Osteogenesis imperfecta due to recurrent point mutations at CpG dinucleotides in the COL1A1 gene of type I collagen

Summary Most individuals with osteogenesis imperfecta (OI) are heterozygous for dominant mutations in one of the genes that encode the chains of type I collagen. Each of the more than 30 mutations characterized to date has been unique to the affected member (s) of the family. We have determined that two individuals with a progressive deforming variety of OI, OI type III, have the same new dominant mutation [1(I)gly154 to arg] and that two unrelated infants with perinatal lethal OI, OI type II, share a second new dominant muation [1(I)gly1003 to ser]. These mutations occurred at CpG dinucleotides, in a manner consistent with deamination of a methylated cytosine residue, and raise the possibility that CpG dinucleotides are common sites of recurrent mutations in collagen genes. Further, these findings confirm that the OI type-III phenotype, previously thought to be inherited in an autosomal recessive manner, can result from new dominant mutations in the COL1A1 gene of type-I collagen.     

Methylation of CpG dinucleotides in the lacI gene of the Big Blue® transgenic mouse

Cytosine residues at CpG dinucleotides can be methylated by endogenous methyltransferases in mammalian cells. The resulting 5-methylcytosine base may undergo spontaneous deamination to form thymine causing G/C to A/T transition mutations. Methylated CpGs also can form preferential targets for environmental mutagens and carcinogens. The Big Blue® transgenic mouse has been used to investigate tissue and organ specificity of mutations and to deduce mutational mechanisms in a mammal in vivo. The transgenic mouse contains approximately 40 concatenated lambda-like shuttle vectors, each of which contains one copy of an Escherichia coli lacI gene as a mutational target. lacI mutations in lambda transgenic mice are characterized by a high frequency of spontaneous mutations targeted to CpG dinucleotides suggesting an important contribution from methylation-mediated events. To study the methylation status of CpGs in the lacI gene, we have mapped the distribution of 5-methylcytosines along the DNA-binding domain and flanking sequences of the lacI gene of transgenic mice. We analyzed genomic DNA from various tissues including thymus, liver, testis, and DNA derived from two thymic lymphomas. The mouse genomic DNAs and methylated and unmethylated control DNAs were chemically cleaved, then the positions of 5-methylcytosines were mapped by ligation-mediated PCR which can be used to distinguish methylated from unmethylated cytosines. Our data show that most CpG dinucleotides in the DNA binding domain of the lacI gene are methylated to a high extent (>98%) in all tissues tested; only a few sites are partially (70–90%) methylated. We conclude that tissue-specific methylation is unlikely to contribute significantly to tissue-specific mutational patterns, and that the occurrence of common mutation sites at specific CpGs in the lacI gene is not related to selective methylation of only these sequences. The data confirm previous suggestions that the high frequency of CpG mutations in lacI transgenes is related to the presence of 5-methylcytosine bases.     

CpG dinucleotides in the hMSH2 and hMLHI genes are hotspots for HNPCC mutations

Hereditary nonpolyposis colon cancer (HN-PCC) is an autosomally inherited predisposition to cancer that has recently been linked to defects in the human mismatch repair genes hMSH2 and hMLHI. The identification of the causative mutations in HNPCC families is desirable, since it confirms the diagnosis and allows the carrier status of unaffected relatives at risk to be determined. We report six different new mutations identified in the hMSH2 and hMLH1 genes of Russian and Moldavian HNPCC families. Three of these mutations occur in CpG dinucleotides and lead to a premature stop codon, a splicing defect or an amino-acid substitution in an evolutionary conserved residue. Analysis of a compilation of published mutations including our new data suggests that CpG dinucleotides within the coding regions of the hMSH2 and hMLH1 genes are hotspots for single base-pair substitutions.     

Methylation detection oligonucleotide microarray analysis: a high-resolution method for detection of CpG island methylation

Methylation of CpG islands associated with genes can affect the expression of the proximal gene, and methylation of non-associated CpG islands correlates to genomic instability. This epigenetic modification has been shown to be important in many pathologies, from development and disease to cancer. We report the development of a novel high-resolution microarray that detects the methylation status of over 25 000 CpG islands in the human genome. Experiments were performed to demonstrate low system noise in the methodology and that the array probes have a high signal to noise ratio. Methylation measurements between different cell lines were validated demonstrating the accuracy of measurement. We then identified alterations in CpG islands, both those associated with gene promoters, as well as non-promoter-associated islands in a set of breast and ovarian tumors. We demonstrate that this methodology accurately identifies methylation profiles in cancer and in principle it can differentiate any CpG methylation alterations and can be adapted to analyze other species.     

Oligonucleotide directed mutagenesis of the human beta-globin gene: a general method for producing specific point mutations in cloned DNA.

A nonadecanucleotide has been used both as a site specific mutagen to introduce a T leads to A transversion mutation in the human beta-globin gene cloned in pBR322 as well as a probe to screen transformed colonies for the desired mutant. The specificity of the oligonucleotide as a mutagen and as a hybridization probe provide a general method for producing site specific mutations in DNA cloned in plasmid vectors such as pBR322.     

Stability of deletion,insertion and point mutations at the bronze locus in maize

Summary Phenotypic revertants from several kinds of mutations, including deletions, have been detected by pollen analysis at the wx and Adh loci in maize. Mutations in these genes give phenotypic revertants with median frequencies of 0.7 and 0.5×10^–5, respectively. However, the nature of such revertants can only be analyzed following their recovery from conventional matings. In the current study large seed populations derived from crosses involving several bz (bronze) locus mutations in maize were examined for reversion to a Bz (purple) expression. Deletion, insertion and point mutations were included in the study. Principally, over 2 million gametes of the bz-R mutation, which is shown here to be associated with a 340 base pair deletion within the transcribed region of the gene, have been screened for reversion. No revertants from it or any of the other bz mutations have been recovered, even though a total of almost 5 million gametes from homoallelic crosses have been examined to date. Results from seed analysis are discussed in reference to those from pollen analysis in maize.     

Tissue-specific methylation patterns and expression of the human apolipoprotein AI gene.

To better understand the tissue-specific expression of the human apolipoprotein (apo)AI gene, we performed a detailed analysis of the pattern of methylation of the gene in various human adult and embryonic tissues and in tissues of transgenic mice harboring the human apo-AI gene. In addition, the gene was analyzed also in liver and intestine-derived human cell lines (HepG2 and Caco2, respectively). Using methyl-sensitive restriction enzymes (HpaII, HhaI, and SmaI) and the appropriate radioactive probes, we were able to determine separately the status of methylation of the 5'-end, the body of the gene, and 3'-end flanking sequences. The apo-AI gene in tissues that express the gene was undermethylated at the 5'-end. However, the 5'-end of the gene in sperm and in all adult tissues that do not express the gene was heavily methylated. The body of the gene which contains a CpG island and the 3'-end flanking sequences were, in general, hypomethylated except for specific sites that showed partial methylation. In contrast, while the gene showed tissue-specific expression already in a 12-week-old embryo, the 5'-end was invariably hypomethylated in all tissues of the embryo. A human apo-AI transgene has recently been shown to be active exclusively in the liver, while the endogenous gene is expressed in both liver and intestine (6). We show here that the 5'-end of the apo-AI transgene was methylated in all tissues of the mouse (including intestine) except liver. The results presented here demonstrate a clear correlation between hypomethylation of the 5'-end and activity of the apo-AI gene. However, the observed methylation pattern of the gene in embryonic tissues suggests that tissue-specific expression precedes formation of the tissue-specific methylation pattern.     

The pattern of spontaneous germ-line mutation: relative rates of mutation at or near CpG dinucleotides in the factor IX gene

Mutations at CpG dinucleotides were delineated in the factor IX gene of 38 hemophilia B patients. When transitions at CpG were considered with those previously reported by us and those compiled in the factor IX mutation database, the following patterns emerged. Many CpG sites were mutated with high frequency, while two CpG sites were infrequently mutated (R²⁹Q and R¹¹⁶ TGA). Of the 6 possible nonsense mutations and the 14 missense mutations that would produce a nonconservative change at conserved amino acids, all have been observed to cause hemophilia B except A^–10T and R³³⁸Q. By contrast, none of the 6 missense changes at nonconserved amino acids have been observed to cause hemophilia B. At those CpG sites that are frequently mutated, the rate of transitions is estimated to be 20-fold higher than transitions at non-CpG sites. Point mutations in close proximity to CpG dinucleotides did not seem elevated.     

Cloning and characterization of the human beta-glucuronidase gene

We have isolated a cosmid clone that contains GUSB, the human gene encoding beta-glucuronidase. The 21-kb gene contains 12 exons ranging from 85 to 376 bp in length. Exon 6 corresponds to the 153-bp deletion in the shorter of two types of cDNAs reported earlier, supporting the hypothesis that this cDNA arose by alternate splicing leading to exon skipping. The insert contains 4.2 kb of sequence upstream from the first exon and 6 kb 3' of the last exon. The clone expresses human beta-glucuronidase in stably transformed rat XCtk- cells. Comparison of the human gene organization with that recently reported for the murine beta-glucuronidase gene revealed that the intron/exon boundaries are identical. In the splice junctions, the most highly conserved regions are those identified as consensus sequences, and these are at least as highly conserved as bases encoding the translated portion of the gene.     

Linked spontaneous CG----TA mutations at CpG sites in the gene for protein kinase regulatory subunit.

CG----TA transitions at CpG sequences account for many human point mutations and are thought to result from hydrolytic deamination of 5-methylcytosine residues in these sites. The gene for regulatory subunit of murine cyclic AMP-dependent protein kinase has two closely linked CpG sites, one of which is a strong hotspot for spontaneous CG----TA mutations leading to cyclic AMP resistance in S49 mouse lymphoma cells. About 5% of mutants with a spontaneous mutation at this CpG site had also acquired a second CG----TA mutation at the nearby CpG site. The two mutations were always at first positions of the Arg codons in which they occurred, and they were always together in a single regulatory subunit allele. Their linked appearance could be attributed to neither the selection conditions nor the preexistence of one mutation in the target cells. The high frequency of these double mutants suggests that their lesions result not from hydrolytic deamination but rather from an endogenous enzymatic mechanism.     

Direct immunologic activities of CpG DNA and implications for gene therapy

Vertebrate immune systems have evolved the ability to detect and be activated by most microbial and viral DNAs by virtue of their content of unmethylated 'CpG motifs', which are selectively suppressed in vertebrate DNA. Because their CpGs are also unmethylated, the DNA in gene therapy vectors routinely induces direct immune stimulation through activating this host defense mechanism. Administration of such 'CpG DNA' by injection or inhalation triggers rapid activation of B cells, monocytes, macrophages, dendritic cells, and natural killer cells, along with the release of pro-inflammatory cytokines. These immune stimulatory effects can be prevented by chloroquine and other drugs that interfere with endosomal maturation or by the presence of certain neutralizing DNA sequences, which block the immune stimulatory CpG motifs. Aside from serving as the genetic code, DNA can have direct immune activities. Vertebrate immune systems have evolved a defense mechanism that is able to broadly detect most microbial and viral DNAs because of differences in the frequency and methylation of CpG dinucleotides in particular base contexts. B cells, monocytes, macrophages, and dendritic cells spontaneously take up DNA of any type. If the DNA contains these immune stimulatory 'CpG-S motifs', the cells become activated within minutes and begin producing pro-inflammatory cytokines such as IL-6 and IL-12 and upregulate expression of co-stimulatory molecules. This results in the activation of both innate and acquired immune responses. The pro-inflammatory effects of CpG-S motifs are opposed by CpG dinucleotides in certain distinct base contexts, termed neutralizing or CpG-N motifs. Increasing the ratio of CpG-S to CpG-N motifs enhances the immune stimulatory effects of DNA, even if the total level of CpGs in the DNA is not altered. While this is useful in generating enhanced genetic vaccines, the opposite strategy is likely to become useful for the generation of gene therapy vectors with reduced inflammatory effects.