Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

I. Introduction
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of genomes of various organisms. If two organisms have a
recent ancestor, their genomes will be related. Comparative
maps display similarities between two organisms by aligning
genes and their order on a chromosome of one species
and then comparing it to the location and order found in
another species. This knowledge is useful for mapping,
identifying and isolating genes, and gaining more information
about principles of evolution. Comparison of the actual
genome sequences of different species allows the detection
of highly conserved regions within or around genes that,
besides representing exonic sequences, most likely serve
as important regulatory elements in gene expression and
function.
C . Disease Gene Mapping
A major objective of genetic research is the identification
of DNA mutations that is involved in disease or genetic
predisposition. A small sequence change located within a
gene can alter or eliminate gene or protein function. These
mutations either arise during imperfect DNA replication or
are caused by mutagens and are distinguished by the type
of change in the nucleotide sequence. A replacement of a
single nucleotide with another base is called a point mutation
, which can either be silent (the amino acid remains
unchanged), a missense (changes the amino acid), or nonsense
point mutation (producing a stop codon) . Insertion
or deletions refer to varied numbers of nucleotides that are
added or deleted, respectively. Nonsense point mutations
and deletions or insertions unequal to an exact multiple of
3 bp can result in an early stop codon and consequently in a
shortened, unstable, or malfunctioning protein. Protein function
can also be impaired by the change or addition/deletion
of amino acids because of a mutation within the coding
region (missense). Additionally, mutations within noncoding
sequences that are necessary for correct gene regulation
and function can also lead to a change in expression
or nonfunctional proteins.
In single gene disorders such a specific mutation that is
severe enough to cause disease by itself and often shows a
simple (Mendelian) inheritance pattern. If the inheritance
is said to be dominant , only one mutant allele is sufficient
for the development of the disease in an affected individual.
Because the second allele is a normal (wild-type)
allele, the affected individual is considered to be heterozygous.
If both alleles have to be mutated to cause clinical
disease, then the inheritance pattern is said to be recessive
and the affected animal is homozygous for the mutant
allele. If the mutation is located on the X chromosome, the
affected male is considered to be hemizygous . Complex
or polygenic disorders are caused by sequence variations
in only a few or numerous genes and are more difficult
to evaluate. The influences of environmental factors are
being recognized and explain some of the variation in
disease presentations of simple and complex inherited
traits. To identify mutant alleles, various methods have been
applied.
1 . Candidate Gene Approach
If the phenotype or metabolic basis of the disease to be
studied is well characterized or previous research has been
done in humans or in other animal species with a similar
disease, there might be potential genes (known as candidate
genes ) that can be suspected to be involved based on the
previous findings or known function. Candidate genes can
be evaluated for their involvement by testing for linkage or
association (discussed later) or direct sequencing of coding
regions, exon/intron boundaries, and promoter regions from
unaffected and affected animals. For example, symptoms
seen in human patients with phosphofructokinase (PFK)
deficiency closely resembled those in other glycogen storage
diseases and extensive biochemical analyses revealed
the deficiency of the key regulatory glycolytic enzyme
muscle-type phosphofructokinase (PFK) ( Tarui et al. ,
1965 ). The gene was then cloned. Based on this information
the canine PFK gene was sequenced in English
springer spaniel dogs affected with PFK deficiency and a
nonsense mutation identified ( Smith et al. , 1996 ), which
is different from published mutations responsible for PFK
deficiency in humans (reviewed in Nakajima et al. , 2002 ).
Protein-based functional assays are another common way
to determine if a candidate gene is involved in the development
of a disease. This approach led to the diagnosis of
PFK deficiency in English springer spaniel dogs experiencing
hemolysis and myopathy ( Giger et al. , 1985 ).
If there is no candidate gene, a linkage approach
involving a whole genome scan utilizing the molecular
tools described earlier is an option to identify a chromosomal
region or gene linked to the disease. This approach
requires medical and pedigree information and a source
to isolate DNA from a fairly large number of affected and
nonaffected animals. Animal breeding data should make
it possible to acquire the necessary data (pedigrees) and
samples from three-generation pedigrees for linkage studies.
If more than one breed is affected with the same disease,
the different genetic background found in different
breeds may further assist in narrowing the DNA region
of interest. Generally, association studies require an equal
number of affected and unaffected (control) animals from
a population.
2 . Genetic Analysis
The development of genome maps allowed for the mapping
of genes without further knowledge of their function.
Thousands of genetic markers mapped throughout the
genome enable genome-wide linkage or association studies