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Explanations on Haplotypes and Haplotype Tests

Haplotypes are pieces of DNA with variation in length that are transmitted entirely over
several generations. Therefore haplotypes can be followed from ancestors to progeny for
generations. It is possible to identify e.g. from what grandparent or great grandparent an
animal got a distinct piece of DNA. But haplotypes are not kept intact for ever. Sooner or
later they are fractured during Meiosis (crossing-over).

Missing Haplotypes
Since the introduction of genomic selection thousands of animals have been genotyped in all
major Holstein populations. The distribution for some haplotypes turned out to be different
than expected by analysing the distribution of haplotypes in big data sets. If two variants (A
and B) of an haplotype exist in a population the expectation is if heterozygous parents are
mated that the offspring are 25% AA, 50% AB, 25% BB like e.g. mating red factor carriers
results in 25% homozygous Red, 50% RDC and 25% homozygous black&white. Despite
hundreds or even thousands of offspring from heterozygous parents only AA and AB
offspring were found and in the ratio 1:2 for some haplotypes. Obviously, the 25% BB
offspring were missing. The only logical explanation for this phenomenon is that individuals
carrying BB already died as embryos i.e. such an animal was never born.

This is how in Holsteins the homozygous missing haplotypes HH1 – HH5 (for Holstein
Haplotype) were discovered that lead to unspecific reduced fertility if carriers are mated.
The marker haplotype does not describes the genetic defect, but the genetic defect must be
somewhere within the haplotype. The haplotype is defined by the SNP markers on the 54K
chip. The gene or gene structure cannot be derived from the SNP markers because in
between two SNP are hundreds or thousands of base pairs. But searching for the underlying
gene defect is effectively possible after narrowing the region by the haplotype. For HH1, HH3
and HH4 the underlying gene defect (mutation) was discovered meanwhile and carriers can
be identified now by a direct gene test. A gene test is valid across populations and does not
lose its predicting power over the generations. In contrast a haplotype test is population
specific and its predictability dependents on total number of genotyped animals, frequency of
the variants, used SNP/chips, relationship structure of population, but on method of genotype
imputing and haplotype definition, too. Reliability of haplotype tests is therefore maximum
99%. And it should be expected that haplotype tests based on different populations give
about 1% differing results.

New gene defect Cholesterol Deficiency

In case of classical genetic recessives analysis starts with the observation of malformed and
dystrophic animals. This was the case, too, for the new genetic cause for calf losses after
non-treatable diarrhoea and calves are leaned completely, called Cholesterol Deficiency
(CD). Via pedigree analysis one tries to find out if there is a joint ancestor on sires and dams
site in the pedigree of all affected for the gene defect homozygous animals. Here a haplotype
analysis helps to detect the region where the mutation must be found. All animals that died
because of “Cholesterol Deficiency (CD)” could be traced back on each sires and dams
pedigree to the Canadian bull Maughlin Storm born 1991. Furthermore all died animals were
homozygous for a specific haplotype on chromosome 11 where Storm is carrier.

In the analysis the entire genotyped population for the CD haplotype some homozygous
animals were found showing no signs of illness and had no Storm in the pedigree. But almost
all of these animals trace back to the 1966 born Fairlea Royal Mark, who himself is the
maternal great grandsire of Storm. So obviously exist in Holsteins two variants of the same
haplotype, the defect Storm variant and the healthy/original F.R.Mark variant. Because the
haplotypes – more precisely the SNP markers on the haplotype – are identical the mutation
must have occurred in the generations between F.R.Mark and Storm somewhere in between
two SNP markers of the haplotype. Therefore based on the 54K SNP the two variants of the
haplotype cannot be differentiated if they carry the mutation or not..

Reliability of CD haplotype test

Because there exist two identical SNP haplotypes with mutation (ill if homozygous) and
healthy/without consequences for the haplotype test are:
- If an animal does not have the haplotype at all it cannot be carrier of the mutation and
therefore negative results have >98% reliability
- If the animal carries the haplotype it might be the variant with mutation or without
mutation. Reliability for positive results is lower i.e. the test gives false-positive
results, too.

Based on the findings from Germany, USA and The Netherlands meanwhile identified a very
similar haplotype. The US haplotype test gave the same result for 99% of the animals as in
Germany, indicating also the same percentage false-positive.

A further differentiation of mutated and healthy/original haplotype within carriers is possible

by pedigree analysis:
- if there is no Storm in the pedigree of a carrier the animal should have the healthy
haplotype i.e. most probably free from the mutation
- if there is only Storm in the pedigree and no F.R.Mark (except as MGGS of Storm)
the animal must have the mutation (true carrier)
- if there are Storm and F.R.Mark found in the pedigree of a carrier the haplotype can
be either the mutated or the healthy and a reliable result is not possible.
Because the separation of mutated and healthy/original haplotype happened many
generations back both variants are spread in the Holstein population and many pedigree
trace back to Storm and F.R.Mark. In the current Holstein population the healthy variant of
the haplotype war spread especially by one famous great granddaughter of F.R.Mark,
Comestar Laurie Sheik. Now it is working in progress to derive a specific probability of having
the mutation by pedigree analysis for every carrier. The probability can range from 50% to

Why no gene test for CD?

By knowing with the haplotype the region, where the mutation must be located in theory it is
possible to find the mutation by sequencing the entire genome and develop a gene test.
Sequencing means enrolling the entire genetic code of 3 billion base pairs and not just the
54,000 SNP. Comparing the sequence of homozygous CD animals with the existing
sequence of known free animals resulted unfortunately in no differences. To understand this,
it is important to know that sequencing is not possible for the entire genome because there
are gaps in cattle reference genome (sequencing is not possible here). Within the region of
the CD haplotype exist 6% gaps. Most probably the mutation has occurred in one of these
gaps. Therefore, chances to find the mutation and develop a gene test with complete
reliability in near future are very low until a new cattle reference genome is available.

Meanwhile the number of SNP in the region of the CD haplotype will be increased on the
customized chip. With the help of these additional SNP markers it might be possible to
further differentiate the two variants of the haplotype. Hopefully the reliability of “animal is
carrier” can be improved significantly. The enhanced chip will be available in October 2015,
so that a refined haplotype test for CD can be developed until end of the year.

vit, Verden
July 2015