Difficulties in recognition of pyruvate dehydrogenase complex deficiency on the basis of clinical and biochemical features. The role of next-generation sequencing.

Pyruvate dehydrogenase complex (PDHc) defect is a well-known cause of mitochondrial disorders (MD) with at least six responsible genes (PDHA1, PDHB, DLAT, DLD, PDHX, PDP1). The aim of this work was to assess the diagnostic value of biochemical methods in recognition of PDHc defect in Polish patients with suspicion of MD. In the first step, Western blot of the E1α subunit was performed on 86 archive muscle bioptates with suspicion of MD. In the second step, Sanger PDHA1 sequencing was performed in 21 cases with low E1α expression. In the third step, 7 patients with negative results of PDHA1 sequencing were subjected to whole-exome sequencing (WES). This protocol revealed 4 patients with PDHA1 and one with DLD mutations. Four additional probands were diagnosed outside the protocol (WES or Sanger sequencing). The molecular characterization of PDHc defect was conducted in a total of 9 probands: 5 according to and 4 off the protocol. Additionally, two affected relatives were recognized by a family study. Altogether we identified seven different PDHA1 changes, including two novel variants [c.464T > C (p.Met155Thr) and c.856_859dupACTT (p.Arg288Leufs*10)] and one DLD variant. The lactate response to glucose load in the PDHA1 subset was compared to a subset of non PDHc-related MD. Opposite responses were observed, with an increase of 23% and decrease of 27%, respectively. The results show that determining lactate response to glucose load and muscle E1α expression may contribute to distinguishing PDHc-related and other MD, however, WES is becoming the method of choice for MD diagnostics.

Novel c.191C>G (p.Pro64Arg) MPV17 mutation identified in two pairs of unrelated Polish siblings with mitochondrial hepatoencephalopathy.

This study reports clinical, biochemical and histopathological findings associated with a novel homozygous MPV17 mutation in four patients with mitochondrial depletion syndrome. The severe course of the disease, which started in the first weeks of life, was dominated by a failure to thrive, hypotonia and liver dysfunction, with relatively mild neurological involvement. All affected infants died by 1 year of age. Laboratory findings included progressive liver failure (hypertransaminasaemia, icterus, and coagulopathy), recurrent hypoglycaemia, lactic acidaemia, hyperferritinaemia, and increased transferrin saturation. Histological and ultrastructural analyses uncovered significant lipid accumulation in hepatocytes and myocytes. A severe decrease in the mitochondrial/nuclear DNA (mtDNA/nDNA) ratio was found post-mortem in the livers (and in one muscle specimen) of both examined patients. Oxidative phosphorylation system (OXPHOS) Western blotting revealed low levels of complexes I, III and IV subunits. The highlights of our findings are as follows: (i) The novel p.Pro64Arg mutation is the second recurrent MPV17 mutation reported. The phenotype associated with the p.Pro64Arg mutation differs from the phenotype of the relatively common p.Arg50Gln mutation, suggesting the existence of a genotype-phenotype correlation. (ii) Tissues collected from patients during autopsy may be useful for both mtDNA/nDNA ratio assessment and OXPHOS Western blotting.

Type of desmin expression in cardiomyocytes - a good marker of heart failure development in idiopathic dilated cardiomyopathy.

AIM: The aim of this study was to determine whether remodelling of the desmin (DES) cytoskeleton affects myocardial function and whether it could be a useful marker of disease progression in patients with idiopathic dilated cardiomyopathy (IDCM). MATERIAL AND METHODS: Endomyocardial biopsy was performed in 195 IDCM patients, and five to six specimens were collected from the left ventricle. DES expression was evaluated using tissue immunostaining and Western blotting. The study population was assigned to four groups according to DES expression type: I, normal DES staining at Z-lines giving a regular pattern of cross-striation (n = 57); IIA, increased DES staining with a regular pattern of cross-striation (n = 40); IIB, increased DES staining with an irregular pattern of cross-striation and/or the presence of aggregates (n = 56); and III, decreased/lack of DES staining (n = 42). Fibrosis, cardiomyocyte hypertrophy and ultrastructure were assessed for the four types of DES expression. RESULTS: The pathological types of DES expression (IIB or III) were associated with pathological changes in mitochondria and the contractile apparatus. Cardiomyocyte diameter and level of fibrosis were both significantly affected. DES expression type correlated with NYHA class, left ventricular end-diastolic diameter, left ventricular ejection fraction and the level of N-terminal pro-brain natriuretic protein. CONCLUSION: The type of immunohistochemical DES expression correlated with the level of myocardial injury at the cellular and organ levels. This correlation was similar to that observed between DES expression and the well-established biochemical, echocardiographic and clinical parameters of heart failure (HF). DES expression type could be used as an important diagnostic feature of HF development.

SURF1 missense mutations promote a mild Leigh phenotype.

UNLABELLED: SURF1 gene mutations are the most common cause of Leigh syndrome (LS), a rare progressive neurodegenerative disorder of infancy, characterized by symmetric necrotizing lesions and hypervascularity in the brainstem and basal ganglia, leading to death before the age of 4 years. Most of the reported mutations create premature termination codons, whereas missense mutations are rare. The aim of the study was to characterize the natural history of LS patients carrying at least one missense mutation in the SURF1 gene. Nineteen such patients (8 own cases and 11 reported in the literature) were compared with a reference group of 20 own c.845_846delCT homozygous patients, and with other LS(SURF-) cases described in the literature. Disease onset in the studied group was delayed. Acute failure to thrive and hyperventilation episodes were rare, respiratory failure did not appear before the age of 4 years. Dystonia, motor regression and eye movement dissociation developed slowly. The number of patients who survived 7 years of life totaled 9 out of 15 (60%) in the 'missense group' and 1 out of 26 (4%) patients with mutations leading to truncated proteins. IN CONCLUSION: (i) The presence of a missense mutation in the SURF1 gene may correlate with a milder course and longer survival of Leigh patients, (ii) normal magnetic resonance imaging (MRI) findings, normal blood lactate value, and only mild decrease of cytochrome c oxidase (COX) activity are not sufficient reasons to forego SURF1 mutation analysis in differential diagnosis.

Light and electron microscopy characteristics of the muscle of patients with SURF1 gene mutations associated with Leigh disease.

AIMS: Leigh syndrome (LS) is characterised by almost identical brain changes despite considerable causal heterogeneity. SURF1 gene mutations are among the most frequent causes of LS. Although deficiency of cytochrome c oxidase (COX) is a typical feature of the muscle in SURF1-deficient LS, other abnormalities have been rarely described. The aim of the present work is to assess the skeletal muscle morphology coexisting with SURF1 mutations from our own research and in the literature. METHODS: Muscle samples from 21 patients who fulfilled the criteria of LS and SURF1 mutations (14 homozygotes and 7 heterozygotes of c.841delCT) were examined by light and electron microscopy. RESULTS: Diffuse decreased activity or total deficit of COX was revealed histochemically in all examined muscles. No ragged red fibres (RRFs) were seen. Lipid accumulation and fibre size variability were found in 14 and 9 specimens, respectively. Ultrastructural assessment showed several mitochondrial abnormalities, lipid deposits, myofibrillar disorganisation and other minor changes. In five cases no ultrastructural changes were found. Apart from slight correlation between lipid accumulation shown by histochemical and ultrastructural techniques, no other correlations were revealed between parameters investigated, especially between severity of morphological changes and the patient's age at the biopsy. CONCLUSION: Histological and histochemical features of muscle of genetically homogenous SURF1-deficient LS were reproducible in detection of COX deficit. Minor muscle changes were not commonly present. Also, ultrastructural abnormalities were not a consistent feature. It should be emphasised that SURF1-deficient muscle assessed in the light and electron microscopy panel may be interpreted as normal if COX staining is not employed.

Are mu-opioid receptors involved in the control of endothelin-1 release from the pituitary gland in normal and dehydrated rats?

UNLABELLED: The objective of the present study was to investigate whether the endogenous opioids are involved in the control of endothelin-1 release from the pituitary gland. To test this hypothesis we have measured the peripheral plasma concentration of ET-1 as well as the content of immunoreactive ET-1 (irET-1) in the pituitary in response to opioid receptors blockade in euhydrated and 24 h water-deprived Wistar-Kyoto rats. Placebo or naltrexone (50 micrograms/kg body wt.) were given i.v. in both groups. Trunk blood was collected to determine hematocrit, plasma sodium and ET-1 levels (RIA). Immunostaining of ET-1 in the whole pituitary glands was performed by colloidal gold labeling. The quantitative analysis of irET-1 was carried out under a light microscope using a computerized image analyzer (MultiScan). RESULTS: (1) Twenty-four-hour dehydration resulted in marked increase of peripheral concentration of ET-1. Naltrexone injection induced a significant elevation of ET-1 plasma concentration in both, dehydrated and control animals. (2) The content of irET-1 in anterior and intermediate lobes of the pituitary in dehydrated rats was markedly higher than in control group. (3) Naltrexone injection caused a rapid and significant reduction irET-1 within the anterior, intermediate and posterior lobes in dehydrated and control animals. CONCLUSIONS: (1) An elevation of irET-1 in the pituitary gland and peripheral circulation in dehydrated animals may play a role in maintaining of water-electrolyte balance. (2) The mu-opioid system appears to control the ET-1 release from the pituitary in normal and dehydrated animals.