Orotic aciduria is a severe, hereditary, life-threatening condition, particularly in newborns. An increased orotic acid (OA) content in urine may be a strong indicator of this condition. In this study, we developed a rapid, simple, highly sensitive diagnostic method for use in monitoring the OA levels in urine samples, which were successfully determined using capillary electrophoresis combined with capacitively coupled contactless conductivity detection (CE-C4D). A straightforward analysis with an increased sensitivity towards OA and an analysis time of approximately 5 min were realized, and the limit of detection of the developed method was 0.014 mg/L in aqueous solution. The optimized composition of the separation electrolyte was 20 mM 2-(N-morpholino)ethanesulfonic acid/histidine with 0.1 mM cetyltrimethylammonium bromide at a pH of 6.5. The sensitivity of the developed method was significantly increased using a sample stacking approach with a 10% acetonitrile (v/v) plug solution. The method was validated, and satisfactory recoveries of 80.0-92.3% were obtained. The amounts of OA in five urine samples were successfully determined.

(1) Background: Atopic dermatitis constitutes one of the most common inflammatory skin manifestations of the pediatric population. The onset of many inborn errors occurs early in life with an AD-like picture associated with a deregulated IgE response. The availability of proteomic tests for the simultaneous evaluation of hundreds of molecules allows for more precise diagnosis in these cases. (2) Methods: Comparative genomic hybridization microarray (Array-CGH) analysis and specific IgE evaluation by using allergenic microarray (ISAC) and microarray (ALEX2) systems were performed. (3) Results: Proteomic investigations that use multiplex methods have proven to be extremely useful to diagnose the sensitization profile in inborn errors with deregulated IgE synthesis. Four patients with rare diseases, such as recessive X-linked ichthyosis (RXLI, OMIM 308100), Comel-Netherton syndrome (NS, OMIM256500), monosomy 1p36 syndrome (OMIM: 607872), and a microduplication of Xp11.4 associated with extremely high levels of IgE: 7.710 kU/L, 5.300 kU/L, 1.826 kU/L, and 10.430 kU/L, respectively, were evaluated by micro- and macroarray multiplex methods. Polyreactivity to both environmental and food allergens was observed in all cases, including the first described case of association of X-chromosome microduplication and HIE. (4) Conclusions: Extensive use of proteomic diagnostics should be included among the procedures to be implemented in inborn errors with hyper-IgE.

Hypophosphatasia (HPP) is the heritable dento-osseous disease caused by loss-of-function mutation(s) of the gene ALPL that encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). TNSALP is a cell-surface homodimeric phosphomonoester phosphohydrolase expressed in healthy people especially in the skeleton, liver, kidneys, and developing teeth. In HPP, diminished TNSALP activity leads to extracellular accumulation of its natural substrates including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5\'-phosphate (PLP), the principal circulating form of vitamin B6 (B6). Autosomal dominant and autosomal recessive inheritance involving >450 usually missense defects scattered throughout ALPL largely explains the remarkably broad-ranging severity of this inborn-error-of-metabolism. In 1985 when we identified elevated plasma PLP as a biochemical hallmark of HPP, all 14 investigated affected children and adults had markedly increased PLP levels. However, pyridoxal (PL), the dephosphorylated form of PLP that enters cells to cofactor many enzymatic reactions, was not low but often inexplicably elevated. Levels of pyridoxic acid (PA), the B6 degradation product quantified to assess B6 sufficiency, were unremarkable. Canonical signs or symptoms of B6 deficiency or toxicity were absent. B6-dependent seizures in infants with life-threatening HPP were later explained by their profound deficiency of TNSALP activity blocking PLP dephosphorylation to PL and diminishing gamma-aminobutyric acid synthesis in the brain. Now, there is speculation that altered B6 metabolism causes further clinical complications in HPP. Herein, we assessed the plasma PL and PA levels accompanying previously reported elevated plasma PLP concentrations in 150 children and adolescents with HPP. Their mean (SD) plasma PL level was nearly double the mean for our healthy pediatric controls: 66.7 (59.0) nM versus 37.1 (22.2) nM (P < 0.0001), respectively. Their PA levels were broader than our pediatric control range, but their mean value was normal; 40.2 (25.1) nM versus 39.3 (9.9) nM (P = 0.7793), respectively. In contrast, adults with HPP often had plasma PL and PA levels suggestive of dietary B6 insufficiency. We discuss why the B6 levels of our pediatric patients with HPP would not cause B6 toxicity or deficiency, whereas in affected adults dietary B6 insufficiency can develop.

Cystathionine β-synthase (CBS) deficiency is a recessive inborn error of sulfur metabolism characterized by elevated blood levels of total homocysteine (tHcy). Patients diagnosed with CBS deficiency are currently treated by a combination of vitamin supplementation and restriction of foods containing the homocysteine precursor methionine, but the effectiveness of this therapy is limited due to poor compliance. A mouse model for CBS deficiency (Tg-I278T Cbs-/- ) was used to evaluate a potential gene therapy approach to treat CBS deficiency utilizing an AAVrh.10-based vector containing the human CBS cDNA downstream of the constitutive, strong CAG promoter (AAVrh.10hCBS). Mice were administered a single dose of virus and followed for up to 1 year. The data demonstrated a dose-dependent increase in liver CBS activity and a dose-dependent decrease in serum tHcy. Liver CBS enzyme activity at 1 year was similar to Cbs+/- control mice. Mice given the highest dose (5.6 × 1011 genomes/mouse) had mean serum tHcy decrease of 97% 1 week after injection and an 81% reduction 1 year after injection. Treated mice had either full- or substantial correction of alopecia, bone loss, and fat mass phenotypes associated with Cbs deficiency in mice. Our findings show that AAVrh.10-based gene therapy is highly effective in treating CBS deficiency in mice and supports additional pre-clinical testing for eventual use human trials.

The purpose of this review is to summarize current knowledge on canine and feline cystinuria from available scientific reports. Cystinuria is an inherited metabolic defect characterized by abnormal intestinal and renal amino acid transport in which cystine and the dibasic amino acids ornithine, lysine, and arginine are involved (COLA). At a normal urine pH, ornithine, lysine, and arginine are soluble, but cysteine forms a dimer, cystine, which is relatively insoluble, resulting in crystal precipitation. Mutations in genes coding COLA transporter and the mode of inheritance were identified only in some canine breeds. Cystinuric dogs may form uroliths (mostly in lower urinary tract) which are associated with typical clinical symptoms. The prevalence of cystine urolithiasis is much higher in European countries (up to 14% according to the recent reports) when compared to North America (United States and Canada) where it is approximately 1-3%. Cystinuria may be diagnosed by the detection of cystine urolithiasis, cystine crystalluria, assessment of amino aciduria, or using genetic tests. The management of cystinuria is aimed at urolith removal or dissolution which may be reached by dietary changes or medical treatment. In dogs with androgen-dependent cystinuria, castration will help. In cats, cystinuria occurs less frequently in comparison with dogs.

Cystathionine beta-synthase (CBS) is a key enzyme of the trans-sulfuration pathway that converts homocysteine to cystathionine. Loss of CBS activity due to mutation results in CBS deficiency, an inborn error of metabolism characterized by extreme elevation of plasma total homocysteine (tHcy). C57BL6 mice containing either a homozygous null mutation in the cystathionine β-synthase (Cbs-/- ) gene or an inactive human CBS protein (Tg-G307S Cbs-/- ) are born in mendelian numbers, but the vast majority die between 18 and 21 days of age due to liver failure. However, adult Cbs null mice that express a hypomorphic allele of human CBS as a transgene (Tg-I278T Cbs-/- ) show almost no neonatal lethality despite having serum tHcy levels similar to mice with no CBS activity. Here, we characterize liver and serum metabolites in neonatal Cbs+/- , Tg-G307S Cbs-/- , and Tg-I278T Cbs-/- mice at 6, 10, and 17 days of age to understand this difference. In serum, we observe similar elevations in tHcy in both Tg-G307S Cbs-/- and Tg-I278T Cbs-/- compared to control animals, but methionine is much more severely elevated in Tg-G307S Cbs-/- mice. Large scale metabolomic analysis of liver tissue confirms that both methionine and methionine-sulfoxide are significantly more elevated in Tg-G307S Cbs-/- animals, along with significant differences in several other metabolites including hexoses, amino acids, other amines, lipids, and carboxylic acids. Our data are consistent with a model that the neonatal lethality observed in CBS-null mice is driven by excess methionine resulting in increased stress on a variety of related pathways including the urea cycle, TCA cycle, gluconeogenesis, and phosphatidylcholine biosynthesis.

Ethylmalonic acid is a metabolic organic acid, and its accumulation in urine is diagnostic of ethylmalonic aciduria. In this study, a simple and fast method employing capillary electrophoresis equipped with capacitively coupled contactless conductivity detection was developed for the detection of ethylmalonic acid in urine samples. The optimized electrophoretic separation was performed in 50 mmol/L 2-(N-morpholino)ethanesulfonic acid solution, buffered at a pH of 6.5, and contained 0.13 mmol/L cetyltrimethylammonium bromide as an electroosmotic modifier. Electrophoresis was run at 28 kV in reversed polarity. The linear range of ethylmalonic acid concentration was between 1 and 100 mg/L with a regression coefficient of 0.9998. This method had good intra- and interday precision with <5% relative standard deviations. The detection limit (signal-to-noise ratio = 3) and the quantification limit (signal-to-noise ratio = 10) values were 0.139 and 0.466 mg/L, respectively. Using our optimized conditions, the method was successfully employed for the detection of ethylmalonic acid in urine sample of ethylmalonic aciduria patient.

Classical homocystinuria is a recessive inborn error of metabolism caused by mutations in the cystathionine beta-synthase (CBS) gene. The highest incidence of CBS deficiency in the world is found in the country of Qatar due to the combination of high rates of consanguinity and the presence of a founder mutation, c.1006C>T (p.R336C). This mutation does not respond to pyridoxine and is considered severe. Here we describe the creation of a mouse that is null for the mouse Cbs gene and expresses human p.R336C CBS from a zinc-inducible transgene (Tg-R336C Cbs -/- ). Zinc-treated Tg-R336C Cbs -/- mice have extreme elevation in both serum total homocysteine (tHcy) and liver tHcy compared with control transgenic mice. Both the steady-state protein levels and CBS enzyme activity levels in liver lysates from Tg-R336C Cbs -/- mice are significantly reduced compared to that found in Tg-hCBS Cbs -/- mice expressing wild-type human CBS. Treatment of Tg-R336C Cbs -/- mice with the proteasome inhibitor bortezomib results in stabilization of liver CBS protein and an increase in activity to levels found in corresponding Tg-hCBS Cbs -/- wild type mice. Surprisingly, serum tHcy did not fully correct even though liver enzyme activity was as high as control animals. This discrepancy is explained by in vitro enzymatic studies of mouse liver extracts showing that p.R336C causes reduced binding affinity for the substrate serine by almost 7-fold and significantly increased dependence on pyridoxal phosphate in the reaction buffer. These studies demonstrate that the p.R336C alteration effects both protein stability and substrate/cofactor binding.

Hypophosphatasia (HPP) is an inheritable disease affecting both skeletal systems and extra-skeletal organs due to mutations of the gene ALPL, which encodes tissue-nonspecific alkaline phosphatase. Recently, an enzyme replacement therapy using asfotase alfa was developed to ameliorate the complications of HPP. However, it requires frequent injections and is expensive to maintain. As an alternative, cell and gene therapy using human induced pluripotent stem cells (iPSCs) after precise correction of the mutation is feasible due to advances in genome-editing technology. In the study, we examined the alkaline phosphatase (ALP) activity and calcification in vitro of two childhood HPP patient-derived iPSCs after the correction of the c.1559delT mutation, which is the most frequent mutation in Japanese patients with HPP, using transcription activator-like effector nucleases (TALENs). The gene correction targeting vector was designed for site-directed mutagenesis using TALEN. After selection with antibiotics, some clones with the selection cassette were obtained. Gene correction was confirmed by Sanger sequencing. The mutation was corrected in one allele of ALPL in homozygous patients and compound heterozygous patients. The correction of ALPL did not result in an increase in ALP when the selection cassette remained. Conversely, iPSCs exhibited ALP activity after the elimination of the cassette using Cre/LoxP. The quantitative analysis showed the half ALP activity in corrected iPSCs of that of control iPSCs, corresponding to heterozygous correction of the mutation. In addition, osteoblasts differentiated from the corrected iPSCs exhibited high ALP activity and some calcification in vitro. Moreover, the osteoblast-like phenotype was confirmed by increased expression of osteoblast-specific genes such as COL1A1 and osteocalcin. These results suggest that gene correction in iPSCs may be a candidate treatment for HPP patients.

Cystathionine β-synthase (CBS) deficiency is a recessive inborn error of metabolism characterized by extremely elevated total homocysteine (tHcy) in the blood. Patients diagnosed with CBS deficiency have a variety of clinical problems, including dislocated lenses, osteoporosis, cognitive and behavioral issues, and a significantly increased risk of thrombosis. Current treatment strategies involve a combination of vitamin supplementation and restriction of foods containing the homocysteine precursor methionine. Here, a mouse model for CBS deficiency (Tg-I278T Cbs-/-) was used to evaluate the potential of minicircle-based naked DNA gene therapy to treat CBS deficiency. A 2.3 kb DNA-minicircle containing the liver-specific P3 promoter driving the human CBS cDNA (MC.P3-hCBS) was delivered into Tg-I278T Cbs-/- mice via a single hydrodynamic tail vein injection. Mean serum tHcy decreased from 351 μM before injection to 176 μM 7 days after injection (p = 0.0005), and remained decreased for at least 42 days. Western blot analysis reveals significant minicircle-directed CBS expression in the liver tissue. Liver CBS activity increased 34-fold (12.8 vs. 432 units; p = 0.0004) in MC.P3-hCBS-injected animals. Injection of MC.P3-hCBS in young mice, subsequently followed for 202 days, showed that the vector can ameliorate the mouse homocystinuria alopecia phenotype. The present findings show that minicircle-based gene therapy can lower tHcy in a mouse model of CBS deficiency.