Severe hepatocellular dysfunction in obstetric cholestasis related to combined genetic variation in hepatobiliary transporters.

Obstetric cholestasis (OC) is a cholestatic disorder with a prominent genetic background including variation in diverse hepatobiliary lipid transporters, such as ABCB4 (phospholipids) and ABCB11 (bile salts). Given a marked hepatocellular dysfunction in an OC patient indicated by > 40-fold rise in alanine aminotransferase activity and minor gamma-glutamyl transpeptidase increases, we performed genotyping of candidate gene variants associated with adult cholestatic phenotypes. Genetic analysis revealed the heterozygous ABCB4 mutation p.R590Q, the ABCB11 variant p.V444A and the lithogenic ABCG8 variant p.D19H. Aggregation of multiple hepatobiliary transporter variants is rare in OC, and may cooperate to negatively modulate hepatobiliary transport capacities.

ATP8B1 mutations in British cases with intrahepatic cholestasis of pregnancy.

BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP) affects approximately 0.7% of pregnancies in the UK and is associated with prematurity, fetal distress, and intrauterine death. Homozygous mutations in the ATP8B1 gene cause cholestasis with a normal serum gamma-glutamyl transpeptidase (gamma-GT), and have been reported in two forms of cholestasis: progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis (BRIC). AIMS: To establish whether mutations in ATP8B1 are associated with ICP in British cases PATIENTS: Sixteen well phenotyped women with ICP without raised gamma-GT were selected for sequence analysis. Subsequently, 182 patients and 120 controls were examined for the presence of the variants detected. METHODS: All coding exons were sequenced in 16 cases. Eight ICP cases, including two women carrying a mutation, were investigated using in vivo hepatic (31)P magnetic resonance spectroscopy (MRS) RESULTS: Two heterozygous ATP8B1 transitions (208G>A and 2599C>T) that resulted in amino acid substitutions were identified; 208G>A was identified in three cases. MRS revealed an increased phosphodiester signal (Mann-Whitney U test, p = 0.03) and a decreased phosphomonoester/phosphodiester ratio (p = 0.04) in ICP cases compared with controls. CONCLUSIONS: We were able to demonstrate ATP8B1 mutations in ICP. MRS studies suggest that susceptibility to ICP is associated with a relative rise in biliary phospholipid. These data also suggest that MRS may be used for non-invasive assessment of the liver and biliary constituents in cholestasis.

Role for CCG-trinucleotide repeats in the pathogenesis of chronic lymphocytic leukemia.

Chromosome 11q deletions are frequently observed in chronic lymphocytic leukemia (CLL) in association with progressive disease and a poor prognosis. A minimal region of deletion has been assigned to 11q22-q23. Trinucleotide repeats have been associated with anticipation in disease, and evidence of anticipation has been observed in various malignancies including CLL. Loss of heterozygosity at 11q22-23 is common in a wide range of cancers, suggesting this is an unstable area prone to chromosome breakage. The location of 8 CCG-trinucleotide repeats on 11q was determined by Southern blot analysis of a 40-Mb YAC and PAC contig spanning 11q22-qter. Deletion breakpoints in CLL are found to co-localize at specific sites on 11q where CCG repeats are located. In addition, a CCG repeat has been identified within the minimal region of deletion. Specific alleles of this repeat are associated with worse prognosis. Folate-sensitive fragile sites are regions of late replication and are characterized by CCG repeats. The mechanism for chromosome deletion at 11q could be explained by a delay in replication. Described here is an association between CCG repeats and chromosome loss suggesting that in vivo "fragile sites" exist on 11q and that the instability of CCG repeats may play an important role in the pathogenesis of CLL.

Extranodal marginal zone B-cell lymphoma genotyping by Alu-polymerase chain reaction.

DNA amplification by polymerase chain reaction (PCR) with primers designed on the widely distributed Alu sequences allows the production of specific inter-Alu DNA-fingerprints. Amplification of tumour and matched normal DNA can show differences due to genetic alterations within the tumour genome. We applied this approach to study low-grade extranodal marginal zone B-cell lymphoma (of MALT type). After digestion with restriction enzymes, DNA samples were separately amplified by PCR with three different Alu-primers. A comparison between the fingerprint pattern from lymphoma and normal samples was made. Inter-Alu bands differing between the two samples were excised from the gel, cloned and sequenced. Nine cases of low-grade MALT-lymphomas have been analysed, giving seventeen different bands between tumour and normal. DNA sequence analysis showed identities for three of them with sequences available at the GenBank. The methodology of Alu-PCR to detect DNA-based abnormalities, in addition or combination with RNA-based methods, is a powerful tool to identify candidate regions frequently altered in tumours. With the increased available genomic sequences through the Human Genome Project, there will be an increasing probability of picking up perfect homologies with these sequences using cloned differential Alu-PCR bands in BLAST searches through genome databases.

Co-localisation of CCG repeats and chromosome deletion breakpoints in Jacobsen syndrome: evidence for a common mechanism of chromosome breakage.

Folate-sensitive fragile sites are associated with the expansion and hypermethylation of CCG-repeats. The fragile site in 11q23.3, FRA11B, has been shown to cause chromosome deletions in vivo, its expression being associated with Jacobsen (11q-) syndrome. However, the majority of Jacobsen deletions are distal to FRA11B and are not related to its expression. To test the hypothesis that other unidentified fragile sites might be located in 11q23.3-24 and may cause these deletions, we have identified and characterised CCG-trinucleotide repeats within a 40 Mb YAC contig spanning distal chromosome 11q. Only eight CCG-repeats were identified within the entire YAC contig (not including FRA11B ), six of which map to the region of 11q23.3-24 that includes Jacobsen deletions. We have previously collated the deletion mapping data of 24 Jacobsen patients with the physical map of chromosome 11q, and accurately localised six breakpoints to short intervals corresponding to individual YAC clones. We now show that in each of these cases, YAC clones found to contain a deletion breakpoint also contain a CCG-repeat. The improved analysis of one of these deletions, together with those of several new Jacobsen cases, further strengthens this association by localising five breakpoints to individual PAC clones containing CCG-repeats. These data provide strong evidence for the non-random clustering of chromosome deletion breakpoints with CCG-repeats, and suggests that they may play an important role in a common mechanism of chromosome breakage.

Cloning of two candidate tumor suppressor genes within a 10 kb region on chromosome 13q14, frequently deleted in chronic lymphocytic leukemia.

Previous studies have indicated the presence of a putative tumor suppressor gene on chromosome 13q14, commonly deleted in patients with B-cell chronic lymphocytic leukemia (B-CLL). We have previously defined a minimally deleted region of 130 kb centromeric to the marker D13S272, and constructed a PAC and cosmid contig encompassing this area. In the present study we have made a detailed restriction and transcriptional map of the region of interest. Using these tools we have screened a panel of 206 primary CLL clones and three cell lines. In five CLL cases we found limited deletions defining the region of interest to an area of no more than 10 kb. Two adjacent genes, termed Leu1 and Leu2 (leukemia-associated gene 1 and 2), were mapped to the minimally deleted region, with several patients showing deletion borders within these genes. The Leu1 and Leu2 genes show little homology to previously published genes at the nucleotide and expected translated amino acid sequence level. Mutational analysis of the Leu1 and 2 genes in 170 CLL samples revealed no small intragenic mutations or point mutations. However, in all cases of 13q14 loss examined, the first exon of both genes, which are only 300 bp apart, were deleted. We conclude that the Leu1 and Leu2 genes are strong candidates as tumor suppressor gene(s) involved in B-CLL leukemogenesis.

A 5.5-Mb high-resolution integrated map of distal 11q13.

The distal part of 11q13, which contains several genes relevant to human diseases, has been poorly mapped as part of genome-wide mapping efforts. In the prospect of drawing a fine-scale integrated map of the area containing KRN1 and OMP, we have established a framework of markers by hybridization to DNA of somatic cell hybrids and by fluorescence in situ hybridization (FISH) on metaphase chromosomes. The probes studied were used to isolate 27 YACs and 16 cosmids that could be organized in three contigs covering approximately 6 Mb. These contigs were separated by two gaps that are likely to contain sequences underrepresented in YAC libraries. They were then integrated based on long-range restriction mapping and DNA-fiber FISH into a high-resolution physical map, which covers a 5.5-Mb region and includes 36 anonymous markers and 10 genes. This map will be used to search for genes within the 2/3 of this region where none have been localized as yet. It will also lay the ground for the characterization of an amplicon surrounding GARP in breast cancer and for the search of disease genes within this region.

Cosmid-derived transcripts and sequence tags mapped to three subregions of human chromosome 22.

Fifty cosmids from the ICRF, London, and Lawrence Livermore Laboratory, California, human chromosome 22 cosmid libraries were isolated, regionally assigned and tested for their ability to detect repeats or single copy sequences. The search resulted in nine cosmids containing repetitive motifs from the pericentric region of chromosome 22. An additional 19 cosmids, that detected single copy sequences in the long arm of chromosome 22q: 7 in the region 22q11.2-q13.1 and 12 in 22q13.1-qter, were mapped more precisely by fluorescence in situ hybridization. Three out of these 19 recombinants displayed restriction fragments containing (CA)n repeats, were subcloned and sequenced. One cosmid, representing a region coding for an ubiquitous 300-bp transcript, is localized 600 kb from PDGFB, and four cosmids contained sequences surrounding the ARSA gene at 22q13.3. Presently, long range physical maps, that may be useful for analysing structural alterations of chromosome 22q13, are being constructed from these additional, regionally assigned markers from chromosome 22q13 employing both existing cosmid and new bacterial artificial chromosome (BAC) libraries.

Distribution and linkage of repetitive clusters from the heterochromatic region of human chromosome 22.

The pericentric regions of eukaryotic chromosomes consist of several types of repetitive DNA families. In human chromosome 22, the organization of such families was studied in more detail. In addition to the known families of alpha and beta repeats, an additional repeat with a 48-bp motif was previously assigned to 22pter-q11. Here, we report in more detail the distribution of these repeat families, applying pulsed-field gel electrophoresis, fluorescence in situ hybridization and physical linkage on cosmid recombinants. At least two clusters of 48-bp repeats are localized on chromosome 22, one on the distal p-arm and one in the region 22cen-q11. Cosmids from a chromosome 22 library, containing both 48-bp and beta-repeats, link both arrays on 22p and define their maximum distances to less than 44 kb. Loss of 48-bp repeat sequences in a Dl-George cell line carrying a deletion in 22q11 suggests the presence of a second cluster in 22q11, a distribution supported by (fluorescene in situ hybridization)-FISH signal analysis. As additional members of the 48-bp repeat family can be found on all acrocentric chromosomes. It remains to be determined whether the distribution seen on chromosome 22 is also common in other human acrocentric chromosomes.

PCR expression analysis of the estrogen-inducible gene BCEI in gastrointestinal and other human tumors.

A polymerase chain reaction (PCR) assay was developed to test for tumor cell specific expression of the BCEI gene. This new marker gene, reported at first for human breast cancer, was found specifically active in various gastrointestinal carcinomas by previously applying immunohistochemistry and RNA (Northern blot) analysis. Presently, by using reverse transcription-PCR analysis, a series of primary tumor tissues and established tumor cell lines were tested for BCEI transcription. This approach was compared to immunostaining achieved by an antibody directed against the BCEI gene's product. The result demonstrate the superior sensitivity of PCR by indicating the gene's expression in cases where immunohistochemical testing remained negative.

Sequence patterns and hybridization analysis of clones generated by Alu-PCR.

A series of clones from an Alu-PCR library were analysed in more detail. Characterization by Southern blot hybridization and sequencing displayed several features common to all probes generated by this approach: Short length of the PCR-products as well as the presence of homologous regions on both ends resulted in a limited feasibility for filter hybridization and a low probability of restriction length polymorphisms. In addition, a series of different short repeats at the 3'-ends of Alu-repeats and present in the generated probes offers a rich source of potential variable sites accessible by PCR.

Debrisoquine hydroxylase gene polymorphism in meningioma.

Cytochrome P450 CYP2D6 polymorphism is an autosomal recessive trait associated with impaired debrisoquine metabolism in 5-10% of caucasian populations. This polymorphism has been associated with susceptibility to Parkinson's disease, bladder cancer, various forms of leukemia and possibly melanoma. In many other cancer forms, the data remained contradictory due to the technical limitations for identifying affected individuals (poor metabolizers). A recently developed polymerase chain reaction-based assay allows convenient screening of approximately 80% of known mutations. We have tested brain tumors correlated with chromosome 22 deviations for genetic polymorphism in the cytochrome P450 CYP2D6 locus localized on chromosome 22q13. Thirty-one meningioma samples were analyzed and the observed frequency of heterozygotes and homozygotes for the G to A mutation did not deviate significantly from the distribution in a normal population. These data are comparable to previous observations in for example breast and colon cancer and indicate that the CYP2D6 locus on chromosome 22q13 is not involved in the pathogenesis of meningiomas.

Cosmid mapping and locus linkage within the human chromosomal region 22q13.1.

Many human meningiomas show loss of heterozygosity at distal loci but retain constitutional heterozygosity at one or more proximal loci of 22q. Molecular analysis indicted deletions involving at least the region 22q12.3-qter. In this region, distal to myoglobin, the putative meningioma locus ought to be expected. Long-range mapping was performed around two loci from 22q12.3-q13.1 (D22S16 and PDGFB, the most proximal locus to be lost in meningioma). D22S16, originally assigned to 22q13-qter by isotopic in situ hybridization, was placed in the vicinity of PDGFB by utilizing a set of somatic cell hybrids, an assignment confirmed by fluorescence in situ hybridization (FISH) of a cosmid clone containing the D22S16 locus. Moreover, pulsed field gel electrophoresis suggests a close linkage of both markers within 630 kb.

Application of DNA filter hybridization and PCR to distinguish between human and non-human tissues of poor quality.

The present report describes a novel approach for the identification of human or non-human specimens after long-term storage in a badly preserved state. The application of the PCR-technique (polymerase chain reaction) using human-specific primers as well as Southern blot (filter) hybridization of the sample DNA to a primate-specific DNA probe enabled us to extend the positive identification beyond the limits of conventional methods such as serological or morphological examinations.

Tumor-specific demethylation of heterochromatic repetitive DNA in gastrointestinal cancer.

DNA methylation appears to play an important role in both physiological and experimentally modified gene expression. Alterations in DNA methylation have been described in animal tumour models, in transformed cells and tumour cell lines, usually for single copy DNA sequences. By applying the isoschizomeres HpaII and MspI the methylation status of two classes of repetitive sequences (D22Z2 - a member of the alphoid repeats and D22Z3 which belongs to the HinfI repeats) in colon and stomach carcinoma were tested. A strong demethylation of both types of repetitive sequences in stomach carcinoma in comparison to healthy stomach mucosa was detected. In colon carcinoma, only minor signs of demethylation occured for the same repetitive DNA.

A non-alphoid repetitive DNA sequence from human chromosome 21.

A non-alphoid repetitive DNA from human chromosome 22, consisting of a 48-bp motif, shows homology to both G-group chromosomes in the gorilla, thus indicating the presence of additional repeat family members on further human chromosomes. Therefore, we screened a chromosome-21-specific cosmid library using this repetitive sequence from chromosome 22 (D22Z3). Some 40-50 cosmid clones were positive in tests for hybridization. One of the clones giving the strongest signals was digested with EcoRI/PstI, which we knew to cut frequently within the repeats; this resulted in fragments containing repeat units only. The fragments were subcloned into plasmid vector pTZ 19. Sequence-analysis of a 500-bp insert showed ten copies of a 48-bp repeat similar to D22Z3, with about 15% sequence deviation from the chromosome 22 consensus sequence. In situ hybridization of the newly isolated recombinant established its chromosome 21 specificity at high stringency. Physical mapping by pulsed field gel electrophoresis placed this new repeat in close vicinity to the chromosome 21 alphoid repeat. No cross-hybridization with other mammalian genomes except for those of apes was observed. The locus has been designated D21Z2 by the Genome Data Base. A gel mobility shift assay indicated that this repetitive motif has protein-binding properties.