Islet biology, the CDKN2A/B locus and type 2 diabetes risk.

Type 2 diabetes SNPs at CDKN2A/B are non-coding, and high-resolution mapping of the CDKN2A/B locus did not reveal SNPs with greater disease association than known SNPs [ 185 ]. Attention has thus turned to the regulation of local genes. Testing whether genomic polymorphisms alter mRNA abundance is performed by expression quantitative trait locus (eQTL) analysis. Thus far, however, eQTL analysis for the CDKN2A/B locus has failed to produce major mechanistic breakthroughs. Type 2 diabetes SNPs at CDKN2A/B were not associated with expression of CDKN2A or CDKN2B in pancreas, liver or colon [ 186 ], nor in pancreatic islets themselves [ 187 ]. CDKN2A/B SNPs impacting cardiovascular risk, outside the type 2 diabetes region, influence ANRIL expression [ 71 , 188 ]. Both type 2 diabetes and non-type 2 diabetes SNPs at this locus appear to have a stronger effect on ANRIL expression than CDKN2A or CDKN2B expression [ 189 ]. Expression of ANRIL is reported to correlate with expression of CDKN2A and CDKN2B in many tissues, suggesting coordinated local regulation [ 69 , 71 , 76 , 189 ], although independent regulation is also reported [ 76 , 126 ]. Deletion of a large region including the type 2 diabetes-risk locus plus part of ANRIL reduced CDKN2A and CDKN2B expression in several vascular-relevant cell types in mice [ 172 ]. ANRIL regulates gene expression at the CDKN2A/B locus by recruiting polycomb proteins chromobox homologue 7 (CBX7 [PRC1]) and suppressor of zeste 12 (SUZ12 [PRC2]) to modulate epigenetic repression by H3K27 methylation [ 70 , 72 , 73 , 90 , 190 ]. ANRIL can also regulate distant genes with retrotransposon Alu repeats in their promoters [ 191 ]. In sum, existing eQTL analyses do not identify a mechanism by which polymorphisms at CDKN2A/B influence type 2 diabetes risk through local gene expression, but studies may have been performed in the wrong cell type, developmental, environmental or nutritional state to identify the point of activity. The requirement to perform these studies in human tissues, the difficulty in obtaining human samples, and caveats introduced through postmortem state, tissue collection and ex vivo culture are barriers to this type of study. Cis -regulatory elements are found near the type 2 diabetes-risk interval [ 192 – 194 ]. Disease-associated SNPs are predicted to disrupt transcription factor binding sites, including some factors with known roles in beta cell development or survival, including, v-maf avian musculoaponeurotic fibrosarcoma oncogene homologue B (MAFB), NK homeobox protein 6.1 (NKX6.1), nuclear factor of activated T lymphocytes (NFAT), FOXA2, forkhead box A2 (FOXA2), nuclear factor κβ (NFκB), hepatocyte nuclear factor 1 (HNF1) and CCAAT-enhancer-binding protein homologous protein (CHOP) [ 90 , 192 , 194 ]. Polymorphisms may also impact microRNA regulation of transcription or translation [ 195 ], although most SNPs at the type 2 diabetes locus are non-coding. An enhancer identified in tumours, RD INK4/ARF , interacts with oncoproteins to silence the CDKN2A/B locus [ 196 , 197 ]. Deletions of RD INK4/ARF have been detected in pancreatic neuroendocrine tumours, implying activity in islet cells [ 198 ]. Epigenetic modification regulates gene expression at this locus in islets [ 116 , 119 ]. The type 2 diabetes-associated rs564398 removes a DNA methylation CpG site, reducing methylation of other local CpG sites and decreasing insulin content in human islets, although without impacting local gene expression [ 199 ]. Histone modifications can be influenced by polymorphisms as well; many epigenetic modifiers act at the CDKN2A/B locus [ 200 ]. Metabolic inputs, such as overfeeding or energy restriction, influence CpG methylation and histone modification at CDKN2A/B in humans [ 201 , 202 ]. Whether this is influenced by type 2 diabetes-risk polymorphisms remains unknown. Taken together, although many mechanisms exist by which polymorphisms at CDKN2A/B might influence local gene expression, none has yet been proved.

The CDKN2A/B locus, at chromosome 9p21, influences diabetes risk across varied ethnicities and geography [ 1 – 3 ], including people of European [ 4 – 9 ], Asian [ 10 – 24 ], Indian [ 25 ], Pakistani [ 26 ], Mexican [ 27 – 29 ] and Arab [ 30 – 32 ] descent. CDKN2A/B polymorphisms impact risk of gestational diabetes [ 33 – 35 ], early progression to type 2 diabetes after gestational diabetes [ 36 ], post-transplant diabetes [ 37 , 38 ] and cystic fibrosis-related diabetes [ 39 ]. CDKN2A/B is not associated with type 1 diabetes risk [ 40 – 42 ], but is associated with rapid decline in beta cell function [ 43 ], and progression to diabetic nephropathy, in individuals with type 1 diabetes [ 44 ]. CDKN2A/B polymorphisms contribute only a fraction of observed heritable type 2 diabetes risk [ 2 ]. How CDKN2A/B influences diabetes risk remains uncertain [ 1 – 3 ]. The CDKN2A/B locus also influences risk for vascular conditions [ 1 ], including coronary artery disease [ 45 – 49 ], aneurysm and ischaemic stroke [ 50 , 51 ], as well as glaucoma [ 52 , 53 ], Alzheimer's disease [ 54 ], endometriosis [ 55 ], periodontitis [ 56 ], ageing-related diseases [ 3 , 57 ] and numerous cancers [ 58 ]. Intriguingly, the region of CDKN2A/B influencing type 2 diabetes risk is physically separated from regions contributing risk for other diseases, even for type 2 diabetes-related disorders such as cancer and cardiovascular disease [ 59 – 61 ].

Great progress has been made in identifying roles played by CDKN2A/B gene products in islets and other metabolic tissues, mostly in rodents but also in humans. p16 INK4A and related proteins are critical regulators of rodent beta cell mass, but whether human CDKN2A/B polymorphisms influence type 2 diabetes risk via islet biology remains uncertain. Gene regulation analyses have not yet proved a relationship between type 2 diabetes SNPs and local gene expression, but the analyses to date may not have been performed in the relevant tissue, developmental stage, metabolic milieu and/or human subpopulation. CDKN2A/B genes impact islet, adipose, muscle, liver and immune cell function, at stages ranging from in utero development to ageing. Human biological variation likely influences CDKN2A/B effects. The availability of human samples across tissues and stages is a serious limitation in this field. We are hopeful that, in the future, CDKN2A/B polymorphisms will improve diabetes understanding and inform clinical decisions.

In order to consider how single nucleotide polymorphisms (SNPs) in the CDKN2A/B region may impact type 2 diabetes risk, an important starting point is the known biology of local genes. The human CDKN2A/B locus ( Fig. 1 ) encodes three proteins: p14 alternate reading frame (p14 ARF ) (p19 ARF in mice), p15 and p16 inhibitors of cyclin dependent kinase 4 (p15 INK4B and p16 INK4A ), and a long non-coding RNA (lncRNA) called ANRIL (also known as CDKN2B-AS) [ 1 , 62 , 63 ]. p16 INK4A and p14 ARF , encoded by the CDKN2A gene, share common second and third exons but have different first exons and promoters about 20 kb apart [ 64 ]. p16 INK4A and p14 ARF are in alternate reading frames, resulting in unrelated peptide sequences despite the common mRNA sequence. CDKN2B , about 30 kb from CDKN2A , encodes p15 INK4B [ 65 , 66 ]. Smaller splice variants of both p16 INK4A (p12) and p15 INK4B (p10) have been described [ 64 , 67 , 68 ]. ANRIL , which overlaps the p14 ARF promoter and two exons of p15 INK4B [ 69 ], transcribed by RNA polymerase II [ 70 ] in the antisense direction to CDKN2B , is spliced into linear or circular isoforms [ 69 , 71 , 72 ]. The mouse Cdkn2a/b locus, on chromosome 4, encodes p16 INK4A , p19 ARF and p15 INK4B in a similar arrangement to the human locus, but with a different lncRNA called AK148321 in a position similar to ANRIL [ 73 ]. CDKN2A/B proteins block cell cycle progression and influence tumorigenesis, senescence and ageing [ 62 , 63 ]. p16 INK4A and p15 INK4B are cyclin-dependent kinase (CDK) inhibitors that prevent activation of CDK4/6 by D-cyclins ( Fig. 2 ). CDK4/6 phosphorylates retinoblastoma (Rb); hypophosphorylated Rb represses early region 2 transcription factor (E2F) to prevent cell cycle entry [ 74 ]. p14 ARF , also antiproliferative, acts by stabilising the tumour suppressor p53 by sequestering its negative regulator, mouse double minute 2 homologue (MDM2) [ 75 ]. p53 reduces cell cycle entry through the CDK-interacting protein/kinase inhibitory protein (CIP/KIP) family inhibitor p21. The p10 and p12 splice variants of p16 INK4A and p15 INK4B also inhibit the cell cycle, but through slightly different mechanisms [ 64 , 67 ]. ANRIL has widespread influences on gene expression, and impacts the cell cycle by regulating the expression of p14 ARF , p15 INK4B and p16 INK4A [ 73 ]. The CDKN2A/B locus is inactivated by deletion, methylation or mutation in many cancers; CDKN2A/B aberrations correlate with advanced tumour stage and reduced overall and disease-free survival [ 58 , 76 ]. Conversely, restoring p16 INK4A suppresses tumour growth [ 77 ]. CDKN2A was the first familial melanoma gene identified [ 78 ]; germline loss-of-function CDKN2A mutations are the most frequent genetic events underlying familial melanoma susceptibility [ 79 ]. Activating CDK4 mutations ( R24C and R24H ) that prevent inhibition by p16 INK4A increase melanoma risk [ 80 ]. Intriguingly, loss of p16 INK4A also contributes to pancreatic neuroendocrine tumours, which include islet tumours such as insulinomas [ 81 – 85 ]. Paradoxically, when not deleted, p16 INK4A may be overexpressed in tumours and transformed cells [ 86 , 87 ]. p14 ARF loss is oncogenic as well, via MDM2/p53 and other mechanisms [ 62 ]. p15 INK4B may be co-deleted with p16 INK4A in haematological malignancies [ 62 ], and plays a role in TGF-β-induced pancreatic cancer [ 88 , 89 ]. ANRIL is also implicated in many cancer pathways, downstream of oncogenic Ras, phospholipase D and specificity protein 1 (SP1), and upstream of Kruppel-like factor 2, p21 and CDKN2A/2B [ 90 – 93 ]. p16 INK4A is an effector of senescence, an irreversible growth arrest that occurs when a cell reaches the end of its replicative lifespan [ 63 , 94 ]. p16 INK4A expression increases with age [ 62 ], triggered by ageing-dependent gene demethylation, telomere shortening and other senescence inducers such as oncogenic activation, oxidative stress, nutrient deprivation and DNA damage [ 95 , 96 ]. Mice and humans with the R24C activating mutation of CDK4 are resistant to senescence [ 97 ]. A recent study has shown that ablating senescent cells, as defined by p16 INK4A expression, extends longevity and forestalls ageing-related tissue functional decline [ 98 ]. p14 ARF is also linked to senescence, independent of p16 INK4A [ 99 ].

Several observations contradict the concept that p16 INK4A impacts diabetes incidence via effects on beta cell mass. The combined loss of p16 INK4A and p14 ARF was not sufficient to restore the glucose induction of mitotic genes lost in aged islets [ 169 ]. Old islets that recovered proliferation when exposed to young mouse circulation did not have reduced p16 INK4A expression [ 111 ]. Perhaps most puzzling from an islet-centric viewpoint are observations from the Super-Ink4/ARF mouse, which contains an extra copy of the entire Cdkn2a/b locus [ 170 ]. Despite modestly increased expression of p15 INK4B , p16 INK4A and p14 ARF in several tissues, these mice have improved glucose tolerance with ageing from enhanced insulin sensitivity in liver and muscle. Beta cell proliferation and islet number are unaffected. Reduced insulin secretion is observed, but is likely to be secondary to the improved insulin sensitivity. Also curious, a survey of islet gene expression at type 2 diabetes-linked loci in diabetes-prone New Zealand Obese (NZO) mice vs diabetes-resistant ob / ob mice revealed increased p15 INK4B and p16 INK4A expression in the diabetes-resistant mice [ 115 ]. Some observations suggest an impact on insulin secretory function independent of the effects on beta cell mass (see the text box `Effects of CDKN2A/B -related proteins on metabolic tissues'). Knockdown of p16 INK4A in the EndoC-bH1 human beta cell line increased insulin secretion [ 168 ]. In mice, haploinsufficiency for telomerase increased p16 INK4A expression and impaired insulin secretion via altered regulation of exocytosis. These mice were glucose intolerant even though beta cell mass was normal, suggesting a primary effect on beta cell function rather than proliferation [ 121 ]. CDK4 also regulates insulin secretion, through Rb-dependent transcriptional regulation of potassium inward rectifying channel 6.2 (Kir6.2) [ 171 ]. In human studies, individuals from familial melanoma kindreds with heterozygous loss of function of CDKN2A had increased insulin secretion, impaired insulin sensitivity and reduced hepatic insulin clearance [ 168 ]. CDKN2A/B locus genes and their CDK targets influence adipose, liver and muscle biology. Deletion of a region of mouse chromosome 4, orthologous to the human 9p21 cardiovascular disease risk interval, reduced local gene expression and increased body weight, linking the region with obesity and metabolic risk [ 172 ]. ANRIL regulates genes involved in glucose and fatty acid metabolism [ 173 ]. p15 INK4B is highly expressed in subcutaneous adipose tissue and may inhibit expandability of the subcutaneous fat depot, a critical protection against overnutrition toxicity [ 174 , 175 ]. Mice lacking CDK4 in all tissues are smaller, with less fat mass, and have insulin resistance in addition to insulin deficiency [ 176 ]. CDK4 regulates adipogenesis, via both Rb-dependent and Rb-independent mechanisms, including direct phosphorylation of insulin signalling intermediates [ 177 – 179 ]. CDKN2A/B locus genes also impact adipose tissue inflammation; p16 INK4A modulates the activation and polarisation of adipose-associated macrophages [ 180 ], although deleting p16 INK4A in bone marrow-derived cells did not impact glucose metabolism, even under obese conditions [ 181 ]. In liver, p16 INK4A regulates hepatic gluconeogenesis independently of the cell cycle [ 182 ]. p16 INK4A deficiency increased liver glucose production via a protein kinase A (PKA)-mediated induction of gluconeogenic gene expression. CDK4 participates in the hepatocyte fasting–fed transition by phosphorylating and activating the general control of amino acid synthesis protein 5-like 2 (GCN5) histone acetyltransferase, which regulates PPARG coactivator 1 α (PGC-1A) and hepatic glucose metabolism [ 183 ]. CDK4 also impacts muscle mitochondrial oxidative metabolism [ 184 ]. These observations, collectively, greatly broaden the potential mechanism(s) by which CDKN2A/B polymorphisms might impact diabetes risk.

CDKN2A/B was not associated with proinsulin conversion to insulin [ 210 ] or the effect of ambient glycaemia on insulin secretion [ 211 ]. Although CDKN2A/B significantly impacted risk of diabetes in a Han Chinese population, this was not related to reduced HOMA-B, although other loci ( CDKAL1 , IGF2BP2 and SLC30A8 ) did impact beta cell function in this population [ 212 ]. In a relatively young cohort of European individuals tested by OGTT, CDKN2A/B was not associated with insulin secretion or glucose sensitivity, although CDKAL1 and HHEX were [ 212 ]; intriguingly, in this cohort two of three CDKN2A/B risk alleles tested (rs10757283 and rs564398 but not rs10811661) showed a trend towards reduced insulin sensitivity. In a hyperglycaemic clamp study, CDKAL1 and IGF2BP2 , but not CDKN2A/B , were associated with reduced first-phase insulin secretion [ 213 ]. Complicating matters, the influence of CDKN2A/B type 2 diabetes polymorphisms on insulin secretion may depend on ambient insulin sensitivity [ 214 ]. In a post-hoc analysis of the Diabetes Prevention Program (DPP), CDKN2A/B identity was not related to baseline insulin secretory capacity; however, unique among the eight loci tested, CDKN2A/B predicted response to therapy. Insulin secretion, but not insulin sensitivity, was improved in protective-allele carriers, but only in the thiazolidinedione treatment arm [ 215 ]. In another pharmacogenetics study, of 27 diabetes loci tested, only CDKN2A/B rs10811661 predicted response to therapy: the protective allele was associated with greater response to sulfonylurea therapy [ 216 ]. The CDKN2A/B locus may [ 217 ] or may not [ 218 ] influence the metabolic response to exercise. The intriguing relationship between CDKN2A/B genotype and response to thiazolidinedione in the DPP cohort suggests activity in adipose tissue; as lipids negatively impact pancreatic beta cell proliferation and function this may represent an adipocyte–beta cell axis [ 123 , 157 , 215 ]. In a Japanese population, CDKN2A/B was not related to visceral fat accumulation [ 219 ], but in an Indian sibling-pair study, CDKN2A/B polymorphisms impacted fasting insulin and HOMA-IR but not HOMA-B, suggesting a primary effect on insulin sensitivity [ 220 ]. The melanoma kindred study also found that individuals haploinsufficient for p16 INK4A had impaired insulin sensitivity [ 168 ]. In the Helsinki Birth Cohort, CDKN2A/B type 2 diabetes-risk polymorphisms were associated with reduced birthweight, suggesting the possibility that CDKN2A/B diabetes risk might be related to developmental impact of this locus, bringing an additional temporal variable to play [ 221 ]. Initial attempts to use type 2 diabetes genome-wide association study (GWAS) information to predict disease risk, optimise therapeutic impact and estimate prognosis have been disappointing. Even combining risk alleles only marginally improves type 2 diabetes prediction over clinical factors [ 222 – 224 ]. As described above, some progress has been made using the CDKN2A/B genotype to predict response to therapy [ 215 , 216 ]. Although CDKN2A/B polymorphisms are not generally associated with longevity [ 225 ], cardiovascular mortality was paradoxically reduced in individuals homozygous for the type 2 diabetes-risk allele at rs10811661 [ 226 ]. In sum, despite progress, genotype information at the CDKN2A/B locus does not yet have meaningful clinical implications for individual patients.