Molecular heterogeneity for bovine α-mannosidosis: PCR based assays for detection of breed-specific mutations

Researchin VeterinaryScience1997,63, 279-282

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Molecular heterogeneity for bovine (x-mannosidosis: PCR based assays for detection of breed-specific mutations T. BERG, Department of Medical Genetics, Institute of Clinical Medicine, University of TromsO, N-9037 TromsO, Norway, P. J. HEALY, Elizabeth Macarthur Agricultural Institute, Private mail bag 8, Camden, NSW 2570 Australia, O. K. TOLLERSRUD, Department of Medical Biochemistry, Institute of Medical Biology, University of TromsO, N-9037 TromsO, Norway, O. NILSSEN*, Department of Medical Genetics, University Hospital of TromsÜ, N-9038 TromsO, Norway

SUMMARY DNA tests, based on the polymerase chain reaction (PCR), were developed for the detection of two breed-specific mutations responsible for the autosomalrecessive disorder bovine a-mannosidosis. The tests involve separate amplification of two exons of the lysosomal ¢¢-manuosidasegene followed by restriction enzyme digestion of the amplicons. We demonstrate that one of the mutations, the 662G---~Atransition, is responsible for c~-mannosidosisin Galloway cattle. The other mutation, the 961T---~Ctransition, is uniquely associated with c~-mannosidosisin Angus, Murray Grey and Brangus cattle from Australia. The 961T--+Cmutation was also detected in Red Angus cattle exported from Canada to Australia as embryos. All 39 animals classified as heterozygotes on the basis of biochemical assays were heterozygous for one of the two mutations. None of 102 animals classified as homozygous-normalon the basis of biochemical assays possessed the mutations. Our results indicate that the two breed-specific mutations may have arisen in Scotland and by the export of animals and germplasm disseminated to America, New Zealand and Australia.

c~-MANNOSIDOSIS is an autosomal recessive lysosomal storage disorder caused by a deficiency of lysosomal (xmannosidase (EC 3.2.1.24). The disease has been identified in man, cat and cattle and is characterised by an accumulation of mannose rich oligosaccharides in various tissues (Thomas and Beaudet 1995). Bovine c~-mannosidosis has been reported in Angus (Whittem and Walker 1957), Murray Grey (Healy and Cole 1976) and Galloway cattle (Borland et al 1984). The majority of affected Angus calves fail to survive the immediate postnatal period, those who do survive show severe, progressive neurological disease characterised by tremors of the head, ataxia, and aggression (Whittem and Walker 1957, Healy et al 1990). The phenotype in Galloways is more severe than in Angus, as most affected Galloway cases are aborted or stillborn (Borland et al 1984, Healy et al 1990). In addkion to the variation in expression across breeds, significant variation has been reported within Angus (Jolly 1975, Healy et al 1990). Twenty years ago o~-mannosidosis carrier frequencies between 0.05 and 0-25 were reported in Angus and Murray Greys in New Zealand and Australia (Jolly and Townsley 1980, Healy et al 1983). After mass screening programs, based on biochemical assays and promoted by the respective breed societies, the disease is now relatively rare in these countries. A carrier frequency of 0-02 was reported among Aberdeen Angus in Scotland (Jolly and Hartley 1977) and cases have been diagnosed in North America (Leipold et al 1979). The disease has only been reported in Galloways in Australia (Borland et al 1984, Embury and Jerret 1985). Healy (1983) reported a carrier frequency of 0- i in a selected population of Australian Galloways. Two missense mutations in the lysosomal c¢-mannosidase gene causing bovine o~-mannosidosis were recently * Correspondingauthor 0034-5288/97/060279 + 04 $18.00/0

identified (Tollersrnd et al 1997). An affected Angus bull calf was homozygous for a 961T---~C transition resulting in Phe321Leu substitution of the lysosomal a-mannosidase polypeptide, whilst a Galloway steer, carrier of o~-mannosidosis, was heterozygous for a 662G---~A transition leading to Arg221His substitution. This study was undertaken to establish the association between o~-mannosidosis phenotype and the mutations. To accomplish this, and to devise a robust and reliable diagnostic test for (x-mannosidosis, we developed DNA based tests for detection of the two transitions.

MATERIALS AND METHODS

Animals The Angus and Murray Greys were sampled in unrelated herds depastnred in different regions of Australia. The Red Angus samples came from subjects exported as embryos from Canada to Australia. The Galloways sampled were born in Australia, with the exception of a Scottish bull whose semen was exported to Australia. With the exception of the Scottish Galloway bull, all subjects were classified as homozygous normal, heterozygous or affected on the basis of plasma and granulocyte (x-mannosidase activity (Healy et al 1983).

Analytical methods With knowledge of genomic sequence of the bovine lysosomal c~-mannosidase (GenBank accession numbers U97686 - U97694) we designed two sets of primers flanking exons 5 and 7. The 662G---)A mutation, located in exon 5, deletes a unique BsaHI restriction site while the 961T-+C mutation in exon 7 creates a Mnll site (Fig la). © 1997W. B. SaundersCompanyLtd

280

T. Berg, P. J. Healy, O. K Tollersrud, O. NiIssen

(a)

EMAI214 ~* ~ -.

mpi_.~6F

EMA1215bsa

mpi 7R

(b)

(c)

g Bp

__M N ..~.__.AC_.. +

+

+

+

Bp

FIG 1: (a) Genomic organisation of the lysosomal c~-mannosidase gene between intron 4 and 7 according to Tollersrud et al (1997). Exons are numbered and shown as boxes. Positions of the primers (EMAI214, EMAI215bsa, mpi6F, mpi7R), Mnll (M) and BsaHI (B) restriction sites of the two amplicons are indicated. B* and M* show the locations of the BsaHI site lost due to the 662G~A mutation and the Mnll site introduced by the 961T-->C mutation, respectively. (b) Banding pattern before (-) or after (+) BsaHI digestion of the 360 bp PCR products amplified from Galloway cattle using the primer combination EMAI214 and EMAI215bsa. M is the molecular weight marker VIII from Boehringer Mannheim (Mannheim, Germany). Lane B (blank) is a negative control where no DNA was added to the PCR. Amplicons from c~-mannosidosis Galloways (A) are digested to 24 bp and 336 bp (the 24 bp fragments are not visible on the gel) while normal amplicons (N) are digested to fragments of 24 bp, 95 bp and 241 bp. Carriers (C) are identified by the presence of three bands. (c) Banding pattern before (-) or after (+) Mnll digestion of 295 bp amplicons from Angus animals amplified by the primers mpi6F and mpi7R. The mass ladder (M) is 100 bp DNA ladder from Gibeo-BRL. N and A are amplified from DNA of normals and affected, respectively. Carriers are characterised by the presence of three bands of 101 bp,

Sequences of the primers are given in Table 1. Restriction sites were introduced into both reverse primers to serve as positive controls for digestion (underlined in Table 1). To screen for the 662G--+A mutation, a 360 bp fragment including exon 5 was amplified by PCR in 10 ~tl containing 100 ng of genomic DNA, 1 gM primer EMAI 214 and EMAI 215 bsa, 100 gM dNTPS, 0.5 U Taq DNA polymerase (Boehringer Mannheim, Mannheim, Germany), 1 x PCR buffer (50 mM KC1, 10 mM Tris-HC1, pH 8.3) and 1-5 mM MgC12. After an initial denaturation at 94°C for two minutes the reaction was cycled 35 times as follows: 92°C for 10 seconds, 61°C for 20 seconds and 72°C for 30 seconds and then a final extension for two minutes at 72°C. The PCR product was incubated with 2 U BsaHI (New England Biolabs, Beverly, MA, USA), 150 pg BSA and 1-5 pl 10 x NEB 4 buffer for two hours at 60°C. The resultant fragments were separated on a 4 per cent agarose gel and visualised with ethidium bromide (Fig lb). The 961T--+C mutation was detected by amplification of exon 7 with flanking intron sequences. The reaction conditions were similar to those described above with the exception that the primers were mpi6F and mpi7R. After an ini-

tial denaturation at 94°C for two minutes the reaction was cycled 35 times at 94°C for 10 seconds, 64°C for 20 seconds and 72°C for 30 seconds followed by an extension step at 72°C for two minutes. The resulting 295bp amplicon was incubated in PCR buffer with 2 U of MnlI (New England Biolabs) at 37°C for two hours and the fragments separated on a 3 per cent agarose gel and visualised by ethidium bromide straining (Fig lc). Minor modifications were made to standard methods for extraction of DNA for blood (Miller et al 1988), hair roots (Thomson et al 1992) and semen (Tammen et al 1996).

RESULTS Amplicons containing exons 5 and 7 were produced with blood, hair roots and semen as the source of target DNA. A total of 38 Galloway animals were tested for the 662G--->A mutation, whereas 73 Angus, 18 Red Angus and 17 Murray Grey cattle were tested for the 961T---~C mutation (Table 2). The exon 5 amplicon from Galloways classified as non-

TABLE 1: PCR primers and restriction enzymes for the DNA based assays for c~-mannosidosis

Mutations

Breeds

PCR primers

Restriction enzymes

662G~A

Galloway

EMAI 214: 5'-GGTTGTAGCGTTGGACTTGC-3' EMAI 215bsa:

BsaHI

961T-->C

Angus, Red Angus, Murray Grey

5'-TCTACCACCTTACTGGTGAAGAC*GTC-3"

mpi6F:

Mnll

5"-CGCAGGACACCCTAGCCTTAG-3"

mpi7R: 5'-CCTTGCTATTG J I I I AAGCCT*CTAAGTTTGTGGT-3' * The A in position 21 of primer mpi7R was substituted with a T and the G in position 23 of EMAI 215bsa was substituted with C to introduce Mnll and BsaHI restriction sites (underlined) serving as positive control for digestion

281

DNA tests for bovine a-mannosidosis

TABLE 2: Correlation between the c~-mannosidosis genotype as determined by enzymatic assays and the presence of a BsaHI restriction site in Galloway cattle or a Mnll restriction site in Angus, Red Angus and Grey cattle

Number of subjects tested (herds)

Breed

Genotype as determined by enzymatic assays

Presence of BsaHI site in Galloway and Mntl site in Angus, Red Angus and Murray Grey

Galloway

38 (3)

Homozygous-normal Heterozygous Homozygous-affected

+BsaHI/+BsaHI -BsaHI/+BsaHI -BsaHI/-BsaHI

(n = 29) (n = 7) (n = 2)

Angus

73 (13)

Homozygous-normal Heterozygous Homozygous-affected

-Mnll/-Mnll -Mnll/+Mnll +Mntl/+Mnii

(n = 58) (n =12) (n = 3)

Red Angus

18 (2)

Homozygous-normal Heterozygous

-Mnll/-Mnll -Mml/+Mnll

(n = 7) (n = 11 )

Murray Grey

17 (6)

Homozygous-normal Heterozygous

-Mnll/-Mnll -Mnll/+Mnll

(n = 8) (n = 9)

carriers (n = 29) was cut by BsaHI whilst that from affected Galloway calves (n = 2) was not. Galloways classified as carriers (n = 7) were found to have one allele that was digested and one that was not (Fig 1). The Scottish Galloway bull was heterozygous for the 662G--+A mutation. MnlI digestion cut the exon 7 amplicon from affected Angus calves (n = 3) yielding two bands not evident in that from Angus and Murray Greys classified as non-carriers (n = 73) (Fig 1). Both cut and uncut bands were evident in MnII digested amplicons from Angus and Murray Grey animals classified as carriers (n = 32).

DISCUSSION National screening programs, supported by breed societies, have been implemented to reduce the prevalence of heterozygotes for o~-mannosidosis in Angus and Murray Grey bull-breeding herds in Australia and New Zealand (Jolly 1978, Healy et al 1983). The programs were based upon estimates of either plasma or serum o~-mannosidase activity to detect heterozygotes. Unfortunately, ill-defined environmental factors complicated the procedure (Jolly et al 1974). Consequently, accurate carrier detection required estimates of relative o~-mannosidase activity in granulocytes isolated from freshly collected blood (Healy et al 1983). The use of the pCR-based assays described in this paper will permit the exploitation of a range of stable samples for detection of animals heterozygous for the breedspecific mutations. Both mutations cause substitutions of highly conserved amino acids (Tollersrud et al 1997). Coupled with the close association between the mutations and (x-mannosidosis genotype determined on the basis of enzyme activity the data we have presented strongly suggested the mutations are those responsible for the disease in the respective breeds. An epidemiological study reported by Jolly (1978) indicated that the original cases in Australian Angus cattle were descendants of bulls imported from New Zealand. It was also shown that one of these bulls was a founder for the dissemination of the defect in the New Zealand Angus herd. Therefore, it is highly likely that the procedure for detection of the 961T--~C mutation would be effective for heterozygote detection in the New Zealand Angus population. Identification of seven heterozygotes for the 961T--~C mutation among Canadian Red Angus exported to Australia

and the presence of heterozygotes in Scotland (Jolly and Hartley 1977) strongly suggests that the mutation may have arisen in Scottish Angus cattle and from there disseminated to North America, New Zealand and Australia. Murray Grey cattle were developed in Australia in recent times from Angus stock, consequently it is not surprising that the 961T--~C mutation was found to be associated with the disease in this breed. (x-Mannosidosis was diagnosed as the cause of severe neurological disease in a Brangus calf on the basis of a cytoplasmic vacuolation of neurones and a profound deficiency of o~-mannosidase activity in blood (Healy unpublished). The subject was found to be homozygous for the 961T--~C mutation indicating that, like Murray Greys, this Brahman-derived breed gained the mutation from Angus ancestors. Anecdotal information from breeders in Australia linked the Galloway cases to the Scottish bull that was found to possess the 662G---~A mutation. Given that both Angus and Galloway breeds were developed in Scotland, in relatively recent times, it was considered more likely that a single mutation arose in a founder common to both breeds, rather than separate mutations arising in these two related breeds (Healy et al 1990). However, genetic heterogeneity for c~-mannosidosis is in agreement with the different clinical and biochemical expression of cz-mannosidosis between Angus and Galloway cattle (Healy et al 1990, Tollersrud et al 1997). If indeed the mutations are truly breed-specific the explanation may lie in development of the two breeds in different parts of Scotland. On the other hand, as the breeds had a common herd book it would be prudent to consider screening important individuals for both the 662G---~A and 961T---~C mutations. Samples used in this study were collected after Australian screening programs were almost completed, and hence the data does not reflect the contemporary prevalence of heterozygotes in the respective breeds. The detection of heterozygotes in North American Angus has implications for countries importing Angus germplasm from the North American herds. Fifteen per cent of a total of 703 Australian Galloway cattle examined by Healy (1983) and Embury and Jerrett (1985) were diagnosed as heterozygotes. Such a high prevalence was acknowledged as a matter of concern to individual breeders. Our finding of a heterozygote in the Scottish population indicates that it would be prudent for breeders to determine the 662G--+A genotype of germplasm imported to augment limited national 'gene pools'. Founder effects have been previously shown to be responsible for widespread international dissemina-

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T. Berg, P. J. Healy, O. K Tollersrud, 0. Nilssen

tion of two other lethal recessive defects in cattle, citrullinaemia (Dennis et al 1989) and bovine leucocyte adhesion deficiency (Shuster et al 1992). Application of the tests we describe in this communication, at both national and international levels, will assist in preventing similar founder effects disseminating c~-mannosidosis mutations via modern artificial breeding technologies that will be increasingly used to amplify beneficial production traits.

ACKNOWLEDGEMENT This work was supported by a graduate fellowship to T. Berg from the Institute of Clinical Medicine, University of TromsO, Norway.

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bovine ~-mannosidosis. Research in Veterinary Science 49~ 82-84 JOLLY, R. D. (1975) Mannosidosis of Angus Cattle: a prototype control program for some genetic diseases. Recent Advances in Veterinary Science and Comparative Medicine 19, 1-21 JOLLY, R. D. (1978) Mannosidosis and its control in Angus and Murray Grey cattle. New Zealand Veterinary Journal 26, i94-198 JOLLY, R. D. & HARTLEY, W. J. (1977) Storage diseases of domestic animals. Australian Veterinary Journal 53, 1-8 JOLLY, R. D., THOMSON, K. G., TSE, C. A., MUNFORD, R. E. & MERRALL, M. (1974) Identification of mannosidosis heterozygotes-factors affecting normal plasma c~-mannosidase levels. New Zealand Veterinary Journal 22, I55-162 JOLLY, R. D. & TOWNSLEY, R. J. (1980) Genetic screening programmes: an analysis of benefits and costs using the bovine rnarmosidosis scheme as a model. New Zealand Veterinary Journal 28, 3-6 LEIPOLD, H. W., SMITH, J. E., JOLLY, R. D. & ELDRIDGE, F. E. (1979) Mannosidosis of Angus calves. Journal American Veterinary Medical Association 175(5), 457-459 MILLER, S. A., DYKES, D. D. & POLENSKY, F. (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acid Research 16, 1215 SHUSTER, D. E., KEHRLI, M. E., Jr, ACKERMANN, M. R. & GILBERT, R. O. (1992) Identification and prevalence of a genetic defect that causes leukocyte adhesion deficiency in Holstein cattle. Proceedings of the National Academy of Sciences of the USA 89, 9225-9229 TAMMEN, I., KLIPPERT, H., KUCZKA, A., TREVIRANUS, A., POHLENZ, J., STOBER, M., SIMON, D. & HARLIZIUS, B. (1996) An improved DNA test for bovine leucocyte adhesion deficiency. Research in Veterinary Science 60, 218-221 THOMAS, G. H. & BEAUDET, A. L. (1995) Disorders of glycoprotein degradation and structure: ~-mannosidosis, ]]-mmmosidosis, fucosidosis, sialidosis, aspartylglucosaminuria, and carbohydrate-deficient glycoprotein syndrome. In The Metabolic and Molecular Bases of Inherited Disease. 7th Edn. New York, McGraw-Hill, Inc. pp 2529-2561 THOMSON, D. M., BROWN, N. N. & CLAGUE, A. E. (1992) Routine use of hair root or buccaI swabs for PCR analysis: advantages over using blood. Clinica Chimica Acta 207, I69-174 TOLLERSRUD, O. K., BERG, T., HEALY, P., EVJEN, G., RAMACHANDRAN, U. & NILSSEN, O. (1997) Purification of bovine lysosomal ~-marmosidase, characterization of its gene and determination of two mutations that cause (zmarmosidosis. European Journal of Biochemistry 246, 410-419 WHITTEM, J. H. & WALKER, D. (1957) 'Neuronopathy' and 'pseudolipidosis' in Aberdeen Angus calves. Journal of Pathology and Bacteriology 74, 281-288 Accepted July 3, 1997