Primary 5-oxoprolinuria (pyroglutamic aciduria) is caused by a genetic defect in the γ-glutamyl cycle, affecting either glutathione synthetase or 5-oxoprolinase. While several dozens of patients with glutathione synthetase deficiency have been reported, with hemolytic anemia representing the clinical key feature, 5-oxoprolinase deficiency due to OPLAH mutations is less frequent and so far has not attracted much attention. This has prompted us to investigate the clinical phenotype as well as the underlying genotype in patients from 14 families of various ethnic backgrounds who underwent diagnostic mutation analysis following the detection of 5-oxoprolinuria. In all patients with 5-oxoprolinuria studied, bi-allelic mutations in OPLAH were indicated. An autosomal recessive mode of inheritance for 5-oxoprolinase deficiency is further supported by the identification of a single mutation in all 9/14 parent sample sets investigated (except for the father of one patient whose result suggests homozygosity), and the absence of 5-oxoprolinuria in all tested heterozygotes. It is remarkable, that all 20 mutations identified were novel and private to the respective families. Clinical features were highly variable and in several sib pairs, did not segregate with 5-oxoprolinuria. Although a pathogenic role of 5-oxoprolinase deficiency remains possible, this is not supported by our findings. Additional patient ascertainment and long-term follow-up is needed to establish the benign nature of this inborn error of metabolism. It is important that all symptomatic patients with persistently elevated levels of 5-oxoproline and no obvious explanation are investigated for the genetic etiology.

Ion channel proteins are required for both the establishment of resting membrane potentials and the generation of action potentials. Hundreds of mutations in genes encoding voltage-gated ion channels responsible for action potential generation have been found to cause severe neurological diseases. In contrast, the roles of voltage-independent "leak" channels, important for the establishment and maintenance of resting membrane potentials upon which action potentials are generated, are not well established in human disease. UNC80 is a large component of the NALCN sodium-leak channel complex that regulates the basal excitability of the nervous system. Loss-of-function mutations of NALCN cause infantile hypotonia with psychomotor retardation and characteristic facies (IHPRF). We report four individuals from three unrelated families who have homozygous missense or compound heterozygous truncating mutations in UNC80 and persistent hypotonia, encephalopathy, growth failure, and severe intellectual disability. Compared to control cells, HEK293T cells transfected with an expression plasmid containing the c.5098C>T (p.Pro1700Ser) UNC80 mutation found in one individual showed markedly decreased NALCN channel currents. Our findings demonstrate the fundamental significance of UNC80 and basal ionic conductance to human health.

Phosphatidylinositol glycan biosynthesis class A protein (PIGA) is one of the enzymes involved in the biosynthesis of glycosylphosphatidylinositol (GPI) anchor proteins, which function as enzymes, adhesion molecules, complement regulators and co-receptors in signal transduction pathways. Until recently, only somatic PIGA mutations had been reported in patients with paroxysmal nocturnal hemoglobinuria (PNH), while germline mutations had not been observed, and were suspected to result in lethality. However, in just two years, whole exome sequencing (WES) analyses have identified germline PIGA mutations in male patients with XLIDD (X-linked intellectual developmental disorder) with a wide spectrum of clinical presentations.

Caenorhabditis elegans, especially the N2 isolate, is an invaluable biological model system. Numerous additional natural C. elegans isolates have been shown to have unexpected genotypic and phenotypic variations which has encouraged researchers to use next generation sequencing methodology to develop a more complete picture of genotypic variations among the isolates. To understand the phenotypic effects of a genomic variation (GV) on a single gene, in a variation-rich genetic background, one should analyze that particular GV in a well understood genetic background. In C. elegans, the analysis is usually done in N2, which requires extensive crossing to bring in the GV. This can be a very time consuming procedure thus it is important to establish a fast and efficient approach to test the effect of GVs from different isolates in N2. Here we use a Mos1-mediated single-copy insertion (MosSCI) method for phenotypic assessments of GVs from the variation-rich Hawaiian strain CB4856 in N2. Specifically, we investigate effects of variations identified in the CB4856 strain on tac-1 which is an essential gene that is necessary for mitotic spindle elongation and pronuclear migration. We show the usefulness of the MosSCI method by using EU1004 tac-1(or402) as a control. or402 is a temperature sensitive lethal allele within a well-conserved TACC domain (transforming acidic coiled-coil) that results in a leucine to phenylalanine change at amino acid 229. CB4856 contains a variation that affects the second exon of tac-1 causing a cysteine to tryptophan change at amino acid 94 also within the TACC domain. Using the MosSCI method, we analyze tac-1 from CB4856 in the N2 background and demonstrate that the C94W change, albeit significant, does not cause any obvious decrease in viability. This MosSCI method has proven to be a rapid and efficient way to analyze GVs.

Increasing genetic and phenotypic differences found among natural isolates of C. elegans have encouraged researchers to explore the natural variation of this nematode species.

Sialic acid storage diseases are neurodegenerative disorders characterized by accumulation of sialic acid in the lysosome. These disorders are caused by mutations in SLC17A5, the gene encoding sialin, a sialic acid transporter located in the lysosomal membrane. The most common form of sialic acid storage disease is the slowly progressive Salla disease, presenting with hypotonia, ataxia, epilepsy, nystagmus and findings of cerebral and cerebellar atrophy. Hypomyelination and corpus callosum hypoplasia are typical as well. We report a 16 year-old boy with an atypically mild clinical phenotype of sialic acid storage disease characterized by psychomotor retardation and a mixture of spasticity and rigidity but no ataxia, and only weak features of hypomyelination and thinning of corpus callosum on MRI of the brain.

Copaxone is an efficacious and safe therapy that has demonstrated clinical benefit for over two decades in patients with relapsing forms of multiple sclerosis (MS). On an individual level, patients show variability in their response to Copaxone, with some achieving significantly higher response levels. The involvement of genes (e.g., HLA-DRB1*1501) with high inter-individual variability in Copaxone's mechanism of action (MoA) suggests the potential contribution of genetics to treatment response. This study aimed to identify genetic variants associated with Copaxone response in patient cohorts from late-phase clinical trials.

Objectives: Chronic primary vasculitis describes a group of complex and rare diseases that are characterized by blood vessel inflammation. Classification of vasculitis subtypes is based predominantly on the size of the involved vessels and clinical phenotype. There is a recognized need to improve classification, especially for small-to-medium sized vessel vasculitides, that, ideally, is based on the underlying biology with a view to informing treatment. Methods: We performed RNA-Seq on blood samples from children (n = 41) and from adults (n = 11) with small-to-medium sized vessel vasculitis, and used unsupervised hierarchical clustering of gene expression patterns in combination with clinical metadata to define disease subtypes. Results: Differential gene expression at the time of diagnosis separated patients into two primary endotypes that differed in the expression of ~3,800 genes in children, and ~1,600 genes in adults. These endotypes were also present during disease flares, and both adult and pediatric endotypes could be discriminated based on the expression of just 20 differentially expressed genes. Endotypes were associated with distinct biological processes, namely neutrophil degranulation and T cell receptor signaling. Conclusions: Phenotypically similar subsets of small-to-medium sized vessel vasculitis may have different mechanistic drivers involving innate vs. adaptive immune processes. Discovery of these differentiating immune features provides a mechanistic-based alternative for subclassification of vasculitis.

Genetic modifiers are variants modulating phenotypic outcomes of a primary detrimental variant. They contribute to rare diseases phenotypic variability, but their identification is challenging. Genetic screening with model organisms is a widely used method for demystifying genetic modifiers. Forward genetics screening followed by whole genome sequencing allows the detection of variants throughout the genome but typically produces thousands of candidate variants making the interpretation and prioritization process very time-consuming and tedious. Despite whole genome sequencing is more time and cost-efficient, usage of computational pipelines specific to modifier identification remains a challenge for biological-experiment-focused laboratories doing research with model organisms. To facilitate a broader implementation of whole genome sequencing in genetic screens, we have developed Model Organism Modifier or MOM, a pipeline as a user-friendly Galaxy workflow. Model Organism Modifier analyses raw short-read whole genome sequencing data and implements tailored filtering to provide a Candidate Variant List short enough to be further manually curated. We provide a detailed tutorial to run the Galaxy workflow Model Organism Modifier and guidelines to manually curate the Candidate Variant Lists. We have tested Model Organism Modifier on published and validated Caenorhabditis elegans modifiers screening datasets. As whole genome sequencing facilitates high-throughput identification of genetic modifiers in model organisms, Model Organism Modifier provides a user-friendly solution to implement the bioinformatics analysis of the short-read datasets in laboratories without expertise or support in Bioinformatics.

The objective of this study was to investigate the genetic etiology of the X-linked disorder "Hypomyelination of Early Myelinating Structures" (HEMS).

Cyclin-dependent kinases (CDK) and their compulsory cofactors, the cyclins, are the two key classes of regulatory molecules that determine the eukaryotic cell's progress through the cell cycle by substrate phosphorylation. Cdk1 forms complexes with B-type cyclins and phosphorylates a number of substrates as cells prepare to enter mitosis. CYB-3 (Cyclin B3) is a B-type cyclin that has been recently shown to be required for the timely metaphase-to-anaphase transition, presumably by alleviating a spindle assembly checkpoint (SAC) block. Previously, we have shown that doubling the CYB-3 dosage suppresses sterility in the absence of the essential SAC component MDF-1/Mad1. Here we demonstrate the importance of the Mos1-mediated single-copy insertion method for understanding the effects of gene dosage by generating strains that have more (two or three) copies of the cyb-3 in wild-type and mdf-1(gk2) backgrounds to investigate dosage effect of CYB-3 on mitotic progression as well as development and fertility in the absence and the presence of the MDF-1 checkpoint component. We show that tripling the dosage of CYB-3 results in a significantly variable metaphase-to-anaphase transition, both in wild-type and mdf-1(gk2) mutant backgrounds. Although a majority of embryos initiate anaphase onset normally, a significant number of embryos initiate anaphase with a delay. We also show that tripling the dosage of CYB-3 has no effect on viability in the wild-type background; however, it does reduce the sterility caused by the absence of MDF-1. Together, these data reveal that proper dosage of CYB-3 is important for precision of timely execution of anaphase onset regardless of the presence of the MDF-1 checkpoint component.

Huntington disease (HD) is a dominant neurodegenerative disorder caused by a CAG repeat expansion in the Huntingtin gene (HTT). Heterozygous polymorphisms in cis with the mutation allow for allele-specific suppression of the pathogenic HTT transcript as a therapeutic strategy. To prioritize target selection, precise heterozygosity estimates are needed across diverse HD patient populations. Here we present the first comprehensive investigation of all common target alleles across the HTT gene, using 738 reference haplotypes from the 1000 Genomes Project and 2364 haplotypes from HD patients and relatives in Canada, Sweden, France, and Italy. The most common HD haplotypes (A1, A2, and A3a) define mutually exclusive sets of polymorphisms for allele-specific therapy in the greatest number of patients. Across all four populations, a maximum of 80% are treatable using these three target haplotypes. We identify a novel deletion found exclusively on the A1 haplotype, enabling potent and selective silencing of mutant HTT in approximately 40% of the patients. Antisense oligonucleotides complementary to the deletion reduce mutant A1 HTT mRNA by 78% in patient cells while sparing wild-type HTT expression. By suppressing specific haplotypes on which expanded CAG occurs, we demonstrate a rational approach to the development of allele-specific therapy for a monogenic disorder.

Early-infantile encephalopathies with epilepsy are devastating conditions mandating an accurate diagnosis to guide proper management. Whole-exome sequencing was used to investigate the disease etiology in four children from independent families with intellectual disability and epilepsy, revealing bi-allelic GOT2 mutations. In-depth metabolic studies in individual 1 showed low plasma serine, hypercitrullinemia, hyperlactatemia, and hyperammonemia. The epilepsy was serine and pyridoxine responsive. Functional consequences of observed mutations were tested by measuring enzyme activity and by cell and animal models. Zebrafish and mouse models were used to validate brain developmental and functional defects and to test therapeutic strategies. GOT2 encodes the mitochondrial glutamate oxaloacetate transaminase. GOT2 enzyme activity was deficient in fibroblasts with bi-allelic mutations. GOT2, a member of the malate-aspartate shuttle, plays an essential role in the intracellular NAD(H) redox balance. De novo serine biosynthesis was impaired in fibroblasts with GOT2 mutations and GOT2-knockout HEK293 cells. Correcting the highly oxidized cytosolic NAD-redox state by pyruvate supplementation restored serine biosynthesis in GOT2-deficient cells. Knockdown of got2a in zebrafish resulted in a brain developmental defect associated with seizure-like electroencephalography spikes, which could be rescued by supplying pyridoxine in embryo water. Both pyridoxine and serine synergistically rescued embryonic developmental defects in zebrafish got2a morphants. The two treated individuals reacted favorably to their treatment. Our data provide a mechanistic basis for the biochemical abnormalities in GOT2 deficiency that may also hold for other MAS defects.

Genetic variation databases provide invaluable information on the presence and frequency of genetic variants in the 'untargeted' human population, aggregated with the primary goal to facilitate the interpretation of clinically important variants. The presence of somatic variants in such databases can affect variant assessment in undiagnosed rare disease (RD) patients. Previously, the impact of somatic mosaicism was only considered in relation to two Mendelian disease-associated genes. Here, we expand the analyses to identify additional mosaicism-prone genes in blood-derived reference population databases.

Genetic balancers in Caenorhabditis elegans are complex variants that allow lethal or sterile mutations to be stably maintained in a heterozygous state by suppressing crossover events. Balancers constitute an invaluable tool in the C. elegans scientific community and have been widely used for decades. The first/traditional balancers were created by applying X-rays, UV, or gamma radiation on C. elegans strains, generating random genomic rearrangements. Their structures have been mostly explored with low-resolution genetic techniques (e.g., fluorescence in situ hybridization or PCR), before genomic mapping and molecular characterization through sequencing became feasible. As a result, the precise nature of most chromosomal rearrangements remains unknown, whereas, more recently, balancers have been engineered using the CRISPR-Cas9 technique for which the structure of the chromosomal rearrangement has been predesigned. Using short-read whole-genome sequencing (srWGS) and tailored bioinformatic analyses, we previously interpreted the structure of four chromosomal balancers randomly created by mutagenesis processes. Here, we have extended our analyses to five CRISPR-Cas9 balancers and 17 additional traditional balancing rearrangements. We detected and experimentally validated their breakpoints and have interpreted the balancer structures. Many of the balancers were found to be more intricate than previously described, being composed of complex genomic rearrangements (CGRs) such as chromoanagenesis-like events. Furthermore, srWGS revealed additional structural variants and CGRs not known to be part of the balancer genomes. Altogether, our study provides a comprehensive resource of complex genomic variations in C. elegans and highlights the power of srWGS to study the complexity of genomes by applying tailored analyses.

Genomic rearrangements cause congenital disorders, cancer, and complex diseases in human. Yet, they are still understudied in rare diseases because their detection is challenging, despite the advent of whole genome sequencing (WGS) technologies. Short-read (srWGS) and long-read WGS approaches are regularly compared, and the latter is commonly recommended in studies focusing on genomic rearrangements. However, srWGS is currently the most economical, accurate, and widely supported technology. In Caenorhabditis elegans (C. elegans), such variants, induced by various mutagenesis processes, have been used for decades to balance large genomic regions by preventing chromosomal crossover events and allowing the maintenance of lethal mutations. Interestingly, those chromosomal rearrangements have rarely been characterized on a molecular level. To evaluate the ability of srWGS to detect various types of complex genomic rearrangements, we sequenced three balancer strains using short-read Illumina technology. As we experimentally validated the breakpoints uncovered by srWGS, we showed that, by combining several types of analyses, srWGS enables the detection of a reciprocal translocation (eT1), a free duplication (sDp3), a large deletion (sC4), and chromoanagenesis events. Thus, applying srWGS to decipher real complex genomic rearrangements in model organisms may help designing efficient bioinformatics pipelines with systematic detection of complex rearrangements in human genomes.

Since Garrod's first description of alkaptonuria in 1902, and newborn screening for phenylketonuria introduced in the 1960s, P4 medicine (preventive, predictive, personalized, and participatory) has been a reality for the clinician serving patients with inherited metabolic diseases. The era of high-throughput technologies promises to accelerate its scale dramatically. Genomics, transcriptomics, epigenomics, proteomics, glycomics, metabolomics, and lipidomics offer an amazing opportunity for holistic investigation and contextual pathophysiologic understanding of inherited metabolic diseases for precise diagnosis and tailored treatment. While each of the -omics technologies is important to systems biology, some are more mature than others. Exome sequencing is emerging as a reimbursed test in clinics around the world, and untargeted metabolomics has the potential to serve as a single biochemical testing platform. The challenge lies in the integration and cautious interpretation of these big data, with translation into clinically meaningful information and/or action for our patients. A daunting but exciting task for the clinician; we provide clinical cases to illustrate the importance of his/her role as the connector between physicians, laboratory experts and researchers in the basic, computer, and clinical sciences. Open collaborations, data sharing, functional assays, and model organisms play a key role in the validation of -omics discoveries. Having all the right expertise at the table when discussing the diagnostic approach and individualized management plan according to the information yielded by -omics investigations (e.g., actionable mutations, novel therapeutic interventions), is the stepping stone of P4 medicine. Patient participation and the adjustment of the medical team's plan to his/her and the family's wishes most certainly is the capstone. Are you ready?

Pathogen immune responses are profoundly attenuated in fetuses and premature infants, yet the mechanisms underlying this developmental immaturity remain unclear. Here we show transcriptomic, metabolic and polysome profiling and find that monocytes isolated from infants born early in gestation display perturbations in PPAR-γ-regulated metabolic pathways, limited glycolytic capacity and reduced ribosomal activity. These metabolic changes are linked to a lack of translation of most cytokines and of MALT1 signalosome genes essential to respond to the neonatal pathogen Candida. In contrast, they have little impact on house-keeping phagocytosis functions. Transcriptome analyses further indicate a role for mTOR and its putative negative regulator DNA Damage Inducible Transcript 4-Like in regulating these metabolic constraints. Our results provide a molecular basis for the broad susceptibility to multiple pathogens in these infants, and suggest that the fetal immune system is metabolically programmed to avoid energetically costly, dispensable and potentially harmful immune responses during ontogeny.

Dramatic improvements in DNA-sequencing technologies and computational analyses have led to wide use of whole exome sequencing (WES) to identify the genetic basis of Mendelian disorders. More than 180 novel rare-disease-causing genes with Mendelian inheritance patterns have been discovered through sequencing the exomes of just a few unrelated individuals or family members. As rare/novel genetic variants continue to be uncovered, there is a major challenge in distinguishing true pathogenic variants from rare benign mutations.

Fatty acid amide hydrolase 2 (FAAH2) is a hydrolase that mediates the degradation of endocannabinoids in man. Alterations in the endocannabinoid system are associated with a wide variety of neurologic and psychiatric conditions, but the phenotype and biochemical characterization of patients with genetic defects of FAAH2 activity have not previously been described. We report a male with autistic features with an onset before the age of 2 years who subsequently developed additional features including anxiety, pseudoseizures, ataxia, supranuclear gaze palsy, and isolated learning disabilities but was otherwise cognitively intact as an adult.