Childhood hypophosphatasia due to a de novo missense mutation in the tissue-nonspecific alkaline phosphatase gene.

Hypophosphatasia is an inherited disorder due to mutations in the bone alkaline phosphatase (ALPL) gene. We report here a patient with childhood hypophosphatasia diagnosed at 1.4 yr because of pectus excavatum, large anterior fontanel, rachitic skeletal changes, and low serum alkaline phosphatase. Sequencing of the ALPL gene produced evidence of two distinct missense mutations, E174K (c.571G>A), of maternal origin, and a de novo mutation, M45I (c.186G>C). The study of various microsatellite polymorphisms ruled out false paternity and therefore confirmed that M45I occurred de novo in the paternal germline or in the early development of the patient. Site-directed mutagenesis showed that M45I results in the absence of in vitro alkaline phosphatase activity, suggesting that the mutation is a severe allele. In conclusion, childhood hypophosphatasia in this patient is the result of compound heterozygosity for the moderate mutation E174K and a novel severe de novo mutation M45I.

Identification of differentially expressed genes in human gliomas by DNA microarray and tissue chip techniques.

New genomic large-scale screening techniques have made the task of establishing an accurate molecular fingerprint of cancer cells feasible. Here, we have used a two-phase strategy for identification of molecular alterations in gliomas. First, cDNA microarrays (Clontech Laboratories, Inc., Research Genetics) were used to pinpoint differentially expressed genes between normal brain and diffuse astrocytomas (grades II-IV), and between a primary tumor and a later tumor reoccurrence in the same patient. More than 200 gene expression alterations were detected from glioblastomas, whereas relatively few changes were seen in grade II and grade III tumors. The most distinct progression-related expression change was the up-regulation of the insulin-like growth factor binding protein 2 (IGFBP2) gene. Second, a high-density tissue microarray of 418 brain tumors was constructed and used for clinical validation of gene expression changes. Strong expression of IGFBP2 was associated with progression and poor patient survival in diffuse astrocytomas (P < 0.0001). Third, comparisons of the data between (a) multiple spots retrieved from one predefined tumor region (IGFBP2 and vimentin immunohistochemistry, 20 tumors) or between (b) standard slides and arrayed tissues (p53 immunohistochemistry, 42 tumors) revealed very little variation. In conclusion, the combined use of DNA microarrays and tissue microarrays offers a powerful strategy for rapid identification and thorough characterization of differentially expressed genes in gliomas.

Genetic aberrations in sporadic and neurofibromatosis 2 (NF2)-associated schwannomas studied by comparative genomic hybridization (CGH).

BACKGROUND: Schwannomas occur sporadically or in association with neurofibromatosis 2 (NF2), an autosomal dominant disorder, which predisposes to multiple schwannomas, meningiomas and spinal ependymomas, with bilateral vestibular schwannomas as the classic hallmark. As NF2 and sporadic schwannomas differ in some respect in their clinical and biological behavior we evaluated whether there are any differences in the distribution of genetic aberrations between NF2 and sporadic schwannomas. Our interest was also to verify whether secondary genetic alterations besides the loss of 22q could be detected in schwannomas. METHODS: We investigated DNA copy number changes in 25 schwannomas (12 NF2 and 13 sporadic schwannomas) using the comparative genomic hybridization (CGH) technique. Some chromosomal regions were further studied by LOH or FISH analysis. FINDINGS: CGH detected genomic abnormalities in 15 of 25 schwannomas (60%). The most common alteration was loss on 22q, found in 32% (8/25) of schwannomas. No consistent changes were detected in other chromosomal regions. The overall number of genetic aberrations was similar in NF2 and in sporadic schwannomas. INTERPRETATION: Our results support the present view that loss of chromosome 22q harboring the NF2 gene plays a universal role in the pathogenesis of schwannomas without consistent involvement of other chromosomal regions.

Chromosome imbalances in familial gliomas detected by comparative genomic hybridization.

Familial occurrence of gliomas, in the absence of well-defined hereditary multisystem disorders, is reported occasionally. We describe 17 families that have been afflicted with two or more gliomas but do not raise suspicion of other inheritable syndromes. The families were identified among 369 consecutive glioma patients operated at the Tampere University Hospital during 1983-1994. We applied comparative genomic hybridization (CGH) analysis on 21 gliomas occurring in these 17 families. The most frequent genetic alterations, detected in over 20% of the tumors, were losses of 6q, 10, 4q, 9p and gains of 7, 19, 20q, 1p. We compared the chromosomal alterations detected in the familial gliomas to those reported previously on 209 sporadic gliomas in nine different CGH studies. In this comparison, the familial gliomas more often showed losses of chromosome arms 4q and 6q and gains of 1p and 22q. The most frequent losses (9/21 tumors) in the familial gliomas resided on chromosome arm 6q (P = 0.005, Fisher's exact test; with Bonferroni correction, P = 0.04). The loss of 6q was also the most common intrafamilial aberration, present in four separate gliomas belonging to two families. The minimal common area of loss on this chromosome resided at 6q14-16. In conclusion, we have found several characteristic aberrations by CGH in the familial gliomas and we present new chromosomal regions possibly involved in the familial predisposition to gliomas.

Grading of diffusely infiltrating astrocytomas by quantitative histopathology, cell proliferation and image cytometric DNA analysis. Comparison of 133 tumours in the context of the WHO 1979 and WHO 1993 grading schemes.

The aim of the study was to evaluate the applicability of quantitative histopathology as an aid for grading diffusely infiltrating astrocytomas. Primary astrocytomas were analysed for parameters (mean nuclear size, mitosis count, area fraction of endothelial cells and tumour necrosis, area fraction of nuclei, and Ki-67 (MIB-1) labelling index), which are closely related to the World Health Organization (WHO) 1979 and WHO 1993 grading criteria. All estimates correlated with the WHO histopathological grade and patient outcome. According to the receiver-operating characteristics curve, the presence of tumour necrosis and mitosis count (cut-off at 3 mitoses/mm2 of neoplastic tissue) showed the best sensitivity and specificity in separating patients with different survival. The multivariate survival analyses confirmed this result. A decision-tree model was constructed based on these two variables: twig I with less than 3 mitoses/mm2, twig II with equal or more than 3 mitoses/mm2 but no necrosis, and twig III with tumour necrosis. This model was found to be more strongly associated with survival than the WHO 1979 or WHO 1993 grading schemes. Low-malignancy astrocytomas (WHO grade II or twig I tumours) could be further divided into two prognostic categories by the image cytometric DNA analysis. The results put an emphasis on astrocytoma grading on mitosis counts (grade II vs. III) and tumour necrosis (grade III vs. IV). To standardize the sampling for mitosis counting, it is suggested that a parallel Ki-67 immunostaining be used for the identification of the most proliferative areas.

Cyclic expression of mRNA transcripts for connective tissue components in the mouse ovary.

In the ovary, differentiation of germinal cells into primordial follicles, functional ovulatory follicles and corpus luteum, all take place in a connective tissue matrix. We postulated that extracellular matrix (ECM) of the ovary participates actively in ovarian functions. To test this, the mRNA levels for several ECM components were determined in the mouse ovary at six distinct stages of the 4-day oestrous cycle. Northern analysis revealed statistically significant cyclic expression patterns for the mRNAs coding for type III, IV and VI collagens as well as for the small proteoglycan, biglycan, and for syndecan-1 and osteonectin. The cyclic changes observed in the mRNAs for these structural components exceeded those for matrix metalloproteinases (MMP)-2, -9 and -13, and for tissue inhibitors of matrix metalloproteinases (TIMP)-1, -2 and -3, where the changes were not statistically significant, despite their apparent role in ECM remodelling in the ovary. These observations support the hypothesis that cyclic changes in the production and degradation of ECM are part of normal ovarian function connected with follicular maturation, rupture and corpus luteum formation.

Cyclin D1 expression in astrocytomas is associated with cell proliferation activity and patient prognosis.

An important positive regulator of the cell cycle, cyclin D1, is often amplified and overexpressed in malignancies. Cyclin D1 aberrations were analysed in grade II-IV astrocytomas by fluorescence in situ hybridization (FISH), mRNA in situ hybridization and immunohistochemistry. Proliferation activity was determined by Ki-67(MIB-1) immunolabelling and mitotic counting. High cyclin D1 expression was observed in grade IV astrocytomas (grades II-III versus grade IV; mRNA expression: p<0.001; immunoexpression: p=0.013), and correlated with poor patient survival (p<0.001, n=46). Upregulated cyclin D1 expression was also closely associated with poor patient prognosis in grade II-III astrocytomas (p<0.001, n=30). Cyclin D1 gene was not found to be amplified (n=7). Cell proliferation activity was significantly increased in tumours exhibiting high cyclin D1 mRNA levels (Ki-67(MIB-1): p<0.001; mitotic count: p<0.001) and high cyclin D1 protein expression (Ki-67(MIB-1): p=0.002; mitotic count: p=0.012). These results indicate that increased production of cyclin D1 is closely associated with high cell proliferation activity and aggressive behaviour in diffusely infiltrating astrocytomas.

Clinicopathological correlation of cell proliferation, apoptosis and p53 in cerebellar pilocytic astrocytomas.

We have analysed 78 cerebellar pilocytic astrocytomas to assess whether histopathology, cell proliferation, apoptosis rate, p53 immunoreactivity, or flow cytometry could predict their long-term behaviour. Classic pilocytic/microcystic pattern was seen in 62 patients and 16 patients had mixed pattern with an additional non-pilocytic glial component. The overall 5-year survival was 93%, complete resection providing 100% survival. The four patients who died during the follow-up were more than 14 years of age, their primary operation had been incomplete and three of them were mixed variants. In 15 cases the tumour recurred giving a recurrence-free 5-year survival of 77%. The proliferation indices were low: Ki-67MIB-1 (median 2.0%), PCNA (1.2%) and S-phase fraction (4.4%). The Ki-67MIB-1-labelling index was significantly higher in young patients, but did not differ between the classic and mixed variants. Twenty-two per cent of the tumours were aneuploid with a significantly higher S-phase fraction than in diploid tumours. p53 seems to act as ardian of the genome' in pilocytic astrocytomas, because aberrant/increased expression of p53 and aneuploidy associated with enhanced apoptosis. Only patient age (P = 0.01), radicality of the primary operation (P = 0.0001) and histology (classic vs mixed, P=0.008) significantly correlated with survival. The poorer prognosis of the mixed variant suggests that this may represent a distinct entity. Although none of the novel parameters significantly predicted recurrence or survival, they indicate substantial biological variation among cerebellar pilocytic astrocytomas.

Accumulation of genetic changes is associated with poor prognosis in grade II astrocytomas.

Unexpectedly aggressive clinical course of some grade II astrocytomas is a diagnostic dilemma for routine histopathology. Because increasing tumor malignancy is a consequence of progressive accumulation of chromosomal alterations, we investigated whether aggressive behavior of grade II astrocytomas could be predicted by the number and type of gross chromosomal aberrations. We used comparative genomic hybridization to analyze 11 grade II astrocytomas with typical (good, n = 7) or poor (n = 4) prognosis. The results were also compared with a reference material of 13 grade III-IV astrocytomas and nine established cell lines. We found a median of two aberrations (range 0 to 4) in tumors with good prognosis and of 15.5 changes (range 8 to 28) in tumors with poor prognosis. Chromosomal gains were present in both groups, whereas chromosomal losses were frequent in tumors with poor prognosis (median 9.5, range 3 to 14) but rare in tumors with good prognosis (range 0 to 2). All chromosomal gains were also found in the high-grade astrocytoma group and the majority of them in cell lines. Chromosomal losses in grade II astrocytomas with poor prognosis were very similar to those in grade III-IV astrocytomas and cell lines. We conclude that an early accumulation of genetic changes in grade II astrocytomas is closely associated with poor patient prognosis, suggesting diagnostic use for comparative genomic hybridization in characterization of grade II astrocytomas.

Increased expression of telomerase RNA component is associated with increased cell proliferation in human astrocytomas.

Deregulation of telomerase, a ribonucleoprotein polymerase that compensates progressive loss of telomeric (TTAGGG)n repeats during DNA replication, has been suggested to facilitate tumorigenesis and cellular immortality by providing unlimited proliferation capacity for cancer cells. We investigated the relationship between tumor proliferation activity and in situ expression of the telomerase RNA component in 46 human grade I to IV astrocytomas. Heterogeneously distributed telomerase RNA expression was detected from all of the tumor samples as well as from normal human brain tissue. However, expression of telomerase RNA was significantly increased in highly malignant tumors (P = 0.024) and in tumors that showed increased proliferation activity determined by MIB-1 immunohistochemistry (P = 0.014). Interestingly, increased telomerase RNA levels were observed in a subgroup of grade II astrocytomas that showed significant increase in proliferation activity (P = 0.047), indicating that the telomerase RNA component is up-regulated already in early states of astrocytoma malignancy. Telomeric repeats amplification assays revealed telomerase activity in 4 of 6 glioblastomas and in 1 rapidly proliferating grade II astrocytoma. These results suggest that increased tumor proliferation activity triggers telomerase activation via mechanisms that involve increased production of the telomerase RNA component.

A real-time microprocessor QRS detector system with a 1-ms timing accuracy for the measurement of ambulatory HRV.

The design, test methods and results of an ambulatory QRS detector are presented. The device is intended for the accurate measurement of heart rate variability (HRV) and reliable QRS detection in both ambulatory and clinical use. The aim of the design work was to achieve high QRS detection performance in terms of timing accuracy and reliability, without compromising the size and power consumption of the device. The complete monitor system consists of a host computer and the detector unit. The detector device is constructed of a commonly available digital signal processing (DSP) microprocessor and other components. The QRS detection algorithm uses optimized prefiltering in conjunction with a matched filter and dual edge threshold detection. The purpose of the prefiltering is to attenuate various noise components in order to achieve improved detection reliability. The matched filter further improves signal-to-noise ratio (SNR) and symmetries the QRS complex for the threshold detection, which is essential in order to achieve the desired performance. The decision for detection is made in real-time and no search-back method is employed. The host computer is used to configure the detector unit, which includes the setting of the matched filter impulse response, and in the retrieval and postprocessing of the measurement results. The QRS detection timing accuracy and detection reliability of the detector system was tested with an artificially generated electrocardiogram (ECG) signal corrupted with various noise types and a timing standard deviation of less than 1 ms was achieved with most noise types and levels similar to those encountered in real measurements. A QRS detection error rate (ER) of 0.1 and 2.2% was achieved with records 103 and 105 from the MIT-BIH Arrhythmia database, respectively.