Clinical Endocrinology (2003) 59, 519– 525 Interleukin-13 gene polymorphisms in patients with Graves’ disease Blackwell Publishing Ltd. Tomasz Bednarczuk*, Grzegorz Placha†, Krystian Jazdzewski*,1, Alina Kurylowicz*, Marta Kloza*, Urszula Makowska‡, Yuji Hiromatsu§ and Janusz Nauman* *Department of Endocrinology, Medical Research Center, Polish Academy of Science, †Departments of Internal Medicine and Hypertension, ‡Internal Medicine and Endocrinology, Medical University of Warsaw, Poland; §Department of Endocrinology and Metabolism, Kurume University School of Medicine, Kurume, Japan (Received ??? 2003; returned for revision ??? 2003; finally revised ??? 2003; accepted ???2003) Summary OBJECTIVE In patients with Graves’ disease (GD), an elevation of serum immunoglobulin E (IgE) has been recently reported to be associated with the severity of hyperthyroidism and ophthalmopathy. Interleukin 13 (IL-13) is a major cytokine involved in IgE synthesis and therefore may be a potential candidate gene contributing to the development of GD or influencing the clinical course of the disease. DESIGN In a case–control study, we examined IL-13 gene single-nucleotide polymorphisms in the 5′′ promoter region at position −1112 (C to T change, termed −1112T) and in exon 4 at position 2044 (G to A as C− change, G2044A, which results in an amino acid exchange Arg130Gln) in 261 patients with GD. The control groups consisted of healthy young subjects (n = 168) and subjects over 100 years old with no history of autoimmune or allergic diseases recruited from the Polish Centenarians Project (n = 50). −1112T and G2044A polymorphisms MEASUREMENTS C− were defined by fluorescent single-strand conformational polymorphism and by restriction fragment length polymorphism analysis, respectively. Correspondence: Dr Tomasz Bednarczuk, Department of Endocrinology, Medical Research Center, Polish Academy of Science, Banacha 1A, 02-097 Warsaw, Poland. Tel./ Fax: +48 22 659 75 62; E-mail: bednar@amwaw.edu.pl 1 Present address: Department of Hypertension and Diabetology, Medical University of Gdansk, Poland. © 2003 Blackwell Publishing Ltd RESULTS In patients with GD, the distribution of IL-13 − 1112T 31%; 2044A 25%) and genotypes alleles (− −1112T/ T 10%; 2044A/A 7%) did not differ significantly (− compared to control groups. Subdividing GD patients according to clinically evident ophthalmopathy (NOSPECS class III or higher, n = 93) revealed no significant differences in the frequencies of −1112T allele (33% vs. 29%; P = 0·4), −1112T/ T genotype (13% vs. 8%; P = 0·3), 2044A allele (27% vs. 24%; P = 0·5) and 2044A/A genotype (9% vs. 7%; P = 0·7) between GD patients with and without eye involvement. In order to analyse the association with the severity of hyperthyroidism, we examined patients with a first onset of GD treated with antithyroid drugs (n = 32). IL-13 genotypes were not associated with the laboratory findings at diagnosis (thyroid volume, serum levels of FT4, TRAb, TPOAb, TGAb) and with the outcome of antithyroid drug treatment. CONCLUSIONS Our results suggest that IL-13 gene polymorphisms at positions −1112 (C → T) and 2044 (G → A): (1) do not confer genetic susceptibility to Graves’ disease; (2) do not contribute to the development of clinically evident ophthalmopathy; (3) are not associated with severity of hyperthyroidism. Graves’ disease (GD) is a heterogeneous autoimmune disorder affecting with varying degrees of severity the thyroid, eyes and skin (Weetman, 2000). There is now considerable evidence that immunoglobulin class E (IgE), which is the principal mediator of allergic reactions, may be associated with the severity of Graves’ hyperthyroidism and ophthalmopathy. Recent studies demonstrated that IgE serum concentrations were increased in 30 –40% of GD patients and that levels of thyroid hormones, antimicrosomal antibodies, TSH receptor antibodies were significantly greater in patients with IgE elevation than in those with normal serum IgE (Sato et al., 1999; Yamada et al., 2000). Furthermore, elevated IgE levels were associated with increased recurrence rates after antithyroid drug treatment (Komiya et al., 2001). IgE levels have also been demonstrated to correlate with the activity and severity of Graves’ ophthalmopathy (Raikow et al., 1992; Molnar et al., 1996). Although thyroid autoantibodies are predominantly of IgG class, TSH receptor and thyroid peroxidase antibodies of IgE isotypes were also detected (Guo et al., 1997; 519 520 T. Bednarczuk et al. Metcalfe et al., 2002). Finally, mast cells and IgE deposits were reported in the thyroid and in eye muscles of GD patients, suggesting a role in the autoimmune inflammation (Werner et al., 1972; Hufnagel et al., 1984; Raikow et al., 1990; Smith & Parikh, 1999). Interleukin 13 (IL-13) is a major cytokine involved in IgE synthesis (Chomarat & Banchereau, 1998). Numerous singlenucleotide polymorphisms (SNP) have been identified in the IL-13 gene and have been found to be associated with IgE levels and /or allergic diseases (Vercelli, 2002). Thus, IL-13 may be a potential candidate gene contributing to the development or influencing the clinical course of GD. Currently, two SNPs in the IL-13 locus are considered to have a biological function. A SNP in the 5′ flanking region, at position −1112 (C to T change, termed as C-1112T) has been shown to regulate gene transcription (van der Pouw Kraan et al., 1999; Howard et al., 2001). A second SNP in exon 4, at position 2044 (G to A change, termed as G2044A) results in an amino acid exchange (arginine, Arg to glutamine, Gln at position 130, termed as Arg130Gln), which may affect ligand–receptor interactions (Graves et al., 2000; Heinzmann et al., 2000). The aim of this study was to analyse the association of IL-13 gene polymorphisms at positions −1112 (C → T) and 2044 (G → A) with GD. We further examined the relationship between IL-13 gene polymorphisms and the development of clinically evident ophthalmopathy or the severity of Graves’ hyperthyroidism. Subjects and methods Subjects We studied 261 unrelated, Polish Caucasian patients with Graves’ disease (201 women, 60 men), aged 14–78 (median 42). The diagnosis of GD was based on the presence of hyperthyroidism, diffuse goiter, detectable TSH receptor autoantibodies and /or increased radioiodine uptake. The severity of ophthalmopathy was assessed according to the NOSPECS classification (Werner, 1977). Patients with proptosis (NOSPECS class III), extraocular muscle dysfunction (class IV), exposure keratitis (class V) and optic neuropathy (class VI) were considered clinically evident (n = 93). The clinical characteristics of the studied group have been described in details previously (Bednarczuk et al., 2003). The control populations consisted of 168 randomly selected anonymous healthy adults recruited from the Blood Transfusion Center and 50 subjects (43 women, 7 men), who were over 100 years old, recruited from the Polish Centenarians Program carried out at the International Institute of Molecular and Cell Biology in Warsaw. These subjects had no personal history of autoimmune or allergic diseases, they were clinically and biochemically euthyroid and were negative for thyroid peroxidase antibodies. The research programme was approved by the Local Ethical Committee and all subjects gave written informed consent for genetic studies. IL-13 gene polymorphisms analysis The C-1112T dimorphism was defined by fluorescent singlestrand conformational polymorphism (SSCP) analysis. Genomic DNA was subjected to PCR to amplify a 280-bp fragment of the promoter region, using the following primers: 5′-TCTGAGCGGGAATCCAGCAT-3′ and 5′-AATGAGTGCTGTGGAGGGCG3′. PCR was performed in a 12·5-µl reaction mixture containing: 100 ng of genomic DNA, 0·3 U Taq polymerase (Life Technologies Ltd, Paisley, UK), 1·6 pmol of each primer, 0·8 mm dNTPs and 1·5 mm MgCl2, under the following conditions: 5 min of denaturation at 94 °C, followed by 35 cycles of 0·5 min at 94 °C, 0·5 min at 64 °C, 0·5 min at 72 °C and a final 7-min extension at 72 °C. SSCP was performed in Genetic Analyser ABI PRISM 310 (Applied Biosystems, Foster City, CA, USA), using 3% GeneScan Polymer as the capillary electrophoresis polymer and 1 × TBE with 10% glycerol as the electrophoresis buffer. Samples were prepared immediately before electrophoresis by adding 2 µl PCR product to 10·5 µl deionized formamide and 1 µl GeneScan-500 TAMRA internal standard, then denatured at 95°C for 3 min followed by quick chilling on ice. Samples were injected electrokinetically for 8 s at 15 kV and electrophoresed at 13 kV for 22 min at 35 °C. The capillaries were 47 cm in length and 50 µm wide. Data were analysed using GeneScan and Genotyper Software (Applied Biosystems). The variant mobilities of conformational fragments corresponded to nucleotide substitutions as defined by direct sequencing. The G2044A dimorphism was genotyped by PCR–restriction fragment length polymorphism (RFLP) analysis, as described previously by Graves et al. (2000). A 236-bp PCR fragment including the polymorphic region was generated with the use of following primers: 5′-CTTCCGTGAGGACTGAATGAGACGGTC-3′ and 5′-GCAAATAATGATGCTTTCGAAGTTTCAGTGGA-3′. The underlined bases were modified to create NlaIV restriction sites. PCR was performed in a 12·5-µl reaction mixture containing: 100 ng of genomic DNA, 0·3 U Taq polymerase, 2·5 pmol of each primer, 0·8 mm dNTPs and 2 mm MgCl2, under the following conditions: 5 min of denaturation at 94 °C, followed by 35 cycles of 0·5 min at 94 °C, 0·5 min at 63 °C, 0·5 min at 72 °C and a final 7-min extension at 72 °C. Subsequently, 3·5 µl of amplified PCR products were digested in a volume of 10 µl with 0·5 U of BspLI (NlaIV) restriction enzyme (Fermentas UAB, Vilnius, Lithuania). After incubation for 2 h at 37 °C, digested fragments were resolved on 2% agarose gel containing 10 mg/ml ethidium bromide, visualized by UV light, and compared with a 100-bp molecular size control ladder. To confirm the accuracy of method employed, randomly selected patients were analysed by direct sequencing. © 2003 Blackwell Publishing Ltd, Clinical Endocrinology, 59, 519– 525 IL-13 gene polymorphisms in Graves’ disease 521 Results Other measurements Human TSH receptor autoantibodies (TRAb) were determined by luminescence receptor assay (B.R.A.H.M.S. Diagnostica GmbH, Hennigsdorf/Berlin Germany: TRAb values above 1·5 IU/ l were defined as positive). Antibodies against human thyroglobulin (TgAb) and antibodies against native human thyroid peroxidase (TPOAb) were determined by luminescence immunoassays (B.R.A.H.M.S. Diagnostica GmbH: positive values > 60 U /l). Total IgE levels were determined by immunoenzymetric assay (Immunotech, Marseilles, France: positive values > 20 kIU /l). Concentration of free T4 was determined by microparticle enzyme immunoassays (Abbott Laboratories, Abbott Park, IL, USA). The thyroid volume was measured by ultrasonography. Statistical analysis Alleles and genotypes frequencies were compared between groups using the Chi-square (χ2) test or Fisher’s exact probability test, where appropriate. A P-value < 0·05 was considered significant. Odds ratios (OR) were calculated as a measurement of strength of association according to Woolf’s (1955) method. The method of Mittal (1976) was used for linkage disequilibrium analysis. The linkage disequilibrium was considered statistically significant, when the t-value was greater than 2. Laboratory data were analysed using a nonparametric Mann– Whitney U-test. Association of IL-13 gene polymorphisms with Graves’ disease Genotypes and allele frequencies of the C-1112T and G2044A polymorphisms in GD patients and controls are shown in Table 1. There were no significant differences in the distribution of −1112T/ T (10% vs. 10%; P = 0·9), 2044A/A (7% vs. 5%; P = 0·3) genotypes or −1112T (31% vs. 28%; P = 0·5), 2044A (25% vs. 24%; P = 0·8) alleles between GD patients and healthy subjects. There were also no significant differences in either the genotypes (−1112T/ T: 10% vs. 10%, P = 0·8; 2044A /A: 7% vs. 6%; P = 0·9) or alleles (−1112T: 31% vs. 27%, P = 0·5; 2044A: 25% vs. 30%; P = 0·3) frequencies between GD patients and centenarians. The −1112T and 2044A alleles were in linkage disequilibrium in all studied groups (GD: t = 10·7; healthy subjects: t = 7·8; centenarians: t = 3·7). The frequency of the T/A haplotype in GD patients (19%) was similar compared to healthy subjects (17%, P = 0·5) and centenarians (18%, P = 0·7). There were no significant differences in −1112T (24% vs. 32%, P = 0·1) and 2044A (28% vs. 25%, P = 0·5) allele frequencies between male and female patients with GD. Association of IL-13 gene polymorphisms with Graves’ ophthalmopathy In patients with Graves’ ophthalmopathy (GO), there was no Table 1 IL-13 gene polymorphisms in patients with Graves’ disease, healthy subjects and centenarians Graves’ disease (n = 261) IL-13 gene polymorphisms C-1112T G2044A Genotype frequencies T/ T T/C C/C Allele frequencies T C Genotype frequencies A /A G/A G/G Allele frequencies A G Healthy subjects (n = 168) Centenarians (n = 50) 26 (10) 108 (41) 127 (49) 16 (10)* 63 (37) 89 (53) 5 (10)† 17 (34) 28 (56) 160 (31) 362 (69) 95 (28)‡ 241 (72) 27 (27)§ 73 (73) 19 (7) 94 (36) 148 (57) 8 (5)¶ 66 (39) 94 (56) 3 (6)** 24 (48) 23 (46) 132 (25) 390 (75) 82 (24)†† 254 (76) 30 (30)‡‡ 70 (70) Values in parentheses are percentages of the group. Odds ratio (OR) was calculated as compared to patients with Graves’ disease (GD). P-values given below were calculated with the use of χ2 test comparing GD with healthy subjects or GD with centenarians. *P = 0·9, OR = 1·1; †P = 0·8, OR = 1·0; ‡P = 0·5, OR = 1·1; §P = 0·5, OR = 1·2; ‡P = 0·8, OR = 1·0; ¶P = 0·3, OR = 1·6; **P = 0·9, OR = 1·2; ††P = 0·8, OR = 1·0; ‡‡P = 0·3, OR = 0·8. © 2003 Blackwell Publishing Ltd, Clinical Endocrinology, 59, 519– 525 522 T. Bednarczuk et al. Table 2 IL-13 gene polymorphisms in patients with Graves’ ophthalmopathy and in patients with Graves’ hyperthyroidism without clinically evident ophthalmopathy Graves’ ophthalmopathy (n = 93) IL-13 gene polymorphisms C-1112T Genotype frequencies T/ T T/C C/C Allele frequencies T C G2044A Genotype frequencies A /A G/A G/G Allele frequencies A G Graves’ hyperthyroidism (n = 168) 12 (13) 37 (40) 44 (47) 14 (8)* 71 (42) 83 (49) 61 (33) 125 (67) 99 (29)† 237 (71) 8 (9) 34 (37) 51 (55) 11 (7)‡ 60 (36) 97 (58) 50 (27) 136 (73) 82 (24)§ 254 (76) Values in parentheses are percentages of the group. Odds ratio (OR) was calculated as compared to patients with Graves’ hyperthyroidism. P-values given below were calculated with the use of χ2 test comparing Graves’ ophthalmopathy with Graves’ hyperthyroidism. *P = 0·3, OR = 1·6; †P = 0·4, OR = 1·2; ‡P = 0·7, OR = 1·3; §P = 0·5, OR = 1·1. Table 3 Association of IL-13 gene polymorphisms with laboratory features of patients with Graves’ hyperthyroidism at the time of diagnosis IL-13 gene polymorphisms Thyroid volume (ml) FT4 (pmol/l) TRAb (IU / l) TPOAb (U/ml) TGAb (U/ml) IgE kIU/ l‡‡‡ C-1112T genotypes T/T + C/T 17 C/C 15 17·4 (7·8– 40·6) 15·3 (7·0– 46·0)* 50·2 (32·2–70·8) 56·6 (30·9 –110·7)† 6·8 (2·1–75·0) 8·1 (2·2–21·6)‡ 1556 (0 –10000) 515·7 (14–3000)§ 55·5 (0 –2000) 54·5 (2·6–7500)¶ 4·1 (1·1–78·8) 3·1 (1·1–11·2)** G2044A genotype A /A + G/A 17 G/G 15 17·4 (7·8– 40·6) 13·4 (7·0– 46·0)†† 47·6 (32·2–70·8) 56·6 (30·9 –110·7)‡‡ 6·5 (2·1–75·0) 9·0 (2·2–36·2)§§ 1146 (0 –8238) 55·5 (0 –666) 1042 (14 –10000)¶¶ 54·5 (2·6–7500)*** n 4·1 (1·1–78·8) 2·9 (1·1–11·2)††† Values are presented as median (range). FT4-free thyroxine. TRAb-TSH receptor autoantibodies; TPOAb-thyroid peroxidase autoantibodies; TGAb, thyroglobulin autoantibodies; Ig, immunoglobulins. P-values given below were calculated with the use of Mann–Whitney U-test: *P = 0·9; †P = 0·3; ‡P = 0·3; §P = 0·2; ¶P = 0·9; **P = 0·3; ††P = 0·7; ‡‡P = 0·1; §§P = 0·1; ¶¶P = 0·9; ***P = 0·6; †††P = 0·3. ‡‡‡IgE levels were determined in 27 patients. significant increase in −1112T/ T genotype (13% vs. 8%, P = 0·3) and −1112T allele (33% vs. 29%, P = 0·4) compared to patients with Graves’ hyperthyroidism without clinically evident ophthalmopathy (Table 2). There was also no correlation between the frequency of −1112T allele and the severity of GO: NOSPECS class ≤ I (n = 140)−30%; class II (n = 28)−23%; class III (n = 50)−33%; class ≥ IV (n = 43)−33%. In GO patients, frequencies of 2044A/A homozygotes (9% vs. 7%; P = 0·7) and 2044A allele (27% vs. 24%, P = 0·5) were similar compared to GD patients without ophthalmopathy. There was also no significant association between the frequency of 2044A allele and the severity of GO: NOSPECS class ≤ I−25%; class II−20%; class III−26%; class ≥ IV−28%. Association of IL-13 gene polymorphisms with the severity of Graves’ hyperthyroidism Finally, we investigated the relationship between IL-13 gene polymorphisms and the severity of hyperthyroidism in 32 patients with a first episode of GD (27 women, 5 men), aged 20– 60 (median 38), treated for 1 year with Thiamazole. The patients were followed for 1 year after cessation of drug therapy. There were no significant associations between C-1112T or G2044A genotypes and laboratory findings (thyroid volume, serum levels of FT4, TRAb, TPOAb, TGAb, IgE) at the time of diagnosis (Table 3). IL-13 gene polymorphisms did not influence the laboratory findings after withdrawal of antithyroid drugs © 2003 Blackwell Publishing Ltd, Clinical Endocrinology, 59, 519– 525 IL-13 gene polymorphisms in Graves’ disease 523 (data not shown). However, 23% of GD patients (three out of 13) with −1112T/ T + C/ T or 2044A/A + G /A genotypes had significantly increased IgE levels (> 20 kIU /l), whereas no patient with a C/C or G/G genotype had significantly elevated IgE levels (P = 0·09). IL-13 gene polymorphisms did not predict the outcome of antithyroid drug treatment (recurrence rates in patients with −1112T/T + C/T vs. C/C genotypes: 19% vs. 33%, P = 0·4; 2044A/A + A /G vs. G/G: 31% vs. 21%, P = 0·7). Discussion Graves’ disease appears to develop as a result of a complex interaction between genetic and environmental factors. Although the specific genes controlling susceptibility to GD have not been identified, recent genome-wide searches have provided evidence for the linkage to loci on multiple chromosomes, which await further characterization (Tomer et al., 1999; Vaidya et al., 2002). Linkage analyses performed in Japanese, Chinese and Amish populations, suggested the presence of an autoimmune thyroid disease (AITD) or GD susceptibility locus on chromosome 5 in regions: 5q31–q33 and 5q11–q14 (Sakai et al., 2001; Allen et al., 2003; Jin et al., 2003). Interestingly, the region 5q31–q33 has been also identified as a susceptibility locus for IgE synthesis, asthma and/or atopic dermatitis (Postma et al., 1995; Lonjou et al., 2000; Soderhall et al., 2001; Xu et al., 2001). This region encodes a cluster of cytokines genes, including IL-3, IL-4, IL5, IL-9 and IL-13, that are involved in inflammation and IgE synthesis. The IL-4 gene has been previously studied in patients with GD. A C/T polymorphism at position −590 in the IL-4 gene showed initially modest protection against the development of GD in a UK population, but a subsequent study could not confirm this association (Hunt et al., 2000; Heward et al., 2001). Multiple genetic analysis across diverse ethnic populations provided strong support for the candidacy of the IL-13 gene (and not IL-4) as an allergy/asthma susceptibility locus on chromosome 5q31–q33 (Shirakawa et al., 2000; Vercelli, 2002). Additionally, studies in both humans and mice, strongly suggest that IL-13 is a central regulator of allergic inflammations (Chomarat & Banchereau, 1998). Although the mRNA expression of IL-13 has been demonstrated in thyroid and orbital tissues of GD patients, the role of this Th2 cytokine in the pathogenesis of hyperthyroidism and ophthalmopathy remains unknown (Ajjan et al., 1997; Wakelkamp et al., 2003). In this case–control study, we analysed the association of IL-13 promoter C-1112T and exon 4 G2044A (which results in an amino acid exchange Arg130Gln) single nucleotide polymorphisms (SNP) with the development and clinical course of Graves’ disease. Because autoimmune thyroid diseases may develop in older patients (Petrone et al., 2001), the control groups consisted of healthy young subjects and subjects over 100 years old with no history of autoimmune diseases. Our results showed © 2003 Blackwell Publishing Ltd, Clinical Endocrinology, 59, 519– 525 that there were no significant differences in either allelic or genotypic distributions of IL-13 gene SNPs between GD patients and control groups. Our study confirmed previous reports that −1112T and 2044A alleles were in linkage disequilibrium (Graves et al., 2000). However, it was impossible to find any significant association with GD resulting from haplotype analysis. It is still of great clinical interest to identify reliable factors predicting the development of ophthalmopathy in patients with GD (Wiersinga & Bartalena, 2002). Subdividing patients with GD for the presence of clinically evident ophthalmopathy revealed no significant differences in either allelic or genotypic frequencies of C-1112T and G2044A polymorphisms. Furthermore, there was no correlation between the severity of GO and the frequency of −1112T and 2044A alleles. It seems therefore unlikely that IL-13 gene polymorphisms analysis may be helpful in predicting the development or severity of ophthalmopathy. The need for reliable predictors of relapse in Graves’ disease is obvious, as these could help to identify patients needing prolonged antithyroid drug or ablative treatments. In addition to generally accepted predictors such as goiter size or TRAb levels, recent reports suggested that elevated serum concentrations of IgE and IL-13 were associated with increased recurrence rates in Japanese GD patients treated with antithyroid drugs (Yamada et al., 2000; Komiya et al., 2001). However, these reports await confirmation in other populations. We analysed the relationship between IL-13 genotypes and the severity of hyperthyroidism in patients with a first onset of Graves’ disease treated for 1 year with an antithyroid drug. IL-13 genotypes were not associated with thyroid volume, serum levels of FT4, TPOAb, TGAb and TRAb antibodies. In addition, IL-13 gene polymorphisms were not associated with the outcome of antithyroid drug treatment. Due to the small number of GD patients with significantly elevated IgE levels, we were unable to analyse the relationship between IgE levels and severity of Graves’ hyperthyroidism. In conclusions, our results suggest that IL-13 gene SNPs at positions −1112 (C → T) and 2044 (G → A): (1) do not confer genetic susceptibility to Graves’ disease; (2) do not contribute to the development of clinically evident Graves’ ophthalmopathy; (3) are not associated with severity of Graves’ hyperthyroidism. Acknowledgements This work was supported by the State Committee for Scientific Research, grant no. 4PO5B13119. We are grateful to J. Kuznicki and M. Mossakowska (International Institute of Molecular and Cell Biology in Warsaw, Poland) for allowing us access to the data-base on Centenarians, and samples for analysis. The Polish Centenarians Program was supported by grants of State Committee for Scientific Research and UNESCO (contract # SC/ 206·623·0). This work was partially presented at the 84th Annual Meeting of the American Endocrine Society (San Francisco, 2002). 524 T. Bednarczuk et al. References Ajjan, R.A., Watson, P.F. & Weetman, A.P. (1997) Detection of IL-12, IL-13, and IL-15 messenger ribonucleic acid in the thyroid of patients with autoimmune thyroid disease. Journal of Clinical Endocrinology and Metabolism, 82, 666 – 669. Allen, E.M., Hsueh, W.C., Sabra, M.M., Pollin, T.I., Ladenson, P.W., Silver, K.D., Mitchell, B.D. & Shuldiner, A.R. 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