Butyrylcarnitine Elevation in Newborn Screening: Reducing False Positives and Distinguishing between Two Rare Diseases through the Evaluation of New Ratios.

One of the main challenges of newborn screening programs, which screen for inherited metabolic disorders, is cutting down on false positives (FPs) in order to avoid family stresses, additional analyses, and unnecessary costs. False positives are partly caused by an insubstantial number of robust biomarkers in evaluations. Another challenge is how to distinguish between diseases which share the same primary marker and for which secondary biomarkers are just as highly desirable. Focusing on pathologies that involve butyrylcarnitine (C4) elevation, such as short-chain acylCoA dehydrogenase deficiency (SCADD) and isobutyrylCoA dehydrogenase deficiency (IBDD), we investigated the acylcarnitine profile of 121 newborns with a C4 increase to discover secondary markers to achieve two goals: reduce the FP rate and discriminate between the two rare diseases. Analyses were carried out using tandem mass spectrometry with whole blood samples spotted on filter paper. Seven new biomarkers (C4/C0, C4/C5, C4/C5DC\C6OH, C4/C6, C4/C8, C4/C14:1, C4/C16:1) were identified using a non-parametric ANOVA analysis. Then, the corresponding cut-off values were found and applied to the screening program. The seven new ratios were shown to be robust (p < 0.001 and p < 0.01, 0.0937 < ε2 < 0.231) in discriminating between FP and IBDD patients, FP and SCADD patients, or SCADD and IBDD patients. Our results suggest that the new ratios are optimal indicators for identifying true positives, distinguishing between two rare diseases that share the same primary biomarker, improving the predictive positive value (PPV) and reducing the false positive rate (FPR).

Detection of Single-Nucleotide and Copy Number Defects Underlying Hyperphenylalaninemia by Next-Generation Sequencing.

Hyperphenylalaninemia (HPA) is the most common inherited amino acid metabolism disorder characterized by serious clinical manifestations, including irreversible brain damage, intellectual deficiency and epilepsy. Due to its extensive genic and allelic heterogeneity, next-generation sequencing (NGS) technology may help to identify the molecular basis of this genetic disease. Herein, we describe the development and validation of a targeted NGS (tNGS) approach for the simultaneous detection of single-nucleotide changes and copy number variations (CNVs) in genes associated with HPA (PAH, GCH1, PTS, QDPR, PCBD1, DNAJC12) or useful for its differential diagnosis (SPR). Our tNGS approach offers the possibility to detail, with a high accuracy and in a single workflow, the combined effect of a broader spectrum of genomic variants in a comprehensive view, providing a significant step forward in the development of optimized patient care and management.

Neuroactive Amino Acid Profile in Autism Spectrum Disorder: Results from a Clinical Sample.

Biological bases of autism spectrum disorder (ASD) include both genetic and epigenetic causes. Patients with ASD show anomalies in the profile of certain plasma amino acids, including neuroactive amino acids. Monitoring plasma amino acids may be relevant for patient care and interventions. We evaluated the plasma amino acid profile in samples extracted from dry blood spots by electrospray ionization-tandem mass spectrometry. Fourteen amino acids and eleven amino acid ratios were examined in patients with ASD and intellectual disability (ID), and neurotypical control subjects (TD). The amino acid profile in the ASD group showed reduced levels of ornithine (p = 0.008), phenylalanine (p = 0.042) and tyrosine (p = 0.013). The statistically significant amino acid ratios were Leu+Val/Phe+Tyr (p = 0.002), Tyr/Leu (p = 0.007) and Val/Phe (p = 0.028), such differences remaining significant only in the comparison between ASD and TD. Finally, a positive correlation emerged between the score of the restricted and repetitive behavior on ADOS-2 and the citrulline levels in the ASD group (p = 0.0047). To conclude, patients with ASD may show a distinguishable metabolic profile useful for studying their metabolic pathways in order to develop screening tests and targeted therapies.

Dihydropyridine Reductase Deficiency: Acute Encephalopathy Related to Folinic Acid Treatment Interruption in a Girl.

We reported the case of acute encephalopathy related to colonic acid treatment interruption in a 12-year-old female child presenting to our unit with episodes of vomiting, headache, irritability, acute confusional state, seizures, and left lower limb hypotonia. Brain magnetic resonance imaging (MRI) showed signs of vasogenic and cytotoxic edema at the cerebellar level bilaterally, and lesions at the temporo-occipito-parietal right level, temporomandibular left, and right thalamic with swelling of the convolutions and reduced differentiation between white and gray matter. The patient had suspended the folinic acid treatment at least 6 months before the present admission. The relation between the clinical signs presented by the girl and folic acid deficiency was confirmed by the result of laboratory assessment and by the answer to the notable clinical improvement with the renewal of folinic acid treatment. Dihydropteridine reductase (DHPR) deficiency is a rare autosomal recessive genetic disorder caused by the quinoid dihydropteridine reductase (QDPR) gene mutations. DHPR deficiency impairs the synthesis of the tetrahydrobiopterin (BH4), an essential cofactor for the hydroxylation of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. When not precociously treated, the disorder may present whit severe neurologic impairment including developmental delay/intellective disability (DD/ID), microcephaly, seizures, movement disorders, cerebral palsy, and other neurological impairments. The clinical and neuroradiologic anomalies observed in our case were unusual, with signs previously unreported in patients with folic acid deficiency. The present case shows that the clinical presentation and MRI anomalies of the cerebral folic acid deficiency may be various and unusual compared with those reported in the literature, and it confirms the usefulness of the continuation of folinic acid treatment during the course of the disorder in patients with DHPR deficiency.

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.

New ratio as a useful marker for early diagnosis of proximal urea cycle disorders.

BACKGROUND AND AIMS: Proximal urea cycle disorders (PUCDs) are not included in most newborn screening programs due to the lack of adequate markers to monitor. Failure to alter citrulline and glutamine levels, the prognostic markers commonly used, can results in high false negative. Therefore, new biomarkers, prognostic of PUCDs, are strongly desirable. MATERIALS AND METHODS: We used tandem mass spectrometry to analyze blood spot from PUCDs patients during their follow up in our referral center focusing on glutamine to glutamate (Gln/Glu) ratio. We reanalyzed the same specimens of three patients after two months and the specimen of a new patient with suspicious of PUCD disorder. RESULTS: Specimens of our patients shown a significant elevation of the ratio Gln/Glu compared to that of a healthy population (p < 0.05) as well as the specimens analyzed after two months, while the glutamine concentration dropped. New patient, showing high value of the ratio, was molecularly confirmed as PUCD patient. We further analyzed the blood spots from a neonatal population in order to fix a cut-off value and include it in a newborn screening panel. CONCLUSION: Our preliminary results suggest that the Gln/Glu ratio could be a very useful diagnostic marker, more stable over time than glutamine, which could improve the performance in early PUCDs identification.

Neonatal Screening on Tandem Mass Spectrometry as a Powerful Tool for the Reassessment of the Prevalence of Underestimated Diseases in Newborns and Their Family Members: A Focus on Short Chain Acyl-CoA Dehydrogenase Deficiency.

Early detection of disabling diseases, prior to clinical manifestations, is the primary goal of newborn screening (NS). Indeed, the required number of core and secondary conditions selected for screening panels is increasing in many countries. Furthermore, newborn screening can lead to diagnosis of maternal diseases such as vitamin B12 deficiency or 3-MethylcrotonylCoA-carboxylase deficiency (3MCC). NS became mandatory in Sicily in December 2017. Here we report NS data collected between December 2017 and April 2020. Our results show that tandem mass spectrometry is a powerful tool for discovery of underestimated disease in newborns and their family members. Our panel included short chain acyl-CoA dehydrogenase deficiency (SCADD). Here, we report that results of our investigation led to reassessment of SCADD prevalence in our population. The infant and adult patients diagnosed in our study had previously not shown overt symptoms.

Expanded Newborn Screening Using Tandem Mass Spectrometry: Seven Years of Experience in Eastern Sicily.

The expanded newborn screening for selected inborn errors of metabolism (IEM) in Sicily was introduced in 2007 by a Regional project entitled "Early detection of congenital metabolic diseases: expanded neonatal screening". It established two newborn screening laboratories, for Western and Eastern Sicily, which started their activity in 2011. Here we present the results of expanded screening (excluding phenylketonuria (PKU)) of the Eastern laboratory from January 2011 to December 2017. Our data highlight the importance of the expanded newborn screening as a basic health program to avoid the underestimation of rare diseases and the need of further investigations even when there are no textbook alterations of the metabolic profiles. We performed our analysis on dried blood spot by tandem mass spectrometry, according to Italian guidelines. A total of 196 samples from 60,408 newborns gave positive screening results (recall rate 0.32%) while 12 babies were true positive, including 2 newborns whose mothers resulted in being affected by a metabolic disease. The overall frequency of IEM found in the screening panel was 1:6041 (mothers excluded) or 1:5034 (mothers included). The introduction of MS/MS technology in Sicily has significantly increased the detection of inherited metabolic disorders, including those not previously covered, with a predictable improved outcome for several disorders.

Hyperprolinemia Type IA: Benign Metabolic Anomaly or a Trigger for Brain Dysfunction?

Abstract Objective: Hyperprolinemia type I (HPI) is a rare and inherited autosomal recessive disorder caused by proline oxidase deficiency. Hyperprolinemia type 1 is biochemically defined as high plasma proline levels without urinary ?-1-pyrroline-5-carboxylate excretion. Hyperprolinemia type 1 has been considered a benign metabolic disorder, but a relationship with neurological disorders has recently been suggested. Study Design: We retrospectively analyzed plasma amino acid values obtained by amino acid analysis from 10 030 children admitted for neurological reasons during the years 1996 to 2010 at the Regional Sicilian Centre for Metabolic Diseases. Patients with proline levels above the normal range of 129 to 245 ?M were identified. Results: Only 2 children showed high levels of proline (450-480 ?M and 380-470 ?M, respectively), but their disorders (tubercular neuroencephalitis and progressive mitochondrial encephalopathy) did not seem to be related to hyperprolinemia as a causative factor. Conclusion: The question of HPI as benign metabolic anomaly or as a direct cause of brain damage is still open. Since HPI is rare, other observations on this regard are necessary.

Long-term follow-up and outcome of phenylketonuria patients on sapropterin: a retrospective study.

OBJECTIVE: Sapropterin dihydrochloride, the synthetic form of 6R-tetrahydrobiopterin (BH4), is an approved drug for the treatment of patients with BH4-responsive phenylketonuria (PKU). The purpose of this study was to assess genotypes and data on the long-term effects of BH4/sapropterin on metabolic control and patient-related outcomes in 6 large European countries. METHODS: A questionnaire was developed to assess phenotype, genotype, blood phenylalanine (Phe) levels, Phe tolerance, quality of life, mood changes, and adherence to diet in PKU patients from 16 medical centers. RESULTS: One hundred forty-seven patients, of whom 41.9% had mild hyperphenylalaninemia, 50.7% mild PKU, and 7.4% classic PKU, were followed up over ≤12 years. A total of 85 different genotypes were reported. With the exception of two splice variants, all of the most common mutations were reported to be associated with substantial residual Phe hydroxylase activity. Median Phe tolerance increased 3.9 times with BH4/sapropterin therapy, compared with dietary treatment, and median Phe blood concentrations were within the therapeutic range in all patients. Compared with diet alone, improvement in quality of life was reported in 49.6% of patients, improvement in adherence to diet was reported in 47% of patients, and improvement in adherence to treatment was reported in 63.3% of patients. No severe adverse events were reported. CONCLUSIONS: Our data document a long-term beneficial effect of orally administered BH4/sapropterin in responsive PKU patients by improving the metabolic control, increasing daily tolerance for dietary Phe intake, and for some, by improving dietary adherence and quality of life. Patient genotypes help in predicting BH4 responsiveness.

Clinical manifestations and management of four children with Pearson syndrome.

Pearson marrow-pancreas syndrome is a fatal disorder mostly diagnosed during infancy and caused by mutations of mitochondrial DNA. We hereby report on four children affected by Pearson syndrome with hematological disorders at onset. The disease was fatal to three of them and the fourth one, who received hematopoietic stem cell transplantation, died of secondary malignancy. In this latter patient transplantation corrected hematological and non-hematological issues like metabolic acidosis, and we therefore argue that it could be considered as a useful option in an early stage of the disease.

Exon deletions of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemics.

A consistent finding of many studies describing the spectrum of mutant phenylalanine hydroxylase (PAH) alleles underlying hyperphenylalaninemia is the impossibility of achieving a 100% mutation ascertainment rate using conventional gene-scanning methods. These methods include denaturing gradient gel electrophoresis (DGGE), denaturing high performance liquid chromatography (DHPLC), and direct sequencing. In recent years, it has been shown that a significant proportion of undetermined alleles consist of large deletions overlapping one or more exons. These deletions have been difficult to detect in compound heterozygotes using gene-scanning methods due to a masking effect of the non-deleted allele. To date, no systematic search has been carried out for such exon deletions in Italian patients with phenylketonuria or mild hyperphenylalaninemia. We used multiplex ligation-dependent probe amplification (MLPA), comparative multiplex dosage analysis (CMDA), and real-time PCR to search for both large deletions and duplications of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemia patients. Four deletions removing different phenylalanine hydroxylase (PAH) gene exons were identified in 12 patients. Two of these deletions involving exons 4-5-6-7-8 (systematic name c.353-?_912+?del) and exon 6 (systematic name c.510-?_706+?del) have not been reported previously. In this study, we show that exon deletion of the PAH gene accounts for 1.7% of all mutant PAH alleles in Italian hyperphenylalaninemics.

Evidence for genetic heterogeneity in D-2-hydroxyglutaric aciduria.

We performed molecular, enzyme, and metabolic studies in 50 patients with D-2-hydroxyglutaric aciduria (D-2-HGA) who accumulated D-2-hydroxyglutarate (D-2-HG) in physiological fluids. Presumed pathogenic mutations were detected in 24 of 50 patients in the D-2-hydroxyglutarate dehydrogenase (D2HGDH) gene, which encodes D-2-hydroxyglutarate dehydrogenase (D-2-HGDH). Enzyme assay of D-2-HGDH confirmed that all patients with mutations had impaired enzyme activity, whereas patients with D-2-HGA whose enzyme activity was normal did not have mutations. Significantly lower D-2-HG concentrations in body fluids were observed in mutation-positive D-2-HGA patients than in mutation-negative patients. These results imply that multiple genetic loci may be associated with hyperexcretion of D-2-HG. Accordingly, we suggest a new classification: D-2-HGA Type I associates with D-2-HGDH deficiency, whereas idiopathic D-2-HGA manifests with normal D-2-HGDH activity and higher D-2-HG levels in body fluids compared with Type I patients. It remains possible that several classifications for idiopathic D-2-HGA patients with diverse genetic loci will be revealed in future studies.

Pharmacokinetics of sapropterin in patients with phenylketonuria.

BACKGROUND AND OBJECTIVE: Untreated phenylketonuria is characterized by neurocognitive and neuromotor impairment, which result from elevated blood phenylalanine concentrations. To date, the recommended management of phenylketonuria has been the use of a protein-restricted diet and the inclusion of phenylalanine-free protein supplements; however, this approach is often associated with poor compliance and a suboptimal clinical outcome. Sapropterin dihydrochloride, herein referred to as sapropterin, a synthetic formulation of 6R-tetrahydrobiopterin (6R-BH4), has been shown to be effective in reducing blood phenylalanine concentrations in patients with phenylketonuria. The objective of the current study was to characterize the pharmacokinetics and pharmacokinetic variability of sapropterin and to identify the characteristics that influence this variability. PATIENTS AND METHODS: This was a 12-week, fixed-dose phase of an open-label extension study. The study was conducted at 26 centres in North America and Europe.Patients with phenylketonuria were eligible to participate if they were > or =8 years of age and had received > or =80% of the scheduled doses in a previous 6-week, randomized, placebo-controlled study or had been withdrawn from that study after exceeding a plasma phenylalanine concentration of > or =1500 micromol/L to > or =1800 micromol/L, depending on the subject's age and baseline plasma phenylalanine concentration. A total of 78 patients participated. Patients received oral once-daily doses of sapropterin (Kuvan) 5, 10 or 20 mg/kg/day. Blood samples for the pharmacokinetic analysis were obtained during weeks 6, 10 and 12. A D-optimal sparse sampling strategy was used, and data were analysed by population-based, nonlinear, mixed-effects modelling methods. MAIN OUTCOME MEASURE: In a prospectively planned analysis, the apparent clearance, apparent volume of distribution, absorption rate constant and associated interindividual variabilities of each parameter were estimated by modelling observed BH4 plasma concentration-time data. RESULTS: The best structural model to describe the pharmacokinetics of sapropterin was a two-compartment model with first-order input, first-order elimination and a baseline endogenous BH4 concentration term. Total bodyweight was the only significant covariate identified, the inclusion of which on both the apparent clearance (mean = 2100 L/h/70 kg) and central volume of distribution (mean = 8350 L/70 kg) substantially improved the model's ability to describe the data. The mean (SD) terminal half-life of sapropterin was 6.69 (2.29) hours and there was little evidence of accumulation, even at the highest dose. CONCLUSION: These findings, taken together with the observed therapeutic effect, support bodyweight-based, once-daily dosing of sapropterin 5-20 mg/kg/day.

Autism and phenylketonuria.

Phenylketonuria (PKU) has been also reported in children with infantile autism (IA); however, the frequency of this association is variably reported. Patients with various forms of hyperphenylalaninemia (HPA) were evaluated applying two methods: the Autism Diagnostic Interview-Revised (ADI-R) and the Childhood Autism Rating Scale (CARS). A total of 243 patients were investigated, 97 with classical PKU, 62 identified by neonatal screening, and 35 late diagnosed. None out of 62 patients with classic PKU diagnosed early met criteria for autism. In the group of 35 patients diagnosed late, two boys (5.71%) ages 16 and 13 years fulfilled the diagnostic criteria for autism. The present study confirms that classical PKU is one of the causes of autism, but the prevalence seems to be very low.

Hyperphenylalaninemia and birth weight.

Hyperphenylalaninemias (HPAs) are due to autosomal recessive inherited deficiency of phenylalanine hydroxylase and include three different biochemical and clinical phenotypes: classic phenylketonuria, mild phenylketonuria and persistent HPA. Recently the relationship between birth weight and HPA has been investigated. We performed an evaluation of birth weight in our 260 HPA patients. Our results do not support the view that birth weight is reduced in HPA patients and we found no correlation between birth weight and severity of the disease. Only a better knowledge of genetic mechanisms involved in HPA can clarify the interaction between HPA and fetal development.