Forensic Science International: Genetics 15 (2015) 98–104

Amelogenin test abnormalities revealed in Belarusian populationduring forensic DNA analysis

Sergey Borovko *, Alena Shyla, Victorya Korban, Alexandra Borovko

State Committee of Forensic Examinations of the Republic of Belarus, Volodarskiy str. 2a, 220030 Minsk, Belarus

A R T I C L E I N F O

Keywords:

Amelogenin X (AMELX)

Amelogenin Y (AMELY)

Null allele

Deletion

Short tandem repeat (STR)

XX male syndrome

A B S T R A C T

Study of gender markers is a part of routine forensic genetic examination of crime scene and reference

samples, paternity testing and personal identification. Amelogenin locus as a gender marker is included

in majority of forensic STR kits of different manufacturers. In current study we report 11 cases of

amelogenin abnormalities identified in males of Belarusian origin: 9 cases of AMELY dropout and 2 cases

of AMELX dropout. Cases were obtained from forensic casework (n = 9) and paternity testing (n = 2)

groups. In 4 out of 9 AMELY-negative cases deletion of AMELY was associated with the loss of DYS458

marker. In addition, we identified 3 males with SRY-positive XX male syndrome. Deletion of the long arm

of the Y-chromosome was detected in two XX males. Loss of the major part of the Y-chromosome was

identified in the third XX male. The presence of two X-chromosomes in XX males was confirmed with the

use of Mentype1 Argus X-8 PCR Amplification Kit. AMELY null allele observed in 2 out of 9 cases with

AMELY dropout can be caused by mutation in the primer-binding site of AMELY allele. Primer-binding

site mutations of AMELX can result in AMELX dropout identified in 2 cases with amplification failure of

AMELX. Our study represents the first report and molecular genetic investigation of amelogenin

abnormalities in the Belarusian population.

� 2014 Elsevier Ireland Ltd. All rights reserved.

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Forensic Science International: Genetics

jou r nal h o mep ag e: w ww .e lsev ier . co m / loc ate / fs ig

1. Introduction

Human gender determination based on the amelogenin gene iswidely used in many fields: prenatal diagnosis of X-linkeddiseases (e.g. Duchenne muscular dystrophy and haemophilia),diagnosis of sex chromosome aneuploidies, preimplantationdiagnosis and monitoring patients after sexmismatched bonemarrow transplant, archaeological analysis, DNA databasing,blood sample storage. The accurate gender determination ofbiological samples is essential in forensic casework, paternitytesting and person identification [1,2].

The amelogenin gene is a single copy gene, homologues ofwhich amelogenin X (AMELX) and amelogenin Y (AMELY) arelocated on Xp22.1–Xp22.3 and Yp11.2, respectively. HomologuesAMELX and AMELY differ in both size and sequence. Theamelogenin locus has been incorporated in various commercialshort tandem repeat (STR) multiplex kits for human genderidentification. The most commonly used amelogenin primer setflanks a 6 bp deletion within intron 1 of AMELX and produces

* Corresponding author. Tel.: +375 29 629 74 91; fax: +375 17 218 74 03.

E-mail address: s_borovko@mail.ru (S. Borovko).

http://dx.doi.org/10.1016/j.fsigen.2014.10.014

1872-4973/� 2014 Elsevier Ireland Ltd. All rights reserved.

fragments of 106 bp and 112 bp for the X and Y-chromosomes,respectively [3–6].

Amplification failure of the AMELY in the male samples cancause misidentification of the biological sample as a female if theother information of the sample is not considered or not available.Similarly, mutations of AMELX in males can be responsible forAMELX dropout although the genotype of the biological samplewould still be identified as a male due to amplification of AMELY.

Cases of amelogenin-negative males have been detectedworldwide. Genetic mechanisms underlying AMELY dropoutinvolve deletions of different size encompassing AMELY locus,mutations in the primer-binding region of AMELY allele in thelesser extent [1,5]. Deletions on Yp11.2 region as a major cause ofAMELY null allele are often combined with the absence of adjacentY-STR loci DYS456 and/or DYS458 [1,2,6–12].

Translocations between distal Xp and Yp result in thegeneration of 46,XX males, the majority of whom display a malephenotype due to transfer of the sex-determining region Y (SRY)gene onto the short arm of the X-chromosome (SRY-positive XXmale syndrome) [13,14]. The XX male syndrome or 46,XXtesticular disorder of sex development (OMIM ID #400045) occursvery rare with a frequency of 1:20,000–1:30,000 male newbornsand was first described by de la Chapelle et al. in 1964

S. Borovko et al. / Forensic Science International: Genetics 15 (2015) 98–104 99

[13–16]. Since then up to 2006 approximately 250 cases with46,XX male syndrome were published in the world literature,mostly as individual cases. On the basis of the SRY gene analysis46,XX males can be divided into SRY-positive (90%) and SRY-negative (10%) groups [16].

Cases of AMELX dropout in males have been reported in severalpapers [5,14,17–19]. A primer-binding site point mutations inAMELX allele have been detected in all males displayed only the Yallele from amelogenin amplification in these studies.

During 15-year expert practice in forensic DNA analysis weanalyzed more than 30,000 forensic cases and more than 4000paternity testing cases. Of this amount of cases we collected 11cases with amelogenin abnormalities (AMELY or AMELX drop-outs) identified in Belarusian population. AMELX and AMELY nullalleles were revealed with either AmpF‘STR1 Identifiler1 PlusPCR Amplification Kit (Identifiler1 Plus Kit) or GlobalFiler1 PCRAmplification Kit (GlobalFiler1 Kit). To elucidate genetic mecha-nisms underlying anomalous amplification of the amelogeninlocus in Belarusian males we used AmpF‘STR1 Yfiler1 PCRAmplification Kit (Yfiler1 Kit) for Y-STRs profiling, Mentype1

Argus X-8 PCR Amplification Kit (Mentype1 Argus X-8 Kit) forX-STRs and amelogenin analysis, Quantifiler1 Y Human MaleDNA Quantification Kit (Quantifiler1 Y Kit) for the detection ofthe SRY gene.

2. Materials and methods

All 11 amelogenin-negative cases we describe here wereanalyzed in different periods of time during our 15-year expertpractice in forensic DNA analysis. 9 cases were obtained fromcrime casework groups and 2 cases (father–son sample pairs) werediscovered during routine paternity testing. The choice of the kitsused for the genetic analysis of these samples was dependent onthe above-mentioned factors (e.g., availability of various kits) andalso on the genetic nature of the revealed amelogenin abnormali-ties in the samples.

Depending on the source of a biological sample (blood stainsamples, buccal swabs, etc.) and its quality genomic DNA wasextracted using one of the following methods: Chelex-100 method(Bio-Rad, USA), phenol–chloroform extraction [20], and such kits

Table 1Results of gender identification in cases with amelogenin abnormalities.

Sample Phenotype AMEL (Identifiler Plus1 Kit) SRY Yfiler1 Kit (16 loci)

Amplified

Deletion AMELY-DYS458

S1 Male M/– + 15 loci

S2.1 (father) Male M/– + 15 loci

S2.2 (son) Male M/– + 15 loci

S3 Male M/– + 15 loci

S4.1 (Father) Male M/–

GlobalFiler X/–

+ 15 loci

S4.2 (son) Male M/– + 15 loci

XX-male, Y short arm inversion and translocation

S5 Male M/– + 4 loci (DYS456, DYS45

S6 Male M/– + 4 loci (DYS456, DYS45

XX-male, Y short arm translocation

S7 Male M/– + 2 loci (DYS456, DYS39

Mutation in primer-binding site

S8 Male M/– + All 16 loci

S9 Male –/I n.a. n.a.

S10 Male –/I n.a. n.a.

S11 Male M/– n.a. All 16 loci

n.a., not available; –, no alleles present (null).

as PrepFiler1 Forensic DNA Extraction Kit (Applied Biosystems),NucleoSpin1 Tissue (Macherey-Nagel, Germany).

Autosomal STRs and amelogenin were amplified using eitherAmpF‘STR1 Identifiler1 Plus PCR Amplification Kit (AppliedBiosystems) or GlobalFiler1 PCR Amplification Kit (AppliedBiosystems). Y-STRs were analyzed using AmpF‘STR1 Yfiler1

PCR Amplification Kit (Applied Biosystems). Analysis of X-STRmarkers including alternative amelogenin marker was done usingMentype1 Argus X-8 Kit (Biotype, Germany).

To confirm the gender of the studied samples, a sex-determining region Y (SRY) specific to males, was amplified. SRYlocus was investigated using the real-time PCR-based DNAquantification kit Quantifiler1 Y Human Male DNA QuantificationKit (Applied Biosystems).

DNA isolation and PCR amplification of above-mentionedgroups of markers including amelogenin and SRY genes wereperformed according to manufacturer’s recommendations. PCRamplifications were carried out on the GeneAmp PCR System 2700(Applied Biosystems) and amplification products were separatedby capillary electrophoresis on the ABI PRISM1 3130xl GeneticAnalyzer, ABI PRISM1 3100 Genetic Analyzer and ABI PRISM1

3500 Genetic Analyzer (Applied Biosystems, Foster City, CA).Fragments were automatically analyzed using GeneMapper ID-Xv.1.1.1 Software (Applied Biosystems). For the quantification ofthe SRY gene expression ABI PRISM1 7000 Sequence DetectionSystem was used.

3. Results

A total of 11 cases with amelogenin abnormalities havebeen detected in Belarusian males: 9 AMELY null and 2 AMELXnull cases. The results of autosomal STRs and amelogenin, Y-STRs,X-STRs (including amelogenin locus) genotyping for all cases areshown in the Supplementary Tables S1–S3, respectively.

3.1. Interstitial Yp11.2 deletion (AMELY-DYS458)

The AMELY-DYS458 deletion pattern has been identified in4 out of 9 AMELY-negative cases (Table 1). Two cases (samples S2.1and S2.2, S4.1 and S4.2) involve related males (father–son sample

Mentype1 Argus X-8 kit

Not amplified AMEL X/Y Number of X-chromosomes

1 locus (DYS458) n.a. n.a.

1 locus (DYS458) M/– One X-chromosome

1 locus (DYS458) M/– One X-chromosome

1 locus (DYS458) n.a. n.a.

1 locus (DYS458) n.a. n.a.

1 locus (DYS458) n.a. n.a.

8, DYS19, DYS393) 12 loci M/– Heterozygous at 7 loci

8, DYS19, DYS393) 12 loci n.a. n.a.

3) 14 loci M/� Heterozygous at 7 loci

– n.a. n.a.

n.a. M/I Hemizygous at 8 loci

n.a. n.a. n.a.

n.a. n.a. n.a.

Fig. 1. Y-STRs haplotype of S2.1. In the case S2.1 amplification of only DYS458 was failed. The rest of Y-STR markers were successfully amplified with Yfiler1 Kit.

S. Borovko et al. / Forensic Science International: Genetics 15 (2015) 98–104100

pairs). The rest of the cases (S1 and S3) were crime scene samplesfrom unrelated males. In all 4 cases absence of AMELY wasdetected with Identifiler1 Plus Kit. In the case S4.1 (our recentexpertise when GlobalFiler1 Kit was available) absence of AMELYwas confirmed with GlobalFiler1 Kit. It is interesting to note thatthe GlobalFiler1 Kit helped to identify the sex of S4.1 sample

Fig. 2. Y-STRs haplotype of S5. Lack of amplification of the most of Yfiler1 Kit

correctly as AMELY dropout was detected in this sample withIdentifiler1 Plus Kit (Supplementary Fig. S1).

Among 16 Y-STRs amplified with Yfiler1 Kit amplification ofonly DYS458 marker was failed in the cases S1–S4 (Fig. 1). All caseswere SRY positive (data not shown). Thus, the complete absence ofAMELY and DYS458 and the presence of SRY gene in all 4 cases let

loci was observed in S5 except for DYS456, DYS458, DYS19 and DYS393.

Fig. 3. X-STR marker profiles of S5. AMELY null allele was detected in S5 with Mentype1 Argus X-8 Kit. Heterozygous profiles were observed at all X-STR loci in S5 except for

DXS8378 indicating the presence of two X-chromosomes in S5.

S. Borovko et al. / Forensic Science International: Genetics 15 (2015) 98–104 101

us to conclude that AMELY dropout in the cases S1–S4 was causedby a deletion in the short arm of Y-chromosome (Yp11.2 region).

In addition, for the samples S2.1 and S2.2 (a father–son samplepair) amelogenin and X-STRs were typed with Mentype1 Argus X-8 Kit. The results of genotyping confirmed the absence of AMELYand the presence of one X-chromosome in the samples. The DNA-samples were hemizygous at all 8 X-STR loci.

3.2. SRY-positive XX male syndrome

We have identified 3 AMELY-negative samples with XX malesyndrome–samples S5, S6 and S7 (Table 1). Karyotyping was notdone for these samples. Therefore, we can only assume their XXstatus. Interestingly, all three samples were SRY-positive (data notshown).

Samples S5 and S6 were amplified successfully for two distal YpSTR markers, DYS393 and DYS456, and two proximal Yp STRmarkers, DYS458 and DYS19, to the absent AMELY. AMELY and12 Y-STRs mapping to the long arm of Y-chromosome wereundetectable in the samples (Fig. 2). Absence of AMELY in sampleS5 was proved with Mentype1 Argus X-8 Kit. Moreover, S5 showedheterozygous profile at 7 out of 8 X-STR markers, indicating the

Fig. 4. Y-STRs haplotype of S7. Only two Y-STR loci DYS45

presence of two X-chromosomes in S5 (Fig. 3). X-STR analysis wasnot done for sample S6.

Sample S7 was another XX man and showed a different Y-STRspattern compared to the samples S5 and S6. Only DYS393 andDYS456 loci were detected with Yfiler1 Kit in S7 (Fig. 4). SRY wasalso identified in S7 (data not shown). These data pinpoint to thepresence of the deletion in S7, encompassing the major part of Y-chromosome from Yp11.2 up to the whole long arm. Profiling withMentype1 Argus X-8 Kit confirmed the absence of AMELY in S7 andrevealed the heterozygous genotype of S7 at 7 out of 8 X-STRmarkers (Supplementary Fig. S2). Thus, these findings support ourassumption that the man, from whom DNA sample S7 wasobtained, has a SRY-positive XX male syndrome.

3.3. Mutations in the primer-binding site of AMELX or AMELY alleles

Samples S8 and S11 were detected as AMELY-negative sampleswith Identifiler1 Plus Kit. However, these samples showedcomplete Y-STR profiles of the Yfiler1 Kit (Table 1). This datalet us to assume that AMELY dropout observed in S8 and S11 iscaused by a mutation occurred in the primer-binding region of theAMELY allele.

6 and DYS393 were amplified in S7 with Yfiler1 Kit.

Fig. 5. Genetic profiles of autosomal STRs, X-STRs and amelogenin of S9 generated with Identifiler1 Plus (panels A and B) and Mentype1 Argus X-8 (panels C and D) Kits.

Absence of AMELX allele and presence of 112 bp fragment from AMELY was detected in S9 with Identifiler1 Plus Kit (panel B). Use of the alternative set of primers for

amelogenin included in Mentype1 Argus X-8 Kit let to detect both AMELX (103 bp) and AMELY (109 bp) fragments in S9 (panel C). S9 was hemizygous for all 8 X-STR loci

included in Mentype1 Argus X-8 Kit (panels C and D).

S. Borovko et al. / Forensic Science International: Genetics 15 (2015) 98–104102

Loss of AMELX has been detected in two male samples S9 andS10 with Identifiler1 Plus Kit (Table 1). Amelogenin amplifica-tion performed with another kit (Mentype1 Argus X-8) revealedboth AMELX and AMELY fragments in sample S9 (Fig. 5).Moreover, sample S9 showed hemizygous X-STR profile withonly one allele at each X-STR locus. These findings support theassumption of the presence of X-chromosome with a primer-binding site mutation at the amelogenin locus in sample S9.Genetic nature of AMELX dropout observed in sample S10 needsto be further investigated as there is only data of amplificationwith Identifiler1 Plus for this sample. Absence of AMELX inS10 can be caused by either a primer-binding site mutation atthe amelogenin locus or a deletion of the amelogenin locus ofX-chromosome.

4. Discussion

In the present study, to our knowledge, we first reported casesof amelogenin abnormalities in Belarusian population. Herein, 11cases of amelogenin abnormalities collected during 15-yearpractice in forensic DNA analysis in Belarus were described.Genetic mechanisms underlying AMELX or AMELY dropoutsidentified in these cases fall into four categories: (1) deletioninvolving AMELY and DYS458 loci (n = 4), (2) loss of the long arm ofthe Y-chromosome with partial X–Y translocation in an XX-man(n = 2), (3) loss of most of the Y-chromosome in an XX man (n = 1),(4) mutation in the primer-binding region of AMELX (n = 2) orAMELY (n = 2) loci.

Deletions in Yp11.2 region are the major cause of the failureof AMELY allele amplification [5,8,12]. AMELY dropout is oftencombined with deletion of the DYS458 locus [7–9,11,12]. Onthe other hand, the DYS458 null allele may serve as a strongindicator of the AMELY-negative sample. High frequency of the

AMELY-DYS458 deletion pattern may be explained by smallphysical distance (1.19 Mb) between these two loci [5,7–9].

Absence of AMELY and DYS458 amplification caused by Yp11.2deletion has been detected in various populations with differentfrequency (Table 2). It can be explained by population-specificdifferences and by different sizes of analyzed population groupsreported in the papers [7,21]. The high percent of AMELY andDYS458 null alleles have been identified in Nepalese males andMalaysian Indians (a migrant male group from India) (Table 2). Atthe same time AMELY and DYS458 null alleles have not beendetected in Malaysian Chinese. In the present study, we detected4 cases with AMELY-DYS458 allelic pattern out of 11 cases ofamelogenin-negative Belarusian males (Table 2).

Large Yp11.2 deletion patterns DYS456-AMELY-DYS458 orAMELY-DYS458-DYS19 have been identified in AMELY-negativemales in several studies [7,8,10]. We have not identified suchdeletion patterns in AMELY-negative Belarusian males. In all 9AMELY-negative cases Y-STR markers DYS456 and DYS19 weresuccessfully amplified with Yfiler1 Kit (Supplementary Table S2).

Overall, the frequency of AMELY dropout in Sri Lankan (2/24,8.333%) [22], Nepalese (9/200, 4.50%) [2], (5/77, 6.490%) [6], andIndian (10/4257, 0.230%) [23], (1/100, 1%) [9], (5/270, 1.852%) [24]populations is notably higher than that observed in someCaucasian population groups from Austria (5/28,182, 0.018%)[21], Israel (1/96, 1.042%) [25], Spain (1/1000, 0.1%) [9], (1/768,0.130%) [26], England (2/2000, 0.1%) [9], and Italy (1/13,000,0.008%) [27]. The data on the frequency of either AMELY dropout orAMELY-DYS458 deletion pattern in the Belarusian populationcannot be directly compared to that of above-mentioned Caucasianpopulation groups on the several reasons: (1) there are no data ofDYS458 marker amplification because this Y-STR marker was notused in typing of the samples [21,25–27]; (2) different amount ofsamples analyzed in the studies.

Table 2Frequency distribution of males with AMELY-DYS458 deletion pattern in different populations.

Population Null AMELY-DYS458/

null AMELY

No. of males

studied

Source of the samples Population

frequency

Frequency (number

of null AMELY)

Reference

Nepalese 9/9 200 Paternity testing group

(n = 200)

9/200 (4.5%) – [2]

Malaysian Chinese 0 331 Y-STR database and forensic

casework groups (n = 980)

– – [9]

Malaysian Indiansa 10/12 315 10/315 (3.175%) –

Malaysian Malaysa 2/12 334 2/334 (0.599%) –

Chineseb 2/3 8087 DNA database (n = 8087) 2/8087 (0.025%) – [10]

(Guangdong province)

Chinesec 3/3 40 DNA database (n = 12,891) 3/12,891 (0.023%) – [8]

(Zheijiang and

Guangdong provinces)

Chinesed 13/18 79,304 DNA database and forensic

casework groups (n = 79,304)

13/79,304 (0.016%) – [7]

(North of China)

Mixed population

groupe

9/45 45 Paternity testing group,

population studies

9/45 (20%) – [1]

Japanese 4/4 4 Forensic casework – 4/4 (100%) [11]

Italian 2/2 2 Paternity testing group – 2/2 (100%) [12]

Belarusian 4/9 13 Forensic casework, paternity

testing groups

– 4/9 (44.4%) Current study

a In all samples with AMELY-DYS458 deletion pattern, the absence of Y-specific MSY1 minisatellite located between AMELY and DYS458 loci was found.b One sample showed AMELY-DYS458 deletion pattern, another one showed DYS456-AMELY-DYS458 deletion pattern.c Two samples showed AMELY-DYS458 deletion pattern, one sample showed DYS456-AMELY-DYS458 deletion pattern.d AMELY-DYS458 deletion pattern was identified in 8 out of 18 AMELY-negative samples. Different deletion patterns including the locus DYS458 were detected in 5

samples with AMELY dropout: DYS456-AMELY-DYS458 null alleles (1 sample); AMELY-DYS458-DYS19 null alleles (1 sample); AMELY-DYS458-GATA_H4 deletion

(2 samples); deletion involving the major part of Y-chromosome except for two Y-STRs DYS456 and DYS393 (1 sample).e Mixed population group comprises 45 AMELY-deficient males from 12 populations.

S. Borovko et al. / Forensic Science International: Genetics 15 (2015) 98–104 103

Paternity testing samples S2.1 and S2.2, S4.1 and S4.2 werecollected from related men (father–son sample pairs). It meansthat the deletion AMELY-DYS458 identified in these samples is nota de novo mutation, but was transmitted from the father tothe son. Therefore, the deletion in the short arm of theY-chromosome Yp11.2, containing AMELY and DYS458 loci, didnot affect fertility as paternity was proven in both cases S2and S4. Similar observations have been done by several authors[2,7,8,11,12,27,28]. Deletions in the long arm of the Y-chromosomeare associated with azoospermia and can cause reproductionfailure in males [1,9,16,27,29,30].

Combined use of different kits for analysis of autosomal STRs,Y-STRs, X-STRs and amelogenin and SRY genes let us to identifythree cases with SRY-positive XX-male syndrome: S5, S6, and S7.We could not perform karyotype analysis for these samples.However, with Mentype1 Argus X-8 Kit samples S5 and S7exhibited a heterozygous profile with two alleles at 7 out of 8X-STR loci. This finding confirms the presence of two X-chromosomes in samples S5 and S7. Although the X-STR analysiswas not done for sample S6, we combined samples S5 and S6 inone group because of the similar genetic findings in Y-STR typingin the samples.

Long arm loss of Y-chromosome has been detected in theAMELY-negative samples S5 and S6. Only four Y-STR markers,DYS393, DYS456, DYS458, and DYS19, could be amplified in thesesamples. In addition, heterozygous profile at 7 out of 8 analyzed X-STR loci has been identified in sample S5. SRY has been detected inboth S5 and S6 samples. Cases with similar genetic findings havebeen described in several studies [7,31]. It has been proposed aputative genetic mechanism explaining the discrepant patternobserved in the above-mentioned samples: DYS19 is transferred tothe distal IR3 element (inverted repeats region) by a paracentricinversion, followed by the translocation of the terminal segment of

Y-chromosome, including SRY, DYS393, DYS456, DYS19, andDYS458, onto the X-chromosome [7,31].

A case with loss of most of Y-chromosome in a man withXX male syndrome similar to S7 (current study) was describedby Ma et al. [7]. Two out of 16 Y-STR loci, DYS393 and DYS456,were amplified in S7. Ma and coauthors proposed a geneticmodel explaining mechanism underlying deletion patternobserved in these samples: AMELY together with the Yp markers,DYS458, DYS19, and the whole long arm of Y-chromosomeundergo deletion, while the distal Yp markers, including SRY,DYS393, and DYS456, are translocated onto the short arm ofX-chromosome [7].

A plausible explanation for AMELY or AMELX dropout in foursamples, S8, S9, S10, S11, can be a mutation in primer-bindingregion of AMELY (samples S8 and S11) or AMELX (samples S9 andS10).

Successful amplification of all 16 Y-STR loci in the AMELY-negative samples S8 and S11 suggested that AMELY dropout inthese samples resulted from the point mutation in the primer-binding site of AMELY. AMELY null allele was detected in S8 withIdentifiler1 Plus Kit. Since different kit (e.g. GlobalFiler1 orMentype1 Argus X-8 Kits) might use different pair of primers forAMELY allele, this sort of amelogenin abnormalities may notappear in other PCR kits.

AMELX locus that was not detected in S9 with Identifiler1

Plus Kit was successfully identified with Mentype1 Argus X-8Kit. In addition, sample S9 showed a hemizygous profile at alleight X-STR loci. These data point towards the presence ofprimer-binding site mutation at AMELX locus. Zehethoferand Rolf described similar genetic findings for a pair of samplesfrom paternity testing group (farther-son) [14]. AMELX alleledropouts are believed to be mainly caused by mutations inthe primer-binding region [5]. Moreover, the loss of AMELX

S. Borovko et al. / Forensic Science International: Genetics 15 (2015) 98–104104

allele is more common in some populations [5]. AMELX dropoutshave been detected in Polish population (1/5534, 0.018%) [17],males from West Africa (10/503, 2%) [18], and African–Americanmales (48/144,391, 0.033%) [5].

In summary, our data indicate that the combined use ofdifferent kits such as Identifiler1 Plus or GlobalFiler1 forautosomal STR loci and amelogenin, Quantifiler1 Y for SRY gene,Yfiler1 for Y-profiling and Mentype1 Argus X-8 or Investigator1

Argus X-12 for X-STRs and amelogenin may help to overcomeproblems related to the wrong gender determination and toelucidate the genetic mechanisms underlying amelogenin abnor-malities. A new PCR multiplex GenderPlex presented by Esteve etal. [32] gives additional possibilities for a forensic DNA expert toavoid sex misinterpretation.

The data on the incidence of amelogenin abnormalities in theglobal population are scarce. Therefore, we believe that the datapresented in current study along with planned study on thefrequency of AMELY and AMELX dropouts in Belarusian populationwould be helpful for forensic community.

Conflict of interest

The authors declare that they have no conflict of interest.

Funding

The study was not funded by a special source.

Acknowledgement

We would like to thank all members of DNA laboratory of StateMedical Forensic Service of the Republic of Belarus.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.fsigen.2014.10.014.

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