Legal Medicine 14 (2012) 320–323

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Legal Medicine

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Case Report

A Case of Amelogenin Y-null: A simple primer binding site mutationor unusual genetic anomaly?

Carey Davis a,⇑, María Illescas b,1, Carmen Tirado c, Roberto Lopez c, Bruce Budowle a, Tracey Dawson Cruz d

a Institute of Applied Genetics, Department of Forensic and Investigative Genetics, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth,TX 76107, United Statesb BIOMICs Research Group, Department of Animal Zoology and Cellular Biology, University of the Basque Country, Vitoria-Gasteiz, Spainc Instituto de Ciencias Forenses de Puerto Rico, P.O. Box 11878, Caparra Heights Station, San Juan 00922, Puerto Ricod Virginia Commonwealth University, Department of Forensic Science and Biology, 1000 W. Cary St., P.O. Box 842012, Richmond, VA 23284, United States

a r t i c l e i n f o

Article history:Received 1 March 2012Received in revised form 7 April 2012Accepted 9 May 2012Available online 20 June 2012

Keywords:Forensic DNA analysisY chromosomeSTRNull allele

1344-6223/$ - see front matter � 2012 Elsevier Irelanhttp://dx.doi.org/10.1016/j.legalmed.2012.05.002

⇑ Corresponding author. Tel.: +1 8177352940; fax:E-mail address: Carey.davis@unthsc.edu (C. Davis)

1 Present address.

a b s t r a c t

A thirteen year old boy was murdered by a gunshot wound to the head. In order to confirm identity of theboy, samples were sent to the Instituto de Ciencias Forenses de Puerto Rico (PR-ICF) DNA laboratory.Autosomal DNA results exhibited only an X at the Amelogenin locus, whereas the autopsy resultsreported the child to be anatomically male. The sample was amplified with four separate YSTR markersystems. While a full Y-STR profile for the father of the boy was obtained, the boy only amplified atSTR markers on the p arm of the Y chromosome. Theories that could account for this large absence ofY-STR results include an X–Y translocation or Yp isochromosome.

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1. Case

A thirteen year old boy was murdered by a gunshot wound tothe head. Due to facial disfiguration, blood from the victim wasplaced on an Flinders Technology Associates (FTA™) card and sentto the DNAs laboratory at the Instituto de Ciencias Forenses dePuerto Rico (PR-ICF) to confirm the identity of the boy. Positiveidentification was obtained by comparing the autosomal STR pro-files of the boy and the boy’s father. Curiously, the boy, who wasdescribed by the medical examiner as anatomically male, pre-sented only an X at the Amelogenin locus whereas the father typedas expected (X, Y). While the case was completed and closed, thisAmelogenin Y null remained of interest to the laboratory.

2. Materials and methods

2.1. Extraction and quantification

Hair, whole blood, and blood on FTA™ paper were extractedusing organic extraction with phenol:chloroform:isoamyl alcoholfollowed by concentration with Microcon� YM-100 according to

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PR-ICF guidelines (Millipore, Bedford, MA, USA). Blood on FTA™paper was extracted using Qiagen’s EZ1 DNA Investigator Kit onthe EZ1 Advanced XL (Qiagen Inc., Valencia, CA, USA) followingthe manufacturer’s protocol. The quantity of DNA was determinedusing the Quantifiler� Human DNA Quantification Kit and theQuantifiler� Duo DNA Quantification Kit on the ABI 7500 Real-Time PCR System (Life Technologies, Carlsbad, CA, USA).

2.2. Amplification and STR analysis

The samples were amplified using AmpFlSTR� Identifiler� kit(Life Technologies). PCR products were separated and detectedon an ABI 3100Avant Genetic Analyzer (Life Technologies) follow-ing the manufacturer’s recommendations.

DNA obtained from the blood on the FTA™ punch was utilizedfor further STR analysis. The sample was amplified usingAmpFlSTR� Yfiler™ kit (Life Technologies) in a reduced reactionvolume of 15 lL (14 lL of master mix and 1 ng (in 1 lL) of DNA).The sample was also amplified using PowerPlex� 16HS system(Promega Corporation, Madison, WI, USA) and PowerPlex� Y sys-tem (Promega Corp.) using the manufacturer’s recommendationsto confirm results obtained from Identifiler� and Yfiler™ kits,respectively.

Finally, the sample was analyzed with the Prototype Power-Plex� Y23 System (Promega Corp.) using the manufacturer’s rec-ommendations and the Y multiplex described by Hedman et al. [1].

C. Davis et al. / Legal Medicine 14 (2012) 320–323 321

2.3. Sequencing

Three Y-STR loci were amplified using AmpFlSTR� Yfiler™ ampli-fication parameters with the following Y forward and reverse prim-ers, respectively: DYS390 50-TATATTTTACACATTTTTGGGCC -30,50-GTGACAGTAAAATGAAAACATTGC -30, DYS391 50-TTCATCATA-CACCCATATCTGTC-30, 50-GATAGAGGGATAGGTAGGCAGGC-30 andfor DYS448 50-TGGGAGAGGCAAGGATCCAA-30, 50-GTCATATTTCTG-GCCGGTCTGG-30. The amplicons were sequenced using Big Dye Ter-minator v3.1 (Life Technologies) using the same primers followedby analysis on the ABI3100Avant Genetic Analyzer (Life Technolo-gies) using manufacturer’s recommended procedures.

3. Results

At PR-ICF, Quantifiler Duo yielded results from the boy’s samplefor both the human (RPPH1) and Y (SRY) targets. The STR profile ofthe boy generated using the AmpFlSTR� Identifiler� kit was com-pared with the STR profile from his father. However, the results ofthe boy’s DNA profile showed only an X allele in the Amelogenin lo-cus. The boy’s DNA was typed using the AmpFlSTR� Yfiler™ kit toconfirm male gender. Only four of the 16 possible loci in the multi-plex (DYS456, DYS458, DYS19, and DYS393) yielded results (Fig. 1).

The samples from the boy and father were then sent to VirginiaCommonwealth University (VCU) for confirmation. There,AmpFlSTR� Yfiler™ analysis was conducted for both samples. The

Fig. 1. Electropherogram of the boy’s sample g

autosomal and Y chromosome results obtained from the PR-ICFwere confirmed for the boy. The father yielded a complete Y STRprofile (Fig. 2). Fluorescent In situ Hybridization (FISH) was at-tempted on the formalin fixed, paraffin embedded tissues fromthe heart and liver using two probes, a Y centromeric and a SRYgene probe. No results were obtained from FISH analysis. Sequenc-ing was attempted for three loci on the q arm of the Y chromosome(DYS390, DYS391, and DYS448) to assess whether the absence ofresults was due to a large deletion or substantial primer bindingsite variations (although the latter was unlikely). No amplificationresults were obtained for the three loci, but all controls workedproperly.

The sample was sent to the University of North Texas HealthScience Center (UNTHSC) to analyze additional Y STR loci to indi-rectly infer the extent of the deletion. Twelve loci DYS481,DYS533, DYS549, DYS576, DYS643, DYS449, DYS460, DYS505,DYS522, DYS612, and DYS627 were typed using the PrototypePowerPlex� Y23 System - further spanning the p and q arm ofthe chromosome. Of these loci, no results were obtained for anyof the loci on the q arm or for the loci DYS570 and DYS576 onthe p arm (Table 1).

4. Discussion

Amelogenin testing is routinely used in autosomal forensic kitsto determine sex [2–5]. Amelogenin Y drop out can occur in male

enerated using the AmpFlSTR� Yfiler™ kit.

Fig. 2. Electropherogram of the father’s sample generated using the AmpFlSTR� Yfiler™ kit.

322 C. Davis et al. / Legal Medicine 14 (2012) 320–323

samples [6–8]. When there is a possibility of Amelogenin drop out,it is common practice to perform Y-STR analysis to verify sex. Inrare cases, portions of the Y chromosome may be missing makingY STR analysis difficult. While the identity of the boy in this casewas confirmed with autosomal STR analysis and comparison withthe father’s genetic data, the Y drop out at the Amelogenin locus aswell as the subsequent number of Y-STR markers that did not am-plify interested the scientists involved. While large deletions of iso-lated portions of the Y chromosome have been reported [9], thiscase involved deletions spanning most of the Y chromosome. Inan attempt to determine how substantial the deletions were, sev-eral processes were attempted.

To confirm the autopsy results of the child being anatomicallymale, the SRY gene was amplified using the Quantifiler� DUO kit.This gene determines testis differentiation and confirms male gen-der. The child in question was positive for the SRY gene. The SRYgene is located on the p arm of the Y chromosome at 2.65–2.66 Mb, confirming at least that portion of the p arm on the Y chro-mosome was present. DNA from the hair, whole blood, and blood onFTA™ paper samples was amplified to reduce the possibility ofmosaicism as a plausible cause [10]. The DNA results were consis-tent among the different samples taken, ruling out this hypothesis.

To determine possible chromosomal abnormalities, FISH wasattempted. No results were obtained from FISH, most likely dueto the high level of formalin used and degradation of the DNA

sample tested. While a substantial number of loci did not amplifyamong the four Y-STR multiplex systems, three Y STR loci were se-quenced to confirm that multiple primer binding mutations werenot the cause of the null alleles (although this hypothesis washighly unlikely) [11]. No sequence data were obtained for the threeloci supporting that a deletion is a more plausible explanation thanmultiple primer binding site mutations.

Taken together, these data indicate that the high number of nullalleles among the Y STR loci analyzed is most likely due to a dele-tion of the entire q arm as well as a large portion of the p arm. Thethree loci on the p arm that did not amplify (DYS570, DYS576, andAmelogenin Y) are located proximal with each other on the p arm(at 6.7–7 Mb). Unfortunately, this large deletion could not be dem-onstrated by karyotyping.

While there are many sex chromosome abnormalities, two pos-sible genetic anomalies could result in loss of the Y STR data de-scribed herein: an X–Y translocation or a Y isochromosome. AnX–Y translocation allows a person to be phenotypically male with-out having a free standing Y chromosome [12]. The Y isochromo-some, or isodicentric chromosome is a duplication of one armalong with the total absence of the other arm [13]. In the case ofan Yp isochromosome, a duplication of the p arm would occur withthe loss of the q arm. If large portions of the q arm of the Y chro-mosome are missing, typical forensic measures involving Y STRtyping may not be adequate for identity testing purposes.

Table 1YSTR loci examined on the case sample. Allele call is the repeat number for the allele atthat locus, if 0, no allelic data were present. Chromosomal location was determinedusing NCBI build 37.3, GRCH37. The box identifies the loci on the p arm that likelyreside within the deleted region of the boy’s Y chromosome.

C. Davis et al. / Legal Medicine 14 (2012) 320–323 323

Alternatively, if the q arm was deleted, either of these twoanomalies (an X-Y translocation or a Y isochromosome) could haveallowed the gender determining portion of the Y chromosome tostill be functional to confer male phenotype. An X–Y translocationcould have occurred where only a portion of the Yp arm translo-cated to the X arm. An isochromosome could also account for theloss of the entire q arm. The male could either have a duplicationof the Yp arm around one centromere, or originally have had twoY chromosomes, which upon rejoining resulted in two Yp armsand a loss of the q arm. With the latter type, the isochromosomewith two centromeres would still be mitotically stable becauseone centromere is suppressed [14]. Unfortunately, the two anom-aly hypotheses presented above could not be proven by karyotyp-ing due to lack of an appropriate sample, but the extent of thedeletion can be inferred indirectly by the absence of loci on the qarm and absence of loci between the loci DYS456 and DYS522 onthe p arm.

Amelogenin typing is a good tool for determining gender of thedonor of a sample. At times, although not common, some males do

not display the Y Amelogenin marker. If the apparent X connoting afemale gender is suspicious or does not fit the circumstances of acase, Y STR typing can be pursued. Because deletions are commonin the Y chromosome, some Y STR typing can be limited. Given thelarge battery of Y STR loci that are available today, it should be pos-sible to obtain one to a few Y STR positive results even with a largeportion of the Y chromosome missing. In this case, the identity ofthe boy was confirmed by autosomal STR typing and subsequentanalysis of partial profiles from the Y STR results. Also, the discrep-ancy of the Amelogenin data had no impact on identification.

Acknowledgments

The authors would like to thank Dr. Jackson-Cook and the staffof the VCU, Department of Pathology, Cytogenetics DiagnosticsLaboratory for their assistance with FISH analysis.

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