Implementation of second-tier tests in newborn screening for the detection of vitamin B12 related acquired and genetic disorders: results on 258,637 newborns

Homocisteïna; Cribratge de nounats; Deficiència de vitamina B12Homocisteína; Cribado de recién nacidos; Deficiencia de vitamina B12Homocysteine; Newborn screening; Vitamin B12 deficiencyBackground Alteration of vitamin B12 metabolism can be genetic or acquired, and can result in anemia, failure to thrive, developmental regression and even irreversible neurologic damage. Therefore, early diagnosis and intervention is critical. Most of the neonatal cases with acquired vitamin B12 deficiency have been detected by clinical symptoms and only few of them trough NBS programs. We aim to assess the usefulness of the second-tier test: methylmalonic acid (MMA), methylcitric acid (MCA) and homocysteine (Hcys) in our newborn screening program and explore the implications on the detection of cobalamin (vitamin B12) related disorders, both genetic and acquired conditions. Methods A screening strategy using the usual primary markers followed by the analysis of MMA, MCA and Hcys as second tier-test in the first dried blood spot (DBS) was developed and evaluated. Results During the period 2015–2018 a total of 258,637 newborns were screened resulting in 130 newborns with acquired vitamin B12 deficiency (incidence 1:1989), 19 with genetic disorders (incidence 1:13,613) and 13 were false positive. No false negatives were notified. Concerning the second-tier test, the percentage of cases with MMA above the cut-off levels, both for genetic and acquired conditions was very similar (58% and 60%, respectively). Interestingly, the percentage of cases with increased levels of Hcys was higher in acquired conditions than in genetic disorders (87% and 47%, respectively). In contrast, MCA was high only in 5% of the acquired conditions versus in 53% of the genetic disorders, and it was always very high in all patients with propionic acidemia. Conclusions When screening for methylmalonic acidemia and homocystinuria, differential diagnosis with acquired vitamin B12 deficiency should be done. The results of our strategy support the inclusion of this acquired condition in the NBS programs, as it is easily detectable and allows the adoption of corrective measures to avoid the consequences of its deficiency

Clinical, biochemical, cellular and molecular characterization of mitochondrial DNA depletion syndrome due to novel mutations in the MPV17 gene

Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are severe autosomal recessive disorders associated with decreased mtDNA copy number in clinically affected tissues. The hepatocerebral form (mtDNA depletion in liver and brain) has been associated with mutations in the POLG, PEO1 (Twinkle), DGUOK and MPV17 genes, the latter encoding a mitochondrial inner membrane protein of unknown function. The aims of this study were to clarify further the clinical, biochemical, cellular and molecular genetic features associated with MDS due to MPV17 gene mutations. We identified 12 pathogenic mutations in the MPV17 gene, of which 11 are novel, in 17 patients from 12 families. All patients manifested liver disease. Poor feeding, hypoglycaemia, raised serum lactate, hypotonia and faltering growth were common presenting features. mtDNA depletion in liver was demonstrated in all seven cases where liver tissue was available. Mosaic mtDNA depletion was found in primary fibroblasts by PicoGreen staining. These results confirm that MPV17 mutations are an important cause of hepatocerebral mtDNA depletion syndrome, and provide the first demonstration of mosaic mtDNA depletion in human MPV17 mutant fibroblast cultures. We found that a severe clinical phenotype was associated with profound tissue-specific mtDNA depletion in liver, and, in some cases, mosaic mtDNA depletion in fibroblasts

Bases bioquímiques i genètiques de les deplecions De mtDNA i de les alteracions de NFU1

Les deplecions de DNA mitocondrial (mtDNA) i les alteracions de NFU1 són dos grups de malalties que afecten vies molt importants dins de la mitocòndria i que alteren el metabolisme energètic. Clínicament, els pacients afectes de depleció de mtDNA es caracteritzen per mostrar un ampli ventall de símptomes, depenent del gen afectat. Els símptomes clínics són molt greus i la majoria de vegades porten a la mort del pacient. Els pacients amb mutacions en el gen NFU1, descrits per primera vegada en aquest treball, presenten una clínica més homogènia, caracteritzada sobretot per presentar una encefalopatia infantil fatal i/o hipertensió pulmonar i un fenotip bioquímic molt particular però ben definit, caracteritzat principalment per presentar acidosi làctica, hiperglicinèmia i deficiència de l’activitat piruvat deshidrogenasa. L’objectiu principal d’aquesta tesis ha consistit en millorar i implementar metodologies per a contribuir al diagnòstic, i millorar la comprensió de la fisiopatologia d’aquestes deficiències. Per a tal fita s’ha posat a punt una tècnica de PCR a temps real per estudiar el número de còpies de mtDNA i poder relacionar-lo amb l’activitat citrat sintasa. A més, la depleció s’ha pogut estudiar en biòpsies incloses en parafina, una important font de material biològic arxivat mai utilitzat fins al moment. D’aquesta forma s’han pogut estudiat 50 pacients amb sospita clínica de depleció de mtDNA i cercar mutacions en els gens relacionats; DGUOK, MPV17, C10orf2, SUCLG1, SUCLA2 i POLG. L’estudi molecular ens ha permès identificar 4 mutacions no descrites prèviament, c.70+5G>A en el gen MPV17, c.1048G>A i c.1049G>T en el gen SUCLA2, i c.531+4A>T en el gen SUCLG1. A més s’han identificat 7 mutacions prèviament descrites en un total de 10 pacients (8 famílies). Quan va ser possible, es va quantificar el ràtio mtDNA/nDNA i la activitat citrat sintasa en la mateixa mostra de teixit, proveint noves dades per l’estudi de les MDS. La depleció relacionada amb la citrat sintasa, (mtDNA/nDNA)/CS, ha donat resposta a certes discrepàncies observades entre els resultats de depleció i els resultats de la cadena respiratòria mitocondrial en alguns pacients. Per altra banda, utilitzant el mapatge per homozigositat, es va identificar una mutació de canvi de sentit en homozigositat en el gen NFU1 (c.622G>T, p.Gly208Cys), que codifica per una proteïna altament conservada en totes les espècies i que està implicada en la biogènesis dels clústers de sulfur de ferro (Fe-S). Aquesta ha esta la primera vegada que s’ha associat mutacions en aquest gen a patologia humana. El fenotip bioquímic dels pacients suggeria una activitat deficient de l’enzima lipoic àcid sintasa (LAS), una proteïna que necessita clústers de Fe-S com a cofactor. Es va poder constatar una disminució del grau de lipoilació de les proteïnes dependents d’àcid lipoic, el que suggeria una manca d’ activitat LAS. Utilitzant models cel·lulars hem demostrat que la proteïna NFU1 és necessària com a donadora de sulfur per la biosíntesi d’àcid lipoic i també ens ha permès conèixer la funció específica en la biosíntesi dels clústers de Fe-S i en la maduració de proteïnes Fe-S, concretament LAS i la succinat deshidrogenasa (SDH). La descripció clínica, bioquímica y genètica d’aquesta malaltia és molt important pel diagnòstic de nous pacients i obre les portes a la cerca d’altres gens implicats en la biosíntesi de l’àcid lipoic, així com al disseny futur de noves estratègies terapèutiques.Mitochondrial DNA (mtDNA) depletion syndromes (MDS) and NFU1 defects are two groups of diseases affecting crucial mitochondrial pathways of energetic metabolism. Clinically, patients affected of mtDNA depletion displayed a wide range of symptoms, depending on the altered gene. The clinical symptoms are severe and in most cases lead to death of the patient. Patients with NFU1 mutations, described for the first time in this paper, present a more homogeneous clinical phenotype, characterized by fatal infantile encephalopathy and / or pulmonary hypertension. NFU1 patients also showed a peculiar, but well defined, biochemical phenotype, presenting with lactic acidosis, hyperglycinemia and deficiency of pyruvate dehydrogenase activity. The main objective of the present thesis is to improve and to implement new methods for the diagnosis and understanding of the pathophysiology of these deficiencies. To this goal, a real-time PCR technique has been developed to study the mtDNA copy number and its relationship to citrate synthase activity in MDS patients. In addition, mtDNA depletion has been studied in formalin-fixed paraffin-embedded tissues, an important source of biological material never used for this purpose. We studied 50 paediatric individuals suspected to have mtDNA depletion and the appropriate MDS genes have been screened according to their clinical and biochemical phenotypes. Mutational study of DGUOK, MPV17, SUCLA2, SUCLG1 and POLG allowed us to identify 4 novel mutations; c.70+5G>A in MPV17, c.1048G>A and c.1049G>T in SUCLA2 and c.531+4A>T in SUCLG1, and 7 already known mutations in 10 patients (8 families). When possible, we quantified mtDNA/nDNA and CS activity in the same tissue sample, providing an additional tool for the study of MDS. The ratio (mtDNA/nDNA)/CS has shed some light in the discrepant results between the mtDNA copy number and the enzymatic respiratory chain activities of some cases. Using homozigosity mapping, we identified a homozygous missense mutation in NFU1 gene (c.622G> T, p.Gly208Cys), which encodes a conserved protein suggested to participate in Fe-S cluster biogenesis. This is the first time that a clinical phenotype has been associated with mutations to NFU1. The biochemical phenotype suggested an impaired activity of the Fe-S enzyme lipoic acid synthase (LAS), a protein that requires Fe-S cluster as a cofactor. Direct measurement of protein-bound lipoic acid in individual tissues indeed showed marked decreases, which suggested a lack of LAS activity. Human cell models studies showed that NFU1 protein is required as sulfur donor for the biosynthesis of lipoic acid and it performs a specific function in mitochondrial Fe-S proteins maturation, particularly succinate dehydrogenase and LAS (SDH). Clinical, biochemical and genetic description of NFU1 disease is very important for the diagnosis of new patients and will allow us to find other genes involved in the biosynthesis of lipoic acid, and provided the basis for the future design of new therapeutic strategies

Bases bioquímiques i genètiques de les deplecions De mtDNA i de les alteracions de NFU1

Les deplecions de DNA mitocondrial (mtDNA) i les alteracions de NFU1 són dos grups de malalties que afecten vies molt importants dins de la mitocòndria i que alteren el metabolisme energètic. Clínicament, els pacients afectes de depleció de mtDNA es caracteritzen per mostrar un ampli ventall de símptomes, depenent del gen afectat. Els símptomes clínics són molt greus i la majoria de vegades porten a la mort del pacient. Els pacients amb mutacions en el gen NFU1, descrits per primera vegada en aquest treball, presenten una clínica més homogènia, caracteritzada sobretot per presentar una encefalopatia infantil fatal i/o hipertensió pulmonar i un fenotip bioquímic molt particular però ben definit, caracteritzat principalment per presentar acidosi làctica, hiperglicinèmia i deficiència de l'activitat piruvat deshidrogenasa. L'objectiu principal d'aquesta tesis ha consistit en millorar i implementar metodologies per a contribuir al diagnòstic, i millorar la comprensió de la fisiopatologia d'aquestes deficiències. Per a tal fita s'ha posat a punt una tècnica de PCR a temps real per estudiar el número de còpies de mtDNA i poder relacionar-lo amb l'activitat citrat sintasa. A més, la depleció s'ha pogut estudiar en biòpsies incloses en parafina, una important font de material biològic arxivat mai utilitzat fins al moment. D'aquesta forma s'han pogut estudiat 50 pacients amb sospita clínica de depleció de mtDNA i cercar mutacions en els gens relacionats; DGUOK, MPV17, C10orf2, SUCLG1, SUCLA2 i POLG. L'estudi molecular ens ha permès identificar 4 mutacions no descrites prèviament, c.70+5G>A en el gen MPV17, c.1048G>A i c.1049G>T en el gen SUCLA2, i c.531+4A>T en el gen SUCLG1. A més s'han identificat 7 mutacions prèviament descrites en un total de 10 pacients (8 famílies). Quan va ser possible, es va quantificar el ràtio mtDNA/nDNA i la activitat citrat sintasa en la mateixa mostra de teixit, proveint noves dades per l'estudi de les MDS. La depleció relacionada amb la citrat sintasa, (mtDNA/nDNA)/CS, ha donat resposta a certes discrepàncies observades entre els resultats de depleció i els resultats de la cadena respiratòria mitocondrial en alguns pacients. Per altra banda, utilitzant el mapatge per homozigositat, es va identificar una mutació de canvi de sentit en homozigositat en el gen NFU1 (c.622G>T, p.Gly208Cys), que codifica per una proteïna altament conservada en totes les espècies i que està implicada en la biogènesis dels clústers de sulfur de ferro (Fe-S). Aquesta ha esta la primera vegada que s'ha associat mutacions en aquest gen a patologia humana. El fenotip bioquímic dels pacients suggeria una activitat deficient de l'enzima lipoic àcid sintasa (LAS), una proteïna que necessita clústers de Fe-S com a cofactor. Es va poder constatar una disminució del grau de lipoilació de les proteïnes dependents d'àcid lipoic, el que suggeria una manca d' activitat LAS. Utilitzant models cel·lulars hem demostrat que la proteïna NFU1 és necessària com a donadora de sulfur per la biosíntesi d'àcid lipoic i també ens ha permès conèixer la funció específica en la biosíntesi dels clústers de Fe-S i en la maduració de proteïnes Fe-S, concretament LAS i la succinat deshidrogenasa (SDH). La descripció clínica, bioquímica y genètica d'aquesta malaltia és molt important pel diagnòstic de nous pacients i obre les portes a la cerca d'altres gens implicats en la biosíntesi de l'àcid lipoic, així com al disseny futur de noves estratègies terapèutiques.Mitochondrial DNA (mtDNA) depletion syndromes (MDS) and NFU1 defects are two groups of diseases affecting crucial mitochondrial pathways of energetic metabolism. Clinically, patients affected of mtDNA depletion displayed a wide range of symptoms, depending on the altered gene. The clinical symptoms are severe and in most cases lead to death of the patient. Patients with NFU1 mutations, described for the first time in this paper, present a more homogeneous clinical phenotype, characterized by fatal infantile encephalopathy and / or pulmonary hypertension. NFU1 patients also showed a peculiar, but well defined, biochemical phenotype, presenting with lactic acidosis, hyperglycinemia and deficiency of pyruvate dehydrogenase activity. The main objective of the present thesis is to improve and to implement new methods for the diagnosis and understanding of the pathophysiology of these deficiencies. To this goal, a real-time PCR technique has been developed to study the mtDNA copy number and its relationship to citrate synthase activity in MDS patients. In addition, mtDNA depletion has been studied in formalin-fixed paraffin-embedded tissues, an important source of biological material never used for this purpose. We studied 50 paediatric individuals suspected to have mtDNA depletion and the appropriate MDS genes have been screened according to their clinical and biochemical phenotypes. Mutational study of DGUOK, MPV17, SUCLA2, SUCLG1 and POLG allowed us to identify 4 novel mutations; c.70+5G>A in MPV17, c.1048G>A and c.1049G>T in SUCLA2 and c.531+4A>T in SUCLG1, and 7 already known mutations in 10 patients (8 families). When possible, we quantified mtDNA/nDNA and CS activity in the same tissue sample, providing an additional tool for the study of MDS. The ratio (mtDNA/nDNA)/CS has shed some light in the discrepant results between the mtDNA copy number and the enzymatic respiratory chain activities of some cases. Using homozigosity mapping, we identified a homozygous missense mutation in NFU1 gene (c.622G> T, p.Gly208Cys), which encodes a conserved protein suggested to participate in Fe-S cluster biogenesis. This is the first time that a clinical phenotype has been associated with mutations to NFU1. The biochemical phenotype suggested an impaired activity of the Fe-S enzyme lipoic acid synthase (LAS), a protein that requires Fe-S cluster as a cofactor. Direct measurement of protein-bound lipoic acid in individual tissues indeed showed marked decreases, which suggested a lack of LAS activity. Human cell models studies showed that NFU1 protein is required as sulfur donor for the biosynthesis of lipoic acid and it performs a specific function in mitochondrial Fe-S proteins maturation, particularly succinate dehydrogenase and LAS (SDH). Clinical, biochemical and genetic description of NFU1 disease is very important for the diagnosis of new patients and will allow us to find other genes involved in the biosynthesis of lipoic acid, and provided the basis for the future design of new therapeutic strategies

A Fatal Mitochondrial Disease Is Associated with Defective NFU1 Function in the Maturation of a Subset of Mitochondrial Fe-S Proteins

We report on ten individuals with a fatal infantile encephalopathy and/or pulmonary hypertension, leading to death before the age of 15 months. Hyperglycinemia and lactic acidosis were common findings. Glycine cleavage system and pyruvate dehydrogenase complex (PDHC) activities were low. Homozygosity mapping revealed a perfectly overlapping homozygous region of 1.24 Mb corresponding to chromosome 2 and led to the identification of a homozygous missense mutation (c.622G>T) in NFU1, which encodes a conserved protein suggested to participate in Fe-S cluster biogenesis. Nine individuals were homozygous for this mutation, whereas one was compound heterozygous for this and a splice-site (c.545+5G>A) mutation. The biochemical phenotype suggested an impaired activity of the Fe-S enzyme lipoic acid synthase (LAS). Direct measurement of protein-bound lipoic acid in individual tissues indeed showed marked decreases. Upon depletion of NFU1 by RNA interference in human cell culture, LAS and, in turn, PDHC activities were largely diminished. In addition, the amount of succinate dehydrogenase, but no other Fe-S proteins, was decreased. In contrast, depletion of the general Fe-S scaffold protein ISCU severely affected assembly of all tested Fe-S proteins, suggesting that NFU1 performs a specific function in mitochondrial Fe-S cluster maturation. Similar biochemical effects were observed in Saccharomyces cerevisiae upon deletion of NFU1, resulting in lower lipoylation and SDH activity. Importantly, yeast Nfu1 protein carrying the individuals' missense mutation was functionally impaired. We conclude that NFU1 functions as a late-acting maturation factor for a subset of mitochondrial Fe-S proteins

Tindjauan sosiografi Indonesia desa Meliling

We evaluated coenzyme Q10 (CoQ) levels in patients studied under suspicion of mitochondrial DNA depletion syndromes (MDS) (n = 39). CoQ levels were quantified by HPLC, and the percentage of mtDNA depletion by quantitative real-time PCR. A high percentage of MDS patients presented with CoQ deficiency as compared to other mitochondrial patients (Mann–Whitney-U test: p = 0.001). Our findings suggest that MDS are frequently associated with CoQ deficiency, as a possible secondary consequence of disease pathophysiology. Assessment of muscle CoQ status seems advisable in MDS patients since the possibility of CoQ supplementation may then be considered as a candidate therapy

X-inactivation of HSD17B10 revealed by cDNA analysis in two female patients with 17β-hydroxysteroid dehydrogenase 10 deficiency

17β-Hydroxysteroid dehydrogenase 10 (HSD10) is a mitochondrial enzyme involved in the degradation pathway of isoleucine and branched-chain fatty acids. The gene encoding HSD10, HSD17B10, has been reported as one of the few genes that escapes X-inactivation. We previously studied two female patients with HSD10 deficiency, one of them was severely affected and the other presented a mild phenotype. To elucidate as to why these two carriers were so differently affected, cDNA analyses were performed. The HSD17B10 cDNA of eight control cell lines, two hemizygous patients and two carriers was obtained from cultured fibroblasts, amplified by PCR and sequenced by standard methods. All HSD17B10 cDNAs were quantified by real-time PCR. In the fibroblasts of the female patient who presented with the severe phenotype, only the mutant allele was identified in the cDNA sequence, which was further confirmed by relative quantification (RQ) of HSD17B10 cDNA. This is in agreement with an unfavourable X-inactivation. The other female patient, with slight clinical affectation, showed the presence of both mutant and wild-type alleles in the cDNA sequence, which was confirmed by RQ of HSD17B10 cDNA in fibroblasts. This is in line with normal X-inactivation and the expression of both alleles in different cells (functional mosaicism). RQ results of HSD17B10 cDNA did not differ significantly between male and female controls, which indicate that the genetic doses of mRNA of HSD17B10 was the same in both sexes. In conclusion, these results suggest that the HSD17B10 gene does not escape X-inactivation as has been reported previously