Identification of human D lactate dehydrogenase deficiency.

Phenotypic and biochemical categorization of humans with detrimental variants can provide valuable information on gene function. We illustrate this with the identification of two different homozygous variants resulting in enzymatic loss-of-function in LDHD, encoding lactate dehydrogenase D, in two unrelated patients with elevated D-lactate urinary excretion and plasma concentrations. We establish the role of LDHD by demonstrating that LDHD loss-of-function in zebrafish results in increased concentrations of D-lactate. D-lactate levels are rescued by wildtype LDHD but not by patients' variant LDHD, confirming these variants' loss-of-function effect. This work provides the first in vivo evidence that LDHD is responsible for human D-lactate metabolism. This broadens the differential diagnosis of D-lactic acidosis, an increasingly recognized complication of short bowel syndrome with unpredictable onset and severity. With the expanding incidence of intestinal resection for disease or obesity, the elucidation of this metabolic pathway may have relevance for those patients with D-lactic acidosis.

GLS hyperactivity causes glutamate excess, infantile cataract and profound developmental delay.

Loss-of-function mutations in glutaminase (GLS), the enzyme converting glutamine into glutamate, and the counteracting enzyme glutamine synthetase (GS) cause disturbed glutamate homeostasis and severe neonatal encephalopathy. We report a de novo Ser482Cys gain-of-function variant in GLS encoding GLS associated with profound developmental delay and infantile cataract. Functional analysis demonstrated that this variant causes hyperactivity and compensatory downregulation of GLS expression combined with upregulation of the counteracting enzyme GS, supporting pathogenicity. Ser482Cys-GLS likely improves the electrostatic environment of the GLS catalytic site, thereby intrinsically inducing hyperactivity. Alignment of +/-12.000 GLS protein sequences from >1000 genera revealed extreme conservation of Ser482 to the same degree as catalytic residues. Together with the hyperactivity, this indicates that Ser482 is evolutionarily preserved to achieve optimal-but submaximal-GLS activity. In line with GLS hyperactivity, increased glutamate and decreased glutamine concentrations were measured in urine and fibroblasts. In the brain (both grey and white matter), glutamate was also extremely high and glutamine was almost undetectable, demonstrated with magnetic resonance spectroscopic imaging at clinical field strength and subsequently supported at ultra-high field strength. Considering the neurotoxicity of glutamate when present in excess, the strikingly high glutamate concentrations measured in the brain provide an explanation for the developmental delay. Cataract, a known consequence of oxidative stress, was evoked in zebrafish expressing the hypermorphic Ser482Cys-GLS and could be alleviated by inhibition of GLS. The capacity to detoxify reactive oxygen species was reduced upon Ser482Cys-GLS expression, providing an explanation for cataract formation. In conclusion, we describe an inborn error of glutamate metabolism caused by a GLS hyperactivity variant, illustrating the importance of balanced GLS activity.

Effective CRISPR/Cas9-based nucleotide editing in zebrafish to model human genetic cardiovascular disorders.

The zebrafish (Danio rerio) has become a popular vertebrate model organism to study organ formation and function due to its optical clarity and rapid embryonic development. The use of genetically modified zebrafish has also allowed identification of new putative therapeutic drugs. So far, most studies have relied on broad overexpression of transgenes harboring patient-derived mutations or loss-of-function mutants, which incompletely model the human disease allele in terms of expression levels or cell-type specificity of the endogenous gene of interest. Most human genetically inherited conditions are caused by alleles carrying single nucleotide changes resulting in altered gene function. Introduction of such point mutations in the zebrafish genome would be a prerequisite to recapitulate human disease but remains challenging to this day. We present an effective approach to introduce small nucleotide changes in the zebrafish genome. We generated four different knock-in lines carrying distinct human cardiovascular-disorder-causing missense mutations in their zebrafish orthologous genes by combining CRISPR/Cas9 with a short template oligonucleotide. Three of these lines carry gain-of-function mutations in genes encoding the pore-forming (Kir6.1, KCNJ8) and regulatory (SUR2, ABCC9) subunits of an ATP-sensitive potassium channel (KATP) linked to Cantú syndrome (CS). Our heterozygous zebrafish knock-in lines display significantly enlarged ventricles with enhanced cardiac output and contractile function, and distinct cerebral vasodilation, demonstrating the causality of the introduced mutations for CS. These results demonstrate that introducing patient alleles in their zebrafish orthologs promises a broad application for modeling human genetic diseases, paving the way for new therapeutic strategies using this model organism.

Intestinal Failure and Aberrant Lipid Metabolism in Patients With DGAT1 Deficiency.

BACKGROUND & AIMS: Congenital diarrheal disorders are rare inherited intestinal disorders characterized by intractable, sometimes life-threatening, diarrhea and nutrient malabsorption; some have been associated with mutations in diacylglycerol-acyltransferase 1 (DGAT1), which catalyzes formation of triacylglycerol from diacylglycerol and acyl-CoA. We investigated the mechanisms by which DGAT1 deficiency contributes to intestinal failure using patient-derived organoids. METHODS: We collected blood samples from 10 patients, from 6 unrelated pedigrees, who presented with early-onset severe diarrhea and/or vomiting, hypoalbuminemia, and/or (fatal) protein-losing enteropathy with intestinal failure; we performed next-generation sequencing analysis of DNA from 8 patients. Organoids were generated from duodenal biopsies from 3 patients and 3 healthy individuals (controls). Caco-2 cells and patient-derived dermal fibroblasts were transfected or transduced with vectors that express full-length or mutant forms of DGAT1 or full-length DGAT2. We performed CRISPR/Cas9-guided disruption of DGAT1 in control intestinal organoids. Cells and organoids were analyzed by immunoblot, immunofluorescence, flow cytometry, chromatography, quantitative real-time polymerase chain reaction, and for the activity of caspases 3 and 7. RESULTS: In the 10 patients, we identified 5 bi-allelic loss-of-function mutations in DGAT1. In patient-derived fibroblasts and organoids, the mutations reduced expression of DGAT1 protein and altered triacylglycerol metabolism, resulting in decreased lipid droplet formation after oleic acid addition. Expression of full-length DGAT2 in patient-derived fibroblasts restored formation of lipid droplets. Organoids derived from patients with DGAT1 mutations were more susceptible to lipid-induced cell death than control organoids. CONCLUSIONS: We identified a large cohort of patients with congenital diarrheal disorders with mutations in DGAT1 that reduced expression of its product; dermal fibroblasts and intestinal organoids derived from these patients had altered lipid metabolism and were susceptible to lipid-induced cell death. Expression of full-length wildtype DGAT1 or DGAT2 restored normal lipid metabolism in these cells. These findings indicate the importance of DGAT1 in fat metabolism and lipotoxicity in the intestinal epithelium. A fat-free diet might serve as the first line of therapy for patients with reduced DGAT1 expression. It is important to identify genetic variants associated with congenital diarrheal disorders for proper diagnosis and selection of treatment strategies.

Synaptic UNC13A protein variant causes increased neurotransmission and dyskinetic movement disorder.

Munc13 proteins are essential regulators of neurotransmitter release at nerve cell synapses. They mediate the priming step that renders synaptic vesicles fusion-competent, and their genetic elimination causes a complete block of synaptic transmission. Here we have described a patient displaying a disorder characterized by a dyskinetic movement disorder, developmental delay, and autism. Using whole-exome sequencing, we have shown that this condition is associated with a rare, de novo Pro814Leu variant in the major human Munc13 paralog UNC13A (also known as Munc13-1). Electrophysiological studies in murine neuronal cultures and functional analyses in Caenorhabditis elegans revealed that the UNC13A variant causes a distinct dominant gain of function that is characterized by increased fusion propensity of synaptic vesicles, which leads to increased initial synaptic vesicle release probability and abnormal short-term synaptic plasticity. Our study underscores the critical importance of fine-tuned presynaptic control in normal brain function. Further, it adds the neuronal Munc13 proteins and the synaptic vesicle priming process that they control to the known etiological mechanisms of psychiatric and neurological synaptopathies.

Heterozygous KIDINS220/ARMS nonsense variants cause spastic paraplegia, intellectual disability, nystagmus, and obesity.

We identified de novo nonsense variants in KIDINS220/ARMS in three unrelated patients with spastic paraplegia, intellectual disability, nystagmus, and obesity (SINO). KIDINS220 is an essential scaffold protein coordinating neurotrophin signal pathways in neurites and is spatially and temporally regulated in the brain. Molecular analysis of patients' variants confirmed expression and translation of truncated transcripts similar to recently characterized alternative terminal exon splice isoforms of KIDINS220 KIDINS220 undergoes extensive alternative splicing in specific neuronal populations and developmental time points, reflecting its complex role in neuronal maturation. In mice and humans, KIDINS220 is alternative spliced in the middle region as well as in the last exon. These full-length and KIDINS220 splice variants occur at precise moments in cortical, hippocampal, and motor neuron development, with splice variants similar to the variants seen in our patients and lacking the last exon of KIDINS220 occurring in adult rather than in embryonic brain. We conducted tissue-specific expression studies in zebrafish that resulted in spasms, confirming a functional link with disruption of the KIDINS220 levels in developing neurites. This work reveals a crucial physiological role of KIDINS220 in development and provides insight into how perturbation of the complex interplay of KIDINS220 isoforms and their relative expression can affect neuron control and human metabolism. Altogether, we here show that de novo protein-truncating KIDINS220 variants cause a new syndrome, SINO. This is the first report of KIDINS220 variants causing a human disease.

Effectiveness of whole-exome sequencing and costs of the traditional diagnostic trajectory in children with intellectual disability.

PURPOSE: This study investigated whole-exome sequencing (WES) yield in a subset of intellectually disabled patients referred to our clinical diagnostic center and calculated the total costs of these patients' diagnostic trajectory in order to evaluate early WES implementation. METHODS: We compared 17 patients' trio-WES yield with the retrospective costs of diagnostic procedures by comprehensively examining patient records and collecting resource use information for each patient, beginning with patient admittance and concluding with WES initiation. We calculated cost savings using scenario analyses to evaluate the costs replaced by WES when used as a first diagnostic tool. RESULTS: WES resulted in diagnostically useful outcomes in 29.4% of patients. The entire traditional diagnostic trajectory average cost was $16,409 per patient, substantially higher than the $3,972 trio-WES cost. WES resulted in average cost savings of $3,547 for genetic and metabolic investigations in diagnosed patients and $1,727 for genetic investigations in undiagnosed patients. CONCLUSION: The increased causal variant detection yield by WES and the relatively high costs of the entire traditional diagnostic trajectory suggest that early implementation of WES is a relevant and cost-efficient option in patient diagnostics. This information is crucial for centers considering implementation of WES and serves as input for future value-based research into diagnostics.Genet Med 18 9, 949-956.

MKS1 regulates ciliary INPP5E levels in Joubert syndrome.

BACKGROUND: Joubert syndrome (JS) is a recessive ciliopathy characterised by a distinctive brain malformation 'the molar tooth sign'. Mutations in >27 genes cause JS, and mutations in 12 of these genes also cause Meckel-Gruber syndrome (MKS). The goals of this work are to describe the clinical features of MKS1-related JS and determine whether disease causing MKS1 mutations affect cellular phenotypes such as cilium number, length and protein content as potential mechanisms underlying JS. METHODS: We measured cilium number, length and protein content (ARL13B and INPP5E) by immunofluorescence in fibroblasts from individuals with MKS1-related JS and in a three-dimensional (3D) spheroid rescue assay to test the effects of disease-related MKS1 mutations. RESULTS: We report MKS1 mutations (eight of them previously unreported) in nine individuals with JS. A minority of the individuals with MKS1-related JS have MKS features. In contrast to the truncating mutations associated with MKS, all of the individuals with MKS1-related JS carry ≥ 1 non-truncating mutation. Fibroblasts from individuals with MKS1-related JS make normal or fewer cilia than control fibroblasts, their cilia are more variable in length than controls, and show decreased ciliary ARL13B and INPP5E. Additionally, MKS1 mutant alleles have similar effects in 3D spheroids. CONCLUSIONS: MKS1 functions in the transition zone at the base of the cilium to regulate ciliary INPP5E content, through an ARL13B-dependent mechanism. Mutations in INPP5E also cause JS, so our findings in patient fibroblasts support the notion that loss of INPP5E function, due to either mutation or mislocalisation, is a key mechanism underlying JS, downstream of MKS1 and ARL13B.

Joubert syndrome: genotyping a Northern European patient cohort.

Joubert syndrome (JBS) is a rare neurodevelopmental disorder belonging to the group of ciliary diseases. JBS is genetically heterogeneous, with >20 causative genes identified to date. A molecular diagnosis of JBS is essential for prediction of disease progression and genetic counseling. We developed a targeted next-generation sequencing (NGS) approach for parallel sequencing of 22 known JBS genes plus 599 additional ciliary genes. This method was used to genotype a cohort of 51 well-phenotyped Northern European JBS cases (in some of the cases, Sanger sequencing of individual JBS genes had been performed previously). Altogether, 21 of the 51 cases (41%) harbored biallelic pathogenic mutations in known JBS genes, including 14 mutations not previously described. Mutations in C5orf42 (12%), TMEM67 (10%), and AHI1 (8%) were the most prevalent. C5orf42 mutations result in a purely neurological Joubert phenotype, in one case associated with postaxial polydactyly. Our study represents a population-based cohort of JBS patients not enriched for consanguinity, providing insight into the relative importance of the different JBS genes in a Northern European population. Mutations in C5orf42 are relatively frequent (possibly due to a Dutch founder mutation) and mutations in CEP290 are underrepresented compared with international cohorts. Furthermore, we report a case with heterozygous mutations in CC2D2A and B9D1, a gene associated with the more severe Meckel-Gruber syndrome that was recently published as a potential new JBS gene, and discuss the significance of this finding.

Chromothripsis in healthy individuals affects multiple protein-coding genes and can result in severe congenital abnormalities in offspring.

Chromothripsis represents an extreme class of complex chromosome rearrangements (CCRs) with major effects on chromosomal architecture. Although recent studies have associated chromothripsis with congenital abnormalities, the incidence and pathogenic effects of this phenomenon require further investigation. Here, we analyzed the genomes of three families in which chromothripsis rearrangements were transmitted from a mother to her child. The chromothripsis in the mothers resulted in completely balanced rearrangements involving 8-23 breakpoint junctions across three to five chromosomes. Two mothers did not show any phenotypic abnormalities, although 3-13 protein-coding genes were affected by breakpoints. Unbalanced but stable transmission of a subset of the derivative chromosomes caused apparently de novo complex copy-number changes in two children. This resulted in gene-dosage changes, which are probably responsible for the severe congenital phenotypes of these two children. In contrast, the third child, who has a severe congenital disease, harbored all three chromothripsis chromosomes from his healthy mother, but one of the chromosomes acquired de novo rearrangements leading to copy-number changes. These results show that the human genome can tolerate extreme reshuffling of chromosomal architecture, including breakage of multiple protein-coding genes, without noticeable phenotypic effects. The presence of chromothripsis in healthy individuals affects reproduction and is expected to substantially increase the risk of miscarriages, abortions, and severe congenital disease.

Effect of vertical sleeve gastrectomy in melanocortin receptor 4-deficient rats.

Bariatric surgery is currently the most effective treatment for obesity. Vertical sleeve gastrectomy (VSG), a commonly applied bariatric procedure, involves surgically incising most of the volume of the stomach. In humans, partial loss of melanocortin receptor-4 (MC4R) activity is the most common monogenic correlate of obesity regardless of lifestyle. At present it is unclear whether genetic alteration of MC4R signaling modulates the beneficial effects of VSG. Following VSG, we analyzed body weight, food intake, glucose sensitivity, and macronutrient preference of wild-type and MC4R-deficient (Mc4r(+/-) and Mc4r(-/-)) rats compared with sham-operated controls. VSG reduced body weight and fat mass and improved glucose metabolism and also shifted preference toward carbohydrates and away from fat. All of this occurred independently of MC4R activity. In addition, MC4R was resequenced in 46 human subjects who underwent VSG. We observed common genetic variations in the coding sequence of MC4R in five subjects. However, none of those variations appeared to affect the outcome of VSG. Taken together, these data suggest that the beneficial effect of VSG on body weight and glucose metabolism is not mediated by alterations in MC4R activity.

Discovery of variants unmasked by hemizygous deletions.

Array-based genome-wide segmental aneuploidy screening detects both de novo and inherited copy number variations (CNVs). In sporadic patients de novo CNVs are interpreted as potentially pathogenic. However, a deletion, transmitted from a healthy parent, may be pathogenic if it overlaps with a mutated second allele inherited from the other healthy parent. To detect such events, we performed multiplex enrichment and next-generation sequencing of the entire coding sequence of all genes within unique hemizygous deletion regions in 20 patients (1.53 Mb capture footprint). Out of the detected 703 non-synonymous single-nucleotide variants (SNVs), 8 represented variants being unmasked by a hemizygous deletion. Although evaluation of inheritance patterns, Grantham matrix scores, evolutionary conservation and bioinformatic predictions did not consistently indicate pathogenicity of these variants, no definitive conclusions can be drawn without functional validation. However, in one patient with severe mental retardation, lack of speech, microcephaly, cheilognathopalatoschisis and bilateral hearing loss, we discovered a second smaller deletion, inherited from the other healthy parent, resulting in loss of both alleles of the highly conserved heat shock factor binding protein 1 (HSBP1) gene. Conceivably, inherited deletions may unmask rare pathogenic variants that may exert a phenotypic impact through a recessive mode of gene action.

Limited contribution of NR5A1 (SF-1) mutations in women with primary ovarian insufficiency (POI).

OBJECTIVE: To evaluate the significance of NR5A1 mutations in a large, well-phenotyped cohort of women with primary ovarian insufficiency (POI). Mutations in the NR5A1 gene (SF-1) were previously described in disorders of sexual development and adrenal insufficiency. Recently, a high frequency of NR5A1 gene mutations was reported in a small group of women with POI. DESIGN: Cross-sectional cohort study. SETTING: University hospital. PATIENT(S): Well-phenotyped women (n = 386) with secondary amenorrhea and diagnosed with POI, including women with familial POI (n = 77). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The entire coding region and splice sites of the NR5A1 gene were PCR-amplified and sequenced. The pathogenicity of identified mutations was predicted in silico by assessing Align-GVGD class and Grantham score. RESULT(S): Sequencing was successful in 356 patients with POI. In total, 9 mutations were identified in 10 patients. Five of these mutations concerned novel nonconservative mutations occurring in 5 patients. Prediction of effect on protein function showed low to intermediate pathogenicity for all nonconservative mutations. The overall NR5A1 gene mutation rate was 1.4%. CONCLUSION(S): The current study demonstrates that mutations in the NR5A1 gene are rare in women with POI. Primary ovarian insufficiency remains unexplained in the great majority of patients; therefore, continued efforts are needed to elucidate its underlying genetic factors.

Chromothripsis as a mechanism driving complex de novo structural rearrangements in the germline.

A variety of mutational mechanisms shape the dynamic architecture of human genomes and occasionally result in congenital defects and disease. Here, we used genome-wide long mate-pair sequencing to systematically screen for inherited and de novo structural variation in a trio including a child with severe congenital abnormalities. We identified 4321 inherited structural variants and 17 de novo rearrangements. We characterized the de novo structural changes to the base-pair level revealing a complex series of balanced inter- and intra-chromosomal rearrangements consisting of 12 breakpoints involving chromosomes 1, 4 and 10. Detailed inspection of breakpoint regions indicated that a series of simultaneous double-stranded DNA breaks caused local shattering of chromosomes. Fusion of the resulting chromosomal fragments involved non-homologous end joining, since junction points displayed limited or no homology and small insertions and deletions. The pattern of random joining of chromosomal fragments that we observe here strongly resembles the somatic rearrangement patterns--termed chromothripsis--that have recently been described in deranged cancer cells. We conclude that a similar mechanism may also drive the formation of de novo structural variation in the germline.

Characterization of COMMD protein-protein interactions in NF-kappaB signalling.

COMMD [copper metabolism gene MURR1 (mouse U2af1-rs1 region 1) domain] proteins constitute a recently identified family of NF-kappaB (nuclear factor kappaB)-inhibiting proteins, characterized by the presence of the COMM domain. In the present paper, we report detailed investigation of the role of this protein family, and specifically the role of the COMM domain, in NF-kappaB signalling through characterization of protein-protein interactions involving COMMD proteins. The small ubiquitously expressed COMMD6 consists primarily of the COMM domain. Therefore COMMD1 and COMMD6 were analysed further as prototype members of the COMMD protein family. Using specific antisera, interaction between endogenous COMMD1 and COMMD6 is described. This interaction was verified by independent techniques, appeared to be direct and could be detected throughout the whole cell, including the nucleus. Both proteins inhibit TNF (tumour necrosis factor)-induced NF-kappaB activation in a non-synergistic manner. Mutation of the amino acid residues Trp24 and Pro41 in the COMM domain of COMMD6 completely abolished the inhibitory effect of COMMD6 on TNF-induced NF-kappaB activation, but this was not accompanied by loss of interaction with COMMD1, COMMD6 or the NF-kappaB subunit RelA. In contrast with COMMD1, COMMD6 does not bind to IkappaBalpha (inhibitory kappaBalpha), indicating that both proteins inhibit NF-kappaB in an overlapping, but not completely similar, manner. Taken together, these data support the significance of COMMD protein-protein interactions and provide new mechanistic insight into the function of this protein family in NF-kappaB signalling.