Identification of a bovine ␤-mannosidosis mutation and detection of
two ␤-mannosidase pseudogenes
Jeffrey R. Leipprandt,
Juliann E. Horvath,
Xiao Tan Qiao,
Margaret Z. Jones,
Karen H. Friderici
Department of Pathology, Michigan State University, East Lansing, Michigan 48824, USA
Department of Microbiology, Michigan State University, 407 Giltner Hall, East Lansing, Michigan 48824, USA
Received: 18 June 1999 / Accepted: 13 August 1999
Abstract. ␤-Mannosidase deficiency results in ␤-mannosidosis, a
severe neurodegenerative lysosomal storage disease identified in
cattle, goats, and humans. To more fully understand the molecular
pathology of this disease, the mutation associated with bovine
␤-mannosidosis was identified by sequence analysis of cDNA
from an affected calf. A transition mutation of G to A at position
2574 of the cDNA coding sequence creates a premature stop codon
near the 3Ј end of the protein coding region. To aid commercial
breeders of Salers cattle, a PCR-based test was developed to detect
the mutation for ␤-mannosidosis carrier screening. Application of
this test also revealed the presence of two ␤-mannosidase pseudo-
genes. Portions of the pseudogenes were amplified with allele-
specific primers and then sequenced. One pseudogene was highly
homologous (>99% sequence identity) to the expressed cDNA
sequence over the 1292 bp that were sequenced, while the other
showed more divergence (83% sequence identity) in the 477 bp
that were sequenced. Both are processed pseudogenes that are not
expressed. The severity of the bovine ␤-mannosidosis phenotype
suggests that the 22 C-terminal amino acids of ␤-mannosidase play
an important role in the function of this enzyme.
␤-Mannosidosis is a lysosomal storage disease caused by lack of
␤-mannosidase activity (EC 220.127.116.11). This autosomal recessive
genetic disease has been identified in humans as well as in goats
and cattle. The enzyme is required for cleavage of the unique
␤-mannoside linkage of the carbohydrate moiety of all N-linked
The disease was originally described in Nubian goats (Hartley
and Blakemore 1973; Jones and Laine 1981; Jones and Dawson
1981; Healy et al. 1981), later in humans (Wenger et al. 1986;
Cooper et al. 1986), and was identified more recently in Salers
cattle (Abbitt et al. 1991; Bryan et al. 1990; Jolly et al. 1991).
These cattle originated in the highlands of France and have been
distributed world-wide, where they are valued, in part, for ease of
calving. Calves affected with ␤-mannosidosis are born with mul-
tiple defects and do not survive the neonatal period. ␤-Mannosi-
dosis in both goats and cattle causes consistent clinical features of
skeletal and facial dysmorphology, intention tremor, and nystag-
mus. Goats are deaf, whereas cattle are not, although cattle do
show some structural auditory system abnormality (Render et al.
1992). Both species show central nervous system (CNS) dysmy-
elination. Affected animals are hypothyroid, possibly accounting
for the CNS dysmelination seen in the ruminant forms of the
disease. All tissues show extensive vacuolation owing to lysosom-
al storage of the trisaccharide Man␤1–4GlcNAc␤1–4GlcNAc and
the disaccharide Man␤1-4ClcNAc. There is no ␤-mannosidase ac-
tivity in the plasma and tissues of affected animals (Abbitt et al.
1991; Jones and Laine, 1981; Jones et al. 1983).
Human ␤-mannosidosis is more variable in presentation, and
the cases reported to date generally have a milder clinical expres-
sion and a later age of onset. The phenotypes range from mild
peripheral neuropathy and depression to dysmorphology, mental
retardation, and speech and hearing defects [reviewed in (Levade
et al., 1994)]. The primary storage and urinary excretion product in
the human disease is the disaccharide, Man␤1-4GlcNAc. In all
cases, plasma ␤-mannosidase enzyme activity is absent or pro-
foundly reduced. Variability in the human disease may be due in
part to the severity of the mutations responsible for inactivation of
the ␤-mannosidase enzyme. In some of the cases, part of the vari-
ability may be owing to coincident diseases such as mucopolysac-
charidosis (Wenger et al. 1986), ethanolaminuria (Wijburg et al.
1992), or other unidentified factors arising from suspected or
proven consanguinity (Kleijer et al. 1990).
It is not known whether the differences in disease expression
between ruminants and humans are primarily related to the types
of mutations, species differences in developmental requirement for
enzyme function, the nature of the storage products, or effects on
thyroid function. Identification of the mutations causing disease is
necessary to understand the molecular basis for ␤-mannosidosis, to
investigate the variation in disease expression between species,
and to understand the relationship between structure and function
of the enzyme.
Bovine ␤-mannosidase protein has been purified (Sopher et al.
1993), and cDNA has been cloned and sequenced (Chen et al.
1995). This information was used to amplify and sequence cDNA
from affected animals and to identify a mutation associated with
bovine ␤-mannosidosis. Analysis of the mutation in genomic DNA
from control and affected animals revealed the presence of two
bovine ␤-mannosidase pseudogenes.
Materials and methods
Bovine cDNA synthesis.
For sequence analysis, RNA was isolated by
published CsCl density gradient purification methods (Ausubel et al. 1987)
from thyroid tissue of a calf affected with ␤-mannosidosis. Total RNA (10
g) was heated at 65°C for 5 min, then reverse transcribed with M-MLV
reverse transcriptase (RT; Life Technologies, Inc., Rockville, MD) accord-
ing to the manufacturer’s protocols and various primers at 0.1 pmol/l
based on bovine ␤-mannosidase cDNA sequence (Chen et al. 1995; see
Table 1). For the mutation assay, RNA was isolated with Trisol Reagent
(Life Technologies, Inc.) following the manufacturer’s protocols. DNase I
treatment to remove any contaminating genomic DNA from the RNA and
Correspondence to: K.H. Friderici
The nucleotide sequence data reported in this paper have been submitted to
GenBank and have been assigned accession numbers AF098512,
AF097936, and AF097937. Commercial use of the mutation assay is cov-
ered under U.S. patent #5,605,797.
Mammalian Genome 10, 1137–1141 (1999).
© Springer-Verlag New York Inc. 1999