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

BACKGROUND: 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.

[50 years of the Neonatal Screening Program in Catalonia.] / 50 años del Programa de Cribado Neonatal en Cataluña.

The Catalonian Newborn Screening Program (CNSP) began in 1969, in Barcelona. It was promoted by Dr. Juan Sabater Tobella and supported by Barcelona Provincial Council and Juan March Foundation. That is how the Institute of Clinical Biochemistry was born, whose aims were diagnosis, research and teaching, along with the spirit of contributing to the prevention of mental retardation. The CNSP began with the detection of phenylketonuria (PKU), and, in 1982, the Program was expanded with the inclusion of congenital hypothyroidism detection. Towards 1990, the Program covered almost 100% of all newborns (NB) in Catalonia. In 1999, the CNSP was expanded with the incorporation of cystic fibrosis. It took fourteen years, until 2013, to make the largest expansion so far, with the incorporation of 19 metabolic diseases to the screening panel. The detection of sickle cell disease began in 2015 and in 2017 the detection of severe combined immunodeficiency was included. Currently, the CNSP includes 24 diseases in its main panel. Since 1969, 2,787,807 NBs have been screened, of whom 1,724 have been diagnosed with any of these diseases, and 252 of other disorders by differential diagnosis with those included in the main panel. The global prevalence is 1: 1,617 NBs affected by any of the diseases included in the CNSP and 1: 1,140 NBs if incidental findings diagnosed through the CNSP are included.

El Programa de Cribado Neonatal de Cataluña (PCNC) se inició en el año 1969, en Barcelona, impulsado por el Dr. Juan Sabater Tobella y apoyado por la Diputación de Barcelona y la Fundación Juan March. Así nació el Instituto de Bioquímica Clínica para acometer funciones asistenciales, de investigación y docencia, con el espíritu de contribuir a la prevención del retraso mental. El PCNC se inició con la detección de la fenilcetonuria (PKU) y en el año 1982 se amplió con la detección del hipotiroidismo congénito. Hacia el año 1990 la cobertura territorial llegó casi al 100% de todos los recién nacidos en Cataluña. En 1999 se amplió el PCNC con la incorporación de la fibrosis quística y tras catorce años, en 2013, se realizó la ampliación más numerosa hasta ahora, con la incorporación de la detección de 19 enfermedades metabólicas hereditarias. En el año 2015 comenzó la detección de la enfermedad de células falciformes y en el 2017 la detección de la inmunodeficiencia combinada grave. Actualmente, el PCNC incluye la detección de 24 enfermedades. Desde su inicio en el año 1969, se han cribado 2.787.807 recién nacidos, de los cuales 1.724 han sido diagnosticados de alguna de las 24 enfermedades que componen nuestro panel principal y 252 por diagnóstico diferencial de las primeras. En total la prevalencia global es de 1:1.617 RN afectos de alguna de las enfermedades incluidas en el PCNC y de 1:1.140 RN si se incluyen los hallazgos incidentales encontrados.

Mutations in the lipoyltransferase LIPT1 gene cause a fatal disease associated with a specific lipoylation defect of the 2-ketoacid dehydrogenase complexes.

Cofactor disorders of mitochondrial energy metabolism are a heterogeneous group of diseases with a wide variety of clinical symptoms, particular metabolic profiles and variable enzymatic defects. Mutations in NFU1, BOLA3, LIAS and IBA57 have been identified in patients with deficient lipoic acid-dependent enzymatic activities and defects in the assembly and activity of the mitochondrial respiratory chain complexes. Here, we report a patient with an early onset fatal lactic acidosis presenting a biochemical phenotype compatible with a combined defect of pyruvate dehydrogenase (PDHC) and 2-ketoglutarate dehydrogenase (2-KGDH) activities, which suggested a deficiency in lipoic acid metabolism. Immunostaining analysis showed that lipoylated E2-PDH and E2-KGDH were extremely reduced in this patient. However, the absence of glycine elevation, the normal activity of the glycine cleavage system and the normal lipoylation of the H protein suggested a defect of lipoic acid transfer to particular proteins rather than a general impairment of lipoic acid biosynthesis as the potential cause of the disease. By analogy with yeast metabolism, we postulated LIPT1 as the altered candidate gene causing the disease. Sequence analysis of the human LIPT1 identified two heterozygous missense mutations (c.212C>T and c.292C>G), segregating in different alleles. Functional complementation experiments in patient's fibroblasts demonstrated that these mutations are disease-causing and that LIPT1 protein is required for lipoylation and activation of 2-ketoacid dehydrogenases in humans. These findings expand the spectrum of genetic defects associated with lipoic acid metabolism and provide the first evidence of a lipoic acid transfer defect in humans.

Exome sequencing identifies a new mutation in SERAC1 in a patient with 3-methylglutaconic aciduria.

3-Methylglutaconic aciduria (3-MGA-uria) is a heterogeneous group of syndromes characterized by an increased excretion of 3-methylglutaconic and 3-methylglutaric acids. Five types of 3-MGA-uria (I to V) with different clinical presentations have been described. Causative mutations in TAZ, OPA3, DNAJC19, ATP12, ATP5E, and TMEM70 have been identified. After excluding the known genetic causes of 3-MGA-uria we used exome sequencing to investigate a patient with Leigh syndrome and 3-MGA-uria. We identified a homozygous variant in SERAC1 (c.202C>T; p.Arg68*), that generates a premature stop codon at position 68 of SERAC1 protein. Western blot analysis in patient's fibroblasts showed a complete absence of SERAC1 that was consistent with the prediction of a truncated protein and supports the pathogenic role of the mutation. During the course of this project a parallel study identified mutations in SERAC1 as the genetic cause of the disease in 15 patients with MEGDEL syndrome, which was compatible with the clinical and biochemical phenotypes of the patient described here. In addition, our patient developed microcephaly and optic atrophy, two features not previously reported in MEGDEL syndrome. We highlight the usefulness of exome sequencing to reveal the genetic bases of human rare diseases even if only one affected individual is available.

Coenzyme Q10 deficiency in mitochondrial DNA depletion syndromes.

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.

Protein expression profiles in patients carrying NFU1 mutations. Contribution to the pathophysiology of the disease.

Cofactor disorders of mitochondrial energy metabolism are a heterogeneous group of diseases with a wide variety of clinical symptoms, particular metabolic profiles and variable enzymatic defects. Mutations in NFU1 were recently identified in patients with fatal encephalopathy displaying a biochemical phenotype consistent with defects in lipoic acid-dependent enzymatic activities and respiratory chain complexes. This discovery highlighted the molecular function of NFU1 as an iron-sulfur(Fe-S) cluster protein necessary for lipoic acid biosynthesis and respiratory chain complexes activities. To understand the pathophysiological mechanisms underlying this disease we have characterized the protein expression profiles of patients carrying NFU1 mutations. Fibroblasts from patients with the p.Gly208Cys mutation showed complete absence of protein-bound lipoic acid and decreased SDHA and SDHB subunits of complex II. In contrast, subunits of other respiratory chain complexes were normal. Protein lipoylation was also decreased in muscle and liver but not in other tissues available (brain, kidney, lung) from NFU1 patients. Although levels of the respiratory chain subunits were unaltered in tissues, BN-PAGE showed an assembly defect for complex II in muscle, consistent with the low enzymatic activity of this complex. This study provides new insights into the molecular bases of NFU1 disease as well as into the regulation of NFU1 protein in human tissues. We demonstrate a ubiquitous expression of NFU1 protein and further suggest that defects in lipoic acid biosynthesis and complex II are the main molecular signature of this disease, particularly in patients carrying the p.Gly208Cys mutation.

Mitochondrial DNA depletion syndrome: new descriptions and the use of citrate synthase as a helpful tool to better characterise the patients.

Mitochondrial DNA depletion syndrome (MDS) is a clinically heterogeneous group of mitochondrial disorders characterised by a quantitative reduction of the mitochondrial DNA copy number. Three main clinical forms of MDS: myopathic, encephalomyopathic and hepatocerebral have been defined, although patients may present with other MDS associated clinical symptoms and signs that cover a wide spectrum of onset age and disease. We studied 52 paediatric individuals suspected to have MDS. These patients have been divided into three different groups, 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 3 novel mutations (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). Seventeen patients presented with mtDNA depletion in liver or muscle, but the cause of mtDNA depletion still remains unknown in 8 of them. 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.

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.

Clinical and inflammatory markers in amniotic fluid as predictors of adverse outcomes in preterm premature rupture of membranes.

OBJECTIVE: We sought to evaluate gestational age, cervical length, amniotic fluid interleukin (IL)-6, and selected proteomic biomarkers as independent predictors of adverse outcome in preterm premature rupture of membranes (PPROM). STUDY DESIGN: This was a prospective cohort study of 65 consecutive women with PPROM (20.0-34.6 weeks). Gestational age, cervical length, amniotic fluid IL-6, and proteomic biomarkers (calgranulins A and C, and neutrophil defensins 1 and 2) were evaluated at diagnosis. The predictive value for intraamniotic infection and neonatal composite morbidity was calculated by logistic regression. RESULTS: Proteomic biomarkers were independent predictors of intraamniotic infection (odds ratio, 22.1; P=.011) and neonatal composite morbidity (odds ratio, 17.6; P=.02). With the exception of a trend between gestational age and neonatal morbidity (P=.054), none of the other parameters were independent predictors of outcome measures. CONCLUSION: Selected proteomic biomarkers were the only independent predictors of adverse outcomes in PPROM. Contrary to what is reported in preterm labor with intact membranes, gestational age, cervical length, and IL-6 were not.

Quantitative Analysis of mtDNA Content in Formalin-Fixed Paraffin-Embedded Muscle Tissue.

Quantification of mitochondrial DNA (mtDNA) content is an essential tool for the diagnosis of mtDNA depletion syndrome (MDS). Samples collected and processed for anatomopathology studies represent a unique source of archived biological material. Thus, the possibility to study mtDNA copy number in these specimens would be a useful way to screen for MDS. In this study, we designed and validated the methodology to determine mtDNA content by quantitative real-time polymerase chain reaction (qRT-PCR) in formalin-fixed paraffin-embedded (FFPE) muscle tissue. We studied 14 frozen muscle biopsies and compared the results with a portion of the same biopsy embedded in paraffin. Our results showed a similar variability among frozen and FFPE muscle biopsies. Patients with MDS detected in frozen muscle were also confirmed in their corresponding FFPE samples, which validate the usefulness of this approach. We conclude that the analysis of mtDNA copy number in FFPE muscle tissue by qRT-PCR is a useful method for the molecular screening of patients suspected to have MDS when frozen biopsies are not available. Analysis of these samples would facilitate retrospective studies and diagnostic procedures.

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

17ß-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.

Functional splicing assay supporting that c.70 + 5G > A mutation in the MPV17 gene is disease causing.

Mitochondrial DNA depletion syndrome (MDS) is a group of disorders characterized by a quantitative reduction of the mitochondrial DNA copy number and inherited as autosomal recessive traits. Patients affected by this group of diseases present with a wide variety of symptoms depending on the altered gene. MPV17 is one of the genes causing combined encephalopathy and liver failure and at present there is no treatment for this devastating disease. The gene codes for an inner mitochondrial membrane protein, but its function is still unknown, and therefore, the only way to offer prenatal diagnosis relies on DNA studies. Consequently, mutations have to be well characterized. We previously described a patient homozygous for a novel intronic mutation in the MPV17 gene (c.70 + 5G > A). Here we report the use of a functional splicing assay based on the use of minigenes to support that c.70 + 5G > A mutation is disease causing. We carried out three prenatal diagnoses on three consecutive pregnancies of the previously described family. After two affected fetuses, a healthy baby was born homozygous for the wild-type allele.

Comparison between high performance liquid chromatography and capillary zone electrophoresis for the diagnosis of congenital disorders of glycosylation.

Transferrin isoelectric focusing (IEF) is the most widely used method to screen for congenital disorders of glycosylation (CDG). Our aim was to compare high performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE) procedures for serum sialotransferrin analysis. 58 serum samples were processed both by CZE and HPLC: 35 were from paediatric controls, 18 from patients with an altered sialotransferrin IEF pattern and 5 were transferrin variant samples. HPLC analysis was performed with an anion-exchange column with spectrophotometric detection at 470 nm. CZE analysis was done using the commercial CEofix-CDT kit with spectrophotometric detection at 200 nm. Passing-Bablok regression analysis showed good agreement for tri-, tetra- and penta-sialotransferrin by both procedures. But for disialotransferrin, higher values were observed by the HPLC procedure. The HPLC and CZE methods allowed reproducible separation and analysis of single transferrin glycoforms with similar peak patterns. All patients presented values outside the range established in our control population either by HPLC or by CZE, even in patients with moderate forms of CDG that had been difficult to detect by IEF. In conclusion, measurement of sialotransferrin isoforms and interpretation using method-specific reference values may offer some advantages for the diagnosis of CDG as compared with the standard IEF procedure.

Predictive value of combined amniotic fluid proteomic biomarkers and interleukin-6 in preterm labor with intact membranes.

OBJECTIVE: To assess proteomic biomarkers and interleukin-6 alone or in combination to predict intraamniotic infection, preterm birth, and neonatal morbidity in preterm labor with intact membranes. STUDY DESIGN: Amniotic fluid interleukin-6 and selected proteomic biomarkers were assayed from 86 patients with preterm labor and intact membranes (22-36 weeks). The predictive value of each marker alone or in combination was evaluated for intraamniotic infection, preterm birth, and neonatal composite morbidity. RESULTS: Both interleukin-6 (odds ratio, 19.5; P = .012) and proteomic biomarkers (odds ratio, 25.2; P = .001) were statistically independent predictors of intraamniotic infection with sensitivity, positive predictive value, and false-positive rates of 25%, 17.6%, and 20% when 1 marker was present and of 75%, 75%, and 4.3% when both were detected. Their combination did not improve prediction of preterm birth or neonatal morbidity. CONCLUSION: The combined use of proteomic biomarkers and interleukin-6 to predict intraamniotic infection shows better accuracy than when used alone.

Screening for congenital disorders of glycosylation (CDG): transferrin HPLC versus isoelectric focusing (IEF).

OBJECTIVES: Transferrin isoelectrofocusing (Tf-IEF) is widely used to screen for Congenital Disorders of Glycosylation (CDG), but it is laborious, time-consuming, and not suitable for automation or accurate quantification. We present our experience and advantages of the implementation of Tf-HPLC. METHODS: Sera were iron saturated, lipid precipitated and filtrated on Microcon-YM10. Glycoforms were separated by HPLC on a SOURCE 15Q anion-exchange column. Detection was at 470 nm. RESULTS: We established reference values and validated the HPLC method by analysing samples with abnormal Tf-IEF. Comparison between both methods is described. CONCLUSIONS: HPLC is useful for CDG screening, especially for laboratories that deal with great number of samples, due to its easy sample processing, the possibility of performing long series of analysis and the advantage of peak quantification, which allows objective interpretations.

Brain injury in glutaric aciduria type I: the value of functional techniques in magnetic resonance imaging.

BACKGROUND: Acute striatal necrosis is a devastating consequence of encephalopathic crisis in patients with glutaric aciduria type I (GA-I), but the mechanisms underlying brain injury are not completely understood. OBJECTIVE: To approach pathophysiological aspects of brain injury in GA-I by means of functional techniques in magnetic resonance imaging (MRI). PATIENTS AND METHODS: Four patients during an acute encephalopathic crisis and three asymptomatic siblings with GA-I underwent single-voxel hydrogen magnetic resonance spectroscopy (MRS) and brain MRI including gradient echo T1-weighted, FLAIR, T2-weighted and diffusion-weighted imaging. RESULTS: The study was performed between three and eight days after the onset of acute encephalopathic crisis. Isotropic diffusion images showed high signal changes with corresponding low apparent diffusion coefficient values within the putamen, caudate nuclei and globus pallidus (four patients), and the cerebral peduncles including the substantia nigra (one patient). The study disclosed normal findings in asymptomatic siblings. MRS showed decreased N-acetyl-aspartate/creatine ratio at the basal ganglia in encephalopathic patients when compared to a group of sex- and age-matched controls. CONCLUSIONS: Brain injury in GA-I is characterized by the presence of cytotoxic edema and reduced neuronal integrity by functional imaging techniques. Involvement of the basal ganglia may be asymmetrical in patients with unilateral motor disorder and may extent to the cerebral peduncles and substantia nigra, which may be responsible for the acute onset dystonia in some patients. Functional techniques failed to demonstrate any abnormalities in asymptomatic patients, which is in agreement with the integrity of basal ganglia structures observed by conventional MRI sequences.

Study of patients and carriers with 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiency: difficulties in the diagnosis.

OBJECTIVES: To search for biochemical and molecular markers for the diagnosis of patients and carriers with 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiency. DESIGN AND METHODS: Organic acids in urine, MHBD activity in fibroblasts, immunoblotting and molecular studies were performed in seven patients. Seven carriers were also studied. RESULTS: Under low protein diet or poor feeding all the patients showed only a slightly altered organic acid profile. Measurement of 2-methyl-3-hydroxybutyric acid and tiglylglycine after an isoleucine loading test, failed to demonstrate the carrier status of one patient. However, measurement of 2-ethylhydracrylic acid (EHA) was positive in all the carriers tested. MHBD activity was clearly deficient in males and in one female patient. We identified four missense mutations, two of them were novel. CONCLUSIONS: Quantification of EHA may be of help for the diagnosis of the heterozygous condition. The carrier females showed the classical biochemical variability of X-linked diseases due to random X-chromosome inactivation.

Coenzyme Q10 deficiency associated with a mitochondrial DNA depletion syndrome: a case report.

OBJECTIVES: To report on a case with a mitochondrial DNA (mtDNA) depletion syndrome. DESIGN AND METHODS: Laboratory studies were done in muscle biopsy and fibroblasts to evaluate coenzyme Q(10) (CoQ(10)) status and quantify mitochondrial DNA. RESULTS: Decreased CoQ(10) values and a 78% of mtDNA depletion were detected in muscle. Mutational studies failed to reveal any pathogenic mutation in nuclear genes related with mtDNA maintenance. CONCLUSIONS: mtDNA depletion syndrome was associated with CoQ(10) deficiency in our patient.

Lethal hepatopathy and leukodystrophy caused by a novel mutation in MPV17 gene: description of an alternative MPV17 spliced form.

It has recently been reported that mutations in MPV17 gene may be causative of mtDNA depletion syndrome (MDS). Patients with this alteration presented with severe liver failure, hypoglycemia, growth retardation and neurological symptoms during the first year of life. We report on the clinical, biochemical and molecular findings of a patient presenting with lethal hepatopathy, polyneuropathy, neurological regression and leukodystrophy associated with mutations in MPV17. Mitochondrial respiratory chain activities were low in liver and within reference values in muscle. However, levels of mtDNA were markedly reduced both in muscle and liver. A novel homozygous mutation in MPV17, c.70+5G>A (IVS1+5G>A), was identified. This intronic change causes the full-length cDNA loss, probably due to loss of strength of the splice donor site of exon 1. Western blot analysis, performed in liver homogenates, further corroborates these results as the amount of patient's protein was highly reduced, or almost absent, compared with that of controls. We also identified an additional alternative spliced form in controls and in the patient, due to exon 2 skipping, that has not previously been reported.