Biomarkers for gene therapy clinical trials of lysosomal storage disorders.

Lysosomal storage disorders (LSDs) are multisystemic progressive disorders caused by defects in proteins involved in lysosomal function. Different gene therapy strategies are under clinical investigation in several LSDs to overcome the limitations of available treatments. However, LSDs are slowly progressive diseases that require long-term studies to establish the efficacy of experimental treatments. Biomarkers can be reliable substitutes for clinical responses and improve the efficiency of clinical trials, especially when long-term disease interventions are evaluated. In this review, we summarize both available and future biomarkers for LSDs and discuss their strengths and weaknesses.

Expanded Newborn Screening for Inborn Errors of Immunity: The Experience of Tuscany.

BACKGROUND: Inborn errors of immunity (IEIs) include 485 inherited disorders characterized by an increased susceptibility to life-threatening infectious diseases, autoimmunity, and malignant diseases with a high mortality rate in the first years of life. Severe combined immunodeficiency is the most severe of the IEIs, and its detection should be a primary goal in a newborn screening (NBS) program. The term "actionable" has recently been used for all IEIs with outcomes that can be demonstrably improved through early specialized intervention. OBJECTIVE: To evaluate the results of the expanded NBS strategy for IEIs in Tuscany Region (Italy), based on T-cell receptor excision circle, kappa recombining excision circle, and tandem mass-based assays. METHODS: This is a retrospective study collecting data from all infants born in Tuscany from October 10, 2018, to October 10, 2022. Tandem mass assay to identify adenosine deaminase and purine nucleoside phosphorylase deficiency, together with T-cell receptor excision circle and kappa recombining excision circle molecular analysis, was conducted on dried blood spot from the newborns' Guthrie Cards. A new dried blood spot and evaluation by an immunologist were carried out when the results of the first test were outside the diagnostic cutoffs. RESULTS: A total of 94,319 newborns were evaluated. Referral rates for T-cell recombining excision circles (0.031%) and kappa recombining excision circles (0.074%) in this study are in line with the data available in literature. The results from the expanded NBS strategy revealed an incidence rate of 1 per 9431 affected newborns. CONCLUSIONS: This work represents the first description of a sustainable and real-life-based expanded NBS program for IEIs with a high diagnostic incidence facilitating prompt management of identified patients.

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.

PRDX1 gene-related epi-cblC disease is a common type of inborn error of cobalamin metabolism with mono- or bi-allelic MMACHC epimutations.

BACKGROUND: The role of epigenetics in inborn errors of metabolism (IEMs) is poorly investigated. Epigenetic changes can contribute to clinical heterogeneity of affected patients but could also be underestimated determining factors in the occurrence of IEMs. An epigenetic cause of IEMs has been recently described for the autosomal recessive methylmalonic aciduria and homocystinuria, cblC type (cblC disease), and it has been named epi-cblC. Epi-cblC has been reported in association with compound heterozygosity for a genetic variant and an epimutation at the MMACHC locus, which is secondary to a splicing variant (c.515-1G > T or c.515-2A > T) at the adjacent PRDX1 gene. Both these variants cause aberrant antisense transcription and cis-hypermethylation of the MMACHC gene promotor with subsequent silencing. Until now, only nine epi-cblC patients have been reported. METHODS: We report clinical/biochemical assessment, MMACHC/PRDX1 gene sequencing and genome-wide DNA methylation profiling in 11 cblC patients who had an inconclusive MMACHC gene testing. We also compare clinical phenotype of epi-cblC patients with that of canonical cblC patients. RESULTS: All patients turned out to have the epi-cblC disease. One patient had a bi-allelic MMACHC epimutation due to the homozygous PRDX1:c.515-1G > T variant transmitted by both parents. We found that the bi-allelic epimutation produces the complete silencing of MMACHC in the patient's fibroblasts. The remaining ten patients had a mono-allelic MMACHC epimutation, due to the heterozygous PRDX1:c.515-1G > T, in association with a mono-allelic MMACHC genetic variant. Epi-cblC disease has accounted for about 13% of cblC cases diagnosed by newborn screening in the Tuscany and Umbria regions since November 2001. Comparative analysis showed that clinical phenotype of epi-cblC patients is similar to that of canonical cblC patients. CONCLUSIONS: We provide evidence that epi-cblC is an underestimated cause of inborn errors of cobalamin metabolism and describe the first instance of epi-cblC due to a bi-allelic MMACHC epimutation. MMACHC epimutation/PRDX1 mutation analyses should be part of routine genetic testing for all patients presenting with a metabolic phenotype that combines methylmalonic aciduria and homocystinuria.

Multicenter evaluation of use of dried blood spot compared to conventional plasma in measurements of globotriaosylsphingosine (LysoGb3) concentration in 104 Fabry patients.

OBJECTIVES: Fabry disease (FD) is an X-linked lysosomal storage disorder, resulting from a deficiency of the enzyme α-galactosidase A, responsible for breaking down glycolipids such as globotriaosylceramide and its deacylated derivative, globotriaosylsphingosine (LysoGb3). Here, we compare the levels of LysoGb3 in dried blood spots (DBS) and plasma in patients with classic and late-onset phenotypes. METHODS: LysoGb3 measurements were performed in 104 FD patients, 39 males and 65 females. Venous blood was collected. A portion was spotted onto filter paper and another portion separated to obtain plasma. The LysoGb3 concentrations in DBS and plasma were determined by highly sensitive electrospray ionization liquid chromatography tandem mass spectrometry. Agreement between different matrices was assessed using linear regression and Bland Altman analysis. RESULTS: The method on DBS was validated by evaluating its precision, accuracy, matrix effect, recovery, and stability. The analytical performances were verified by comparison of a total of 104 paired DBS and plasma samples from as many FD patients (representing 46 GLA variants). There was a strong correlation between plasma and the corresponding DBS LysoGb3 concentrations, with few exceptions. Discrepancies were observed in anemic patients with typically low hematocrit levels compared to the normal range. CONCLUSIONS: The method proved to be efficient for the rapid analysis of LysoGb3. DBS provides a convenient, sensitive, and reproducible method for measuring LysoGb3 levels for diagnosis, initial phenotypic assignment, and therapeutic monitoring in patients with FD.

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.

Development of Strategies to Decrease False Positive Results in Newborn Screening.

The expansion of national newborn screening (NBS) programmes has provided significant benefits in the diagnosis and early treatment of several rare, heritable conditions, preventing adverse health outcomes for most affected infants. New technological developments have enabled the implementation of testing panel covering over 50 disorders. Consequently, the increment of false positive rate has led to a high number of healthy infants recalled for expensive and often invasive additional testing, opening a debate about the harm-benefit ratio of the expanded newborn screening. The false-positive rate represents a challenge for healthcare providers working in NBS systems. Here, we give an overview on the most commonly used strategies for decreasing the adverse effects due to inconclusive screening results. The focus is on NBS performance improvement through the implementation of analytical methods, the application of new and more informative biomarkers, and by using post-analytical interpretive tools. These strategies, used as part of the NBS process, can to enhance the positive predictive value of the test and reduce the parental anxiety and healthcare costs related to the unnecessary tests and procedures.

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.

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.

Expanded newborn screening by mass spectrometry: New tests, future perspectives.

Tandem mass spectrometry (MS/MS) has become a leading technology used in clinical chemistry and has shown to be particularly sensitive and specific when used in newborn screening (NBS) tests. The success of tandem mass spectrometry is due to important advances in hardware, software and clinical applications during the last 25 years. MS/MS permits a very rapid measurement of many metabolites in different biological specimens by using filter paper spots or directly on biological fluids. Its use in NBS give us the chance to identify possible treatable metabolic disorders even when asymptomatic and the benefits gained by this type of screening is now recognized worldwide. Today the use of MS/MS for second-tier tests and confirmatory testing is promising especially in the early detection of new disorders such as some lysosomal storage disorders, ADA and PNP SCIDs, X-adrenoleucodistrophy (X-ALD), Wilson disease, guanidinoacetate methyltransferase deficiency (GAMT), and Duchenne muscular dystrophy. The new challenge for the future will be reducing the false positive rate by using second-tier tests, avoiding false negative results by using new specific biomarkers and introducing new treatable disorders in NBS programs.

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.

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.

Delivery of doxorubicin across the blood-brain barrier by ondansetron pretreatment: a study in vitro and in vivo.

Doxorubicin (Dox) has got a limited efficacy in the treatment of central nervous system tumors because of its poor penetration through blood-brain barrier mediated by MDR efflux transporters. We investigated the possibility that ondansetron (Ond) enhances Dox cytotoxicity in cell lines interfering with P-glycoprotein and increases Dox concentration in rat brain tissues. The MDR phenotype was studied using human hepatocellular carcinoma cell line PLC/PRF/5 (P5 and P1(0.5) clones), two subclones of NIH 3T3 cells (PSI-2 and PN1A) and two glioblastoma cell lines (A172, U87MG). Rats were pretreated with Ond before injection of Dox. Quantitative analysis of Dox was performed by mass spectrometry. Our in vitro experiments demonstrated that Ond at 10 µg/ml is not toxic to all cell lines. However, Ond reverses the MDR phenotype in P1(0.5) and PN1A cell lines. In addition, we showed that pretreatment with Ond increases Dox concentration in rat brain tissues, without increasing acute heart and renal toxicity.

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.