Barth syndrome: an X-linked cause of fetal cardiomyopathy and stillbirth.

OBJECTIVE: Barth Syndrome (BTHS) is an X-linked multisystem disorder (OMIM 302060) usually diagnosed in infancy and characterized by cardiac problems [dilated cardiomyopathy (DCM) ± endocardial fibroelastosis (EFE) ± left ventricular non-compaction (LVNC)], proximal myopathy, feeding problems, growth retardation, neutropenia, organic aciduria and variable respiratory chain abnormalities. We wished to determine whether BTHS had a significant impact on fetal and perinatal health in a large cohort of family groups originating from a defined region. METHOD: Case note review on 19 families originating from the UK and known to the Barth Syndrome Service of the Bristol Royal Hospital for Children. RESULTS: Details are presented on six kindreds (32%) with genetically and biochemically proven BTHS that demonstrate a wider phenotype including male fetal loss, stillbirth and severe neonatal illness or death. In these families, 9 males were stillborn and 14 died as neonates or infants but there were no losses of females. BTHS was definitively proven in five males with fetal onset of DCM ± hydrops/EFE/LVNC. CONCLUSION: These findings stress the importance of considering BTHS in the differential diagnosis of unexplained male hydrops, DCM, EFE, LVNC or pregnancy loss, as well as in neonates with hypoglycemia, lactic acidosis and idiopathic mitochondrial disease.

Vasoactive intestinal peptide receptor expression in chronic periapical lesions.

AIM: To use radioreceptor analysis for evaluating whether vasoactive intestinal peptide (VIP) receptors are present in chronic periapical lesions and to determine whether differences in its expression are found according to the size of the lesions. METHODOLOGY: Twelve periapical lesions were obtained from teeth diagnosed with chronic apical periodontitis and indicated for endodontic surgery; they were classified according to the size of the lesion in two groups of six samples (lesion size greater or smaller than 5 mm), and then processed and labelled with (125)I-VIP. Binding sites were identified by (125)I-VIP and standard VIP competition assays. Mann-Whitney's test was used to establish statistically significant differences in the VIP receptor expression between groups. RESULTS: Vasoactive intestinal peptide receptor expression was found in all periapical lesion samples. There was a statistically significantly higher expression in periapical lesions <5 mm (P < 0.001). CONCLUSION: Vasoactive intestinal peptide receptors were expressed in chronic periapical lesions with levels inversely proportional to lesion size.

Effect of triplet repeat expansion on chromatin structure and expression of DMPK and neighboring genes, SIX5 and DMWD, in myotonic dystrophy.

Myotonic dystrophy (DM), an autosomal dominant neuromuscular disease, is associated with expansion of a polymorphic (CTG)n repeat in the 3'-untranslated region of the DM protein kinase (DMPK) gene. The repeat expansion results in decreased levels of DMPK mRNA and protein, but the mechanism for this decreased expression is unknown. Loss of a nuclease-hypersensitive site in the region of the repeat expansion has been observed in muscle and skin fibroblasts from DM patients, indicating a change in local chromatin structure. This change in chromatin structure has been proposed as a mechanism whereby the expression of DMPK and neighboring genes, sine oculis homeobox (Drosophila) homolog 5 (SIX5) and dystrophia myotonica-containing WD repeat motif (DMWD), might be affected. We have developed a polymerase chain reaction (PCR)-based method to assay the chromatin sensitivity of the region adjacent to the repeat expansion in somatic cell hybrids carrying either normal or affected DMPK alleles and show that hybrids carrying expanded alleles exhibit decreased sensitivity to PvuII digestion in this region. Semiquantitative multiplex reverse transcriptase PCR (RT/PCR) assays of gene expression from the chromosomes carrying the expanded alleles showed marked reduction of DMPK mRNA, partial inhibition of SIX5 expression from a congenital DM chromosome, and no reduction of DMWD mRNA. Nested RT/PCR analysis of DMPK mRNA from somatic cell hybrids carrying the repeat expansions revealed that most of the DMPK transcripts expressed from the expanded alleles lacked exons 13 and 14, whereas full-length transcripts were expressed predominantly from the normal alleles. These results suggest that the CTG repeat expansion leads to a decrease in DMPK mRNA levels by affecting splicing at the 3' end of the DMPK pre-mRNA transcript.

Human rDNA: evolutionary patterns within the genes and tandem arrays derived from multiple chromosomes.

Human rDNA forms arrays on five chromosome pairs and is homogenized by concerted evolution through recombination and gene conversion (loci RNR1, RNR2, RNR3, RNR4, RNR5, OMIM: 180450). Homogenization is not perfect, however, so that it becomes possible to study its efficiency within genes, within arrays, and between arrays by measuring and comparing DNA sequence variation. Previous studies with randomly cloned genomic DNA fragments showed that different parts of the gene evolve at different rates but did not allow comparison of rDNA sequences derived from specific chromosomes. We have now cloned and sequenced rDNA fragments from specific acrocentric chromosomes to (1) study homogenization along the rDNA and (2) compare homogenization within chromosomes and between homologous and nonhomologous chromosomes. Our results show high homogeneity among regulatory and coding regions of rDNA on all chromosomes, a surprising homogeneity among adjacent distal non-rDNA sequences, and the existence of one to three very divergent intergenic spacer classes within each array.

Control of ticks resistant to immunization with Bm86 in cattle vaccinated with the recombinant antigen Bm95 isolated from the cattle tick, Boophilus microplus.

The recombinant Bm86-containing vaccine Gavac(TM) against the cattle tick Boophilus microplus has proved its efficacy in a number of experiments, especially when combined with acaricides in an integrated manner. However, tick isolates such as the Argentinean strain A, show low susceptibility to this vaccine. In this paper we report on the isolation of the Bm95 gene from the B. microplus strain A, which was cloned and expressed in the yeast Pichia pastoris producing a glycosylated and particulated recombinant protein. This new antigen was effective against different tick strains in a pen trial, including the B. microplus strain A, resistant to vaccination with Bm86. A Bm95-based vaccine was used to protect cattle against tick infestations under production conditions, lowering the number of ticks on vaccinated animals and, therefore, reducing the frequency of acaricide treatments. The Bm95 antigen from strain A was able to protect against infestations with Bm86-sensitive and Bm86-resistant tick strains, thus suggesting that Bm95 could be a more universal antigen to protect cattle against infestations by B. microplus strains from different geographical areas.

Sequence variations in the Boophilus microplus Bm86 locus and implications for immunoprotection in cattle vaccinated with this antigen.

Cattle tick infestations constitute a major problem for the cattle industry in tropical and subtropical regions of the world. Traditional control methods have been only partially successful, hampered by the selection of chemical-resistant tick populations. The Boophilus microplus Bm86 protein was isolated from tick gut epithelial cells and shown to induce a protective response against tick infestations in vaccinated cattle. Vaccine preparations including the recombinant Bm86 are used to control cattle tick infestations in the field as an alternative measure to reduce the losses produced by this ectoparasite. The principle for the immunological control of tick infestations relies on a polyclonal antibody response against the target antigen and, therefore, should be difficult to select for tick-resistant populations. However, sequence variations in the Bm86 locus, among other factors, could affect the effectiveness of Bm86-containing vaccines. In the present study we have addressed this issue, employing data obtained with B. microplus strains from Australia, Mexico, Cuba, Argentina and Venezuela. The results showed a tendency in the inverse correlation between the efficacy of the vaccination with Bm86 and the sequence variations in the Bm86 locus (R2 = 0.7). The mutation fixation index in the Bm86 locus was calculated and shown to be between 0.02 and 0.1 amino acids per year. Possible implications of these findings for the immunoprotection of cattle against tick infestations employing the Bm86 antigen are discussed.

Beyond ribosomal DNA: on towards the telomere.

We have sequenced and analyzed 8.3 kb of sequence adjacent and distal to the human ribosomal DNA (rDNA); this distal sequence connects to the rDNA cluster just 4 kb upstream of the first promoter and is shared among the acrocentric chromosomes and, at least in part, it is also present in other primates. The sequence differs in character from that of the rDNA intergenic spacer (IGS) in that it does not contain long stretches of either polypyrimidine or polypurine. However, just like the IGS, it contains numerous repetitive elements, including retroposed fragments of 28S rRNA and large pieces of the IGS. In addition, we show that the rDNA clusters are not interrupted by other sequences and do not recombine with this distal segment.

Incognito rRNA and rDNA in databases and libraries.

Both ribosomal DNA (rDNA) and ribosomal RNA (rRNA) are over-represented in the starting material for genomic and cDNA libraries; thus, their sequences have the potential of repeatedly entering the various databases. When DNA (both transcribed and intergenic spacer regions) is used as query sequence, a great number of matches are found in the databases, particularly in the EST database, and to a lesser extent among genomic sequences and STSs, which are not identified as rDNA. We discuss the following explanations for the widespread occurrence of rDNA in cDNA and genomic DNA libraries: pseudogenes of rRNA in other genomic locations, mRNA-derived pseudogenes that reside in rDNA, cDNAs derived from rRNA [either by self-priming or by internal oligo(dT) priming], cDNAs derived from actual transcripts of the rDNA intergenic spacer, and genomic DNA contamination of RNA preparations. Because so many database entries contain unidentified rDNA, we recommend that all sequence submissions be checked (by the submitters) for the presence of structural RNAs in addition to repetitive sequences.

Human ribosomal RNA variants from a single individual and their expression in different tissues.

We have investigated the extent of sequence variation in human ribosomal RNA (rRNA) genes and the expression of specific rRNA gene variants in different tissues of an individual. Focusing on the fifth variable region (V5; nt 2065-2244) of the 28S rRNA gene, we find that sequence differences between rRNA genes of a single individual are characterized by differences in number of repeats of simple sequences at four specific sites. These data support and extend previous findings which show similar V5 sequence variation in rRNA genes from a group of individuals. We performed experiments to determine if there is differential gene expression within the rRNA multigene family. From the analysis of data of six variant V5 probes protected from RNase digestion by rRNAs isolated from different tissues of the individual, we conclude that each variant rRNA is present in a similar proportion in these tissues, whereas the actual contributions of variants differ, their relative proportion is maintained from tissue to tissue in an individual. We favor the explanation of a gene dosage effect over that of a regulated gene effect to account for this pattern of rRNA gene expression. In addition, computer generated secondary structure models of each V5 clone structure predict the same three helix structure with the regions of sequence variation contained in one stem-loop structure.

Studies of the inheritance of human ribosomal DNA variants detected in two-dimensional separations of genomic restriction fragments.

We have investigated the variation in human ribosomal DNA repeat units as revealed in two-dimensional electrophoretic separates of genomic restriction fragments that were end-labeled at NotI cleavage sites. The transcribed portion of the ribosomal DNA results in approximately 20 labeled fragments visible on each gel as multicopy spots. We have mapped these spots to the sequences responsible for their appearance on the gels, based on their migration positions and direct sequencing of spots, and describe several previously unreported sources of variation. By studying mother/father/child families we gained information on how much of the between-repeats variation is due to differences between and within repeat arrays on homologous chromosomes. Two instances in which a child exhibited more copies of a particular fragment than were present in the parents are described and hypothesized to be due to events such as multiple unequal sister-chromatid exchanges or gene conversions.

Complete sequence of the 43-kb human ribosomal DNA repeat: analysis of the intergenic spacer.

We have sequenced the remaining 12.4 kb of the 30-kb human ribosomal DNA intergenic spacer (IGS), which allows us to piece together both a complete IGS and a 43-kb rDNA unit. The sequence of the complete IGS reveals a collection of sequence motifs that can be correlated with functions known or expected to reside in the rDNA repeat: modulation of transcription, recombination, initiation of DNA replication, and chromosomal organization. Finally, we find that IGS accumulates variation at a much higher rate than the transcribed regions. This finding leads us to correlate sequence character with types of mutation and sequence context with rate of mutation.

Fixation times of retroposons in the ribosomal DNA spacer of human and other primates.

We have investigated the presence/absence of two types of retroposed sequences found in human ribosomal DNA in equivalent positions in chimpanzee, gorilla, orangutan, gibbon, and rhesus monkey rDNA. These sequences are one pseudogene derived from the single-copy cdc27hs gene and seven complete Alu elements. The 2-kb pseudogene is present in the apes but not in Old World monkeys, indicating fixation in an ape ancestor. Five of the Alu elements are shared by the whole set of primates studied, indicating insertion and fixation prior to the split of the ape and Old World monkey lineages. One is absent only from the rhesus monkey rDNA, and another is absent from both gibbon and rhesus rDNA, indicating fixation at different times in primate evolutionary history. Since branching times for the primate phylogenetic tree are known from a combination of the fossil record and multiple molecular data sets, it is possible to compare Alu fixation times determined from the phylogenetic information with those calculated from Alu element mutation rates.

The topographic organization of repetitive DNA in the human nucleolus.

The nucleolus is a highly specialized nuclear domain where ribosomal DNA (rDNA) is transcribed and preribosomes are assembled. We investigated the molecular organization of the human lymphocyte nucleolus by fluorescence in situ hybridization and confocal laser scanning microscopy and found that transcribed rDNA and nontranscribed ribosomal intergenic spacer (IGS) sequences colocalized to discrete regions frequently on the nucleolar periphery of phytohemagglutinin-stimulated cells. The 5S rDNA gene cluster located on the long arm of chromosome 1 was not regularly associated with the nucleolus. Short interspersed (SINE) Alu elements detected by BLUR 11 were distributed diffusely throughout the nucleus but were severely underrepresented in the nucleolus, whereas an Alu element subcloned from the IGS detected sequences enriched in the nucleolus but sparsely represented in the remainder of the nucleus. In contrast, long interspersed (LINE) Kpn elements, which were located at the nucleolus, were not found in rDNA but were identified outside the ribosomal gene complex on the short arm of at least one acrocentric chromosome. A human chromosome 21-derived alphoid sequence that hybridized to the centromere was localized outside but near the nucleolus, and nonribosomal DNA consisting of a tandemly repeated simple sequence cluster derived from the short arm of chromosome 15 was organized in a compact fashion in the nucleolus. Our study provides new insight into the content and structure of the human nucleolus and illustrates that the unique organization of repetitive DNA on the acrocentric chromosome short arms is reflected in the topographic organization of the nucleolus.

Ribosomal RNA gene sequences and hominoid phylogeny.

Sequences totaling 3,500 bases from the 28S rRNA gene and from one of the ribosomal internal transcribed spacers (ITS1) have been determined for human, chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), and orangutan (Pongo pygmaeus). Analyses of the rRNA alignments show (1) a clustering of substitutions in the "variable regions" of the 28S gene, (2) a 1.5-3-fold increase in divergence in the transcribed spacer over that in the exon, and (3) that human and chimpanzee are the most closely related pair, in agreement with the results of Miyamoto et al., Sibley and Ahlquist, and Caccone and Powell.

Sequence and structure correlation of human ribosomal transcribed spacers.

We report the sequences of the transcribed spacers of human rRNA that now allow us to piece together the entire primary transcript sequence of approximately 13.3 x 10(3) base-pairs. Comparison of transcribed spacer sequences with those of variable regions of rRNA and with those of the non-transcribed spacers supports the hypothesis that the variable regions are descended from transcribed spacers. Nucleotide sequence-derived secondary structures for the 5' external transcribed spacer and for internal transcribed spacers 1 and 2 match both the sizes and shapes of the structures that were visualized 15 years ago on electron micrographs. Parts of these structures are conserved in mammals and may be related to transcript processing.

Independent insertion of Alu elements in the human ribosomal spacer and their concerted evolution.

A 2,700-bp segment of human ribosomal DNA (rDNA) spacer upstream of the rRNA promoter contains a set of four Alu elements, two in the direction of rRNA transcription and two in the opposite orientation. We report and compare the sequences of these Alu elements found in three rDNA clones and seek to determine the origin of the cluster, either from a single insertion followed by duplications or from multiple simultaneous or independent insertions. The high (20%-27%) divergence among members of a set and the lack of similarity/complementarity of sequences flanking different members of the set demonstrate the independent insertion of each of the four Alu elements into A-rich sequences on the appropriate strand of the rDNA. We also demonstrate that the Alu sets found in different rDNA repeats are subject to concerted evolution, yielding divergences of only 0.4%-3% between Alu elements in equivalent positions. However, the pairs of adjacent similarly oriented Alu elements do not show reduced divergence, indicating that there is no recombination or gene conversion between similarly oriented but not equivalently positioned Alu elements. Finally, crossing-over must occur in the rDNA junction region between Alu element 3 and the nonribosomal sequences at the telomere end of the acrocentric chromosome, so that the Alu elements of the terminal rDNA repeats and the terminal repeats themselves evolve in concert with the rDNA repeats located internally in the tandem array.

Definition of a second dimeric subfamily of human alpha satellite DNA.

We describe a new human subfamily of alpha satellite DNA. The restriction endonuclease XbaI cleaves this subfamily into a collection of fragments which are heterogeneous with respect to size. We compared the sequences of 6 clones from four different XbaI size classes. Clones from a single size class were not necessarily more related than clones from different classes. Clones from different size classes were found to produce almost identical hybridization patterns with XbaI-digested human genomic DNA. All clones were found to share a common dimeric repeat organization, with dimers exhibiting about 84% sequence identities, indicating that the clones evolved from a common progenitor alphoid dimer. We show that this subfamily, and the EcoRI dimer subfamily originally described by Wu and Manuelidis, evolved from different progenitor alphoid dimers, and therefore represent distinct human alphoid subfamilies.

Human 28S ribosomal RNA sequence heterogeneity.

DNA sequencing of several cloned human 28S ribosomal RNA gene fragments has revealed sequence heterogeneity (1) but it was not clear whether these are inactive pseudogenes or are active genes that are transcribed and represented in ribosomes. S1 nuclease analysis allowed us to examine the population of ribosomal RNA molecules of a cell, and we found that 28S rRNA is a heterogeneous assortment of molecules in both mono- and polysomal preparations. Sequence variation, although largely concentrated in variable regions of the molecule, apparently also occurs in the conserved regions.

Molecular analysis of a constitutional X-autosome translocation in a female with muscular dystrophy.

The gene responsible for Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) maps to the X chromosome short arm, band Xp21. In a few females with DMD or BMD, the Xp21 region is disrupted by an X-autosome translocation. Accumulating evidence suggests that the exchange has physically disrupted the DMD/BMD locus to cause the disease. One affected female with a t(X;21)(p21;p12) translocation was studied in detail. The exchange points from both translocation chromosomes were cloned, restriction-mapped, and sequenced. The translocation is reciprocal, but not conservative. A small amount of DNA is missing from the translocated chromosomes; 71 to 72 base pairs from the X chromosome and 16 to 23 base pairs from the 28S ribosomal gene on chromosome 21.