Animal medical genetics: a historical perspective on more than 50 years of research into genetic disorders of animals at Massey University.

Over the last 50 years, there have been major advances in knowledge and technology regarding genetic diseases, and the subsequent ability to control them in a cost-effective manner. This review traces these advances through research into genetic diseases of animals at Massey University (Palmerston North, NZ), and briefly discusses the disorders investigated during that time, with additional detail for disorders of major importance such as bovine α-mannosidosis, ovine ceroid-lipofuscinosis, canine mucopolysaccharidosis IIIA and feline hyperchylomicronaemia. The overall research has made a significant contribution to veterinary medicine, has provided new biological knowledge and advanced our understanding of similar disorders in human patients, including testing various specific therapies prior to human clinical trials.

Prevalence of factor V Leiden and prothrombin variant G20210A in patients age <50 years with no significant stenoses at angiography three to four weeks after myocardial infarction.

OBJECTIVES: We sought to determine the frequencies of factor V Leiden and prothrombin variant G20210A in patients age <50 years with no significant coronary stenoses three to four weeks after myocardial infarction (MI). BACKGROUND: Factor V Leiden and prothrombin variant G20210A occur frequently in patients with venous thromboembolism. However, the contribution of these mutations to the development of MI requires clarification. METHODS: The frequencies of factor V Leiden and prothrombin variant G20210A were determined in 41 patients age <50 years who had "normal" or "near normal" coronary arteries (no stenosis >50%) at angiography three to four weeks after MI (the study group) and compared with those in 114 patients who had at least one angiographic stenosis >50% after MI (the control group). Patients age > or =50 years with, or without, stenoses were also studied. RESULTS: The frequency of factor V Leiden was 14.6% in patients age <50 years in the study group compared with 3.6% in patients in the control group (odds ratio [OR] 4.7 [95% confidence interval (CI) 1.3-17.7], p = 0.02). The frequency of the prothrombin variant G20210A was 7.3% in the study group compared with 1.8% in the control group (OR 4.4 [95% CI 0.7-27.5], p = 0.12). One or both mutations were present in 8 of the 41 patients (19.5%) age <50 years in the study group compared with 6 of the 114 patients (5.5%) in the control group (OR 4.4 [95% CI 1.4-13.5], p = 0.01). In all 271 patients (irrespective of age) with normal arteries, the frequency of factor V Leiden was 11.7% (7/60) compared with 4.3% (9/211) in patients with at least one >50% stenosis (OR 2.9 [95% CI 1.1-8.3], p = 0.04), and the frequency of prothrombin variant G20210A was 6.7% (4/60) compared with 1.4% (3/211) (OR 4.9 [95% CI 1.1-22.8], p = 0.04), respectively. CONCLUSIONS: The frequencies of factor V Leiden and/or prothrombin variant G20210A are increased in patients age <50 years with normal or near normal coronary arteries after MI.

Colorectal neoplasms detected colonoscopically in at-risk members of colorectal cancer families stratified by the demonstration of DNA microsatellite instability.

This study compared colonoscopic findings in families meeting the Amsterdam criteria (A) for hereditary non-polyposis colorectal cancer (HNPCC) but stratified according to whether the familial cancers showed DNA microsatellite instability. DNA was extracted from paired samples of normal and cancer, and microsatellite instability was analysed at up to six loci. Families were termed replication error positive (RER+) when at least 50% of tumours tested per family were positive. Of 26 families studied 17 were RER+ and 9 were RER-. Cancers in the A/RER- families showed no right-sided predilection (P < 0.001). Colonoscopies have been performed on 182 at-risk members of A/RER+ families and 60 members of A/RER- families. More of the at-risk members of A/RER-families were found to have adenomas at colonoscopy (P = 0.095), but these were smaller than those of A/RER+ families (P = 0.19). The adenoma:carcinoma ratio was twice as high in A/RER- families (13:1) as in A/RER+ families (7:1). One of the A/RER- families had hyperplastic polyposis. The others do not appear to have attenuated familial adenomatous polyposis and are similar to the adenoma families or late-onset colorectal cancer families described by others. This study illustrates the importance of molecular technology in separating HNPCC from syndromes with overlapping phenotypes.

Graft-versus-host disease following interferon therapy for relapsed chronic myeloid leukaemia post-allogeneic bone marrow transplantation.

The combination of donor leucocytes, with or without interferon, has produced encouraging responses in patients with haematological relapse following allogeneic BMT for chronic myeloid leukaemia (CML). A 25-year-old male received low-dose interferon-alpha alone for haematological relapse occurring 10 months following an allogeneic BMT for Ph-positive CML. Interferon therapy was complicated by severe GVHD requiring immunosuppressive therapy. The patient was subsequently found to be in complete haematological and cytogenetic remission, raising the possibility of an immune-mediated antileukaemic action.

Factor VIII gene inversions in severe hemophilia A patients.

The mutations causing hemophilia A are very heterogeneous with the exception of a large inversion involving intron 22 in the factor VIII (FVIII) gene which appears to be the underlying defect in approximately 45% of all severely affected patients (FVIII < or = 1%). In these patients it is thought that the factor VIII gene is disrupted within intron 22 due to inappropriate recombination of FVIIIA with one of 2 homologous regions upstream of the factor VIII gene resulting in a large (approximately 500 kb) inversion. The inversion can be detected by Southern blot analysis and greatly enhances the accuracy of genetic counselling services available to families with severe hemophilia A. We report here the presence of this mutation in a study of 27 unrelated families with severe hemophilia. The factor VIII inversion was identified in 12 of 27 (44%) severe hemophilia A patients and has been successfully used for direct carrier analysis and prenatal diagnosis.

Haemophilia management: the application of DNA analysis for prenatal diagnosis.

The haemophilias are chronic debilitating disorders which cause significant morbidity for the patient and may affect the whole family. An important part of the management of these disorders is the provision of accurate carrier detection and prenatal diagnosis in conjunction with genetic counselling services. We report the results of carrier detection and prenatal diagnosis obtained over a two year period using recombinant DNA techniques. Eighty-seven individuals from 15 families with either haemophilia A or B have been evaluated using informative intragenic factor VIII or IX restriction fragment length polymorphisms. Chorionic villi biopsies for prenatal diagnosis have been performed in four subjects, revealing two female carriers, one normal male, one normal of unknown sex and one haemophiliac male. The use of genotypic diagnosis of haemophilia A and B, in conjunction with conventional assays, is now a routine part of the modern management of haemophilia and many other inherited disorders.

Analysis of Ha-ras 1 allele frequencies in hereditary non-polyposis colorectal cancer.

Restriction enzyme digests of genomic DNA show multiple alleles of common, intermediate, and rare frequencies at the minisatellite locus of Ha-ras. It has been suggested that a higher frequency of rare alleles is associated with the presence of colorectal and other types of cancer. This study investigated the distribution of Ha-ras alleles in 40 members of hereditary non-polyposis colorectal carcinoma (HNPCC) families and in 34 cancer free subjects (spouses). There was no difference in rare allele frequency between the cancer group and cancer free group (chi2 = 0.25, not significant).

Possible misdiagnosis using the Xba I polymorphism for prenatal diagnosis of haemophilia A.

A mother of two haemophilia A sons presented at 7 weeks pregnancy for a prenatal diagnosis. Southern blot analysis of haemophilia DNA at the factor VIII intron 22 Xba I restriction fragment length polymorphism (RFLP) site revealed an Xba I haplotype of A-B+C-. This haplotype has been alluded to but not reported before, and when a 1.6 kb Bst XI fragment of p482.6 is used as a probe the resulting band pattern is similar to that of females heterozygous at site A.

Canine Gaucher disease--the enzymic defect.

beta-Glucosidase activity was investigated in tissues from a case of canine Gaucher disease and from a normal dog. In the latter, enzyme activity showed two pH optima at pH 4-0-4-25 and pH 5-0-5-5. In Gaucher disease tissues, negligible activity could be measured at the mouse acidic pH.

Restriction fragment length polymorphisms associated with the factor VIII and factor IX genes in Polynesians.

New Zealand Maoris (72 X chromosomes) have been compared with Pacific Island Polynesians (121 X chromosomes) and Caucasian New Zealanders (51 X chromosomes) as a control group to determine the allelic frequency of six RFLPs associated with the genes for two X linked diseases (haemophilia A and haemophilia B). RFLPs examined were BclI, XbaI, and BglI within the factor VIII gene, the factor VIII extragenic TaqI system, and the factor IX intragenic TaqI and XmnI sites. The information obtained facilitates the design of strategies for both carrier detection and prenatal diagnosis of haemophilia A within these groups. Strong linkage disequilibrium was observed between the factor VIII BclI and XbaI sites in Polynesians. Genetic counselling for Polynesians with haemophilia B continues, however, to rely on phenotypic diagnosis. The RFLP data from the two separate loci on the X chromosome in Polynesians show similarities with Chinese and Japanese populations, reinforcing theories of an early Polynesian ancestry originating in east Asia.

Generalized glycogenosis in beef shorthorn cattle--heterozygote detection.

A preliminary study of acidic alpha-glucosidase in a variety of tissues was carried out in an attempt to develop a test which might be used to detect individuals heterozygous for the genetype associated with generalized glycogenosis in beef Shorthorn cattle. Of the tissues readily available peripheral lymphocytes were chosen as being likely to be the most suitable. It was concluded that, when coupled with genealogical information, assays of alpha-glucosidase in extracts of lymphocytes were useful for identifying heterozygous individuals with a reasonably high degree of probability.

The nature of the residual alpha-mannosidase in plasma in bovine mannosidosis.

Acidic alpha-mannosidase (EC 3.2.1.24), optimum pH 4.25, is absent from the plasma of Angus calves with mannosidosis, and the residual alpha-mannosidase activity has an optimum pH of 5.5, intermediate between that of the acidic and neutral alpha-mannosidases. This 'intermediate' alpha-mannosidase differs from the acidic form in its kinetic properties, its lack of marked inhibition by EDTA and its thermolability at 55 degrees C and physiological pH. Isoelectric focusing and ion-exchange chromatography show that it exists in at least two forms. The presence of a secondary peak at pH 5.5 in the pH/activity profile of normal plasma and the effect of heating at 55 degrees C indicate that such a form is present in normal plasma. The residual activity in the plasma of a calf with mannosidosis is therefore probably not the product of the defective gene. A differential assay, based on their different stabilities at 55 degrees C, has been developed for measuring the acidic and intermediate alpha-mannosidases in plasma. There was no correlation between the concentrations of the two enzymes in the plasma of Angus cows heterozygous for mannosidosis or in the plasma of normal animals. This precludes the use of the intermediate form as a reference enzyme for the acidic activity in a test for heterozygosity for mannosidosis based on the gene-dosage phenomenon. The concentrations of the intermediate activity were comparable in normal animals and animals homozygous or heterozygous for mannosidosis.

Haemophilia B Leyden arising de novo by point mutation in the putative factor IX promoter region.

Haemophilia B Leyden is characterized by severe factor IX deficiency during childhood with partial resolution at puberty or following the administration of anabolic steroids. The disorder has recently been associated with point mutations in the putative factor IX promoter region, which contains an imperfect direct repeat spanning a possible start site of transcription. We have identified a T to C transition at position +8 in the promoter region of a patient with the haemophilia B Leyden phenotype. A mutation at this site has not been previously reported and occurs within the repeat consensus sequence in the transcribed but untranslated portion of the gene. There is no family history of haemophilia and sequence analysis of his mother and other family members indicates that the mutation has arisen de novo in this patient. This observation provides further support for a causal relationship between point mutations in the presumptive promoter region of the factor IX gene and the Leyden phenotype.

Factor IX gene mutations in haemophilia B: a New Zealand population-based study.

Haemophilia B (Christmas disease) is an X-linked bleeding disorder resulting from an inherited deficiency of coagulation factor IX activity. Due to the heterogeneity of mutations within the factor IX gene there is a marked clinical variability in disease severity. By applying techniques of mutational analysis and direct sequencing of PCR products it is now potentially possible to determine the pathogenic gene defect in entire haemophilia B populations. We report here characterization of the factor IX gene defect in all the haemophilia B patients in New Zealand as part of a nationwide approach towards providing efficient and cost-effective haemophilia B genetic counselling services for these families. Twenty-six different mutations were identified in 32 unrelated haemophilia B families. Three defects at nucleotide positions +8,6659 and 17696 are novel mutations which have not been reported by other laboratories. A PCR-based diagnostic screening test for direct mutational analysis could be performed in most cases; 17 of the 26 mutations altered a restriction enzyme recognition sequence and, with the exception of the total gene deletion, base changes not affecting a restriction enzyme site could be detected by allele-specific PCR.

Familial giant hyperplastic polyposis predisposing to colorectal cancer: a new hereditary bowel cancer syndrome.

A mother and five of her ten offspring developed colonic cancers, the mother and one of the offspring being younger than 50 years of age at diagnosis. Despite fulfilling the Amsterdam criteria for hereditary non-polyposis colorectal cancer (HNPCC), several features pointed towards the possibility that this represented a different syndrome of familial cancer. Most notable was the presence of large, multiple hyperplastic polyps and mixed polyps in four of the subjects whose pathology was available for review. In addition, three of the four subjects had cancers that were negative for DNA replication errors (RER-). The subject with an RER+ cancer had a second RER+ cancer and three adenomas, one in contiguity with the second cancer. This subject also had multiple, large hyperplastic polyps, thereby combining hyperplastic polyposis and a proneness to multiple RER+ tumours. One of the hyperplastic polyps was also RER+. Two of five young asymptomatic descendants have been found to harbour multiple colorectal polyps. It is suggested that giant hyperplastic polyposis is a new familial syndrome predisposing to colorectal cancer.