Restless Legs Syndrome in NKX2-1-related chorea: An expansion of the disease spectrum.

BACKGROUND: Molecular technologies are expanding our knowledge about genetic variability underlying early-onset non-progressive choreic syndromes. Focusing on NKX2-1-related chorea, the clinical phenotype and sleep related disorders have been only partially characterized. METHODS: We propose a retrospective and longitudinal observational study in 7 patients with non-progressive chorea due to NKX2-1 mutations. In all subjects sleep and awake EEG, brain MRI with study of pituitary gland, chest X-rays, endocrinological investigations were performed. Movement disorders, pattern of sleep and related disorders were investigated using structured clinical evaluation and several validated questionnaires. RESULTS: In patients carrying NKX2-1 mutations, chorea was mainly distributed in the upper limbs and tended to improve with age. All patients presented clinical or subclinical hypothyroidism and delayed motor milestones. Three subjects had symptoms consistent with Restless Legs Syndrome (RLS) that improved with Levodopa. CONCLUSIONS: Patients with NKX2-1 gene mutations should be investigated for RLS, which, similarly to chorea, can sometimes be ameliorated by Levodopa.

Review article: the potential of interferon and nucleos(t)ide analogue combination therapy in chronic hepatitis B infection.

BACKGROUND: A short-term course of pegylated-interferon (Peg-IFN), or a long-term treatment with a third generation nucleot(s)ide analogue (NUC), of chronic hepatitis B (CHB) infection achieves viral suppression and may prevent disease progression. Owing to different mechanisms of action of the two regimens, a Peg-IFN and NUC combination treatment may be an attractive approach to enhance the off-treatment rates of virological and serological response. AIM: To review the literature on combinations of Peg-IFN plus NUC, including the simultaneous initiation of Peg-IFN and NUC in naïve patients; an 'add-on' combination, where Peg-IFN is started at variable times after the beginning of NUC; or a 'switch-to' strategy usually from NUC to Peg-IFN. METHODS: We performed a PubMed literature search using the following terms individually or in combination: NUC, hepatitis B virus, chronic hepatitis, interferon, pegylated-interferon, nucleos(t)ide analogues, entecavir, tenofovir. English-language articles published up to December 2015, as well as conference proceedings from international meetings were reviewed. References from selected papers were reviewed and used if relevant. RESULTS: While combination and NUC pre-treatment failed to increase HBsAg clearance rates, more promising results were achieved in patients under long-term effective NUC therapy. CONCLUSION: While Peg-IFN and nucleos(t)ide analogue combination therapy should not be recommended currently, the addition of or the switch to Peg-IFN in nucleos(t)ide analogue-treated patients with chronic hepatitis B infection may be useful option.

Liver stiffness measurement reliability and main determinants of point shear-wave elastography in patients with chronic liver disease.

BACKGROUND: Liver stiffness (LS) measured by transient elastography (TE) accurately predicts the severity of chronic liver diseases (CLD). Point quantification shear-wave elastography (pSWE) is a new technique incorporated into a conventional ultrasound system for measuring LS. We evaluated pSWE feasibility, reproducibility and diagnostic accuracy in consecutively recruited CLD patients who concomitantly underwent TE and liver biopsy. AIM: To evaluate pSWE feasibility, reproducibility and diagnostic accuracy in consecutively recruited CLD patients who concomitantly underwent TE and liver biopsy. METHODS: Over 2 years 186 CLD patients (116 males, 132 viral hepatitis) consecutively underwent pSWE (10 valid measurements by ElastPQ) blindly performed by two raters. A further operator performed TE. Inter-observer agreement for pSWE was analysed by intraclass correlation coefficient (ICC) and correlated with histological liver fibrosis (METAVIR). Main determinants of pSWE were investigated by linear regression model. RESULTS: Three hundred and seventy-two (100%) reliable measurements were obtained by pSWE and 184 by TE (99%). LS was 8.1 ± 4.5 kPa for pSWE with the first rater and 8.0 ± 4.2 kPa with the second one vs. 8.8 ± 3.6 kPa for TE. pSWE ICC was 0.89 (95% CI 0.85-0.91), not influenced by age, sex, BMI, liver enzymes, liver aetiology. ICC increased over time with year 1 at 0.86 and 95% CI 0.81-0.90 vs. year 2 at 0.92 and 95% CI 0.87-0.95. Liver fibrosis was the only independent determinant of LS on pSWE. The AUROCs for diagnosing F ≥ 2, F ≥ 3 and F = 4 were 0.77, 0.85 and 0.88 for pSWE vs. 0.81, 0.88 and 0.94 for TE. After 1-year training they were 0.86, 0.94 and 0.91. CONCLUSION: Point quantification shear-wave elastography reliably and reproducibly evaluates liver stiffness, matching transient elastography for accuracy after a 1-year learning curve or 130 examinations.

Energy harvesting from human motion: materials and techniques.

Energy harvesting from human motion is a research field under rapid development. In this tutorial review we address the main physical and physico-chemical processes which can lead to energy generation, including electromagnetism, piezoelectricity, and electrostatic generation. Emphasis is put on the relationships among material properties and device efficiency. Some new and relatively less known approaches, such as triboelectric nanogeneration (TENG) and reverse electrowetting (REWOD), are reported in more detail.

Mitochondrial Diseases in Childhood.

Mitochondrial disorders are a group of heterogeneous diseases associated with abnormalities of the oxidative phosphorylation (OXPHOS), the most important source of energy for the cell. The number of mitochondrial syndromes and of identified causative genes is constantly increasing. Taken as a whole they are among the most frequent genetic diseases in humans at any age. The respiratory chain is the only metabolic pathway under double genome control and molecular genetics of these disorders is complicated by the existence of strict interactions between mitochondrial DNA and nuclear DNA. In childhood and infancy, clinical presentation differs from mitochondrial disorders with adult onset. The phenotypes are much more severe, often involving brain, frequently presenting as multisystemic disorders and seldom as isolated myopathy. Mutations in nDNA are more frequent than in adulthood. The major phenotypes presenting in infancy are here correlated with genetic defects and biochemical data with the aim to facilitate diagnosis work-up.

A multi-center comparison of diagnostic methods for the biochemical evaluation of suspected mitochondrial disorders.

A multicenter comparison of mitochondrial respiratory chain and complex V enzyme activity tests was performed. The average reproducibility of the enzyme assays is 16% in human muscle samples. In a blinded diagnostic accuracy test in patient fibroblasts and SURF1 knock-out mouse muscle, each lab made the correct diagnosis except for two complex I results. We recommend that enzyme activities be evaluated based on ratios, e.g. with complex IV or citrate synthase activity. In spite of large variations in observed enzyme activities, we show that inter-laboratory comparison of patient sample test results is possible by using normalization against a control sample.

Microscale oxygraphy reveals OXPHOS impairment in MRC mutant cells.

Given the complexity of the respiratory chain structure, assembly and regulation, the diagnostic workout for the identification of defects of oxidative phosphorylation (OXPHOS) is a major challenge. Spectrophotometric assays, that measure the activity of individual respiratory complexes in tissue and cell homogenates or isolated mitochondria, are highly specific, but their utilization is limited by the availability of sufficient biological material and intrinsic sensitivity. A further limitation is tissue specificity, which usually determines attenuation, or disappearance, in cultured fibroblasts, of defects detected in muscle or liver. We used numerous fibroblast cell lines derived from patients with OXPHOS deficiencies to set up experimental protocols required for the direct readout of cellular respiration using the Seahorse XF96 apparatus, which measures oxygen consumption rate (OCR) and extra-cellular acidification rate (ECAR) in 96 well plates. Results demonstrate that first level screening based on microscale oxygraphy is more sensitive, cheaper and rapid than spectrophotometry for the biochemical evaluation of cells from patients with suspected mitochondrial disorders.

Clinical and molecular features of mitochondrial DNA depletion syndromes.

Mitochondrial DNA depletion syndromes (MDSs) form a group of autosomal recessive disorders characterized by profoundly decreased mitochondrial DNA copy numbers in affected tissues. Three main clinical presentations are known: myopathic, encephalomyopathic and hepatocerebral. The first is associated with mutations in thymidine kinase 2 (TK2) and p53-induced ribonucleotide reductase B subunit (RRM2B); the second with mutations in succinate synthase A (SUCLA2) and B (SUCLG1); the third with mutations in Twinkle (PEO1), pol-gammaA (POLG1), deoxyguanosine kinase (DGUOK) and MPV17 (MPV17). In this work, we review the MDS-associated phenotypes and present our own experience of 32 MDS patients, with the aim of defining the mutation frequency of the known genes, the clinical spectrum of the diseases, and the genotype-phenotype correlations. Five of our patients carried previously unreported mutations in one of the eight MDS genes.

Identification of new mutations in the ETHE1 gene in a cohort of 14 patients presenting with ethylmalonic encephalopathy.

BACKGROUND: Ethylmalonic encephalopathy (EE) is a rare autosomal recessive metabolic disorder characterised by progressive encephalopathy, recurrent petechiae, acrocyanosis and chronic diarrhoea, with a fatal outcome in early in life. METHODS: 14 patients with EE were investigated for mutations in the ETHE1 gene. RESULTS: Of the 14 patients, 5 were found to carry novel mutations. CONCLUSIONS: This work expands our knowledge of the causative mutations of EE.

Clinical and molecular findings in children with complex I deficiency.

Isolated complex I deficiency, the most frequent OXPHOS disorder in infants and children, is genetically heterogeneous. Mutations have been found in seven mitochondrial DNA (mtDNA) and eight nuclear DNA encoded subunits, respectively, but in most of the cases the genetic basis of the biochemical defect is unknown. We analyzed the entire mtDNA and 11 nuclear encoded complex I subunits in 23 isolated complex I-deficient children, classified into five clinical groups: Leigh syndrome, progressive leukoencephalopathy, neonatal cardiomyopathy, severe infantile lactic acidosis, and a miscellaneous group of unspecified encephalomyopathies. A genetic definition was reached in eight patients (35%). Mutations in mtDNA were found in six out of eight children with Leigh syndrome, indicating a prevalent association between this phenotype and abnormalities in ND genes. In two patients with leukoencephalopathy, homozygous mutations were detected in two different nuclear-encoded complex I genes, including a novel transition in NDUFS1 subunit. In addition to these, a child affected by mitochondrial encephalomyopathy had heterozygous mutations in NDUFA8 and NDUFS2 genes, while another child with neonatal cardiomyopathy had a complex rearrangement in a single NDUFS7 allele. The latter cases suggest the possibility of unconventional patterns of inheritance in complex I defects.

GTP-cyclohydrolase I gene mutations in patients with autosomal dominant and recessive GTP-CH1 deficiency: identification and functional characterization of four novel mutations.

GTP-cyclohydrolase I (GTP-CH1, EC 3.5.4.16) is encoded by the GCH1 gene. Mutations in the GCH1 gene cause both dopa-responsive dystonia (McKusick 128230) and recessive GTP-CH1 deficiency (McKusick 600225). The exact molecular mechanism resulting in decreased GTP-CH1 activity in the patients is still obscure. We report the clinical features and molecular and functional study of the GCH1 gene in eight Italian patients affected by dominant and recessive GTP-CH1 deficiency. All the studied patients had mutations in the GCH1 gene. Three missense mutations (V205G, K224R, P199A), a frameshift mutation (Delta G693), and a splice-site mutation (ivs5 + 1g > c) were found. Except for K224R these are all novel mutations. To analyse the defect caused by the novel mutations, an in vivo functional assay in a Saccharomyces cerevisiae strain lacking the endogenous gene encoding GTP-CH1 ( FOL2 ) was performed. Complementation analysis showed that the Delta G693 and V205G mutations abolish the enzymatic function, while the P199A mutation causes a conditional defect. In conclusion, the clinical phenotypes displayed by our patients confirm the wide clinical spectrum of the disease and further support the lack of correlation between a given mutation and a clinical phenotype. Complementation analysis in yeast is a useful tool for confirming the pathogenetic effect of GCH1 mutations.

Variable penetrance of a familial progressive necrotising encephalopathy due to a novel tRNA(Ile) homoplasmic mutation in the mitochondrial genome.

INTRODUCTION: We present a family comprising a clinically normal mother and two daughters, each with severe encephalopathy with onset in late childhood. A third daughter had died previously of an earlier onset but neuropathologically similar disease. METHODS: Sequence analysis of the entire mtDNA was carried out in muscle, fibroblasts, and lymphocytes of the affected daughters and unaffected mother. Biochemical analysis of individual respiratory chain enzymes was performed on the same tissues, and on several transmitochondrial cybrid clones containing the nucleus of a 143B.206 osteosarcoma cell line and the mutant mtDNA. RESULTS: Genetic analyses revealed in both daughters and mother the presence of a novel mutation in the tRNA(Ile) gene of mtDNA, which was homoplasmic in fibroblasts, lymphocytes, and skeletal muscle of the two patients. It was also homoplasmic in fibroblast and skeletal muscle samples of the mother, and approximately 97% heteroplasmic in her lymphocytes. Combined defects of complexes I and IV of the mitochondrial respiratory chain were found not only in fibroblasts of the two probands, but surprisingly also in those of their clinically unaffected mother. The respiratory chain defect segregated in transmitochondrial cybrids containing the nucleus of a 143B.206 osteosarcoma cell line and the mutant mtDNA, indicating that the latter was responsible for the biochemical phenotype. DISCUSSION: Our results support the concept that homoplasmic mutations in tRNA genes can be responsible for mitochondrial disorders characterised by extremely variable penetrance. Albeit still unexplained, this phenomenon has important consequences in the nosological characterisation, clinical management, and genetic counselling of mitochondrial disorders.

Neonatal dopa-responsive extrapyramidal syndrome in twins with recessive GTPCH deficiency.

The authors report two twin sisters, age 15 years, with recessive GTP cyclohydrolase deficiency, who presented with neonatal onset of rigidity, tremor, and dystonia but with no other symptoms suggestive of a diffuse CNS involvement. The plasma phenylalanine levels were normal. Treatment with L-dopa/carbidopa, started at age 1 year, was associated with sustained recovery from all neurologic signs. The patients were homozygous for a new recessive mutation in the GHI gene.

Lethal neonatal presentation of carnitine palmitoyltransferase I deficiency.

A neonate subsequently diagnosed with carnitine palmitoyltransferase I deficiency died at 34 h of untreatable bradycardia. There was fatty infiltration of the liver and increased free carnitine and reduced acylcarnitines in the blood.

Clinical and molecular heterogeneity in very-long-chain acyl-coenzyme A dehydrogenase deficiency.

Very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency is an increasingly recognized defect of mitochondrial fatty acid beta-oxidation manifesting with episodes of metabolic decompensation or isolated recurrent myoglobinuria. In this report the clinical, biochemical, and molecular studies in a series of five patients (four Italian and one Spanish) with this disorder are discussed. Biochemical studies included the determination of fibroblast substrate oxidation rates and enzyme activity and Western blot analysis of VLCAD protein. Molecular analysis was performed by sequencing the VLCAD gene from the genomic DNA. Clinical features were within the spectrum previously reported. Four patients presented in infancy or childhood with episodes of severe metabolic decompensation and dicarboxylic aciduria. Two exhibited cardiomyopathy. The fifth patient presented with isolated recurrent rhabdomyolysis, with no cardiomyopathy or dicarboxylic aciduria. In all patients a significant loss of VLCAD activity associated with a marked reduction of VLCAD protein levels occurred. Molecular analysis disclosed one novel missense mutation (Cys437Tyr) and four previously reported mutations, including two missense substitutions (Phe418Leu and Arg419Trp), a single amino acid deletion (Lys258del), and one splice site mutation (IVS8-C(-2)), which was present in all four Italian patients. All patients exhibited compound heterozygosity. The phenotypic variability and the high genotypic heterogeneity of this hereditary metabolic disorder is reported.

Five-year follow-up of 165 Italian patients with undifferentiated connective tissue diseases.

OBJECTIVE: To study those conditions with a proven or hypothesised immunologic pathogenesis and denominated under a working definition of undifferentiated connective tissue diseases (UCTD). METHODS: A multicentre prospective study was organised involving 10 tertiary referral centers of internal medicine in Italy, with the aim of describing the natural history of UCTD and the prevalence of its different clinical and immunological manifestations. RESULTS: After a five-year follow-up period, data on 165 patients were available for analysis. UCTDs occur mainly in females in their fourth decade of life. Articular and mucocutaneous features and Raynaud's phenomenon represent the most common findings. Nevertheless, we also detected a relatively high incidence of permanent major organ damage. Regarding the immunologic parameters, we documented some conflicting results in the correlation between serologic abnormalities and clinical features. In 10 patients UCTD evolved to a major disease, generally systemic lupus erythematosus or Sjögren's syndrome. CONCLUSION: A low rate of evolution to a defined autoimmune disease, the limited use of steroid or immunosuppressive therapy, and a favourable course in the majority of cases are the main characteristics of patients with UCTDs.

Medium-chain triglyceride loading test in carnitine-acylcarnitine translocase deficiency: insights on treatment.

The results of a medium-chain triglyceride loading test in a patient with severe carnitine-acylcarnitine translocase deficiency clearly demonstrated impaired in vivo utilization of medium-chain triglycerides. The loading test was performed at the ages of 7 and 36 months. The diet was adjusted accordingly. The clinical course has been favourable and the child is now in very good condition at age 4 years. We conclude that the utilization of medium-chain triglycerides is only partial in carnitine-acylcarnitine translocase deficiency and cannot reasonably be considered an optimal source of energy for these patients. Careful adjustment of dietetic treatment may help to improve prognosis.

Two-dimensional electrophoretic analysis of cryoproteins: a report of 335 samples.

Cryoproteins are defined as proteins precipitating at low temperature. Most frequently, the precipitate contains immunoglobulins (Igs), and are therefore called cryoglobulins. Three types of cryoglobulins have been described: type I contains a single monoclonal Ig, whereas type II is a mixture of a monoclonal Ig with polyclonal Igs, and type III is a mixture of polyclonal Igs of different isotypes, most frequently IgG and IgM. Type II and type III are also called mixed cryoglobulins. A new type of cryoglobulins, containing polyclonal IgG associated with a mixture of polyclonal and monoclonal IgM has recently been described after two-dimensional polyacrylamide gel electrophoresis (2-DE). This type of cryoglobulin has been called type II-III cryoglobulin. In this study, we report on 2-DE analysis of 335 cryoproteins from patients with heterogeneous clinical conditions. In 69 out of 335 samples (20.7%), 2-DE revealed patterns that were inadequate to characterize the cryoproteins. Out of 335 (79.3%) cryoproteins, 266 were identified according to their two-dimensional patterns: 265 samples contained Igs and were diagnosed as cryoglobulins, and one sample consisted of fibrinogen, and was identified as cryofibrinogen. Among the 265 cryoglobulins, types I, II, and III were observed in 9 (3.4%), 69 (26%), and 116 (43.8%) cases, respectively, whereas type II-III was detected in 71 (26.8%) cases. Eleven of the latter consisted of oligoclonal Igs (IgM in 10 cases, IgA in 1 case) mixed with traces of polyclonal IgG. These cryoproteins were tentatively named type II-IIIvariant cryoglobulins. Taken together, our result clearly show that 2-DE is a suitable technique to analyze cryoproteins.