CIAO1 and MMS19 deficiency: A lethal neurodegenerative phenotype caused by cytosolic Fe-S cluster protein assembly disorders.

PURPOSE: The functionality of many cellular proteins depends on cofactors; yet, they have only been implicated in a minority of Mendelian diseases. Here, we describe the first 2 inherited disorders of the cytosolic iron-sulfur protein assembly system. METHODS: Genetic testing via genome sequencing was applied to identify the underlying disease cause in 3 patients with microcephaly, congenital brain malformations, progressive developmental and neurologic impairments, recurrent infections, and a fatal outcome. Studies in patient-derived skin fibroblasts and zebrafish models were performed to investigate the biochemical and cellular consequences. RESULTS: Metabolic analysis showed elevated uracil and thymine levels in body fluids but no pathogenic variants in DPYD, encoding dihydropyrimidine dehydrogenase. Genome sequencing identified compound heterozygosity in 2 patients for missense variants in CIAO1, encoding cytosolic iron-sulfur assembly component 1, and homozygosity for an in-frame 3-nucleotide deletion in MMS19, encoding the MMS19 homolog, cytosolic iron-sulfur assembly component, in the third patient. Profound alterations in the proteome, metabolome, and lipidome were observed in patient-derived fibroblasts. We confirmed the detrimental effect of deficiencies in CIAO1 and MMS19 in zebrafish models. CONCLUSION: A general failure of cytosolic and nuclear iron-sulfur protein maturation caused pleiotropic effects. The critical function of the cytosolic iron-sulfur protein assembly machinery for antiviral host defense may well explain the recurrent severe infections occurring in our patients.

Children with atopic dermatitis show increased activity of ß-glucocerebrosidase and stratum corneum levels of glucosylcholesterol that are strongly related to the local cytokine milieu.

BACKGROUND: Atopic dermatitis (AD) is characterized by immune dysregulations and an impaired skin barrier, including abnormalities in lipid organization. In the stratum corneum (SC), ß-glucocerebrosidase (GBA) mediates transformation of glucosylceramide (GlcCER) into ceramide (CER) and cholesterol into glucosylcholesterol (GlcChol). Alteration in GBA activity might contribute to skin barrier defects in AD. OBJECTIVES: To investigate GBA activity in the SC of children with AD before and after topical corticosteroid therapy and to compare it with healthy controls; to determine SC levels of GlcCER- and CER-containing hydroxysphingosine base (GlcCER[H] and CER[H], respectively) and GlcChol; and to relate them to disease severity, skin barrier function and the local cytokine milieu. METHODS: Lipid markers and cytokines of innate, T helper 1 and T helper 2 immunity were determined in SC collected from healthy children and from clinically unaffected skin of children with AD, before and after 6 weeks of therapy with topical corticosteroids. AD severity was assessed by Scoring Atopic Dermatitis and skin barrier function by transepidermal water loss (TEWL). RESULTS: Baseline GBA activity and GlcChol levels were increased in children with AD but declined after therapy. CER[H] levels and the CER[H] to GlcCER[H] ratio were increased in AD. GBA activity and GlcChol correlated with TEWL and levels of multiple cytokines, especially interleukin-1α and interleukin-18. GlcChol was strongly associated with disease severity. CONCLUSIONS: We show increased GBA activity and levels of GlcChol in AD. Our data suggest an important role of inflammation in disturbed lipid processing. GBA activity or GlcChol might be useful biomarkers in the monitoring of therapeutic responses in AD. What is already known about this topic? Patients with atopic dermatitis (AD) have a reduced skin barrier, mainly caused by altered lipid organization. The mechanisms underlying these lipid anomalies are not fully understood but likely reflect both genetic abnormalities in AD skin and the local cutaneous inflammatory environment. What does this study add? We show increased activity of the ceramide-generating enzyme ß-glucocerebrosidase in AD. Activity of this enzyme was correlated with the local cytokine milieu and declined after local corticosteroid therapy. We show that glucosylcholesterol levels in the stratum corneum are increased in AD. The function of glucosylcholesterol and the physiological consequences of increased levels are not clear yet; however, its levels were strongly correlated with skin barrier function: high transepidermal water loss strongly correlated with high levels of glucosylcholesterol. What is the translational message? Correction of cutaneous inflammation largely restores alterations in lipid metabolism in the stratum corneum of infants with AD.

The pathophysiology of human obstructive cholestasis is mimicked in cholestatic Gold Syrian hamsters.

Obstructive cholestasis causes liver injury via accumulation of toxic bile acids (BAs). Therapeutic options for cholestatic liver disease are limited, partially because the available murine disease models lack translational value. Profiling of time-related changes following bile duct ligation (BDL) in Gold Syrian hamsters revealed a biochemical response similar to cholestatic patients in terms of BA pool composition, alterations in hepatocyte BA transport and signaling, suppression of BA production, and adapted BA metabolism. Hamsters tolerated cholestasis well for up to 28days and progressed relatively slowly to fibrotic liver injury. Hepatocellular necrosis was absent, which coincided with preserved intrahepatic energy levels and only mild oxidative stress. The histological response to cholestasis in hamsters was similar to the changes seen in 17 patients with prolonged obstructive cholestasis caused by cholangiocarcinoma. Hamsters moreover upregulated hepatic fibroblast growth factor 15 (Fgf15) expression in response to BDL, which is a cytoprotective adaptation to cholestasis that hitherto had only been documented in cholestatic human livers. Hamster models should therefore be added to the repertoire of animal models used to study the pathophysiology of cholestatic liver disease.

Phenotypic and clinical implications of variants in the dihydropyrimidine dehydrogenase gene.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases uracil, thymine and the antineoplastic agent 5-fluorouracil. Genetic variations in the gene encoding DPD (DPYD) have emerged as predictive risk alleles for 5FU-associated toxicity. Here we report an in-depth analysis of genetic variants in DPYD and their consequences for DPD activity and pyrimidine metabolites in 100 Dutch healthy volunteers. 34 SNPs were detected in DPYD and 15 SNPs were associated with altered plasma concentrations of pyrimidine metabolites. DPD activity was significantly associated with the plasma concentrations of uracil, the presence of a specific DPYD mutation (c.1905+1G>A) and the combined presence of three risk variants in DPYD (c.1905+1G>A, c.1129-5923C>G, c.2846A>T), but not with an altered uracil/dihydrouracil (U/UH2) ratio. Various haplotypes were associated with different DPD activities (haplotype D3, a decreased DPD activity; haplotype F2, an increased DPD activity). Functional analysis of eight recombinant mutant DPD enzymes showed a reduced DPD activity, ranging from 35% to 84% of the wild-type enzyme. Analysis of a DPD homology model indicated that the structural effect of the novel p.G401R mutation is most likely minor. The clinical relevance of the p.D949V mutation was demonstrated in a cancer patient heterozygous for the c.2846A>T mutation and a novel nonsense mutation c.1681C>T (p.R561X), experiencing severe grade IV toxicity. Our studies showed that the endogenous levels of uracil and the U/UH2 ratio are poor predictors of an impaired DPD activity. Loading studies with uracil to identify patients with a DPD deficiency warrants further investigation.

Dihydropyrimidinase deficiency in four East Asian patients due to novel and rare DPYS mutations affecting protein structural integrity and catalytic activity.

Dihydropyrimidinase (DHP) is the second enzyme of the pyrimidine degradation pathway and catalyzes the ring opening of 5,6-dihydrouracil and 5,6-dihydrothymine. To date, only 31 genetically confirmed patients with a DHP deficiency have been reported and the clinical, biochemical and genetic spectrum of DHP deficient patients is, therefore, still largely unknown. Here, we show that 4 newly identified DHP deficient patients presented with strongly elevated levels of 5,6-dihydrouracil and 5,6-dihydrothymine in urine and a highly variable clinical presentation, ranging from asymptomatic to infantile spasm and reduced white matter and brain atrophy. Analysis of the DHP gene (DPYS) showed the presence of 8 variants including 4 novel/rare missense variants and one novel deletion. Functional analysis of recombinantly expressed DHP mutants carrying the p.M250I, p.H295R, p.Q334R, p.T418I and the p.R490H variant showed residual DHP activities of 2.0%, 9.8%, 9.7%, 64% and 0.3%, respectively. The crystal structure of human DHP indicated that all point mutations were likely to cause rearrangements of loops shaping the active site, primarily affecting substrate binding and stability of the enzyme. The observation that the identified mutations were more prevalent in East Asians and the Japanese population indicates that DHP deficiency may be more common than anticipated in these ethnic groups.

Novel PRPS1 gain-of-function mutation in a patient with congenital hyperuricemia and facial anomalies.

Phosphoribosylpyrophosphate synthetase (PRPPS) superactivity (OMIM 300661) is a rare inborn error of purine metabolism that is caused by gain-of-function mutations in the X-chromosomal gene PRPS1 (Xq22.3). Clinical characteristics include congenital hyperuricemia and hyperuricosuria, gouty arthritis, urolithiasis, developmental delay, hypotonia, recurrent infections, short stature, and hearing loss. Only eight families with PRPPS superactivity and PRPS1 gain-of-function mutations have been reported to date. We report on a 7-year-old boy with congenital hyperuricemia, urolithiasis, developmental delay, short stature, hypospadias, and facial dysmorphisms. His mother also suffered from hyperuricemia that was diagnosed at age 13 years. A novel PRPS1 missense mutation (c.573G>C, p.[Leu191Phe]) was detected in the proband and his mother. Enzyme activity analysis confirmed superactivity of PRPP synthetase. Analysis of the crystal structure of human PRPPS suggests that the Leu191Phe mutation affects the architecture of both allosteric sites, thereby preventing the allosteric inhibition of the enzyme. The family reported here broadens the clinical spectrum of PRPPS superactivity and indicates that this rare metabolic disorder might be associated with a recognizable facial gestalt.

Clinical, biochemical and molecular analysis of 13 Japanese patients with ß-ureidopropionase deficiency demonstrates high prevalence of the c.977G > A (p.R326Q) mutation [corrected].

ß-ureidopropionase (ßUP) deficiency is an autosomal recessive disease characterized by N-carbamyl-ß-amino aciduria. To date, only 16 genetically confirmed patients with ßUP deficiency have been reported. Here, we report on the clinical, biochemical and molecular findings of 13 Japanese ßUP deficient patients. In this group of patients, three novel missense mutations (p.G31S, p.E271K, and p.I286T) and a recently described mutation (p.R326Q) were identified. The p.R326Q mutation was detected in all 13 patients with eight patients being homozygous for this mutation. Screening for the p.R326Q mutation in 110 Japanese individuals showed an allele frequency of 0.9 %. Transient expression of mutant ßUP enzymes in HEK293 cells showed that the p.E271K and p.R326Q mutations cause profound decreases in activity (≤ 1.3 %). Conversely, ßUP enzymes containing the p.G31S and p.I286T mutations possess residual activities of 50 and 70 %, respectively, suggesting we cannot exclude the presence of additional mutations in the non-coding region of the UPB1 gene. Analysis of a human ßUP homology model revealed that the effects of the mutations (p.G31S, p.E271K, and p.R326Q) on enzyme activity are most likely linked to improper oligomer assembly. Highly variable phenotypes ranging from neurological involvement (including convulsions and autism) to asymptomatic, were observed in diagnosed patients. High prevalence of p.R326Q in the normal Japanese population indicates that ßUP deficiency is not as rare as generally considered and screening for ßUP deficiency should be included in diagnosis of patients with unexplained neurological abnormalities.

Phosphoribosylpyrophosphate synthetase superactivity and recurrent infections is caused by a p.Val142Leu mutation in PRS-I.

We identified a novel missense mutation, c.424G>C (p.Val142Leu) in PRPS1 in a patient with uric acid overproduction without gout but with developmental delay, hypotonia, hearing loss, and recurrent respiratory infections. The uric acid overproduction accompanying this combination of symptoms suggests that the patient presented with phosphoribosylpyrophosphate (PRPP) synthetase superactivity, but recurrent infections have not been associated with superactivity until now. However, recurrent infections are a prominent feature of patients with Arts syndrome, which is caused by PRPS1 loss-of-function mutations, indicating that the patient reported here has an intermediate phenotype. Molecular modeling predicts that the p.Val142Leu change affects both allosteric sites that are involved in inhibition of PRPS1 and the ATP-binding site, which suggests that this substitution can result both in a gain-of-function and loss-of-function of PRPP synthetase. This finding is in line with the normal PRPP synthetase activity in fibroblasts and the absence of activity in erythrocytes of the present patient. We postulate that the overall effect of the p.Val142Leu change on protein activity is determined by the cell type, being a gain-of-function in proliferating cells and a loss-of-function in postmitotic cells. Our results show that missense mutations in PRPS1 can cause a continuous spectrum of features ranging from progressive non-syndromic postlingual hearing impairment to uric acid overproduction, neuropathy, and recurrent infections depending on the functional sites that are affected.

6-mercaptopurine inhibits atherosclerosis in apolipoprotein e*3-leiden transgenic mice through atheroprotective actions on monocytes and macrophages.

OBJECTIVE: 6-Mercaptopurine (6-MP), the active metabolite of the immunosuppressive prodrug azathioprine, is commonly used in autoimmune diseases and transplant recipients, who are at high risk for cardiovascular disease. Here, we aimed to gain knowledge on the action of 6-MP in atherosclerosis, with a focus on monocytes and macrophages. METHODS AND RESULTS: We demonstrate that 6-MP induces apoptosis of THP-1 monocytes, involving decreased expression of the intrinsic antiapoptotic factors B-cell CLL/Lymphoma-2 (Bcl-2) and Bcl2-like 1 (Bcl-x(L)). In addition, we show that 6-MP decreases expression of the monocyte adhesion molecules platelet endothelial adhesion molecule-1 (PECAM-1) and very late antigen-4 (VLA-4) and inhibits monocyte adhesion. Screening of a panel of cytokines relevant to atherosclerosis revealed that 6-MP robustly inhibits monocyte chemoattractant chemokine-1 (MCP-1) expression in macrophages stimulated with lipopolysaccharide (LPS). Finally, local delivery of 6-MP to the vessel wall, using a drug-eluting cuff, attenuates atherosclerosis in hypercholesterolemic apolipoprotein E*3-Leiden transgenic mice (P<0.05). In line with our in vitro data, this inhibition of atherosclerosis by 6-MP was accompanied with decreased lesion monocyte chemoattractant chemokine-1 levels, enhanced vascular apoptosis, and reduced macrophage content. CONCLUSIONS: We report novel, previously unrecognized atheroprotective actions of 6-MP in cultured monocytes/macrophages and in a mouse model of atherosclerosis, providing further insight into the effect of the immunosuppressive drug azathioprine in atherosclerosis.

Co-therapy with S-adenosylmethionine and nicotinamide riboside improves t-cell survival and function in Arts Syndrome (PRPS1 deficiency).

Arts syndrome or phosphoribosyl-pyrophosphate-synthetase-1 (PRPS1) deficiency is caused by loss-of-function mutations in the PRPS1 gene (Xq22.3). PRPS1 is an initial and essential step for the synthesis of the nucleotides of purines, pyrimidines, and nicotinamide. Classically, affected males present with sensorineural hearing loss, optic atrophy, muscular hypotonia, developmental impairment, and recurrent severe respiratory infections early in life. Treatment of a 3-year old boy with S-adenosylmethionine (SAM) replenished erythrocyte purine nucleotides of adenosine and guanosine, while SAM and nicotinamide riboside co-therapy further improved his clinical phenotype as well as T-cell survival and function.

Atypical presentation of Arts syndrome due to a novel hemizygous loss-of-function variant in the PRPS1 gene.

The PRPS1 gene, located on Xq22.3, encodes phosphoribosyl-pyrophosphate synthetase (PRPS), a key enzyme in de novo purine synthesis. Three clinical phenotypes are associated with loss-of-function PRPS1 variants and decreased PRPS activity: Arts syndrome (OMIM: 301835), Charcot-Marie-Tooth disease type 5 (CMTX5, OMIM: 311070), and nonsyndromic X-linked deafness (DFN2, OMIM: 304500). Hearing loss is present in all cases. CMTX5 patients also show peripheral neuropathy and optic atrophy. Arts syndrome includes developmental delay, intellectual disability, ataxia, and susceptibility to infections, in addition to the above three features. Gain-of-function PRPS1 variants result in PRPS superactivity (OMIM: 300661) with hyperuricemia and gout. We report a 6-year-old boy who presented with marked generalized muscular hypotonia, global developmental delay, lack of speech, trunk instability, exercise intolerance, hypomimic face with open mouth, oropharyngeal dysphagia, dysarthria, and frequent upper respiratory tract infections. However, his nerve conduction velocity, audiologic, and funduscopic investigations were normal. A novel hemizygous variant, c.130A > G p.(Ile44Val), was found in the PRPS1 gene by panel sequencing. PRPS activity in erythrocytes was markedly reduced, confirming the pathogenicity of the variant. Serum uric acid and urinary purine and pyrimidine metabolite levels were normal. In conclusion, we present a novel PRPS1 loss-of-function variant in a patient with some clinical features of Arts syndrome, but lacking a major attribute, hearing loss, which is congenital/early-onset in all other reported Arts syndrome patients. In addition, it is important to acknowledge that normal levels of serum and urinary purine and pyrimidine metabolites do not exclude PRPS1-related disorders.

Prenatal growth restriction, retinal dystrophy, diabetes insipidus and white matter disease: expanding the spectrum of PRPS1-related disorders.

PRPS1 codes for the enzyme phosphoribosyl pyrophosphate synthetase-1 (PRS-1). The spectrum of PRPS1-related disorders associated with reduced activity includes Arts syndrome, Charcot-Marie-Tooth disease-5 (CMTX5) and X-linked non-syndromic sensorineural deafness (DFN2). We describe a novel phenotype associated with decreased PRS-1 function in two affected male siblings. Using whole exome and Sanger sequencing techniques, we identified a novel missense mutation in PRPS1. The clinical phenotype in our patients is characterized by high prenatal maternal α-fetoprotein, intrauterine growth restriction, dysmorphic facial features, severe intellectual disability and spastic quadraparesis. Additional phenotypic features include macular coloboma-like lesions with retinal dystrophy, severe short stature and diabetes insipidus. Exome sequencing of the two affected male siblings identified a shared putative pathogenic mutation c.586C>T p.(Arg196Trp) in the PRPS1 gene that was maternally inherited. Follow-up testing showed normal levels of hypoxanthine in urine samples and uric acid levels in blood serum. The PRS activity was significantly reduced in erythrocytes of the two patients. Nucleotide analysis in erythrocytes revealed abnormally low guanosine triphosphate and guanosine diphosphate. This presentation is the most severe form of PRPS1-deficiency syndrome described to date and expands the spectrum of PRPS1-related disorders.

Hydrogen sulfide donor NaHS reduces organ injury in a rat model of pneumococcal pneumosepsis, associated with improved bio-energetic status.

Sepsis is characterized by a generalized inflammatory response and organ failure, associated with mitochondrial dysfunction. Hydrogen sulfide donor NaHS has anti-inflammatory properties, is able to reduce metabolism and can preserve mitochondrial morphology and function. Rats were challenged with live Streptococcus pneumonia or saline and infused with NaHS (36 µmol/kg/h) or vehicle. Lung and kidney injury markers were measured as well as mitochondrial function, viability and biogenesis. Infusion of NaHS reduced heart rate and body temperature, indicative of a hypo-metabolic state. NaHS infusion reduced sepsis-related lung and kidney injury, while host defense remained intact, as reflected by unchanged bacterial outgrowth. The reduction in organ injury was associated with a reversal of a fall in active oxidative phosphorylation with a concomitant decrease in ATP levels and ATP/ADP ratio. Preservation of mitochondrial respiration was associated with increased mitochondrial expression of α-tubulin and protein kinase C-ε, which acts as regulators of respiration. Mitochondrial damage was decreased by NaHS, as suggested by a reduction in mitochondrial DNA leakage in the lung. Also, NaHS treatment was associated with upregulation of peroxisome proliferator-activated receptor-γ coactivator 1α, with a subsequent increase in transcription of mitochondrial respiratory subunits. These findings indicate that NaHS reduces organ injury in pneumosepsis, possibly via preservation of oxidative phosphorylation and thereby ATP synthesis as well as by promoting mitochondrial biogenesis. Further studies on the involvement of mitochondria in sepsis are required.

Cholestasis is associated with hepatic microvascular dysfunction and aberrant energy metabolism before and during ischemia-reperfusion.

AIMS: The aim was to investigate the impact of ischemia-reperfusion (I/R) on intrahepatic oxidative stress, oxidative phosphorylation, and nucleotide metabolism in relation to liver damage and inflammation in cholestatic rats to elucidate the molecular mechanisms responsible for post-I/R pathogenesis during cholestasis. RESULTS: Pre-I/R cholestatic livers exhibited mild hepatopathology in the form of oxidative/nitrosative stress, perfusion defects, necrosis and apoptosis, inflammation, and fibrosis. Plasma bilirubin concentration in cholestatic livers was 190 µM. I/R in cholestatic livers exacerbated hepatocellular damage and leukocyte infiltration. However, myeloperoxidase activity in neutrophils at 6 h reperfusion was not elevated in cholestatic livers compared to pre-I/R levels and to control (Ctrl) livers. At 6 h reperfusion, cholestatic livers exhibited severe histological damage, which was absent in Ctrl livers. Despite a lower antioxidative capacity after I/R, no cardiolipin peroxidation and equivalent reduced glutathione/oxidized glutathione ratios and Hsp70 levels were found in cholestatic livers versus Ctrls. Bilirubin acted as a potent and protective antioxidant. Postischemic resumption of oxidative phosphorylation in Ctrl livers proceeded rapidly and encompassed reactive hyperemia, which was significantly impaired in cholestatic livers owing to extensive vasoconstriction and perfusion defects. Normalization of intrahepatic energy status and nucleotide-based metabolic cofactors was delayed in cholestatic livers during reperfusion. Innovation and CONCLUSIONS: Cholestatic livers possess sufficient antioxidative capacity to ameliorate radical-mediated damage during I/R. I/R-induced damage in cholestatic livers is predominantly caused by microvascular perfusion defects rather than exuberant oxidative/nitrosative stress. The forestalled rate of oxidative phophorylation and recovery of bioenergetic and possibly metabolic parameters during the early reperfusion phase are responsible for extensive liver damage.

Mycophenolate mofetil inhibits T-cell proliferation in kidney transplant recipients without lowering intracellular dGTP and GTP.

To study if mycophenolic acid (MPA), the active metabolite of mycophenolate mofetil (MMF), indeed inhibits T-cell proliferation in kidney transplant recipients by lowering intracellular deoxyguanosine triphosphate (dGTP) and guanosine triphosphate (GTP) levels. Blood was drawn from 11 kidney transplant recipients. Ex vivo T-cell proliferation was measured by stimulation with phytohemagglutin (PHA) and anti-CD3 monoclonal antibody (mAb). Plasma MPA levels and intracellular dGTP and GTP in peripheral blood mononuclear cells were measured. MMF induces a significant decrease in T-lymphocyte proliferation at all time points (i.e. 24 h, 10 days and 8 weeks) after stimulation with both PHA (P = 0.001, 0.002 and 0.013 respectively) and anti-CD3 mAb (P = 0.004, 0.004 and 0.005 respectively). There was no significant change in intracellular dGTP (P = 0.31, 0.16 and 0.35) or GTP levels (P = 0.99, 0.32 and 0.49) between baseline and day 1, day 10 or week 8. All MPA levels were above the minimal required concentration for the inhibition of lymphocyte proliferation. MMF inhibits T-lymphocyte proliferation in kidney transplant recipients without lowering intracellular dGTP or GTP levels. This suggests another mechanism underlying its immunosuppressive capacity.