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A Poustka

Publications and source records attributed to A Poustka.

At least 73 records · Page 4Linked to original sources
PubMed1998

Primer design for large scale sequencing.

We have developed PRIDE, a primer design program that automatically designs primers in single contigs or whole sequencing projects to extend the already known sequence and to double strand single-stranded regions. The program is fully integrated into the Staden package (GAP4) and accessible with a graphical user interface. PRIDE uses a fuzzy logic-based system to calculate primer qualities. The computational performance of PRIDE is enhanced by using suffix trees to store the huge amount of data being produced. A test set of 110 sequencing primers and 11 PCR primer pairs has been designed on genomic templates, cDNAs and sequences containing repetitive elements to analyze PRIDE's success rate. The high performance of PRIDE, combined with its minimal requirement of user interaction and its fast algorithm, make this program useful for the large scale design of primers, especially in large sequencing projects.

Algorithms↗
PubMed1998

A database of germline p53 mutations in cancer-prone families.

We created a comprehensive database covering all published cases of germline p53 mutations. The current version lists 580 tumours in 448 individuals belonging to 122 independent pedigrees. The database describes each p53 mutation (type of the mutation, exon and codon affected by the mutation, nucleotide and amino acid change), each family (family history of cancer, diagnosis of Li-Fraumeni syndrome), each affected individual (sex, generation, p53 status, from which parent the mutation was inherited) and each tumour (type, age of onset, p53 status-loss of heterozygosity, immunostaining). Each entry contains the original reference(s). The database is freely available and can be obtained from http://www.lf2.cuni.cz

Computer Communication Networks↗
PubMed1998

Two Li-Fraumeni syndrome families with novel germline p53 mutations: loss of the wild-type p53 allele in only 50% of tumours.

We describe two Li-Fraumeni syndrome families. Family A was remarkable for two early childhood cases of adrenocortical tumours, family B for a high incidence of many characteristic cancers, including a childhood case of choroid plexus tumour. Using direct sequencing, we analysed exons 5-9 of the p53 gene in constitutional DNA of individuals from both families and found two novel germline mutations in exon 5. In family A, we detected a point substitution in codon 138 (GCC to CCC), which resulted in the replacement of the alanine by a proline residue. Family B harboured a single-base pair deletion in codon 178 (CAC to -AC), resulting in a frameshift and premature chain termination. Three out of six tumours examined from both families, a renal cell carcinoma, a rhabdomyosarcoma and a breast cancer, showed loss of heterozygosity and contained only the mutant p53 allele. The remaining three neoplasms, both adrenocortical tumours and the choroid plexus tumour retained heterozygosity. Immunohistochemistry with anti-p53 antibody confirmed accumulation of p53 protein in tumours with loss of heterozygosity, while the remaining tumours were p53 negative. These results support the view that complete loss of activity of the wild-type p53 need not be the initial event in the formation of all tumours in Li-Fraumeni individuals.

Adolescent↗
PubMed1998

X-linked dyskeratosis congenita is caused by mutations in a highly conserved gene with putative nucleolar functions.

X-linked recessive dyskeratosis congenita (DKC) is a rare bone-marrow failure disorder linked to Xq28. Hybridization screening with 28 candidate cDNAs resulted in the detection of a 3' deletion in one DKC patient with a cDNA probe (derived from XAP101). Five different missense mutations in five unrelated patients were subsequently identified in XAP101, indicating that it is the gene responsible for X-linked DKC (DKC1). DKC1 is highly conserved across species barriers and is the orthologue of rat NAP57 and Saccharomyces cerevisiae CBF5. The peptide dyskerin contains two TruB pseudouridine (psi) synthase motifs, multiple phosphorylation sites, and a carboxy-terminal lysine-rich repeat domain. By analogy to the function of the known dyskerin orthologues, involvement in the cell cycle and nucleolar function is predicted for the protein.

Amino Acid Sequence↗
PubMed1998

1.4 Mb candidate gene region for X linked dyskeratosis congenita defined by combined haplotype and X chromosome inactivation analysis.

Dyskeratosis congenita (DC) is a rare inherited disorder characterised by the early onset of reticulate skin pigmentation, nail dystrophy, and mucosal leucoplakia. In over 80% of cases bone marrow failure develops and this is the main cause of early mortality. The DC1 gene responsible for the X linked form (MIM 305000) of dyskeratosis congenita has been mapped to Xq28. In order to narrow the candidate gene region, genetic linkage analysis was performed in eight X linked pedigrees using a set of markers spanning Xq28. A maximum lod score of 5.31 with no recombinations was achieved with marker DXS1073. Two recombination events were identified; one of these uses X chromosome inactivation pattern analysis to determine carrier status and haplotype analysis to fine map the recombination breakpoint. The fine mapping of these recombination events has enabled the candidate gene region for X linked dyskeratosis congenita to be defined as the 1.4 Mb interval between Xq3274 and DXS1108.

Child↗
PubMed1997

A gene for FG syndrome maps in the Xq12-q21.31 region.

FG syndrome is an X-linked recessive condition in which mental retardation is associated with congenital hypotonia, macrocephaly, characteristic face, and constipation. This syndrome was mapped by Zhu et al. [Cytogenet Cell Genet 1991;58:2091A] to Xq21.31-q22 by linkage analysis with a max lod score of 1.2 for the DXYS1X, DXS178, DXS101, and DXS94 loci and crossovers at DXS16 (Xp22.31) and DXS287 (Xq22.3). However, this mapping was only provisional and needed to be refined. In this paper, we report the results of a new linkage analysis performed on 10 families including that studied by Zhu et al. [1991]. Two-point analysis demonstrated linkage with DXS441 (Zmax = 3.39 at theta = 0.12) at Xq13. In addition, separate analysis of the lod scores obtained for the Xq13 markers suggested linkage exclusion for three families. Genetic heterogeneity was confirmed by analysis of the linkage results with the HOMOG program (max logL = 4.07, theta = 0, alpha = 0.65). Localization of one FG gene between DXS135 and DXS1066 was suggested by analysis of crossovers found in those three families which were assumed to be linked to Xq13 with a probability of 0.95 or more. This region could be reduced to the DXS135-DXS72 interval after combining our data with those from deletions previously described in males in the Xq13-q21 region.

Abnormalities, Multiple↗
PubMed1997

A gene in human chromosome band Xq28 (GABRE) defines a putative new subunit class of the GABAA neurotransmitter receptor.

We have isolated and sequenced a novel human gene (GABRE) of the GABAA neurotransmitter receptor family. A cDNA sequence of the gene coding for a 506 amino acid protein was identified, representing a member of a putative new class (epsilon) of the GABAA receptor. The gene is transcribed at least at low level in several different tissues, with the highest levels being detected in adult heart and placenta. Alternative splicing of GABRE transcripts isolated from different tissues was observed at multiple positions of the gene, yielding an unusually complex variety of cDNA variants. The structure of the 5' region of most cDNAs is compatible with expression of protein sequence epsilon only in adult brain, whereas in other tissues, the majority of transcripts code for truncated protein sequences. The GABRE gene extends over 14 kb and is clustered together with the alpha 3 and the putative beta 4 GABAA receptor subunit genes in an approximately 0.8-Mb interval in chromosome band Xq28, located in the candidate regions of two different neurologic diseases. Based on features of conservation of protein sequences, gene structure, and genomic organization of GABAA receptor gene clusters, we propose that the epsilon and gamma subunit genes have a common ancestor and that GABAA receptor gene clusters in the human genome have diverged by multiple duplication events of an ancestral gene cluster containing one each alpha, beta, and gamma/epsilon precursor gene.

Adult↗
PubMed1997

Genomic structure of a novel chloride channel gene, CLIC2, in Xq28.

A transcript map was previously constructed in the 1200-kb telomeric region of Xq28. One of the cDNAs, XAP121, displayed homology to a p64 bovine chloride channel and to a human chloride channel (p64CLCP, NCC27) at both the nucleotide and the peptide levels. In addition, all three sequences exhibited homologies to numerous ESTs derived from human, mouse, rat and pig. While the human NCC27 and XAP121 homologs encode small peptides of 241 and 243 amino acids, respectively, the bovine peptide has a length of 437 amino acids. This suggests that the human genes represent a novel and separate class of small chloride channels. Unlike other chloride channels, the NCC27 peptide was recently shown to localize intracellularly in the cytoplasm and nucleus. The NCC27 and XAP121 genes have thus been designated CLIC1 and CLIC2 for chloride intracellular channel genes 1 and 2, respectively. Since a direct association exists between a number of human chloride channel genes and a range of hereditary diseases, CLIC2 possibly represents a candidate for one of the many diseases linked to Xq28. To facilitate defined mutation analyses, we determined the genomic structure of the CLIC2 gene.

Amino Acid Sequence↗
PubMed1997

Isolation of human and mouse HMG2a cDNAs: evidence for an HMG2a-specific 3' untranslated region.

We have isolated cDNAs of the human gene for high mobility group protein HMG2a, using the method of direct cDNA selection. The gene maps to chromosome band Xq28, and is located within 40 kb from marker DXS1684, at a distance of 5.4 Mb from the telomere. The deduced human HMG2a protein sequence has a length of 199 amino acids and is 97% identical to the sequence of chicken HMG2a. The 3' untranslated regions of the HMG2a gene in both species are highly homologous (87% identical nucleotides), and are even more conserved than the coding sequences (84% identical nucleotides). In addition, a partial cDNA sequence of the putative HMG2a gene from mouse was identified. The 3' untranslated regions from human and mouse are 90% identical. We conclude that the 3' untranslated sequences have been under strong selective pressure during evolution. Whereas expression of the chicken HMG2a gene has previously been demonstrated in liver of newly hatched chicken, the human HMG2a gene is transcribed mainly in placenta.

Amino Acid Sequence↗
PubMed1997

Genomic structure of a novel LIM domain gene (ZNF185) in Xq28 and comparisons with the orthologous murine transcript.

Construction of a transcript map in the DXS52 region in Xq28 had previously led to the isolation of a cDNA with a LIM zinc finger domain in the carboxyl terminus. In parallel, the orthologous murine transcript was isolated from the syntenic region. The human and mouse cDNAs have been designated ZNF185 and Zfp185, respectively. By integrating the cDNA sequence with the cosmid-derived genomic sequence the exon-intron structure of the 3' end of the ZNF185 gene was resolved. Comparative sequence analyses of the human genomic sequence with the full-length murine cDNA facilitated prediction of the 5' end of the gene. The selective expression of three transcripts corresponding to the ZNF185 gene and a related gene was shown by Northern and Southern blots. In situ hybridizations revealed a nonubiquitous and stage-specific expression of Zfp185, especially in differentiating connective tissue. Since LIM proteins regulate cellular proliferation and/or differentiation by diverse mechanisms, and some have directly been associated with disease, conceivably ZNF185 may represent a candidate for a disease-causing gene linked to Xq28. Knowledge of the genomic structure will permit detailed mutation analyses.

Amino Acid Sequence↗
PubMed1997

Molecular characterization of a genetically unstable region containing the SMS critical area and a breakpoint cluster for human PNETs.

Recently we demonstrated the clustering of deletion breakpoints in the pericentromeric region of human chromosome 17p in human primitive neuroectodermal tumors (PNETs). Chromosomal disruption was shown to occur between the two markers D17S805 and D17S953, a region previously shown to be deleted in the Smith-Magenis syndrome. To characterize the molecular basis of this genomic instability, we established clone contigs covering this region. An initial physical map of chromosome 17p has been constructed with overlapping sets of YACs. YAC clones were transformed into five clone contigs according to their content of 30 previously known and 16 newly established sequence-tagged sites (STSs). To circumvent the complications inherent in YAC technologies, such as internal deletions, chimerism, and complex rearrangements, we then converted the YAC contigs to PAC and cosmid contigs. Thirty-nine individual PAC/cosmid clones were identified and were used to construct six different PAC/cosmid contigs ranging from 130 to 1200 kb in size and covering approximately 2.5 Mb of genomic DNA. The composite YAC/PAC/cosmid map covers a region of > 6 Mb of genomic DNA consisting of four different clone contigs of up to 2.9 Mb in size. We have demonstrated that three STSs (D17S58, PS1, and D17S842) are duplicated, suggesting the occurrence of low abundant repetitive sequences in this region. By integration of publicly available information we further mapped 10 genes and ESTs to their precise chromosomal positions and thus could exclude or identify them as candidate genes for PNET and/or the Smith-Magenis syndrome.

Base Sequence↗
PubMed1997

Cloning and characterization of an alternatively spliced gene in proximal Xq28 deleted in two patients with intersexual genitalia and myotubular myopathy.

We have identified a novel human gene that is entirely deleted in two boys with abnormal genital development and myotubular myopathy (MTM1). The gene, F18, is located in proximal Xq28, approximately 80 kb centromeric to the recently isolated MTM1 gene. Northern analysis of mRNA showed a ubiquitous pattern and suggested high levels of expression in skeletal muscle, brain, and heart. A transcript of 4.6 kb was detected in a range of tissues, and additional alternate forms of 3.8 and 2.6 kb were present in placenta and pancreas, respectively. The gene extends over 100 kb and is composed of at least seven exons, of which two are noncoding. Sequence analysis of a 4.6-kb cDNA contig revealed two overlapping open reading frames (ORFs) that encode putative proteins of 701 and 424 amino acids, respectively. Two alternative spliced transcripts affecting the large open reading frame were identified that, together with the Northern blot results, suggest that distinct proteins are derived from the gene. No significant homology to other known proteins was detected, but segments of the first ORF encode polyglutamine tracts and proline-rich domains, which are frequently observed in DNA-binding proteins. The F18 gene is a strong candidate for being implicated in the intersexual genitalia present in the two MTM1-deleted patients. The gene also serves as a candidate for other disorders that map to proximal Xq28.

Alternative Splicing↗
PubMed1997

Human histone gene organization: nonregular arrangement within a large cluster.

We have previously located the genes of the five human main type H1 genes and the gene encoding the testicular subtype H1t to the region 21.1 to 22.2 on the short arm of chromosome 6. To investigate the organization of the histone genes in this region, we isolated two YACs from a human YAC library by PCR screening with primers specific for histone H1.1. This screen revealed two YAC clones, YAC Y23 (corresponding to ICRFy901D1223) contains an insert of about 480 kb, whereas the smaller YAC 4A (corresponding to ICRFy900C104) spans about 340 kb and is completely covered by YAC Y23. We have subcloned the YAC inserts in cosmids, determined the linear orientation of the cosmids by cosmid walking, and constructed a restriction map of the entire region by mapping the individual cosmids using partial digests and hybridization with labeled oligonucleotides complementary to the cos site of the vector. Hybridization analysis, subcloning, restriction mapping, and sequencing revealed that most of the previously isolated phage and cosmid clones containing histone genes are part of this YAC including the clones containing the four human main type H1 histone genes H1.1 to H1.4, the H1t gene, and core histone genes. Thirty-five histone genes map within 260 kb of the YAC Y23 insert. All newly identified histone genes were sequenced, and the sequences were deposited with the EMBL nucleotide sequence database. The histone H1.5 gene is not part of this region, and we therefore conclude that the H1.5 gene and the associated core histone genes form a separate subcluster within this chromosomal region.

Chromosome Mapping↗
PubMed1997

High-resolution deletion mapping of chromosome arm 17p in childhood primitive neuroectodermal tumors reveals a common chromosomal disruption within the Smith-Magenis region, an unstable region in chromosome band 17p11.2.

Loss of heterozygosity (LOH) on chromosome arm 17p is the most common genetic aberration in childhood primitive neuroectodermal tumors (PNETs). To determine the frequency and extent of 17p deletions, 29 loci on 17p were investigated in 24 tumors by using restriction fragment length polymorphism (RFLP) and microsatellite analysis. LOH on 17p was found in 9 of 24 tumors. In all tumors with LOH, a continuous stretch from the telomere to chromosome band 17p11.2 was completely deleted, and no interstitial or terminal small-scale deletions were detected in the remaining 15 tumors. In four tumors with LOH on 17p, the chromosomal breakpoint was located between D17S953 and D17S805. To identify this deletion breakpoint on the cytogenetic map of chromosome 17 and to exclude uniparental disomy, we verified our data by using fluorescence in situ hybridization (FISH) analyses. By using two yeast artificial chromosome (YAC) clones that were positive for D17S689 and D17S953, the same breakpoint was confirmed in two specimens of cerebrospinal fluid (CSF) metastases by using FISH on interphase preparations. We demonstrate that, in most childhood PNETs with LOH on 17p, the breakpoint is close to, but not within, the centromere. It varies, and it occurs predominantly between the two markers D17S689 and D17S953, which is an unstable chromosomal region that is deleted or duplicated in the Smith-Magenis syndrome. Because LOH of 17p is associated with the formation of isochromosome 17q in the majority of PNETs, this study provides entry points to determine the molecular nature of this phenomenon.

Adolescent↗
PubMed1997

The 5-HT transporter gene-linked polymorphic region (5-HTTLPR) in evolutionary perspective: alternative biallelic variation in rhesus monkeys. Rapid communication.

By conferring allele-specific transcriptional activity on the 5-HT transporter gene promoter in humans, the 5-HT transporter gene-linked polymorphic region (5-HTTLPR) influences a constellation of personality traits related to anxiety and increases the risk for neurodevelopmental, neurodegenerative, and psychiatric disorders. Here we have analyzed the presence and variability of the 5-HTTLPR in several species of primates including humans, and other mammals. PCR, Southern blot, and sequence analyses of the 5-HT transporter gene's 5'-flanking region in different mammalian species confirmed the presence of the 5-HTTLPR in platyrrhini and catarrhini (hominoids, cercopithecoids) but not in prosimian primates and other mammals. Since the 5-HTTLPR is unique to humans and simian primates, a progenitor 5-HTTLPR sequence may have been introduced into the genome some 40 Mio, years ago. In humans the majority of alleles are composed of either 14 or 16 repeat elements, while alleles with 18 or 20 repeat elements are rare. In contrast, great apes including orang-utan, gorilla, and chimpanzee display a high prevalence of alleles with 18 and 20 repeat elements. In hominoids all alleles originate from variation at a single locus (polymorphic locus 1). In the 5-HTTLPR of rhesus monkeys (rh5-HTTLPR) we found an alternative locus for length variation (polymorphic locus 2) generated by a 21 bp insertion/deletion event. The existence of a distinct biallelic variation of the 5-HTTLPR in rhesus monkeys but similar allele and genotype frequencies in this species and humans supports the notion that there may be a relationship between functional 5-HT transporter expression, anxiety-related traits, and the complexity of socialization in human and non-human primate populations.

Alleles↗
PubMed1997

Molecular genetic analysis of the FMR-1 gene in a large collection of autistic patients.

A genetic etiology in autism is now strongly supported by family and twin studies. A 3:1 ratio of affected males to females suggests the involvement of at least one X-linked locus in the disease. Several reports have indicated an association of the fragile X chromosomal anomaly at Xq27.3 (FRAXA) with autism, whereas others have not supported this finding. We have so far collected blood from 105 simplex and 18 multiplex families and have assessed 141 patients by using the Autism Diagnostic Interview-Revised (ADI-R), the Autism Diagnostic Observation Scale, and psychometric tests. All four ADI-R algorithm criteria were met by 131 patients (93%), whereas 10 patients (7%) showed a broader phenotype of autism. Southern blot analysis was performed with three different enzymes, and filters were hybridized to an FMR-1-specific probe to detect amplification of the CCG repeat at FRAXA, to the complete FMR-1 cDNA probe, and to additional probes from the neighborhood of the gene. No significant changes were found in 139 patients (99%) from 122 families, other than the normal variations in the population. In the case of one multiplex family with three children showing no dysmorphic features of the fragile X syndrome (one male meeting 3 out of 4 ADI-algorithm criteria, one normal male with slight learning disability but negative ADI-R testing, and one fully autistic female), the FRAXA full-mutation-specific CCG-repeat expansion in the genotype was not correlated with the autism phenotype. Further analysis revealed a mosaic pattern of methylation at the FMR-1 gene locus in the two sons of the family, indicating at least a partly functional gene. Therefore, we conclude that the association of autism with fragile X at Xq27.3 is non-existent and exclude this location as a candidate gene region for autism.

Autistic Disorder↗