Integrated analysis of metabolomic profiling and exome data supplements sequence variant interpretation, classification, and diagnosis.

PURPOSE: A primary barrier to improving exome sequencing diagnostic rates is the interpretation of variants of uncertain clinical significance. We aimed to determine the contribution of integrated untargeted metabolomics in the analysis of exome sequencing data by retrospective analysis of patients evaluated by both exome sequencing and untargeted metabolomics within the same clinical laboratory. METHODS: Exome sequencing and untargeted metabolomic data were collected and analyzed for 170 patients. Pathogenic variants, likely pathogenic variants, and variants of uncertain significance in genes associated with a biochemical phenotype were extracted. Metabolomic data were evaluated to determine if these variants resulted in biochemical abnormalities that could be used to support their interpretation using current American College of Genetics and Genomics (ACMG) guidelines. RESULTS: Metabolomic data contributed to the interpretation of variants in 74 individuals (43.5%) over 73 different genes. The data allowed for the reclassification of 9 variants as likely benign, 15 variants as likely pathogenic, and 3 variants as pathogenic. Metabolomic data confirmed a clinical diagnosis in 21 cases, for a diagnostic rate of 12.3% in this population. CONCLUSION: Untargeted metabolomics can serve as a useful adjunct to exome sequencing by providing valuable functional data that may not otherwise be clinically available, resulting in improved variant classification.

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.

Mutations in X-linked PORCN, a putative regulator of Wnt signaling, cause focal dermal hypoplasia.

Focal dermal hypoplasia is an X-linked dominant disorder characterized by patchy hypoplastic skin and digital, ocular and dental malformations. We used array comparative genomic hybridization to identify a 219-kb deletion in Xp11.23 in two affected females. We sequenced genes in this region and found heterozygous and mosaic mutations in PORCN in other affected females and males, respectively. PORCN encodes the human homolog of Drosophila melanogaster porcupine, an endoplasmic reticulum protein involved in secretion of Wnt proteins.

Aromatic L-amino acid decarboxylase deficiency diagnosed by clinical metabolomic profiling of plasma.

Aromatic L-amino acid decarboxylase (AADC) deficiency is an inborn error of metabolism affecting the biosynthesis of serotonin, dopamine, and catecholamines. We report a case of AADC deficiency that was detected using the Global MAPS platform. This is a novel platform that allows for parallel clinical testing of hundreds of metabolites in a single plasma specimen. It uses a state-of-the-art mass spectrometry platform, and the resulting spectra are compared against a library of ~2500 metabolites. Our patient is now a 4 year old boy initially seen at 11 months of age for developmental delay and hypotonia. Multiple tests had not yielded a diagnosis until exome sequencing revealed compound heterozygous variants of uncertain significance (VUS), c.286G>A (p.G96R) and c.260C>T (p.P87L) in the DDC gene, causal for AADC deficiency. CSF neurotransmitter analysis confirmed the diagnosis with elevated 3-methoxytyrosine (3-O-methyldopa). Metabolomic profiling was performed on plasma and revealed marked elevation in 3-methoxytyrosine (Z-score +6.1) consistent with the diagnosis of AADC deficiency. These results demonstrate that the Global MAPS platform is able to diagnose AADC deficiency from plasma. In summary, we report a novel and less invasive approach to diagnose AADC deficiency using plasma metabolomic profiling.

The development and implementation of an in-service exam for medical genetics residency programs.

PURPOSE: In-service exams are a commonly used educational tool in postgraduate medical education. Although most specialties utilize such an exam, medical genetics did not. It was decided in the spring of 2009 at the inaugural Medical Genetics Residency Program Directors (PDs) Group meeting to develop and implement such a test. METHODS: Using questions sent in from PDs, a 125-question exam was created, with 125 multiple-choice questions according to the format of the National Board of Medical Examiners. The exam covered genetics in the following areas: basic/molecular (~45 questions), cancer and adult (20), prenatal (20), biochemical (20), pediatric/dysmorphology (20). The exam was administered for the first time in February 2010, and again with modifications in 2011. RESULTS: In total, 174 trainees from 35 programs completed the exam in 2010; in 2011 the number increased to 214, representing 39 US programs, and 4 Canadian programs. For both years, most participants were medical genetics residents (106 in 2010; 127 in 2011), but a substantial number of clinical laboratory fellows also participated (68 in 2010; 85 in 2011). CONCLUSION: The development and implementation of this test were an overall success, in that in two years we were able to secure almost 100% participation from medical genetics residency programs, and that we created an infrastructure to develop and implement this exam on a yearly basis. There is need for improvement, notably in the relatively low mean score and relatively narrow spread of scores. However, we believe that, with efforts under way to improve the quality of the questions, the in-service exam will become a fundamental tool in medical genetics residency education.

Parkes Weber syndrome occurring in a family with capillary malformations.

Parkes Weber syndrome is a disorder characterized by cutaneous blush, arteriovenous fistula, and overgrowth of the affected limb. It has been differentiated from Klippel-Trenaunay syndrome on the basis of the presence of arteriovenous fistula that are always absent in the latter. We report a case of Parkes Weber syndrome with diffuse arteriovenous high flow leading to hemodynamic complications but without radiographic evidence of arteriovenous fistula. There are multiple individuals in the family with capillary malformations inherited in an autosomal dominant pattern. These observations reinforce the suggestions that Parkes Weber syndrome and capillary malformations may share a common pathogenetic pathway.

Guidelines for evaluating and managing children born with disorders of sexual development.

Children born with disorders of sexual differentiation (DSD) pose numerous challenges for the parents, family, and treating physicians. The pediatrician is usually the first medical contact for newborns with DSD or for toddlers and children who present with DSD at a later time. Several years ago, we formed a Gender Medicine Team (GMT) at Baylor College of Medicine and Texas Children's Hospital (TCH) to explore and evaluate the most appropriate management strategies, which had long been a matter of concern and contention. Subsequently, the GMT, composed of experts in the fields of endocrinology, ethics, genetics, gynecology, psychology, pediatric surgery, and urology, formed a Task Force to evaluate the information available from our own experiences and from reviews of the literature. Utilizing the Grading of Recommendation, Assessment, Development and Evaluation (GRADE) system to assess the evidence and recommendations, the Task Force developed a consensus statement for clinical management of DSD and for making appropriate sex assignments.

Allele-specific methylation of a functional CTCF binding site upstream of MEG3 in the human imprinted domain of 14q32.

The gene MEG3 is located in the imprinted human chromosomal region on 14q32. Imprinting of a structurally homologous region IGF2/H19 on 11p15 is mediated through cytosine methylation-controlled binding of the protein CTCF to target sites upstream of H19. We identified five new CTCF binding sites around the promoter of MEG3. Using an electrophoretic mobility shift assay, we showed that these sites bind CTCF in vitro. Using one of these sites, chromatin immunoprecipitation (ChIP) analysis confirmed CTCF binding in-vivo, and differential allele-specific methylation was demonstrated in seven individuals with either maternal or paternal uniparental disomy 14 (UPD14). The site was unmethylated on the maternally inherited chromosomes 14 and methylated on the paternally inherited chromosomes 14, suggesting parent-specific methylation of sequences upstream of MEG3. We speculate that this CTCF-binding region may provide a mechanism for the transcriptional regulation of MEG3 and DLK1.

A mouse model of argininosuccinic aciduria: biochemical characterization.

Argininosuccinate lyase (AL) has several roles in intermediary metabolism. It is an essential component of the urea cycle, providing a pathway for the disposal of excess nitrogen in mammals. AL links the urea cycle to the tricarboxylic acid (TCA) cycle by generating fumarate. Finally, AL is required for the endogenous production of arginine. In this latter role it may function outside ureagenic organs to provide arginine as a substrate for nitric oxide synthases (NOS). Increasing evidence suggests that argininosuccinate synthetase (AS) and AL are more globally expressed, and the coordinate regulation of AS and AL gene expression with that of the inducible form of NOS (iNOS) provides evidence that this may facilitate the regulation of NOS activity. Deficiency of AL leads to the human urea cycle disorder argininosuccinic aciduria. We produced an AL deficient mouse by gene targeting in order to investigate the role of AL in endogenous arginine production. This mouse also provides a model of the human disorder to explore the pathogenesis of the disorder and possible new treatments. Metabolic studies of these mice demonstrated that they have the same biochemical phenotype as humans, with hyperammonemia, elevated plasma argininosuccinic acid and low plasma arginine. Plasma nitrites, derived from NO, were not reduced in AL deficient mice and there was no significant difference is the level of cyclic GMP, the second messenger induced by NO.