Amelogenin test: From forensics to quality control in clinical and biochemical genomics

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Amelogenin test: From forensics to quality control in clinical and biochemical genomics
  Amelogenin test: From forensics to quality control in clinical and biochemical genomics ☆ F. Francès  a,b, ⁎ , O. Portolés  c , J.I. González  a,b , O. Coltell  c , F. Verdú  a  , A. Castelló  a  , D. Corella  a,b a   Department of Preventive Medicine, Public Health and Legal Medicine, University of València, València, Spain  b CIBER Fisiopatología de la obesidad y nutrición, Instituto de Salud Carlos III, Madrid, Spain c  Departamento de Lenguajes y Sistemas Informáticos, Universitat Jaume I, Castellón, Spain Received 18 April 2007; received in revised form 23 July 2007; accepted 23 July 2007Available online 31 July 2007 Abstract  Background:  The increasing number of samples from the biomedical genetic studies and the number of centers participating in the same involvesincreasing risk of mistakes in the different sample handling stages. We have evaluated the usefulness of the amelogenin test for quality control insample identification.  Methods:  Amelogenin test (frequently used in forensics) was undertaken on 1224 individuals participating in a biomedical study. Concordance between referred sex in the database and amelogenin test was estimated. Additional sex-error genetic detecting systems were developed.  Results:  The overall concordance rate was 99.84% (1222/1224). Two samples showed a female amelogenin test outcome, being codified as malesin the database. The first, after checking sex-specific biochemical and clinical profile data was found to be due to a codification error in thedatabase. In the second, after checking the database, no apparent error was discovered because a correct male profile was found. False negatives inamelogenin male sex determination were discarded by additional tests, and feminine sex was confirmed. A sample labeling error was revealedafter a new DNA extraction. Conclusion:  The amelogenin test is a useful quality control tool for detecting sex-identification errors in large genomic studies, and can contributeto increase its validity.© 2007 Published by Elsevier B.V.  Keywords:  Amelogenin; Sex; Quality control; DNA, Genomics 1. Introduction  Nowadays, thousands of biomedical studies are undertakenin which genetic, environmental, clinical and biochemicalvariables are collected, and analyzed together in researchingseveral pathologies [1 – 3]. This involves obtaining a great number of biomedical samples (blood, buffy-coat, saliva, etc)from which DNA is extracted. In addition a great number of  biological determinations as well as lifestyle variables areincluded in these studies. Although DNA identification is acarefully controlled factor in biomedical laboratories, mislabel-ing still occurs. This problem increases in the case of multi-center studies where the sample-obtaining center is different from that of genetic analysis, and where, therefore, densesample traffic between research centers takes place. Moreover,the development of high throughput technologies will extendDNA analysis from thousands to tens of thousands of samples per laboratory per day. Thus, there are many stages in thesample handling where errors can occur: sample obtaining,DNA extraction, sample labeling, genotyping and databasecodification. It is, therefore, necessary to introduce qualitycontrol tools throughout the process in order to minimize the probability of sample confusion [4]. Clinica Chimica Acta 386 (2007) 53 –  Abbreviations:  Bp, base pairs; PCR, Polymerase Chain Reaction; STR,Short tandem Repeat; TTY2, testis-specific transcript Y-linked 2. ☆ This work was supported by the Generalitat Valenciana (Grupos 2004-43),the Fondo de Investigaciones Sanitarias; Instituto de Salud Carlos III(PI061326), and the CIBER (CB/06/03/0035), Spain. ⁎  Corresponding author. Department of Preventive Medicine, Public Healthand Legal Medicine, València University, Avda, Blasco Ibáñez no 15 46010València, Spain. Tel.: +34 96 3983107; fax: +34 96 3864166.  E-mail address: (F. Francès).0009-8981/$ - see front matter © 2007 Published by Elsevier B.V.doi:10.1016/j.cca.2007.07.020  Given that many studies include random samples withindividuals from both sexes, a sex determination test, like theamelogenin test, could be useful for this quality control.Amelogenin is a protein present in dental enamel, codified bygenes located in chromosomes X (Xp22.1 – Xp22.3) and Y(Yp11.2) [5,6]. Deletion of this gene causes  X-linked amelogen-esisimperfecta, ageneticdisorderaffectingenamelformation[7].These genes, called AMEX and AMEY, although presentingsize and sequence differences, have homologous regions that allow simultaneous amplification of AMEX and AMEYusing asingle pair of primers. These two characteristics justify its use asa sex-typing marker. However its use has been limited toforensics, archeological analysis and prenatal diagnoses [8 – 10].Sex identification by the amelogenin test is based on thedetection of different sizes in the amplified products from thetwo sexual chromosomes. Several variants of the amelogenintest have been published; the most widely used being themethod of Sullivan et al [11]. This technique generatesfragments X and Y of 106 and 112 bp respectively and these products are identified after capillary electrophoresis [11]. The protocol designed by Eng et al [12] generates products of 977 pb in chromosome X, and 790 in chromosome Y, that can be easily separated using electrophoresis in agarose gels. In both protocols, the presence of two different sizes of DNA fragmentswould indicate male sex, while the presence of only one size of fragments would correspond to a female sex.Thus, our aim is to evaluate the usefulness of the amelogenintest (commonly used in forensics) in sample identificationquality control in a gene bank, where samples are obtainedwithin the framework of a biomedical study in which clinical, biochemical,andlifestylevariablesarealsoobtained.Inadditionwe develop complementary methods in order to widen our knowledge of this technique's possible sources of error. 2. Materials and methods 2.1. Individuals Samples from 1224 individuals (50% from each sex) were randomlyselected from the general population of València (Spain). They were participating in a study on genetic and environmental cardiovascular risk factors [13]. The study was approved by the Ethics Committee on Human Research of Valencia University. Peripheral blood samples were obtained fromeach participant in order to extract their DNA. Informed consent was alsoobtained. 2.2. Genotyping  DNA was obtained from leucocytes of peripheral venous blood samples,using standard methods [13]. For the amelogenin test, DNAwas amplified using Polymerase Chain Reaction (PCR) in a 96-plate thermocycler (Eppendorf,Hamburg). The reaction mixture used in the PCR consisted of 15 ng of genomicDNA, 0.2  μ M of each primer  [12]: AME-F: 5 ′ -ctgatggttggcctcaagcctgtg-3 ′  andAME-R: 5 ′ -taaagagattcattaacttgactg-3 ′ , 5 U of Go Taq Polymerase (Promega,Madison USA), 2.5 mM of MgCl 2 , 10 mM of Tris – HCl (10×) and 200 μ M eachdeoxynocleotide triphosphate (dNTPs) (Roche Diagnostics GMBH, Germany),in a total volume of 25  μ l. After an initial denaturation at 94 °C for 6 min, 27PCR cycles were performed with 45 s of denaturation at 94 °C and 45 s of annealing at 64.2 °C, with a 45 s extension at 72 °C, and a final extension of 72 °C/10 min. Amplified products were separated by horizontal electrophoresisin a 2% agarose gel. After a 10 min exposition to ethidium bromide, DNAfragments were visualized using a ultraviolet light. In the case of male sex, two bands (977 and 790 bp) were visible, whereas in female sex, a single 977 bp band is present.In order to explore the possible causes of eventual discrepancy betweenreferred phenotype and amelogenin test, we set up two additional genetic tests.Two pair of primers were designed in order to specifically amplify thehybridization regions of amelogenin forward and reverse primers in the Ychromosome, and by sequencing, to confirm the presence of mutations affectingthis hybridization regions. The reaction mixture used in these two PCRsconsisted of 15 ng of genomic DNA, 0.1  μ M of each pair of primers: AMFOR-F: 5 ′ -tcactgtttgcattagcagtcc-3 ′ , AMFOR-R: 5 ′ -cctaatctttacattttaccggatg-3 ′  andAMREV-F: 5 ′ -ttgcggcataattttatgtttg-3 ′  and AMREV-R: 5 ′ -tcctgtgtgtcaggcactgt-3 ′ , 5 U of Go Taq Polymerase (Promega,Madison USA), 1.5 mM of MgCl 2, 10 mM of Tris – HCl (10×) and 200  μ M each deoxynocleotide triphosphate(dNTPs) (Roche Diagnostics GMBH, Germany), in a total volume of 25  μ l.After an initial denaturation at 94 °C for 6 minutes, 27 PCR cycles were performed with 45 s of denaturation at 94 °C and 45 s of annealing at 61.4 °C,with a 45 s extension at 72 °C, and a final extension of 72 °C for 10 min.Amplified products were: for AMFOR amplicon, 324 bp and for the AMREVfragment, 226 bp. In each PCR mix, an additional pair of primers: 5 ′ -ggaacagctcaggcagaaac-3 ′  and 5 ′ -ttgggacagacagacaggtg-3 ′  were added (0.2 μ M)in order to generate an autosomal 297 bp control amplification that would verifythe correct function of the PCR, in the case of females, when there is an absenceof amplification of the amelogenin primer forward and reverse target sequences.Additionally, two controls (male and female) were included in each test.Lastly, in order to confirm the existence of the Y chromosome in the case of large deletions in the amelogenin gene, an amplification protocol of a holandricgene was set up. We chose testis-specific transcript Y-linked 2 (TTY2) gene, aholandric gene expressed in testis. The TTY2 reaction mixture consisted of 15 ng of genomic DNA, 0.2  μ M of each primer: TTY2-F: 5 ′ -aacccaggatgaat-gagtgc-3 ′ , TTY2-R: 5 ′ -ttcctgaaaggggttttgtg-3 ′ , 5 U of Go Taq Polymerase(Promega,Madison USA), 2.5 mM of MgCl 2,  10 mM of Tris – HCl (10×) and200  μ M each deoxynocleotide triphosphate (dNTPs) (Roche DiagnosticsGMBH, Germany), in a total volume of 25  μ l. After an initial denaturation at 94 °C for 6 min, 27 PCR cycles were performed with 45 s of denaturation at 94 °C and 45 s of annealing at 58.5 °C, with a 45 s extension at 72 °C, and a finalextension of 72 °C for 10 min. The size of the amplified TTY2 gene fragment was 169 bp. As in the case of forward and reverse hybridisation regionamplifications, two additional primers were included: 5 ′ -cttgacccgaattcttggaa-3 ′ and 5 ′ -cctgaacctgagccttttgt-3 ′  that generated an autosomal 273 bp controlfragment (0.2 μ M) in order to verify the success of the PCR. In each test, femaleand male controls were genotyped. 3. Results The amelogenin test were undertaken on the 1224 indivi-duals (622 from each sex) randomly selected from the general population of València. In the case of males, this test generatestwo bands (977 and 790 bp) as opposed to the single 977 bp band in females (Fig. 1).After undertaking the amelogenin test and testing theconcordance between genotype and referred sex in thedatabase, we found four non-concordant results. These sampleswere codified as male in the database, whereas the amelogenintest revealed a female pattern of bands. On repeating theamelogenin test in these samples, two of them showedconcordant (masculine) results. Therefore, we attributed thisdiscrepancy to genotyping errors. After checking the potentialcauses of the two remaining amelogenin discrepant results, wefound that this first non-concordant result was due to adatabase codification error. This was discovered after checkingall the values of the demographic, biochemical and clinicalvariables from that particular individual (including variablessuch as number of pregnancies, age of menarche, menopause 54  F. Francès et al. / Clinica Chimica Acta 386 (2007) 53  –  56   and sex-specific biochemical profile) which could onlycorrespond to a female, but the sex variable had been wronglycodified as a male.In the case of the second discrepant sample, no error could bediscovered after checking the sex-specific variables in thedatabase due to the fact that its masculine biochemical profilewas concordant with the gender variable in the database (male).Then, we designed additional genetic tests in order to explorethe possibility of mutations affecting the hybridation regions inthe AMEY gene of the amelogenin primers, and total or partialdeletions in the AMEY gene in this particular sample.PCR did not produce any DNA fragment when we tried toamplify the hybridization regions of the amelogenin primers.This absence of amplification could be attributable to total or  partial deletions of the AMEY gene. Thus, we tried to amplify aTTY2 gene fragment, with identical negative result, confirmingthe absence of Y chromosome and thereby, the female sex of thesample. Given the consistency of the genetic evidence about thefemalesex ofthissample,a new DNA extractionwas carriedout,using the remaining blood of this subject. The new amelogenintest carried out in this newly isolated DNA resulted in a malediagnostic, indicating the presence of a previous mistake in the post-DNA extraction processing of the first sample.Overall, the concordance between the amelogenin test andthe database codified sex was 1222/1224 (99.84%). 4. Discussion In the present study we have investigated the application of the amelogenin test in sex identification and we have exploredthe usefulness of this test in sample identification quality controlin a gene bank, where samples are obtained within theframework of a biomedical study in which clinical, biochem-ical, and lifestyle variables are also obtained, by detectingerroneous sex adjudications. We have found two non-concordant results when comparing the amelogenin outcomeand the registered sex in the database. One of them was easilyidentified as a codification error in the database. However, noapparent database codification error was discovered for thesecond one.Several failures of this technique have been previouslyreported, resulting in a female amelogenin test outcome in a phenotypically normal male, due to the absence of the Yfragment  [14 – 19]. One of the causes of this phenomenon is the presence of deletions of different sizes involving the AMEYgene that range from between 2.5 Mb and 304 Kb [14,15]. These deletions are especially frequent in the Indian population,having frequencies ranging from 1.8% to 3.6% [16 – 18].Another source of failure in the male sex diagnostic using theamelogenin test is the presence of mutations in sequenceshomologous to those of the primers used in the test, which mayinhibit hybridization and amplification of the AMEY sequence[19]. In our discrepant sample, we did not find any genetic causeof amelogenin failure in the genetic sex determination. However we cannot assess the impact of these potential genetic causes inthe Spanish population because, at the moment, there are nostudies that have evaluated the frequency of these geneticanomalies (both deletions and mutations). However, taking inmind that Steinlechner et al. [20] studied 29,432 phenotypicmale individual from the Austrian National DNA Database, andonly found 6 individuals (0.018%) lacking the AMEY gene, wecan hypothesize that in our Caucasian population the frequencywould also have to be very low.Finally, a third cause of discrepancy between referred sexand amelogenin test is the existence of identification errors inthe samples during the different stages of its handling: DNAextraction, sample labeling, storage, as well as during itsdatabase codification. It was precisely this kind of error that wasfound in our study, suggesting that this source of amelogenin-database discrepancy is, by far, the most frequent in Caucasian populations. It is for this reason that we propose the amelogenintest as a quality control tool to detect misclassification of individuals in the database and subsequent mistakes instatistical analysis. The risk of misclassification is greater when very large sample sizes are used and even more in multi-center studies with a dense sample traffic. To the best of our knowledge, this is the first time that this specific amelogenintest has been proposed as a quality control tool in biomedicalstudies carried out in the field of clinical and biochemicalgenomics. Glock et al. [21], did, however suggest the use of the amelogenin test in the quality control of biological samples in a blood donation center. They applied the DNA profiling,consisting of a panel of nine different short tandem repeats(STRs) plus amelogenin that has been used for routine identitytesting in forensics. However, the complexity and cost of thistest makes its use in quality control studies in geneticepidemiology which handles thousands of samples.Our proposal, in which only amelogenin is included and it isvisualized in agarose gels (its later development being possibleusing fluorescent probes), is much more economic and faster.Therefore, we believe that the use of the amelogenin test as part of a wider system of quality control in clinical and biochemicalgenomic studies will contribute to increase the validity of results. Fig. 1. Typical male and female results of the amelogenin test. Lines 1, 2 and 3correspond to three different DNA samples form males; line 4: DNA marker;lines 5, 6 and 7 correspond to three different DNA samples from females. Malesshow two bands of 977 and 790 bp. Females only present the heavy 977 bp band.55  F. Francès et al. / Clinica Chimica Acta 386 (2007) 53  –  56   Acknowledgements The authors gratefully acknowledge the contributions of F.Gimenez-Fernández for laboratory assistance. References [1] RonningenKS, PaltielL,MeltzerHM,et al.The biobankofthe NorwegianMother and Child Cohort Study: a resource for the next 100 years. Eur JEpidemiol 2006;21:619 – 25.[2] Danesh J, Saracci R, Berglund G, et al. EPIC-Heart: The cardiovascular component of a prospective study of nutritional, lifestyle and biologicalfactors in 520,000 middle-aged participants from 10 European countries.Eur J Epidemiol 2007;22:129 – 41.[3] Splansky GL, Corey D, Yang Q, et al. The Third Generation Cohort of the National Heart, Lung, and Blood Institute's Framingham Heart Study:design, recruitment, and initial examination. Am J Epidemiol 2007;165:1328 – 35.[4] Steinberg K, Beck J, Nickerson D, et al. DNA banking for epidemiologicstudies: a review of current practices. Epidemiology 2002;13:246 – 54.[5] Snead ML, Lau EC, Fincham AG, Zeichner-David M, Davis C, SlavkinHC. Of mice and men: anatomy of the amelogenin gene. Connect TissueRes 1989;22:101 – 9.[6] NakahoriY, TakenakaO, Nakagome Y. A humanX – Y homologous regionencodes amelogenin. Genomics 1991;9:264 – 9.[7] Lagestrom M, Dahl N, Nakahori Y, et al. A deletion in the amelogeningene (AMG) causes X-linked amelogenesis imperfecta (AIH1). Genomics1991;10:971 – 5.[8] Mannucci A, Sullivan KM, Ivanov PL, Gill P. Forensic application of arapid and quantitative DNA sex test by amplification of the X – Yhomologous gene amelogenin. Int Legal Med 1994;106:190 – 3.[9] Faerman M, Filon D, Kahila G, Greenblatt CL, Smith P, Oppenheim A.Sex identification of archeological human remains based on amplificationof the X and Yamelogenin alleles. Gene 1995;167:327 – 32.[10] Caenazzo L, Ponzano E, Greggio NA, Cortivo P. Prenatal sexing and sexdetermination in infants with amboguous genitalia by polymerase chainreaction. Genet test 1997;1:289 – 91.[11] Sullivan KM, Mannucci A, Kimpton CP, Gill P. A rapid and quantitativeDNA sex test: fluorescence-based PCR analysis of X – Y homologousamelogenin. Biotechniques 1993;15:636 – 41.[12] Eng B, Ainsworth P, Waye JS. Anomalous migration of PCR productsusing nondenaturing polyacrylamide gel electrophoresis: the amelogeninsex-typing system. J Forensic Sci 1994;39:1356 – 9.[13] Qi L, Corella D, Sorli JV, et al. Genetic variation at the perilipin (PLIN)locus is associated with obesity-related phenotypes in White women. ClinGenet 2004;66:299 – 310.[14] Lattanzi W, Di Giacomo MC, Lenato GM, et al. A large interstitial deletionencompassing the amelogenin geneon the shortarmof the Y chromosome.Hum Genet 2005;116:395 – 401.[15] Mitchell RJ, Kreskas M, Baxter E, Buffalino L, Van Oorschot RA. Aninvestigation of sequence deletions of amelogenin (AMELY), a Y-chromosome locus commonly used for gender determination. Ann HumBiol 2006;33:227 – 40.[16] Chang YM, Burgoyne LA, Both K. Higher failures of amelogenin sex test in an Indian population group. J Forensic Sci 2003;48:1309 – 13.[17] Thangaraj K, Reddy AG, Singh L. Is the amelogenin gene reliable for gender identification in forensic casework and prenatal diagnosis? Int JLegal Med 2002;116:121 – 3.[18] Santos FR, Pandya A, Tyler-Smith C. Reliability of DNA-based sex tests. Nat Genet 1998;18:103.[19] Shadrach B, Commane M, Hren C, Warshawsky I. A rare mutation in the primer binding region of the amelogenin gene can interfere with gender identification. J Mol Diagn 2004;6:401 – 5.[20] Steinlechner M, Berger B, Niederstatter H, Parson W. Rare failures in theamelogenin sex test. Int J Legal Med 2002;116:117 – 20.[21] Glock B, Reisacher RB, Schock MA, et al. DNA profiling: a valuable toolfor quality control of sample logistics including occurrences of suspectedsample confusion in a blood donation centre. Vox Sang 2002;82:137 – 40.56  F. Francès et al. / Clinica Chimica Acta 386 (2007) 53  –  56 
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