Hugo Rocha

Four years of expanded newborn screening in Portugal with tandem mass spectrometry

IntroductionThe Portuguese Neonatal Screening Programme (PNSP) was started in 1979 for phenylketonuria (2,590,700 newborns screened; prevalence 1:11,031) and, shortly after, for congenital hypothyroidism (2,558,455 newborns screened; prevalence 1:3,174). In 2004, expanded neonatal screening was implemented in the National Laboratory. The programme is not mandatory and has 99.8% coverage of the country (including Madeira and the Azores islands).Material and methodsIn the past 4xa0years, 316,243 neonates were screened with the use of tandem mass spectrometry (MS/MS) to test for selected amino acids and acylcarnitines.ResultsDuring this time, 132 patients were identified with 24 different inherited metabolic diseases (classic forms and variants). To date, the global frequency for all disorders integrated into the PNSP is estimated to be 1:1,380, with 1:2,396 for metabolic disorders. A total of 379 tests (0.12%) were classified as having false positive results, yielding an overall specificity of 99.9%. Despite the low frequency of several disorders, the positive predictive value of the overall MS/MS screening was found to be 26%, reflecting high diagnostic specificity of the method. Diagnostic sensitivity of extended screening for the different groups of disorders was 100%. Eight cases of maternal disorders [three glutaric aciduria typexa0I, one carnitine transporter defect, and four 3-methylcrotonyl coenzymexa0A (CoA) carboxylase deficiency] were also detected through newborn screening.ConclusionsOur data support the advantage of a centralised laboratory for screening an elevated number of samples and making decisions if relying on a clinical network able to provide fast treatment and a good outcome in the screened cases.

About the “Pathological” Role of the mtDNA T3308C Mutation…

To the Editor: n nNumerous mtDNA mutations have been associated with the mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS) syndrome (MIM 540000). These include transitions at nucleotide positions (nt) 1642, 3243, 3252, 3256, 3271, 3291, 3308, and 9957, and a 4-bp deletion beginning at nt 14787. For some of these mutations (A3243G, C3256T, and T3271C), the causal relationship with the phenotype has been confirmed, whereas for others, the status is still provisional (MITOMAP). The T3308C mutation in the NADH dehydrogenase subunit 1 (ND1) is a member of the “provisional” group and was described in a Spanish subject affected by MELAS and bilateral striatal necrosis. This mutation changes the highly conserved methionine 1 to a threonine, was heteroplasmic in both the proband and her asymptomatic mother, and was absent in 130 normal and other-disease controls (Campos et al. 1997). More recently, a homoplasmic T3308C mutation has also been reported in a colorectal tumor, in which it was associated with two other somatic homoplasmic transitions, T710C and T1738C. It has been suggested that these mutations could have a functional effect in mitochondrial selection (Polyak et al. 1998). However, doubts about the pathological significance of the T3308C mutation have been raised by a study involving 37 Portuguese patients with a clinical phenotype of mitochondrial encephalomyopathies and 150 Portuguese control subjects. The T3308C mutation was observed in two patients and in four controls (Vilarinho et al. 1999). In all cases it was homoplasmic. n nTo better define the role of this putative pathological mutation, we did a detailed analysis of the mtDNA background on which the T3308C had been reported. By sequence analysis of several tRNA genes and their surrounding sequences, we determined that, in addition to the T3308C mutation, the mtDNA of both Portuguese patients harbored the combination of mutations T1738C, T5655C, G7521A, A10398C, and A14769G and a dinucleotide deletion at nt 514–515. We observed the same mutations in the two Spanish patients (in the meantime a second Spanish patient had been found) and in the four Portuguese controls who tested positive for the mutation. Thus, these results indicated that all these mtDNAs were members of the same mtDNA haplogroup and that most likely they shared the T3308C mutation by descent. Intriguingly, this haplogroup harbored the combination of mutations T3308C and T1738C, similar to the case reported by Polyak et al. (1998). The search in our samples for the third somatic mutation (T710C) found in the colorectal tumor was negative. n nTo identify the mtDNA haplogroup harboring the mutation T3308C, sequence analysis of the mtDNA control region between nt 16090 and 16375 was performed in the eight T3308C samples (table 1). This analysis revealed a consensus motif (16126–16187–16189–16223–16264–16270–16278–16293–16311) that is typical of the West African haplogroup L1b (Watson et al. 1997; Rando et al. 1998), thus allowing us to classify Portuguese and Spanish mtDNAs with the T3308C mutation within this haplogroup. It has been determined elsewhere, by high-resolution restriction analysis (Torroni et al. 1996, 1997), that haplogroup L1b is defined by the RFLP motif: +185 TaqI, +2349 MboI, −2758 RsaI, +3592 HpaI, −3693 MboI, −7055 AluI, +10394 DdeI, +10806 HinfI (Chen et al. 1995; Rando et al. 1998; A. Torroni, unpublished data). Therefore, we selected, among our African population samples, all those (a total of 48) who either by RFLP analysis or by control region sequencing had been classified as members of haplogroup L1b. Analysis of their status at nt 3308 revealed that all of them harbored the mutation. In contrast, control samples belonging to African haplogroups L1a, L1c, and L2 were found to lack the mutation. These results indicate that the T3308C mutation defines exclusively by descent haplogroup L1b mtDNAs, and it is very ancient since L1b probably originated in western Africa ∼12,000–19,000 years ago (Watson et al. 1997; Rando et al. 1998). Thus, Spanish and Portuguese mtDNAs with the T3308C mutation are of African origin, and their presence probably reflects the arrival of North Africans during the Mesolithic Age (8000 b.c.) and/or during the Arabic rule that started at ∼800 a.d. (Arnaiz-Villena et al. 1997). If we take into account that haplogroup L1b frequencies in populations of western Africa are in the range of 10%–20% (Watson et al. 1997; Rando et al. 1998), the observed frequency in the Portuguese population (∼2%–3%) indicates a significant influence of North Africans in the Iberian gene pool. n n n nTable 1 n nmtDNA Control Region Variation in Iberian Patients and Controls n n n nIn conclusion, the T3308C mutation is an ancient marker of a common West African haplogroup, and all Iberian subjects with this mutation who were affected by mitochondrial encephalomyopathies harbored haplogroup L1b mtDNAs. This finding is difficult to reconcile with a role of this mutation in disease expression and further indicates that haplogroup classification of patients mtDNAs, followed by a search for the putative disease mutation in phylogenetically closely related control mtDNAs, is a crucial step in the identification of mtDNA disease mutations. Furthermore, the observation that the elimination of the methionine codon AUA at position 1 of the ND1 subunit is common in some human populations suggests that the maintenance of that codon is not so critical in our species. Possibly this is because the third codon (AUG) of the human ND1 subunit also encodes for a methionine, and the ND1 subunit of L1b mtDNAs, although it might be shortened by two amino acids, apparently still retains its functionality. However, it is intriguing that the same combination, T3308C–T1738C, that characterizes haplogroup L1b has also occurred in a colorectal tumor as new somatic mutations. This is especially noteworthy when it is taken into account that T1738C occurs in the 16S rRNA, a gene involved in the translation process, and that the T3308C mutation might indeed affect the translation process of ND1 on non-L1b mtDNA backgrounds. This observation raises again the possibility of polygenic models in which certain mtDNA mutations can be functional and maintained in the population only if they occur in combination with other specific mtDNA mutations.

Outcome of three cases of untreated maternal glutaric aciduria type I

We report, for the first time, the outcome of three children born to two women with untreated glutaric aciduria type I (GA I). Isolated hypocarnitinemia in neonatal screening in one baby allowed the identification of the disease in his mother, who was undiagnosed so far and had had a previous daughter. The other baby was born to an already diagnosed mother who was not treated; newborn screening in the child reflected the metabolic state of the mother. Biochemical abnormalities returned to normal within one week. At the age of 4xa0months, neuroimaging showed Sylvian enlargement in both infants and bilateral temporal arachnoid cysts in one. Physical and neurological developments were normal for the three patients at ages 2 and 5xa0years. We conclude that long-term follow up will determine the true impact of GA I in such children.

Characterization of mitochondrial proteome in a severe case of ETF-QO deficiency.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a mitochondrial fatty acid oxidation disorder caused by mutations that affect electron transfer flavoprotein (ETF) or ETF:ubiquinone oxidoreductase (ETF-QO) or even due to unidentified disturbances of riboflavin metabolism. Besides all the available data on the molecular basis of FAO disorders, including MADD, the pathophysiological mechanisms underlying clinical phenotype development, namely at the mitochondrial level, are poorly understood. In order to contribute to the elucidation of these mechanisms, we isolated mitochondria from cultured fibroblasts, from a patient with a severe MADD presentation due to ETF-QO deficiency, characterize its mitochondrial proteome and compare it with normal controls. The used approach (2-DE-MS/MS) allowed the positive identification of 287 proteins in both patient and controls, presenting 35 of the significant differences in their relative abundance. Among the differentially expressed are proteins associated to binding/folding functions, mitochondrial antioxidant enzymes as well as proteins associated to apoptotic events. The overexpression of chaperones like Hsp60 or mitochondrial Grp75, antioxidant enzymes and apoptotic proteins reflects the mitochondrial response to a complete absence of ETF-QO. Our study provides a global perspective of the mitochondrial proteome plasticity in a severe case of MADD and highlights the main molecular pathways involved in its pathogenesis.

Mitochondria proteome profiling: a comparative analysis between gel- and gel-free approaches.

Mitochondrial proteomics emerged aiming to disclose the dynamics of mitochondria under various pathophysiological conditions. In the present study we investigated the relative merits of gel-based (2DE and SDS-LC) and gel-free (2D-LC) protein separation approaches and protein identification algorithms (Mascot and Paragon) in the proteome profiling of mitochondria isolated from cultured fibroblasts, a sample traditionally used for diagnosis purposes. Combining data retrieved from 2DE, 2D-LC and SDS-LC and search methods, a total of 696 non-redundant proteins were identified. An overlap of only 19% between the proteins identified by the three different methods was observed when Mascot and Paragon were used. Regarding protein ID, a consistency in the number of identified proteins per sample was noticed for 2DE approach. Independent of the methodological approach chosen, it was noticed that the predominance in mitochondria of hydrophilic proteins with 20-50 kDa and pI 5-6 and 8-9; however, 2D-LC and SDS-LC allowed the enrichment of proteins with a mass below 30 kDa and of basic proteins with pI values above 8. In conclusion, data from the present study highlight the power of integrating different separation technologies and protein identification algorithms.

The ND1 T3308C mutation may be a mtDNA polymorphism. Report of two Portuguese patients

The ND1 T3308C mutation may be a mtDNA polymorphism. Report of two Portuguese patients L . V ilarinho1*, R. M. L . Cardoso1, H. Rocha1, C. Nogueira1, Chora8 o2, and F. M. Santorelli3,4 1 Department of Clinical Biology, Instituto de Genex8d tica Mex8d dica, Porto ; 2 Department of Neurology, Hospital Geral de S. Antox8d nio, Porto, Portugal ; 3 Laboratory of Molecular Neurobiology, Institute of Neurology “C. MondinoÏ, University of Pavia, Italy ; 4 Department of Neurology, CPMC, Columbia University, New York, USA Correspondence : Dept. of Clinical Biology, Instituto de Genex8d tica Mex8d dica, Pr. Pedro Nunes, 88 4050 Porto, Portugal. E-mail : vilarinho.fam=mail.telepac.pt

Mutations at the flavin binding site of ETF:QO yield a MADD-like severe phenotype in Drosophila

Following a screening on EMS-induced Drosophila mutants defective for formation and morphogenesis of epithelial cells, we have identified three lethal mutants defective for the production of embryonic cuticle. The mutants are allelic to the CG12140 gene, the fly homologue of electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). In humans, inherited defects in this inner membrane protein account for multiple acyl-CoA dehydrogenase deficiency (MADD), a metabolic disease of β-oxidation, with a broad range of clinical phenotypes, varying from embryonic lethal to mild forms. The three mutant alleles carried distinct missense mutations in ETF:QO (G65E, A68V and S104F) and maternal mutant embryos for ETF:QO showed lethal morphogenetic defects and a significant induction of apoptosis following germ-band elongation. This phenotype is accompanied by an embryonic accumulation of short- and medium-chain acylcarnitines (C4, C8 and C12) as well as long-chain acylcarnitines (C14 and C16:1), whose elevation is also found in severe MADD forms in humans under intense metabolic decompensation. In agreement the ETF:QO activity in the mutant embryos is markedly decreased in relation to wild type activity. Amino acid sequence analysis and structural mapping into a molecular model of ETF:QO show that all mutations map at FAD interacting residues, two of which at the nucleotide-binding Rossmann fold. This structural domain is composed by a β-strand connected by a short loop to an α-helix, and its perturbation results in impaired cofactor association via structural destabilisation and consequently enzymatic inactivation. This work thus pinpoints the molecular origins of a severe MADD-like phenotype in the fruit fly and establishes the proof of concept concerning the suitability of this organism as a potential model organism for MADD.

Intermittent rhabdomyolysis with adult onset associated with a mutation in the ACADVL gene

Deficiency of very-long-chain acyl-CoA dehydrogenase (VLCAD) is an autosomal recessive disease. Most common phenotypes occur in the neonatal period or in childhood with cardiomyopathy, hepatomegaly, and hypoketogenic hypoglycemia. Juvenile/adult-onset is characterized by exercise intolerance and recurrent rhabdomyolysis triggered by prolonged exercise or fasting. This article reports a patient with the homozygous mutation c.1097G>A (p.R366H) in the ACADVL gene. In Portugal, VLCAD deficiency became part of the neonatal screening plan in 2004, and as of 2012, 8 early-onset cases have been diagnosed, giving an incidence rate of 1:97.238 per 737.902 newborns. This patient was diagnosed outside of the neonatal screening plan. Beta-oxidation defects pose a diagnostic challenge because of their transient clinical and laboratorial manifestations and the absence of morphological changes in muscle biopsy further complicate matters, especially in the late-onset forms of the disease. The adult phenotype of VLCAD deficiency is highlighted, emphasizing the need for a high suspicion index and the value of tandem mass spectrometry for the diagnosis.

NeoScreen: A Software Application for MS/MS Newborn Screening Analysis

The introduction of the Tandem Mass Spectrometry (MS/MS), in neonatal screening laboratories, has opened the doors to innovative newborn screening analysis. With this technology the number of metabolic disorders, that can be detected, from dried blood-spot species, increases significantly. However, the amount of information obtained with this technique and the pressure for quick and accurate diagnostics raises serious difficulties in the daily data analysis. To face this challenge we developed a software system, NeoScreen, which simplifies and allow speeding up newborn screening diagnostics.

Birth Prevalence of Fatty Acid β-Oxidation Disorders in Iberia

Mitochondrial fatty acid β-oxidation disorders (FAOD) are main targets for newborn screening (NBS) programs, which are excellent data sources for accurate estimations of disease birth prevalence. Epidemiological data is of key importance for the understanding of the natural history of the disorders as well as to define more effective public health strategies. In order to estimate FAOD birth prevalence in Iberia, the authors collected data from six NBS programs from Portugal and Spain, encompassing the screening of more than 1.6 million newborns by tandem mass spectrometry (MS/MS), and compared it with available data from other populations. The participating NBS programs are responsible for the screening of about 46% of all Iberian newborns. Data reveals that Iberia has one of the highest FAOD prevalence in Europe (1:7,914) and that Portugal has the highest birth prevalence of FAOD reported so far (1:6,351), strongly influenced by the high prevalence of medium-chain acyl-CoA dehydrogenase deficiency (MCADD; 1:8,380), one of the highest ever reported. This is justified by the fact that more than 90% of Portuguese MCADD patients are of Gypsy origin, a community characterized by a high degree of consanguinity. From the comparative analysis of various populations with comparable data other differences emerge, which points to the existence of significant variations in FAOD prevalences among different populations, but without any clear European variation pattern. Considering that FAOD are one of the justifications for MS/MS NBS, the now estimated birth prevalences stress the need to screen all Iberian newborns for this group of inherited metabolic disorders.