Functional analysis of CDKN2A/p16INK4a 5'-UTR variants predisposing to melanoma

Functional analysis of CDKN2A/p16INK4a 50-UTRvariants predisposing to melanoma

Alessandra Bisio1, Sabina Nasti3, Jennifer J. Jordan4,{, Sara Gargiulo3, Lorenza Pastorino3,

Alessandro Provenzani4,{, Alessandro Quattrone4,{, Paola Queirolo2,

Giovanna Bianchi-Scarra3,{, Paola Ghiorzo3,�,{ and Alberto Inga1,{

1Unit of Molecular Mutagenesis and DNA Repair and 2Medical Oncology Unit, National Institute for Cancer Research

IST, 16132 Genoa, Italy, 3Laboratory of Genetics of Rare Hereditary Cancers, DOBIG, University of Genoa, 16132

Genoa, Italy and 4Centre for Integrative Biology, CIBIO, University of Trento, 38123 Trento, Italy

Received September 22, 2009; Revised November 23, 2009; Accepted January 19, 2010

Germline CDKN2A mutations are observed in 20–50% of melanoma-prone families. We identified melanomapatients that were heterozygous for non-coding germline variants in the 50-UTR of CDKN2A (c.-21C > T; c.-25C > T&c.-180G > A; c.-56G > T; c.-67G > C) and examined their impact on the p16INK4a 50-UTR activityusing two luciferase-based reporter vectors that differ in basal transcription level and that were transfectedinto the melanoma-derived WM266-4 and in the breast cancer-derived MCF7 cells. The wild-type 50-UTRsequence, containing a reported SNP (c.-33G > C) and a known melanoma-predisposing mutation (c.-34G >T), was included as controls. Results revealed that the variants at -21 and -34 severely reduced the reporteractivity. The variants at -56 and at -25&-180 exhibited a milder impact, while results with c.-67G > C weredependent on the plasmid type. Quantification of the luciferase mRNA indicated that the effects of the var-iants were mainly post-transcriptional. Using a bicistronic dual-luciferase reporter plasmid, we confirmedthat c.-21C > T and c.-34G > T had a severe negative impact in both cell lines. We also applied a polysomalprofiling technique to samples heterozygous for the 50-UTR variants, including patient-derived lymphoblasts.Analysis of allelic imbalance indicated that in addition to the c.-21C > T variant, the c.-56T > G and c.-67G > Cvariants also reduced mRNA translation efficiency. Overall, our results suggest that the c.-21C > T sequencevariant is a melanoma-predisposing mutation. The c.-25C > T&c.-180G > A and particularly the c.-56G > T var-iants showed a range of intermediate functional defects in the different assays, and were not observed in thecontrol population. We propose that these variants should be considered as potential mutations.

INTRODUCTION

Melanoma is a malignant cancer originating from the neoplas-tic progression of cutaneous melanocytes whose incidence hasbeen increasing in the last 25 years. Early melanoma remainseminently curable if excised appropriately. However, despitedecades of both simple and complex therapeutic approaches,little progress has been made in the treatment of metastaticmelanoma. Identifying people at risk is crucial for early diag-nosis and potential intervention (1).

The most significant risk factor for melanoma is a familyhistory of the disease; it is estimated that �10% of melanomacases report a first- or second-degree relative with melanoma.

The CDKN2A gene (cyclin-dependent kinase inhibitor 2A;MIM 600160), located on chromosome 9p21, is presently con-sidered the main melanoma-predisposing gene. The CDKN2Alocus is unique in that through overlapping coding regions, itencodes two structurally unrelated proteins involved in growthregulation, p16INK4a and p14ARF (Alternative Reading Frame).The locus comprises four exons: exon1a and exon1b are

†Present address: Center of Environmental Health Sciences, MIT, Cambridge, MA 02139, USA.‡The last three authors equally contributed to the study and are listed in alphabetical order.

�To whom correspondence should be addressed at: Laboratory of Genetics of Rare Hereditary Cancers, DOBIG, University of Genoa, V.le BenedettoXV, 6, 16132 Genoa, Italy. Tel: þ39 0103538982; Fax: þ39 0103538978; Email: [email protected]

# The Author 2010. Published by Oxford University Press. All rights reserved.For Permissions, please email: [email protected]

Human Molecular Genetics, 2010 1–13doi:10.1093/hmg/ddq022

HMG Advance Access published February 3, 2010

specific for p16INK4a and p14ARF, respectively, while exons2 and 3 are shared. Interestingly, loss of p16INK4a leads toinactivation of the Rb pathway while inactivation of ARFaffects the p53 pathway; thus, alteration of this single locusis a highly efficient means of compromising two importanttumor suppressor pathways (1). Overlapping reading framescreate a strong codependency between the proteins: forexample, a synonymous change in one frame often leads toan amino acid replacement in the other. The high intrinsicmutation rate of the locus amplifies this effect. INK4a/ARFis one of the most frequently mutated loci in human cancers.Mutations, deletions and promoter methylation of INK4a/ARF are common in a variety of human tumors (2–4), withthe frequencies ranging from 30% in esophageal tumors to100% in pancreatic carcinomas (5,6).

Germline mutations of CDKN2A are responsible for predis-position to melanoma in 20–50% of melanoma familiesworldwide (7). However, evidence of linkage to 9p21 hasbeen demonstrated in a significant proportion of kindredswho exhibit no detectable mutations in CDKN2A. Therefore,it has been hypothesized that an additional disease gene atthe locus or mutations in non-coding regions may be respon-sible for the predisposition to melanoma in these families (8).

Since p16INK4a was first described as a melanoma suscepti-bility gene, an increasing number of mutations at the locushave been described, including a deep intronic mutation(IVS2-105 A/G) common in England (9). A comprehensivescreen of the intronic regions of CDKN2A identified twoadditional putative intronic mutations. However, these specificmutations did not appear to explain the predisposition tomelanoma in a significant proportion of English pedigreefamilies (10).

Recently, causative mutations have been described in exon1b which only impact p14ARF. Specifically, these include agermline deletion not affecting p16INK4a (11), a 16 base pairinsertion in exon 1b in a Spanish melanoma family (12,13)and a number of pedigrees with exon 1b splice site variants(14) have been described.

Furthermore, recent studies performed in English, Frenchand Italian populations identified a small number of pedigreeswith germline deletions at the 9p21 locus (15–18).

Germline mutations in the CDKN2A 50 untranslated region(50-UTR) and promoter have also been investigated. The first,and thus far only, finding of a causative mutation for mela-noma was the 50-UTR 34G . T mutation, which gives riseto an alternative translation initiating codon able to decreasetranslation from the wild-type AUG, and probably arosefrom a common founder in the UK (19). Two extensivestudies that screened more than 1 kb of the p16INK4a untrans-lated and promoter regions in search of mutations in English,Italian, American (20) and Australian families (8) identifiedpolymorphisms at positions -33, -191, -493 and -735, as wellas three novel variants at positions, -252, -347 and -981.However, these novel mutations showed no segregation withthe disease and thus were classified as rare polymorphisms(numbers are relative to the AUG start codon). These studiesalso confirmed the English origin of the c.-34G . T mutationthat was also found in one Australian family. Allele-specificexpression analysis was performed revealing an absence oftranscriptional silencing in the germline of this cohort of

families and suggesting that overall mutations in theCDKN2A promoter appear to have a limited role in the predis-position to melanoma (8).

While functional tests for determining the pathogenicity ofmissense germline mutations in the coding region have beendeveloped (13,21–23), there are no studies addressing thepossible impact of 50-UTR/promoter variants on the transcrip-tion or translation of the protein. Rather, rare polymorphismsor variants at the CDKN2A 50-UTR encountered duringroutine screening are usually defined as variants withunknown significance after determining their frequency incontrol population and the cosegregation analysis in thefamily, when possible.

In this study, we developed reporter assays to study a panelof p16INK4a 50-UTR variants recently identified in a hospital-based series of melanoma cases selected within an ongoingcase-control study from our Italian population. We also usedpolysomal profiling as a means to determine the relativeimpact of the 50-UTR variants on mRNA translation efficiencystarting from heterozygous patients’ cells. Overall, our resultsprovide tools to assess the functional significance of non-coding 50-UTR mutations and strongly suggest that the ident-ified non-coding p16INK4a variants, particularly the c.-21C .T, can be of clinical significance in melanoma-pronenessdue to their negative impact on the post-transcriptionaldynamics of the CDKN2A/p16INK4a mRNA.

RESULTS

Identification of germline p16INK4a 50-UTR variantsin melanoma patients

We surveyed probands from 250 melanoma families and 782hospital-based sporadic cases for variants in the 50-UTR ofp16INK4a. The frequency of four novel 50-UTR variants thatwe recently identified in the Italian cases (c.-21C . T,c.-56G . T, c.-67G . C) (24–26), one variant previouslyreported and classified as a variant with uncertain functionalsignificance (c.-25C . T) (27,28) which we found in associ-ation with the c.-180G . A and a known rare polymorphism(c.-33G . C) were determined. No additional variants wereobserved in our patient cohort.

Both the c.-21C . T and the c.-56G . T variants wereobserved in familial patients, but cosegregation analysis waspossible only for -21 C . T. As described in Figure 1A, thisvariant segregates in the two affected patients (diagnosed atage 30 and 47, respectively) in a small family. It does not orig-inate from the unaffected branch and is also present in theunaffected sister. The c.-56G . T variant was found in aproband diagnosed at age 53 (Fig. 1B). The mother, diagnosedat age 71, was deceased. Two other cancers (endometrial andlarynx) were diagnosed in first-degree relatives from theaffected branch of this family. Neither the c.-56G . T, northe c.-21C . T variant was found in the controls.

The c.-25C . T variant was found in two non-familialpatients, who were diagnosed at ages 37 and 64. Furthersequencing of the 50-UTR upstream of this variant showedin both patients the presence of an additional variant(c.-180G . A) which had not been previously described.Cloning and sequencing of the 50-UTR amplified from the

2 Human Molecular Genetics, 2010

patients’ DNA showed that these two variants were on thesame allele. Neither the c.-25C . T nor the c.-180G . Avariant was found in healthy controls.

The c.-67G . C variant was found in one non-familial andone familial patient. The latter also carried a CDKN2A codingmutation (V126D) and was diagnosed with multiple mela-noma at a young age. This variant was also found in twohealthy controls. The other patients who carried p16INK4a

50-UTR variants were wild-type at the CDKN2A codingregions (p14ARF and p16INK4a).

The known c.-33G . C and c.-191G . A polymorphismswere found at similar frequencies in cases and controls(Table 1).

The melanoma-associated p16INK4a 50-UTR variantsare functionally defective in gene reporter assays

We developed luciferase-based reporter assays to investigate thepotential clinical significance of the 50-UTR alleles found as het-erozygous variants in our patients. To this aim, we fused the50-UTR upstream of the firefly cDNA both in the pGL3-basicand pGL3-promoter vectors. We chose to compare these twovector types as they differ in the basal transcription level of theluciferase, which is very low in the case of the pGL3-basicbecause it does not contain a constitutive promoter sequence,and higher in the pGL3-promoter due to the presence of anSV40-derived promoter. In the latter plasmid type, the

p16INK4a 50-UTR was cloned just outside and downstream ofthe viral promoter, thus it is expected to be transcribed entirelyand to act as the 50-UTR of the firefly luciferase. In the case ofpGL3-basic, the precise transcription start site (TSS) cannot beaccurately predicted, but one proposed site would be upstreamof the cloned 50-UTR. However, the low transcription from thisvector was evaluated for the potential to tease out small effectsof the cloned 50-UTR sequences on transcription or post-transcription processing of the reporter gene. Luciferase assayswere performed upon transient transfection of the variousplasmid constructs along with the control luciferase pRL-SV40plasmid in MCF7 and WM266-4 cells. These cell lines—bothnull for p16INK4a protein expression—were chosen to compareeffects in two different cell types, where one was directlyrelated to the cancer susceptibility observed in the patients thatinherited these 50-UTR variants. In MCF7 cells, luciferaseassays using the pGL3-basic plasmids revealed that the introduc-tion of the wild-type 50-UTR led to a much higher reporteractivity (Fig. 2A). The 50-UTR variants had a variable negativeimpact on the luciferase activity in comparison with wild-type,ranging from severe (c.-21C . T and c.-34G . T) to modest(c.-56G . T and c.-67G . C). The c.-33G . C SNP variantand the double mutant c.-25C . T & c.-180G . A were equiv-alent to the wild-type 50-UTR. Similar results were obtainedupon transient transfection of WM266-4 cells (Fig. 2B), but theimpact of c.-21C . T was less severe and the c.-33G . C SNPseemed to be slightly more active than the wild-type 50-UTR instimulating reporter activity. Although a decrease in activitywas observed for the c.-56G . T and c.-67G . C variants, thiseffect was not significant.

In MCF7 cells, cloning of the 50-UTR in the pGL3-promoterbackbone resulted in a subtle increase in reporter activity(Fig. 3A), while the c.-34G . T and c.-21C . T variants ledto a severe and moderate reduction in reporter activity,respectively. The c.-33G . C SNP as well as the other var-iants were equivalent to the wild-type while, surprisingly,the c.-67G . C variant appeared to be more active. Theimpact of pGL3-promoter constructs was somewhat differentin the WM266-4 cells (Fig. 3B) where the presence of thewild-type 50-UTR resulted in a considerable increase in trans-activation of the reporter. All the variants except the c.-33G .C SNP showed a significant alteration in luciferase activity. Inparticular, all the variants led to a reduction in reporter activityexcept for the c.-67G . C, which was confirmed to be moreactive than the wild-type in this cell type as well. Theassays with the pGL3-promoter vectors were extended totwo additional melanoma-derived cell lines, G361 andSK-Mel-5. Results confirmed the negative impact of thec.-34G . T and c.-21C . T variants, the intermediate effectof c.-56G . T and the enhanced activity of c.-67G . C (Sup-plementary Material, Fig. S1). Interestingly, in SK-Mel-5 thec.-25C . T & c.-180G . A variant showed a significantlyreduced activity.

Quantitative RT–PCR experiments point to apost-transcriptional impact of the p16INK4a 50-UTRvariants

To investigate whether the impact of the 50-UTR on thereporter activity could be related to transcriptional or

Figure 1. Melanoma pedigrees carrying the CDKN2A (p16INK4a) 50-UTRc.-21C . T (A) and c.-56G . T (B) germline variants. Age at diagnosis indi-cates confirmation of cancer diagnosis and is provided below each symbol.Melanomas are indicated with black symbols. End. ca. ¼ endometrialcancer; Larynx ca. ¼ larynx cancer. The probands are indicated by an arrow.

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post-transcriptional effects, we quantified the Firefly luciferasemRNA using a real-time PCR approach. To take into accountdifferences in transfection efficiencies, we extracted both totalRNA and genomic DNA (gDNA)—containing the transfectedplasmid DNA—from the same culture dish and amplified aspecific fragment of the firefly luciferase sequence oncDNA, as well as on plasmid-DNA-containing gDNA prep-arations. This procedure enabled us to compare the relativeamounts of luciferase mRNA obtained upon transfection ofthe various plasmids containing the 50-UTR variants takinginto account the potential for small variation in transfectionefficiency obtained with the different plasmids. With

pGL3-basic backbone in MCF7 cells, the empty vector andthose with the wild-type, c.-21C . T, c.-34G . T andc.-67G . C 50-UTR were examined. Comparable mRNAexpression levels were observed, with relative differences inthe 50% range that were not consistent with the reporteractivity (Fig. 2C, compare with Fig. 2A). These results indi-cated that there was only a subtle impact, if any, of thep16INK4a 50-UTR wild-type or variant on transcription.Similar results were obtained in WM266-4 transfected cells(Fig. 2D) where the presence of the wild-type or variant50-UTR led to a 40–60% reduction in relative mRNA levelswhich, again, were not consistent with the results of the

Table 1. Frequency of p16INK4a 50-UTR variants in melanoma patients and in the control population

p16INK4a 50-UTRvariants and SNPs

Allele frequency incontrols (n ¼ 150)

Allele frequency in familialmelanoma (n ¼ 250)

P-value; OR (95% CI)relative to controls

Allele frequency innon-familial melanoma(n ¼ 782)

P-value; OR (95% CI)relative to controls

c.-191G . Aa 0.59 0.53 0.13; 0.77 (0.55 -1.07) 0.51 0.06; 0.72 (0.52–1.01)c.-21C . T 0 0.002 – 0 –c.-25C . Tb 0 0 – 0.0013 –c.-33G . C 0.003 0.004 1; 1.2 (0.11–13.6) 0.002 1; 1.1 (0.12–11.42)c.-56G . T 0 0.002 – 0 –c.-67G . C 0.006 0.002 0.56; 0.3 (0.03–3.4) 0.006 0.07; 0.09 (0.09–1.08)

aTested in 150 controls, 150 familial and 150 non-familial melanomas.bfound in association with c.-180G . A.

Figure 2. Impact of the p16INK4a 50-UTR variants in pGL3 basic-derived constructs. Gene reporter assays in MCF7 (A) and WM266-4 (B). Cells were transientlyco-transfected with the reporter constructs containing the wild-type or variant 50-UTR, as indicated, along with the pRL-SV40 control vector. Luciferase activitywas measured 24 h post-transfection and normalized for transfection efficiency. Presented are the average fold-induction relative to the empty vector and thestandard deviation of at least three independent biological replicates. Significant differences in activity relative to the wild-type 50-UTR are shown (�P ,0.01; VP , 0.05, Student’s t-test). Real-time PCR experiments in MCF7 (C) and WM266-4 (D). Cells were transiently transfected with the indicatedvectors and harvested 24 h later. RNA and genomic DNA extraction, cDNA synthesis and PCR were performed as described in Materials and Methods.Bars represent the average luciferase mRNA expression normalized for the average plasmid copy number and for relative cDNA synthesis efficiency as revealedby the amplification of the b2M housekeeping mRNA.


luciferase assays (Fig. 2D, compare with Fig. 2B). The sameanalysis was conducted in both cell lines transfected withpGL3-promoter vectors (Fig. 3C and 3D). With this type ofvector, there was no apparent decrease in relative mRNAlevels due to the presence of the 50-UTR and the small differ-ences (50% or less) observed with the variants did not corre-late with the reporter activity. Taken together, the luciferaseactivity and mRNA quantification results indicated that theimpact of the 50-UTR variants was mainly post-transcriptional.Cell-type-dependent effects were also evident.

A bicistronic dual-luciferase reporter plasmid confirms thepost-transcriptional effect of the p16INK4a 50-UTR variants

The combined results of the luciferase assays and the quantitat-ive PCR encouraged us to study the impact of the 50-UTRmutations in a bicistronic vector named pRuF. We specificallydeveloped pRuF to investigate post-transcriptional/translationalregulation mechanisms. In this vector, the p16INK4a 50-UTR wascloned as an intervening sequence between the Renilla renifor-mis and the Photinus pyralis (Firefly) luciferase cDNAs:transcription would start at a viral promoter site upstream ofRenilla, and stop at a polyA signal downstream of the Firefly.pRuF vectors containing wild-type or variant 50-UTRs weretransfected in MCF7 (Fig. 4A) or WM266-4 (Fig. 4B) cells.In MCF7 cells, we observed a modest negative impact on theratio between Firefly and Renilla luciferase activities with thec.-21C . T, c.-34G . T and, surprisingly, also with c.-67G .C. In WM266-4 cells, c.-21C . T and c.-34G . T exhibiteda similar, severe reduction in the dual-luciferase ratio, whilec.-33G . C was not different from the wild-type UTRand c.-67G . C resulted in a small reduction that was not

statistically significant. Quantitative PCR was also used tomeasure the relative amount of the mRNA with the constructsdiffering for the 50-UTR variants. Two regions of the bicistronicmRNA were evaluated separately, one within the Renilla cDNAand a second in Firefly. Unlike the Firefly/Renilla ratio forenzymatic activity, the quantitative PCR results indicated thatthe 50-UTR variants had no apparent differential impact at thetranscriptional level or on the processing of this bicistronicmRNA (Fig. 4B and 4D).

Allelic imbalance in translated mRNAs from p16INK4a

50-UTR heterozygous cells

We attempted to develop an assay that could distinguish thewild-type and variant 50-UTR allele in the heterozygousstate at the level of the endogenous gene. To this aim, wetook advantage of all available patient-derived lymphoblastcells and attempted to establish whether the 50-UTR variantswould affect translation potential that could be visualized aslower engagement of the mutant allele on ribosomes. Epstein-Barr Virus (EBV)-transformed cells were available only forthe c.-56G . T and c.-67G . C variants and IL2-stimulatedlymphocytes were available for c.-21C . T. We also usedMCF7 cells and mimicked the heterozygous state by transfect-ing equal amounts of pGL3 promoter reporter plasmids con-taining wild-type and variant 50-UTR. Cytoplasmic lysateswere prepared to separate RNA and polysomes that were frac-tionated using sucrose gradients. Subpolysomal and polysomalRNA fractions were then purified separately from each sample(Supplementary Material, Fig. S2A). The p16INK4a 50-UTRwas PCR amplified from these two fractions and sequenced.The relative proportion of the wild-type and mutant alleles

Figure 3. Impact of the p16INK4a 50-UTR variants in pGL3 promoter-derived constructs. Gene reporter assays as well as relative mRNA quantification in MCF7(A, C) and WM266-4 (B, D) cells. Experiments were performed and analyzed as described in Figure 2.



in subpolysomal and polysomal RNA was measured from thesequencing electropherograms (Supplementary Material,Fig. S2B). Results of this analysis are presented in Figure 5as relative ratios of wild-type over variant allele in thecomparison between polysomal and subpolysomal RNA.The impact of the c.-21C . T and c.-56G . T variants wasexamined both at the level of the endogenous gene and intransfected plasmids. Results consistently showed an under-representation of the variant allele in the polysomal RNA,although the magnitude of imbalance was higher when theendogenous gene was analyzed and was not directly pro-portional to the extent of functional defect observed in theluciferase-based assays. The two alleles of the polymorphismat -33 had a similar distribution although the rare C alleleappeared to be slightly more prevalent in polysomal RNA.Interestingly, the -34T mutant allele that introduces anupstream ATG preceded by a Kozak consensus sequencesimilar to the one surrounding the þ1 ATG (19) was over-represented in the polysomal RNA fraction, suggesting thatit does not impact, and may even enhance, ribosome loading.

DISCUSSION

Assessing the pathogenicity of non-synonymous mutations ina high-penetrance melanoma susceptibility gene such asCDKN2A is critical to evaluate disease risk in carriers.

While functional studies for determining the clinical signifi-cance of mutations in the coding regions are well described(13,21–23,29), it is harder to draw solid conclusions and com-municate information during genetic counseling for carriers ofvariants in the non-coding regions, with the exception of spli-cing variants whose relation to the disease may be clearlydemonstrated (9,10). Currently, there are no systematicfunctional studies addressing the role of the 50-UTR variantsidentified during routine CDKN2A mutation screeningin familial melanoma patients. To date, only one provenmutation in the p16INK4a 50-UTR (c.-34G . T) has beendescribed and its mechanism of action was related to the gen-eration of an alternative AUG translation start site (19). Recentstudies highlight the importance of upstream ORFs (uORFs)-mRNA elements defined by a start codon in the 50-UTR,that are out-of-frame with the main coding sequence and canresults in a reduction of protein synthesis from the nativeAUG (30). In addition to common polymorphisms, rare uORF-generating mutations are examples of alterations in untrans-lated regions that can alter the levels of essential proteinsand may be causative of human diseases (30,31).

In this study, we have evaluated in greater detail the fre-quency of occurrence of germline sequence variants in thep16INK4a 50-UTR and near-promoter region among 250 mela-noma families and 782 hospital-based sporadic cases (Table 1)and their co-segregation with the disease (Fig. 1). We thendeveloped plasmid-based assays to test the impact of four

Figure 4. Impact of the p16INK4a 50-UTR variants in the bicistronic pRuF reporter vector. MCF7 (A) and WM266-4 (B) cells were transiently transfected with thenewly developed bicistronic vector pRuF containing the wild-type or the indicated p16INK4a 50-UTR variants cloned in-between the Renilla and Firefly cDNAs.In pRuF, transcription is driven by an SV40-derived promoter located upstream of Renilla, while a polyA is present downstream of the Luciferase sequence. Eachbar represents the average light units and the standard deviation of three independent replicates. Significant differences in activity relative to the wild-type50-UTR are shown (�P , 0.01, Student’s t-test). Real-time PCR experiments on cDNAs prepared from MCF7 (C) and WM266-4 (D) transiently transfectedwith pRuF constructs. Bars represent the average and standard deviation of firefly/renilla luciferase mRNA ratios.




identified sequence variants in the 50-UTR that were clonedupstream of the luciferase reporter gene (at c.-21C . T;c.-25C . T & c.-180G . A; c.-56G . T; c.-67G . C). Aknown, albeit rare c.-33G . C SNP and the c.-34 G . Tmutation were included as controls. Two plasmid types(pGL3-basic and promoter) that differ in basal transcriptionlevels were used and expression was compared after transienttransfection in two different p16-null cancer-derived cell lines:MCF7 (breast adenocarcinoma) and WM266-4 (metastaticmelanoma). The p16INK4a 50-UTR variants cloned into thepGL3-promoter plasmid type were also tested in twoadditional p16-null, melanoma-derived cell lines G361 andSK-Mel-5. All the variants led to a significant reduction inluciferase activity with respect to the wild-type sequence,even if the magnitude of the impact differed, rangingfrom severe (those at -21 and -34) to moderate/weak (at -25,-56, -67).

To gain more insights into the molecular impact of thosep16INK4a 50-UTR sequence variants, we compared results ofgene reporter assays with the quantification of luciferasemRNA levels. We observed only small variations in the rela-tive mRNA levels with the pGL3-basic and promoter vectorsthat did not correlate with the relative reporter activities.

Because the luciferase activity was strongly stimulatedby the cloning of the wild-type p16INK4a 50-UTR, with noapparent steady-state changes in the low-level transcriptionprovided by the pGL3-basic vector (Fig. 2), we attempted toexplore more directly the impact of the 50-UTR on mRNAtranslation potential. We thus developed a third plasmid type(pRuF), which can transcribe a bicistronic dual-luciferase

reporter mRNA from a viral promoter where the p16INK4a

50-UTR was cloned as intervening sequence between theRenilla and Firefly luciferases. Similar bicistronic vectorshave been used to investigate mechanisms, regulatorysequences and proteins involved in CAP-independent trans-lation (32–35). In pRuF, cloning of the wild-type p16INK4a

50-UTR strongly stimulated the activity of the Firefly relativeto the Renilla luciferase, suggesting that the 270 nt sequencecould stimulate ribosome binding and translation. Understand-ing the stimulatory effect lies beyond the scope of this study;however, we have used the pRuF backbone to investigate theimpact of the sequence variants and observed that thec.-21C . T and c.-34G . T had a negative impact whichwas more evident in WM266-4 cells. There was also a trendfor a negative impact of the c.-67G . C variant, particularlyin MCF7 cells. The introduction of the sequence variants didnot appear to alter the relative steady-state level of the bicis-tonic mRNA. Taken together, our results suggest that themelanoma-associated p16INK4a 50-UTR variants can be func-tionally distinguished from the wild-type in different genereporter assays and appear to have an effect mainly at the post-transcriptional/translational level.

It is important to note that our functional assays, even thosewith the pGL3 vector backbones, were developed to examinethe 50-UTR activity of the wild-type and variant sequencescloned upstream of the luciferase cDNA and thus could havelimited sensitivity in revealing a potential impact on transacti-vation. Although multiple TSSs in the CDKN2A internal(p16INK4a) promoter have been reported (36), even the tran-script from the nearest TSS to the AUG site will comprisethe region where the variants are located. Nonetheless, wecannot rule out a contribution to gene transcription, giventhat functional enhancer elements can also be located down-stream of the TSS, as revealed, for example, by unbiased invivo transcription factors binding studies (37,38).

p16INK4a transcription has been shown to be affected by Rbstatus (39) and the INK4a promoter resides within a CpGisland, whose abnormal hypermethylation has been observedin many tumor types and related to the silencing of the gene(3). CpG islands are regions rich in the CpG dinucleotidewhich are often associated with genes and are normally keptunmethylated in cells through unknown mechanisms butwhich may include binding of the Sp1 transcription factor(40). The INK4a promoter/50-UTR sequence contains fourpotential Sp1 sites upstream of the AUG site, one of whichoverlaps and is predicted to be affected by the c.-67G . Cvariant (http://www.genomatix.de). Indeed, we were able toshow that the c.-67G . C change can inhibit Sp1 binding inan in vitro gel shift assay, confirming the potential for Sp1interaction at this site and the possibility of a transcriptionaleffect of the c.-67G . C variant (Supplementary Material,Fig. S3). Furthermore, binding of RNA helicase A (RHA)has been mapped using gel shift assays to a 14 nt sequencecomprising the c.-56G . T variant (41). An engineeredmutation at -57 was shown to affect RHA binding and resultedin a �50% reduction in activity in a luciferase-based reporterassay where a 0.9 Kb fragment upstream the AUG sequencewas cloned as a promoter element (41). Furthermore, RHAbinding appears to be inhibited by CpG methylation.Overall, these data suggest that the identified 50-UTR sequence

Figure 5. Evaluation of allelic balance in heterozygous cells among polysomaland sub-polysomal RNA fractions. MCF7 cells were co-transfected with pairof reporter plasmids containing the wild-type and one of the indicatedp16INK4a 50-UTR variants to mimic the heterozygous allele situation foundin melanoma patients (grey bars). Twenty-four hours after transfection, poly-somal and subpolysomal RNA fractions were collected using sucrose gradientfractionation, as described in Materials and Methods. Where available,patients’-derived lymphoblast cells were used as source of polysomal and sub-polysomal RNAs (darker bars). From each sample, cDNA was synthesizedfrom the two separated RNA fractions and the p16INK4a 50-UTR sequencewas amplified and sequenced on both DNA strands. Presented are theaverage relative ratios of wild-type over variant allele in the comparisonbetween polysomal-associated and subpolysomal RNAs obtained from thequantification of sequencing electropherograms. Two to three independentsubpolysomal/polysomal RNA preparations were obtained and analyzed foreach p16INK4a variant. Error bars report the standard deviation amongrepeats. Numbers above zero indicate an over-representation of the wild-typeallele among mRNAs bound to the polysomes. Conversely, numbers belowzero indicate the over-representation observed with the -33 C and -34 Tvariant alleles in polysomal RNAs.




variants may also have transcriptional effects, which howeverneed to be confirmed in vivo, at the level of the native chromo-somal context, in order to take into proper account theadditional complexity deriving from the chromatin landscapeof the CDKN2A gene.

To examine the impact of the p16INK4a 50-UTR variants atthe level of the endogenous gene, we used polysomal profil-ing. Previous studies have shown that western blot analysison proteins extracted from patient-derived lymphoblast celllines and normal controls was sensitive enough to conclus-ively establish a quantitative reduction in p16INK4a proteinlevels caused by a heterozygous truncating mutation (29).However, we doubt it would be appropriate to discriminatequantitative differences in post-transcriptional regulation atthe heterozygous state, especially for those variants thatwere also detected in controls, considering the low expressionlevels of p16INK4a as well. Therefore, based on the evidencederived from our gene reporter assays showing that theimpact of the 50-UTR variants would be mainly post-transcriptional, we chose polysomal profiling as a surrogatefor protein analysis. Polysomal profiling, also called transla-tional profiling, takes advantage of the isolation from cyto-plasmic lysates of mRNAs engaged in translation by meansof polysome isolation, usually achieved by density fraction-ation on sucrose gradients. This technique has been success-fully used to study the translation potential of specificmRNAs and regulatory mechanisms in the context of differentstress responses. It has also been employed to distinguish cellsubpopulations through their specific sets of translatedmRNAs (42–46), as also reported in a recent study inwhich some of us used it to profile the translatome of color-ectal cancer cells progressing from invasive to metastatic car-cinoma (47). Unlike the case of protein analysis, the twop16INK4a mRNA alleles that differ at the 50-UTR can be dis-tinguished into heterozygous cells and quantified by molecu-lar analysis, an added advantage of this method which wesought to explore in this study. Polysomal profiling enabledus to address the hypothesis that a 50-UTR variant negativelyaffecting translation potential would be revealed by examin-ing relative allelic imbalance in polysomal RNA fractionscompared with sub-polysomal RNAs. Given that patient-derived cells were not available for all the p16INK4a variants,

we also used MCF7 cells transfected with equal amounts oftwo pGL3-promoter vectors containing the wild-type andvariant 50-UTR to mimic the heterozygous state, followedby polysome isolation. Indeed, the CDKN2A wild-typeallele was more represented in the polysomal fraction com-pared with the c.-21C . T, c.-56G . T and c.-67G . T50-UTR variants. In the case of the c.-33G . C SNP, the wild-type and the mutated allele were similarly distributed on thepolysomes with a slight preference for the rare allele, whilethe c.-34G . T mutant alleles appeared to be over-represented in polysomes. This opposite pattern might berelated to the alternative AUG site (uORF) introduced bythis mutation as well as the close match to the Kozak consen-sus of the surrounding sequence (19), and is consistent withthe distinct mechanism of action of this mutation. Althoughtrends were similar, the degree of allelic imbalance washigher when evaluated at the level of the endogenous tran-scripts from patient-derived lymphoblast cells (Fig. 4).

In conclusion, we have developed functional assays to studythe clinical significance of heterozygous germline variants atthe p16INK4a 50-UTR which can be applied in general to thestudy of 50-UTR sequence changes of uncertain significanceidentified during mutation screening of disease genes. Theassays rely both on plasmid reporter vectors and on analysisof allelic imbalance conducted directly from patient-derivedcells heterozygous for the variants and were tailored to inves-tigate post-transcriptional and translational impact. A knownSNP (c.-33G . C) and a previously reported mutation(c.-34G . T) were included as controls in the analysis of thefour variants identified in our patient cohort (c.-21C . T;c.-25C . T & c.-180G . A; c.-56G . T; c.-67G . C) (24)(Table 2). All variants consisted in single point mutations inthe proximal region of the p16INK4a 50-UTR, with the excep-tion of the c.-25C . T variant that was found associatedin-cis with the c.-180G . A, change in two unrelated patientsbut not found in the controls. While the c.-21C . T and thec.-56G . T variants have only been observed in our seriesof patients, the c.-25C . T variant was also identified inother populations (27,28), although no data are currently avail-able on its possible association with c.-180G . A. Thec.-25C . T & c.-180G . A variant only showed a weak nega-tive impact in the reporter assays in MCF7, WM266-4 and

Table 2. Summary of the functional analyses of the p16INK4a 50-UTR variants

p16INK4a 50-UTRvariant

Familyco-segregation

Presence incontrolpopulation

Relative (%) luciferase reporter activity compared with the wild-type 50-UTRsequence

PolysomalRNA allelicimbalancepGL3-b pGL3-p pRuF

MCF7 WMa MCF7 Melb MCF7 WMa

c.-21C . T Yes No 18 51 39 46 61 46 62 33 Yesc.-25C . Tc No No 82 NA 111 70 96 55 82 NA NAc.-33G . C No Yes 112 155 109 90 139 122 NA 114 Noc.-34G . Td Yes No 15 21 6 11 20 10 47 27 Yese

c.-56G . T No No 63 72 114 45 61 64 79 NA Yesc.-67G . C No Yes 69 67 277 144 206 158 44 65 Yes

aWM266-4.bSeparate results for WM266-4, G361 and SK-Mel-5, respectively.cfound in association with c.-180G . A.dOriginally reported in Ref. (19).eMutant allele over-represented in polysome-associated RNA compared with sub-polysomal RNA.


G361 cells, while in SK-Mel-5 the impact was more severeand similar to that of the c.-21C . T variant. The effect ofthe individual sequence changes and their possible functionalinteractions deserve further study.

Overall, the impact of the variants was similar in the differ-ent cell lines, although quantitative differences were apparentand suggestive of tissue-specific modifiers (Table 2). Thec.-34G . T mutation had a severe impact in the reporterassays in every cell lines tested. Instead, the rare SNP at -33was not functionally significant in all assays. Surprisingly,the c.-67G . C variant was somewhat defective in thepGL3-basic and pRuF reporter plasmids, but led instead tohigher luciferase activity when cloned in pGL3-promoter(Table 2). Unlike the other melanoma-associated variants,the c.-67G . C was also found in two controls and can thusbe considered a rare polymorphism, although a transcriptionaleffect cannot be excluded as described above (SupplementaryMaterial, Fig. S2). Further studies are necessary to investigateand draw solid conclusions on this variant. The c.-56G . Tvariant exhibited a modest negative impact in the reporterassays. While a general correlation between reduction in luci-ferase activity and lower abundance in polysomal RNA wasapparent, with the noted exception of the c.-34G . Tmutation, the allelic imbalance of c.-56G . T heterozygoustranscripts exceeded that of c.-21C . T and c.-67G . C.However, at the present stage of our study it is unclearwhether quantitation of allelic imbalance can be directlyrelated to degree of severity of the different p16INK4a variants.

The c.-21C . T variant exhibited the strongest negativeimpact overall (Table 2). This variant co-segregated with mel-anoma in the studied family, was not found in the controlpopulation, and could thus be proposed to be a melanoma-predisposing mutation. The precise mechanisms of action ofthis and the other p16INK4a variants examined in this studyremain to be elucidated but appear to be at least in partrelated to translation regulation and to differ from that of thec.-34G . T mutation, as they do not generate an upstreamalternative reading frame.

We can conclude from these data that the c.-21C . Tvariant appears to be a causal mutation, and that the other var-iants we tested, aside from c.-67G . C for which, as pre-viously mentioned, further studies are necessary to drawsolid conclusions, appear as potential mutations based on therange of intermediate effects they exhibited in the functionalassays. While there were quantitative differences in theresults obtained depending on the cell line used and plasmidtype (Table 2), there was a consistent trend for the c.-25C .T & c.-180G . A and for the c.-56G . T variants to exhibitan intermediate effect. It is worth mentioning that differencesamong assays could be in part related to the fact that eachspecific variant may have a stronger impact oncap-independent translation (analyzed with the pRuF con-structs) than on other post-transcriptional regulations (asassayed with quantitative mRNA and polysomal profiling).

Overall, the functional assays are in agreement with the dataobtained by screening normal controls for the presence ofthese variants (Table 2) in that the two rare polymorphismsc.-33G . C and c.-67G . C had no or a small impact, respect-ively. Also, the functional evaluation adds information if weconsider that it is difficult to discriminate between mutation

and rare polymorphism based on the absence in the controlpopulation, and that even established polymorphisms couldhave a functional consequence and act as modifiers ofcancer risk. With regard to patients’ counseling for these var-iants it has to be noted that our counseling protocol for mela-noma recommends that mutation-positive individuals as wellthose who belong to mutation-negative families be offeredthe same kind of follow-up, as susceptibility to melanoma inthe family may derive from mutations in genes that are stillunknown (26). We suggest this recommendation, that includesoffering additional encouragement to perform self skin exam-ination, follow sun protection measures and undergo more fre-quent (i.e. six monthly rather than yearly) clinical skinexamination, should be extended to patients presenting var-iants classified as potential mutations. We propose that thecombined approach of reporter assays and polysomal profilingcan be applied to studying the impact of other 50-UTR variantsdescribed in the literature for p16INK4a or other SNPs/variantsin the 50-UTR of novel candidate genes/loci for susceptibilityto melanoma and other diseases.

MATERIALS AND METHODS

Patients and design

Two sets of patients were considered: one cohort of 250 mel-anoma families who underwent genetic testing from 2000 to2009 and 782 melanoma patients selected within an ongoinghospital-based case-control study. A subset of these patientswas previously described (24–26). The study was approvedby the local Ethics Committee. All melanoma patients(whose diagnoses were confirmed either by histology ormedical records) and controls signed an informed consentform, provided a blood sample and answered a questionnairewith the aid of a trained interviewer.

Mutation analysis

The CDKN2A coding region, including splice junctions, the 50

and 30-UTRs, the intronic sequence described to contain theIVS2-105 A/G mutation and exon 1b were entirely sequenced,as well as CDK4 exon 2. The primers and procedures forsequencing have been previously described (29). Briefly, stan-dard PCR conditions were applied, with an annealing tempera-ture of 608C for all primer sets. Sequencing reactions wereperformed using an ABI PRISM Big Dye Terminator CycleSequencing Kit (Applied Biosystems, Milan, Italy) and theobtained products obtained were analyzed on an ABI 3100DNA sequencer (Applied Biosystems). DNA sequencing wascarried out in both directions on independent DNA extractionsusing the same primers that were used in the initial PCRamplification.

The 50-UTR/promoter region was screened up to the 420thbase upstream of the ATG in 150 familial melanoma patients,150 sporadic cases and 150 controls. For an additional groupof 100 families and 782 controls, DNA sequencing waslimited to the 150 base pairs upstream of the ATG.

Differences in CDKN2A variant frequencies between casesand controls were evaluated by Fisher’s exact or WilcoxonMann–Whitney (WMW) test.




Point estimates and 95% confidence intervals (CI) of unad-justed odds ratio (OR) were calculated and used as themeasure of association between melanoma risk and specificindividual CDKN2A variants.

Cell lines and culture conditions

The human breast adenocarcinoma-derived MCF-7, the humanneuroblastoma-derived SH-SY5Y cells and the humanmelanoma-derived G361 and SK-Mel-5 cells were obtainedfrom the InterLab Cell Line Collection bank (ICLC, Genoa,Italy). The WM266-4 cell line derived from a human meta-static melanoma was kindly provided by Dr L. Lanfrancone(European Institute of Oncology, Milan, Italy). Cells weremaintained in DMEM or MEM medium supplemented with10% FCS and antibiotics (100 units/ml penicillin plus100 mg/ml streptomycin). MEM medium was also sup-plemented with þ1% non-essential amino acids and 1%Sodium Pyruvate (Invitrogen, Milan, Italy).

Lymphoblastoid cells derived from melanoma patients het-erozygous for the CDKN2A 50-UTR mutations/variants weretransformed by EBV and established cell lines are stored atthe Galliera Genetic Bank Repository (Genoa, Italy). EBV-transformed lymphoblastoid cells were cultured using RPMImedium supplemented with 10% FBS and antibiotics. WhereEBV-transformed lymphoblasts from patients were not avail-able, frozen lymphoblasts were obtained from the same Repo-sitory and cultured for 48–72 hrs in RPMI medium in thepresence of the stimulatory cytokine IL2.

Plasmids

Several p16INK4a TSSs, spanning from -247 to -306 have beendetermined by RNase protection assays (36). In this study, weconsidered the wild-type p16INK4a 50-UTR, a 271bp fragmentas annotated in the Ensembl Genome Browser (http://www.ensembl.org/index.html) at the time of cloning. The wild-typep16INK4a 50-UTR was PCR amplified from genomic DNAobtained from SH-SY5Y cells using primers that contained endo-nuclease restriction sites at their 50end (HindIII-XbaI-EcoRI andNcoI-NdeI-ClaI; for Fw-C and Rev-C primer, respectively; seeSupplementary Material, Table S1A). The amplicon was thencloned into the commercial pGL3-basic and pGL3-promotervectors (Promega, Milan, Italy) using the HindIII-NcoIrestriction enzymes. In the case of the pGL3-promoter vector,the p16INK4a 50-UTR was cloned immediately upstream ofthe Firefly cDNA start codon and just downstream of theSV40-derived promoter to enable the assessment of its impactas 50-UTR sequence for the reporter gene. With the pGL3-basicvector, there is low-level transcription of the reporter in theabsence of cloned promoter fragments, although the preciseTSS has not been mapped.

A site-directed mutagenesis approach based on a two-roundPCR protocol was developed to obtain the c.-21 C . T and thec.-34 G . T p16INK4a 50-UTR mutations (coordinates arerelative to the ATG codon, where the A nucleotide is þ1).Briefly, a pair of complementary 30 nt primers containing thedesired mutations was used each in combination with the appro-priate external Fw-C or Rev-C primer to obtain two PCR pro-ducts comprising the 271bp p16INK4a 50-UTR overlapping by

30bp, corresponding to the sequence of the internal primerscontaining the desired mutation. The two PCR products werethen used as template for a second PCR that utilized the externalpair of primers. Mutated CDKN2A 50-UTR were then clonedinto the reporter vectors using the EcoRI-NdeI restrictionsites. Four additional p16INK4a 50-UTR variants identifiedin our population (c.-25 C . T&c.-180G . A, c.-33 G . C,c.-56 G . T and c.-67 G . C) were cloned in the pGL3-plasmid backbones using the cloning primers Fw-C andRev-C starting with genomic DNA obtained from patient-derived lymphocytic DNA, heterozygous for the sequence var-iants. pGL3-promoter plasmids containing the different 50-UTRsequences were also used to construct a new reporter vector,herein referred to as pRuF. Briefly, the wild-type p16INK4a

50-UTR-Firefly cDNA fragment was extracted from the pre-viously developed pGL3-promoter-based vector exploiting thenew Xba I site introduced by primer Fw-C and a second XbaI site present immediately downstream of the Firefly cDNAstop codon. This fragment was cloned into the pRL-SV40vector (Promega) at the unique Xba I site located downstreamof the Renilla reniformis cDNA stop codon and upstream ofthe SV40-derived poly-A sequence. Thus, in the pRuF vectorthe Renilla and Firefly cDNAs would be transcribed as partof a single transcript separated by the p16INK4a 50-UTRsequence. p16INK4a 50-UTR mutations/sequence variants wereintroduced in the pRuF vector using the EcoRI-NdeI restrictionsites flanking the 50-UTR from the previously developedpGL3-based vectors. All plasmid clones were checked byrestriction mapping followed by direct DNA sequencing.

Luciferase assays in cell lines

5 � 104 cells were seeded in 24-well plates 24 h before trans-fection. Cells were transfected at �80% confluence usingFuGENE 6 (Roche, Milan, Italy) or Myrus LT-1 (TemaRicerca, Milan, Italy) transfection reagents, according to themanufacturer’s instructions. Specifically, 350 ng of thep16INK4a 50-UTR reporter vectors were used along with 50 ngof the control pRLSV40 plasmid introduced to normalize thetransfection efficiency, except for the cases when the pRuFplasmid was tested, as this vector contains both Renilla andFirefly luciferases. Cells were harvested 24 h after transfectionand luciferase assays were carried out using the dual-luciferasereagent (Promega) as previously described (48). Presented arethe average relative light units and the standard deviations ofat least three independent biological repeats.

Quantification of luciferase mRNA expression byreal-time PCR

MCF7 and WM266-4 cells were seeded into 6-well plates andtransfected at 70–80% confluence with 1.2 mg of thepGL3-based or pRuF luciferase vectors containing the wild-type or the mutated p16INK4a 50-UTR. Twenty-four hoursafter transfection, cells were harvested and washed oncewith phosphate buffer saline (PBS). Total RNA and genomicDNA were extracted using the AllPrep DNA/RNA Mini Kit(Qiagen, Milan, Italy) according to the manufacturer’s instruc-tions. In-column DNAse treatment (Qiagen) was performed toremove DNA contamination during total RNA extraction.



Purity and concentration of RNA and genomic DNA wereevaluated using a NanoDdrop ND1000 spectrophotometer.cDNA was generated starting from 1 mg of RNA by usingthe AffinityScript cDNA Synthesis Kit (Stratagene, Milan,Italy). Real-time PCR was performed using a cDNA aliquotequivalent to 50 ng of converted RNA or with 10 ng ofgenomic DNA using a RotorGene 6000 thermal cycler(Corbett Life Science, Ancona, Italy) and the 5PRIME Sybr-Green MasterMix (Eppendorf, Milan, Italy). Relative mRNAquantification was obtained using the DCt method, takinginto account the efficiency of cDNA synthesis by the quantifi-cation of the Beta2Microglobulin (b2M) housekeeping genefrom the same cDNA. The average plasmid copy number inthe transfected cell population, quantified using the samePCR primers (listed in Supplementary Material, Table S1B)on genomic DNA extracted from the same transfection plate,was also used to normalize mRNA quantities for the pRuF-based transfections, total RNA was extracted using theRNAeasy mini Kit (Qiagen) and in-column DNAse treatment.cDNA was synthesized and quantified as described above.Real-time PCR was performed using a cDNA aliquot equival-ent to 50 ng of converted RNA and two pairs of primers ampli-fying a portion of the Renilla and of the Firefly luciferases,respectively (Supplementary Material, Table S1B).

Polysomal RNA extraction

1–1.2 � 106 MCF7 cells were seeded into 10 cm Petri dishesand cotransfected the following day with 2.5 mg of a reportervector containing the wild-type p16INK4a 50-UTR along with2.5 mg of the equivalent vector containing a mutant/variant50-UTR, thus mimicking the heterozygous condition seen inthe melanoma patients. These experiments were performedusing the pGL3-promoter backbone. Polysome preparationswere also obtained from patient-derived lymphoblast cells,EBV-immortalized or IL2-stimulated, as indicated. Forty-eight hours after seeding patient-derived cells or 24 h aftertransfection, cells were treated with cycloheximide (final con-centration of 10 mg/ml) for 5 min at 378C in order to stabilizethe polysomes. Cells were then washed twice with PBScontaining the same concentration of cycloheximide andlysed by adding directly on the plate with 300 ml of lysisbuffer [10 mM NaCl, 10 mM MgCl2, 10 mMTris–HCl, pH7.5, 1% Triton X-100, 1% sodium deoxycholate, 0.2 U/mlRNase inhibitors (Fermentas, Milan, Italy), 1 mM dithiothreitoland 10 mg/ml cycloheximide]. Lysates were transferred toEppendorf tubes using cell scrapers, incubated on ice for5 min with occasional vortexing, and centrifuged for 10 minat 12 000 g at 48C. The supernatant was frozen in liquid nitro-gen and stored at 2808C or loaded directly onto a 15–50%linear sucrose gradient (15 ml total volume) containing30 mM Tris–HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2, andcentrifuged in a SW41 rotor for 110 min at 180 000 g. Onemilliliter fractions were collected using a Teledine ISCOgradient maker with automated fraction collector. Thedevice is coupled to a computer-controlled peristaltic pumpthat uses a 50% sucrose solution injected by a needle at thebottom of the gradient tube, recovered after the ultracentrifu-gation, to achieve the transfer of the lighter fractions of thegradient to collection tubes passing through an optical unit

equipped with UV (260 nm) detector. Fractions containingsubpolysomal and polysomal RNA were identified based onthe quantification of UV fluorescence. Fractions were treatedwith proteinase K (100 mg/ml) in 1% SDS for 3 h at 378C fol-lowed by extraction with 0.25 volumes of phenol–chloroformand 10 mM NaCl and by precipitation in one volume of isopro-panol for 30 min at 14 000 g and 48C. The recovered RNApellet was resuspended in 30 ml of RNase-free water andfurther purified using the RNA clean-up protocol and theRNeasy mini spin column (Qiagen). For each transfected orlymphoblast cell sample, cDNAs were prepared startingfrom RNA preparations corresponding to subpolysomal andpolysomal fractions using the AffinityScript cDNA SynthesisKit (Stratagene). The p16INK4a 50-UTR was then amplifiedby PCR using specific primers (Supplementary Material,Table S1C). The relative contribution of the two p16INK4a

50-UTR alleles in the transfected construct or at the level ofthe endogenous gene was then assessed by double strandDNA sequencing on two to three independent subpolysomal/polysomal RNA preparations (BMR Genomics, Padova,Italy) and quantification of the electropherograms was con-ducted using the image J software (http://rsbweb.nih.gov/ij/).Average signal intensities for each dideoxynucleotide in theelectropherograms were taken into account in the comparisonof individual sequencing runs. To further control for technicalsources of allele imbalance in the sequencing runs, we alsocompared the relative signal intensity of the same nucleotidetype found at the heterozygous mutation site at specific pos-itions surrounding the position of the mutation.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.

ACKNOWLEDGEMENTS

We are grateful to Chiara Baldo at the Galliera GeneticBank—Network of Telethon Genetic Biobanks (projectGTB07001) for providing lymphoblastoid cell-lines and toYari Ciribilli, Veronica de Sanctis and Linda Battistuzzi forcritical reading and suggestions.

A. Bisio is a fellow of the Doctorate School in Biology andGenetics, University of Genoa.

Conflict of Interest statement. None declared.

FUNDING

The authors acknowledge the support of the EU FP6 GenoMELNetwork of Excellence (2005–2010) to G.B.S. and the Auton-omous Province of Trento, Italy (PAT) for the PROTRA grantto A.P. This work was partially supported by the ACC funding2007 to P.Q, by the 2007 Italian Ministry of Health DGRST.4/4235-P1.9.A.B. to G.B.S. and by PRIN 2009.

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Functional analysis of CDKN2A/p16INK4a 5'-UTR variants predisposing to melanoma

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