Marcus J. Miller

Untargeted metabolomic analysis for the clinical screening of inborn errors of metabolism

Global metabolic profiling currently achievable by untargeted mass spectrometry-based metabolomic platforms has great potential to advance our understanding of human disease states, including potential utility in the detection of novel and known inborn errors of metabolism (IEMs). There are few studies of the technical reproducibility, data analysis methods, and overall diagnostic capabilities when this technology is applied to clinical specimens for the diagnosis of IEMs. We explored the clinical utility of a metabolomic workflow capable of routinely generating semi-quantitative z-score values for ~900 unique compounds, including ~500 named human analytes, in a single analysis of human plasma. We tested the technical reproducibility of this platform and applied it to the retrospective diagnosis of 190 individual plasma samples, 120 of which were collected from patients with a confirmed IEM. Our results demonstrate high intra-assay precision and linear detection for the majority compounds tested. Individual metabolomic profiles provided excellent sensitivity and specificity for the detection of a wide range of metabolic disorders and identified novel biomarkers for some diseases. With this platform, it is possible to use one test to screen for dozens of IEMs that might otherwise require ordering multiple unique biochemical tests. However, this test may yield false negative results for certain disorders that would be detected by a more well-established quantitative test and in its current state should be considered a supplementary test. Our findings describe a novel approach to metabolomic analysis of clinical specimens and demonstrate the clinical utility of this technology for prospective screening of IEMs.

Use of Exome Sequencing for Infants in Intensive Care Units: Ascertainment of Severe Single-Gene Disorders and Effect on Medical Management

Importance While congenital malformations and genetic diseases are a leading cause of early infant death, to our knowledge, the contribution of single-gene disorders in this group is undetermined. Objective To determine the diagnostic yield and use of clinical exome sequencing in critically ill infants. Design, Setting, and Participants Clinical exome sequencing was performed for 278 unrelated infants within the first 100 days of life who were admitted to Texas Children’s Hospital in Houston, Texas, during a 5-year period between December 2011 and January 2017. Exome sequencing types included proband exome, trio exome, and critical trio exome, a rapid genomic assay for seriously ill infants. Main Outcomes and Measures Indications for testing, diagnostic yield of clinical exome sequencing, turnaround time, molecular findings, patient age at diagnosis, and effect on medical management among a group of critically ill infants who were suspected to have genetic disorders. Results The mean (SEM) age for infants participating in the study was 28.5 (1.7) days; of these, the mean (SEM) age was 29.0 (2.2) days for infants undergoing proband exome sequencing, 31.5 (3.9) days for trio exome, and 22.7 (3.9) days for critical trio exome. Clinical indications for exome sequencing included a range of medical concerns. Overall, a molecular diagnosis was achieved in 102 infants (36.7%) by clinical exome sequencing, with relatively low yield for cardiovascular abnormalities. The diagnosis affected medical management for 53 infants (52.0%) and had a substantial effect on informed redirection of care, initiation of new subspecialist care, medication/dietary modifications, and furthering life-saving procedures in select patients. Critical trio exome sequencing revealed a molecular diagnosis in 32 of 63 infants (50.8%) at a mean (SEM) of 33.1 (5.6) days of life with a mean (SEM) turnaround time of 13.0 (0.4) days. Clinical care was altered by the diagnosis in 23 of 32 patients (71.9%). The diagnostic yield, patient age at diagnosis, and medical effect in the group that underwent critical trio exome sequencing were significantly different compared with the group who underwent regular exome testing. For deceased infants (n = 81), genetic disorders were molecularly diagnosed in 39 (48.1%) by exome sequencing, with implications for recurrence risk counseling. Conclusions and Relevance Exome sequencing is a powerful tool for the diagnostic evaluation of critically ill infants with suspected monogenic disorders in the neonatal and pediatric intensive care units and its use has a notable effect on clinical decision making.

Chronic Oral l-Carnitine Supplementation Drives Marked Plasma TMAO Elevations in Patients with Organic Acidemias Despite Dietary Meat Restrictions

Recent studies have implicated trimethylamine N-oxide (TMAO) in atherosclerosis, raising concern about L-carnitine, a common supplement for patients with inborn errors of metabolism (IEMs) and a TMAO precursor metabolized, in part, by intestinal microbes. Dietary meat restriction attenuates carnitine-to-TMAO conversion, suggesting that TMAO production may not occur in meat-restricted individuals taking supplemental L-carnitine, but this has not been tested. Here, we mine a metabolomic dataset to assess TMAO levels in patients with diverse IEMs, including organic acidemias. These data were correlated with clinical information and confirmed using a quantitative TMAO assay. Marked plasma TMAO elevations were detected in patients treated with supplemental L-carnitine, including those on a meat-free diet. On average, patients with an organic acidemia had ~45-fold elevated [TMAO], as compared to the reference population. This effect was mitigated by metronidazole therapy lasting 7 days each month. Collectively, our data show that TMAO production occurs at high levels in patients with IEMs receiving oral L-carnitine. Further studies are needed to determine the long-term safety and efficacy of chronic oral L-carnitine supplementation and whether suppression or circumvention of intestinal bacteria may improve L-carnitine therapy.

Diagnosis of adenylosuccinate lyase deficiency by metabolomic profiling in plasma reveals a phenotypic spectrum.

Adenylosuccinate lyase (ADSL) deficiency is a rare autosomal recessive neurometabolic disorder that presents with a broad-spectrum of neurological and physiological symptoms. The ADSL gene produces an enzyme with binary molecular roles in de novo purine synthesis and purine nucleotide recycling. The biochemical phenotype of ADSL deficiency, accumulation of SAICAr and succinyladenosine (S-Ado) in biofluids of affected individuals, serves as the traditional target for diagnosis with targeted quantitative urine purine analysis employed as the predominate method of detection. In this study, we report the diagnosis of ADSL deficiency using an alternative method, untargeted metabolomic profiling, an analytical scheme capable of generating semi-quantitative z-score values for over 1000 unique compounds in a single analysis of a specimen. Using this method to analyze plasma, we diagnosed ADSL deficiency in four patients and confirmed these findings with targeted quantitative biochemical analysis and molecular genetic testing. ADSL deficiency is part of a large a group of neurometabolic disorders, with a wide range of severity and sharing a broad differential diagnosis. This phenotypic similarity among these many inborn errors of metabolism (IEMs) has classically stood as a hurdle in their initial diagnosis and subsequent treatment. The findings presented here demonstrate the clinical utility of metabolomic profiling in the diagnosis of ADSL deficiency and highlights the potential of this technology in the diagnostic evaluation of individuals with neurologic phenotypes.

Recurrent ACADVL molecular findings in individuals with a positive newborn screen for very long chain acyl-coA dehydrogenase (VLCAD) deficiency in the United States

Very long chain acyl-coA dehydrogenase deficiency (VLCADD) is an autosomal recessive inborn error of fatty acid oxidation detected by newborn screening (NBS). Follow-up molecular analyses are often required to clarify VLCADD-suggestive NBS results, but to date the outcome of these studies are not well described for the general screen-positive population. In the following study, we report the molecular findings for 693 unrelated patients that sequentially received Sanger sequence analysis of ACADVL as a result of a positive NBS for VLCADD. Highlighting the variable molecular underpinnings of this disorder, we identified 94 different pathogenic ACADVL variants (40 novel), as well as 134 variants of unknown clinical significance (VUSs). Evidence for the pathogenicity of a subset of recurrent VUSs was provided using multiple in silico analyses. Surprisingly, the most frequent finding in our cohort was carrier status, 57% all individuals had a single pathogenic variant or VUS. This result was further supported by follow-up array and/or acylcarnitine analysis that failed to provide evidence of a second pathogenic allele. Notably, exon-targeted array analysis of 131 individuals screen positive for VLCADD failed to identify copy number changes in ACADVL thus suggesting this test has a low yield in the setting of NBS follow-up. While no genotype was common, the c.848T>C (p.V283A) pathogenic variant was clearly the most frequent; at least one copy was found in ~10% of all individuals with a positive NBS. Clinical and biochemical data for seven unrelated patients homozygous for the p.V283A allele suggests that it results in a mild phenotype that responds well to standard treatment, but hypoglycemia can occur. Collectively, our data illustrate the molecular heterogeneity of VLCADD and provide novel insight into the outcomes of NBS for this disorder.

Elucidation of the complex metabolic profile of cerebrospinal fluid using an untargeted biochemical profiling assay

We sought to determine the molecular composition of human cerebrospinal fluid (CSF) and identify the biochemical pathways represented in CSF to understand the potential for untargeted screening of inborn errors of metabolism (IEMs). Biochemical profiles for each sample were obtained using an integrated metabolomics workflow comprised of four chromatographic techniques followed by mass spectrometry. Secondarily, we wanted to compare the biochemical profile of CSF with those of plasma and urine within the integrated mass spectrometric-based metabolomic workflow. Three sample types, CSF (N=30), urine (N=40) and EDTA plasma (N=31), were analyzed from retrospectively collected pediatric cohorts of equivalent age and gender characteristics. We identified 435 biochemicals in CSF representing numerous biological and chemical/structural families. Sixty-three percent (273 of 435) of the biochemicals detected in CSF also were detected in urine and plasma, another 32% (140 of 435) were detected in either plasma or urine, and 5% (22 of 435) were detected only in CSF. Analyses of several metabolites showed agreement between clinically useful assays and the metabolomics approach. An additional set of CSF and plasma samples collected from the same patient revealed correlation between several biochemicals detected in paired samples. Finally, analysis of CSF from a pediatric case with dihydropteridine reductase (DHPR) deficiency demonstrated the utility of untargeted global metabolic phenotyping as a broad assessment to screen samples from patients with undifferentiated phenotypes. The results indicate a single CSF sample processed with an integrated metabolomics workflow can be used to identify a large breadth of biochemicals that could be useful for identifying disrupted metabolic patterns associated with IEMs.

Analytes related to erythrocyte metabolism are reliable biomarkers for preanalytical error due to delayed plasma processing in metabolomics studies.

BACKGROUND Delaying plasma separation after phlebotomy (processing delay) can cause perturbations of numerous small molecule analytes. This poses a major challenge to the clinical application of metabolomics analyses. In this study, we further define the analyte changes that occur during processing delays and generate a model for the post hoc detection of this preanalytical error. METHODS Using an untargeted metabolomics platform we analyzed EDTA-preserved plasma specimens harvested after processing delays lasting from minutes to days. Identified biomarkers were tested on (i) a test-set of samples exposed to either minimal (n=28) or long delays (n=40) and (ii) samples collected in a clinical setting for metabolomics analysis (n=141). RESULTS A total of 149 of 803 plasma analytes changed significantly during processing delays lasting 0-20h. Biomarkers related to erythrocyte metabolism, e.g., 5-oxoproline, lactate, and an ornithine/arginine ratio, were the strongest predictors of plasma separation delays, providing 100% diagnostic accuracy in the test set. Together these biomarkers could accurately predict processing delays >2h in a pilot study and we found evidence of sample mishandling in 4 of 141 clinically derived specimens. CONCLUSIONS Our study highlights the widespread effects of processing delays and proposes that erythrocyte metabolism creates a reproducible signal that can identify mishandled specimens in metabolomics studies.

Analyses of SLC13A5-epilepsy patients reveal perturbations of TCA cycle

OBJECTIVE To interrogate the metabolic profile of five subjects from three families with rare, nonsense and missense mutations in SLC13A5 and Early Infantile Epileptic Encephalopathies (EIEE) characterized by severe, neonatal onset seizures, psychomotor retardation and global developmental delay. METHODS Mass spectrometry of plasma, CSF and urine was used to identify consistently dysregulated analytes in our subjects. RESULTS Distinctive elevations of citrate and dysregulation of citric acid cycle intermediates, supporting the hypothesis that loss of SLC13A5 function alters tricarboxylic acid cycle (TCA) metabolism and may disrupt metabolic compartmentation in the brain. SIGNIFICANCE Our results indicate that analysis of plasma citrate and other TCA analytes in SLC13A5 deficient patients define a diagnostic metabolic signature that can aid in diagnosing children with this disease.

Biallelic Variants in OTUD6B Cause an Intellectual Disability Syndrome Associated with Seizures and Dysmorphic Features

Ubiquitination is a posttranslational modification that regulates many cellular processes including protein degradation, intracellular trafficking, cell signaling, and protein-protein interactions. Deubiquitinating enzymes (DUBs), which reverse the process of ubiquitination, are important regulators of the ubiquitin system. OTUD6B encodes a member of the ovarian tumor domain (OTU)-containing subfamily of deubiquitinating enzymes. Herein, we report biallelic pathogenic variants in OTUD6B in 12 individuals from 6 independent families with an intellectual disability syndrome associated with seizures and dysmorphic features. In subjects with predicted loss-of-function alleles, additional features include global developmental delay, microcephaly, absent speech, hypotonia, growth retardation with prenatal onset, feeding difficulties, structural brain abnormalities, congenital malformations including congenital heart disease, and musculoskeletal features. Homozygous Otud6b knockout mice were subviable, smaller in size, and had congenital heart defects, consistent with the severity of loss-of-function variants in humans. Analysis of peripheral blood mononuclear cells from an affected subject showed reduced incorporation of 19S subunits into 26S proteasomes, decreased chymotrypsin-like activity, and accumulation of ubiquitin-protein conjugates. Our findings suggest a role for OTUD6B in proteasome function, establish that defective OTUD6B function underlies a multisystemic human disorder, and provide additional evidence for the emerging relationship between the ubiquitin system and human disease.

Lysinuric protein intolerance presenting with multiple fractures

Lysinuric protein intolerance (LPI) is a rare autosomal recessive inborn error of metabolism caused by mutations in SLC7A7, which encodes a component of the dibasic amino acid transporter found in intestinal and renal tubular cells. Patients typically present with vomiting, diarrhea, irritability, failure to thrive, and symptomatic hyperammonemia after protein-rich meals. Long-term complications may include pulmonary alveolar proteinosis, renal disease, and osteoporosis. We present a 5-year-old male who was followed in our skeletal dysplasia clinic for 3 years for multiple fractures, idiopathic osteoporosis, and short stature in the absence of typical features of LPI. Whole exome sequencing performed to determine the etiology of the osteoporosis and speech delay identified a nonsense mutation in SLC7A7. Chromosome microarray analysis identified a deletion involving the second allele of the same gene, and biochemical analysis supported the diagnosis of LPI. Our patients atypical presentation underscores the importance of maintaining a high index of suspicion for LPI in patients with unexplained fractures and idiopathic osteoporosis, even in the absence of clinical symptoms of hyperammonemia after protein rich meals or other systemic features of classical LPI. This case further demonstrates the utility of whole exome sequencing in diagnosis of unusual presentations of rare disorders for which early intervention may modify the clinical course.