PANORAIA KYRIAZOPOULOU

ER A SM U S + P L A C E M E N T 2 0 15 - 2 0 1 6

Analysis of 2 unique Greek MHC haplotypes

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).

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.

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.

MHC Structural Classification

Schematic presentation of Ovar-MHC structure- Dukkipati et al., 2006

MHC Genes

CLASS I GENESClassical 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).

MHC Genes

CLASS II GENESClass 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).

MHC GenesOvar-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 genesDRA 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).

MHC Genes

Ovar-DR genes

DRB genesDRB 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.

MHC Genes

Ovar-DQ genes

DQA genesTwo loci, DQA1 and DQA2, exon 2A 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).

MHC Genes

Ovar-DQ genes

DQB genesDQB1 and DQB2- exon 2 (Wright and Ballingall,

1994). Difficulty in assigning sequences to separate loci

because of high similarity between the two DQB genes.

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).

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.

Materials and methods

Breed details:Officially recognised breedArea of distribution: Messinia, PeloponnesePopulation size: 100 animalsRisk status: endangeredColour: white with black headFat tailedWeight ram: 70 kg; ewe: 59 kgHeight ram: 85 cm; ewe: 70 cmUse: milk, meatProductivity milkyield: 140-160kg; littersize: 1.5-1.8

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.

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.

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.

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

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.

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.

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.

Materials and methods

Patterns when cut with RSAI:

DQB patterns

DQA patternsClass I patterns

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%.

Agarose gel with PCR sample removed after 12, 14 and 16 cycles

12 cycles

14 cycles

16 cycles

Click icon to add pictureAgarose gel with PCR sample removed after 18, 20 and 22 cycles

18 cycles

20 cycles

22 cycles

Click icon to add pictureAgarose gel with PCR sample removed after 25 and 28 cycles

25 cycles

28 cycles

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.

178 minipreps later..........

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

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.

Thank you for your attention!