20p12.3 microdeletion predisposes to Wolff-Parkinson-White syndrome with variable neurocognitive deficits.

BACKGROUND: Wolff-Parkinson-White syndrome (WPW) is a bypass re-entrant tachycardia that results from an abnormal connection between the atria and ventricles. Mutations in PRKAG2 have been described in patients with familial WPW syndrome and hypertrophic cardiomyopathy. Based on the role of bone morphogenetic protein (BMP) signalling in the development of annulus fibrosus in mice, it has been proposed that BMP signalling through the type 1a receptor and other downstream components may play a role in pre-excitation. METHODS AND RESULTS: Using the array comparative genomic hybridisation (CGH), we identified five individuals with non-recurrent deletions of 20p12.3. Four of these individuals had WPW syndrome with variable dysmorphisms and neurocognitive delay. With the exception of one maternally inherited deletion, all occurred de novo, and the smallest of these harboured a single gene, BMP2. In two individuals with additional features of Alagille syndrome, deletion of both JAG1 and BMP2 were identified. Deletion of this region has not been described as a copy number variant in the Database of Genomic Variants and has not been identified in 13 321 individuals from other cohort examined by array CGH in our laboratory. CONCLUSIONS: Our findings demonstrate a novel genomic disorder characterised by deletion of BMP2 with variable cognitive deficits and dysmorphic features and show that individuals bearing microdeletions in 20p12.3 often present with WPW syndrome.

Immunofluorescent studies of human chromosomes with antibodies against phosphorylated H1 histone.

One of the prominent cell cycle-related modifications of histone proteins whose function remains unresolved is the phosphorylation of linker histone H1. In this work we have used indirect immunofluorescence on human cells with antibodies that are specific for phosphorylated histone H1 to examine the cellular distribution and chromosome association patterns of this protein. With confocal microscopy on whole cells, strong immunofluorescence was seen in association with mitotic chromosomes as well as a prominent punctate pattern of labeling throughout the mitotic cell, whereas interphase cells showed very little, if any, specific fluorescence. Multiple patterns of fluorescence distribution were detected with metaphase chromosomes, ranging from apparent tight colocalization with the DNA to expanded "puffy" mitotic figures to an amorphous network of staining. It was also shown that the ability to label chromosomes could vary drastically with different fixation procedures, adding further complications to interpretation of the potentially complex role of phosphorylated histone H1 in chromatin condensation or decondensation.

Analysis of DNA replication by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) has been shown to discriminate between unreplicated and replicated regions of the genome in interphase nuclei, based on the number of specific fluorescent signals that can be detected. By examining the replication status of hybridizing sequences in large numbers of individual cells from an asynchronously growing population, it is possible to deduce a relative order of replication of different sequences. The availability of well-mapped genomic probes and the ability to compare results from different cell lines make this a convenient approach with which to map domains of replication timing control at any chromosomal position and to relate this to various patterns of gene expression. Since there appear to be important but poorly understood correlations among replication timing, chromatin structure, and transcriptional competence in mammalian cells, this provides a valuable approach to understanding these interrelationships at the molecular level. The procedures for using FISH to examine replication timing in mammalian nuclei are described here in detail, and the advantages and limitations of the approach are discussed. Some other strategies for using high-resolution FISH on chromatin fibers to examine replication properties of specific sequences in situ are also described.

Reduced levels of histone H3 acetylation on the inactive X chromosome in human females.

Novel antibodies were generated that are highly selective for either acetylated or unacetylated isoforms of histone H3, or the acetylated form of histone H4 in organisms as diverse as Tetrahymena and humans. Using these antibodies as pair-wise sets in immunocytological analyses, we demonstrate that the inactive X chromosome is hypoacetylated for both histone H3 and H4 in female mammalian cells, whereas the antibody that recognizes the unacetylated form of histone H3 identifies all chromosomes uniformly. These data verify and extend previous results and suggest that hypoacetylation of core histones may be a general feature of the chromatin along the inactive X chromosome.

Significant divergence in nucleotide sequences for beta-tubulin from different laboratory strains of Chinese hamster ovary cells.

The nucleotide sequences for isotype 1 beta-tubulin cDNAs cloned from different laboratory strains of Chinese hamster ovary (CHO) cells were compared and found to contain an unexpected number of sequence differences in both translated and untranslated regions of the gene. The results indicate significant changes in the DNA, but not protein, sequence while the cells have been in culture and reveal sequences in the 5' and 3' untranslated regions that have resisted these changes.

Analysis of replication timing properties of human X-chromosomal loci by fluorescence in situ hybridization.

We have used fluorescence in situ hybridization on interphase nuclei of normal female cells to compare the replication timing patterns of genes on the human X chromosome that are known to escape X inactivation with those that are inactivated. By this procedure it was possible not only to determine the relative time of replication of the earlier-replicating allele for different loci but also to estimate the degree of asynchrony of replication of the two alleles for each individual locus. Loci such as HPRT and FRAXA, which are normally inactivated, displayed a high degree of replication asynchrony, whereas loci that are not inactivated (ZFX and RPS4X) were found to replicate very synchronously. Interestingly, examination of XIST, which is expressed only from the inactive X chromosome, by this procedure revealed that it also replicated asynchronously, with the expressed copy apparently replicating first. Therefore, by examining different loci from the X chromosome it was determined that there is a strict correlation between the expression and relative time of replication of individual genes.

Identification of methionine-containing tryptic peptides of unstable beta-tubulin separated by reverse-phase high-performance liquid chromatography.

Methods for examining altered regions in unstable mutant proteins are described. The strategy is illustrated using assembly defective Chinese hamster beta-tubulin subunits that are rapidly degraded in the cell. These unstable proteins are metabolically labeled to high specific activity and isolated as spots on two-dimensional gels. Conditions for the generation of tryptic peptides from gel pieces containing beta-tubulin and their subsequent resolution by HPLC have been worked out. Through a combination of dual labeling with various tritiated amino acids and [35S]methionine as well as partial sequence analysis, the identification of several HPLC peaks with the known sequence of beta-tubulin has been accomplished. This technique should greatly aid attempts to map the sites of mutational alterations in beta-tubulin polypeptides, and the general strategy should be readily applicable to other mutant proteins.

Mutations affecting assembly of beta-tubulin localize to a region near the carboxyl terminus.

The generation of Chinese hamster ovary cell lines that express assembly defective forms of beta-tubulin were isolated using selections based on reversion of conditional lethal or drug resistance phenotypes. Two such cell lines, D2 and 6H3, were chosen for further characterization because they contain beta-tubulin polypeptides that exhibit decreases in apparent molecular weight on two-dimensional gel electrophoresis. Analysis of the nucleic acid from these cell lines using both Southern and Northern procedures suggests a deletion in one of the beta-tubulin genes in each cell line. Localization of the missing sequence in D2 was first determined by tryptic peptide mapping by high performance liquid chromatography. Subsequently, the assignment was confirmed by constructing appropriate subclones of a wild type Chinese hamster ovary beta-tubulin cDNA for Southern analysis to demonstrate a failure to recognize characteristic hybridization patterns of the mutant tubulin gene. In the other revertant, 6H3, the deletion was detected on a Northern blot by differential hybridization of a 3' fragment of the cDNA to the beta-tubulin messages. The results indicate that D2 has an internal deletion whose approximate limits extend from amino acid residues 250 through 345. Cell line 6H3 has a deletion that begins near amino acid residue 330 and extends into the 3'-untranslated region of the gene.

Determination of clonality in acute nonlymphocytic leukemia by restriction fragment length polymorphism and methylation analysis.

Determination of cellular clonality in hematological malignancies provides fundamental information that is important in understanding the pathogenesis of these disorders. We present here an extension of one approach to accomplish this that is based on the interpretation of different methylation patterns on active and inactive X chromosomes within the region of the hypoxanthine-guanine phosphoribosyltransferase gene spanned by a restriction fragment length polymorphism. The successful application of the method to determine clonality is described for three female patients with acute nonlymphocytic leukemia.

New mutation and prenatal diagnosis in ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (OTC) (E.C.2.1.3.3) is an X-linked hepatic enzyme in the urea cycle necessary for ammonia detoxification. Deficiency of OTC results in neonatal hyperammonemia, coma, and death in childhood. Because fibroblasts do not express OTC, prenatal diagnosis in the past has required fetal liver biopsy. Using a complementary DNA (cDNA) for OTC for Southern blot analysis of genomic DNA, we have found probands with complete OTC deficiency from two unrelated families in whom the same TaqI restriction endonuclease site has been altered because of independent, but not necessarily identical, mutations in the OTC gene, suggesting that this site may be a relative hotspot for mutation at a location that is critical for normal gene function. This TaqI alteration has allowed the identification of the individual in each family in whom the mutation originated as well as the exclusion of a recurrence of OTC deficiency in a male fetus at risk for the disease. OTC deficiency joins the growing list of genetic disorders for which Southern blot analysis allows accurate heterozygote detection and prenatal diagnosis in conditions for which they were not previously available.

Two anonymous X-specific human sequences detecting restriction fragment length polymorphisms in region Xq26----qter.

Two anonymous X-specific sequences isolated from a genomic library of flowsorted X chromosomal DNA were selected for study because they revealed restriction fragment length polymorphisms in the region Xq26----qter. One sequence, DXS10, detected a two-allele TaqI polymorphic system with allele frequencies of 0.33 and 0.67. The other, 4D-8, defined an MspI polymorphism with allele frequencies of 0.18 and 0.82. DXS10 is tightly linked to the hypoxanthine phosphoribosyltransferase (HPRT) locus with recombination distance theta = O cM at LOD = 5.55 (95% probability limit theta less than 15 cM). DXS10 maps to Xq26 but is not contained within the HPRT locus itself. 4D-8 shows no detectable linkage to the HPRT locus, with maximum likelihood estimate for theta = 50 cM and a LOD score of -2.61 at theta = 5 cM. These two polymorphisms provide additional chromosomal loci for gene mapping by linkage at the distal end of the long arm of the human X chromosome.