Development of a high-resolution melting genotyping assay for the angiotensin I converting enzyme insertion/deletion polymorphism and establishment of genotype-specific reference intervals in a Danish population.
BACKGROUND
The serum-angiotensin I converting enzyme (s-ACE) activity is influenced by a genetic insertion/deletion (I/D) polymorphism in the ACE gene, and the resulting large interindividual variation in s-ACE limits the use of normal reference intervals in the evaluation of sarcoidosis. In this study, we developed a new method for genotyping the I/D polymorphism in ACE and established genotype-specific reference intervals in order to improve the diagnostic accuracy and the value for treatment of sarcoidosis.


METHODS
The new genotyping assay is based on high-resolution melting (HRM) using LCGreen + and was used to genotype 400 healthy Danish individuals. The assay was compared to a real-time polymerase chain reaction (RT-PCR) assay in a validation set of 86 samples. Enzyme activity in serum was measured using the Infinity™ ACE Liquid Stable Reagent from Thermo adapted for the ABX Pentra analyzer.


RESULTS
There was full concordance between genotyping assays. The three genotypes II, ID and DD were present with a frequency of 0.23, 0.51 and 0.26. The distribution of s-ACE values in the total population was non-Gaussian (non-parametric 95% reference interval 12.0-60.0 U/L). The median activities of the genotypes differed significantly (P<0.0001). Ninety-five per cent non-parametric reference intervals for the subpopulations were determined to 6.3-38.5, 14.0-56.0 and 23.3-71.2 U/L for II, ID and DD, respectively.


CONCLUSIONS
We have developed a simple and robust method for ACE genotyping and determined genotype-specific reference intervals for s-ACE concentrations in the Danish population. The new reference intervals may increase the value of s-ACE measurements.
Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark peter.h.nissen@aarhus.rm.dk.
Peter H
PH
Nissen
Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark.
Nina Buntzen
NB
Campbell
Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark.
Carsten S
CS
Højskov
Department of Respiratory Diseases, Aarhus University Hospital, Aarhus, Denmark.
Andreas
A
Fløe
Department of Respiratory Diseases, Aarhus University Hospital, Aarhus, Denmark.
Hans Jürgen
HJ
Hoffmann
Department of Respiratory Diseases, Aarhus University Hospital, Aarhus, Denmark.
Ole
O
Hilberg
Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark.
Søren A
SA
Ladefoged
Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark.
Holger J
HJ
Møller
D001681:Biological Assay / Q000592:standards*; D001782:Blood Donors; D003718:Denmark; D005260:Female; D005787:Gene Frequency; D005838:Genotype; D060005:Genotyping Techniques; D006801:Humans; D054643:INDEL Mutation; D008297:Male; D009691:Nucleic Acid Denaturation; D007703:Peptidyl-Dipeptidase A / Q000097:blood / Q000235:genetics*; D011110:Polymorphism, Genetic; D060888:Real-Time Polymerase Chain Reaction; D012016:Reference Values; D012507:Sarcoidosis / Q000097:blood / Q000175:diagnosis / Q000235:genetics
C488034:ACE protein, human; D007703:Peptidyl-Dipeptidase A
Angiotensin I converting enzyme; I/D polymorphism; genotype-specific reference intervals; high-resolution melting; molecular diagnostics; peptidyl-dipeptidase; sarcoidosis