X-linked hypophosphatemic rickets (HYP) is a dominant disorder characterised by impaired phosphate uptake in the kidney, which is likely to be caused by abnormal regulation of sodium phosphate cotransport in the proximal tubules. By positional cloning, we have isolated a candidate gene from the HYP region in Xp22.1. This gene exhibits homology to a family of endopeptidase genes, members of which are involved in the degradation or activation of a variety of peptide hormones. This gene (which we have called PEX) is composed of multiple exons which span at least five cosmids. Intragenic non-overlapping deletions from four different families and three mutations (two splice sites and one frameshift) have been detected in HYP patients, which suggest that the PEX gene is involved in the HYP disorder.
Idiopathic infantile hypercalcemia (IIH) is characterized by severe hypercalcemia with failure to thrive, vomiting, dehydration, and nephrocalcinosis. Recently, mutations in the vitamin D catabolizing enzyme 25-hydroxyvitamin D 3 -24-hydroxylase (CYP24A1) were described that lead to increased sensitivity to vitamin D due to accumulation of the active metabolite 1,25-(OH) 2 D 3 . In a subgroup of patients who presented in early infancy with renal phosphate wasting and symptomatic hypercalcemia, mutations in CYP24A1 were excluded. Four patients from families with parental consanguinity were subjected to homozygosity mapping that identified a second IIH gene locus on chromosome 5q35 with a maximum logarithm of odds (LOD) score of 6.79. The sequence analysis of the most promising candidate gene, SLC34A1 encoding renal sodium-phosphate cotransporter 2A (NaPi-IIa), revealed autosomal-recessive mutations in the four index cases and in 12 patients with sporadic IIH. Functional studies of mutant NaPi-IIa in Xenopus oocytes and opossum kidney (OK) cells demonstrated disturbed trafficking to the plasma membrane and loss of phosphate transport activity. Analysis of calcium and phosphate metabolism in Slc34a1-knockout mice highlighted the effect of phosphate depletion and fibroblast growth factor-23 suppression on the development of the IIH phenotype. The human and mice data together demonstrate that primary renal phosphate wasting caused by defective NaPi-IIa function induces inappropriate production of 1,25-(OH) 2 D 3 with subsequent symptomatic hypercalcemia. Clinical and laboratory findings persist despite cessation of vitamin D prophylaxis but rapidly respond to phosphate supplementation. Therefore, early differentiation between SLC34A1 (NaPi-IIa) and CYP24A1 (24-hydroxylase) defects appears critical for targeted therapy in patients with IIH.
Using exome sequencing, we identify SERAC1 mutations as the cause of MEGDEL syndrome, a recessive disorder of dystonia and deafness with Leigh-like syndrome, impaired oxidative phosphorylation and 3-methylglutaconic aciduria. We localized SERAC1 at the interface between the mitochondria and the endoplasmic reticulum in the mitochondria-associated membrane fraction that is essential for phospholipid exchange. A phospholipid analysis in patient fibroblasts showed elevated concentrations of phosphatidylglycerol-34:1 (where the species nomenclature denotes the number of carbon atoms in the two acyl chains:number of double bonds in the two acyl groups) and decreased concentrations of phosphatidylglycerol-36:1 species, resulting in an altered cardiolipin subspecies composition. We also detected low concentrations of bis(monoacyl-glycerol)-phosphate, leading to the accumulation of free cholesterol, as shown by abnormal filipin staining. Complementation of patient fibroblasts with wild-type human SERAC1 by lentiviral infection led to a decrease and partial normalization of the mean ratio of phosphatidylglycerol-34:1 to phosphatidylglycerol-36:1. Our data identify SERAC1 as a key player in the phosphatidylglycerol remodeling that is essential for both mitochondrial function and intracellular cholesterol trafficking.
BackgroundWhole-exome sequencing (WES) has led to an exponential increase in identification of causative variants in mitochondrial disorders (MD).MethodsWe performed WES in 113 MD suspected patients from Polish paediatric reference centre, in whom routine testing failed to identify a molecular defect. WES was performed using TruSeqExome enrichment, followed by variant prioritization, validation by Sanger sequencing, and segregation with the disease phenotype in the family.ResultsLikely causative mutations were identified in 67 (59.3 %) patients; these included variants in mtDNA (6 patients) and nDNA: X-linked (9 patients), autosomal dominant (5 patients), and autosomal recessive (47 patients, 11 homozygotes). Novel variants accounted for 50.5 % (50/99) of all detected changes. In 47 patients, changes in 31 MD-related genes (ACAD9, ADCK3, AIFM1, CLPB, COX10, DLD, EARS2, FBXL4, MTATP6, MTFMT, MTND1, MTND3, MTND5, NAXE, NDUFS6, NDUFS7, NDUFV1, OPA1, PARS2, PC, PDHA1, POLG, RARS2, RRM2B, SCO2, SERAC1, SLC19A3, SLC25A12, TAZ, TMEM126B, VARS2) were identified. The ACAD9, CLPB, FBXL4, PDHA1 genes recurred more than twice suggesting higher general/ethnic prevalence. In 19 cases, variants in 18 non-MD related genes (ADAR, CACNA1A, CDKL5, CLN3, CPS1, DMD, DYSF, GBE1, GFAP, HSD17B4, MECP2, MYBPC3, PEX5, PGAP2, PIGN, PRF1, SBDS, SCN2A) were found. The percentage of positive WES results rose gradually with increasing probability of MD according to the Mitochondrial Disease Criteria (MDC) scale (from 36 to 90 % for low and high probability, respectively). The percentage of detected MD-related genes compared with non MD-related genes also grew with the increasing MD likelihood (from 20 to 97 %). Molecular diagnosis was established in 30/47 (63.8 %) neonates and in 17/28 (60.7 %) patients with basal ganglia involvement. Mutations in CLPB, SERAC1, TAZ genes were identified in neonates with 3-methylglutaconic aciduria (3-MGA) as a discriminative feature. New MD-related candidate gene (NDUFB8) is under verification.ConclusionsWe suggest WES rather than targeted NGS as the method of choice in diagnostics of MD in children, including neonates with 3-MGA aciduria, who died without determination of disease cause and with limited availability of laboratory data. There is a strong correlation between the degree of MD diagnosis by WES and MD likelihood expressed by the MDC scale.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0930-9) contains supplementary material, which is available to authorized users.
We studied a group of individuals with elevated urinary excretion of 3-methylglutaconic acid, neutropenia that can develop into leukemia, a neurological phenotype ranging from nonprogressive intellectual disability to a prenatal encephalopathy with progressive brain atrophy, movement disorder, cataracts, and early death. Exome sequencing of two unrelated individuals and subsequent Sanger sequencing of 16 individuals with an overlapping phenotype identified a total of 14 rare, predicted deleterious alleles in CLPB in 14 individuals from 9 unrelated families. CLPB encodes caseinolytic peptidase B homolog ClpB, a member of the AAA+ protein family. To evaluate the relevance of CLPB in the pathogenesis of this syndrome, we developed a zebrafish model and an in vitro assay to measure ATPase activity. Suppression of clpb in zebrafish embryos induced a central nervous system phenotype that was consistent with cerebellar and cerebral atrophy that could be rescued by wild-type, but not mutant, human CLPB mRNA. Consistent with these data, the loss-of-function effect of one of the identified variants (c.1222A>G [p.Arg408Gly]) was supported further by in vitro evidence with the mutant peptides abolishing ATPase function. Additionally, we show that CLPB interacts biochemically with ATP2A2, known to be involved in apoptotic processes in severe congenital neutropenia (SCN) 3 (Kostmann disease [caused by HAX1 mutations]). Taken together, mutations in CLPB define a syndrome with intellectual disability, congenital neutropenia, progressive brain atrophy, movement disorder, cataracts, and 3-methylglutaconic aciduria.
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