Peter Kay

C4 allotyping on plasma or serum: application to routine laboratories

Allotypes of the fourth component of complement (C4) can be detected by electrophoresis and immunofixation after treatment of EDTA plasma with neuraminidase (NAse). We have assessed the value of additional treatment with carboxypeptidase B (CPseB). Following treatment with CPseB + NAse, each allele is resolved into a single band, permitting clear definition of overlapping bands seen following treatment with NAse alone. More importantly, C4 allotypes can be determined using stored heparinized plasma or serum. Most C4 null alleles can be assigned without requiring family studies. The approach described is suitable for routine use by tissue typing laboratories.

Alternate Pax7 transcripts are expressed specifically in skeletal muscle, brain and other organs of adult mice

Pax7 is associated with formation of skeletal muscle and the neural tube in developing embryos. Interestingly, in adult mice, rearrangements of Pax7 are associated with differences in the efficiency of skeletal muscle regrowth between mouse strains. The aim of this study was to investigate the possibility that Pax7 is expressed in skeletal muscle or other tissues from adult mice. Total RNA was isolated from adult mouse tissues and the polymerase chain reaction was performed on reverse transcribed mRNA using primers specific for regions that encode the paired and homeodomain of Pax7. At least four different Pax7 transcripts were found. A full-length transcript similar in sequence to that published previously was identified in skeletal muscle, brain and spleen cells of adult mice. Further putative full-length Pax7 transcripts, including one that contains a hexanucleotide insertion in the paired box and one in which approximately 10 bp have been deleted in the homeobox, were found to be expressed in skeletal muscle and brain of adult mice, respectively. A truncated Pax7 splice product comprising the paired box only was found to be expressed in most adult tissues except liver. Results of these studies demonstrate that there are alternate transcripts of Pax7, some of which are expressed exclusively in adult skeletal muscle and brain. It is possible that one of these transcripts may specify an alternate myogenic pathway involved in regeneration of damaged skeletal muscle in adult mice.

Comparative mapping of the human major histocompatibility complex in different racial groups by pulsed field gel electrophoresis

The molecular map of the human major histocompatibility complex was examined in multiple examples of various Caucasoid and Japanese major histocompatibility complex supratypes using pulsed field gel electrophoresis. Extensive differences in restriction fragment lengths were observed. However, each supratype showed specific genomic characteristics including deletions, duplications, or insertions supporting the hypothesis that these supratypes are markers of conserved ancestral haplotypes. Some of the gene arrangements are consistent with the deletions or duplications previously described or suggested by conventional DNA techniques and protein typing, while others have not been recognized previously. Characterization of the gene organization within disease-associated ancestral haplotypes will provide new insights into the functional role and evolution of the major histocompatibility complex.

B-cell target DNA quantity is a critical factor in the interpretation of B-cell clonality by PCR

Summary Criteria for the assessment of clonality by Southern blotting are well established but this is not the case for polymerase chain reaction (PCR)‐based assays. Our studies, and infrequent reports in the literature, indicate that B‐cell clonality may be erroneously inferred if only small numbers of polyclonal B‐cells are present in test samples. In order to establish criteria to minimise the false positive assignment of B‐cell clonality, DNA was analysed in a semi‐nested PCR to detect rearrangement of the immunoglobulin heavy chain gene using a range (1 μg‐0.1 ng) of target DNA amounts from four tonsils and five lymph nodes showing reactive follicular hyperplasia, and from six B‐cell lymphomas. A discrete, narrow band of PCR product of constant size was detected throughout the range of target DNA amounts in most lymphomas indicating the presence of a monoclonal B‐cell population. In contrast, from the non‐malignant tonsils and lymph nodes, larger target amounts generated a broad band of PCR products indicating populations of polyclonal B‐cells, but smaller target amounts generated discrete, narrow PCR product bands of inconstant size indicating oligoor monoclonal B‐cell populations. Results of this study demonstrate that a range of DNA target amounts should be tested when the proportion of B‐cells in a sample is unknown, thus preventing the analysis of insufficient target DNA which may lead to the false assignment of clonality.Abbreviations: LPDs, lymphoproliferative disorders; IgH, immunoglobulin heavy chain gene; B‐NHL, B‐cell non‐Hodgkins lymphoma.

Differential expression of four alternate Pax7 paired box transcripts is influenced by organ- and strain-specific factors in adult mice.

The developmental paired-type gene Pax7 is expressed in skeletal muscle and brain during development and in the adult mouse. In this study, RNA was isolated from the brains and skeletal muscles of the limbs of adult BALB/c and SJL/J mice to determine whether there were alternate transcripts which could account for the biological diversity of Pax7. Four alternate transcripts have been identified, each of which differs by the presence and/or absence of a trinucleotide or a hexanucleotide in the region of Pax7 which encodes the paired domain. Inclusion of the trinucleotide and the hexanucleotide results in insertion of the amino acids glutamine (Q) and glycine (GL), respectively, into the paired domain. The insertion of GL is predicted to influence the binding specificity, whereas insertion of Q may affect the relative binding affinity of the N and C termini of the paired domain. The transcripts which include the hexanucleotide are more frequent in RNA from the hind-limb skeletal muscles of adult mice compared with the brain, suggesting that they may effect some form of myogenic function. By contrast, it is possible that transcripts which lack the hexanucleotide encode factors which are involved in neurogenesis. The proportion of the potential myogenic transcript Pax7b was found to be increased in RNA from limb skeletal muscles of adult SJL/J mice compared with that of BALB/c mice. These results provide a new basis for examination of the genetic control of skeletal-muscle formation and neurogenesis.

Taq I Polymorphism of the Epidermal Growth Factor Receptor Gene in Caucasoids and Japanese

Genetic polymorphism of the epidermal growth factor (EGF) receptor gene following Taq I digestion was compared between samples of genomic DNA from glioma-derived cell lines and Caucasoid and Japanese subjects. The same three allelic forms of the EGF receptor gene, marked by variant fragments of approximately 12.8, 11.6 and 10.8 kb in size were common to both ethnic groups and the 12.8- and 11.6-kb fragments were found in the glioma-derived cell line DNA. A further variant fragment of approximately 13.8 kb in size has been shown to be thus far restricted to the Japanese. These data suggest that most allelic forms of the EGF receptor gene recognized by Taq I restriction fragment length polymorphism have a long evolutionary history and probably do not predispose to development of malignant glioma.

The diagnostic significance of Myf-3 hypermethylation in malignant lymphoproliferative disorders

Deregulated methylation of cytosine in DNA is a frequent finding in malignancy that is reflected by general genomic hypomethylation and regional hypermethylation that includes the myogenic gene Myf-3. In this study of 198 DNA samples from 186 patients with a wide range of lymphoproliferative disorders (LPD), the methylation status of Myf-3 was assessed to evaluate its significance in the diagnosis of malignant LPD. DNA was digested with the restriction endonucleases HpaII and MspI, and using the Southern blot (SB) technique, the size and density of fragments that hybridized with a Myf-3 probe were used to assign the methylation status. None of the samples from 45 patients from a wide age range with benign LPDs had evidence of altered Myf-3 methylation and there was no age-related methylation change. By contrast, 115/123 (93%) of samples from patients with non-Hodgkin lymphoma (NHL) or lymphoid leukemia had increased Myf-3methylation. There was no methylation alteration in 22/24 (92%) of samples from patients with Hodgkin lymphoma (HL), nor in five of six samples from LPDs that had atypical histopathologic features which were not diagnostic of lymphoma, while the remaining sample of atypical LPD had hypermethylated Myf-3 fragments. There was an association between increasing Myf-3 methylation and higher histopathologic grade of malignancy within specific lymphoma categories. It is concluded that the detection of increased Myf-3 methylation is a sensitive and specific test of malignancy which may complement other molecular methods that are currently used for the assessment of clonality. It may be of particular diagnostic use in natural killer (NK) and null cell malignancies for which other indicators of clonality are lacking. Furthermore, methylation status may prove to be of potential prognostic value.

VARIATION IN THE METHYLATION PROFILE AND STRUCTURE OF PAX3 AND PAX7 AMONG DIFFERENT MOUSE STRAINS AND DURING EXPRESSION

Structural alterations within the myogenic and neurogenic developmental gene Pax7 which involve TaqI recognition sequences have previously been reported. These alterations are associated with differences in the efficiency of regrowth of damaged skeletal muscle. To identify other structural features of Pax genes which may influence skeletal muscle regrowth, variation in the structure and methylation status of Pax7 and the closely related gene Pax3 has been sought among different mouse strains and during gene expression using the restriction endonucleases MspI and HpaII. Following MspI digestion, RFLPs within Pax7 have been found which most likely reflect intron size variability within the paired box. Differences in the size of MspI and HpaII fragments hybridising with Pax7 and Pax3 region specific sub-probes indicate that the paired boxes are hypomethylated, whereas the region encoding the homeodomain of each gene is highly methylated in the spleen and other tissues from adult mice. In the skeletal muscle precursor cell line C2C12, which expresses Pax7 but not Pax3, the homeodomain encoding region of Pax7 is hypomethylated. In spleen cells, the Pax7 paired box is transcribed but the homeodomain encoding region is not. By contrast, both the paired box and the homeobox of Pax3 are hypermethylated in C2C12 cells indicating that generation of alternate transcripts from Pax genes may be controlled by DNA methylation. In contrast to Pax3, reference to the size of fragments hybridising with a Pax7 homeobox specific probe provides evidence for CpNpG methylation within and immediately downstream from the region encoding the homeodomain. Interestingly, CpNpG methylation remains when the Pax7 homeobox is expressed. Structural variation recognised by MspI digestion and differences in the methylation profile of Pax7 are not associated with the ability to regrow damaged skeletal muscle.

Different allotypes of C3 degrade at different rates

To determine whether different forms of C3 degrade at different rates, we compared two strains of mice with a B10 background. The only difference was that one is C3A, while the other is OR These strains allow comparison of C3A and C3B without the added complication of differing C3 convertases. Sera from the two strains were incubated with zymosan and the degradation products were detected by immunofixation following electrophoresis in agarose. The rate of degradation of mouse C3B was more rapid than that of C3A. Differences in the rates of degradation could not be explained by differing concentrations of C3. We suggest that the genetic differences in C3 determine the decay rate following activation via the alternate pathway.

Genomic hypomethylation in neoplastic cells from dogs with malignant lymphoproliferative disorders

DNA methylation is an epigenetic modification in which a methyl group is added usually to the fifth carbon position of a cytosine residue. Dysregulation of this process is an important molecular event which has been shown to be associated with neoplastic transformation and tumour progression in humans and mice. Features of methylation dysregulation in many different types of neoplasms include general genomic hypomethylation, focal hypermethylation, and altered expression of genes which encode a series of DNA (cytosine-5) methyltransferases. Interestingly, many types of neoplasia that are recognised in humans also develop spontaneously in the dog. By comparing the restriction patterns of MspI and HpaII, this study demonstrates that as in human, genomic hypomethylation is a feature of neoplastic cells in the majority of canine lymphoma cases and approximately one-third of canine leukemia cases confirming that dysregulation of the DNA methylating machinery is implicated in malignant transformation of lymphoid cells in some dogs.