Feature clustering of intracranial pressure time series for alarm function estimation in traumatic brain injury.

OBJECTIVE: The conventional application of intracranial pressure (ICP) monitoring of traumatic brain injury (TBI) patients consists merely in the acquisition of ICP values in discrete time and their comparison to the established ICP threshold. An exceeding of this threshold triggers a special emergency treatment protocol. This paper addresses the possibility of making use of the rich information latent in the ICP records of known vital and fatal outcomes gathered during real clinical practice of treating TBI patients. Our assumption was that the proposed algorithmic procedure derived from this information could, in addition to ICP monitoring itself, provide a complementary added value. This might help clinicians to make better decisions during a patient's treatment. APPROACH: We concentrated on studying specific clustering schemes for subsequences of ICP time series. The clusterization problem was formulated for feature vectors which are introduced to represent ICP time subsequences. The ICP transformation to a feature space uses global and local definitions of time subsequences. For clusterization itself, we adopted hierarchical Gaussian mixture models (hGMMs). By using posterior probabilities of the clusters, we introduced three novel alarm functions. We explored two alternative methods of searching for optimum alarm function thresholds (ROC analysis and a novel efficiency measure). MAIN RESULTS: We performed extensive cross-validation experiments on a clinical retrospective data set. The results of the optimization over several hGMMs, various feature space dimensionality and all the types of the novel alarm functions show the potential of the novel alarm functions for supplementing conventional ICP monitoring. SIGNIFICANCE: In conclusion, the paper provides a prospective extended ICP monitoring technique for real TBI patients, based on the proposed methodology of ICP subsequence clustering and thresholding of the optimum novel alarm function.

Evaluation of mutation screening by heteroduplex analysis in acute intermittent porphyria: comparison with denaturing gradient gel electrophoresis.

Acute intermittent porphyria is the major autosomal dominant form of acute hepatic porphyrias. The disease is due to mutations in the gene encoding for porphobilinogen deaminase (PBGD). Many different strategies have been developed to screen for mutations. However the high prevalence (0.6 per thousand) of PBGD gene defect, the large allelic heterogeneity of mutations (n = 130), and the limitations of the PBGD enzymatic assay for asymptomatic patients' detection, require for diagnosis an efficient and easy to handle strategy for locating mutations within the PBGD gene. In a recent study the sensitivity of the denaturing gradient gel electrophoresis (DGGE) technique was 100%. However DGGE requires the preparation of gradient gels and the use of primers with long GC-clamps; thus alternative methods should be preferable in the clinical laboratory. We have compared the detection rate of DGGE with heteroduplex analysis (HA) using 16 characterized PBGD gene mutations. Six different HA conditions were used to determine the efficiency of the method, including: (1) MDE (mutation detection enhancement) gel concentration; (2) addition of urea and sodium dodecyl sulfate (SDS); (3) radioactive labelling. The sensitivity of each HA condition varied from 31 to 81% vs. 100% in DGGE analysis. HA using 1 x MDE with 15% urea with or without 0.55% SDS was the most sensitive condition. This first comparative study of DGGE and HA mutation screening methods suggests that DGGE is a more sensitive screening assay than optimized HA. However, because of its simplicity HA should be considered as an efficient alternative mutation screening method.

[Congenital disorder of type Ia protein glycosylation: clinical, biochemical and molecular characteristics in 2 siblings with cerebellar hypoplasia]. / Dedicné poruchy glykosylace proteinu typ Ia: klinická, biochemická a molekulární charakteristika u dvou sourozencu s hypoplazií mozecku.

BACKGROUND: Congenital disorders of glycosylation (CDG syndrome) represent a newly delineated group of inherited diseases of glycoprotein synthesis. We present results of biochemical and molecular analyses in two Czech patients with CDG Ia syndrome. METHODS AND RESULTS: Serum concentrations of the nonglycosylated and hypoglycosylated transferrin were measured using turbidimetric immunoassay. In positive patients, the isoelectric focusing of serum transferrin and molecular analyses of the gene for phosphomannomutase 2 were performed. The disease manifested in both children in infancy with failure to thrive, inverted nipples, strabismus, epilepsy, muscle hypotonia, microcephaly, psychomotor retardation and hypoplasia of the cerebellum. The biochemical investigation revealed elevated liver enzymes, low concentration of factor XI and protein S. In one child lower concentration of the antithrombin III and protein C were found. Activities of arylsulfatase A and beta-glucuronidase in serum were higher and activity of alpha-mannosidase in leucocytes was lower in comparison with controls. Molecular analyses revealed that both children are compound heterozygotes for the mutation 422G > A and 357C > A in gene for phosphomanomutase 2. Both siblings are also homozygotes for polymorfism IVS5 + 19 C-->T and heterozygygotes for polymorfism IVS5 + 22 T-->A. CONCLUSIONS: The prognosis of children with CDG Ia is unfavourable. Enzymatic and/or molecular studies are necessary for genetic counselling and the prenatal diagnosis.

[Analysis of the most frequent mutations in girls with Rett syndrome]. / Analýza nejcastejsích mutací u dívek s Rettovým syndromem.

BACKGROUND: Rett syndrome is an X-linked dominant neurodevelopmental disorder affecting 1 from 10,000 to 15,000 females worldwide. The responsible gene, encoding methyl-CpG binding protein 2 was recently identified. Methyl-CpG binding protein 2 is thought to act as a global transcriptional repressor. In the methyl-CpG binding protein 2 gene are known 5 prevalent mutations that cause Rett syndrome. Four of them are detectable by restriction analysis. In this study we present the results of the molecular study of four prevalent mutations in the gene for methyl-CpG binding protein 2 in Czech and Slovak patients with Rett syndrome. METHODS AND RESULTS: 22 females with Rett syndrome were investigated by methods of molecular biology. Restriction analysis and direct sequencing of PCR products revealed in methyl-CpG binding protein 2 gene 3 different mutations (T158M, R168X, R270X) in six unrelated patients with Rett syndrome. Mutation R306C, frequent in Great Britain and Sweden, was not detected in our group of patients with Rett syndrome. CONCLUSIONS: The diagnosis of Rett syndrome and genetic counselling in affected families should go out from the close cooperation of the pediatric, neurologic, and genetic departments with the specialized laboratories dealing with the molecular biological diagnosis.

Protoporphyrinogen oxidase: complete genomic sequence and polymorphisms in the human gene.

Variegate porphyria (VP) is an autosomal dominant disorder of heme synthesis caused by a partial deficiency of protoporphyrinogen oxidase (PPOX). Human cDNA encoding PPOX has been recently sequenced and the gene has been cloned, assigned to chromosome 1q23, and its exon/intron organization has been characterized. We report here the complete nucleotide sequence of the Human PPOX gene. Including 660 bp of its promotor region, the PPOX gene spans 5.5 kb. Introns vary in size from 84 bp to 507 bp. Two exonic and 3 intronic biallelic sequence variations have been characterized.

Molecular analysis of porphobilinogen (PBG) deaminase gene mutations in acute intermittent porphyria: first study in patients of Slavic origin.

Acute intermittent porphyria (AIP) is an autosomally dominant inherited metabolic disorders caused by decreased activity of porphobilinogen deaminase, the third enzyme in the human heme biosynthetic pathway. We report here the first mutations in the human porphobilinogen deaminase gene in seven unrelated patients from the Czech and Slovak Republics with acute intermittent porphyria. We used denaturing gradient gel electrophoresis to screen all 15 exons and exon/intron boundaries of the porphobilinogen deaminase gene. Polymerase chain reaction products of abnormal migration patterns were subjected to direct sequencing to identify the causative mutations. Thus we revealed four novel mutations and three which have been previously described. Of the four novel mutations, two were mis-sense (G24S, V267M), one was a single base insertion (158insA) that produced a stop codon 12 codons downstream, and one was a single base substitution in intron 12 (771 + 1) resulting in a splicing defect. The three previously detected mutations were mis-sense mutations (R26C, R26H, G111R). These results suggest a high allelic heterogeneity in Czech and Slovak patients.

Systematic analysis of coproporphyrinogen oxidase gene defects in hereditary coproporphyria and mutation update.

Hereditary coproporphyria (HC) is an acute hepatic porphyria with autosomal dominant inheritance caused by deficient activity of coproporphyrinogen III oxidase (CPO). Clinical manifestations of the disease are characterized by acute attacks of neurological dysfunction often precipitated by drugs, fasting, cyclical hormonal changes, or infectious diseases. Skin photosensitivity may also be present. The seven exons, the exon/intron boundaries and part of 3' noncoding sequence of the CPO gene were systematically analyzed by an exon-by-exon denaturing gradient gel electrophoresis (DGGE) strategy followed by direct sequencing in seven unrelated heterozygous HC patients from France, Holland, and Czech Republic. Seven novel mutations and two new polymorphisms were detected. Among these mutations: two are missense (G197W, W427R), two are nonsense (Q306X, Q385X), two are small deletions (662de14bp; 1168del3bp removing a glycine at position 390), and one is a splicing mutation (IVS1-15c-->g) which creates a new acceptor splice site. The pathological significance of the point mutations G197W, W427R, and the in-frame deletion 390delGly were assessed by their respective expression in a prokaryotic system using site-directed mutagenesis. These mutations resulted in the absence or a dramatic decrease of CPO activity. The two polymorphisms were localized in noncoding part of the gene: 1) a C/G polymorphism in the promotor region, 142 bp upstream from the transcriptional initiation site (-142C/G), and 2) a 6 bp deletion polymorphism in the 3' noncoding part of the CPO gene, 574 bp downstream of the last base of the normal termination codon (+574 delATTCTT). Five intragenic dimorphisms are now well characterized and the high degree of allelic heterogeneity in HC is demonstrated with seven new different mutations making a total of nineteen CPO gene defects reported so far.

Neonatal hemolytic anemia due to inherited harderoporphyria: clinical characteristics and molecular basis.

Porphyrias, a group of inborn errors of heme synthesis, are classified as hepatic or erythropoietic according to clinical data and the main site of expression of the specific enzymatic defect. Hereditary coproporphyria (HC) is an acute hepatic porphyria with autosomal dominant inheritance caused by deficient activity of coproporphyrinogen III oxidase (COX). Typical clinical manifestations of the disease are acute attacks of neurological dysfunction; skin photosensitivity may also be present. We report a variant form of HC characterized by a unifying syndrome in which hematologic disorders predominate: harderoporphyria. Harderoporphyric patients exhibit jaundice, severe chronic hemolytic anemia of early onset associated with hepatosplenomegaly, and skin photosensitivity. Neither abdominal pain nor neuropsychiatric symptoms are observed. COX activity is markedly decreased. In a first harderoporphyric family, with three affected siblings, a homozygous K404E mutation has been previously characterized. In the present study, molecular investigations in a second family with neonatal hemolytic anemia and harderoporphyria revealed two heterozygous point mutations in the COX gene. One allele bore the missense mutation K404E previously described. The second allele bore an A-->G transition at the third position of the donor splice site in intron 6. This new COX gene mutation resulted in exon 6 skipping and the absence of functional protein production. In contrast with other COX gene defects that produce the classical hepatic porphyria presentation, our data suggest that the K404E substitution (either in the homozygous or compound heterozygous state associated with a mutation leading to the absence of functional mRNA or protein) is responsible for the specific hematologic clinical manifestations of harderoporphyria.