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Calpain-10 Haplotype Combination and Association With Gestational Diabetes Mellitus

Heinz Leipold, MD^*, Martin Knöfler, PhD^*, Christian Gruber, MD, Peter Haslinger^*, Dagmar Bancher-Todesca, MD^* and Christof Worda, MD^*

From the Department of Obstetrics and Gynaecology, *Division of Obstetrics and Division of Gynecological Endocrinology and Reproductive Medicine, University of Vienna Medical School, Waehringer Guertel 18–20, Vienna, Austria.

Address reprint requests to: Christof Worda, MD, Department of Obstetrics and Gynaecology, University of Vienna Medical School, Waehringer Guertel 18–20, A-1090 Vienna, Austria; e-mail: christof.worda{at}akh-wien.ac.at.

	ABSTRACT

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OBJECTIVE: Gestational diabetes mellitus (GDM) is a frequent complication of pregnancy. Epidemiologic and pathophysiologic data suggest a close link of this disease to non-insulin-dependent diabetes mellitus. Within the calpain-10 gene various single-nucleotide polymorphisms have been identified that increased the risk for non-insulin-dependent diabetes mellitus. Therefore, we examined single-nucleotide exchanges of this gene in women with GDM.

METHODS: A total of 875 unselected women were prospectively screened for GDM. Eighty women of this population, 40 patients with an abnormal oral glucose tolerance test and 40 normal controls, were randomly selected. DNA samples isolated from sera of the control and study groups were analyzed with respect to single-nucleotide polymorphisms of the calpain-10 gene at positions 43, 19, and 63 using polymerase chain reaction amplification and restriction analysis.

RESULTS: Women with GDM were more likely to be homozygous for the allele 1 of single-nucleotide polymorphism 63 (P = .02 by ² test). With respect to single-nucleotide polymorphisms 19 and 43, no significant differences in allele distribution were detected between controls and women with GDM. When comparing the different haplotypes for calpain-10 (single-nucleotide polymorphisms 43, 19, and 63), all women with the haplotype combination 121/221 (n = 8) had gestational diabetes (P = .005 by Fisher exact test).

CONCLUSION: Our results indicate that the haplotype 121/221 of the calpain-10 gene may be associated with disturbances of glucose metabolism during pregnancy.

LEVEL OF EVIDENCE: II-1

In women with GDM the free fatty acids-mediated insulin resistance, which is physiological during pregnancy, cannot be compensated because of a ß-cell defect.^3,4 Additionally, women who once had GDM display an increased risk for developing non-insulin-dependent diabetes mellitus in their later life.^5,6 GDM as well as non-insulin-dependent diabetes mellitus are polygenic, multifactorial diseases.³ In 1996, Hanis et al⁷ identified a region on chromosome 2q37 with potential relevance to non-insulin-dependent diabetes mellitus. The calpain-10 gene was found to be located within this particular region, and several of its single-nucleotide polymorphisms have been related to the development of non-insulin-dependent diabetes mellitus.^8–10

The calpain-10 gene encodes a calcium-dependent protease that is present in most tissue cells catalyzing the oxidation of glucose in skeletal muscle cells.⁸ Various studies showed that the allele combination 112/121 (single-nucleotide polymorphisms 43, 19, and 63) in the calpain-10 gene increased the risk of non-insulin-dependent diabetes mellitus up to 3.6-fold in a Mexican-American population.⁸ Additionally, the allele combination 112/121 was associated with impaired glucose tolerance and impaired fasting glucose.⁸ Given the possible pathophysiologic and etiologic link between GDM and non-insulin-dependent diabetes mellitus, we were interested in learning whether the gene encoding calpain-10 plays a role in GDM. We therefore examined the 10 most common haplotype combinations (single-nucleotide polymorphisms 43, 19, and 63) of this gene associated with non-insulin-dependent diabetes mellitus in women suffering from GDM.

	MATERIALS AND METHODS

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The study was conducted with the approval of our institutional review board, and informed consent was obtained from all participants according to Austrian gene technology law. An oral glucose tolerance test (OGTT) was performed between the 24th and 28th weeks of gestation in all women who intended to deliver at the Vienna University Hospital, Department of Obstetrics and Gynecology. Preceded by an 8-hour period of fasting, a standardized 75-g glucose solution (GLUCO-DRINK 75; Unipack GmbH, Wiener Neustadt, Austria) was ingested orally. Venous blood samples were drawn before glucose ingestion, then after 1 hour and after 2 hours. On the same day a measurement for glycosylated hemoglobin (Hb A_1C) was performed, and we stored venous blood for DNA analysis from the mothers at –20°C until extraction. Before the test a precise medical and obstetric history was taken from the women and entered into a computerized database. We followed the guidelines of the German Society for Diabetes to evaluate the results of the OGTT. The upper normal limit for the fasting serum glucose was set at 90 mg/dL, 180 mg/dL 1 hour after glucose ingestion and 155 mg/dL 2 hours after glucose ingestion. If at least 1 value was exceeded, we classified the woman as having gestational diabetes and admitted her to the program, which started with dietary instruction lessons. After the instructions were given, we asked the women to measure their capillary blood glucose concentrations at home daily for 1 week before and 1 hour after meal. Upper limits of 90 mg/dL for fasting glucose concentration and 130 mg/dL for 1 hour postprandially glucose concentration were considered acceptable. If a woman exceeded these limits 5 or more times a week, insulin therapy was initiated.

We included only Caucasian women living in Vienna in this study, and entered a total of 875 subjects into our database between June 2002 and February 2003. All women received examination in short time intervals with an initial contact normally between the fifth and eighth week of pregnancy. The examinations include ultrasound examinations, complete gynecologic examination with Pap test, and blood tests. This program (Mother Child Pass) is paid for by the Austrian government. Immediately after delivery, cord blood was obtained from the umbilical vein of all newborns enrolled in the study. Of this cohort, we randomly selected 40 women with physiologic OGTT results (group I) and 40 women with insulin-requiring GDM (group II) by using a computer-generated table of random numbers. We excluded women with non-insulin-dependent GDM, a history of multiple pregnancies, fetal abnormalities, preexisting hypertension, preexisting diabetes mellitus, or other chronic disease. For calculating the risk for GDM, we used the same calpain-10 haplotype combinations (single-nucleotide polymorphism 43: allele 1 = guanine, allele 2 = adenine; single-nucleotide polymorphism 19: allele 1 = 2 repeats of 32 base pairs sequence, allele 2 = 3 repeats of this sequence; single-nucleotide polymorphism 63: allele 1 = cytosine, allele 2 = thymine) for analysis as reported by Horikawa et al.⁸

Genomic DNA was isolated from anticoagulated blood by using the QiAmp Blood Midi Kit, as described by the manufacturer (Quiagen, Hilden, Germany), and stored at –20°C. Glucose was determined immediately after sampling with an electrochemiluminescence immunoassay on an Elecsys 2010 immunoassay analyzer (Boehringer-Mannheim GmbH, Mannheim, Germany).

For analyzing single-nucleotide polymorphism 43 (CAPN10-g.4852G/A), we genotyped the women by using 1 forward primer and 2 different allele-specific reverse primers in independent polymerase chain reactions (PCRs): forward primer 5'-CATCCATAGCTTCCACGCCTC-3'; reverse primer allele 1 (G) 5'-GCTTAGCCTCACCTTCAATC-3', and reverse primer allele 2 (A) 5'-ATCCTCACCAAGTCAAGCGTTAGCCTCACCTTCAAGT-3'. We conducted the PCR in a volume of 25 µL, consisting of 1 x PCR buffer, 200 µmol of dNTP, 1.5 mmol of MgCl₂, 0.5 U TaqDNA Polymerase (Invitrogen, Carlsbad, CA), and 40 ng of genomic DNA. The concentration of each primer was 20 pmol/µL. Cycle conditions were 94°C for 1 minute; 35 cycles at 94°C for 30 seconds, 53°C for 30 seconds, 72°C for 30 seconds, and 72°C for 10 minutes (allele 1). Allele 2 was amplified by using 94°C for 1 minutes; 35 cycles at 94°C for 30 seconds, 64°C for 30 seconds, 72°C for 30 seconds and 72°C for 10 minutes. DNA amplified by PCR was separated on a 3% agarose gel (50% NuSieve [FMC-Bioproducts, Rockland, ME], 50% LE agarose [Invitrogen, Carlsbad, CA]) and stained with ethidium bromide. Allele 1 (G) measured 134 base pairs and allele 2 (A) 152 base pairs.

Single-nucleotide polymorphism 19 (CAPN10-g.7920indel32bp) was detected by forward and reverse primers 5'- GTTTGGTTCTCTTCAGCGTGGAG-3' and 5'- ATGAACCCTGGCAGGGTCTAAG-3'. We conducted the PCR in a volume of 25 µL, consisting of 1 x PCR buffer, 200 µmol of dNTP/L, 1.5 mmol of MgCl₂/L, 0.5 U TaqDNA Polymerase (Invitrogen), and 40 ng of genomic DNA. Cycle conditions were 94°C for 1 minute; 35 cycles at 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, and 72°C for 10 minutes. DNA amplified by PCR was separated on a 3% agarose gel (50% NuSieve, 50% LE agarose). Allele 1 (2 repeats of the 32-base pairs sequence) measured 155 base pairs and allele 2 (3 repeats) 187 base pairs.

Single-nucleotide polymorphism 63 (CAPN10-g.16378C/T) was detected by using the forward and reverse primers 5'-AGGGGGGCCAGGGCCTGACGGGGGTGGCG-3' and 5'-AGCACTCCCAGCTCCTGATC-3'. PCR conditions were the same as for single-nucleotide polymorphism 19 with the exception of the annealing temperature, which was set at 62°C. We purified the PCR products using QIAquick PCR purification kit as described by the supplier (Qiagen). PCR fragments were digested with 25 U Hhal in 1x NEB4 buffer plus 1 x calf-albumin at 37°C for 4 hours. DNA products amplified by PCR were separated on a 3% agarose gel (50% NuSieve, 50% LE agarose). Allele 1 (C) and allele 2 (T) measured 162 base pairs and 192 base pairs, respectively.

Data were evaluated by using SPSS 10.0 for Windows (SPSS Inc, Chicago, IL). We performed a Kolmogorov-Smirnov test to verify the use of tests for normally distributed variables. Values are given as mean plus/minus standard deviation (sd). Chi-square, Fisher exact, and t tests were used accordingly. Bonferroni-Holm correction was used for the calpain-10 haplotype comparisons.¹¹ Our calculation using Fisher exact test with a 5% 2-sided significance level has a 80% power to detect the difference between a control group (women with physiologic OGTT) proportion of 1% and a study group (women with GDM) proportion of 20%. All tests were 2-tailed, and we considered a P .05 statistically significant.

	RESULTS

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We assessed a total of 875 women by OGTT. Among them, 713 women (81.5%) displayed a physiologic OGTT. We classified 162 women (18.5%) as having gestational diabetes and treated them with human insulin. The high rate of women with GDM may be a result of the fact that our department is a tertiary care center. We analyzed 80 women for the calpain-10 genotype. The distribution of their genotypes (single-nucleotide polymorphisms 19, 43, and 63) and allele frequencies are shown in Table 1.

Women with GDM were significantly older (33.6 ± 4.8 years versus 31.0 ± 6.2 years; P = .04 by t test), had a higher body mass index (BMI) (27.6 ± 6.1 kg/m² versus 24.9 ± 5.7 kg/m²; P =.04 by t test) and displayed higher Hb A_1C values at the time of the OGTT (5.8 ± 1.0 versus 4.8 ± 0.5; P .001 by t test). The newborns of mothers with GDM had higher cord blood insulin concentrations (14.11 ± 10.8 µU/mL versus 7.6 ± 6.7 µU/mL; P = .02 by t test) than newborns of women with physiologic OGTT results (Table 2). Women with GDM were more likely to be homozygous for the allele 1 of single-nucleotide polymorphism 63 than women without GDM (29 [59%] versus 20 [41%]; P = .02 by ² test). With respect to single-nucleotide polymorphisms 19 and 43, no significant differences in allele distribution were detected between the 2 groups of women (Table 1).

When comparing the different haplotypes for calpain-10 (single-nucleotide polymorphisms 43, 19, and 63), all women with the haplotype combination 121/221 (n = 8) had GDM (Table 3). Additionally, women with the haplotype combination 121/221 displayed significantly higher fasting glucose concentrations and 1-hour values in the OGTT. However, no significant differences were seen with respect to Hb A_1C and 2-hour OGTT values in women with this haplotype compared with others (Table 4).

	DISCUSSION

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With a reported prevalence of 1–20% in all pregnancies, GDM accounts for one of the most frequent gestational complications.¹ Various risk factors are currently known and include maternal obesity, age, ethnic origin, and a family history as well as individual history of previous disturbances in glucose metabolism.^12,13 Although genetic factors are likely to be involved in the onset of GDM, no gene loci have been identified to date.

A pathophysiologic link between GDM and non-insulin-dependent diabetes mellitus seems likely and is substantiated by the fact that women with GDM have up to a 50% chance of developing non-insulin-dependent diabetes mellitus over the next 10 years.¹⁴ From 6 controlled follow-up studies, the overall relative risk for developing diabetes after GDM was calculated to be 6.0 (95% confidence interval 4.1, 8.8).¹⁵ Consequently, one would expect gene alterations, such as single-nucleotide changes, that are associated with non-insulin-dependent diabetes mellitus to be present in women with GDM. Horikawa et al,⁸ Cassell et al,⁹ and Malecki et al¹⁰ have shown that single-nucleotide polymorphisms in the gene encoding calpain-10 are associated with an increased risk for non-insulin-dependent diabetes mellitus, although other studies do not agree.^16–18 In October 2003, we searched the medical databases MEDLINE and EMBASE using the key words "calpain" and "gestational diabetes" to identify trials in the English literature. We did not find studies that examined the role of mutations of the calpain-10 gene in the development of GDM.

In our study, we investigated 3 single-nucleotide polymorphisms of the gene encoding calpain-10 known to increase the risk for non-insulin-dependent diabetes mellitus in women with and without GDM. Horikawa et al⁸ demonstrated a significant association between the haplotype 112/121 and the risk for non-insulin-dependent diabetes mellitus in Mexican-Americans. Subsequent studies partially confirmed these results.^9,10 Because there are presently no studies that examined these single-nucleotide polymorphisms in the calpain-10 gene with respect to GDM risk and because a relationship between these two diseases is likely, we compared our results with those dealing with non-insulin-dependent diabetes mellitus.

Interestingly, the haplotype 112/121, highlighted by Horikawa et al⁸ for non-insulin-dependent diabetes mellitus, did not yield an increased risk of GDM in our study cohort. This difference could be explained by the limited number of cases in our study but could also be due to an only partially shared genetic background for non-insulin-dependent diabetes mellitus and GDM. Non-insulin-dependent diabetes mellitus will develop in some but not all women with GDM in their later life. From a genetic point of view, one might speculate that the same genes are affected in both diseases, but in different regions.

In our study, however, all women with the 121/221 haplotype had GDM and displayed pathologic OGTT results without exception. Even when we used the 75-g glucose load OGTT criteria as recommended by the American Diabetes Association, all women with this haplotype combination would be classified as having GDM.¹⁹

Although an increased BMI is one of the most important risk factors for GDM,²⁰ women with the high-risk 121/221 haplotype had somewhat lower BMI scores than women with other haplotypes, but this difference was not statistically significant. We further demonstrated that women homozygous for allele 1 of single-nucleotide polymorphism 63 had a higher risk of developing GDM. This allele is also homozygously present in the 121/221 haplotype. Although our cohort is too small for an exact analysis of allele distribution, the frequencies of the different haplotypes are comparable with those in other studies.⁹

Horikawa et al⁸ calculated an attributable risk of 14% for the highest-risk haplotype 112/121 in Mexican-Americans, whereas Europeans displayed a 4% attributable risk of developing non-insulin-dependent diabetes mellitus with this haplotype. This is explainable because of the much lower frequency of haplotype 112 in Europeans. Haplotypes 121 and 221 are relatively frequent in most populations.⁸ Taken from this, a relatively high population-attributable risk of GDM could be expected for this haplotype combination.

	Footnotes

 
Received December 1, 2003. Received in revised form February 20, 2004. Accepted February 20, 2004.

10.1097/01.AOG.0000127790.15556.3d

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