrial α- and ß-oxidation.

Four-dimensional lipidomics profiling in X-linked adrenoleukodystrophy using trapped ion mobility mass spectrometry.

Lipids play pivotal roles in an extensive range of metabolic and physiological processes. In recent years, the convergence of trapped ion mobility spectrometry and MS has enabled 4D-lipidomics, a highly promising technology for comprehensive lipid analysis. 4D-lipidomics assesses lipid annotations across four distinct dimensions-retention time, collisional cross section, m/z (mass-to-charge ratio), and MS/MS spectra-providing a heightened level of confidence in lipid annotation. These advantages prove particularly valuable when investigating complex disorders involving lipid metabolism, such as adrenoleukodystrophy (ALD). ALD is characterized by the accumulation of very-long-chain fatty acids (VLCFAs) due to pathogenic variants in the ABCD1 gene. A comprehensive 4D-lipidomics strategy of ALD fibroblasts demonstrated significant elevations of various lipids from multiple classes. This indicates that the changes observed in ALD are not confined to a single lipid class and likely impacts a broad spectrum of lipid-mediated physiological processes. Our findings highlight the incorporation of mainly saturated and monounsaturated VLCFA variants into a range of lipid classes, encompassing phosphatidylcholines, triacylglycerols, and cholesterol esters. These include ultra-long-chain fatty acids with a length of up to thirty carbon atoms. Lipid species containing C26:0 and C26:1 were the most frequently detected VLCFA lipids in our study. Furthermore, we report a panel of 121 new candidate biomarkers in fibroblasts, exhibiting significant differentiation between controls and individuals with ALD. In summary, this study demonstrates the capabilities of a 4D-lipid profiling workflow in unraveling novel insights into the intricate lipid modifications associated with metabolic disorders like ALD.

Lethal Capecitabine Toxicity in Patients With Complete Dihydropyrimidine Dehydrogenase Deficiency Due to Ultra-Rare DPYD Variants.

A DPD deficiency should be considered in case of severe toxicity even in the absence of common risk variants in DPYD.

Sulfate: a neglected (but potentially highly relevant) anion.

Sulfate is an important anion as sulfonation is essential in modulation of several compounds, such as exogens, polysaccharide chains of proteoglycans, cholesterol or cholesterol derivatives and tyrosine residues of several proteins. Sulfonation requires the presence of both the sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) and a sulfotransferase. Genetic disorders affecting sulfonation, associated with skeletal abnormalities, impaired neurological development and endocrinopathies, demonstrate the importance of sulfate. Yet sulfate is not measured in clinical practice. This review addresses sulfate metabolism and consequences of sulfonation defects, how to measure sulfate and why we should measure sulfate more often.

Long term follow-up in GAMT deficiency - Correlation of therapy regimen, biochemical and in vivo brain proton MR spectroscopy data.

GAMT deficiency is a rare autosomal recessive disease within the group of cerebral creatine deficiency syndromes. Cerebral creatine depletion and accumulation of guanidinoacetate (GAA) lead to clinical presentation with intellectual disability, seizures, speech disturbances and movement disorders. Treatment consists of daily creatine supplementation to increase cerebral creatine, reduction of arginine intake and supplementation of ornithine for reduction of toxic GAA levels. This study represents the first long-term follow-up over a period of 14 years, with detailed clinical data, biochemical and multimodal neuroimaging findings. Developmental milestones, brain MRI, quantitative single voxel 1H magnetic resonance spectroscopy (MRS) and biochemical analyses were assessed. The results reveal insights into the dose dependent effects of creatine/ornithine supplementation and expand the phenotypic spectrum of GAMT deficiency. Of note, the creatine concentrations, which were regularly monitored over a long follow-up period, increased significantly over time, but did not reach age matched control ranges. Our patient is the second reported to show normal neurocognitive outcome after an initial delay, stressing the importance of early diagnosis and treatment initiation.

Discovery of novel diagnostic biomarkers for Sjögren-Larsson syndrome by untargeted lipidomics.

AIM: Sjögren-Larsson syndrome (SLS) is a rare neurometabolic disorder that mainly affects brain, eye and skin and is caused by deficiency of fatty aldehyde dehydrogenase. Our recent finding of a profoundly disturbed brain tissue lipidome in SLS prompted us to search for similar biomarkers in plasma as no functional test in blood is available for SLS. METHODS AND RESULTS: We performed plasma lipidomics and used a newly developed bioinformatics tool to mine the untargeted part of the SLS plasma and brain lipidome to search for SLS biomarkers. Plasma lipidomics showed disturbed ether lipid metabolism in known lipid classes. Untargeted lipidomics of both plasma and brain (white and grey matter) uncovered two new endogenous lipid classes highly elevated in SLS. The first biomarker group were alkylphosphocholines/ethanolamines containing different lengths of alkyl-chains where some alkylphosphocholines were > 600-fold elevated in SLS plasma. The second group of biomarkers were a set of 5 features of unknown structure. Fragmentation studies suggested that they contain ubiquinol and phosphocholine and one feature was also found as a glucuronide conjugate in plasma. The plasma features were highly distinctive for SLS with levels >100-1000-fold the level in controls, if present at all. We speculate on the origin of the alkylphosphocholines/ethanolamines and the nature of the ubiquinol-containing metabolites. CONCLUSIONS: The metabolites identified in this study represent novel endogenous lipid classes thus far unknown in humans. They represent the first plasma metabolite SLS-biomarkers and may also yield more insight into SLS pathophysiology.

Ophthalmic acid is a glutathione regulating tripeptide.

Since its discovery in 1958 in the lens of cows, ophthalmic acid (OPH) has stood in the shadow of its anti-oxidant analog: glutathione (GSH). Lacking the thiol group that gives GSH many of its important properties, ophthalmic acid's function has remained elusive, and it has been widely presumed to be an accidental product of the same enzymes. In this review, we compile evidence demonstrating that OPH is a ubiquitous metabolite found in bacteria, plants, fungi, and animals, produced through several layers of metabolic regulation. We discuss the limitations of the oft-repeated suggestions that aberrations in OPH levels should solely indicate GSH deficiency or oxidative stress. Finally, we discuss the available literature and suggest OPH's role in metabolism as a GSH-regulating tripeptide; controlling both cellular and organelle influx and efflux of GSH, as well as modulating GSH-dependent reactions and signaling. Ultimately, we hope that this review reinvigorates and directs more research into this versatile metabolite.

Newborn screening for Cerebrotendinous Xanthomatosis: A retrospective biomarker study using both flow-injection and UPLC-MS/MS analysis in 20,000 newborns.

BACKGROUND AND AIMS: Cerebrotendinous Xanthomatosis (CTX) is a treatable disorder of bile acid synthesis caused by deficiency of 27-sterol hydroxylase -encoded by CYP27A1- leading to gastrointestinal and progressive neuropsychiatric symptoms. Biochemically, CTX is characterized by accumulation of the bile alcohol cholestanetetrol glucuronide (GlcA-tetrol) and the deficiency of tauro-chenodeoxycholic acid (t-CDCA) and tauro-trihydroxycholestanoic acid (t-THCA). MATERIALS AND METHODS: To ascertain the feasibility of CTX newborn screening (NBS) we performed a study with deidentified Dutch dried blood spots using reagents and equipment that is frequently used in NBS laboratories. 20,076 deidentified newborn blood spots were subjected to flow-injection (FIA)-MS/MS and UPLC-MS/MS analysis to determine the concentration of GlcA-tetrol and calculate the GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios. RESULTS: Using UPLC-MS/MS analysis both GlcA-tetrol concentration and/or metabolite ratios GlcA-tetrol/t-CDCA proved to be informative biomarkers; newborn DBS results did not overlap with those of the CTX patients. For FIA-MS/MS, GlcA-tetrol also was an excellent marker but when using the combination of the GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios also did not yield any screen positives. CONCLUSION: Newborn screening for CTX using only metabolite ratios following the measurement of three CTX biomarkers is possible using either FIA-MS/MS or UPLC-MS/MS, which paves the way for introduction of CTX NBS.

Sex-specific newborn screening for X-linked adrenoleukodystrophy.

Males with X-linked adrenoleukodystrophy (ALD) are at high risk for developing adrenal insufficiency and/or progressive leukodystrophy (cerebral ALD) at an early age. Pathogenic variants in ABCD1 result in elevated levels of very long-chain fatty acids (VLCFA), including C26:0-lysophosphatidylcholine (C26:0-LPC). Newborn screening for ALD enables prospective monitoring and timely therapeutic intervention, thereby preventing irreversible damage and saving lives. The Dutch Health Council recommended to screen only male newborns for ALD without identifying untreatable conditions associated with elevated C26:0-LPC, like Zellweger spectrum disorders and single peroxisomal enzyme defects. Here, we present the results of the SCAN (Screening for ALD in the Netherlands) study which is the first sex-specific newborn screening program worldwide. Males with ALD are identified based on elevated C26:0-LPC levels, the presence of one X-chromosome and a variant in ABCD1, in heel prick dried bloodspots. Screening of 71 208 newborns resulted in the identification of four boys with ALD who, following referral to the pediatric neurologist and confirmation of the diagnosis, enrolled in a long-term follow-up program. The results of this pilot show the feasibility of employing a boys-only screening algorithm that identifies males with ALD without identifying untreatable conditions. This approach will be of interest to countries that are considering ALD newborn screening but are reluctant to identify girls with ALD because for girls there is no direct health benefit. We also analyzed whether gestational age, sex, birth weight and age at heel prick blood sampling affect C26:0-LPC concentrations and demonstrate that these covariates have a minimal effect.

Impact of COVID-19 on Pediatric Laboratory Medicine: An IFCC C-ETPLM, SSIEM, ISNS Global Survey.

Objective: Pediatric laboratory medicine is a unique practice serving a vulnerable group of patients including highly specialized testing aiming to detect and treat inherited conditions early to avoid adverse outcomes. Data on the actual impact of COVID-19 pandemic on this speciality is lacking. Methods: A survey was conducted by the IFCC Committee on Emerging Technologies in Pediatric Laboratory Medicine in partnership with the Society for the Study of Inborn Errors of Metabolism and International Society for Neonatal Screening, to assess the impact on the clinical service provision during the initial wave (January to July 2020) of the COVID-19 pandemic and to gather experiences learned in order to improve laboratory preparedness for future outbreaks. Results: 217 survey responses were received from 69 regions. Sixty-three laboratories (29%) reported a restriction or suspension of service for a median period of four months. The common tests/ services suspended were new-born screening program, body fluids and sweat testing. The reasons for the suspension were related to bio-safety risks of COVID-19 transmission, manpower constraints and supplies disruption. A minority (9-10%) of laboratories did observe delayed/missed diagnoses or a more severe presentation of a clinical disorder. The critical operational decisions that helped manage the initial wave of COVID-19 included modifying work shift patterns, split-teams arrangement, use of personal protection equipment and social distancing. Conclusion: The provision and delivery of pediatric laboratories services were affected during the initial wave of the COVID-19 pandemic. Manpower preparedness for future potential disruptions to pediatric laboratory services is a key finding and recommendation from this survey.

WARS1 and SARS1: Two tRNA synthetases implicated in autosomal recessive microcephaly.

Aminoacylation of transfer RNA (tRNA) is a key step in protein biosynthesis, carried out by highly specific aminoacyl-tRNA synthetases (ARSs). ARSs have been implicated in autosomal dominant and autosomal recessive human disorders. Autosomal dominant variants in tryptophanyl-tRNA synthetase 1 (WARS1) are known to cause distal hereditary motor neuropathy and Charcot-Marie-Tooth disease, but a recessively inherited phenotype is yet to be clearly defined. Seryl-tRNA synthetase 1 (SARS1) has rarely been implicated in an autosomal recessive developmental disorder. Here, we report five individuals with biallelic missense variants in WARS1 or SARS1, who presented with an overlapping phenotype of microcephaly, developmental delay, intellectual disability, and brain anomalies. Structural mapping showed that the SARS1 variant is located directly within the enzyme's active site, most likely diminishing activity, while the WARS1 variant is located in the N-terminal domain. We further characterize the identified WARS1 variant by showing that it negatively impacts protein abundance and is unable to rescue the phenotype of a CRISPR/Cas9 wars1 knockout zebrafish model. In summary, we describe two overlapping autosomal recessive syndromes caused by variants in WARS1 and SARS1, present functional insights into the pathogenesis of the WARS1-related syndrome and define an emerging disease spectrum: ARS-related developmental disorders with or without microcephaly.

Polar metabolomics in human muscle biopsies using a liquid-liquid extraction and full-scan LC-MS.

We describe here a user-friendly analysis protocol for semi-targeted polar metabolomics in human muscle biopsies using Zwitterionic Hydrophilic Interaction Liquid Chromatography and high-resolution full-scan mass spectrometry. Previously, this protocol has been used for Caenorhabditis elegans. Here we show that it can be successfully applied to human muscle biopsies with minor adjustments. Summarized instructions for other matrices are also provided. As peak integration in metabolomics can be challenging, we provide expected retention times and extensive peak descriptions to aid this process. For complete details on the use and execution of this protocol, please refer to Molenaars et al. (2021).

Identification of three novel pathogenic mutations in cystathionine beta-synthase gene of Pakistani intellectually disabled patients.

BACKGROUND: Classical homocystinuria (HCU) is an autosomal recessive inborn error of metabolism, which is caused by the cystathionine-ß-synthase (CBS: encoded by CBS) deficiency. Symptoms of untreated classical HCU patients include intellectual disability (ID), ectopia lentis and long limbs, along with elevated plasma methionine, and homocysteine. METHODS: A total of 429 ID patients (age range: 1.6-23 years) were sampled from Northern areas of Punjab, Pakistan. Biochemical and genetic analyses were performed to find classical HCU disease in ID patients. RESULTS: Biochemically, nine patients from seven unrelated families were identified with high levels of plasma methionine and homocysteine. Targeted exonic analysis of CBS confirmed seven causative homozygous mutations; of which three were novel missense mutations (c.451G>T; p.Gly151Trp, c.975G>C; p.Lys325Asn and c.1039 + 1G>T splicing), and four were recurrent variants (c.451 + 1G>A; IVS4 + 1 splicing, c.770C>T; p.Thr257Met, c.808_810del GAG; p.Glu270del and c.752T>C; p.Leu251Pro). Treatment of patients was initiated without further delay with pyridoxine, folic acid, cobalamin, and betaine as well as dietary protein restriction. The immediate impact was noticed in behavioral improvement, decreased irritability, improved black hair color, and socialization. Overall, health outcomes in this disorder depend on the age and symptomatology at the time of treatment initiation. CONCLUSIONS: With personalized treatment and care, such patients can reach their full potential of living as healthy a life as possible. This screening study is one of the pioneering initiatives in Pakistan which would help to minimize the burden of such treatable inborn errors of metabolism in the intellectually disabled patients.

Heterogenous Clinical Landscape in a Consanguineous Malonic Aciduria Family.

Malonic aciduria is an extremely rare inborn error of metabolism due to malonyl-CoA decarboxylase deficiency. This enzyme is encoded by the MLYCD (Malonyl-CoA Decarboxylase) gene, and the disease has an autosomal recessive inheritance. Malonic aciduria is characterized by systemic clinical involvement, including neurologic and digestive symptoms, metabolic acidosis, hypoglycemia, failure to thrive, seizures, developmental delay, and cardiomyopathy. We describe here two index cases belonging to the same family that, despite an identical genotype, present very different clinical pictures. The first case is a boy with neonatal metabolic symptoms, abnormal brain MRI, and dilated cardiomyopathy. The second case, the cousin of the first patient in a consanguineous family, showed later symptoms, mainly with developmental delay. Both patients showed high levels of malonylcarnitine on acylcarnitine profiles and malonic acid on urinary organic acid chromatographies. The same homozygous pathogenic variant was identified, c.346C > T; p. (Gln116*). We also provide a comprehensive literature review of reported cases. A review of the literature yielded 52 cases described since 1984. The most common signs were developmental delay and cardiomyopathy. Increased levels of malonic acid and malonylcarnitine were constant. Presentations ranged from neonatal death to patients surviving past adolescence. These two cases and reported patients in the literature highlight the inter- and intrafamilial variability of malonic aciduria.

Fetal akinesia deformation sequence and massive perivillous fibrin deposition resulting in fetal death in six fetuses from one consanguineous couple, including literature review.

BACKGROUND: Massive perivillous fibrin deposition (MPFD) is associated with adverse pregnancy outcomes and is mainly caused by maternal factors with limited involvement of fetal or genetic causes. We present one consanguineous couple with six fetuses developing Fetal Akinesia Deformation Sequence (FADS) and MPFD, with a possible underlying genetic cause. This prompted a literature review on prevalence of FADS and MPFD. METHODS: Fetal ultrasound examination, motor assessment, genetic testing, postmortem examination, and placenta histology are presented (2009-2019). Literature was reviewed for the association between congenital anomalies and MPFD. RESULTS: All six fetuses developed normally during the first trimester. Thereafter, growth restriction, persistent flexed position, abnormal motility, and contractures in 4/6, consistent with FADS occurred. All placentas showed histologically confirmed MPFD. Genetic analyses in the five available cases showed homozygosity for two variants of unknown significance in two genes, VARS1 (OMIM*192150) and ABCF1 (OMIM*603429). Both parents are heterozygous for these variants. From 63/1999 manuscripts, 403 fetal outcomes were mobilized. In 14/403 fetuses, congenital abnormalities in association with MPFD were seen of which two fetuses with contractures/FADS facial anomalies. CONCLUSION: The low prevalence of fetal contractures/FADS facial anomalies in association with MPFD in the literature review supports the possible fetal or genetic contribution causing FADS and MPFD in our family. This study with literature review supports the finding that fetal, fetoplacental, and/or genetic components may play a role in causing a part of MPFDs.

A bi-allelic loss-of-function SARS1 variant in children with neurodevelopmental delay, deafness, cardiomyopathy, and decompensation during fever.

Aminoacyl-tRNA synthetases (aaRS) are ubiquitously expressed enzymes responsible for ligating amino acids to their cognate tRNA molecules through an aminoacylation reaction. The resulting aminoacyl-tRNA is delivered to ribosome elongation factors to participate in protein synthesis. Seryl-tRNA synthetase (SARS1) is one of the cytosolic aaRSs and catalyzes serine attachment to tRNASer . SARS1 deficiency has already been associated with moderate intellectual disability, ataxia, muscle weakness, and seizure in one family. We describe here a new clinical presentation including developmental delay, central deafness, cardiomyopathy, and metabolic decompensation during fever leading to death, in a consanguineous Turkish family, with biallelic variants (c.638G>T, p.(Arg213Leu)) in SARS1. This missense variant was shown to lead to protein instability, resulting in reduced protein level and enzymatic activity. Our results describe a new clinical entity and expand the clinical and mutational spectrum of SARS1 and aaRS deficiencies.

Is impaired energy production a novel insight into the pathogenesis of pyridoxine-dependent epilepsy due to biallelic variants in ALDH7A1?

BACKGROUND: Pyridoxine-dependent epilepsy (PDE) is due to biallelic variants in ALDH7A1 (PDE-ALDH7A1). ALDH7A1 encodes α-aminoadipic semialdehyde dehydrogenase in lysine catabolism. We investigated the gamma aminobutyric acid (GABA) metabolism and energy production pathways in human PDE-ALDH7A1 and its knock-out aldh7a1 zebrafish model. METHODS: We measured GABA pathway, and tricarboxylic acid cycle metabolites and electron transport chain activities in patients with PDE-ALDH7A1 and in knock-out aldh7a1 zebrafish. RESULTS: We report results of three patients with PDE-ALDH7A1: low paired complex I+II and complex II+III and individual complex IV activities in muscle biopsy in patient 1 (likely more severe phenotype); significantly elevated CSF glutamate in the GABA pathway and elevated CSF citrate, succinate, isocitrate and α-ketoglutarate in the TCA cycle in patient 3 (likely more severe phenotype); and normal CSF GABA pathway and TCA cycle metabolites on long-term pyridoxine therapy in patient 2 (likely milder phenotype). All GABA pathway metabolites (γ-hydroxybutyrate, glutamine, glutamate, total GABA, succinic semialdehyde) and TCA cycle metabolites (citrate, malate, fumarate, isocitrate, lactate) were significantly low in the homozygous knock-out aldh7a1 zebrafish compared to the wildtype zebrafish. Homozygous knock-out aldh7a1 zebrafish had decreased electron transport chain enzyme activities compared to wildtype zebrafish. DISCUSSION: We report impaired electron transport chain function, accumulation of glutamate in the central nervous system and TCA cycle dysfunction in human PDE-ALDH7A1 and abnormal GABA pathway, TCA cycle and electron transport chain in knock-out aldh7a1 zebrafish. Central nervous system glutamate toxicity and impaired energy production may play important roles in the disease neuropathogenesis and severity in human PDE-ALDH7A1.

Expanded phenotype of AARS1-related white matter disease.

PURPOSE: Recent reports of individuals with cytoplasmic transfer RNA (tRNA) synthetase-related disorders have identified cases with phenotypic variability from the index presentations. We sought to assess phenotypic variability in individuals with AARS1-related disease. METHODS: A cross-sectional survey was performed on individuals with biallelic variants in AARS1. Clinical data, neuroimaging, and genetic testing results were reviewed. Alanyl tRNA synthetase (AlaRS) activity was measured in available fibroblasts. RESULTS: We identified 11 affected individuals. Two phenotypic presentations emerged, one with early infantile-onset disease resembling the index cases of AARS1-related epileptic encephalopathy with deficient myelination (n = 7). The second (n = 4) was a later-onset disorder, where disease onset occurred after the first year of life and was characterized on neuroimaging by a progressive posterior predominant leukoencephalopathy evolving to include the frontal white matter. AlaRS activity was significantly reduced in five affected individuals with both early infantile-onset and late-onset phenotypes. CONCLUSION: We suggest that variants in AARS1 result in a broader clinical spectrum than previously appreciated. The predominant form results in early infantile-onset disease with epileptic encephalopathy and deficient myelination. However, a subgroup of affected individuals manifests with late-onset disease and similarly rapid progressive clinical decline. Longitudinal imaging and clinical follow-up will be valuable in understanding factors affecting disease progression and outcome.

Treatment of ARS deficiencies with specific amino acids.

PURPOSE: Recessive cytosolic aminoacyl-tRNA synthetase (ARS) deficiencies are severe multiorgan diseases, with limited treatment options. By loading transfer RNAs (tRNAs) with their cognate amino acids, ARS are essential for protein translation. However, it remains unknown why ARS deficiencies lead to specific symptoms, especially early life and during infections. We set out to increase pathophysiological insight and improve therapeutic possibilities. METHODS: In fibroblasts from patients with isoleucyl-RS (IARS), leucyl-RS (LARS), phenylalanyl-RS-beta-subunit (FARSB), and seryl-RS (SARS) deficiencies, we investigated aminoacylation activity, thermostability, and sensitivity to ARS-specific amino acid concentrations, and developed personalized treatments. RESULTS: Aminoacylation activity was reduced in all patients, and further diminished at 38.5/40 °C (PLARS and PFARSB), consistent with infectious deteriorations. With lower cognate amino acid concentrations, patient fibroblast growth was severely affected. To prevent local and/or temporal deficiencies, we treated patients with corresponding amino acids (follow-up: 1/2-2 2/3rd years), and intensified treatment during infections. All patients showed beneficial treatment effects, most strikingly in growth (without tube feeding), head circumference, development, coping with infections, and oxygen dependency. CONCLUSION: For these four ARS deficiencies, we observed a common disease mechanism of episodic insufficient aminoacylation to meet translational demands and illustrate the power of amino acid supplementation for the expanding ARS patient group. Moreover, we provide a strategy for personalized preclinical functional evaluation.