Use of inverse PCR to amplify and sequence breakpoints of HPRT deletion and translocation mutations.

Deletion and translocation mutations have been shown to play a significant role in the genesis of many cancers. The hprt gene located at Xq26 is a frequently used marker gene in human mutational studies. In an attempt to better understand potential mutational mechanisms involved in deletions and translocations, inverse PCR (IPCR) methods to amplify and sequence the breakpoints of hprt mutants classified as translocations and large deletions were developed. IPCR involves the digestion of DNA with a restriction enzyme, circularization of the fragments produced, and PCR amplification around the circle with primers oriented in a direction opposite to that of conventional PCR. The use of this technique allows amplification into an unknown region, in this case through the hprt breakpoint into the unknown joined sequence. Through the use of this procedure, two translocation, one inversion, and two external deletion hprt breakpoint sequences were isolated and sequenced. The isolated IPCR products range in size from 0.4 to 1.8 kb, and were amplified from circles ranging in size from 0.6 to 7.7 kb. We have shown that inverse PCR is useful to sequence translocation and large deletion mutant breakpoints in the hprt gene.

Development of long PCR techniques to analyze deletion mutations of the human hprt gene.

DNA primers and reaction conditions for long polymerase chain reaction amplification (PCR) of portions of the human hprt gene are presented. Use of these primers with DNA from previously defined hprt deletion mutants demonstrated production of the expected smaller size products as compared to DNA from wild type cells. These primers will be of value in the rapid analysis and subsequent sequencing of hprt deletion mutations in human cells.

Large deletions partially external to the human hprt gene result in chimeric transcripts.

We postulated that gene fusions sometimes occur in normal cells as a result of gene rearrangements as have been observed involving oncogene loci in tumours. To test this, we searched for fusion-gene transcripts in selected human T-lymphocyte large deletion mutations of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene using the 3' rapid amplification of cDNA ends (RACE) technique. Aberrant hprt-containing transcripts were observed in seven out of 19 mutants (approximately 36%) indicating that a surprising number of these rearrangements code for processed mRNAs. RNA splicing and polyadenylation occurred downstream of the non-deleted hprt sequence in chimeric transcripts and the majority resulted from mutants with fusions of hprt into regions containing a repetitive element (Alu, LINE or microsatellite).

Breakpoints and junctional regions of intragenic deletions in the HPRT gene in human T-Cells.

DNA sequences of the deletion breakpoints of 24 human T-lymphocyte hprt gene mutations are reported. These independent deletions ranged in size from 18 to 15655 base pairs. Seven of the 21 in vivo mutations arose in normal adults, three in normal children, eight in radioimmunotherapy patients and three in platinum chemotherapy patients. One in vitro mutation was isolated after 93cGy radon exposure and two after 300cGy gamma radiation. The breakpoints were found to be non-random and a cluster of small deletions in exon 6 is reported. Ten of the mutations had 2-5bp direct repeats at the breakpoints. There was no excess of "deletion-associated" motifs over that expected by chance. Some breakpoints do occur at consensus topoisomerase II cleavage sites and the centromeric end of a Donehower sequence occurs exactly at a telomeric breakpoint. Three mutants had breakpoints at hairpins expected by the model of Glickman and Ripley.

Homozygous type I protein C deficiency in two unrelated families exhibiting thrombophilia related to Ala136-->Pro or Arg286-->His mutations.

Separate single nucleotide mutations have been identified in two unrelated homozygous type I protein C deficient individuals suffering from thrombophilia. Each mutation, initially established by direct DNA sequencing of polymerase chain reaction amplification products, results in an amino acid substitution. The first mutation (PCClamart) results in an Ala136 to Pro substitution in the protein's second epidermal growth factor-like domain. The second mutation (PCMünchen) results in an Arg286 to His substitution in the serine protease domain. Comparison of the location of these two mutations and the relative conservation of the two regions in homologous vitamin K-dependent plasma proteins is consistent with the difference in severity of protein C deficiency and disease in the two individuals. Both mutations result in the abolition of a naturally occurring restriction endonuclease site, thereby allowing independent confirmation of the mutations and rapid and unambiguous genetic analysis of protein C deficiency in family members. In both families, the genetic analysis has proven useful in cases where an assignment of the protein C status based upon clinical laboratory measurements was either ambiguous or incorrect.

Protein CVermont: symptomatic type II protein C deficiency associated with two GLA domain mutations.

This study investigates type II protein C deficiency in a family with manifestations of both arterial and venous thrombosis. Of 64 members of the kindred, 14 have been tested and 7 have PC deficiency. Among affected individuals (n = 7), mean protein C levels by different assays were as follows: enzyme-linked immunosorbent assay (ELISA), 3.8 micrograms/mL (2.1 to 4.3 micrograms/mL); amidolytic with venom activator, 115% (60% to 140%); clotting with venom activator, 42% (23% to 59%). The mean ratio of clotting to amidolytic assays for the affected individuals was 0.37 compared with a normal range of 0.8 to 1.2. Thus, the affected individuals have normal total protein C and their activated protein C has a normal active site assessed by chromogenic substrate; however, they have markedly diminished clotting activity. Immunoassay and chromatography data suggested an abnormality of carboxylation in the gamma carboxyglutamic acid (Gla) domain. Polymerase chain reaction amplification and direct DNA sequencing of exon 2 from genomic DNA of affected individuals showed two nucleotide substitutions. One of the mutations (A----C) results in Glu20----Ala, thereby eliminating a site for vitamin K-dependent gamma-carboxylation. The other substitution (G----A) results in a Val34----Met mutation. DNA sequencing of the other exons from affected individuals has shown no further difference from that of the wild-type gene. The former mutation also removes a Bgl II restriction endonuclease site, which has allowed us to confirm the mutation in affected individuals by direct digestion and Southern hybridization of genomic DNA from family members. This is the first reported family with documented Gla domain mutations in the protein C gene.