Absolute quantification reveals the stable transmission of a high copy number variant linked to autoinflammatory disease.
BACKGROUND
Dissecting the role copy number variants (CNVs) play in disease pathogenesis is directly reliant on accurate methods for quantification. The Shar-Pei dog breed is predisposed to a complex autoinflammatory disease with numerous clinical manifestations. One such sign, recurrent fever, was previously shown to be significantly associated with a novel, but unstable CNV (CNV_16.1). Droplet digital PCR (ddPCR) offers a new mechanism for CNV detection via absolute quantification with the promise of added precision and reliability. The aim of this study was to evaluate ddPCR in relation to quantitative PCR (qPCR) and to assess the suitability of the favoured method as a genetic test for Shar-Pei Autoinflammatory Disease (SPAID).


RESULTS
One hundred and ninety-six individuals were assayed using both PCR methods at two CNV positions (CNV_14.3 and CNV_16.1). The digital method revealed a striking result. The CNVs did not follow a continuum of alleles as previously reported, rather the alleles were stable and pedigree analysis showed they adhered to Mendelian segregation. Subsequent analysis of ddPCR case/control data confirmed that both CNVs remained significantly associated with the subphenotype of fever, but also to the encompassing SPAID complex (p < 0.001). In addition, harbouring CNV_16.1 allele five (CNV_16.1|5) resulted in a four-fold increase in the odds for SPAID (p < 0.001). The inclusion of a genetic marker for CNV_16.1 in a genome-wide association test revealed that this variant explained 9.7 % of genetic variance and 25.8 % of the additive genetic heritability of this autoinflammatory disease.


CONCLUSIONS
This data shows the utility of the ddPCR method to resolve cryptic copy number inheritance patterns and so open avenues of genetic testing. In its current form, the ddPCR test presented here could be used in canine breeding to reduce the number of homozygote CNV_16.1|5 individuals and thereby to reduce the prevalence of disease in this breed.
Department of Medicine, Rheumatology Unit, Karolinska Institute, Stockholm, Sweden.
M
M
Olsson
Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
M
M
Kierczak
Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
Å
Å
Karlsson
Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
J
J
Jabłońska
Department of Clinical Sciences of Companion Animals, Utrecht University, Utrecht, Netherlands.
P
P
Leegwater
Broad Institute of MIT and Harvard, Boston, MA, USA.
M
M
Koltookian
LUNAM University, Oniris, AMaROC Unit, Nantes, F-44307, France.
J
J
Abadie
ANTAGENE Animal Genetics Laboratory, La Tour de Salvagny, Lyon, 69, France.
C Dufaure
CD
De Citres
ANTAGENE Animal Genetics Laboratory, La Tour de Salvagny, Lyon, 69, France.
A
A
Thomas
Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden.
Å
Å
Hedhammar
Wurtsboro Veterinary Clinic, Wurtsboro, New York, USA.
L
L
Tintle
Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.|Broad Institute of MIT and Harvard, Boston, MA, USA.
K
K
Lindblad-Toh
Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden. jennifer.meadows@imbim.uu.se.
J R S
JR
Meadows
D000483:Alleles; D000818:Animals; D001327:Autoimmune Diseases / Q000235:genetics / Q000662:veterinary*; D056915:DNA Copy Number Variations; D004283:Dog Diseases / Q000235:genetics*; D004285:Dogs; D014644:Genetic Variation; D055106:Genome-Wide Association Study; D060005:Genotyping Techniques; D006720:Homozygote; D010375:Pedigree; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results
Autoinflammation; Copy number variation; Droplet digital PCR; Quantitative PCR