Muscular dystrophies and myopathies: the spectrum of mutated genes in the Czech Republic.

Inherited neuromuscular disorder (NMD) is a wide term covering different genetic disorders affecting muscles, nerves, and neuromuscular junctions. Genetic and clinical heterogeneity is the main drawback in a routine gene-by-gene diagnostics. We present Czech NMD patients with a genetic cause identified using targeted next-generation sequencing (NGS) and the spectrum of these causes. Overall 167 unrelated patients presenting NMD falling into categories of muscular dystrophies, congenital muscular dystrophies, congenital myopathies, distal myopathies, and other myopathies were tested by targeted NGS of 42 known NMD-related genes. Pathogenic or probably pathogenic sequence changes were identified in 79 patients (47.3%). In total, 37 novel and 51 known disease-causing variants were detected in 23 genes. In addition, variants of uncertain significance were suspected in 7 cases (4.2%), and in 81 cases (48.5%) sequence changes associated with NMD were not found. Our results strongly indicate that for molecular diagnostics of heterogeneous disorders such as NMDs, targeted panel testing has a high-clinical yield and should therefore be the preferred first-tier approach. Further, we show that in the genetic diagnostic practice of NMDs, it is necessary to take into account different types of inheritance including the occurrence of an autosomal recessive disorder in two generations of one family.

Two patients with clinically distinct manifestation of pyruvate dehydrogenase deficiency due to mutations in PDHA1 gene.

The most common cause of pyruvate dehydrogenase complex (PDHc) deficiency is the deficit of the E1α-subunit. The aim of this study was to describe distinct course of the disease in two boys with mutations in PDHA1 gene and illustrate the possible obstacles in measurement of PDHc activity. Clinical data and metabolic profiles were collected and evaluated. PDHc and E1α-subunit activities were measured using radiometric assay. Subunits of PDHc were detected by Western blot. PDHA1 gene was analysed by direct sequencing. In patient 1, the initial hypotonia with psychomotor retardation was observed since early infancy. The child gradually showed symptoms of spasticity and arrest of psychomotor development. In patient 2, the disease manifested by seizures and hyporeflexia in the toddler age. The diagnosis was confirmed at the age of seven years after attacks of dystonia and clinical manifestation of myopathy with normal mental development. Brain MRI of both patients revealed lesions typical of Leigh syndrome. Enzymatic analyses revealed PDHc deficiency in isolated lymphocytes in the first but not in the second patient. The direct measurement of PDH E1-subunit revealed deficiency in this individual. In patient 1, a novel hemizigous mutation c.857C>T (Pro250Leu) was detected in the X-linked PDHA1 gene. Mutation c.367C>T (Arg88Cys) was found in patient 2. We present first two patients with PDHc deficit due to mutations in PDHA1 gene in the Czech Republic. We document the broad variability of clinical symptoms of this disease. We proved that normal PDHc activity may not exclude the disease.

[Mechanical ventilation on paediatric intensive care units in Czech Republic]. / Kontrollierte Beatmung auf pädiatrischen Intensivstationen in der Tschechischen Republik.

OBJECTIVE: The aims of this study were to evaluate the incidence of mechanically ventilated children in participating units, to find out the demographic data of the patients, to evaluate ventilator settings and to assess the mortality of ventilated children. DESIGN: Prospective observational multicenter study between 1. 2. 2002 and 30. 4. 2002. SETTING: Seven paediatric intensive care units in tertiary hospitals in the Czech Republic. PATIENTS: All children between 1 month and 18 years admitted to the participating paediatric intensive care units who required intubation and mechanical ventilation were enrolled. METHOD: Following parameters were recorded in all patients: demographic data (age, weight, gender), the origin of the admitting diagnosis, severity of illness (Pediatric Risk of Mortality Score - PRISM, Multiorgan System Failure - MOSF, Lung Injury Score - LIS), the origin of respiratory failure, presence of chronic disease and immunosuppression, length of ventilation, length of stay, ventilator setting, the use of unconventional ventilation, outcome (mortality), blood gas analyses and indices (alveoloarterial oxygen difference - AaDO (2), oxygenation index - OI, hypoxemia score - PaO (2)/FiO (2) and ventilation index - VI), deadspace to tidal volume ratio-Vd/Vt and dynamic respiratory system compliance (Cdyn). RESULTS: One hundred and forty four children (42 % girls) were enrolled in total which represent 23 % of all admitted children. The mean age of the patients was 70 months and mean weight was 23 kg. PRISM score and the length of stay were twofold against mean values (11.7 vs. 5.7 and 10.4 vs. 4.8 days respectively). The mean length of ventilation was 117 hours, 66 % of the patients had an extrapulmonary origin of respiratory failure, 19 % of the patients were chronically ill, and 0,7 % had the evidence of immunosuppression. Pressure regulated volume controlled and Biphasic positive airway pressure were the most frequently used ventilator settings. Unconventional ventilation in all was used in 13 % of the patients. Mortality was 3.5 %. CONCLUSION: Children on mechanical ventilation create 23 % of all patients admitted to paediatric intensive care units. The severity of illness and length of stay were twofold against mean values. Mortality rate was 3.5 % and hypoxia was not a cause of death in any patient.

[Molecular analysis of Wilson disease]. / Molekulární analýza Wilsonovy choroby.

BACKGROUND: Wilson disease is an autosomal recessive disorder, characterized by cooper accumulation and intoxication of the organism. Molecular basis of the disease represent mutations in the gene for the copper-transporting ATPase (ATP7B). METHODS AND RESULTS: The submitted paper deals with results of molecular-genetic examination in 130 unrelated families in which Wilson disease was diagnosed. By denaturing gradient gel electrophoresis (DGGE), the exons with abnormal sequences were detected. Followed by sequencing, 17 causal mutations and 9 silent polymorphism were found. Five novel mutations were detected. After analysis of 260 mutant alleles, 214 (82.3%) were identified. The most frequent mutation, H1069Q, occurred in our population with the frequency of 65.8%. Incidence of other mutations, however, did not exceed 5%. CONCLUSIONS: DNA analysis of the Wilson disease offers prompt and reliable results in affected families. It can help to identify asymptomatic and heterozygote siblings at genetic counselling.

[Metabolic complications and neurologic manifestations of vitamin B12 deficiency in children of vegetarian mothers]. / Metabolické komplikace a neurologické projevy pri deficitu vitaminu B12 u detí vegetariánských matek.

BACKGROUND: Serious hematological, metabolic and neurological complications owing to the nutritional deficiency of vitamin B12 may occur in infants of mothers on a strict vegetarian diet. METHODS AND RESULTS: The mother of the first child was a strict vegetarian. She had an elevated urinary methylmalonic acid level and a low concentration of serum vitamin B12. Her 13-month-old daughter was exclusively breast-fed until the age of 9 month and then she was fed only vegetables. Physical examination revealed psychomotoric retardation, apathy, muscular hypotonia, abnormal movements and failure to thrive. Laboratory analysis showed a megaloblastic anaemia, a low level of vitamin B12 and methylmalonic aciduria. MRI of the brain revealed diffuse frontotemporoparietal atrophy and retardation of myelination. After treatment with vitamin B12 supplements, abnormal movements disappeared and development improved, but a mild generalised hypotonia continued. A cranial MRI 9 months after treatment still showed signs of retardation of myelination. The second patient, an 8 month-old male, son of a strict vegetarian mother too, was referred for investigation of psychomotoric retardation, hypotonia, dyskinesia, failure to thrive and microcephaly. He was breast-fed and from 6 month of age he had also received fruit juices. Laboratory analysis revealed megaloblastic anaemia, high methylmalonic aciduria and homocystinuria. The patient's and his mother's serum level of vitamin B12 were low. After treatment with vitamin B12 supplements, biochemical and metabolic markers of disease were normal but there continued a generalised hypotonia, microcephaly and language delay. CONCLUSION: Our observations emphasize the health complications of nutritional cobalamine deficiency and a requirement of clinical, biochemical and metabolic monitoring in infants within strict vegetarian families.

[The hepatic form of Wilson's disease in young patients]. / Jaterní forma Wilsonovy nemoci u mladých pacientu.

UNLABELLED: Wilson's disease (WD) is a hereditary disorder of the copper metabolism with very varied clinical and biochemical symptoms. Hepatic and neurological forms are the most frequent manifestations of this rare disease. In schoolchildren and adolescents symptoms of liver damage predominate. In a retrospective study 19 patients were evaluated with biochemical signs of hepatopathy manifested before the age of 18 years. The diagnosis of WD was established at the age of 7 to 27 years. One female patient was admitted with fulminant hepatic failure which was treated by acute transplantation of the liver in the Institute of Clinical and Experimental Medicine in Prague. Only 9 of 18 patients with chronic hepatic affection at the time of diagnosis met the Sternlieb diagnostic criteria. These patients had reduced ceruloplasmin levels (0.08-0.18 g/l) and a high copper content in the hepatic dry matter (783 ug/g +/- 323 [SD]). In the remaining 9 patients the ceruloplasmin level was normal, however, in 8 a high copper content of the hepatic dry matter was found (696 ug/g (+)- 352[SD]. The last patient from this group had Kayser-Fleischer's (K-F) ring. It was possible to confirm the high copper content in the hepatic dry matter only after one year's penicillinamine treatment because at the time of the diagnosis poor coagulation did not permit to perform a liver biopsy. There was a statistically significant difference in the copper content of the hepatic dry matter in patients meeting and not meeting Sternlieb's criteria. Statistically significant differences between both groups were found in the plasma copper levels and in the 24-hour urinary copper excretion. Histological examination of the liver under a light microscope revealed findings from minimal changes associated with the presence of glycogen nuclei in hepatocytes to the picture of active chronic hepatitis. In all 19 patients the gene mutation H1069Q was examined and the results were positive in 39.8%. In 3 asymptomatic patients it was present in the homozygous form. CONCLUSION: Early detection of the atypical form of WD remains very difficult. The gold standard is still in all cases assessment of copper in the dry liver tissue. In the near future an important place will be held also by direct DNA analysis although its use is limited not only by the large number of known mutations but also by the financial costs of the method.