Compound heterozygosity for loss-of-function GARS variants results in a multisystem developmental syndrome that includes severe growth retardation.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes that ligate amino acids onto tRNA molecules. Genes encoding ARSs have been implicated in myriad dominant and recessive disease phenotypes. Glycyl-tRNA synthetase (GARS) is a bifunctional ARS that charges tRNAGly in the cytoplasm and mitochondria. GARS variants have been associated with dominant Charcot-Marie-Tooth disease but have not been convincingly implicated in recessive phenotypes. Here, we describe a patient from the NIH Undiagnosed Diseases Program with a multisystem, developmental phenotype. Whole-exome sequence analysis revealed that the patient is compound heterozygous for one frameshift (p.Glu83Ilefs*6) and one missense (p.Arg310Gln) GARS variant. Using in vitro and in vivo functional studies, we show that both GARS variants cause a loss-of-function effect: the frameshift variant results in depleted protein levels and the missense variant reduces GARS tRNA charging activity. In support of GARS variant pathogenicity, our patient shows striking phenotypic overlap with other patients having ARS-related recessive diseases, including features associated with variants in both cytoplasmic and mitochondrial ARSs; this observation is consistent with the essential function of GARS in both cellular locations. In summary, our clinical, genetic, and functional analyses expand the phenotypic spectrum associated with GARS variants.

Defining Disease, Diagnosis, and Translational Medicine within a Homeostatic Perturbation Paradigm: The National Institutes of Health Undiagnosed Diseases Program Experience.

Traditionally, the use of genomic information for personalized medical decisions relies on prior discovery and validation of genotype-phenotype associations. This approach constrains care for patients presenting with undescribed problems. The National Institutes of Health (NIH) Undiagnosed Diseases Program (UDP) hypothesized that defining disease as maladaptation to an ecological niche allows delineation of a logical framework to diagnose and evaluate such patients. Herein, we present the philosophical bases, methodologies, and processes implemented by the NIH UDP. The NIH UDP incorporated use of the Human Phenotype Ontology, developed a genomic alignment strategy cognizant of parental genotypes, pursued agnostic biochemical analyses, implemented functional validation, and established virtual villages of global experts. This systematic approach provided a foundation for the diagnostic or non-diagnostic answers provided to patients and serves as a paradigm for scalable translational research.

Abnormal glycosylation in Joubert syndrome type 10.

BACKGROUND: The discovery of disease pathogenesis requires systematic agnostic screening of multiple homeostatic processes that may become deregulated. We illustrate this principle in the evaluation and diagnosis of a 5-year-old boy with Joubert syndrome type 10 (JBTS10). He carried the OFD1 mutation p.Gln886Lysfs*2 (NM_003611.2: c.2656del) and manifested features of Joubert syndrome. METHODS: We integrated exome sequencing, MALDI-TOF mass spectrometry analyses of plasma and cultured dermal fibroblasts glycomes, and full clinical evaluation of the proband. Analyses of cilia formation and lectin staining were performed by immunofluorescence. Measurement of cellular nucleotide sugar levels was performed with high-performance anion-exchange chromatography with pulsed amperometric detection. Statistical analyses utilized the Student's and Fisher's exact t tests. RESULTS: Glycome analyses of plasma and cultured dermal fibroblasts identified abnormal N- and O-linked glycosylation profiles. These findings replicated in two unrelated males with OFD1 mutations. Cultured fibroblasts from affected individuals had a defect in ciliogenesis. The proband's fibroblasts also had an abnormally elevated nuclear sialylation signature and increased total cellular levels of CMP-sialic acid. Ciliogenesis and each glycosylation anomaly were rescued by expression of wild-type OFD1. CONCLUSIONS: The rescue of ciliogenesis and glycosylation upon reintroduction of WT OFD1 suggests that both contribute to the pathogenesis of JBTS10.

Neurologic involvement in patients with atypical Chediak-Higashi disease.

OBJECTIVE: To delineate the developmental and progressive neurodegenerative features in 9 young adults with the atypical form of Chediak-Higashi disease (CHD) enrolled in a natural history study. METHODS: Patients with atypical clinical features, but diagnostically confirmed CHD by standard evaluation of blood smears and molecular genotyping, underwent complete neurologic evaluation, MRI of the brain, electrophysiologic examination, and neuropsychological testing. Fibroblasts were collected to investigate the cellular phenotype and correlation with the clinical presentation. RESULTS: In 9 mildly affected patients with CHD, we documented learning and behavioral difficulties along with developmental structural abnormalities of the cerebellum and posterior fossa, which are apparent early in childhood. A range of progressive neurologic problems emerge in early adulthood, including cerebellar deficits, polyneuropathies, spasticity, cognitive decline, and parkinsonism. CONCLUSIONS: Patients with undiagnosed atypical CHD manifesting some of these wide-ranging yet nonspecific neurologic complaints may reside in general and specialty neurology clinics. The absence of the typical bleeding or infectious diathesis in mildly affected patients with CHD renders them difficult to diagnose. Identification of these individuals is important not only for close surveillance of potential CHD-related systemic complications but also for a full understanding of the natural history of CHD and the potential role of the disease-causing protein, LYST, to the pathophysiology of other neurodevelopmental and neurodegenerative disorders.

Novel Biomarkers of Human GM1 Gangliosidosis Reflect the Clinical Efficacy of Gene Therapy in a Feline Model.

GM1 gangliosidosis is a fatal neurodegenerative disease that affects individuals of all ages. Favorable outcomes using adeno-associated viral (AAV) gene therapy in GM1 mice and cats have prompted consideration of human clinical trials, yet there remains a paucity of objective biomarkers to track disease status. We developed a panel of biomarkers using blood, urine, cerebrospinal fluid (CSF), electrodiagnostics, 7 T MRI, and magnetic resonance spectroscopy in GM1 cats-either untreated or AAV treated for more than 5 years-and compared them to markers in human GM1 patients where possible. Significant alterations were noted in CSF and blood of GM1 humans and cats, with partial or full normalization after gene therapy in cats. Gene therapy improved the rhythmic slowing of electroencephalograms (EEGs) in GM1 cats, a phenomenon present also in GM1 patients, but nonetheless the epileptiform activity persisted. After gene therapy, MR-based analyses revealed remarkable preservation of brain architecture and correction of brain metabolites associated with microgliosis, neuroaxonal loss, and demyelination. Therapeutic benefit of AAV gene therapy in GM1 cats, many of which maintain near-normal function >5 years post-treatment, supports the strong consideration of human clinical trials, for which the biomarkers described herein will be essential for outcome assessment.

Neurologic involvement in patients with atypical Chediak-Higashi disease.

OBJECTIVE: To delineate the developmental and progressive neurodegenerative features in 9 young adults with the atypical form of Chediak-Higashi disease (CHD) enrolled in a natural history study. METHODS: Patients with atypical clinical features, but diagnostically confirmed CHD by standard evaluation of blood smears and molecular genotyping, underwent complete neurologic evaluation, MRI of the brain, electrophysiologic examination, and neuropsychological testing. Fibroblasts were collected to investigate the cellular phenotype and correlation with the clinical presentation. RESULTS: In 9 mildly affected patients with CHD, we documented learning and behavioral difficulties along with developmental structural abnormalities of the cerebellum and posterior fossa, which are apparent early in childhood. A range of progressive neurologic problems emerge in early adulthood, including cerebellar deficits, polyneuropathies, spasticity, cognitive decline, and parkinsonism. CONCLUSIONS: Patients with undiagnosed atypical CHD manifesting some of these wide-ranging yet nonspecific neurologic complaints may reside in general and specialty neurology clinics. The absence of the typical bleeding or infectious diathesis in mildly affected patients with CHD renders them difficult to diagnose. Identification of these individuals is important not only for close surveillance of potential CHD-related systemic complications but also for a full understanding of the natural history of CHD and the potential role of the disease-causing protein, LYST, to the pathophysiology of other neurodevelopmental and neurodegenerative disorders.

A novel AVPR2 splice site mutation leads to partial X-linked nephrogenic diabetes insipidus in two brothers.

UNLABELLED: X-linked nephrogenic diabetes insipidus (NDI, OMIM#304800) is caused by mutations in the arginine vasopressin (AVP, OMIM*192340) receptor type 2 (AVPR2, OMIM*300538) gene. A 20-month-old boy and his 8-year-old brother presented with polyuria, polydipsia, and failure to thrive. Both boys demonstrated partial DDAVP (1-desamino-8-D AVP or desmopressin) responses; thus, NDI diagnosis was delayed. While routine sequencing of AVPR2 showed a potential splice site variant, it was not until exome sequencing confirmed the AVPR2 splice site variant and did not reveal any more likely candidates that the patients' diagnosis was made and proper treatment was instituted. Both patients were hemizygous for two AVPR2 variants predicted in silico to affect AVPR2 messenger RNA (mRNA) splicing. A minigene assay revealed that the novel AVPR2 c.276A>G mutation creates a novel splice acceptor site leading to 5' truncation of AVPR2 exon 2 in HEK293 human kidney cells. Both patients have been treated with high-dose DDAVP with a remarkable improvement of their symptoms and accelerated linear growth and weight gain. CONCLUSION: We present here a unique case of partial X-linked NDI due to an AVPR2 splice site mutation; patients with diabetes insipidus of unknown etiology may harbor splice site mutations that are initially underestimated in their pathogenicity on sequence analysis. WHAT IS KNOWN: • X-linked nephrogenic diabetes insipidus is caused by AVPR2 mutations, and disease severity can vary depending on the functional effect of the mutation. What is New: • We demonstrate here that a splice site mutation in AVPR2 leads to partial X-linked NDI in two brothers. • Treatment with high-dose DDAVP led to improvement of polyuria and polydipsia, weight gain, and growth.

The phenotype of the musculocontractural type of Ehlers-Danlos syndrome due to CHST14 mutations.

The musculocontractural type of Ehlers-Danlos syndrome (MC-EDS) has been recently recognized as a clinical entity. MC-EDS represents a differential diagnosis within the congenital neuromuscular and connective tissue disorders spectrum. Thirty-one and three patients have been reported with MC-EDS so far with bi-allelic mutations identified in CHST14 and DSE, respectively, encoding two enzymes necessary for dermatan sulfate (DS) biosynthesis. We report seven additional patients with MC-EDS from four unrelated families, including the follow-up of a sib-pair originally reported with the kyphoscoliotic type of EDS in 1975. Brachycephaly, a characteristic facial appearance, an asthenic build, hyperextensible and bruisable skin, tapering fingers, instability of large joints, and recurrent formation of large subcutaneous hematomas are always present. Three of seven patients had mildly elevated serum creatine kinase. The oldest patient was blind due to retinal detachment at 45 years and died at 59 years from intracranial bleeding; her affected brother died at 28 years from fulminant endocarditis. All patients in this series harbored homozygous, predicted loss-of-function CHST14 mutations. Indeed, DS was not detectable in fibroblasts from two unrelated patients with homozygous mutations. Patient fibroblasts produced higher amounts of chondroitin sulfate, showed intracellular retention of collagen types I and III, and lacked decorin and thrombospondin fibrils compared with control. A great proportion of collagen fibrils were not integrated into fibers, and fiber bundles were dispersed into the ground substance in one patient, all of which is likely to contribute to the clinical phenotype. This report should increase awareness for MC-EDS.

Computational evaluation of exome sequence data using human and model organism phenotypes improves diagnostic efficiency.

PURPOSE: Medical diagnosis and molecular or biochemical confirmation typically rely on the knowledge of the clinician. Although this is very difficult in extremely rare diseases, we hypothesized that the recording of patient phenotypes in Human Phenotype Ontology (HPO) terms and computationally ranking putative disease-associated sequence variants improves diagnosis, particularly for patients with atypical clinical profiles. METHODS: Using simulated exomes and the National Institutes of Health Undiagnosed Diseases Program (UDP) patient cohort and associated exome sequence, we tested our hypothesis using Exomiser. Exomiser ranks candidate variants based on patient phenotype similarity to (i) known disease-gene phenotypes, (ii) model organism phenotypes of candidate orthologs, and (iii) phenotypes of protein-protein association neighbors. RESULTS: Benchmarking showed Exomiser ranked the causal variant as the top hit in 97% of known disease-gene associations and ranked the correct seeded variant in up to 87% when detectable disease-gene associations were unavailable. Using UDP data, Exomiser ranked the causative variant(s) within the top 10 variants for 11 previously diagnosed variants and achieved a diagnosis for 4 of 23 cases undiagnosed by clinical evaluation. CONCLUSION: Structured phenotyping of patients and computational analysis are effective adjuncts for diagnosing patients with genetic disorders.Genet Med 18 6, 608-617.

MRI/MRS as a surrogate marker for clinical progression in GM1 gangliosidosis.

Background GM1 gangliosidosis is a lysosomal storage disorder caused by mutations in GLB1, encoding ß-galactosidase. The range of severity is from type I infantile disease, lethal in early childhood, to type III adult onset, resulting in gradually progressive neurological symptoms in adulthood. The intermediate group of patients has been recently classified as having type II late infantile subtype with onset of symptoms at one to three years of age or type II juvenile subtype with symptom onset at 2-10 years. To characterize disease severity and progression, six Late infantile and nine juvenile patients were evaluated using magnetic resonance imaging (MRI), and MR spectroscopy (MRS). Since difficulties with ambulation (gross motor function) and speech (expressive language) are often the first reported symptoms in type II GM1, patients were also scored in these domains. Deterioration of expressive language and ambulation was more rapid in the late infantile patients. Fourteen MRI scans in six Late infantile patients identified progressive atrophy in the cerebrum and cerebellum. Twenty-six MRI scans in nine juvenile patients revealed greater variability in extent and progression of atrophy. Quantitative MRS demonstrated a deficit of N-acetylaspartate in both the late infantile and juvenile patients with greater in the late infantile patients. This correlates with clinical measures of ambulation and expressive language. The two subtypes of type II GM1 gangliosidosis have different clinical trajectories. MRI scoring, quantitative MRS and brain volume correlate with clinical disease progression and may serve as important minimally-invasive outcome measures for clinical trials.

Expanding the clinical and molecular characteristics of PIGT-CDG, a disorder of glycosylphosphatidylinositol anchors.

PIGT-CDG, an autosomal recessive syndromic intellectual disability disorder of glycosylphosphatidylinositol (GPI) anchors, was recently described in two independent kindreds [Multiple Congenital Anomalies-Hypotonia-Seizures Syndrome 3 (OMIM, #615398)]. PIGT encodes phosphatidylinositol-glycan biosynthesis class T, a subunit of the heteropentameric transamidase complex that facilitates the transfer of GPI to proteins. GPI facilitates attachment (anchoring) of proteins to cell membranes. We describe, at ages 7 and 6 years, two children of non-consanguineous parents; they had hypotonia, severe global developmental delay, and intractable seizures along with endocrine, ophthalmologic, skeletal, hearing, and cardiac anomalies. Exome sequencing revealed that both siblings had compound heterozygous variants in PIGT (NM_015937.5), i.e., c.918dupC, a novel duplication leading to a frameshift, and c.1342C > T encoding a previously described missense variant. Flow cytometry studies showed decreased surface expression of GPI-anchored proteins on granulocytes, consistent with findings in previous cases. These siblings further delineate the clinical spectrum of PIGT-CDG, reemphasize the neuro-ophthalmologic presentation, clarify the endocrine features, and add hypermobility, low CSF albumin quotient, and hearing loss to the phenotypic spectrum. Our results emphasize that GPI anchor-related congenital disorders of glycosylation (CDGs) should be considered in subjects with early onset severe seizure disorders and dysmorphic facial features, even in the presence of a normal carbohydrate-deficient transferrin pattern and N-glycan profiling. Currently available screening for CDGs will not reliably detect this family of disorders, and our case reaffirms that the use of flow cytometry and genetic testing is essential for diagnosis in this group of disorders.

MED23-associated intellectual disability in a non-consanguineous family.

Intellectual disability (ID) is a heterogeneous condition arising from a variety of environmental and genetic factors. Among these causes are defects in transcriptional regulators. Herein, we report on two brothers in a nonconsanguineous family with novel compound heterozygous, disease-segregating mutations (NM_015979.3: [3656A > G];[4006C > T], NP_057063.2: [H1219R];[R1336X]) in MED23. This gene encodes a subunit of the Mediator complex that modulates the expression of RNA polymerase II-dependent genes. These brothers, who had profound ID, spasticity, congenital heart disease, brain abnormalities, and atypical electroencephalography, represent the first case of MED23-associated ID in a non-consanguineous family. They also expand upon the clinical features previously reported for mutations in this gene.

Loss-of-function alanyl-tRNA synthetase mutations cause an autosomal-recessive early-onset epileptic encephalopathy with persistent myelination defect.

Mutations in genes encoding aminoacyl-tRNA synthetases are known to cause leukodystrophies and genetic leukoencephalopathies-heritable disorders that result in white matter abnormalities in the central nervous system. Here we report three individuals (two siblings and an unrelated individual) with severe infantile epileptic encephalopathy, clubfoot, absent deep tendon reflexes, extrapyramidal symptoms, and persistently deficient myelination on MRI. Analysis by whole exome sequencing identified mutations in the nuclear-encoded alanyl-tRNA synthetase (AARS) in these two unrelated families: the two affected siblings are compound heterozygous for p.Lys81Thr and p.Arg751Gly AARS, and the single affected child is homozygous for p.Arg751Gly AARS. The two identified mutations were found to result in a significant reduction in function. Mutations in AARS were previously associated with an autosomal-dominant inherited form of axonal neuropathy, Charcot-Marie-Tooth disease type 2N (CMT2N). The autosomal-recessive AARS mutations identified in the individuals described here, however, cause a severe infantile epileptic encephalopathy with a central myelin defect and peripheral neuropathy, demonstrating that defects of alanyl-tRNA charging can result in a wide spectrum of disease manifestations.

The implications of familial incidental findings from exome sequencing: the NIH Undiagnosed Diseases Program experience.

PURPOSE: Using exome sequence data from 159 families participating in the National Institutes of Health Undiagnosed Diseases Program, we evaluated the number and inheritance mode of reportable incidental sequence variants. METHODS: Following the American College of Medical Genetics and Genomics recommendations for reporting of incidental findings from next-generation sequencing, we extracted variants in 56 genes from the exome sequence data of 543 subjects and determined the reportable incidental findings for each participant. We also defined variant status as inherited or de novo for those with available parental sequence data. RESULTS: We identified 14 independent reportable variants in 159 (8.8%) families. For nine families with parental sequence data in our cohort, a parent transmitted the variant to one or more children (nine minor children and four adult children). The remaining five variants occurred in adults for whom parental sequences were unavailable. CONCLUSION: Our results are consistent with the expectation that a small percentage of exomes will result in identification of an incidental finding under the American College of Medical Genetics and Genomics recommendations. Additionally, our analysis of family sequence data highlights that genome and exome sequencing of families has unavoidable implications for immediate family members and therefore requires appropriate counseling for the family.

GRIN2A mutation and early-onset epileptic encephalopathy: personalized therapy with memantine.

OBJECTIVE: Early-onset epileptic encephalopathies have been associated with de novo mutations of numerous ion channel genes. We employed techniques of modern translational medicine to identify a disease-causing mutation, analyze its altered behavior, and screen for therapeutic compounds to treat the proband. METHODS: Three modern translational medicine tools were utilized: 1) high-throughput sequencing technology to identify a novel de novo mutation; 2) in vitro expression and electrophysiology assays to confirm the variant protein's dysfunction; and 3) screening of existing drug libraries to identify potential therapeutic compounds. RESULTS: A de novo GRIN2A missense mutation (c.2434C>A; p.L812M) increased the charge transfer mediated by NMDA receptors containing the mutant GluN2A-L812M subunit. In vitro analysis with NMDA receptor blockers indicated that GLuN2A-L812M-containing NMDARs retained their sensitivity to the use-dependent channel blocker memantine; while screening of a previously reported GRIN2A mutation (N615K) with these compounds produced contrasting results. Consistent with these data, adjunct memantine therapy reduced our proband's seizure burden. INTERPRETATION: This case exemplifies the potential for personalized genomics and therapeutics to be utilized for the early diagnosis and treatment of infantile-onset neurological disease.

Three rare diseases in one Sib pair: RAI1, PCK1, GRIN2B mutations associated with Smith-Magenis Syndrome, cytosolic PEPCK deficiency and NMDA receptor glutamate insensitivity.

The National Institutes of Health Undiagnosed Diseases Program evaluates patients for whom no diagnosis has been discovered despite a comprehensive diagnostic workup. Failure to diagnose a condition may arise from the mutation of genes previously unassociated with disease. However, we hypothesized that this could also co-occur with multiple genetic disorders. Demonstrating a complex syndrome caused by multiple disorders, we report two siblings manifesting both similar and disparate signs and symptoms. They shared a history of episodes of hypoglycemia and lactic acidosis, but had differing exam findings and developmental courses. Clinical acumen and exome sequencing combined with biochemical and functional studies identified three genetic conditions. One sibling had Smith-Magenis Syndrome and a nonsense mutation in the RAI1 gene. The second sibling had a de novo mutation in GRIN2B, which resulted in markedly reduced glutamate potency of the encoded receptor. Both siblings had a protein-destabilizing homozygous mutation in PCK1, which encodes the cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK-C). In summary, we present the first clinically-characterized mutation of PCK1 and demonstrate that complex medical disorders can represent the co-occurrence of multiple diseases.

Functional analysis of a de novo GRIN2A missense mutation associated with early-onset epileptic encephalopathy.

NMDA receptors (NMDARs), ligand-gated ion channels, play important roles in various neurological disorders, including epilepsy. Here we show the functional analysis of a de novo missense mutation (L812M) in a gene encoding NMDAR subunit GluN2A (GRIN2A). The mutation, identified in a patient with early-onset epileptic encephalopathy and profound developmental delay, is located in the linker region between the ligand-binding and transmembrane domains. Electrophysiological recordings revealed that the mutation enhances agonist potency, decreases sensitivity to negative modulators including magnesium, protons and zinc, prolongs the synaptic response time course and increases single-channel open probability. The functional changes of this amino acid apply to all other NMDAR subunits, suggesting an important role of this residue on the function of NMDARs. Taken together, these data suggest that the L812M mutation causes overactivation of NMDARs and drives neuronal hyperexcitability. We hypothesize that this mechanism underlies the patient's epileptic phenotype as well as cerebral atrophy.

Multilineage somatic activating mutations in HRAS and NRAS cause mosaic cutaneous and skeletal lesions, elevated FGF23 and hypophosphatemia.

Pathologically elevated serum levels of fibroblast growth factor-23 (FGF23), a bone-derived hormone that regulates phosphorus homeostasis, result in renal phosphate wasting and lead to rickets or osteomalacia. Rarely, elevated serum FGF23 levels are found in association with mosaic cutaneous disorders that affect large proportions of the skin and appear in patterns corresponding to the migration of ectodermal progenitors. The cause and source of elevated serum FGF23 is unknown. In those conditions, such as epidermal and large congenital melanocytic nevi, skin lesions are variably associated with other abnormalities in the eye, brain and vasculature. The wide distribution of involved tissues and the appearance of multiple segmental skin and bone lesions suggest that these conditions result from early embryonic somatic mutations. We report five such cases with elevated serum FGF23 and bone lesions, four with large epidermal nevi and one with a giant congenital melanocytic nevus. Exome sequencing of blood and affected skin tissue identified somatic activating mutations of HRAS or NRAS in each case without recurrent secondary mutation, and we further found that the same mutation is present in dysplastic bone. Our finding of somatic activating RAS mutation in bone, the endogenous source of FGF23, provides the first evidence that elevated serum FGF23 levels, hypophosphatemia and osteomalacia are associated with pathologic Ras activation and may provide insight in the heretofore limited understanding of the regulation of FGF23.

1,25-(OH)2D-24 Hydroxylase (CYP24A1) Deficiency as a Cause of Nephrolithiasis.

BACKGROUND AND OBJECTIVES: Elevated serum vitamin D with hypercalciuria can result in nephrocalcinosis and nephrolithiasis. This study evaluated the cause of excess 1,25-dihydroxycholecalciferol (1α,25(OH)2D3) in the development of those disorders in two individuals. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Two patients with elevated vitamin D levels and nephrocalcinosis or nephrolithiasis were investigated at the National Institutes of Health (NIH) Clinical Center and the NIH Undiagnosed Diseases Program, by measuring calcium, phosphate, and vitamin D metabolites, and by performing CYP24A1 mutation analysis. RESULTS: Both patients exhibited hypercalciuria, hypercalcemia, low parathyroid hormone, elevated vitamin D (1α,25(OH)2D3), normal 25-OHD3, decreased 24,25(OH)2D, and undetectable activity of 1,25(OH)2D-24-hydroxylase (CYP24A1), the enzyme that inactivates 1α,25(OH)2D3. Both patients had bi-allelic mutations in CYP24A1 leading to loss of function of this enzyme. On the basis of dbSNP data, the frequency of predicted deleterious bi-allelic CYP24A1 variants in the general population is estimated to be as high as 4%-20%. CONCLUSIONS: The results of this study show that 1,25(OH)2D-24-hydroxylase deficiency due to bi-allelic mutations in CYP24A1 causes elevated serum vitamin D, hypercalciuria, nephrocalcinosis, and renal stones.

Exome sequencing as a diagnostic tool in a case of undiagnosed juvenile-onset GM1-gangliosidosis.

OBJECTIVE: To utilize high-throughput sequencing to determine the etiology of juvenile-onset neurodegeneration in a 19-year-old woman with progressive motor and cognitive decline. METHODS: Exome sequencing identified an initial list of 133,555 variants in the proband's family, which were filtered using segregation analysis, presence in dbSNP, and an empirically derived gene exclusion list. The filtered list comprised 52 genes: 21 homozygous variants and 31 compound heterozygous variants. These variants were subsequently scrutinized with predicted pathogenicity programs and for association with appropriate clinical syndromes. RESULTS: Exome sequencing data identified 2 GLB1 variants (c.602G>A, p.R201H; c.785G>T, p.G262V). ß-Galactosidase enzyme analysis prior to our evaluation was reported as normal; however, subsequent testing was consistent with juvenile-onset GM1-gangliosidosis. Urine oligosaccharide analysis was positive for multiple oligosaccharides with terminal galactose residues. CONCLUSIONS: We describe a patient with juvenile-onset neurodegeneration that had eluded diagnosis for over a decade. GM1-gangliosidosis had previously been excluded from consideration, but was subsequently identified as the correct diagnosis using exome sequencing. Exome sequencing can evaluate genes not previously associated with neurodegeneration, as well as most known neurodegeneration-associated genes. Our results demonstrate the utility of "agnostic" exome sequencing to evaluate patients with undiagnosed disorders, without prejudice from prior testing results.