1. PANORAIA KYRIAZOPOULOU
E R A S M U S + P L A C E M E N T 2 0 1 5 - 2 0 1 6
Analysis of 2 unique Greek
MHC haplotypes
2. What is the MHC?
The MHC – Major Histocompatibility Complex is a
genetic region comprised of tightly linked genes.
It has been referred to as ovine leukocyte antigen
(OLA) or sheep lymphocyte antigen and now
following the nomenclature system for the MHC of
vertebrates (Klein et al., 1990), it is designated as
‘Ovar-Mhc’ (‘Ovar’ representing Ovis aries).
3. What is the purpose of the MHC?
Purpose:
Encoding molecules playing a central role in
immunological response.
Codes for specialised antigen-presenting receptor
glycoproteins, known as histocompatibility
molecules or MHC molecules.
These molecules bind processed peptide antigens
and present them to T lymphocytes triggering
immune responses.
Communication between cells during immune
response.
4. Why do we study the MHC?
• Alleles of different MHC genes have been found to
contribute in disease resistance/susceptibility.
• The MHC alleles are markers of biodiversity.
• Selection for productivity traits/ disease research.
• Mate choice for many vertebrates through olfactory
cues.
6. MHC Genes
CLASS I GENES
Classical and non-classical genes.
Present peptides to CD8+ cytotoxic T cells.
Interact with natural killer (NK) cells to prevent NK-
mediated cell lysis (Reyburn et al., 1997).
Controversy over the number of classical class I loci - at
least four distinct polymorphic loci have been identified
(Miltiadou et al., 2005).
7. MHC Genes
CLASS II GENES
Class II genes have antigen peptide presenting role to the
TCR on CD4+ helper T cells.
In the HLA complex, these include five sets of the
classical genes DP, DM, DO, DQ, and DR and non-
classical genes such as LMP, TAP and TAPBP.
Not all sets contain genes for both chains, although some
contain many pseudogenes (Tizard, 2004).
8. MHC Genes
Ovar-DR genes
DR genes highly polymorphic
molecules encoded by these genes are expressed in higher
levels.
The DR heterodimer consists of an α-chain and a β-chain,
encoded by DRA and DRB genes.
Class II region of the MHC of sheep (OLA).
DRA genes
DRA gene is considered to be almost monomorphic.
A significantly divergent DRB1 allele (Ovar-DRB1*0901), was
linked to polymorphism at the DRA locus in domestic sheep
(Ballingall et al., 2010).
9. MHC Genes
Ovar-DR genes
DRB genes
DRB locus is the most polymorphic among the MHC
genes.
Ovar-DRB genes exist in multiple copies, some
functional and others non-functional.
Four Ovar-DRB loci have been described (Scott et al.
1991b).
Majority of nucleotide polymorphism at Class II loci
locates in the second exon and adjoining intron 2.
10. MHC Genes
Ovar-DQ genes
DQA genes
Two loci, DQA1 and DQA2, exon 2
A number of haplotypes the DQA1 gene appears absent
(DQA1 null; Fabb et al. 1993) and is replaced by a second
locus more closely related to DQA2 (DQA2-like; Hickford
et al. 2004)
DQA2-like allelic lineage appears functional (Ballingall
et al., 2015).
11. MHC Genes
Ovar-DQ genes
DQB genes
DQB1 and DQB2- exon 2 (Wright and Ballingall, 1994).
Difficulty in assigning sequences to separate loci because
of high similarity between the two DQB genes.
12. Purpose of study
Characterising the polymorphism in the Ovar-MHC
region in a rare Greek sheep breed ~Argos.
Genes studied: DRB1, DRA, DQA and DQB (partial as
well as full-length allelic sequences).
13. Materials and methods
Argos breed:
Originally from Asia Minor.
Single pure flock in Messinia.
Six flocks near Argos with over 50% purity.
Isolated pure specimens in mixed flocks.
14. Materials and methods
Breed details:
Officially recognised breed
Area of distribution: Messinia, Peloponnese
Population size: 100 animals
Risk status: endangered
Colour: white with black head
Fat tailed
Weight ram: 70 kg; ewe: 59 kg
Height ram: 85 cm; ewe: 70 cm
Use: milk, meat
Productivity milkyield: 140-160kg; littersize: 1.5-1.8
15. Materials and methods
At the start of this study, blood was taken from the 2
Argos sheep.
Genomic DNA was prepared.
RNA was extracted and brought to the Moredun
Research Institute.
16. Materials and methods
DRB1 genotyping:
Primers used for sequence based genotyping were 455/329
(Ballingall and Tassi, 2010, and unpublished data).
PCR cycling profile:
1 cycle 5 minutes, 94o C,
35 cycles of 30 s at 94°C,
30 s at 60°C,
30 s at 72°C
followed by 4 min at 72°C.
17. Materials and methods
DRB1 genotyping:
For the mother (Animal 251 or Argos 28) the 3-prime end
was amplified by designing a forward allele-specific
primer 462 (Unpublished data).
Nested approach :primers 455 and 201
annealing temperature at 55°C for 30 cycles
then 2ul PCR product added into the second round with the
new primer and 201
annealing temperature of 60 °C for 35 cycles.
18. Materials and methods
DRB1 Full-length genotyping:
primers 204/207 and 205/207 (Ballingall et al., 2008)
internal primers 222/223
control primers 414/415
PCR cycling profile:
1 cycle 5 minutes, 94o C,
55 cycles of 1min at 94°C,
1 min at 60°C,
1 Min at 72°C
followed by 4 min at 72°C
19. Materials and methods
DQA/DQB genotyping
Primers:
DQA1: 314/315 DQA2: 316/317
DQA2-like: 316/317
DQB1: 363n/ 365n
DQB2:363n/406
PCR cycling profile:
1 cycle 5 minutes, 94o C,
35 cycles of 30 s at 94°C,
30 s at 58°C,
30 s at 72°C
followed by 4 min at 72°C.
DQA/DQB full-length
genotyping:
Primers:
DQA1/2: 283/241
244/241
347/349
DQB1/2: 245/247
246/248
PCR cycling profile:
1 cycle 3 minutes, 94o C,
35 cycles of 30 s at 94°C,
30 s at 55°C,
30 s at 72°C
followed by 3 min at 72°C.
20. Materials and methods
Class I genotyping:
Primers: 415/409
PCR cycling profile:
1 cycle 5 minutes, 94o C,
30 cycles of 30 s at 94°C,
30 s at 55°C,
30 s at 72°C
followed by 4 min at 72°C.
Class I full-length
genotyping:
Primers: 417/411 417/412
410/412 413/411 413/412
PCR cycling profile:
1 cycle 5 minutes, 94o C,
30 cycles of 30 s at 94°C,
30 s at 55°C,
30 s at 72°C
followed by 4 min at 72°C.
21. Materials and methods
Efficiency of PCR was checked on a 1% agarose .
Remaining PCR product purified.
Bidirectional sequencing.
After receiving the first results the selected PCR
fragment was cloned, screened and digested with
RSAI and sent for sequencing.
22. Materials and methods
Patterns when cut with RSAI:
DQB patternsDQA patterns
Class I patterns
23. Materials and methods
Levels of hybridisation were very high for the Class I
using a PCR profile of 30 cycles.
New cDNA was prepared and PCR using primers
416/409 was repeated.
The samples were removed after 12, 14, 16, 18, 20,
22, 25 and 28 cycles and ran on agarose gels 1%.
27. Materials and methods
The gel fragment for 16, 18 and 20 cycles was cut out
and cleaned up.
PCR screening showed that the transformation
worked effectively and samples from colonies
selected were sent for sequencing.
The results received after this procedure showed
significantly lower levels of hybridisation.
29. Results
New DQA1 and DQA2 alleles found.
New DQB1 and 2 DQB2 alleles found.
5 new classical Class I alleles found.
2 new non-classical Class I alleles found.
High levels of diversity
30. Results
Optimal PCR cycles for lower levels of hybridisation
in Class I : 16-20
Hybrids at 30 cycles: 11/30 and PCR errors
Hybrids at 16-20 cycles: 1/40, few PCR errors
This may mean some alleles that appear in low
frequencies might be missed.
MHC molecules act by binding peptide fragments of antigens that have been processed in specialized antigen-presenting cells. Clonally determined antigen receptors on T cells then recognize and bind to specific peptide-MHC complexes, setting into motion the appropriate immune response. Segments of MHC molecules show sequence homologies with immunoglobulins, T cell antigen receptors, and T cell interaction molecules such as CD4 and CD8, which suggests that all these molecules share a common evolutionary ancestry.
(intracellular proteins and parasites)
(mainly derived from extracellular proteins and parasites)
genotyping studies usually focus on the DRB1 gene to associate MHC diversity with resistance or susceptibility to disease, as the DRB1 exon 2 encodes the b1 domain, which constitutes part of the PBR of the DR molecules which in turn are in close contact with the peptides presented in the PBR or the TCR
. Previous analyses described the duplication of the DQA and DQB genes within the MHC of sheep (Scott et al. 1987) with the majority of haplotypes having both DQA1 andDQA2 loci (Hickford et al. 2007). However, in
The efficiency of the PCR was checked by analyzing 10 µl of the PCR product on a 1% agarose gel and the remaining PCR product was then purified to remove excess nucleotides and primers, and the samples were sent for bidirectional sequencing
using the Wizard® SV Gel and PCR Clean-Up System by Promega and ligation and transformation followed. In order to achieve the formation of clones higher concentration was spread on agarose plates (50 μl, 200μl and 250μl).
making it easier to distinguish new alleles avoiding false interpretations of the sequencing results.
