Biomarkers to predict disease progression and therapeutic response in isolated methylmalonic acidemia.

Methylmalonic Acidemia (MMA) is a heterogenous group of inborn errors of metabolism caused by a defect in the methylmalonyl-CoA mutase (MMUT) enzyme or the synthesis and transport of its cofactor, 5'-deoxy-adenosylcobalamin. It is characterized by life-threatening episodes of ketoacidosis, chronic kidney disease, and other multiorgan complications. Liver transplantation can improve patient stability and survival and thus provides clinical and biochemical benchmarks for the development of hepatocyte-targeted genomic therapies. Data are presented from a US natural history protocol that evaluated subjects with different types of MMA including mut-type (N = 91), cblB-type (15), and cblA-type MMA (17), as well as from an Italian cohort of mut-type (N = 19) and cblB-type MMA (N = 2) subjects, including data before and after organ transplantation in both cohorts. Canonical metabolic markers, such as serum methylmalonic acid and propionylcarnitine, are variable and affected by dietary intake and renal function. We have therefore explored the use of the 1-13 C-propionate oxidation breath test (POBT) to measure metabolic capacity and the changes in circulating proteins to assess mitochondrial dysfunction (fibroblast growth factor 21 [FGF21] and growth differentiation factor 15 [GDF15]) and kidney injury (lipocalin-2 [LCN2]). Biomarker concentrations are higher in patients with the severe mut0 -type and cblB-type MMA, correlate with a decreased POBT, and show a significant response postliver transplant. Additional circulating and imaging markers to assess disease burden are necessary to monitor disease progression. A combination of biomarkers reflecting disease severity and multisystem involvement will be needed to help stratify patients for clinical trials and assess the efficacy of new therapies for MMA.

Organic acidurias in Egyptian children: The urge for high-risk screening.

BACKGROUND: Organic acidurias are a group of inborn errors of metabolism. They present a significant diagnostic challenge and are associated with serious morbidity and mortality. They are considered the most frequent inborn errors of metabolism among high-risk children. Gas chromatography-mass spectrometry is a reliable diagnostic technique for organic acidurias. This hospital-based study aimed to quantify the frequency of organic acidurias among a group of high-risk Egyptian pediatric patients and to highlight the importance of high-risk screening for such disorders. METHODS: One hundred and fifty high-risk children who presented to the inherited metabolic disease unit and the pediatric intensive care units of Cairo University Children Hospital were tested for urine organic acids using gas chromatography-mass spectrometry. RESULTS: Thirty percent (45/150) of the patients were confirmed as having an altered organic acids profile. Neurological manifestations were the most common presentation. Glutaric aciduria type I and maple-syrup urine disease were the most common disorders encountered among the group that was studied. CONCLUSION: Organic acid detection by gas chromatography-mass spectrometry is key to the diagnosis of many metabolic disorders. Until a national expanded newborn screening program is established, high-risk screening is strongly encouraged for the early detection of, and proper intervention for such disorders among Egyptian children.

Exploiting in silico structural analysis to introduce emerging genotype-phenotype correlations in DHCR24-related sterol biosynthesis disorder: a case study.

Desmosterolosis is a rare sterol biosynthesis disorder characterized by multiple congenital anomalies, failure to thrive, severe developmental delay, progressive epileptic encephalopathy, and elevated levels of desmosterol caused by biallelic mutations of DHCR24 encoding 3-ß-hydroxysterol Δ-24-reductase. DHCR24 is regarded as the key enzyme of cholesterol synthesis in the metabolism of brain cholesterol as it catalyzes the reduction of the Δ-24 double bond of sterol intermediates during cholesterol biosynthesis. To date, 15 DHCR24 variants, detected in 2 related and 14 unrelated patients, have been associated with the desmosterolosis disorder. Here, we describe a proband harboring the never-described DHCR24 homozygous missense variant NM_014762.4:c.506T>C, NP_055577.1:p.M169T, whose functional validation was confirmed through biochemical assay. By using molecular dynamics simulation techniques, we investigated the impact of this variant on the protein stability and interaction network with the flavin adenine dinucleotide cofactor, thereby providing a preliminary assessment of its mechanistic role in comparison to all known pathogenic variants, the wild-type protein, and a known benign DHCR24 variant. This report expands the clinical and molecular spectra of the DHCR24-related disorder, reports on a novel DHCR24 deleterious variant associated with desmosterolosis, and gives new insights into genotype-phenotype correlations.

High Incidence of Partial Biotinidase Deficiency in the First 3 Years of a Regional Newborn Screening Program in Italy.

Biotinidase deficiency (BD) is an autosomal recessive inherited disorder in which the enzyme biotinidase is totally or partially defective and the vitamin biotin is not recycled. BD meets the major criteria for a population screening program. Newborn bloodspot screening (NBS) allows early diagnosis of BD, thus preventing the high morbidity and mortality associated with untreated disease. Both profound and partial BD variant can be detected by NBS test, and serum enzyme activity and/or mutational analysis are required for definitive diagnosis. In Italy, BD is included in the screening panel for inborn errors of metabolism (IEMs) that has been declared mandatory in 2016. We analyzed the data of the first 3 years of the NBS for BD in our region (Abruzzo, Italy), with the aim to describe the outcomes of this recently introduced screening program. In over 26,393 newborns screened, we found 2 carriers and 16 cases with genotype associated with partial BD. Since the serum biotinidase assay has been recently introduced in our algorithm, only three of our newborns met the criteria of genetic and biochemical confirmation, with an incidence of 1:8797, which is in the high range of what has been reported in the literature. All affected infants carried the 1330G>C (D444H) variant in compound heterozygosis, with variants known to be associated with profound BD. A variant previously not described and likely pathogenic was found in one newborn. None of the infants had signs or symptoms. The study of the distribution of the enzyme activity in our population allowed us to validate the adopted cutoff with which the program has a positive predictive value of 18% and to analyze some preanalytical factors influencing biotinidase activity: A correlation of the enzyme activity with gestational age and time at specimen collection was found. Lower mean values of enzyme activity were found in infants born in the summer.

ABCD1 gene mutation in an Italian family with X-linkedadrenoleukodystrophy: case series.

SUMMARY: Adrenoleukodystrophy is a peroxisomal X-linked recessive disease caused by mutations in the ABCD1 gene, located on the X-chromosome (Xq28). Gene mutations in patient with adrenoleukodystrophy induce metabolic alterations characterized by impaired peroxisomal beta-oxidation and accumulation of very long chain fatty acid (VLCFA) in plasma and in all tissues. Although nutritional intervention associated with a various mixture of oil prevents the accumulation of VLCFA, to date no causal treatment is available. Therefore, haematopoietic stem cell transplantation (HSCT) and gene therapy are allowed only for very early stages of cerebral forms diagnosed during childhood.We reported a case series describing five family members affected by X-linked adrenoleukodystrophy caused by a novel mutation of the ABCD1 gene. Particularly, three brothers were affected while the sister and mother carried the mutation of the ABCD1 gene. In this family, the disease was diagnosed at different ages and with different clinical pictures highlighting the wide range of phenotypes related to this novel mutation. In addition, these characteristics stress the relevant role of early diagnosis to properly set a patient-based follow-up. LEARNING POINTS: We report a novel mutation in the ABCD1 gene documented in a family group associated to an X-ALD possible Addison only phenotype. All patients present just Addison disease but with different phenotypes despite the presence of the same mutations. Further follow-up is necessary to complete discuss the clinical development. The diagnosis of ALD needs to be included in the differential diagnosis in all patients with idiopathic PAI through accurate evaluation of VLCFA concentrations and genetic confirmation testing. Early diagnosis of neurological manifestation is important in order to refer timely to HSCT. Further follow-up of these family members is necessary to characterize the final phenotype associated with this new mutation.

Vitamin D status in Hashimoto's thyroiditis and its association with vitamin D receptor genetic variants.

BACKGROUND: Hashimoto's thyroiditis (HT) is considered the predominant cause of hypothyroidism in iodine sufficient countries. The deficiency of 25-OH-vitamin D3 serum level and the variation of vitamin D receptor (VDR) gene were implicated in a number of autoimmune disorders. This study aimed to test the hypothesis linking between VDR FokI and BsmI variants and HT, in addition to explain their impact on 25-OH-vitamin D3 serum level. MATERIALS AND METHODS: Cross sectional study included 160 hypothyroid subjects, 112 patients with HT and 48 hypothyroid non-HT controls. They were diagnosed based on anti-TPO Ab and or anti-TG Ab results. All cases were subjected to full history taking, thyroid ultrasound examination and a panel of assays (TSH, f.T3, f.T4, anti-TPO Ab, anti-TG Ab, calcium, alkaline phosphatase and phosphate). Serum 25-OH-vitamin D3 was assayed using HPLC-UV method. VDR variants (FokI and BsmI) were genotyped using real-time PCR. RESULTS: FokI AA genotype was statistically higher in HT patients than control group (P value = 0.02) with subsequently higher serum 25-OH-vitamin D3 level in comparison to all other genotypes (P value = 0.039). Serum 25-OH-vitamin D3 level was statistically indifferent between HT and control group (P value = 0.223). A statistically significant increase in total thyroid volume was observed in HT group (P value = 0.002). CONCLUSION: FokI AA genotype is more associated with HT in Egyptian patients compared to hypothyroid non-HT controls. Moreover, patients with FokI AA genotype have statistically higher levels of 25-OH-vitamin D3 suggesting VDR dysfunction even in patients expressing normal level of vitamin D.

Partial Biotinidase Deficiency Revealed Imbalances in Acylcarnitines Profile at Tandem Mass Spectrometry Newborn Screening.

Biotinidase (BTD) deficiency is an autosomal recessive inherited neurocutaneous disorder. BTD recycles the vitamin biotin, a coenzyme essential for the function of four biotin-dependent carboxylases, including propionyl-CoA carboxylase, 3-methylcrotonyl-CoA carboxylase, pyruvate carboxylase, and acetyl-CoA carboxylase. Due to deficient activities of the carboxylases, BTD deficiency is also recognized as late-onset multiple carboxylase deficiency and is associated with secondary alterations in the metabolism of amino acids, carbohydrates, and fatty acids. BTD deficiency can be classified as "profound", with less than 10% of mean normal activity, and as "partial" with 10-30% of mean normal activity. Newborn screening (NBS) of BTD deficiency is performed in most countries and is able to detect both variants. Moreover, mild metabolic alterations related to carboxylase deficiency in profound BTD deficiency could result and possibly be revealed in the metabolic profile by tandem mass spectrometry (MS/MS) NBS. Here, we report the case of a newborn female infant with an initial suspected BTD deficiency at the NBS test, finally confirmed as a partial variant by molecular testing. Although BTD deficiency was partial, interestingly her metabolic profile at birth and during the follow-up tests revealed, for the first time, alterations in specific acylcarnitines as a possible result of the deficient activity of biotin-dependent carboxylases.

A new UHPLC-MS/MS method for the screening of urinary oligosaccharides expands the detection of storage disorders.

BACKGROUND: Oligosaccharidoses are storage disorders due to enzymatic defects involved in the breakdown of the oligosaccharidic component of glycosylated proteins. The defect cause the accumulation of oligosaccharides (OS) and, depending on the lacking enzyme, results in characteristic profiles which are helpful for the diagnosis. We developed a new tandem mass spectrometry method for the screening of urinary OS which was applied to identify a large panel of storage disorders. METHODS: The method was set-up in urine and dried urine spots (DUS). Samples were analysed, without derivatization and using maltoheptaose as internal standard, by UHPLC-MS/MS with MRM acquisition of target OS transitions, including Glc4, the biomarker of Pompe disease. The chromatographic run was < 30 min. Samples from patients with known storage disorders were used for clinical validation. RESULTS: The method allowed to confirm the diagnosis of oligosaccharidoses (sialidosis, α-/ß-mannosidosis, fucosidosis, aspartylglucosaminuria) and of GM1 and GM2 (Sandhoff type) gangliosidosis, by detecting specific OS profiles. In other storage disorders (mucolipidosis II and III, mucopolysaccharidosis type IVB) the analyisis revealed abnormal OS excretion with non-specific profiles. Besides Pompe disease, the tetrasaccharide Glc4 was increased also in disorders of autophagy (Vici syndrome, Yunis-Varon syndrome, and Danon disease) presenting cardiomuscular involvement with glycogen storage. Overall, results showed a clear separation between patients and controls, both in urine and in DUS. CONCLUSION: This new UHPLC/MS-MS method, which is suitable for rapid and easy screening of OS in urine and DUS, expands the detection of storage disorders from oligosaccharidoses to other diseases, including the novel category of inherited disorders of autophagy.

Delineating the neurological phenotype in children with defects in the ECHS1 or HIBCH gene.

The neurological phenotype of 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) and short-chain enoyl-CoA hydratase (SCEH) defects is expanding and natural history studies are necessary to improve clinical management. From 42 patients with Leigh syndrome studied by massive parallel sequencing, we identified five patients with SCEH and HIBCH deficiency. Fourteen additional patients were recruited through collaborations with other centres. In total, we analysed the neurological features and mutation spectrum in 19 new SCEH/HIBCH patients. For natural history studies and phenotype to genotype associations we also included 70 previously reported patients. The 19 newly identified cases presented with Leigh syndrome (SCEH, n = 11; HIBCH, n = 6) and paroxysmal dystonia (SCEH, n = 2). Basal ganglia lesions (18 patients) were associated with small cysts in the putamen/pallidum in half of the cases, a characteristic hallmark for diagnosis. Eighteen pathogenic variants were identified, 11 were novel. Among all 89 cases, we observed a longer survival in HIBCH compared to SCEH patients, and in HIBCH patients carrying homozygous mutations on the protein surface compared to those with variants inside/near the catalytic region. The SCEH p.(Ala173Val) change was associated with a milder form of paroxysmal dystonia triggered by increased energy demands. In a child harbouring SCEH p.(Ala173Val) and the novel p.(Leu123Phe) change, an 83.6% reduction of the protein was observed in fibroblasts. The SCEH and HIBCH defects in the catabolic valine pathway were a frequent cause of Leigh syndrome in our cohort. We identified phenotype and genotype associations that may help predict outcome and improve clinical management.

Plasma methylcitric acid and its correlations with other disease biomarkers: The impact in the follow up of patients with propionic and methylmalonic acidemia.

Methylcitric acid (MCA) analysis has been mainly utilized for the diagnosis of propionate disorders or as a second-tier test in newborn screening, but its utility for patients monitoring still needs to be established. We explored the potential contribution of MCA in the long-term management of organic acidurias. We prospectively evaluated plasma MCA and its relationship with disease biomarkers, clinical status, and disease burden in 22 patients, 13 with propionic acidemia (PA) and nine with methylmalonic acidemia (MMA) on standard treatment and/or after transplantation. Samples were collected at scheduled routine controls or during episodes of metabolic decompensation (MD), 10 patients were evaluated after transplantation (six liver, two combined liver and kidney, 2 kidney). MCA levels were higher in PA compared to MMA and its levels were not influenced by the clinical status (MD vs well state). In MMA, MCA was higher in elder patients and, along with fibroblast growth factor 21 (FGF21) and plasma methylmalonic acid, negatively correlated with GFR. In both diseases, MCA correlated with ammonia, glycine, lysine, C3, and the C3/C2, C3/C16 ratios. The disease burden showed a direct correlation with MCA and FGF21, for both diseases. All transplanted patients showed a significant reduction of MCA in comparison to baseline values, with some differences dependent on the type of transplantation. Our study provided new insights in understanding the disease pathophysiology, showing similarities between MCA and FGF21 in predicting disease burden, long-term complications and in evaluating the impact of organ transplantation.

A False-Positive Case of Methylmalonic Aciduria by Tandem Mass Spectrometry Newborn Screening Dependent on Maternal Malnutrition in Pregnancy.

Methylmalonic Acidurias (MMAs) are a group of inborn errors of metabolism (IEMs), specifically of propionate catabolism characterized by gastrointestinal and neurometabolic manifestations resulting from a deficiency in the function of methylmalonyl-CoA mutase, methylmalonyl-CoA epimerase, and cobalamin metabolism. In Expanded Newborn Screening (NBS), increased levels of propionylcarnitine (C3) and/or of its ratios by MS/MS analysis of dried blood spots (DBS) samples are suggestive for either Propionic Acidemia or MMAs. C3 elevation is not considered a specific marker for these disorders, resulting in high false-positive rates. The use of analyte ratios improves specificity, but it still cannot resolve the diagnostic issue. Second-tier testing are strongly recommended as confirmation of primary NBS results and for a differential diagnosis. LC-MS/MS analysis allows the quantification of more specific markers of the disorder. Here, we report the case of a newborn with a suspected MMA at Expanded NBS and at second-tier test. Given the urgent situation, in-depth diagnostic investigations were performed. Further investigations surprisingly revealed a Vitamin B12 deficiency due to a maternal malnutrition during pregnancy. This case emphasized that metabolic alterations at NBS may not only be influenced by genome and related to IEMs, but also to external factors and to maternal conditions.

Delayed appearance of 3-methylglutaconic aciduria in neonates with early onset metabolic cardiomyopathies: A potential pitfall for the diagnosis.

Infantile onset cardiomyopathies are highly heterogeneous with several phenocopies compared with adult cardiomyopathies. Multidisciplinary management is essential in determining the underlying etiology in children's cardiomyopathy. Elevated urinary excretion of 3-methylglutaconic acid (3-MGA) is a useful tool in identifying the etiology in some metabolic cardiomyopathy. Here, we report the delayed appearance of 3-MGA-uria, between 6 and 18 months in three patients (out of 100 childhood onset cardiomyopathy) with neonatal onset cardiomyopathy, secondary to TMEM70 mutations and TAZ mutations (Barth syndrome), in whom extensive metabolic investigations, performed in the first weeks of life, did not display 3-MGA-uria. Serial retrospective evaluations showed full characteristic features of TMEM70 and TAZ mutations (Barth syndrome) in these three patients, including a clearly abnormal monolysocardiolipin/cardiolipin ratio in the two Barth syndrome patients. Serially repeated metabolic investigations finally discovered the 3-MGA-uria biomarker in all three patients between the age of 6 and 18 months. Our observation provides novel insights into the temporal appearance of 3-MGA-uria in TMEM70 and TAZ mutations (Barth syndrome) and focus the importance of multidisciplinary management and careful evaluation of family history and red flag signs for phenocopies in infantile onset cardiomyopathies.

Gut microbiota signatures in cystic fibrosis: Loss of host CFTR function drives the microbiota enterophenotype.

BACKGROUND: Cystic fibrosis (CF) is a disorder affecting the respiratory, digestive, reproductive systems and sweat glands. This lethal hereditary disease has known or suspected links to the dysbiosis gut microbiota. High-throughput meta-omics-based approaches may assist in unveiling this complex network of symbiosis modifications. OBJECTIVES: The aim of this study was to provide a predictive and functional model of the gut microbiota enterophenotype of pediatric patients affected by CF under clinical stability. METHODS: Thirty-one fecal samples were collected from CF patients and healthy children (HC) (age range, 1-6 years) and analysed using targeted-metagenomics and metabolomics to characterize the ecology and metabolism of CF-linked gut microbiota. The multidimensional data were low fused and processed by chemometric classification analysis. RESULTS: The fused metagenomics and metabolomics based gut microbiota profile was characterized by a high abundance of Propionibacterium, Staphylococcus and Clostridiaceae, including Clostridium difficile, and a low abundance of Eggerthella, Eubacterium, Ruminococcus, Dorea, Faecalibacterium prausnitzii, and Lachnospiraceae, associated with overexpression of 4-aminobutyrate (GABA), choline, ethanol, propylbutyrate, and pyridine and low levels of sarcosine, 4-methylphenol, uracil, glucose, acetate, phenol, benzaldehyde, and methylacetate. The CF gut microbiota pattern revealed an enterophenotype intrinsically linked to disease, regardless of age, and with dysbiosis uninduced by reduced pancreatic function and only partially related to oral antibiotic administration or lung colonization/infection. CONCLUSIONS: All together, the results obtained suggest that the gut microbiota enterophenotypes of CF, together with endogenous and bacterial CF biomarkers, are direct expression of functional alterations at the intestinal level. Hence, it's possible to infer that CFTR impairment causes the gut ecosystem imbalance.This new understanding of CF host-gut microbiota interactions may be helpful to rationalize novel clinical interventions to improve the affected children's nutritional status and intestinal function.

The urinary organic acids profile in single large-scale mitochondrial DNA deletion disorders.

Single large-scale mitochondrial DNA deletions disorders are classified into three main phenotypes with frequent clinical overlap: Pearson marrow-pancreas syndrome (PMS), Kearns-Sayre syndrome (KSS) and chronic progressive external ophtalmoplegia (PEO). So far, only few anecdotal studies have reported on the urinary organic acids profile in this disease class. In this single-center retrospective study, we performed quantitative evaluation of urinary organic acids in a series of 15 pediatric patients, 7 with PMS and 8 with KSS. PMS patients showed an organic acids profile almost constantly altered, whereas KSS patients frequently presented with normal profiles. Lactate, 3-hydroxybutyrate, 3-hydroxyisobutyrate, fumarate, pyruvate, 2-hydroxybutyrate, 2-ethyl-3-hydroxypropionate, and 3-methylglutaconate represented the most frequent metabolites observed in PMS urine. We also found novel metabolites, 3-methylglutarate, tiglylglycine and 2-methyl-2,3-dihydroxybutyrate, so far never reported in this disease. Interestingly, patients with a disease onset as PMS evolving overtime into KSS phenotype, presented persistent and more pronounced alterations of organic acid signature than in patients with a pure KSS phenotype. Our study shows that the quantitative analysis of urinary organic acid profile represents a helpful tool for the diagnosis of PMS and for the differential diagnosis with other inherited diseases causing abnormal organic acidurias.

3-Methylglutaconic aciduria, a frequent but underrecognized finding in carbamoyl phosphate synthetase I deficiency.

The urea cycle disorder carbamoyl phosphate synthetase I deficiency is an important differential diagnosis in the encephalopathic neonate. This intoxication type inborn error of metabolism often leads to neonatal death or severe and irreversible damage of the central nervous system, even despite appropriate treatment. Timely diagnosis is crucial, but can be difficult on routine metabolite level. Here, we report ten neonates from eight families (finally) diagnosed with CPS1 deficiency at three tertiary metabolic centres. In seven of them the laboratory findings were dominated by significantly elevated urinary 3-methylglutaconic acid levels which complicated the diagnostic process. Our findings are both important for the differential diagnosis of patients with urea cycle disorders and also broaden the differential diagnosis of hyperammonemia associated with 3-methylglutaconic aciduria, which was earlier only reported in TMEM70 and SERAC1 defect.

Expanding the molecular diversity and phenotypic spectrum of glycerol 3-phosphate dehydrogenase 1 deficiency.

Transient infantile hypertriglyceridemia (HTGT1; OMIM #614480) is a rare autosomal recessive disorder, which manifests in early infancy with transient hypertriglyceridemia, hepatomegaly, elevated liver enzymes, persistent fatty liver and hepatic fibrosis. This rare clinical entity is caused by inactivating mutations in the GPD1 gene, which encodes the cytosolic isoform of glycerol-3-phosphate dehydrogenase. Here we report on four patients from three unrelated families of diverse ethnic origins, who presented with hepatomegaly, liver steatosis, hypertriglyceridemia, with or without fasting ketotic hypoglycemia. Whole exome sequencing revealed the affected individuals to harbor deleterious biallelic mutations in the GPD1 gene, including the previously undescribed c.806G > A (p.Arg269Gln) and c.640T > C (p.Cys214Arg) mutations. The clinical features in three of our patients showed several differences compared to the original reports. One subject presented with recurrent episodes of fasting hypoglycemia along with hepatomegaly, hypetriglyceridemia, and elevated liver enzymes; the second showed a severe liver disease, with intrahepatic cholestasis associated with kidney involvement; finally, the third presented persistent hypertriglyceridemia at the age of 30 years. These findings expand the current knowledge of this rare disorder, both with regard to the phenotype and molecular basis. The enlarged phenotypic spectrum of glycerol-3-phosphate dehydrogenase 1 deficiency can mimic other inborn errors of metabolism with liver involvement and should alert clinicians to recognize this entity by considering GPD1 mutations in appropriate clinical settings.

A new multiplex method for the diagnosis of peroxisomal disorders allowing simultaneous determination of plasma very-long-chain fatty acids, phytanic, pristanic, docosahexaenoic and bile acids by high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry.

Peroxisomal disorders (PDs) present with wide phenotypic variability. An appropriate diagnosis requires a complete analysis of peroxisomal metabolites. We developed a multiplex LC-MS/MS method, using atmospheric pressure chemical ionization allowing the simultaneous determination in plasma of very-long-chain fatty acids, phytanic, pristanic, docosahexaenoic acids and di- and tri-hydroxycolestanoic bile acids. Two hundred microliters of plasma extracted with acetonitrile and 200µl extracted with hexane after an acid hydrolysis were combined, evaporated, dissolved in 10µl of methanol and analyzed. The acquisition was in negative-ion mode using multiple reaction monitoring. The method was validated analytically and clinically. Linearity was 0.1-200µmol/l for docosanoic, cis-13-docosenoic, tetracosanoic, cis-15-tetracosenoic and phytanic acids; 0.01-10µmol/l for hexacosanoic acid; 0.02-20µmol/l for di-hydroxycolestanoic, tri-hydroxycolestanoic and pristanic acids; 0.3-300µmol/l for docosahexaenoic acid. Intra-day and inter-day CVs were below 3.88 and 3.98 respectively for all compounds. Samples from patients with known peroxisomal disorders were compared with controls and the method allowed to confirm the diagnosis in all subjects with a 100% sensitivity. The advantage of this multiplex method is to allow in a single chromatographic run the simultaneous determination of a large number of peroxisome biomarkers with a simple preparative phase without derivatization.

Liver transplant in ethylmalonic encephalopathy: a new treatment for an otherwise fatal disease.

Ethylmalonic encephalopathy is a fatal, rapidly progressive mitochondrial disorder caused by ETHE1 mutations, whose peculiar clinical and biochemical features are due to the toxic accumulation of hydrogen sulphide and of its metabolites, including thiosulphate. In mice with ethylmalonic encephalopathy, liver-targeted adeno-associated virus-mediated ETHE1 gene transfer dramatically improved both clinical course and metabolic abnormalities. Reasoning that the same achievement could be accomplished by liver transplantation, we performed living donor-liver transplantation in an infant with ethylmalonic encephalopathy. Unlike the invariably progressive deterioration of the disease, 8 months after liver transplantation, we observed striking neurological improvement with remarkable achievements in psychomotor development, along with dramatic reversion of biochemical abnormalities. These results clearly indicate that liver transplantation is a viable therapeutic option for ETHE1 disease.

Evaluation of plasma cholestane-3ß,5α,6ß-triol and 7-ketocholesterol in inherited disorders related to cholesterol metabolism.

Oxysterols are intermediates of cholesterol metabolism and are generated from cholesterol via either enzymatic or nonenzymatic pathways under oxidative stress conditions. Cholestan-3ß,5α,6ß-triol (C-triol) and 7-ketocholesterol (7-KC) have been proposed as new biomarkers for the diagnosis of Niemann-Pick type C (NP-C) disease, representing an alternative tool to the invasive and time-consuming method of fibroblast filipin test. To test the efficacy of plasma oxysterol determination for the diagnosis of NP-C, we systematically screened oxysterol levels in patients affected by different inherited disorders related with cholesterol metabolism, which included Niemann-Pick type B (NP-B) disease, lysosomal acid lipase (LAL) deficiency, Smith-Lemli-Opitz syndrome (SLOS), congenital familial hypercholesterolemia (FH), and sitosterolemia (SITO). As expected, NP-C patients showed significant increase of both C-triol and 7-KC. Strong increase of both oxysterols was observed in NP-B and less pronounced in LAL deficiency. In SLOS, only 7-KC was markedly increased, whereas in both FH and in SITO, oxysterol concentrations were normal. Interestingly, in NP-C alone, we observed that plasma oxysterols correlate negatively with patient's age and positively with serum total bilirubin, suggesting the potential relationship between oxysterol levels and hepatic disease status. Our results indicate that oxysterols are reliable and sensitive biomarkers of NP-C.

Thiamine Deficiency in a Developed Country: Acute Lactic Acidosis in Two Neonates Due to Unsupplemented Parenteral Nutrition.

Thiamine is a water-soluble vitamin implicated in several metabolic processes. Its deficiency, due to prolonged parenteral nutrition without adequate vitamin supplementation, can lead to multiorgan failure characterized by cardiovascular impairment and metabolic acidosis refractory to bicarbonate administration. Only thiamine administration allows the remission of symptoms. We report 2 preterm infants with acute thiamine deficiency due to prolonged parenteral nutrition without adequate vitamin supplementation.