Functional ADAM12 Variants Modulate Proteolytic Activity and Influence Metabolic Traits

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Although the genetic contribution to insulin resistance and metabolic syndrome is established, the role of a disintegrin and metalloprotease 12 (ADAM12) in insulin resistance and T2D remains unclear. Our GWAS has identified four novel ADAM12 variants, namely, ADAM12-K170R rs112264074 [NP_003465.3:p.Lys170Arg], ADAM12-R176W rs140497576 [NP_003465.3:p.Arg176Trp], ADAM12-M662V rs115100580 [NP_003465.3:p.Met662Val], and ADAM12-I908V rs41303603 [NP_003465.3:p.Ile908Val]. These variants were associated with different metabolic traits, including FBG, HbA1c, low- (LDL) and high-density lipoprotein (HDL), total cholesterol (TC), and diastolic and systolic blood pressure. Using an independent replication cohort, the association of ADAM12-I908V with FBG was confirmed, and an association with HbA1c, LDL, and TC was found. In addition, ADAM12-K170R showed associations with waist-hip ratio in diabetic patients, and ADAM12-M662V associated with HDL, TC, and HbA1c in healthy controls, while ADAM12-R176W variant was not detected in the replication cohort. Moreover, we examined the impact of ADAM12 variants on its proteolytic activity and results show that 176W and 662V variants had higher activity in cell lysate supernatants, but only 662V and 908V variants had higher activity in intact cells, suggesting that enzyme activity dysregulation may contribute to the development of metabolic syndrome andT2D. Health sciences/Biomarkers Health sciences/Diseases Health sciences/Endocrinology Biological sciences/Genetics ADAM12 Metabolic syndrome insulin resistance genetic variant Figures Figure 1 Figure 2 Figure 3 1. Introduction A disintegrin and metalloproteinase (ADAM)-12, is a type-I transmembrane, Zn 2+ -dependent metalloprotease that belongs to the Metzincins superfamily of metalloproteases [ 1 ]. It is composed of multiple domains, including an N-terminal signal sequence, a prodomain, a metalloprotease domain, a disintegrin-like domain, a cysteine-rich region, and an epidermal growth factor (EGF)-like domain, followed by a transmembrane domain and a cytoplasmic tail [ 2 ]. ADAMs are synthesized as zymogens that are activated through prodomain cleavage by Furin [ 3 ]. Their physiological role involves ectodomain processing of membrane-bound proteins resulting in modulation of growth factors’ activity, cytoskeletal reorganization, and cell-cell and cell-extracellular matrix (ECM) contacts through their proteolytic and adhesive potentials [ 4 ]. These functions are critical in various biological and pathological processes, including development, inflammation, cancer, and Alzheimer’s disease [ 5 ]. ADAM12 may also regulate cellular signalling through its cytoplasmic tail, which contains potential recognition motifs for signalling and adaptor proteins; thus, it may regulate its metalloprotease activity and ADAM12’s subcellular localization [ 6 , 7 ]. ADAM12 is expressed in various tissues, including adipose tissue [ 8 ], where it was shown to regulate adipogenesis using knockout mice models [ 8 – 12 ]. While these mice appear phenotypically normal at birth, approximately 30% of them die during the first week of life [ 11 ], and some exhibited abnormalities in interscapular brown adipose tissue and in the skeletal muscles surrounding the brown adipose tissue during embryogenesis. The impairment in brown adipose tissue causes failure of thermoregulation and accounts for the mortality rate in newborns [ 4 ]. Conversely, overexpression of ADAM12 increased adipogenesis in mice via multiple pathways, including its proteolytic activity and its interaction with the ECM [ 10 ]. Likewise, ADAM12 was found to promote new adipocyte formation in high-fat diet-induced obesity [ 12 ], as it is most abundant in preadipocytes that are responsive to adipogenic differentiation [ 8 ]. Since ADAM12 can promote adipocyte formation leading to increased fat mass and altered adipose tissue function, it can indirectly influence insulin resistance, which is a key feature in metabolic syndrome and type 2 diabetes (T2D). Kuwait ranks among the countries with the highest and fastest-growing diabetes burden in the world, with almost 40% of adults affected by prediabetes or T2D as of 2018 [ 13 ]. Contributing to this alarming trend, around 80% of Kuwaiti adults were reported to be overweight or obese in 2014 [ 14 ], a major risk factor for the development of insulin resistance and metabolic syndrome. This in turn, increases the risk of T2D and cardiovascular diseases development [ 15 , 16 ]. The high rates of consanguinity in Kuwait and other Arab populations allowed several studies to identify a strong familial and genetic basis for insulin resistance and metabolic syndrome in these populations [ 17 , 18 ]. Such a population structure increases the prevalence of recessive and familial disorders, including those affecting metabolic traits [ 19 – 24 ]. For example, a recent study identified regions within the TNKS genes associated with T2D, obesity, and hypertension in Kuwaitis [ 25 ]. In support of these conclusions, genome-wide association (studies (GWAS) carried out on Kuwaiti cohorts have revealed novel genetic variants associated with metabolic traits that have not been documented in other populations, indicating the existence of distinct, population-specific genetic risk profiles [ 26 ]. Moreover, familial genetic studies and GWAS support the genetic basis of insulin resistance and individual components of the metabolic syndrome [ 27 – 34 ]. More specifically, studies have consistently reported that a significant amount of genetic variation affects b-cell function and insulin secretion [ 35 ]. The integration of these variations with pancreatic islet epigenomic and transcriptomic datasets enabled the identification of potential effector genes, such as ACSL1 and FAM46C involved in insulin release [ 36 ]. Notably, most of the genetic variants associated with b-cells’ dysfunction are mainly located within non-coding regulatory elements, emphasizing the importance of transcription regulatory mechanisms in the genetic framework of T2D susceptibility and supporting the hypothesis that b-cell dysfunction is a key mechanism in T2D pathogenesis [ 36 ]. Due to ADAM12's documented role in modulating adipogenesis, we examined its correlation with metabolic syndrome traits in a GWAS conducted at our institute. This study identified four ADAM12 variants, namely ADAM12-K170R rs112264074 [NP_003465.3:p.Lys170Arg], ADAM12-R176W rs140497576 [NP_003465.3:p.Arg176Trp], ADAM12-M662V rs115100580 [NP_003465.3:p.Met662Val], and ADAM12-I908V rs41303603 [NP_003465.3:p.Ile908Val] that associated with various metabolic traits. We also validated these findings in a separate replication study and examined the impact of the identified variants on enzyme kinetics. 2. Materials and Methods 2.1. Study Participants of the discovery GWAS and replication study A discovery GWAS was conducted as part of the Kuwait Obesity Genome Project (KOGP) performed at Dasman Diabetes Institute (DDI) [37, 38]. Briefly, this study included 1298 randomly selected Arab adults from the six governorates of Kuwait. The participants were recruited according to procedures authorized by the scientific and ethics advisory boards at DDI. Multiple anthropometric (body mass index (BMI) and waist circumference) and metabolic biomarkers including fasting blood glucose (FBG), Haemoglobin A1c (HbA1c), total cholesterol (TC), triglycerides (TGs), high-density lipoprotein (HDL), low-density lipoprotein (LDL), systolic and diastolic blood pressure (SBP and DBP) were ascertained in the study in relation to the assessed variants. Resultant variants that were significantly associated with biomarkers relevant to metabolic syndrome traits were assessed in a replication study. The replication study participants were selected from the Kuwait Diabetes Epidemiology Program (KDEP) at DDI following the guidelines of the scientific and advisory boards at DDI. These participants were randomly selected to represent the adults of Kuwait using a stratified random sampling technique from a computerized register of the Public Authority of Civil Information, a depository of personal information on Kuwaiti citizens and expatriates [39]. The discovery phase inclusion criteria adhered to established methodological standards used in large-scale GWAS including verification of Arab ethnicity by a questionnaire about parental lineage, which considered parental lineages up to the third generation to mitigate population stratification effects as recommended by Petersen et al. [40]. On the other hand, the replication study allowed the inclusion of Arab, South Asian, and Southeast Asian ancestries. In accordance with standard GWAS methodology our exclusion criteria for both the discovery and the replication studies included age < 18 years, the presence of limiting serious complications of diabetes, mental illness, or cognitive limitation. Furthermore, pregnant women and patients undergoing weight reduction medication/surgery or fitness programs were also excluded [39, 41, 42] . Before blood sample collection, the patients fasted overnight and signed an informed consent form. Simultaneously, their SBP and DBP were measured, and the nationality and ethnicity of each participant were confirmed using a detailed questionnaire. Participants were classified as having T2D based on self-reported diagnosis or in accordance with American Diabetes Association (ADA) criteria using FBG ≥ 7 mmol/l; 126 mg/dL, and HbA1c ≥ 6.5% measurements, while healthy controls were free of diabetes. Other illnesses, including cardiovascular complications, were verified and recorded. The clinical assays conducted for trait outcome measurements were performed at a College of American Pathologists (CAP) accredited laboratory at DDI [38]. All protocols and procedures conducted in this study were reviewed and approved by DDI Ethical Review Committee, which adheres to the guidelines of the Declaration of Helsinki and the US Federal Policy for the Protection of Human Subjects (ERB/ERC numbers: RA HM 2010-005 for KOGP and RA HM 2010-004 for KDEP). Participants were categorized into healthy control and T2D patient groups based on clinical results. This classification was preserved for the examination of genotype associations. 2.2. ADAM12 variants genotyping The discovery GWAS was conducted on the Illumina Human Omni-Express and the iSCAN system (Illumina) using the Infinium®HD ultra genotyping assay method. Genotype calling was performed after pooling the intensity data of all genotyped samples. A prospective power analysis was conducted using the discovery GWAS frequencies for the four variants with the assumption that the diabetic cohort would have higher frequencies than the healthy controls in the replication cohort. We found that a sample size of 400 in each group has a 90% power to detect an increase of 0.03 with a significance level (alpha) of 0.05 (two-tailed) for ADAM12-K170R, ADAM12-R176W, and ADAM12-M662V. As for ADAM12-I908V, a sample size of 400 in each group has a 90% power to detect an increase of 0.05 with a significance level (alpha) of 0.05 (two-tailed). The replication phase genotyping was conducted by targeted genotyping of the selected ADAM12 single nucleotide polymorphisms (SNPs), including ADAM12-K170R (rs112264074 [NP_003465.3:p.Lys170Arg]), ADAM12-R176W (rs140497576 [NP_003465.3:p.Arg176Trp]), ADAM12-M662V (rs115100580 [NP_003465.3:p.Met662Val]), and ADAM12-I908V (rs41303603 [NP_003465.3:p.Ile908Val]). For simplicity, the variant amino acid change will be used throughout the remaining text. These missense variants were prioritized for further investigation based on their associations with metabolic syndrome and T2D-related traits, as well as their predicted functional consequences and biological significance to metabolic regulation pathways. The TaqMan® SNP genotyping assays (Applied Biosystems, MA, USA) were run on a QuantStudio 5 real-time polymerase chain reaction (PCR) system (Applied Biosystems, MA, USA). Genotyping PCR reactions contained 20 ng of genomic DNA, 10µl of 2X TaqMan Genotyping master mix, 1µl of 20X TaqMan SNP genotyping assay, and nuclease-free water to make up the volume to 20µl. The thermal cycling conditions included a pre-PCR read at 60°C for 1 minute followed by polymerase activation at 95°C for 10 minutes and 40 cycles of denaturation at 95°C for 15s and annealing/extension at 60°C for 1 minute. Finally, a post-PCR read was performed at 60°C for 1 minute, and genotype calls were determined. Sanger sequencing of PCR products for selected cases of homozygotes and heterozygotes was conducted to confirm the obtained genotypes. Sequencing reactions were conducted using the BigDye terminator cycle sequencing kit v.3.1 (Applied Biosystems, MA, USA) according to the manufacturer’s instructions on an ABI PRISM 3730xl genetic analyser (Applied Biosystems, MA, USA). 2.3. Structural analysis The ADAM12 variants identified were mapped, modelled, and analysed using PyMOL modelling software (The PyMOL Molecular Graphics System, Version 3.0 Schrödinger, LLC). To examine the impact of the genetic variants on ADAM12 stability and flexibility, the DynaMut web server (https://biosig.lab.uq.edu.au/dynamut/ ) was used. Both analyses were based on the predicted crystal structure (UniProt accession ID O43184). 2.4. DNA constructs and mutagenesis Human-tagged ORF clone plasmid encoding ADAM12 was purchased from OriGene Technologies (RG212501, MD, USA). Site-directed mutagenesis of studied SNPs was conducted using the QuickChange II site-directed Mutagenesis kit (Agilent Technologies, CA, USA) according to the manufacturer’s instructions. Sanger sequencing was conducted to verify successful incorporation of the intended nucleotide substitutions. The sequencing reactions were conducted using the BigDye terminator cycle sequencing v.3.1 kit (Applied Biosystems, MA, USA) according to the manufacturer’s instructions on an ABI PRISM 3730 xl genetic analyser (Applied Biosystems, MA, USA). The primers used to generate the nucleotide substitutions and to verify them by sequencing are listed in Table 1. 2.5. Cell culture, DNA transfections, and Western Blotting HEK293 cells were purchased from ATCC (CRL-1573, VA, USA). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen Corporation, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen Corporation, CA, USA) and 100 U/ml of penicillin and 100 µg/ml streptomycin (Gibco, CA, USA) and incubated at 37°C in a humidified atmosphere containing 5% CO 2 . The cells (2x10 5 or 5x10 4 ) were seeded into 6- or 96-well plates, respectively, for 24 hours prior to transfection. HEK293 cells in 6- or 96-well plates were transfected with 2 µg or 200 ng, respectively, of wild-type (WT) or variant constructs using lipofectamine 3000 reagent (DNA:lipofectamine ratio 1:2) (Invitrogen, MA, USA) in an antibiotic-free medium. Twenty-four hours after transfection, the medium was changed to complete DMEM medium, in which cells were grown for another 24 hours before being harvested for protein analysis. Overexpression of WT and variant proteins was confirmed using western blotting. For western blot analysis, the cells were harvested and lysed in an extraction buffer containing 0.5% NP-40 and 2% CHAPS in phosphate-buffered saline (PBS) (Gibco, CA, USA). The lysates were centrifuged at 4°C for 15 minutes at 13,000 rpm. The protein concentration in the supernatant was quantified using the Bradford assay (Thermo Fischer Scientific, Driesch, Germany). Then, 5 mg of total protein was resolved on 8% denaturing polyacrylamide gel and transferred to a nitrocellulose membrane (EMD Millipore Corporation, MA, USA). Membranes were stained with Ponceau S stain to verify proteins transfer, then it was cut into two pieces to allow probing for multiple targets separately (Figure S1). After that, the membranes were blocked using 5% fat-free milk and blotted with the corresponding primary and horseradish peroxidase (HRP)-linked secondary antibodies. The primary antibodies used were b-actin (ab3700, Abcam, MA, USA), and ADAM12 (ab223745, Abcam, MA, USA). Chemiluminescent detection of HRP activity was done using a chemiluminescent substrate (Thermo Scientific, MA, USA) according to the manufacturer’s instructions, and the membranes were visualized using a ChemiDoc Imaging system and analysed by Image Lab software (Bio-Rad, CA, USA). 2.6. Enzyme kinetic activity of wild-type ADAM and its variants The calibration fluorescent peptide (5`-FAM-Pro-Leu-Arg-Arg-Thr-Leu-Ser-Val-Ala-Ala-OH) was purchased from CPC Scientific (Sunnyvale, CA, USA), while the ADAM12 fluorogenic substrate PEPDAB005 (Dabcyl-LAQAPhe(homo)RSK(5-FAM)-NH 2 ) peptide was purchased from BioZyme Inc. (St. Joseph, MO, USA). The calibration fluorogenic peptide was used at different concentrations (0-10 µM) to generate a fluorescent calibration curve. Cleavage assays of PEPDAB005 were performed in 96-well plates with 10 mg of the total protein lysate supernatant from cells transfected with either the WT or variant ADAM12 in a reaction buffer containing 1 mM CaCl 2 , 10 mM NaCl, 20 mM Tris pH 8.0, and 0.5% Brij-35. Protein extracts from untransfected cells or cells transfected with a GFP-expressing plasmid were used as negative controls. Reactions were conducted at 37°C, and fluorescence emission was measured for a total of 3 hours at 5-minute intervals using a multimode microplate reader (Synergy H4 Hybrid, Bio Tek Instruments, VT, USA) with excitation and emission wavelengths of 485 nm and 530 nm, respectively. Western blot analysis was used to normalize enzyme activities and Lineweaver-Burk plot enzyme kinetic model was used to determine the maximum velocity (Vmax) and the Michaelis-Menten constant (Km) [43]. 2.7. Statistical analysis Clinical and demographic data were analysed using student’s t-test or the non-parametric Mann-Whitney test. Hardy-Weinberg equilibrium was assessed in the healthy control cohort for the four variants using expected genotype frequencies by c 2 -test and linear regression analysis was performed to determine the allele effect size (b coefficient). The associations were considered statistically significant at p < 0.05. To correct for multiple comparisons, a Bonferroni correction was applied, adjusting the significance threshold to p < 0.0045. The ADAM12 activity assays were conducted using two biological replicates and the data are presented as mean ± standard deviation. Significance was tested using the student t-test and P-values £0.05 were considered statistically significant. Biological replicates refer to data obtained from different transfection reactions of cultured cells. All statistical analyses were performed using SPSS v.25 (IBM, NY, USA). 3. Results 3.1. ADAM12 Variants identified in the discovery phase A cohort comprised of 1298 Arab participants that included healthy controls (n=697) and T2D patients (n=601) was used to conduct a discovery GWAS as part of the KOGP study at DDI (Table 2). This study showed a significant association of four ADAM12 variants, namely ADAM12-K170R, ADAM12-R176W, ADAM12-M662V, and ADAM12-I908V, with several metabolic traits (Table 3), including FBG, HbA1c, SBP, DBP, and lipids including TC, HDL, and LDL. The location of the variants on ADAM12 gene and protein are illustrated in (Figure 1A). 3.2. Replication study The ADAM12 variants’ association with metabolic syndrome traits/T2D identified in the discovery cohort was verified in a separate cohort from the KDEP study, which included healthy controls (n=409) and T2D patients (n=450). This cohort consisted of adults of Arab, South Asian and Southeast Asian ethnicities in Kuwait. A summary of the demographic and clinical data of the replication cohort is summarized in Table 4. The minor allele frequencies (MAFs) of ADAM12-K170R and ADAM12-M662V variants were higher (0.031 and 0.044, respectively) in the replication cohort compared to those detected in the discovery cohort (0.003436 and 0.006873, respectively), as well as compared to the total MAFs reported in GnomAd database (0.001106, and 0.002337, respectively) (GnomAd v2.1.1). The MAF of the ADAM12-I908V variant, on the other hand, was lower in the replication cohort (0.018) compared to the discovery cohort (0.029), and the total MAF reported in GnomAd database (0.02375) (GnomAd v2.1.1). In contrast, the minor allele of the ADAM12-R176W variant was not detected among participants in the replication cohort. All variants were in Hardy-Weinberg equilibrium in the healthy cohort (P>0.05). Associations of ADAM12-K170R, and ADAM12-M662V detected in the discovery cohort were not confirmed in the replication cohort (Table S1), however, new associations were detected (Table 5). ADAM12-K170R was found to be associated with waist:hip ratio in T2D cohort ( p = 0.044) suggesting a potential but modest association. ADAM12-M662V was found to be associated with HDL in both healthy control ( p = 0.012) and T2D ( p = 0.017) cohorts suggesting a moderately strong and potentially meaningful effect, which sustained after adjustment for confounding factors, including age, sex, and BMI (β = 0.098; 95%CI: 0.024 - 0.172; p = 0.009) and also after adjusting for ethnicity (β = 0.080; 95%CI: 0.014 – 0.145; p = 0.018) (Table S2). In contrast to the significant association of ADAM12-M662Vwith TC:HDL ratio ( p = 0.005), HbA1c level showed a borderline association ( p = 0.044) in the healthy control cohort . Also, ADAM12-M662V association with waist:hip ratio was revealed after adjustment for the confounding factors mentioned above (β =0.447; 95%CI:0.120 - 0.774; p = 0.007) (Table S2). While most of these associations reached nominal significance ( p < 0.05), Bonferroni correction was applied to account for multiple testing (adjusted threshold: p < 0.0045). After correction, only the association between ADAM12-M662V and HDL and TC:HDL remained statistically significant, supporting the robustness of these findings. In addition, the association between ADAM12-I908V and FBG identified in the discovery cohort was confirmed in healthy control and T2D cohorts, with a stronger association observed in the healthy controls ( p = 0.017) compared to the T2D group ( p = 0.044). This association was sustained after adjustment for confounding factors (β = -0.007; 95%CI:-0.014- (-0.001); p = 0.02) and ethnicity (β = -0.009 ; 95%CI: -0.015 - -0.003; p = 0.004) (Table S3). Also, ADAM12-I908V showed a new association with HbA1c in the replication cohort (β = -0.010; 95%CI: -0.019 - 0; p = 0.04). When analysing healthy controls and T2D patients separately, ADAM12-I908V showed a significant association with LDL ( p = 0.001) and TC ( p = 0.006) in the T2D cohort, which remained statistically significant after Bonferroni correction . Further scrutiny of genotypes found three participants (0.35%) carried dual variants in the ADAM12 gene, specifically the ADAM12-K170R and ADAM12-M662V variants’in heterozygosity. Similarly, another three participants exhibited double-variants of the ADAM12-K170R and ADAM12-I908V alleles, while only a single participant (0.12%) carried the ADAM12-M662V and ADAM12-I908V variants. All variants were detected in heterozygosity; no homozygous individuals were detected for any of the assessed ADAM12 variants. 3.3. Effect of ADAM12 variants on protein structure and stability The variants detected in the ADAM12 gene, namely ADAM12-K170R, ADAM12-R176W, ADAM12-M662V, and ADAM12-I908V are missense variants that cause the substitution of an amino acid in the prodomain (170R, 176W), EGF-like domain (662V) and the cytoplasmic (908V) tail (Figure 1A). The detected variants 170R, 662V and 908V affect amino acid residues that are conserved across species (Figure 1B, C, and D), whereas the variant 176W affects an amino acid that is not conserved across species (Figure 1B). Pymol and DynaMut software assessed how these variants impact the ADAM12 structure’s stability and flexibility (Figure 2). The assessment predicts that 170R, 662V, and 908V variants destabilize the protein and increase its flexibility, while the 176W variant stabilizes the protein and decreases its flexibility (Figure 2A). The molecular mechanisms causing the observed changes in ADAM12 variants’ stability and flexibility were investigated by analysing the intramolecular interactions within the WT and variant protein structures. This analysis revealed that 170R variant may introduce new interactions with glutamic acid (Glu150 within the prodomain and Glu374 of the metalloprotease domain) residues that are absent in the WT (Figure 2B). Similarly, the 176W variant stabilizes the proteins’ structure and possibly introduces new intramolecular interactions with nearby glycine and tyrosine residues [Gly385 and Tyr386] (Figure 2C). In contrast, 662V variant may reduce the intramolecular interactions providing the enzyme with increased flexibility (Figure 2D). Finally, the structural analysis of 908V reveals the interaction of 908V with valine and proline [V899 and P900] that are in close proximity (Figure 2E). Enzyme Activity and Kinetic Parameters of the ADAM12 variants Since the detected ADAM12 variants are in the prodomain, the EGF-like domain, and the cytoplasmic tail, we evaluated the impact of these variants on ADAM12’s proteolytic function. ADAM12 WT and variants were generated through site-directed mutagenesis and expressed separately in HEK-293 cells. Western blotting confirmed that these proteins were overexpressed in the HEK-293 cell lysates (Figure 3A). Enzymatic assays using the fluorogenic substrate PEPDAB005 revealed that ADAM12 variants 176W and 662V demonstrated a significantly increased proteolytic activity compared to ADAM12 WT (176W 55.4±7.2%; p=0.013 and 662V 93.5±25.7; p=0.045; Figure 3B). No significant change in proteolytic activity was observed using 170R and 908V variants’ cell lysates’ supernatants. To exclude any potential interference of the intracellular environment with enzyme activity, HEK-293 cells seeded on a 96-well black plate were transfected with ADAM12 WT and variants as previously described. Enzyme activity was measured in intact cells, allowing the assessment of the enzyme activity present at the cell surface. The results showed that 170R significantly reduced the proteolytic activity of ADAM12 at the cell surface (18.9%; p=0.018). Both ADAM12 662V and 908V variants showed a statistically significant increase in proteolytic activity, which was very slight for the 662V variant (0.5%; p=0.006), while ADAM12 908V variant exhibited a much higher increase in activity at the cell surface (42.5%; p=0.028). No significant change in the proteolytic activity of ADAM12 variant 176W was observed at the cell surface (Figure 3C). Further, the effect of ADAM12 variants on its proteolytic activity was studied by determining the kinetic parameters, Vmax and Km . As illustrated in Figure 3 D&E, ADAM12 WT and variants followed the Michaelis-Menten equation, so the Lineweaver-Burk plot was used to calculate Vmax and Km values. Only 662V variant exhibited a significant reduction in Vmax value by 33.6% (p<0.01) compared to the WT (Figure 3E). Whereas the Km values of 176W and 662V variants were significantly reduced by 41.1% (p<0.001) and 76.05% (p<0.001) respectively, compared to WT (Figure 3E). 4. Discussion ADAM12 plays an important role in various cellular functions and is associated with different pathological conditions [44]. Several GWA studies have identified loci associated with the risk of developing T2D [34, 45, 46]. However, most of the identified loci are associated with insulin secretion and B-cell function [34-36]. Our discovery GWAS identified the association of four novel rare/low-frequency (MAF £0.5%) ADAM12 variants, namely ADAM12-K170R, ADAM12-R176W, ADAM12-M662V, and ADAM12-I908V with different metabolic syndrome and T2D traits including FBG, HbA1c, lipids including TC, HDL and LDL, and hypertension (DBP and SBP). However, these findings were not confirmed in our replication study except for the association of ADAM12-I908V with FBG. Nonetheless, our replication study found new associations with other traits, including FBG, HbA1c, waist:hip ratio, and lipids (TC, HDL, LDL, and TC:HDL ratio). This study reveals an unprecedented identification of associations between ADAM12 variants and metabolic characteristics. Due to their rarity or low frequency, these variants are excluded from the genotyping arrays utilized in global GWA studies. However, the analysis of the GWAS catalogue, which presents GWAS findings from global studies, revealed associations between common variants of the ADAM12 gene and metabolic traits. Specifically, rs10901513 (an intergenic variant FANK1-ADAM12) with visceral fat in men, rs11244839 (an intergenic variant RNA5SP328-ADAM12) with BMI in 0.5-1.5-year-old children, rs7920091 (among others) with height, and rs1551678 associated with thiazide-induced adverse metabolic effects in hypertensive patients (affecting fasting blood glucose measurement). The findings from our research on associations with T2D traits, together with those from global GWA studies on related traits, validate the role of ADAM12 in metabolic processes. Despite the differences between the ethnic origins of the discovery and replication cohorts and their lifestyles, adjustment for ethnicity did not affect the observed associations between ADAM12 variants and the studied traits. However, the differences in MAFs of some of the reported ADAM12 variants in the replication cohort, compared to the discovery cohort, may still reflect underlying population differences [34]. Additionally, the biological significance and effect size of the identified variants may be more specific to the Arab ethnicity of the discovery cohort, which was diluted in the replication cohort as it had different ethnicities. The identified ADAM12 variants result in amino acid changes within the prodomain (170R, 176W), the EGF-like domain (662V), and the cytoplasmic tail (908V). Thus, we expected that protein structure and function may be affected. The type of amino acid change in the two prodomain variants, 170R and 176W may have contributed to the difference in proteolytic activity in cell lysates and intact cells. The conserved change of lysine to arginine in the 170R variant did not affect the proteolytic activity in cell lysates but was decreased at the cell surface of intact cells. Although 170R variant possibly introduces new intramolecular interaction patterns, as observed in our structural analysis, these alterations did not significantly affect the enzyme’s proteolytic activity in cell lysates. Hence, the increased stability introduced by this alteration may not influence the active site architecture or substrate accessibility. However, these alterations could affect the prodomain’s function. It is established that the prodomain acts as an intramolecular chaperone facilitating proper protein folding [5] and regulating ADAMs’ intracellular traffic [47, 48]. Therefore, the increased stability of 170R variant due to the potential intramolecular interactions introduced between R170, E150 within the prodomain, and E374 of the metalloprotease domain, possibly hindered the translocation of ADAM12 to the plasma membrane, which was reflected by a reduced proteolytic activity at the cell surface. Moreover, the ADAM prodomain maintains the enzyme in its latent form by a cysteine switch mechanism [5, 49]. The activation mechanism involves cleavage of the prodomain by the furin enzyme [50]. Therefore, the non-conserved amino acid change of arginine to tryptophan in the R176W variant may have increased the proteolytic activity of this variant in cell lysates by disrupting the prodomain-active site’s interactions while paradoxically increasing the overall protein stability. In the WT, the enzyme is maintained in an autoinhibited state through the interactions of C179 within the prodomain with the catalytic Zn 2+ ion [51]. In contrast, the R176W variant introduces a bulky, hydrophobic tryptophan side chain [the indole ring] near the C179. The indole ring of the tryptophan may form van der Waals repulsive forces with neighbouring residues [G385, Y386], which possibly destabilize the local prodomain conformation and disrupt the inhibitory contact with the active site. This conformational alteration possibly increases substrate accessibility to the catalytic Zn 2+ site consistent with the reduced Km value [7, 51, 52]. Interestingly, while this variant exhibits locally disruptive interactions, Dynamut analysis revealed increased overall protein structural stability. This apparent paradox may be explained by compensatory stabilizing interactions that cause local structural rearrangements, reducing steric hindrance and promoting better hydrophobic packing within adjacent regions of the protein core. This variant may also reduce the translocation of ADAM12 to the plasma membrane, reflected by a non-significant change in activity within the intact cells, despite the increased activity in cell lysates. Moreover, the M662V variant that occurs within the EGF-like domain showed a potential increase in flexibility and proteolytic activity. Although the function of the EGF-like domain is not fully understood [50], it may affect the conformational state of the enzyme and its ability to interact with the substrate. Especially that the reduced intramolecular interactions observed in structural analyses may affect the secondary structure of ADAM12, which plays a vital role in substrate recognition [53]; hence, increasing the flexibility of the protein may allow easier access of the substrate to the active site. The increased proteolytic activity of the cytoplasmic tail variant, I908V at the cell surface of intact cells compared to cell lysates’ supernatants, may be caused by cytoplasmic tail modifications or interactions with different proteins leading to its translocation to the plasma membrane. Because the tail of ADAM proteins contains binding motifs for SH3-domain containing proteins and potential phosphorylation sites, it may modulate their function or provide binding sites for SH3-domain containing proteins [53-55]. The majority of ADAMs are located in the Golgi apparatus under steady-state conditions [56, 57], with only a minority found at the plasma membrane [58]. The translocation to the plasma membrane is induced by the phosphorylation of ADAM12 by PKCƐ [10, 59], whereas endocytosis of ADAMs depends on the binding of PACSIN3 to the SH3-binding domain [60]. Structural analysis suggests that, in the WT, I908 is possibly involved in an interaction with A906. However, the substitution of Ile with Val at position 908 of the variant possibly allows the interaction of V908 with V899 and P900, creating a local loop-like conformation, which exposes nearby serine and tyrosine [S902, Y903, Y905] residues, making them more accessible for potential phosphorylation. This conformational change may facilitate easier access for kinases or adaptor proteins such as Tks5/FISH [61], PI3K, and actinin-1/2 [7], thereby increasing the possibility of phosphorylation-dependent translocation to the plasma membrane. Hence, this structural rearrangement may partially elucidate the increased surface proteolytic activity observed for the I908V variant. Although ADAM12 knockout mice exhibit normal glucose tolerance, the increased activity of the identified ADAM12 variants may contribute to the development of metabolic syndrome/T2D-associated traits such as obesity (Figure S2) [44]. While direct evidence associating ADAM12 to insulin resistance is lacking, several mechanistic links are plausible. Evidence supports the role of ADAM12 in the development of high-fat diet-induced obesity by stimulating new adipocyte formation (hyperplasia) rather than by enhancing differentiation and/or maturation (hypertrophy) of preexisting adipocytes via mechanisms involving its proteolytic activity and its interaction with b1 integrins [8, 10-12]. In addition, Coles et al., reported that the two most affected signalling pathways by ADAM12 knockdown included IGF/IGFBP/mTOR growth pathway and PPARγ signalling pathway, both of which are critical regulators of adipogenesis and insulin sensitivity. ADAM12 can cleave IGFBP3 and IGFBP5 [9]; therefore, increased ADAM12 activity may increase the bioavailability of IGF and stimulate preadipocyte proliferation. Hence, the increased ADAM12 variants’ proteolytic activity may enhance IGF bioavailability and subsequently stimulate preadipocyte proliferation. Therefore, the increased proteolytic activity of ADAM12 variants identified in this study may promote white adipose tissue (WAT) mass expansion. Besides, ADAM12 can remodel the cytoskeleton via b1 integrin and alter the dense fibronectin-rich matrix, allowing the flat preadipocytes to take a round shape during differentiation since fibronectin can be cleaved by ADAM12 [62]. The increase in WAT mass could act as a double-edged sword, while initial hyperplasia may transiently maintain insulin sensitivity, prolonged adipose tissue expansion often leads to hypertrophy, adipose tissue dysfunction, chronic low-grade inflammation, and ultimately, systemic insulin resistance development [63, 64]. However, while these mechanistic links are biologically plausible, direct evidence associating increased ADAM12 activity to insulin resistance and metabolic syndrome traits remains limited. Thus, the association of ADAM12 with metabolic disorders remains exploratory. To address this knowledge gap, further in vitro and in vivo studies that integrate functional metabolic phenotyping that assess metabolic syndrome and T2D traits in models overexpressing hyperactive ADAM12 variants are needed to elucidate how the increased ADAM12 variants’ activity reported herein may contribute to insulin resistance and T2D development. Moreover, evaluating how hyperactive ADAM12 may affect adipose tissue remodelling, inflammation, and insulin sensitivity may aid in elucidating its role in T2D pathogenesis. In vitro experiments studying the role of ADAM12-driven IGF/IGFBP axis modulation in adipocyte insulin sensitivity and glucose metabolism may also be of value. Altogether, these approaches will explain how the increased ADAM12 variants activity reported herein may contribute to the pathophysiology of insulin resistance and T2D. There are several limitations to our study including the relatively small sample size in the two cohorts. However, despite the relatively small sizes of our discovery and replication cohorts, the differences in the ethnic origins of the participants, and their demographic and clinical characteristics we were able to identify rare ADAM12 variants and confirm their association with several metabolic traits relevant to T2D. Another limitation is that the replication results may have been influenced by the heterogeneity of the tested cohorts; with the replication cohort including multiple ethnicities, while the discovery cohort being limited to individuals of Arab descent, through differences in allele frequencies and genetic backgrounds. In addition, the limited sample sizes within individual subgroups impeded ethnicity-stratified analyses or adjustment for population structure, which would have been valuable, and their consideration remains important for future studies. Furthermore, detailed clinical information regarding diabetes duration, treatment, and end-organ complications was not available, which represents another limitation of this study. In addition, the absence of direct or surrogate insulin resistance measurements, such as Homeostatic Model Assessment of Insulin Resistance, HOMA-IR, which would provide a more accurate assessment of T2D status, may added an additional limitation. The use of HEK293 cells in this study, although advantageous for initial enzymatic characterization due to their high transfection efficiency and minimal endogenous ADAM12 activity, lack the physiological context of adipose or muscle cells. Therefore, further in vitro and in vivo studies using more relevant cellular models, such as primary adipocytes, or myoblasts are required to confirm their role in metabolic syndrome and T2D pathophysiology. 5. Conclusion Our GWAS suggested the association of four ADAM12 gene variants (namely ADAM12-K170R, ADAM12-R176W, ADAM12-M662V, and ADAM12-I908V) with metabolic syndrome/T2D traits. Using a replication cohort, the association of ADAM12-I908V with FBG was confirmed, and more associations of the variants with different metabolic traits were found, confirming the potential role of ADAM12 variants in metabolic syndrome/T2D development. The functional study revealed a variant-specific difference in the activity of ADAM12 in cell lysates and the cell surface, suggesting that dysregulation of ADAM12 activity at the cell surface may play a role in the development of T2D. However, the exact mechanisms and tissues involved are not fully elucidated yet. Therefore, further studies are required to enable targeting ADAM12 for the potential treatment of these conditions. Declarations Competing interests: The authors declare that they have no competing interests. Funding: This study was supported and funded by Kuwait University, Graduate Research Project No. YM07/21, and by Kuwait Foundation for the Advancement of Sciences (KFAS), as part of research project (RA AM-2023-010). Author Contribution H.D. conducted genotyping and enzyme activity assays, data collection and analysis, and wrote the original draft of manuscript. A.M. conducted enzyme activity assays, data collection, and manuscript writing. T.A.T. was involved in GWAS data collection and analysis and manuscript editing. A.C. and P.H. assisted with GWAS and genotyping data analysis. S.S. and R.A. provided guidance for enzyme assays and manuscript writing. F.M. and R.T. were involved in conceptualization, methodology development, supervision, and manuscript writing. Data Availability The data supporting the findings of this study are available in public repositories and within the manuscript or supplementary materials. The SNP data is available via the following accession numbers ADAM12-K170R (rs112264074), ADAM12-R176W (rs140497576), ADAM12-M662V (rs115100580), and ADAM12-I908V (rs41303603). Moreover, the ADAM12 crystal structure accession ID (O43184) is a UniProt accession number that is composed of only 6 characters. References Giebeler, N. & Zigrino, P. A Disintegrin and Metalloprotease (ADAM): Historical Overview of Their Functions. Toxins (Basel) . 8 (4), 122 (2016). Duffy, M. J. et al. The role of ADAMs in disease pathophysiology. Clin. Chim. Acta . 403 (1–2), 31–36 (2009). Loechel, F. et al. Human ADAM 12 (meltrin alpha) is an active metalloprotease. J. Biol. Chem. 273 (27), 16993–16997 (1998). Jacobsen, J. & Wewer, U. M. Targeting ADAM12 in human disease: head, body or tail? Curr. Pharm. Des. 15 (20), 2300–2310 (2009). Wong, E. et al. The Functional Maturation of A Disintegrin and Metalloproteinase (ADAM) 9, 10, and 17 Requires Processing at a Newly Identified Proprotein Convertase (PC) Cleavage Site. J. Biol. Chem. 290 (19), 12135–12146 (2015). Huovila, A. P. et al. Shedding light on ADAM metalloproteinases. Trends Biochem. Sci. 30 (7), 413–422 (2005). Kveiborg, M. et al. Cellular roles of ADAM12 in health and disease. Int. J. Biochem. Cell. Biol. 40 (9), 1685–1702 (2008). Kawaguchi, N. et al. ADAM12 induces actin cytoskeleton and extracellular matrix reorganization during early adipocyte differentiation by regulating beta1 integrin function. J. Cell. Sci. 116 (Pt 19), 3893–3904 (2003). Coles, C. A. et al. Knockdown of a disintegrin A metalloprotease 12 (ADAM12) during adipogenesis reduces cell numbers, delays differentiation, and increases lipid accumulation in 3T3-L1 cells. Mol. Biol. Cell. 29 (15), 1839–1855 (2018). Kawaguchi, N. et al. ADAM 12 protease induces adipogenesis in transgenic mice. Am. J. Pathol. 160 (5), 1895–1903 (2002). Kurisaki, T. et al. Phenotypic analysis of Meltrin alpha (ADAM12)-deficient mice: involvement of Meltrin alpha in adipogenesis and myogenesis. Mol. Cell. Biol. 23 (1), 55–61 (2003). Masaki, M. et al. Role of meltrin {alpha} (ADAM12) in obesity induced by high- fat diet. Endocrinology 146 (4), 1752–1763 (2005). Alkandari, A. et al. The prevalence of pre-diabetes and diabetes in the Kuwaiti adult population in 2014. Diabetes Res. Clin. Pract. 144 , 213–223 (2018). Weiderpass, E. et al. The Prevalence of Overweight and Obesity in an Adult Kuwaiti Population in 2014. Front. Endocrinol. (Lausanne) . 10 , 449 (2019). Guembe, M. J. et al. Risk for cardiovascular disease associated with metabolic syndrome and its components: a 13-year prospective study in the RIVANA cohort. Cardiovasc. Diabetol. 19 (1), 195 (2020). Marott, S. C. W. et al. Components of the Metabolic Syndrome and Risk of Type 2 Diabetes. J. Clin. Endocrinol. Metabolism . 101 (8), 3212–3221 (2016). Al-Awadi, S. A. et al. Consanguinity among the Kuwaiti population. Clin. Genet. 27 (5), 483–486 (1985). Warsy, A. S. et al. Is consanguinity prevalence decreasing in Saudis? A study in two generations. Afr. Health Sci. 14 (2), 314–321 (2014). Gosadi, I. M., Goyder, E. C. & Teare, M. D. Investigating the potential effect of consanguinity on type 2 diabetes susceptibility in a Saudi population. Hum. Hered . 77 (1–4), 197–206 (2014). Bener, A. & Hussain, R. Consanguineous unions and child health in the State of Qatar. Paediatr. Perinat. Epidemiol. 20 (5), 372–378 (2006). Benrahma, H. et al. Maternal effect and familial aggregation in a type 2 diabetic Moroccan population. J. Community Health . 36 (6), 943–948 (2011). Arfa, I. et al. Familial aggregation and excess maternal transmission of type 2 diabetes in Tunisia. Postgrad. Med. J. 83 (979), 348–351 (2007). Al-Sinani, S. et al. Familial Clustering of Type 2 Diabetes among Omanis. Oman Med. J. 29 (1), 51–54 (2014). Bener, A. et al. Parental transmission of type 2 diabetes mellitus in a highly endogamous population. World J. Diabetes . 4 (2), 40–46 (2013). Eaaswarkhanth, M. et al. Genome-Wide Selection Scan in an Arabian Peninsula Population Identifies a TNKS Haplotype Linked to Metabolic Traits and Hypertension. Genome Biol. Evol. 12 (3), 77–87 (2020). Hebbar, P. et al. A Perception on Genome-Wide Genetic Analysis of Metabolic Traits in Arab Populations. Front. Endocrinol. (Lausanne) . 10 , 8 (2019). Sakul, H. et al. Familiality of physical and metabolic characteristics that predict the development of non-insulin-dependent diabetes mellitus in Pima Indians. Am. J. Hum. Genet. 60 (3), 651–656 (1997). Elbein, S. C. et al. Heritability of pancreatic beta-cell function among nondiabetic members of Caucasian familial type 2 diabetic kindreds. J. Clin. Endocrinol. Metab. 84 (4), 1398–1403 (1999). Watanabe, R. M. et al. Familiality of quantitative metabolic traits in Finnish families with non-insulin-dependent diabetes mellitus. Finland-United States Investigation of NIDDM Genetics (FUSION) Study investigators. Hum. Hered . 49 (3), 159–168 (1999). Lehtovirta, M. et al. Insulin sensitivity and insulin secretion in monozygotic and dizygotic twins. Diabetologia 43 (3), 285–293 (2000). Lin, H. F. et al. Heritabilities of the metabolic syndrome and its components in the Northern Manhattan Family Study. Diabetologia 48 (10), 2006–2012 (2005). Bellia, A. et al. The Linosa Study: epidemiological and heritability data of the metabolic syndrome in a Caucasian genetic isolate. Nutr. Metab. Cardiovasc. Dis. 19 (7), 455–461 (2009). Carmelli, D., Cardon, L. R. & Fabsitz, R. Clustering of hypertension, diabetes, and obesity in adult male twins: same genes or same environments? Am. J. Hum. Genet. 55 (3), 566–573 (1994). Brown, A. E. & Walker, M. Genetics of Insulin Resistance and the Metabolic Syndrome. Curr. Cardiol. Rep. 18 (8), 75 (2016). Kim, H. et al. High-throughput genetic clustering of type 2 diabetes loci reveals heterogeneous mechanistic pathways of metabolic disease. Diabetologia 66 (3), 495–507 (2023). Madsen, A. L. et al. Genetic architecture of oral glucose-stimulated insulin release provides biological insights into type 2 diabetes aetiology. Nat. Metab. 6 (10), 1897–1912 (2024). Hebbar, P. et al. Genome-wide association study identifies novel recessive genetic variants for high TGs in an Arab population. J. Lipid Res. 59 (10), 1951–1966 (2018). Hebbar, P. et al. Genome-wide landscape establishes novel association signals for metabolic traits in the Arab population. Hum. Genet. 140 (3), 505–528 (2021). Hebbar, P. et al. The TCN2 variant of rs9606756 [Ile23Val] acts as risk loci for obesity-related traits and mediates by interacting with Apo-A1. Obes. (Silver Spring) . 25 (6), 1098–1108 (2017). Peterson, R. E. et al. Genome-wide Association Studies in Ancestrally Diverse Populations: Opportunities, Methods, Pitfalls, and Recommendations. Cell 179 (3), 589–603 (2019). Biobank, U. UK Biobank: protocol for a large-scale prospective epidemiological resource. Accessed May, 7(2016): pp. 1–112. (2007). Xue, A. et al. Genome-wide association analyses identify 143 risk variants and putative regulatory mechanisms for type 2 diabetes. Nat. Commun. 9 (1), 2941 (2018). Bisswanger, H. Practical Enzymology (Wiley-VCH Verlag GmbH & Co. KGaA, 2011). Nyren-Erickson, E. K. et al. A disintegrin and metalloproteinase-12 (ADAM12): function, roles in disease progression, and clinical implications. Biochim. Biophys. Acta . 1830 (10), 4445–4455 (2013). Albrechtsen, A. et al. Exome sequencing-driven discovery of coding polymorphisms associated with common metabolic phenotypes. Diabetologia 56 (2), 298–310 (2013). Steinthorsdottir, V. et al. Identification of low-frequency and rare sequence variants associated with elevated or reduced risk of type 2 diabetes. Nat. Genet. 46 (3), 294–298 (2014). Anders, A. et al. Regulation of the alpha-secretase ADAM10 by its prodomain and proprotein convertases. Faseb j. 15 (10), 1837–1839 (2001). Gonzales, P. E. et al. Inhibition of the tumor necrosis factor-alpha-converting enzyme by its pro domain. J. Biol. Chem. 279 (30), 31638–31645 (2004). Roghani, M. et al. Metalloprotease-disintegrin MDC9: intracellular maturation and catalytic activity. J. Biol. Chem. 274 (6), 3531–3540 (1999). Klein, T. & Bischoff, R. Active metalloproteases of the A Disintegrin and Metalloprotease (ADAM) family: biological function and structure. J. Proteome Res. 10 (1), 17–33 (2011). Loechel, F. et al. Regulation of human ADAM 12 protease by the prodomain. Evidence for a functional cysteine switch. J. Biol. Chem. 274 (19), 13427–13433 (1999). Van Wart, H. E. & Birkedal-Hansen, H. The cysteine switch: a principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family. Proc. Natl. Acad. Sci. U S A . 87 (14), 5578–5582 (1990). Seals, D. F. & Courtneidge, S. A. The ADAMs family of metalloproteases: multidomain proteins with multiple functions. Genes Dev. 17 (1), 7–30 (2003). Reiss, K. & Saftig, P. The a disintegrin and metalloprotease (ADAM) family of sheddases: physiological and cellular functions. Semin Cell. Dev. Biol. 20 (2), 126–137 (2009). Kang, Q., Cao, Y. & Zolkiewska, A. Metalloprotease-disintegrin ADAM 12 binds to the SH3 domain of Src and activates Src tyrosine kinase in C2C12 cells. Biochem J, 352 Pt 3(Pt 3): pp. 883–92. (2000). Lammich, S. et al. Constitutive and regulated alpha-secretase cleavage of Alzheimer's amyloid precursor protein by a disintegrin metalloprotease. Proc. Natl. Acad. Sci. U S A . 96 (7), 3922–3927 (1999). Schlöndorff, J., Becherer, J. D. & Blobel, C. P. Intracellular maturation and localization of the tumour necrosis factor alpha convertase (TACE). Biochem. J. 347 (Pt 1), 131–138 (2000). Gutwein, P. et al. ADAM10-mediated cleavage of L1 adhesion molecule at the cell surface and in released membrane vesicles. Faseb j. 17 (2), 292–294 (2003). Sundberg, C. et al. Regulation of ADAM12 cell-surface expression by protein kinase C epsilon. J. Biol. Chem. 279 (49), 51601–51611 (2004). Mori, S. et al. PACSIN3 binds ADAM12/meltrin alpha and up-regulates ectodomain shedding of heparin-binding epidermal growth factor-like growth factor. J. Biol. Chem. 278 (46), 46029–46034 (2003). Abram, C. L. et al. The adaptor protein fish associates with members of the ADAMs family and localizes to podosomes of Src-transformed cells. J. Biol. Chem. 278 (19), 16844–16851 (2003). Roy, R. et al. ADAM 12 cleaves extracellular matrix proteins and correlates with cancer status and stage. J. Biol. Chem. 279 (49), 51323–51330 (2004). Guilherme, A. et al. Adipocyte dysfunctions linking obesity to insulin resistance and type 2 diabetes. Nat. Rev. Mol. Cell. Biol. 9 (5), 367–377 (2008). Zatterale, F. et al. Chronic Adipose Tissue Inflammation Linking Obesity to Insulin Resistance and Type 2 Diabetes. Front. Physiol. 10 , 1607 (2019). Tables Table 1 to 5 are available in the Supplementary Files section. Additional Declarations No competing interests reported. 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06:58:43","extension":"xml","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":172593,"visible":true,"origin":"","legend":"","description":"","filename":"04c97a380ad9440ba41e227456e5117b1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8138455/v1/15aa156bde54e520cf329763.xml"},{"id":97114498,"identity":"d262fadd-2838-4f13-8760-de83166f81bf","added_by":"auto","created_at":"2025-12-01 06:58:43","extension":"html","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":187429,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8138455/v1/6de6372bc1f35e628b166346.html"},{"id":97114486,"identity":"d6415bb3-e5fa-4059-8151-28d0f3d2b5b5","added_by":"auto","created_at":"2025-12-01 06:58:43","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1147083,"visible":true,"origin":"","legend":"\u003cp\u003eADAM12 Genetic variants and their positional mapping on the protein structure. The location of the metabolic traits associated SNPs identified by GWAS on the \u003cem\u003eADAM12\u003c/em\u003e gene and protein is shown (A). The shaded boxes indicate SNPs associated with increased trait values, while white boxes indicate SNPs associated with reduced trait values. Sequence alignment of the ADAM12 prodomain (B), EGF-like domain (C), and cytoplasmic tail (D) highlights the positions of the missense mutations K170R, R176W, M662V, and I908V, respectively compared to other species.\u003c/p\u003e\n\u003cp\u003eDBP: Diastolic blood pressure; FBG: Fasting blood glucose, HbA1c: Glycated Haemoglobin; HDL: High density lipoprotein; LDL: Low density lipoprotein, SBP: Systolic blood pressure; TC: total cholesterol.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8138455/v1/baaa4d76f9e9e4178199f2de.jpeg"},{"id":97114484,"identity":"2af59df2-ca43-487d-8f39-07725ebc8325","added_by":"auto","created_at":"2025-12-01 06:58:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":293076,"visible":true,"origin":"","legend":"\u003cp\u003eStructural and functional impact of \u003cem\u003eADAM12\u003c/em\u003e missense variants. ADAM12 variants (yellow) are mapped onto the structure of the wild-type (WT) protein (blue). DynaMut analysis predicted that the K170R, M662V, and I908V variants destabilize the protein and increase its flexibility, while the R176W variant stabilizes the structure and decreases flexibility (A). Structural models (B,C,D) illustrate the positions of these mutations and their effects on local intramolecular interactions, highlighting disrupted or strengthened contacts that underlie the observed changes in stability and function.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8138455/v1/4ab38318e17be15ce12011df.png"},{"id":97114496,"identity":"c144ec13-4f5e-4601-a7d1-efdfc22ca7f9","added_by":"auto","created_at":"2025-12-01 06:58:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":67893,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of ADAM12 variants on enzyme kinetic parameters. (A) Western blot analysis of ADAM12 variants (170R, 176W, 662V, 908V) using anti-ADAM12 and β-actin as a loading control. ADAM12-GFP tagged constructs of WT and variants were transfected in HEK293 cells. Control cells were transfected with empty vectors. Before blotting with antibodies, the nitrocellulose membrane was cut into two pieces according to the marker molecular weight to enable blotting with anti-ADAM12 and anti-Actin antibodies separately. Original blots are presented in Supplementary Figure (S1). The immunoblots confirm overexpression of ADAM12 WT and variants in the cells. (B) ADAM12 enzymatic activity was measured using cell lysate containing 10 µg of total protein and 40 mM fluorogenic substrate PEPDAB005, at excitation and emission wavelengths of 485 and 530 nm. The fluorescence was detected using Synergy H4 reader and normalized to a reaction containing the substrate alone. Enzyme activity was determined using a calibration standard curve and was normalized to Western blots. Two-tailed unpaired Student’s T-test was used to determine significance. Experiments were done in duplicates using two different cell extracts. Values with P\u0026lt;0.05 were considered significant. (C) ADAM12 enzyme activity was measured in intact cells, using the same approach as described in B. Enzyme kinetics were studied by drawing the Michaelis-Menten plot (D) and Lineweaver Burk Plot (E) to calculate Vmax and Km.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8138455/v1/a6aaf1c95352e1a109dca68f.png"},{"id":100069049,"identity":"51530e5a-9249-4c5f-b0fa-e1a24275d623","added_by":"auto","created_at":"2026-01-12 16:07:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2324761,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8138455/v1/dc2ef9aa-da53-423f-b3f2-e8f5510296d6.pdf"},{"id":97141831,"identity":"990bad7c-e563-4f69-8bb4-24ae1a5051de","added_by":"auto","created_at":"2025-12-01 10:07:04","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":287974,"visible":true,"origin":"","legend":"","description":"","filename":"2.SupplementaryMaterial03.08.2025Cleanversion.docx","url":"https://assets-eu.researchsquare.com/files/rs-8138455/v1/2ac2c46fbf5e4c69e1438b4f.docx"},{"id":97142175,"identity":"956f9051-f539-4781-9bb5-f421dd035675","added_by":"auto","created_at":"2025-12-01 10:07:23","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":37646,"visible":true,"origin":"","legend":"","description":"","filename":"Draft7ADAM12Paper19.07.2025Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8138455/v1/f2f23c1096e43dfb29709917.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Functional ADAM12 Variants Modulate Proteolytic Activity and Influence Metabolic Traits","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eA disintegrin and metalloproteinase (ADAM)-12, is a type-I transmembrane, Zn\u003csup\u003e2+\u003c/sup\u003e-dependent metalloprotease that belongs to the Metzincins superfamily of metalloproteases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is composed of multiple domains, including an N-terminal signal sequence, a prodomain, a metalloprotease domain, a disintegrin-like domain, a cysteine-rich region, and an epidermal growth factor (EGF)-like domain, followed by a transmembrane domain and a cytoplasmic tail [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. ADAMs are synthesized as zymogens that are activated through prodomain cleavage by Furin [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Their physiological role involves ectodomain processing of membrane-bound proteins resulting in modulation of growth factors\u0026rsquo; activity, cytoskeletal reorganization, and cell-cell and cell-extracellular matrix (ECM) contacts through their proteolytic and adhesive potentials [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These functions are critical in various biological and pathological processes, including development, inflammation, cancer, and Alzheimer\u0026rsquo;s disease [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. ADAM12 may also regulate cellular signalling through its cytoplasmic tail, which contains potential recognition motifs for signalling and adaptor proteins; thus, it may regulate its metalloprotease activity and ADAM12\u0026rsquo;s subcellular localization [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eADAM12 is expressed in various tissues, including adipose tissue [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], where it was shown to regulate adipogenesis using knockout mice models [\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. While these mice appear phenotypically normal at birth, approximately 30% of them die during the first week of life [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and some exhibited abnormalities in interscapular brown adipose tissue and in the skeletal muscles surrounding the brown adipose tissue during embryogenesis. The impairment in brown adipose tissue causes failure of thermoregulation and accounts for the mortality rate in newborns [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Conversely, overexpression of ADAM12 increased adipogenesis in mice via multiple pathways, including its proteolytic activity and its interaction with the ECM [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Likewise, ADAM12 was found to promote new adipocyte formation in high-fat diet-induced obesity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], as it is most abundant in preadipocytes that are responsive to adipogenic differentiation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Since ADAM12 can promote adipocyte formation leading to increased fat mass and altered adipose tissue function, it can indirectly influence insulin resistance, which is a key feature in metabolic syndrome and type 2 diabetes (T2D).\u003c/p\u003e\u003cp\u003eKuwait ranks among the countries with the highest and fastest-growing diabetes burden in the world, with almost 40% of adults affected by prediabetes or T2D as of 2018 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Contributing to this alarming trend, around 80% of Kuwaiti adults were reported to be overweight or obese in 2014 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], a major risk factor for the development of insulin resistance and metabolic syndrome. This in turn, increases the risk of T2D and cardiovascular diseases development [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe high rates of consanguinity in Kuwait and other Arab populations allowed several studies to identify a strong familial and genetic basis for insulin resistance and metabolic syndrome in these populations [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Such a population structure increases the prevalence of recessive and familial disorders, including those affecting metabolic traits [\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. For example, a recent study identified regions within the TNKS genes associated with T2D, obesity, and hypertension in Kuwaitis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In support of these conclusions, genome-wide association (studies (GWAS) carried out on Kuwaiti cohorts have revealed novel genetic variants associated with metabolic traits that have not been documented in other populations, indicating the existence of distinct, population-specific genetic risk profiles [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMoreover, familial genetic studies and GWAS support the genetic basis of insulin resistance and individual components of the metabolic syndrome [\u003cspan additionalcitationids=\"CR28 CR29 CR30 CR31 CR32 CR33\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. More specifically, studies have consistently reported that a significant amount of genetic variation affects b-cell function and insulin secretion [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The integration of these variations with pancreatic islet epigenomic and transcriptomic datasets enabled the identification of potential effector genes, such as ACSL1 and FAM46C involved in insulin release [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Notably, most of the genetic variants associated with b-cells\u0026rsquo; dysfunction are mainly located within non-coding regulatory elements, emphasizing the importance of transcription regulatory mechanisms in the genetic framework of T2D susceptibility and supporting the hypothesis that b-cell dysfunction is a key mechanism in T2D pathogenesis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Due to ADAM12's documented role in modulating adipogenesis, we examined its correlation with metabolic syndrome traits in a GWAS conducted at our institute. This study identified four \u003cem\u003eADAM12\u003c/em\u003e variants, namely ADAM12-K170R rs112264074 [NP_003465.3:p.Lys170Arg], ADAM12-R176W rs140497576 [NP_003465.3:p.Arg176Trp], ADAM12-M662V rs115100580 [NP_003465.3:p.Met662Val], and ADAM12-I908V rs41303603 [NP_003465.3:p.Ile908Val] that associated with various metabolic traits. We also validated these findings in a separate replication study and examined the impact of the identified variants on enzyme kinetics.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003ch3\u003e2.1. Study Participants of the discovery GWAS and replication study \u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eA discovery GWAS was conducted as part of the Kuwait Obesity Genome Project (KOGP) performed at Dasman Diabetes Institute (DDI) [37, 38]. Briefly, this study included 1298 randomly selected Arab adults from the six governorates of Kuwait. The participants were recruited according to procedures authorized by the scientific and ethics advisory boards at DDI. Multiple anthropometric (body mass index (BMI) and waist circumference) and metabolic biomarkers including fasting blood glucose (FBG), Haemoglobin A1c (HbA1c), total cholesterol (TC), triglycerides (TGs), high-density lipoprotein (HDL), low-density lipoprotein (LDL), systolic and diastolic blood pressure (SBP and DBP) were ascertained in the study in relation to the assessed variants. Resultant variants that were significantly associated with biomarkers relevant to metabolic syndrome traits were assessed in a replication study. The replication study participants were selected from the Kuwait Diabetes Epidemiology Program (KDEP) at DDI following the guidelines of the scientific and advisory boards at DDI. These participants were randomly selected to represent the adults of Kuwait using a stratified random sampling technique from a computerized register of the Public Authority of Civil Information, a depository of personal information on Kuwaiti citizens and expatriates [39].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe discovery phase inclusion criteria adhered to established methodological standards used in large-scale GWAS including verification of \u0026nbsp;Arab ethnicity by a questionnaire about parental lineage, which considered parental lineages up to the third generation to mitigate population stratification effects as recommended by Petersen et al. [40]. On the other hand, the replication study allowed the inclusion of Arab, South Asian, and Southeast Asian ancestries. In accordance with standard GWAS methodology our exclusion criteria for both the discovery and the replication studies included age \u0026lt; 18 years, the presence of limiting serious complications of diabetes, mental illness, or cognitive limitation. Furthermore, pregnant women and patients undergoing weight reduction medication/surgery or fitness programs were also excluded [39, 41, 42] . Before blood sample collection, the patients fasted overnight and signed an informed consent form. Simultaneously, their SBP and DBP were measured, and the nationality and ethnicity of each participant were confirmed using a detailed questionnaire. Participants were classified as having T2D based on self-reported diagnosis or in accordance with American Diabetes Association (ADA) criteria using FBG \u0026ge; 7 mmol/l; 126 mg/dL, and HbA1c \u0026ge; 6.5% measurements, while healthy controls were free of diabetes. Other illnesses, including cardiovascular complications, were verified and recorded. The clinical assays conducted for trait outcome measurements were performed at a College of American Pathologists (CAP) accredited laboratory at DDI [38].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll protocols and procedures conducted in this study were reviewed and approved by DDI Ethical Review Committee, which adheres to the guidelines of the Declaration of Helsinki and the US Federal Policy for the Protection of Human Subjects (ERB/ERC numbers: RA HM 2010-005 for KOGP and RA HM 2010-004 for KDEP). Participants were categorized into healthy control and T2D patient groups based on clinical results. This classification was preserved for the examination of genotype associations.\u003c/p\u003e\n\u003ch3\u003e2.2. ADAM12 variants genotyping\u003c/h3\u003e\n\u003cp\u003eThe discovery GWAS was conducted on the Illumina Human Omni-Express and the iSCAN system (Illumina) using the Infinium\u0026reg;HD ultra genotyping assay method. Genotype calling was performed after pooling the intensity data of all genotyped samples.\u003c/p\u003e\n\u003cp\u003eA prospective power analysis was conducted using the discovery GWAS frequencies for the four variants with the assumption that the diabetic cohort would have higher frequencies than the healthy controls in the replication cohort. We found that \u0026nbsp;a sample size of 400 in each group has a 90% power to detect an increase of 0.03 with a significance level (alpha) of 0.05 (two-tailed) for ADAM12-K170R, ADAM12-R176W, and ADAM12-M662V. As for ADAM12-I908V, a sample size of 400 in each group has a 90% power to detect an increase of 0.05 with a significance level (alpha) of 0.05 (two-tailed).\u003c/p\u003e\n\u003cp\u003eThe replication phase genotyping was conducted by targeted genotyping of the selected \u003cem\u003eADAM12\u003c/em\u003e single nucleotide polymorphisms (SNPs), including ADAM12-K170R (rs112264074 [NP_003465.3:p.Lys170Arg]), ADAM12-R176W (rs140497576 [NP_003465.3:p.Arg176Trp]), ADAM12-M662V (rs115100580 [NP_003465.3:p.Met662Val]), and ADAM12-I908V (rs41303603 [NP_003465.3:p.Ile908Val]). For simplicity, the variant amino acid change will be used throughout the remaining text. These missense variants were prioritized for further investigation based on their associations with metabolic syndrome and T2D-related traits, as well as their predicted functional consequences and biological significance to metabolic regulation pathways. The TaqMan\u0026reg; SNP genotyping assays (Applied Biosystems, MA, USA) were run on a QuantStudio 5 real-time polymerase chain reaction (PCR) system (Applied Biosystems, MA, USA). Genotyping PCR reactions contained 20 ng of genomic DNA, 10\u0026micro;l of 2X TaqMan Genotyping master mix, 1\u0026micro;l of 20X TaqMan SNP genotyping assay, and nuclease-free water to make up the volume to 20\u0026micro;l. The thermal cycling conditions included a pre-PCR read at 60\u0026deg;C for 1 minute followed by polymerase activation at 95\u0026deg;C for 10 minutes and 40 cycles of denaturation at 95\u0026deg;C for 15s and annealing/extension at 60\u0026deg;C for 1 minute. \u0026nbsp;Finally, a post-PCR read was performed at 60\u0026deg;C for 1 minute, and genotype calls were determined. Sanger sequencing of PCR products for selected cases of homozygotes and heterozygotes was conducted to confirm the obtained genotypes. Sequencing reactions were conducted using the BigDye terminator cycle sequencing kit v.3.1 (Applied Biosystems, MA, USA) according to the manufacturer\u0026rsquo;s instructions on an ABI PRISM 3730xl genetic analyser (Applied Biosystems, MA, USA).\u003c/p\u003e\n\u003ch3\u003e2.3. Structural analysis\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eADAM12\u003c/em\u003e variants identified were mapped, modelled, and analysed using PyMOL modelling software (The PyMOL Molecular Graphics System, Version 3.0 Schr\u0026ouml;dinger, LLC). To examine the impact of the genetic variants on ADAM12 stability and flexibility, the DynaMut web server (https://biosig.lab.uq.edu.au/dynamut/ ) was used. Both analyses were based on the predicted crystal structure (UniProt accession ID O43184).\u003c/p\u003e\n\u003ch3\u003e2.4. DNA constructs and mutagenesis\u003c/h3\u003e\n\u003cp\u003eHuman-tagged ORF clone plasmid encoding \u003cem\u003eADAM12\u0026nbsp;\u003c/em\u003ewas purchased from OriGene Technologies (RG212501, MD, USA). Site-directed mutagenesis of studied SNPs was conducted using the QuickChange II site-directed Mutagenesis kit (Agilent Technologies, CA, USA) according to the manufacturer\u0026rsquo;s instructions. Sanger sequencing was conducted to verify successful incorporation of the intended nucleotide substitutions. The sequencing reactions were conducted using the BigDye terminator cycle sequencing v.3.1 kit (Applied Biosystems, MA, USA) according to the manufacturer\u0026rsquo;s instructions on an ABI PRISM 3730 xl genetic analyser (Applied Biosystems, MA, USA). The primers used to generate the nucleotide substitutions and to verify them by sequencing are listed in Table 1.\u003c/p\u003e\n\u003ch3\u003e2.5. Cell culture, DNA transfections, and Western Blotting\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eHEK293 cells were purchased from ATCC (CRL-1573, VA, USA). The cells were grown in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) (Invitrogen Corporation, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen Corporation, CA, USA) and 100 U/ml of penicillin and 100 \u0026micro;g/ml streptomycin (Gibco, CA, USA) and incubated at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e. The cells (2x10\u003csup\u003e5\u003c/sup\u003e or 5x10\u003csup\u003e4\u003c/sup\u003e) were seeded into 6- or 96-well plates, respectively, for 24 hours prior to transfection. HEK293 cells in 6- or 96-well plates were transfected with 2 \u0026micro;g or 200 ng, respectively, of wild-type (WT) or variant constructs using lipofectamine 3000 reagent (DNA:lipofectamine ratio 1:2) (Invitrogen, MA, USA) in an antibiotic-free medium. Twenty-four hours after transfection, the medium was changed to complete DMEM medium, in which cells were grown for another 24 hours before being harvested for protein analysis. Overexpression of WT and variant proteins was confirmed using western blotting.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor western blot analysis, the cells were harvested and lysed in an extraction buffer containing 0.5% NP-40 and 2% CHAPS in phosphate-buffered saline (PBS) (Gibco, CA, USA). The lysates were centrifuged at 4\u0026deg;C for 15 minutes at 13,000 rpm. The protein concentration in the supernatant was quantified using the Bradford assay (Thermo Fischer Scientific, Driesch, Germany). Then, 5\u0026nbsp;mg of total protein was resolved on 8% denaturing polyacrylamide gel and transferred to a nitrocellulose membrane (EMD Millipore Corporation, MA, USA). Membranes were stained with Ponceau S stain to verify proteins transfer, then it was cut into two pieces to allow probing for multiple targets separately (Figure S1). After that, the membranes were blocked using 5% fat-free milk and blotted with the corresponding primary and horseradish peroxidase (HRP)-linked secondary antibodies. The primary antibodies used were b-actin (ab3700, Abcam, MA, USA), and ADAM12 (ab223745, Abcam, MA, USA). Chemiluminescent detection of HRP activity was done using a chemiluminescent substrate (Thermo Scientific, MA, USA) according to the manufacturer\u0026rsquo;s instructions, and the membranes were visualized using a ChemiDoc Imaging system and analysed by Image Lab software (Bio-Rad, CA, USA).\u003c/p\u003e\n\u003ch3\u003e2.6. Enzyme kinetic activity of wild-type ADAM and its variants\u003c/h3\u003e\n\u003cp\u003eThe calibration fluorescent peptide (5`-FAM-Pro-Leu-Arg-Arg-Thr-Leu-Ser-Val-Ala-Ala-OH) was purchased from CPC Scientific (Sunnyvale, CA, USA), while the ADAM12 fluorogenic substrate PEPDAB005 (Dabcyl-LAQAPhe(homo)RSK(5-FAM)-NH\u003csub\u003e2\u003c/sub\u003e) peptide was purchased from BioZyme Inc. (St. Joseph, MO, USA). The calibration fluorogenic peptide was used at different concentrations (0-10 \u0026micro;M) to generate a fluorescent calibration curve. Cleavage assays of PEPDAB005 were performed in 96-well plates with 10 mg of the total protein lysate supernatant from cells transfected with either the WT or variant \u003cem\u003eADAM12\u003c/em\u003e in a reaction buffer containing 1 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 10 mM NaCl, 20 mM Tris pH 8.0, and 0.5% Brij-35. Protein extracts from untransfected cells or cells transfected with a GFP-expressing plasmid were used as negative controls. Reactions were conducted at 37\u0026deg;C, and fluorescence emission was measured for a total of 3 hours at 5-minute intervals using a multimode microplate reader (Synergy H4 Hybrid, Bio Tek Instruments, VT, USA) with excitation and emission wavelengths of 485 nm and 530 nm, respectively. Western blot analysis was used to normalize enzyme activities and Lineweaver-Burk plot enzyme kinetic model was used to determine the maximum velocity (Vmax) and the Michaelis-Menten constant (Km) [43].\u003c/p\u003e\n\u003ch3\u003e2.7. Statistical analysis\u003c/h3\u003e\n\u003cp\u003eClinical and demographic data were analysed using student\u0026rsquo;s t-test or the non-parametric Mann-Whitney test. Hardy-Weinberg equilibrium was assessed in the healthy control cohort for the four variants using expected genotype frequencies by\u0026nbsp;c\u003csup\u003e2\u003c/sup\u003e-test and linear regression analysis was performed to determine the allele effect size (b coefficient). The associations were considered statistically significant at p \u0026lt; 0.05. To correct for multiple comparisons, a Bonferroni correction was applied, adjusting the significance threshold to p \u0026lt; 0.0045. The ADAM12 activity assays were conducted using two biological replicates and the data are presented as mean \u0026plusmn; standard deviation. Significance was tested using the student t-test and P-values \u0026pound;0.05 were considered statistically significant. Biological replicates refer to data obtained from different transfection reactions of cultured cells. All statistical analyses were performed using SPSS v.25 (IBM, NY, USA).\u003c/p\u003e"},{"header":"3. Results","content":"\u003ch3\u003e3.1. ADAM12 Variants identified in the discovery phase\u003c/h3\u003e\n\u003cp\u003eA cohort comprised of 1298 Arab participants that included healthy controls (n=697) and T2D patients (n=601) was used to conduct a discovery GWAS as part of the KOGP study at DDI (Table 2). This study showed a significant association of four \u003cem\u003eADAM12\u003c/em\u003e variants, namely ADAM12-K170R, ADAM12-R176W, ADAM12-M662V, and ADAM12-I908V, with several metabolic traits (Table 3), including FBG, HbA1c, SBP, DBP, and lipids including TC, HDL, and LDL. The location of the variants on \u003cem\u003eADAM12\u003c/em\u003e gene and protein are illustrated in (Figure 1A).\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003e3.2. Replication study\u003c/h3\u003e\n\u003cp\u003eThe\u003cem\u003e\u0026nbsp;ADAM12\u003c/em\u003e variants\u0026rsquo; association with metabolic syndrome traits/T2D identified in the discovery cohort was verified in a separate cohort from the KDEP study, which included healthy controls (n=409) and T2D patients (n=450). This cohort consisted of adults of Arab, South Asian and Southeast Asian ethnicities in Kuwait. A summary of the demographic and clinical data of the replication cohort is summarized in Table 4. \u0026nbsp;The minor allele frequencies (MAFs) of ADAM12-K170R and ADAM12-M662V variants were higher (0.031 and 0.044, respectively) in the replication cohort compared to those detected in the discovery cohort (0.003436 and 0.006873, respectively), as well as compared to the total MAFs reported in GnomAd database (0.001106, and 0.002337, respectively) (GnomAd v2.1.1). The MAF of the ADAM12-I908V variant, on the other hand, was lower in the replication cohort (0.018) compared to the discovery cohort (0.029), and the total MAF reported in GnomAd database (0.02375) (GnomAd v2.1.1). In contrast, the minor allele of the ADAM12-R176W variant was not detected among participants in the replication cohort. All variants were in Hardy-Weinberg equilibrium in the healthy cohort (P\u0026gt;0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAssociations of ADAM12-K170R, and ADAM12-M662V detected in the discovery cohort were not confirmed in the replication cohort (Table S1), however, new associations were detected (Table 5). ADAM12-K170R was found to be associated with waist:hip ratio in T2D cohort (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.044) suggesting a potential but modest association. ADAM12-M662V was found to be associated with HDL in both healthy control (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.012) and T2D (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.017) cohorts suggesting a moderately strong and potentially meaningful effect, which sustained after adjustment for confounding factors, including age, sex, and BMI (\u0026beta;\u0026nbsp;= 0.098; 95%CI: 0.024 - 0.172; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.009) and also after adjusting for ethnicity (\u0026beta;\u0026nbsp;= 0.080; 95%CI: 0.014 \u0026ndash; 0.145; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.018) (Table S2). In contrast to the significant association of ADAM12-M662Vwith TC:HDL ratio (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.005), \u0026nbsp;HbA1c level showed a borderline association (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.044) in the healthy control cohort . Also, ADAM12-M662V association with waist:hip ratio was revealed after adjustment for the confounding factors mentioned above (\u0026beta;\u0026nbsp;=0.447; 95%CI:0.120 - 0.774; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.007) (Table S2). While most of these associations reached nominal significance ( \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05), Bonferroni correction was applied to account for multiple testing (adjusted threshold: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0045). After correction, only the association between ADAM12-M662V and HDL and TC:HDL remained statistically significant, supporting the robustness of these findings. In addition, the association between ADAM12-I908V and FBG identified in the discovery cohort was confirmed in healthy control and T2D cohorts, with a stronger association observed in the healthy controls (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.017) compared to the T2D group (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.044). This association was sustained after adjustment for confounding factors (\u0026beta;\u0026nbsp;= -0.007; 95%CI:-0.014- (-0.001); \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.02) and ethnicity (\u0026beta;\u0026nbsp;= -0.009 ; 95%CI: -0.015 - -0.003; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.004) (Table S3). Also, ADAM12-I908V showed a new association with HbA1c in the replication cohort (\u0026beta;\u0026nbsp;= -0.010; 95%CI: -0.019 - 0; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.04). When analysing healthy controls and T2D patients separately, ADAM12-I908V showed a significant association with LDL (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.001) and TC (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.006) in the T2D cohort, which remained statistically significant after Bonferroni correction . Further scrutiny of genotypes found three participants (0.35%) carried dual variants in the \u003cem\u003eADAM12\u003c/em\u003e gene, specifically the ADAM12-K170R and ADAM12-M662V variants\u0026rsquo;in heterozygosity. Similarly, another three participants exhibited double-variants of the ADAM12-K170R and ADAM12-I908V alleles, while only a single participant (0.12%) carried the ADAM12-M662V and ADAM12-I908V variants. All variants were detected in heterozygosity; no homozygous individuals were detected for any of the assessed \u003cem\u003eADAM12\u003c/em\u003e variants.\u003c/p\u003e\n\u003ch3\u003e3.3. Effect of ADAM12 variants on protein structure and stability\u003c/h3\u003e\n\u003cp\u003eThe variants detected in the \u003cem\u003eADAM12\u003c/em\u003e gene, namely ADAM12-K170R, ADAM12-R176W, ADAM12-M662V, and ADAM12-I908V are missense variants that cause the substitution of an amino acid in the prodomain (170R, 176W), EGF-like domain (662V) and the cytoplasmic (908V) tail (Figure 1A). The detected variants 170R, 662V and 908V affect amino acid residues that are conserved across species (Figure 1B, C, and D), whereas the variant 176W affects an amino acid that is not conserved across species (Figure 1B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePymol and DynaMut software assessed how these variants impact the ADAM12 structure\u0026rsquo;s stability and flexibility (Figure 2). The assessment predicts that 170R, 662V, and 908V variants destabilize the protein and increase its flexibility, while the 176W variant stabilizes the protein and decreases its flexibility (Figure 2A). The molecular mechanisms causing the observed changes in ADAM12 variants\u0026rsquo; stability and flexibility were investigated by analysing the intramolecular interactions within the WT and variant protein structures. This analysis revealed that 170R variant may introduce new interactions with glutamic acid (Glu150 within the prodomain and Glu374 of the metalloprotease domain) residues that are absent in the WT (Figure 2B). Similarly, the 176W variant stabilizes the proteins\u0026rsquo; structure and possibly introduces new intramolecular interactions with nearby glycine and tyrosine residues [Gly385 and Tyr386] (Figure 2C). In contrast, 662V variant may reduce the intramolecular interactions providing the enzyme with increased flexibility (Figure 2D). Finally, the structural analysis of 908V reveals the interaction of 908V with valine and proline [V899 and P900] that are in close proximity (Figure 2E).\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eEnzyme Activity and Kinetic Parameters of the ADAM12 variants\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSince the detected \u003cem\u003eADAM12\u003c/em\u003e variants are in the prodomain, the EGF-like domain, and the cytoplasmic tail, we evaluated the impact of these variants on ADAM12\u0026rsquo;s proteolytic function. \u003cem\u003eADAM12\u003c/em\u003e WT and variants were generated through site-directed mutagenesis and expressed separately in HEK-293 cells. Western blotting confirmed that these proteins were overexpressed in the HEK-293 cell lysates (Figure 3A). Enzymatic assays using the fluorogenic substrate PEPDAB005 revealed that ADAM12 variants 176W and 662V demonstrated a significantly increased proteolytic activity compared to ADAM12 WT (176W 55.4\u0026plusmn;7.2%; p=0.013 and 662V 93.5\u0026plusmn;25.7; p=0.045; Figure 3B). No significant change in proteolytic activity was observed using 170R and 908V variants\u0026rsquo; cell lysates\u0026rsquo; supernatants. To exclude any potential interference of the intracellular environment with enzyme activity, HEK-293 cells seeded on a 96-well black plate were transfected with \u003cem\u003eADAM12\u003c/em\u003e WT and variants as previously described. Enzyme activity was measured in intact cells, allowing the assessment of the enzyme activity present at the cell surface. The results showed that 170R significantly reduced the proteolytic activity of ADAM12 at the cell surface (18.9%; p=0.018). Both ADAM12 662V and 908V variants showed a statistically significant increase in proteolytic activity, which was very slight for the 662V variant (0.5%; p=0.006), while ADAM12 908V variant exhibited a much higher increase in activity at the cell surface (42.5%; p=0.028). No significant change in the proteolytic activity of ADAM12 variant 176W was observed at the cell surface (Figure 3C). Further, the effect of ADAM12 variants on its proteolytic activity was studied by determining the kinetic parameters, \u003cem\u003eVmax\u003c/em\u003e and \u003cem\u003eKm\u003c/em\u003e. As illustrated in Figure 3 D\u0026amp;E, ADAM12 WT and variants followed the Michaelis-Menten equation, so the Lineweaver-Burk plot was used to calculate \u003cem\u003eVmax\u003c/em\u003e and \u003cem\u003eKm\u003c/em\u003e values. Only 662V variant exhibited a significant reduction in \u003cem\u003eVmax\u003c/em\u003e value by 33.6% (p\u0026lt;0.01) compared to the WT (Figure 3E). Whereas the \u003cem\u003eKm\u003c/em\u003e values of 176W and 662V variants were significantly reduced by 41.1% (p\u0026lt;0.001) and 76.05% (p\u0026lt;0.001) respectively, compared to WT (Figure 3E).\u0026nbsp;\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eADAM12 plays an important role in various cellular functions and is associated with different pathological conditions [44]. Several GWA studies have identified loci associated with the risk of developing T2D [34, 45, 46]. However, most of the identified loci are associated with insulin secretion and B-cell function [34-36]. Our discovery GWAS identified the association of four novel rare/low-frequency (MAF \u0026pound;0.5%) \u003cem\u003eADAM12\u003c/em\u003e variants, namely ADAM12-K170R, ADAM12-R176W, ADAM12-M662V, and ADAM12-I908V with different metabolic syndrome and T2D traits including FBG, HbA1c, lipids including TC, HDL and LDL, and hypertension (DBP and SBP). However, these findings were not confirmed in our replication study except for the association of ADAM12-I908V with FBG. Nonetheless, our replication study found new associations with other traits, including FBG, HbA1c, waist:hip ratio, and lipids (TC, HDL, LDL, and TC:HDL ratio). This study reveals an unprecedented identification of associations between \u003cem\u003eADAM12\u003c/em\u003e variants and metabolic characteristics. Due to their rarity or low frequency, these variants are excluded from the genotyping arrays utilized in global GWA studies. However, the analysis of the GWAS catalogue, which presents GWAS findings from global studies, revealed associations between common variants of the \u003cem\u003eADAM12\u003c/em\u003e gene and metabolic traits. Specifically, rs10901513 (an intergenic variant FANK1-ADAM12) with visceral fat in men, rs11244839 (an intergenic variant RNA5SP328-ADAM12) with BMI in 0.5-1.5-year-old children, rs7920091 (among others) with height, and rs1551678 associated with thiazide-induced adverse metabolic effects in hypertensive patients (affecting fasting blood glucose measurement). The findings from our research on associations with T2D traits, together with those from global GWA studies on related traits, validate the role of ADAM12 in metabolic processes. Despite the differences between the ethnic origins of the discovery and replication cohorts and their lifestyles, adjustment for ethnicity did not affect the observed associations between \u003cem\u003eADAM12\u003c/em\u003e variants and the studied traits. However, the differences in MAFs of some of the reported \u003cem\u003eADAM12\u003c/em\u003e variants in the replication cohort, compared to the discovery cohort, may still reflect underlying population differences [34]. Additionally, the biological significance and effect size of the identified variants may be more specific to the Arab ethnicity of the discovery cohort, which was diluted in the replication cohort as it had different ethnicities.\u003c/p\u003e\n\u003cp\u003eThe identified \u003cem\u003eADAM12\u003c/em\u003e variants result in amino acid changes within the prodomain (170R, 176W), the EGF-like domain (662V), and the cytoplasmic tail (908V). Thus, we expected that protein structure and function may be affected. The type of amino acid change in the two prodomain variants, 170R and 176W may have contributed to the difference in proteolytic activity in cell lysates and intact cells. The conserved change of lysine to arginine in the 170R variant did not affect the proteolytic activity in cell lysates but was decreased at the cell surface of intact cells. Although 170R variant possibly introduces new intramolecular interaction patterns, as observed in our structural analysis, these alterations did not significantly affect the enzyme\u0026rsquo;s proteolytic activity in cell lysates. Hence, the increased stability introduced by this alteration may not influence the active site architecture or substrate accessibility. \u0026nbsp;However, these alterations could affect the prodomain\u0026rsquo;s function. It is established that the prodomain acts as an intramolecular chaperone facilitating proper protein folding [5] and regulating ADAMs\u0026rsquo; intracellular traffic [47, 48]. Therefore, the increased stability of 170R variant due to the potential intramolecular interactions introduced between R170, E150 within the prodomain, and E374 of the metalloprotease domain, possibly hindered the translocation of ADAM12 to the plasma membrane, which was reflected by a reduced proteolytic activity at the cell surface.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMoreover, the ADAM prodomain maintains the enzyme in its latent form by a cysteine switch mechanism [5, 49]. The activation mechanism involves cleavage of the prodomain by the furin enzyme [50]. Therefore, the non-conserved amino acid change of arginine to tryptophan in the R176W variant may have increased the proteolytic activity of this variant in cell lysates by disrupting the prodomain-active site\u0026rsquo;s interactions while paradoxically increasing the overall protein stability. In the WT, the enzyme is maintained in an autoinhibited state through the interactions of C179 within the prodomain with the catalytic Zn\u003csup\u003e2+\u003c/sup\u003e ion [51]. In contrast, the R176W variant introduces a bulky, hydrophobic tryptophan side chain [the indole ring] near the C179. The indole ring of the tryptophan may form van der Waals repulsive forces with neighbouring residues [G385, Y386], which possibly destabilize the local prodomain conformation and disrupt the inhibitory contact with the active site. This conformational alteration possibly increases substrate accessibility to the catalytic Zn\u003csup\u003e2+\u003c/sup\u003e site consistent with the reduced Km value [7, 51, 52]. Interestingly, while this variant exhibits locally disruptive interactions, Dynamut analysis revealed increased overall protein structural stability. This apparent paradox may be explained by compensatory stabilizing interactions that cause local structural rearrangements, reducing steric hindrance and promoting better hydrophobic packing within adjacent regions of the protein core. This variant may also reduce the translocation of ADAM12 to the plasma membrane, reflected by a non-significant change in activity within the intact cells, despite the increased activity in cell lysates. Moreover, the M662V variant that occurs within the EGF-like domain showed a potential increase in flexibility and proteolytic activity. Although the function of the EGF-like domain is not fully understood [50], it may affect the conformational state of the enzyme and its ability to interact with the substrate. Especially that the reduced intramolecular interactions observed in structural analyses may affect the secondary structure of ADAM12, which plays a vital role in substrate recognition [53]; hence, increasing the flexibility of the protein may allow easier access of the substrate to the active site.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe increased proteolytic activity of the cytoplasmic tail variant, I908V at the cell surface of intact cells compared to cell lysates\u0026rsquo; supernatants, may be caused by cytoplasmic tail modifications or interactions with different proteins leading to its translocation to the plasma membrane. Because the tail of ADAM proteins contains binding motifs for SH3-domain containing proteins and potential phosphorylation sites, it may modulate their function or provide binding sites for SH3-domain containing proteins [53-55]. The majority of ADAMs are located in the Golgi apparatus under steady-state conditions [56, 57], with only a minority found at the plasma membrane [58]. The translocation to the plasma membrane is induced by the phosphorylation of ADAM12 by PKCƐ [10, 59], whereas endocytosis of ADAMs depends on the binding of PACSIN3 to the SH3-binding domain [60]. Structural analysis suggests that, in the WT, I908 is possibly involved in an interaction with A906. However, the substitution of Ile with Val at position 908 of the variant possibly allows the interaction of V908 with V899 and P900, creating a local loop-like conformation, which exposes nearby serine and tyrosine [S902, Y903, Y905] residues, making them more accessible for potential phosphorylation. This conformational change may facilitate easier access for kinases or adaptor proteins such as Tks5/FISH [61], PI3K, and actinin-1/2 [7], thereby increasing the possibility of phosphorylation-dependent translocation to the plasma membrane. Hence, this structural rearrangement may partially elucidate the increased surface proteolytic activity observed for the I908V variant.\u003c/p\u003e\n\u003cp\u003eAlthough ADAM12 knockout mice exhibit normal glucose tolerance, the increased activity of the identified \u003cem\u003eADAM12\u003c/em\u003e variants may contribute to the development of metabolic syndrome/T2D-associated traits such as obesity (Figure S2) [44]. While direct evidence associating ADAM12 to insulin resistance is lacking, several mechanistic links are plausible. Evidence supports the role of ADAM12 in the development of high-fat diet-induced obesity by stimulating new adipocyte formation (hyperplasia) rather than by enhancing differentiation and/or maturation (hypertrophy) of preexisting adipocytes via mechanisms involving its proteolytic activity and its interaction with b1 integrins [8, 10-12]. In addition, Coles et al., reported that the two most affected signalling pathways by ADAM12 knockdown included IGF/IGFBP/mTOR growth pathway and PPAR\u0026gamma; signalling pathway, both of which are critical regulators of adipogenesis and insulin sensitivity. ADAM12 can cleave IGFBP3 and IGFBP5 [9]; therefore, increased ADAM12 activity may increase the bioavailability of IGF and stimulate preadipocyte proliferation. Hence, the increased \u003cem\u003eADAM12\u003c/em\u003e variants\u0026rsquo; proteolytic activity may enhance IGF bioavailability and subsequently stimulate preadipocyte proliferation. Therefore, the increased proteolytic activity of \u003cem\u003eADAM12\u003c/em\u003e variants identified in this study may promote white adipose tissue (WAT) mass expansion. Besides, ADAM12 can remodel the cytoskeleton via b1 integrin and alter the dense fibronectin-rich matrix, allowing the flat preadipocytes to take a round shape during differentiation since fibronectin can be cleaved by ADAM12 [62]. The increase in WAT mass could act as a double-edged sword, while initial hyperplasia may transiently maintain insulin sensitivity, prolonged adipose tissue expansion often leads to hypertrophy, adipose tissue dysfunction, chronic low-grade inflammation, and ultimately, systemic insulin resistance development [63, 64]. However, while these mechanistic links are biologically plausible, direct evidence associating increased ADAM12 activity to insulin resistance and metabolic syndrome traits remains limited. Thus, the association of ADAM12 with metabolic disorders remains exploratory. To address this knowledge gap, further in vitro and in vivo studies that integrate functional metabolic phenotyping that assess metabolic syndrome and T2D traits in models overexpressing hyperactive \u003cem\u003eADAM12\u003c/em\u003e variants are needed to elucidate how the increased \u003cem\u003eADAM12\u003c/em\u003e variants\u0026rsquo; activity reported herein may contribute to insulin resistance and T2D development. Moreover, evaluating how hyperactive ADAM12 may affect adipose tissue remodelling, inflammation, and insulin sensitivity may aid in elucidating its role in T2D pathogenesis. In vitro experiments studying the role of ADAM12-driven IGF/IGFBP axis modulation in adipocyte insulin sensitivity and glucose metabolism may also be of value. Altogether, these approaches will explain how the increased \u003cem\u003eADAM12\u003c/em\u003e variants activity reported herein may contribute to the pathophysiology of insulin resistance and T2D.\u003c/p\u003e\n\u003cp\u003eThere are several limitations to our study including the relatively small sample size in the two cohorts. However, despite the relatively small sizes of our discovery and replication cohorts, the differences in the ethnic origins of the participants, and their demographic and clinical characteristics we were able to identify rare \u003cem\u003eADAM12\u003c/em\u003e variants and confirm their association with several metabolic traits relevant to T2D. Another limitation is that the replication results may have been influenced by the heterogeneity of the tested cohorts; with the replication cohort including multiple ethnicities, while the discovery cohort being limited to individuals of Arab descent, through differences in allele frequencies and genetic backgrounds. In addition, the limited sample sizes within individual subgroups impeded ethnicity-stratified analyses or adjustment for population structure, which would have been valuable, and their consideration remains important for future studies. Furthermore, detailed clinical information regarding diabetes duration, treatment, and end-organ complications was not available, which represents another limitation of this study. In addition, the absence of direct or surrogate insulin resistance measurements, such as Homeostatic Model Assessment of Insulin Resistance, HOMA-IR, which would provide a more accurate assessment of T2D status, may added an additional limitation. The use of HEK293 cells in this study, although advantageous for initial enzymatic characterization due to their high transfection efficiency and minimal endogenous ADAM12 activity, lack the physiological context of adipose or muscle cells. Therefore, further in vitro and in vivo studies using more relevant cellular models, such as primary adipocytes, or myoblasts are required to confirm their role in metabolic syndrome and T2D pathophysiology. \u0026nbsp;\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eOur GWAS suggested the association of four \u003cem\u003eADAM12\u003c/em\u003e gene variants (namely ADAM12-K170R, ADAM12-R176W, ADAM12-M662V, and ADAM12-I908V) with metabolic syndrome/T2D traits. Using a replication cohort, the association of ADAM12-I908V with FBG was confirmed, and more associations of the variants with different metabolic traits were found, confirming the potential role of \u003cem\u003eADAM12\u003c/em\u003e variants in metabolic syndrome/T2D development. The functional study revealed a variant-specific difference in the activity of ADAM12 in cell lysates and the cell surface, suggesting that dysregulation of ADAM12 activity at the cell surface may play a role in the development of T2D. However, the exact mechanisms and tissues involved are not fully elucidated yet. Therefore, further studies are required to enable targeting ADAM12 for the potential treatment of these conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests:\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis study was supported and funded by Kuwait University, Graduate Research Project No. YM07/21, and by Kuwait Foundation for the Advancement of Sciences (KFAS), as part of research project (RA AM-2023-010).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eH.D. conducted genotyping and enzyme activity assays, data collection and analysis, and wrote the original draft of manuscript. A.M. conducted enzyme activity assays, data collection, and manuscript writing. T.A.T. was involved in GWAS data collection and analysis and manuscript editing. A.C. and P.H. assisted with GWAS and genotyping data analysis. S.S. and R.A. provided guidance for enzyme assays and manuscript writing. F.M. and R.T. were involved in conceptualization, methodology development, supervision, and manuscript writing.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data supporting the findings of this study are available in public repositories and within the manuscript or supplementary materials. The SNP data is available via the following accession numbers ADAM12-K170R (rs112264074), ADAM12-R176W (rs140497576), ADAM12-M662V (rs115100580), and ADAM12-I908V (rs41303603). Moreover, the ADAM12 crystal structure accession ID (O43184) is a UniProt accession number that is composed of only 6 characters.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGiebeler, N. \u0026amp; Zigrino, P. A Disintegrin and Metalloprotease (ADAM): Historical Overview of Their Functions. \u003cem\u003eToxins (Basel)\u003c/em\u003e. \u003cb\u003e8\u003c/b\u003e (4), 122 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDuffy, M. J. et al. The role of ADAMs in disease pathophysiology. \u003cem\u003eClin. Chim. Acta\u003c/em\u003e. \u003cb\u003e403\u003c/b\u003e (1\u0026ndash;2), 31\u0026ndash;36 (2009).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLoechel, F. et al. Human ADAM 12 (meltrin alpha) is an active metalloprotease. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cb\u003e273\u003c/b\u003e (27), 16993\u0026ndash;16997 (1998).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJacobsen, J. \u0026amp; Wewer, U. M. Targeting ADAM12 in human disease: head, body or tail? \u003cem\u003eCurr. Pharm. Des.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e (20), 2300\u0026ndash;2310 (2009).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWong, E. et al. The Functional Maturation of A Disintegrin and Metalloproteinase (ADAM) 9, 10, and 17 Requires Processing at a Newly Identified Proprotein Convertase (PC) Cleavage Site. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cb\u003e290\u003c/b\u003e (19), 12135\u0026ndash;12146 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuovila, A. P. et al. Shedding light on ADAM metalloproteinases. \u003cem\u003eTrends Biochem. Sci.\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e (7), 413\u0026ndash;422 (2005).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKveiborg, M. et al. Cellular roles of ADAM12 in health and disease. \u003cem\u003eInt. J. Biochem. Cell. Biol.\u003c/em\u003e \u003cb\u003e40\u003c/b\u003e (9), 1685\u0026ndash;1702 (2008).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKawaguchi, N. et al. ADAM12 induces actin cytoskeleton and extracellular matrix reorganization during early adipocyte differentiation by regulating beta1 integrin function. \u003cem\u003eJ. Cell. Sci.\u003c/em\u003e \u003cb\u003e116\u003c/b\u003e (Pt 19), 3893\u0026ndash;3904 (2003).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eColes, C. A. et al. Knockdown of a disintegrin A metalloprotease 12 (ADAM12) during adipogenesis reduces cell numbers, delays differentiation, and increases lipid accumulation in 3T3-L1 cells. \u003cem\u003eMol. Biol. Cell.\u003c/em\u003e \u003cb\u003e29\u003c/b\u003e (15), 1839\u0026ndash;1855 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKawaguchi, N. et al. ADAM 12 protease induces adipogenesis in transgenic mice. \u003cem\u003eAm. J. Pathol.\u003c/em\u003e \u003cb\u003e160\u003c/b\u003e (5), 1895\u0026ndash;1903 (2002).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKurisaki, T. et al. Phenotypic analysis of Meltrin alpha (ADAM12)-deficient mice: involvement of Meltrin alpha in adipogenesis and myogenesis. \u003cem\u003eMol. Cell. Biol.\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e (1), 55\u0026ndash;61 (2003).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMasaki, M. et al. Role of meltrin {alpha} (ADAM12) in obesity induced by high- fat diet. \u003cem\u003eEndocrinology\u003c/em\u003e \u003cb\u003e146\u003c/b\u003e (4), 1752\u0026ndash;1763 (2005).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlkandari, A. et al. The prevalence of pre-diabetes and diabetes in the Kuwaiti adult population in 2014. \u003cem\u003eDiabetes Res. Clin. Pract.\u003c/em\u003e \u003cb\u003e144\u003c/b\u003e, 213\u0026ndash;223 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWeiderpass, E. et al. The Prevalence of Overweight and Obesity in an Adult Kuwaiti Population in 2014. \u003cem\u003eFront. Endocrinol. (Lausanne)\u003c/em\u003e. \u003cb\u003e10\u003c/b\u003e, 449 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuembe, M. J. et al. Risk for cardiovascular disease associated with metabolic syndrome and its components: a 13-year prospective study in the RIVANA cohort. \u003cem\u003eCardiovasc. Diabetol.\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e (1), 195 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarott, S. C. W. et al. Components of the Metabolic Syndrome and Risk of Type 2 Diabetes. \u003cem\u003eJ. Clin. Endocrinol. Metabolism\u003c/em\u003e. \u003cb\u003e101\u003c/b\u003e (8), 3212\u0026ndash;3221 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAl-Awadi, S. A. et al. Consanguinity among the Kuwaiti population. \u003cem\u003eClin. Genet.\u003c/em\u003e \u003cb\u003e27\u003c/b\u003e (5), 483\u0026ndash;486 (1985).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWarsy, A. S. et al. Is consanguinity prevalence decreasing in Saudis? A study in two generations. \u003cem\u003eAfr. Health Sci.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e (2), 314\u0026ndash;321 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGosadi, I. M., Goyder, E. C. \u0026amp; Teare, M. D. Investigating the potential effect of consanguinity on type 2 diabetes susceptibility in a Saudi population. \u003cem\u003eHum. Hered\u003c/em\u003e. \u003cb\u003e77\u003c/b\u003e (1\u0026ndash;4), 197\u0026ndash;206 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBener, A. \u0026amp; Hussain, R. Consanguineous unions and child health in the State of Qatar. \u003cem\u003ePaediatr. Perinat. Epidemiol.\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e (5), 372\u0026ndash;378 (2006).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBenrahma, H. et al. Maternal effect and familial aggregation in a type 2 diabetic Moroccan population. \u003cem\u003eJ. Community Health\u003c/em\u003e. \u003cb\u003e36\u003c/b\u003e (6), 943\u0026ndash;948 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eArfa, I. et al. Familial aggregation and excess maternal transmission of type 2 diabetes in Tunisia. \u003cem\u003ePostgrad. Med. J.\u003c/em\u003e \u003cb\u003e83\u003c/b\u003e (979), 348\u0026ndash;351 (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAl-Sinani, S. et al. Familial Clustering of Type 2 Diabetes among Omanis. \u003cem\u003eOman Med. J.\u003c/em\u003e \u003cb\u003e29\u003c/b\u003e (1), 51\u0026ndash;54 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBener, A. et al. Parental transmission of type 2 diabetes mellitus in a highly endogamous population. \u003cem\u003eWorld J. Diabetes\u003c/em\u003e. \u003cb\u003e4\u003c/b\u003e (2), 40\u0026ndash;46 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEaaswarkhanth, M. et al. Genome-Wide Selection Scan in an Arabian Peninsula Population Identifies a TNKS Haplotype Linked to Metabolic Traits and Hypertension. \u003cem\u003eGenome Biol. Evol.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e (3), 77\u0026ndash;87 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHebbar, P. et al. A Perception on Genome-Wide Genetic Analysis of Metabolic Traits in Arab Populations. \u003cem\u003eFront. Endocrinol. (Lausanne)\u003c/em\u003e. \u003cb\u003e10\u003c/b\u003e, 8 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSakul, H. et al. Familiality of physical and metabolic characteristics that predict the development of non-insulin-dependent diabetes mellitus in Pima Indians. \u003cem\u003eAm. J. Hum. Genet.\u003c/em\u003e \u003cb\u003e60\u003c/b\u003e (3), 651\u0026ndash;656 (1997).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eElbein, S. C. et al. Heritability of pancreatic beta-cell function among nondiabetic members of Caucasian familial type 2 diabetic kindreds. \u003cem\u003eJ. Clin. Endocrinol. Metab.\u003c/em\u003e \u003cb\u003e84\u003c/b\u003e (4), 1398\u0026ndash;1403 (1999).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWatanabe, R. M. et al. Familiality of quantitative metabolic traits in Finnish families with non-insulin-dependent diabetes mellitus. Finland-United States Investigation of NIDDM Genetics (FUSION) Study investigators. \u003cem\u003eHum. Hered\u003c/em\u003e. \u003cb\u003e49\u003c/b\u003e (3), 159\u0026ndash;168 (1999).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLehtovirta, M. et al. Insulin sensitivity and insulin secretion in monozygotic and dizygotic twins. \u003cem\u003eDiabetologia\u003c/em\u003e \u003cb\u003e43\u003c/b\u003e (3), 285\u0026ndash;293 (2000).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLin, H. F. et al. Heritabilities of the metabolic syndrome and its components in the Northern Manhattan Family Study. \u003cem\u003eDiabetologia\u003c/em\u003e \u003cb\u003e48\u003c/b\u003e (10), 2006\u0026ndash;2012 (2005).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBellia, A. et al. The Linosa Study: epidemiological and heritability data of the metabolic syndrome in a Caucasian genetic isolate. \u003cem\u003eNutr. Metab. Cardiovasc. Dis.\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e (7), 455\u0026ndash;461 (2009).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCarmelli, D., Cardon, L. R. \u0026amp; Fabsitz, R. Clustering of hypertension, diabetes, and obesity in adult male twins: same genes or same environments? \u003cem\u003eAm. J. Hum. Genet.\u003c/em\u003e \u003cb\u003e55\u003c/b\u003e (3), 566\u0026ndash;573 (1994).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrown, A. E. \u0026amp; Walker, M. Genetics of Insulin Resistance and the Metabolic Syndrome. \u003cem\u003eCurr. Cardiol. Rep.\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e (8), 75 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim, H. et al. High-throughput genetic clustering of type 2 diabetes loci reveals heterogeneous mechanistic pathways of metabolic disease. \u003cem\u003eDiabetologia\u003c/em\u003e \u003cb\u003e66\u003c/b\u003e (3), 495\u0026ndash;507 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMadsen, A. L. et al. Genetic architecture of oral glucose-stimulated insulin release provides biological insights into type 2 diabetes aetiology. \u003cem\u003eNat. Metab.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e (10), 1897\u0026ndash;1912 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHebbar, P. et al. Genome-wide association study identifies novel recessive genetic variants for high TGs in an Arab population. \u003cem\u003eJ. Lipid Res.\u003c/em\u003e \u003cb\u003e59\u003c/b\u003e (10), 1951\u0026ndash;1966 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHebbar, P. et al. Genome-wide landscape establishes novel association signals for metabolic traits in the Arab population. \u003cem\u003eHum. Genet.\u003c/em\u003e \u003cb\u003e140\u003c/b\u003e (3), 505\u0026ndash;528 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHebbar, P. et al. The TCN2 variant of rs9606756 [Ile23Val] acts as risk loci for obesity-related traits and mediates by interacting with Apo-A1. \u003cem\u003eObes. (Silver Spring)\u003c/em\u003e. \u003cb\u003e25\u003c/b\u003e (6), 1098\u0026ndash;1108 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePeterson, R. E. et al. Genome-wide Association Studies in Ancestrally Diverse Populations: Opportunities, Methods, Pitfalls, and Recommendations. \u003cem\u003eCell\u003c/em\u003e \u003cb\u003e179\u003c/b\u003e (3), 589\u0026ndash;603 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBiobank, U. \u003cem\u003eUK Biobank: protocol for a large-scale prospective epidemiological resource.\u003c/em\u003e Accessed May, 7(2016): pp. 1\u0026ndash;112. (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXue, A. et al. Genome-wide association analyses identify 143 risk variants and putative regulatory mechanisms for type 2 diabetes. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e (1), 2941 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBisswanger, H. \u003cem\u003ePractical Enzymology\u003c/em\u003e (Wiley-VCH Verlag GmbH \u0026amp; Co. KGaA, 2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNyren-Erickson, E. K. et al. A disintegrin and metalloproteinase-12 (ADAM12): function, roles in disease progression, and clinical implications. \u003cem\u003eBiochim. Biophys. Acta\u003c/em\u003e. \u003cb\u003e1830\u003c/b\u003e (10), 4445\u0026ndash;4455 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlbrechtsen, A. et al. Exome sequencing-driven discovery of coding polymorphisms associated with common metabolic phenotypes. \u003cem\u003eDiabetologia\u003c/em\u003e \u003cb\u003e56\u003c/b\u003e (2), 298\u0026ndash;310 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSteinthorsdottir, V. et al. Identification of low-frequency and rare sequence variants associated with elevated or reduced risk of type 2 diabetes. \u003cem\u003eNat. Genet.\u003c/em\u003e \u003cb\u003e46\u003c/b\u003e (3), 294\u0026ndash;298 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnders, A. et al. Regulation of the alpha-secretase ADAM10 by its prodomain and proprotein convertases. \u003cem\u003eFaseb j.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e (10), 1837\u0026ndash;1839 (2001).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGonzales, P. E. et al. Inhibition of the tumor necrosis factor-alpha-converting enzyme by its pro domain. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cb\u003e279\u003c/b\u003e (30), 31638\u0026ndash;31645 (2004).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoghani, M. et al. Metalloprotease-disintegrin MDC9: intracellular maturation and catalytic activity. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cb\u003e274\u003c/b\u003e (6), 3531\u0026ndash;3540 (1999).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKlein, T. \u0026amp; Bischoff, R. Active metalloproteases of the A Disintegrin and Metalloprotease (ADAM) family: biological function and structure. \u003cem\u003eJ. Proteome Res.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e (1), 17\u0026ndash;33 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLoechel, F. et al. Regulation of human ADAM 12 protease by the prodomain. Evidence for a functional cysteine switch. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cb\u003e274\u003c/b\u003e (19), 13427\u0026ndash;13433 (1999).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVan Wart, H. E. \u0026amp; Birkedal-Hansen, H. The cysteine switch: a principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family. \u003cem\u003eProc. Natl. Acad. Sci. U S A\u003c/em\u003e. \u003cb\u003e87\u003c/b\u003e (14), 5578\u0026ndash;5582 (1990).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSeals, D. F. \u0026amp; Courtneidge, S. A. The ADAMs family of metalloproteases: multidomain proteins with multiple functions. \u003cem\u003eGenes Dev.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e (1), 7\u0026ndash;30 (2003).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eReiss, K. \u0026amp; Saftig, P. The a disintegrin and metalloprotease (ADAM) family of sheddases: physiological and cellular functions. \u003cem\u003eSemin Cell. Dev. Biol.\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e (2), 126\u0026ndash;137 (2009).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKang, Q., Cao, Y. \u0026amp; Zolkiewska, A. \u003cem\u003eMetalloprotease-disintegrin ADAM 12 binds to the SH3 domain of Src and activates Src tyrosine kinase in C2C12 cells.\u003c/em\u003e Biochem J, 352 Pt 3(Pt 3): pp. 883\u0026ndash;92. (2000).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLammich, S. et al. Constitutive and regulated alpha-secretase cleavage of Alzheimer's amyloid precursor protein by a disintegrin metalloprotease. \u003cem\u003eProc. Natl. Acad. Sci. U S A\u003c/em\u003e. \u003cb\u003e96\u003c/b\u003e (7), 3922\u0026ndash;3927 (1999).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchl\u0026ouml;ndorff, J., Becherer, J. D. \u0026amp; Blobel, C. P. Intracellular maturation and localization of the tumour necrosis factor alpha convertase (TACE). \u003cem\u003eBiochem. J.\u003c/em\u003e \u003cb\u003e347\u003c/b\u003e (Pt 1), 131\u0026ndash;138 (2000).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGutwein, P. et al. ADAM10-mediated cleavage of L1 adhesion molecule at the cell surface and in released membrane vesicles. \u003cem\u003eFaseb j.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e (2), 292\u0026ndash;294 (2003).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSundberg, C. et al. Regulation of ADAM12 cell-surface expression by protein kinase C epsilon. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cb\u003e279\u003c/b\u003e (49), 51601\u0026ndash;51611 (2004).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMori, S. et al. PACSIN3 binds ADAM12/meltrin alpha and up-regulates ectodomain shedding of heparin-binding epidermal growth factor-like growth factor. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cb\u003e278\u003c/b\u003e (46), 46029\u0026ndash;46034 (2003).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbram, C. L. et al. The adaptor protein fish associates with members of the ADAMs family and localizes to podosomes of Src-transformed cells. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cb\u003e278\u003c/b\u003e (19), 16844\u0026ndash;16851 (2003).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoy, R. et al. ADAM 12 cleaves extracellular matrix proteins and correlates with cancer status and stage. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cb\u003e279\u003c/b\u003e (49), 51323\u0026ndash;51330 (2004).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuilherme, A. et al. Adipocyte dysfunctions linking obesity to insulin resistance and type 2 diabetes. \u003cem\u003eNat. Rev. Mol. Cell. Biol.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e (5), 367\u0026ndash;377 (2008).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZatterale, F. et al. Chronic Adipose Tissue Inflammation Linking Obesity to Insulin Resistance and Type 2 Diabetes. \u003cem\u003eFront. Physiol.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 1607 (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 5 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ADAM12, Metabolic syndrome, insulin resistance, genetic variant","lastPublishedDoi":"10.21203/rs.3.rs-8138455/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8138455/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe high prevalence of insulin resistance and metabolic syndrome in Kuwait increases the risk of type 2 diabetes (T2D) and cardiovascular diseases development. Although the genetic contribution to insulin resistance and metabolic syndrome is established, the role of a disintegrin and metalloprotease 12 (ADAM12) in insulin resistance and T2D remains unclear. Our GWAS has identified four novel \u003cem\u003eADAM12\u003c/em\u003e variants, namely, ADAM12-K170R rs112264074 [NP_003465.3:p.Lys170Arg], ADAM12-R176W rs140497576 [NP_003465.3:p.Arg176Trp], ADAM12-M662V rs115100580 [NP_003465.3:p.Met662Val], and ADAM12-I908V rs41303603 [NP_003465.3:p.Ile908Val]. These variants were associated with different metabolic traits, including FBG, HbA1c, low- (LDL) and high-density lipoprotein (HDL), total cholesterol (TC), and diastolic and systolic blood pressure. Using an independent replication cohort, the association of ADAM12-I908V with FBG was confirmed, and an association with HbA1c, LDL, and TC was found. In addition, ADAM12-K170R showed associations with waist-hip ratio in diabetic patients, and ADAM12-M662V associated with HDL, TC, and HbA1c in healthy controls, while ADAM12-R176W variant was not detected in the replication cohort. Moreover, we examined the impact of \u003cem\u003eADAM12\u003c/em\u003e variants on its proteolytic activity and results show that 176W and 662V variants had higher activity in cell lysate supernatants, but only 662V and 908V variants had higher activity in intact cells, suggesting that enzyme activity dysregulation may contribute to the development of metabolic syndrome andT2D.\u003c/p\u003e","manuscriptTitle":"Functional ADAM12 Variants Modulate Proteolytic Activity and Influence Metabolic Traits","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-01 06:58:38","doi":"10.21203/rs.3.rs-8138455/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-23T07:10:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-19T00:03:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"76451266985446278491208921866140595039","date":"2025-12-16T17:42:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"233855451656447405964762634416933427212","date":"2025-11-20T19:19:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-20T13:23:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-18T13:51:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-18T13:49:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-17T18:48:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7299ff22-49c6-4919-bf58-47c410018efd","owner":[],"postedDate":"December 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":58347183,"name":"Health sciences/Biomarkers"},{"id":58347184,"name":"Health sciences/Diseases"},{"id":58347185,"name":"Health sciences/Endocrinology"},{"id":58347186,"name":"Biological sciences/Genetics"}],"tags":[],"updatedAt":"2026-01-12T16:00:25+00:00","versionOfRecord":{"articleIdentity":"rs-8138455","link":"https://doi.org/10.1038/s41598-025-34827-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-07 15:57:11","publishedOnDateReadable":"January 7th, 2026"},"versionCreatedAt":"2025-12-01 06:58:38","video":"","vorDoi":"10.1038/s41598-025-34827-1","vorDoiUrl":"https://doi.org/10.1038/s41598-025-34827-1","workflowStages":[]},"version":"v1","identity":"rs-8138455","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8138455","identity":"rs-8138455","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}