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Technical Briefs |
Departments of
1
Surgical Research and
2 Endocrinopathies and Metabolic Diseases, Universitätsklinikum Carl Gustav Carus, Dresden, University of Technology, Fetscherstraße 74, D-01307 Dresden, Germany
aauthor for correspondence; fax 49-351-458-4350, e-mail schacker{at}rcs.urz.tu-dresden.de
CAPN10, a gene that encodes a nonlysosomal cysteine protease, has been recently proposed as a type 2 diabetes susceptibility gene in the non-insulin-dependent diabetes mellitus 1 (NIDDM1) region. Numerous single nucleotide polymorphisms (SNPs) in CAPN10 were identified, but only haplotypes based on four SNPs have been associated with type 2 diabetes in Mexican Americans and two Northern-European populations (from Botnia, Finland and Dresden, Germany) (1). Two major haplotypes in CAPN10 show an association with altered mRNA expression, increased risk of type 2 diabetes in multiple ethnic groups, and findings of insulin resistance in nondiabetic individuals (1)(2)(3).
Studies investigating genetic variations in CAPN10 in different populations have yielded variable results. Some studies confirmed the association of the at-risk haplotype of CAPN10 112/121 with type 2 diabetes in Caucasian populations (3)(4). Further support of the concept that genetic variation in CAPN10 is involved in type 2 diabetes has recently been found in Caucasian populations in which an association of the homozygous 121 (5) and 111/221 haplotype combination with type 2 diabetes has been reported (6). Additional studies have found associations between the homozygosity of the G-allele of UCSNP-43 and, respectively, higher fasting plasma glucose and lower glucose turnover during a low-insulin euglycemic clamp in Pima Indians (2), type 2 diabetes in African-Americans (7), and increased free fatty acid levels in Caucasians (4). In other studies, the population frequency of the 112/121 risk haplotype of CAPN10 was low, so that demonstrating significant associations between the CAPN10 variants and type 2 diabetes in Chinese (8), Samoans(9), Oji-Crees(10), or Caucasians from Finland and Scandinavia (11)(12) was not possible.
To rapidly and cost-effectively screen for the already known CAPN10 SNPs representing genetic variants of the CAPN10 gene, we have established an assay based on the LightCyclerTM technique (Roche Molecular Systems). Template DNA amplification was performed with real-time PCR, and fluorescence resonance energy transfer (FRET) technology was used to facilitate the online melting-curve analysis of oligonucleotide probes bound to the target SNPs (2)(13)(14)(15). We hereby report the use of a LightCycler assay in the determination of the five known haplotypes found in the vast majority of individuals based on the genotyping of four SNPs.
After PCR on the LightCycler, we used hybridization probes in combination with the LightCycler DNA Master Hybridisation Probes Kit (Roche Diagnostics). PCR primers and hybridization probes were synthesized by TIB MOLBIOL (Berlin). Oligonucleotide sequences and hybridization probes sequences are shown in Table 1A
. PCR was performed in a total volume of 20 µL in glass capillaries. The reaction mixture used in each PCR consisted of 50 ng of genomic DNA, 2 µL of each primer (5 µmol/L), 2 µL of the DNA Master Hybridisation Probes Kit reagent, 2µL of each hybridization probe (1.5 µmol/L), and 2.4 µL of MgCl2 (25 mmol/L). After 30 s of an initial denaturation at 94 °C, 40 PCR cycles were performed with 1 s of denaturation at 94 °C and 10 s of annealing at 58 °C for SNPs 44, 43, and 56, and at 63 °C for SNP 63, with a 9-s extension at 72 °C.
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Both the PCR and the melting procedures were detected online with the LightCycler instrument. The fluorescence signal (F) was plotted in real time against the temperature (T) to generate melting curves for each sample (F vs T); the curves were then converted to melting peaks by plotting the negative derivative of F with respect to T against T (-dF/dT against T) (13)(14). Melting peaks generated from fully homologous sequences during the LightCycler-based detection of sequence variants have a higher melting temperature (Tm) than the melting peaks generated from the mismatch sequences. In heterozygous samples containing both sequences, both temperature peaks can be detected (16). The estimated Tm from the derivative melting curve is highly reproducible under constant reaction conditions of heating rate, salt concentration, and probe-target concentrations. The within-run variation between multiple replicate samples is only 0.1 °C, and the between-run variation is within 0.5 °C (17).
All of the genotypes were verified independently by DNA sequence analysis using Sanger sequencing and fragment length polymorphism analysis using standard protocols.
Our hybridization probes perfectly matched the alleles regarded as the common alleles in the Mexican-American population (3); therefore, in a homozygous state (1/1), our probes produced melting curves at higher temperatures than with a variant sequence (2/2). The heterozygous genotype (1/2) showed both melting curves at different temperatures (Fig. 1
). For technical reasons, we used SNP 56 instead of SNP 19 because the former is in nearly perfect linkage disequilibrium with SNP 19 in the various populations tested to date (18).
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The analysis of genotypes of all four SNPs has enabled the construction of five different CAPN10 haplotypes found in more than 99.9% of all tested individuals (1)(18) (Table 1B
). Haplotype combinations 121/112(1) and 121/121 (5) have been associated with an increased risk for diabetes type 2. In addition, haplotype combination 1111/2111 has been shown to increase the risk for type 2 diabetes in Caucasians (3).
To validate our approach, we have genotyped 336 randomly selected type 2 diabetic patients seen at our department. Diabetes was diagnosed using the criteria from WHO. All of the patients came from the city of Dresden or surrounding areas, and written informed consent for the genetic analysis was obtained. The protocol for this study was approved by the Ethics Committee of the Medical Faculty of the Technical University Dresden. The mean (SD) age of the probands was 59.8 (8.3) years; the mean (SD) body mass index was 29.0 (4.2) kg/m2; the mean (SD) age of disease onset was 52.1 (7.1) years; and treatment consisted of only diet in 13.2% of cases, of oral antihyperglycemic agents in 33.4% of cases, and of insulin in 52.4% of cases. Diabetes-associated complications were found as follows: retinopathy in 36.4%; nephropathy in 26%; neuropathy in 17%; and macroangiopathy in 8% of the patients.
The genotyping of SNPs 44, 43, 56, and 63 in CAPN10 of 336 patients by the LightCycler assay revealed genotype combinations that were consistent with the 15 possible haplotype combinations derived from the 5 known haplotypes, without exception. The frequencies of haplotype combinations identified in our study are presented in Table 1C
. Frequencies were similar to those reported in other genotyping studies of CAPN10 in Caucasian populations (1)(3).
Because only 5 of 16 possible haplotypes comprise more than 99.9% of all haplotypes found in various populations, genoptyping of the SNPs 44, 43, 56, and 63 enables haplotype construction. It cannot be excluded that, in rare cases, this approach will not identify the true haplotype, although it has been impossible to demonstrate unambiguously the existence of missing haplotypes (18).
A rapid screening assay is warranted to analyze the association of the haplotypes with additional clinical indices of diabetes and to test the findings in different ethnic groups. Predictive testing for diabetes with CAPN10 haplotypes can be established only if large scale and reproducible data generated by reliable genotyping assays are available. The LightCycler assay for the detection of CAPN10 polymorphisms offers a rapid and reliable method for association studies.
Acknowledgments
We are grateful to Dr. Olfert Landt (TIB MOLBIOL, Berlin, Germany) for design of the probes. We thank Dr. Alexandre Serra for assistance in the preparation of the manuscript.
Footnotes
1 these authors contributed equally to this project 
References
The following articles in journals at HighWire Press have cited this article:
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  H. Gorgens, G. Fitze, D. Roesner, and H. K. Schackert One-Step Analysis of Ten Functional Haplotype Combinations of the Basic RET Promoter with a LightCycler Assay Clin. Chem., September 1, 2004; 50(9): 1693 - 1695. [Full Text] [PDF]
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