Expanded Newborn Screening in Italy Using Tandem Mass Spectrometry: Two Years of National Experience.

Newborn screening (NBS) for inborn errors of metabolism is one of the most advanced tools for secondary prevention in medicine, as it allows early diagnosis and prompt treatment initiation. The expanded newborn screening was introduced in Italy between 2016 and 2017 (Law 167/2016; DM 13 October 2016; DPCM 12-1-2017). A total of 1,586,578 infants born in Italy were screened between January 2017 and December 2020. For this survey, we collected data from 15 Italian screening laboratories, focusing on the metabolic disorders identified by tandem mass spectrometry (MS/MS) based analysis between January 2019 and December 2020. Aminoacidemias were the most common inborn errors in Italy, and an equal percentage was observed in detecting organic acidemias and mitochondrial fatty acids beta-oxidation defects. Second-tier tests are widely used in most laboratories to reduce false positives. For example, second-tier tests for methylmalonic acid and homocysteine considerably improved the screening of CblC without increasing unnecessary recalls. Finally, the newborn screening allowed us to identify conditions that are mainly secondary to a maternal deficiency. We describe the goals reached since the introduction of the screening in Italy by exchanging knowledge and experiences among the laboratories.

Liver-Directed Adeno-Associated Virus-Mediated Gene Therapy for Mucopolysaccharidosis Type VI.

BACKGROUND: Mucopolysaccharidosis type VI (MPS VI) is an inherited multisystem lysosomal disorder due to arylsulfatase B (ARSB) deficiency that leads to widespread accumulation of glycosaminoglycans (GAG), which are excreted in increased amounts in urine. MPS VI is characterized by progressive dysostosis multiplex, connective tissue and cardiac involvement, and hepatosplenomegaly. Enzyme replacement therapy (ERT) is available but requires life-long and costly intravenous infusions; moreover, it has limited efficacy on diseased skeleton and cardiac valves, compromised pulmonary function, and corneal opacities. METHODS: We enrolled nine patients with MPS VI 4 years of age or older in a phase 1/2 open-label gene therapy study. After ERT was interrupted, patients each received a single intravenous infusion of an adeno-associated viral vector serotype 8 expressing ARSB. Participants were sequentially enrolled in one of three dose cohorts: low (three patients), intermediate (two patients), or high (four patients). The primary outcome was safety; biochemical and clinical end points were secondary outcomes. RESULTS: The infusions occurred without severe adverse events attributable to the vector, meeting the prespecified end point. Participants in the low and intermediate dose cohorts displayed stable serum ARSB of approximately 20% of the mean healthy value but returned to ERT by 14 months after gene therapy because of increased urinary GAG. Participants in the high-dose cohort had sustained serum ARSB of 30% to 100% of the mean healthy value and a modest urinary GAG increase that did not reach a concentration at which ERT reintroduction was needed. In the high-dose group, there was no clinical deterioration for up to 2 years after gene therapy. CONCLUSIONS: Liver-directed gene therapy for participants with MPS VI did not have a dose-limiting side-effect and adverse event profile; high-dose treatment resulted in ARSB expression over at least 24 months with preliminary evidence of disease stabilization. (Funded by the Telethon Foundation ETS, the European Commission Seventh Framework Programme, and the Isaac Foundation; ClinicalTrials.gov number, NCT03173521; EudraCT number, 2016-002328-10.)

High frequency of biotinidase deficiency in Italian population identified by newborn screening.

The biotinidase (BTD) enzyme is essential for recycling biotin, a water-soluble B-complex vitamin that is the coenzyme of four carboxylases involved in fatty acid synthesis, amino acid catabolism and gluconeogenesis. If untreated, total or partial BTD deficiencies lead to an autosomal recessive inherited organic aciduria whose clinical features, mainly presenting in the first years of life, include, seizures, skin rash, and alopecia. Based on residual BTD enzyme activity it is possible to identify partial or total biotinidase deficiency. The incidence of profound and partial biotinidase deficiency worldwide is estimated to be about 1 in 60.000. We report twelve years of experience in the newborn screening of biotinidase deficiency on 466.182 neonates. When a positive screening result occurred, a clinical evaluation was made of the patient and genetic counselling was offered to the family. Molecular analysis the BTD gene was carried out in all recalled neonates. Newborn screening lead to the identification of 75 BTD deficiencies with an incidence of about 1:6.300 births, ten times higher than the reported worldwide incidence. BTD deficiency was confirmed at a genomic level in all patients, demonstrating a high frequency of the p.(Asp444His) amino acid substitution and the complex allele p.(Ala171Thr)/p.(Asp444His) in the analyzed Italian newborns. Four new mutations (two small deletions, one stop mutation and one missense mutation) and a new combined allelic alteration were identified. Our data suggests that there is a high incidence of the biotinidase defect in the Italian population, most likely due to the high frequency of certain mutations.

A new strategy implementing mass spectrometry in the diagnosis of congenital disorders of N-glycosylation (CDG).

Objectives: Congenital disorders of N-glycosylation (CDG) are a large group of rare metabolic disorders caused by defects in the most common post-translational modification of proteins. CDGs are often difficult to diagnose as they are manifested with non-specific symptoms and signs. Analysis of serum transferrin (TRF) isoforms, as the classical procedure used to identify a CDG patient, enables to predict pathological steps in the N-linked glycosylation process. Methods: We devised a new strategy based on liquid chromatography-mass spectrometry (LC-MS) for the analysis of TRF isoforms by combining a simple and fast sample preparation with a specific chromatographic cleanup/separation step followed by mass-spectrometric measurement. Single TRF isoform masses were obtained through reconstruction of multiply charged electrospray data collected by quadrupole-MS technology. Hereby, we report the first analyzed serum samples obtained from 20 CDG patients and 100 controls. Results: The ratio of desialylated isoforms to total TRF was calculated for patients and controls. CDG-Type I patients showed higher amounts of bi-sialo isoform (range: 6.7-29.6%) compared to controls (<5.5%, mean percentage 3.9%). CDG-Type II pattern showed an increased peak of tri-sialo isoforms. The mean percentage of tri-sialo-TRF was 9.3% (range: 2.9-12.9%) in controls, which was lower than that obtained from two patients with COG5-CDG and MAN1B1-CDG (18.5 and 24.5%). Intraday and between-day imprecisions were less than 9 and 16%, respectively, for bi-sialo- and less than 3 and 6% for tri-sialo-TRF. Conclusions: This LC-MS-based approach provides a simple, sensitive and fast analytical tool for characterizing CDG disorders in a routine clinical biochemistry while improving diagnostic accuracy and speeding clinical decision-making.

LC-MS/MS method for simultaneous quantification of heparan sulfate and dermatan sulfate in urine by butanolysis derivatization.

Mucopolysaccharidoses are a group of lysosomal storage disorders (LSDs) characterized by the accumulation of glycosaminoglycans (GAGs). Recently, LC-MS/MS has been widely applied in GAGs analysis combined with different sample preparations for cleaving GAGs to disaccharide units. The aim of the present is paper is to present a new method for the simultaneous quantification of urinary dermatan sulfate (DS) and heparan sulfate (HS) by LC-MS/MS, after butanolysis reaction. Chromatographic separation was achieved with a gradient of acetonitrile and water in 0.1% formic acid on a Kinetex Biphenyl analytical column in 21 min. Calibration curves ranging from 0.78 to 50 µg/mL for HS and from 1.56 to 100 µg/mL for DS were prepared and the coefficient of determination (r2) was higher than 0.99 for both analytes. Intra-day and inter-day imprecisions and the bias for both compounds were <10.0%. Up to now, most analytical procedures for quantifying GAGs have not had a high level of reproducibility among laboratories, despite the availability of various techniques. The adoption of a new protocol incorporating the methods outlined in this paper could significantly improve the quality and reproducibility of MS results. A procedure using simple steps for preparing samples and reagents that are easily available on the market could promote the standardization of analytical procedures and increase the use of these measurements in clinical practice.

Development of a fast LC-MS/MS protocol for combined measurement of six LSDs on dried blood spot in a newborn screening program.

New treatment options and improved strategies for Lysosomal Storage Disorders (LSDs) diagnosis on dried blood spot (DBS) have led to the development of several pilot newborn screening programs. Building on a previously published protocol, we devised a new 6-plex assay based on a single DBS punch incubated into a buffer containing a combination of substrates (GAA, GLA, ASM, GALC, ABG and IDUA). This new protocol incorporates a new trapping and clean-up procedure using perfusion chromatography connected on-line with an analytical column for analyte separation, after enzymatic reaction. Results are available after 4.5 min. Several incubation times were tested in order to reduce sample preparation times and to improve accuracy and reproducibility, also regarding the quenching of the reaction within the time window of linear product accumulation. The collected data demonstrate that an incubation time of 4 h is enough to achieve good reaction efficiency without any impact on sensitivity. The method proved versatile and robust for various instrument configurations. The fast sample preparation and running times allow a high sample throughput; an advantage in newborn screening procedures. This method can also be used for diagnostic purposes, allowing a rapid diagnosis in a few hours.

Data in support for the measurement of heparan sulfate and dermatan sulfate by LC-MS/MS analysis.

This article provides supplementary data for the paper "LC-MS/MS method for simultaneous quantification of heparan sulfate and dermatan sulfate in urine by butanolysis derivatization" (Forni et al., 2018). Several parameters were tested to optimize sample preparation by butanolysis in order to carry out simultaneous quantifications of HS and DS by tandem mass spectrometry. Here we describe step-by-step instructions to perform HS and DS analysis in urine samples using external calibration curves of standards of known concentration. Sample are quantified by interpolation from the calibration curve and reported in µg/mL. Then, HS and DS are normalized to creatinine concentration and reported as mg/g uCr.

Late-Onset N-Acetylglutamate Synthase Deficiency: Report of a Paradigmatic Adult Case Presenting with Headaches and Review of the Literature.

N-acetylglutamate synthase deficiency (NAGSD) is an extremely rare urea cycle disorder (UCD) with few adult cases so far described. Diagnosis of late-onset presentations is difficult and delayed treatment may increase the risk of severe hyperammonemia. We describe a 52-year-old woman with recurrent headaches who experienced an acute onset of NAGSD. As very few papers focus on headaches in UCDs, we also report a literature review of types and pathophysiologic mechanisms of UCD-related headaches. In our case, headaches had been present since puberty (3-4 days a week) and were often accompanied by nausea, vomiting, or behavioural changes. Despite three previous episodes of altered consciousness, ammonia was measured for the first time at 52 years and levels were increased. Identification of the new homozygous c.344C>T (p.Ala115Val) NAGS variant allowed the definite diagnosis of NAGSD. Bioinformatic analysis suggested that an order/disorder alteration of the mutated form could affect the arginine-binding site, resulting in poor enzyme activation and late-onset presentation. After optimized treatment for NAGSD, ammonia and amino acid levels were constantly normal and prevented other headache bouts. The manuscript underlies that headache may be the presenting symptom of UCDs and provides clues for the rapid diagnosis and treatment of late-onset NAGSD.

SSADH deficiency in an Italian family: a novel ALDH5A1 gene mutation affecting the succinic semialdehyde substrate binding site.

SSADH deficiency (SSADHD) is a rare autosomal recessively inherited metabolic disorder. It is associated with mutations of ALDH5A1 gene, coding for the homotetrameric enzyme SSADH. This enzyme is involved in γ-aminobutyric acid (GABA) catabolism, since it oxidizes succinic semialdehyde (SSA) to succinate. Mutations in ALDH5A1 gene result in the abnormal accumulation of γ-hydroxybutyrate (GHB), which is pathognomonic of SSADHD. In the present report, diagnosis of SSADHD in a three-month-old female was achieved by detection of high levels of GHB in urine. Sequence analysis of ALDH5A1 gene showed that the patient was a compound heterozygote for c.1226G > A (p.G409D) and the novel missense mutation, c.1498G > C (p.V500 L). By ALDH5A1 gene expression in transiently transfected HEK293 cells and enzyme activity assays, we demonstrate that the p.V500 L mutation, despite being conservative, produces complete loss of enzyme activity. In silico protein modelling analysis and evaluation of tetramer destabilizing energies suggest that structural impairment and partial occlusion of the access channel to the active site affect enzyme activity. These findings add further knowledge on the missense mutations associated with SSADHD and the molecular mechanisms underlying the loss of the enzyme activity.

Clinical relevance of short-chain acyl-CoA dehydrogenase (SCAD) deficiency: Exploring the role of new variants including the first SCAD-disease-causing allele carrying a synonymous mutation.

Short-chain acyl-coA dehydrogenase deficiency (SCADD) is an autosomal recessive inborn error of mitochondrial fatty acid oxidation caused by ACADS gene alterations. SCADD is a heterogeneous condition, sometimes considered to be solely a biochemical condition given that it has been associated with variable clinical phenotypes ranging from no symptoms or signs to metabolic decompensation occurring early in life. A reason for this variability is due to SCAD alterations, such as the common p.Gly209Ser, that confer a disease susceptibility state but require a complex multifactorial/polygenic condition to manifest clinically. Our study focuses on 12 SCADD patients carrying 11 new ACADS variants, with the purpose of defining genotype-phenotype correlations based on clinical data, metabolite evaluation, molecular analyses, and in silico functional analyses. Interestingly, we identified a synonymous variant, c.765G > T (p.Gly255Gly) that influences ACADS mRNA splicing accuracy. mRNA characterisation demonstrated that this variant leads to an aberrant splicing product, harbouring a premature stop codon. Molecular analysis and in silico tools are able to characterise ACADS variants, identifying the severe mutations and consequently indicating which patients could benefit from a long term follow- up. We also emphasise that synonymous mutations can be relevant features and potentially associated with SCADD.

Development and validation of a 2nd tier test for identification of purine nucleoside phosphorylase deficiency patients during expanded newborn screening by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Purine nucleoside phosphorylase (PNP) deficiency has been recently introduced in the newborn screening program in Tuscany. In order to improve the PNP screening efficiency, we developed a 2nd tier test to quantify PNP primary markers deoxyguanosine (dGuo) and deoxyinosine (dIno). METHODS: Dried blood spots (DBS) samples were extracted with 200 µL of methanol and 100 µL of water (by two steps). Internal standards were added at a final concentration of 10 µmol/L. After extraction, samples were analysed by LC-MS/MS. The chromatographic run was performed in gradient mode by using a Synergi Fusion column. RESULTS: The assay was linear over a concentration range of 0.05-50 µmol/L (R2>0.999) for dGuo and 0.5-50 µmol/L (R2>0.998) for dIno. Intra- and interassay imprecision (mean CVs) for dIno and dGuo ranged from 2.9% to 12%. Limit of quantitaion (LOQ) were found to be 0.05 µmol/L and 0.5 µmol/L for dGuo and dIno, respectively. The reference ranges, obtained by measuring dGuo and dIno concentrations on DBS, were close to zero for both biomarkers. Moreover, DBS samples from seven patients with confirmed PNP were retrospectively evaluated and correctly identified. CONCLUSIONS: The LC-MS/MS method can reliably measure dIno and dGuo in DBS for the diagnosis of PNP. Validation data confirm the present method is characterised by good reproducibility, accuracy and imprecision for the quantitation of dIno and dGuo. The assay also appears suitable for use in monitoring treatment of PNP patients.

Reducing the False-Positive Rate for Isovalerylcarnitine in Expanded Newborn Screening: The Application of a Second-Tier Test

Abstract The isodecyl neopentanoate is an ingredient used in the cosmetic industry to prepare a nipple fissure balm. We report on 12 newborns that showed elevated C5-acylcarnitine levels upon newborn screening following treatment with balm. The first 3 neonates were immediately recalled for confirmatory tests and resulted negative for both isovaleric acidemia and short/branched chain acyl-CoA dehydrogenase deficiency. In the other 9 cases, the immediate recall was avoided by applying a new second-tier test able to confirm the presence of pivaloylcarnitine. The concentration of C5-acylcarnitine was measured in the days following the suspension of balm application. Abnormal concentrations of C5-acylcarnitine did not seem to be associated with free carnitine deficiency, probably due to the short time of exposure. A direct correlation between balm ingestion and the elevation in pivaloylcarnitine has been demonstrated in 10 adult volunteers. The commercial balm containing a pivalic acid derivative is causal of false-positive results during newborn screening, and it could have the potential to cause secondary carnitine deficiency when used chronically.

Heptadecanoylcarnitine (C17) a novel candidate biomarker for newborn screening of propionic and methylmalonic acidemias.

BACKGROUND: 3-Hydroxypalmitoleoyl-carnitine (C16:1-OH) has recently been reported to be elevated in acylcarnitine profiles of patients with propionic acidemia (PA) or methylmalonic acidemia (MMA) during expanded newborn screening (NBS). High levels of C16:1-OH, combined with other hydroxylated long chain acylcarnitines are related to long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) and trifunctional protein (TFP) deficiency. METHODS: The acylcarnitine profile of two LCHADD patients was evaluated using liquid chromatography-tandem mass spectrometric method. A specific retention time was determined for each hydroxylated long chain acylcarnitine. The same method was applied to some neonatal dried blood spots (DBSs) from PA and MMA patients presenting abnormal C16:1-OH concentrations. RESULTS: The retention time of the peak corresponding to C16:1-OH in LCHADD patients differed from those in MMA and PA patients. Heptadecanoylcarnitine (C17) has been identified as the novel biomarker specific for PA and MMA patients through high resolution mass spectrometry (Orbitrap) experiments. We found that 21 out of 23 neonates (22 MMA, and 1PA) diagnosed through the Tuscany region NBS program exhibited significantly higher levels of C17 compared to controls. Twenty-three maternal deficiency (21 vitamin B12 deficiency, 1 homocystinuria and 1 gastrin deficiency) samples and 82 false positive for elevated propionylcarnitine (C3) were also analyzed. CONCLUSIONS: We have characterized a novel biomarker able to detect propionate disorders during expanded newborn screening (NBS). The use of this new biomarker may improve the analytical performances of NBS programs especially in laboratories where second tier tests are not performed.

Biotinidase deficiency due to a de novo mutation or gonadal mosaicism in a first child.

Biotinidase deficiency (BD), which is caused by BTD genetic lesions, if untreated, can result in neurological and cutaneous manifestations. Biotin supplementation can improve or prevent symptoms. We herewith present a family, which we studied at biochemical and molecular level, after identifying the proband through a newborn screening programme. BTD gene molecular analysis showed the proband to be compound heterozygous for the c.1330G>C p.(Asp444His) mild known variant, and for the c.1475 C>T p.(Thr492Ile) new variant. Bioinformatic analysis allowed us to confirm the pathogenic role of the newly identified variant. The proband's father, who exhibited low biotinidase (BTD) enzyme activity, was homozygous for the mild variant, whereas the proband's mother, who exhibited borderline BTD values, the BTD mutation carrier status could not be detected. This is the first description of a patient with BD harbouring a variant whose origin is either de novo or the consequence of gonadal mosaicism. BTD molecular analysis and bioinformatic tools for the evaluation of pathogenicity of newly identified variants are necessary for diagnostic purposes (i.e., clarifying borderline enzyme assays and the carrier status of parents), and for genetic counselling.

Intra-individual plasticity of the TAZ gene leading to different heritable mutations in siblings with Barth syndrome.

Infantile-onset skeletal myopathy Barth syndrome (OMIM #302060) is caused by mutations in the X-linked TAZ gene and hence usually manifests itself only in hemizygous males. Confirmatory testing is provided by mutational analysis of the TAZ gene and/or by biochemical dosage of the monolysocardiolipin/tetralinoleoyl cardiolipin ratio. Heterozygous females do not usually display a clinical phenotype but may undergo molecular genetic prenatal diagnosis during pregnancy. We characterized two novel and non-identical TAZ gene rearrangements in the offspring of a single female carrier of Barth syndrome. The hg19chrX:g.153634427_153644361delinsKP_123427.1 TAZ gene rearrangement was identified in her affected son, whereas the NM_000116.3(TAZ)c.-72_109+51del TAZ gene deletion was identified in a male foetus during a subsequent pregnancy. The unaffected mother was surprisingly found to harbour both variants in addition to a wild-type TAZ allele. A combination of breakpoint junction sequencing, linkage analysis and assessment of allelic dosage revealed that the two variants had originated independently from an apparently unstable/mutable TAZ maternal allele albeit via different mutational mechanisms. We conclude that molecular prenatal diagnosis in Barth syndrome families with probands carrying TAZ gene rearrangements should include investigation of the entire coding region of the TAZ gene. The identification of the breakpoint junctions of such gross gene rearrangements is important to ensure accurate ascertainment of carriership with a view to providing appropriate genetic counselling.

Therapeutic drug monitoring of carbamazepine and its metabolite in children from dried blood spots using liquid chromatography and tandem mass spectrometry.

Carbamazepine (CBZ) is a first-line drug for the treatment of different forms of epilepsy and the first choice drug for trigeminal neuralgia. CBZ is metabolized in the liver by oxidation into carbamazepine-10,11-epoxide (CBZE), its major metabolite which is equipotent and known to contribute to the pharmacological activity of CBZ. The aim of the present study was to develop and validate a reliable, selective and sensitive liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of CBZ and its active metabolite in dried blood spots (DBS). The extraction process was carried out from DBS using methanol-water-formic acid (80:20:0.1, v/v/v). Chromatographic elution was achieved by using a linear gradient with a mobile phase consisting of acetonitrile-water-0.1% formic acid at a flow rate of 0.50mL/min. The method was linear over the range 1-40mg/L and 0.25-20mg/L for CBZ and CBZE, respectively. The limit of quantification was 0.75mg/L and 0.25mg/L for CBZ and CBZE. Intra-day and inter-day assay precisions were found to be lower than 5.13%, 6.46% and 11.76%, 4.72% with mean percentage accuracies of 102.1%, 97.5% and 99.2%, 97.8% for CBZ and CBZE. We successfully applied the method for determining DBS finger-prick samples in paediatric patients and confirmed the results with concentrations measured in matched plasma samples. This novel approach allows quantification of CBZ and its metabolite from only one 3.2mm DBS disc by LC-MS/MS thus combining advantages of DBS technique and LC-MS/MS in clinical practice.

Dried blood spot assay for the quantification of phenytoin using Liquid Chromatography-Mass Spectrometry.

Phenytoin (PHT) is one of the most commonly used anticonvulsant drugs for the treatment of epilepsy and bipolar disorders. The large amount of plasma required by conventional methods for drug quantification makes mass spectrometry combined with dried blood spot (DBS) sampling crucial for pediatric patients where therapeutic drug monitoring or pharmacokinetic studies may be difficult to realize. DBS represents a new convenient sampling support requiring minimally invasive blood drawing and providing long-term stability of samples and less expensive shipment and storage. The aim of this study was to develop a LC-MS/MS method for the quantification of PHT on DBS. This analytical method was validated and gave good linearity (r(2)=0.999) in the range of 0-100mg/l. LOQ and LOD were 1.0mg/l and 0.3mg/l, respectively. The drug extraction from paper was performed in a few minutes using a mixture composed of organic solvent for 80%. The recovery ranged from 85 to 90%; PHT in DBS showed to be stable at different storage temperatures for one month. A good correlation was also obtained between PHT plasma and DBS concentrations. This method is both precise and accurate and appears to be particularly suitable to monitor treatment with a simple and convenient sample collection procedure.

Sudden unexpected fatal encephalopathy in adults with OTC gene mutations-Clues for early diagnosis and timely treatment.

BACKGROUND: X-linked Ornithine Transcarbamylase deficiency (OTCD) is often unrecognized in adults, as clinical manifestations are non-specific, often episodic and unmasked by precipitants, and laboratory findings can be normal outside the acute phase. It may thus be associated with significant mortality if not promptly recognized and treated. The aim of this study was to provide clues for recognition of OTCD in adults and analyze the environmental factors that, interacting with OTC gene mutations, might have triggered acute clinical manifestations. METHODS: We carried out a clinical, biochemical and molecular study on five unrelated adult patients (one female and four males) with late onset OTCD, who presented to the Emergency Department (ED) with initial fatal encephalopathy. The molecular study consisted of OTC gene sequencing in the probands and family members and in silico characterization of the newly detected mutations. RESULTS: We identified two new, c.119G>T (p.Arg40Leu) and c.314G>A (p.Gly105Glu), and three known OTC mutations. Both new mutations were predicted to cause a structural destabilization, correlating with late onset OTCD. We also identified, among the family members, 8 heterozygous females and 2 hemizygous asymptomatic males. Patients' histories revealed potential environmental triggering factors, including steroid treatment, chemotherapy, diet changes and hormone therapy for in vitro fertilization. CONCLUSIONS: This report raises awareness of the ED medical staff in considering OTCD in the differential diagnosis of sudden neurological and behavioural disorders associated with hyperammonemia at any age and in both genders. It also widens the knowledge about combined effect of genetic and environmental factors in determining the phenotypic expression of OTCD.

Diagnosis of immunodeficiency caused by a purine nucleoside phosphorylase defect by using tandem mass spectrometry on dried blood spots.

BACKGROUND: Purine nucleoside phosphorylase (PNP) deficiency is a rare form of autosomal recessive combined primary immunodeficiency caused by a enzyme defect leading to the accumulation of inosine, 2'-deoxy-inosine (dIno), guanosine, and 2'-deoxy-guanosine (dGuo) in all cells, especially lymphocytes. Treatments are available and curative for PNP deficiency, but their efficacy depends on the early approach. PNP-combined immunodeficiency complies with the criteria for inclusion in a newborn screening program. OBJECTIVE: This study evaluate whether mass spectrometry can identify metabolite abnormalities in dried blood spots (DBSs) from affected patients, with the final goal of individuating the disease at birth during routine newborn screening. METHODS: DBS samples from 9 patients with genetically confirmed PNP-combined immunodeficiency, 10,000 DBS samples from healthy newborns, and 240 DBSs from healthy donors of different age ranges were examined. Inosine, dIno, guanosine, and dGuo were tested by using tandem mass spectrometry (TMS). T-cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) levels were evaluated by using quantitative RT-PCR only for the 2 patients (patients 8 and 9) whose neonatal DBSs were available. RESULTS: Mean levels of guanosine, inosine, dGuo, and dIno were 4.4, 133.3, 3.6, and 3.8 µmol/L, respectively, in affected patients. No indeterminate or false-positive results were found. In patient 8 TREC levels were borderline and KREC levels were abnormal; in patient 9 TRECs were undetectable, whereas KREC levels were normal. CONCLUSION: TMS is a valid method for diagnosis of PNP deficiency on DBSs of affected patients at a negligible cost. TMS identifies newborns with PNP deficiency, whereas TREC or KREC measurement alone can fail.