1. Amelogenin test abnormalities revealed in Belarusian population
during 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
1. Introduction
Human gender determination based on the amelogenin gene is
widely used in many fields: prenatal diagnosis of X-linked
diseases (e.g. Duchenne muscular dystrophy and haemophilia),
diagnosis of sex chromosome aneuploidies, preimplantation
diagnosis and monitoring patients after sexmismatched bone
marrow transplant, archaeological analysis, DNA databasing,
blood sample storage. The accurate gender determination of
biological samples is essential in forensic casework, paternity
testing and person identification [1,2].
The amelogenin gene is a single copy gene, homologues of
which amelogenin X (AMELX) and amelogenin Y (AMELY) are
located on Xp22.1–Xp22.3 and Yp11.2, respectively. Homologues
AMELX and AMELY differ in both size and sequence. The
amelogenin locus has been incorporated in various commercial
short tandem repeat (STR) multiplex kits for human gender
identification. The most commonly used amelogenin primer set
flanks a 6 bp deletion within intron 1 of AMELX and produces
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 can
cause misidentification of the biological sample as a female if the
other information of the sample is not considered or not available.
Similarly, mutations of AMELX in males can be responsible for
AMELX dropout although the genotype of the biological sample
would still be identified as a male due to amplification of AMELY.
Cases of amelogenin-negative males have been detected
worldwide. Genetic mechanisms underlying AMELY dropout
involve deletions of different size encompassing AMELY locus,
mutations in the primer-binding region of AMELY allele in the
lesser extent [1,5]. Deletions on Yp11.2 region as a major cause of
AMELY null allele are often combined with the absence of adjacent
Y-STR loci DYS456 and/or DYS458 [1,2,6–12].
Translocations between distal Xp and Yp result in the
generation of 46,XX males, the majority of whom display a male
phenotype due to transfer of the sex-determining region Y (SRY)
gene onto the short arm of the X-chromosome (SRY-positive XX
male syndrome) [13,14]. The XX male syndrome or 46,XX
testicular disorder of sex development (OMIM ID #400045) occurs
very rare with a frequency of 1:20,000–1:30,000 male newborns
and was first described by de la Chapelle et al. in 1964
Forensic Science International: Genetics 15 (2015) 98–104
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.
ß 2014 Elsevier Ireland Ltd. All rights reserved.
* Corresponding author. Tel.: +375 29 629 74 91; fax: +375 17 218 74 03.
E-mail address: s_borovko@mail.ru (S. Borovko).
Contents lists available at ScienceDirect
Forensic Science International: Genetics
journal homepage: www.elsevier.com/locate/fsig
http://dx.doi.org/10.1016/j.fsigen.2014.10.014
1872-4973/ß 2014 Elsevier Ireland Ltd. All rights reserved.

2. [13–16]. Since then up to 2006 approximately 250 cases with
46,XX male syndrome were published in the world literature,
mostly as individual cases. On the basis of the SRY gene analysis
46,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 several
papers [5,14,17–19]. A primer-binding site point mutations in
AMELX allele have been detected in all males displayed only the Y
allele from amelogenin amplification in these studies.
During 15-year expert practice in forensic DNA analysis we
analyzed more than 30,000 forensic cases and more than 4000
paternity testing cases. Of this amount of cases we collected 11
cases with amelogenin abnormalities (AMELY or AMELX drop-
outs) identified in Belarusian population. AMELX and AMELY null
alleles were revealed with either AmpF‘STR1
Identifiler1
Plus
PCR Amplification Kit (Identifiler1
Plus Kit) or GlobalFiler1
PCR
Amplification Kit (GlobalFiler1
Kit). To elucidate genetic mecha-
nisms underlying anomalous amplification of the amelogenin
locus in Belarusian males we used AmpF‘STR1
Yfiler1
PCR
Amplification Kit (Yfiler1
Kit) for Y-STRs profiling, Mentype1
Argus X-8 PCR Amplification Kit (Mentype1
Argus X-8 Kit) for
X-STRs and amelogenin analysis, Quantifiler1
Y Human Male
DNA Quantification Kit (Quantifiler1
Y Kit) for the detection of
the SRY gene.
2. Materials and methods
All 11 amelogenin-negative cases we describe here were
analyzed in different periods of time during our 15-year expert
practice in forensic DNA analysis. 9 cases were obtained from
crime casework groups and 2 cases (father–son sample pairs) were
discovered during routine paternity testing. The choice of the kits
used for the genetic analysis of these samples was dependent on
the above-mentioned factors (e.g., availability of various kits) and
also on the genetic nature of the revealed amelogenin abnormali-
ties in the samples.
Depending on the source of a biological sample (blood stain
samples, buccal swabs, etc.) and its quality genomic DNA was
extracted using one of the following methods: Chelex-100 method
(Bio-Rad, USA), phenol–chloroform extraction [20], and such kits
as PrepFiler1
Forensic DNA Extraction Kit (Applied Biosystems),
NucleoSpin1
Tissue (Macherey-Nagel, Germany).
Autosomal STRs and amelogenin were amplified using either
AmpF‘STR1
Identifiler1
Plus PCR Amplification Kit (Applied
Biosystems) or GlobalFiler1
PCR Amplification Kit (Applied
Biosystems). Y-STRs were analyzed using AmpF‘STR1
Yfiler1
PCR Amplification Kit (Applied Biosystems). Analysis of X-STR
markers including alternative amelogenin marker was done using
Mentype1
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. SRY
locus was investigated using the real-time PCR-based DNA
quantification kit Quantifiler1
Y Human Male DNA Quantification
Kit (Applied Biosystems).
DNA isolation and PCR amplification of above-mentioned
groups of markers including amelogenin and SRY genes were
performed according to manufacturer’s recommendations. PCR
amplifications were carried out on the GeneAmp PCR System 2700
(Applied Biosystems) and amplification products were separated
by capillary electrophoresis on the ABI PRISM1
3130xl Genetic
Analyzer, ABI PRISM1
3100 Genetic Analyzer and ABI PRISM1
3500 Genetic Analyzer (Applied Biosystems, Foster City, CA).
Fragments were automatically analyzed using GeneMapper ID-X
v.1.1.1 Software (Applied Biosystems). For the quantification of
the SRY gene expression ABI PRISM1
7000 Sequence Detection
System was used.
3. Results
A total of 11 cases with amelogenin abnormalities have
been detected in Belarusian males: 9 AMELY null and 2 AMELX
null cases. The results of autosomal STRs and amelogenin, Y-STRs,
X-STRs (including amelogenin locus) genotyping for all cases are
shown in the Supplementary Tables S1–S3, respectively.
3.1. Interstitial Yp11.2 deletion (AMELY-DYS458)
The AMELY-DYS458 deletion pattern has been identified in
4 out of 9 AMELY-negative cases (Table 1). Two cases (samples S2.1
and S2.2, S4.1 and S4.2) involve related males (father–son sample
Table 1
Results of gender identification in cases with amelogenin abnormalities.
Sample Phenotype AMEL (Identifiler Plus1
Kit) SRY Yfiler1
Kit (16 loci) Mentype1
Argus X-8 kit
Amplified Not amplified AMEL X/Y Number of X-chromosomes
Deletion AMELY-DYS458
S1 Male M/– + 15 loci 1 locus (DYS458) n.a. n.a.
S2.1 (father) Male M/– + 15 loci 1 locus (DYS458) M/– One X-chromosome
S2.2 (son) Male M/– + 15 loci 1 locus (DYS458) M/– One X-chromosome
S3 Male M/– + 15 loci 1 locus (DYS458) n.a. n.a.
S4.1 (Father) Male M/–
GlobalFiler X/–
+ 15 loci 1 locus (DYS458) n.a. n.a.
S4.2 (son) Male M/– + 15 loci 1 locus (DYS458) n.a. n.a.
XX-male, Y short arm inversion and translocation
S5 Male M/– + 4 loci (DYS456, DYS458, DYS19, DYS393) 12 loci M/– Heterozygous at 7 loci
S6 Male M/– + 4 loci (DYS456, DYS458, DYS19, DYS393) 12 loci n.a. n.a.
XX-male, Y short arm translocation
S7 Male M/– + 2 loci (DYS456, DYS393) 14 loci M/À Heterozygous at 7 loci
Mutation in primer-binding site
S8 Male M/– + All 16 loci – n.a. n.a.
S9 Male –/I n.a. n.a. n.a. M/I Hemizygous at 8 loci
S10 Male –/I n.a. n.a. n.a. n.a. n.a.
S11 Male M/– n.a. All 16 loci n.a. n.a. n.a.
n.a., not available; –, no alleles present (null).
S. Borovko et al. / Forensic Science International: Genetics 15 (2015) 98–104 99

3. pairs). The rest of the cases (S1 and S3) were crime scene samples
from unrelated males. In all 4 cases absence of AMELY was
detected with Identifiler1
Plus Kit. In the case S4.1 (our recent
expertise when GlobalFiler1
Kit was available) absence of AMELY
was confirmed with GlobalFiler1
Kit. It is interesting to note that
the GlobalFiler1
Kit helped to identify the sex of S4.1 sample
correctly as AMELY dropout was detected in this sample with
Identifiler1
Plus Kit (Supplementary Fig. S1).
Among 16 Y-STRs amplified with Yfiler1
Kit amplification of
only DYS458 marker was failed in the cases S1–S4 (Fig. 1). All cases
were SRY positive (data not shown). Thus, the complete absence of
AMELY and DYS458 and the presence of SRY gene in all 4 cases let
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.
Fig. 2. Y-STRs haplotype of S5. Lack of amplification of the most of Yfiler1
Kit loci was observed in S5 except for DYS456, DYS458, DYS19 and DYS393.
S. Borovko et al. / Forensic Science International: Genetics 15 (2015) 98–104100

4. us to conclude that AMELY dropout in the cases S1–S4 was caused
by 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 sample
pair) amelogenin and X-STRs were typed with Mentype1
Argus X-
8 Kit. The results of genotyping confirmed the absence of AMELY
and 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 male
syndrome–samples S5, S6 and S7 (Table 1). Karyotyping was not
done for these samples. Therefore, we can only assume their XX
status. Interestingly, all three samples were SRY-positive (data not
shown).
Samples S5 and S6 were amplified successfully for two distal Yp
STR markers, DYS393 and DYS456, and two proximal Yp STR
markers, DYS458 and DYS19, to the absent AMELY. AMELY and
12 Y-STRs mapping to the long arm of Y-chromosome were
undetectable in the samples (Fig. 2). Absence of AMELY in sample
S5 was proved with Mentype1
Argus X-8 Kit. Moreover, S5 showed
heterozygous profile at 7 out of 8 X-STR markers, indicating the
presence of two X-chromosomes in S5 (Fig. 3). X-STR analysis was
not done for sample S6.
Sample S7 was another XX man and showed a different Y-STRs
pattern compared to the samples S5 and S6. Only DYS393 and
DYS456 loci were detected with Yfiler1
Kit in S7 (Fig. 4). SRY was
also identified in S7 (data not shown). These data pinpoint to the
presence of the deletion in S7, encompassing the major part of Y-
chromosome from Yp11.2 up to the whole long arm. Profiling with
Mentype1
Argus X-8 Kit confirmed the absence of AMELY in S7 and
revealed the heterozygous genotype of S7 at 7 out of 8 X-STR
markers (Supplementary Fig. S2). Thus, these findings support our
assumption that the man, from whom DNA sample S7 was
obtained, 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 samples
with Identifiler1
Plus Kit. However, these samples showed
complete Y-STR profiles of the Yfiler1
Kit (Table 1). This data
let us to assume that AMELY dropout observed in S8 and S11 is
caused by a mutation occurred in the primer-binding region of the
AMELY allele.
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.
Fig. 4. Y-STRs haplotype of S7. Only two Y-STR loci DYS456 and DYS393 were amplified in S7 with Yfiler1
Kit.
S. Borovko et al. / Forensic Science International: Genetics 15 (2015) 98–104 101

5. Loss of AMELX has been detected in two male samples S9 and
S10 with Identifiler1
Plus Kit (Table 1). Amelogenin amplifica-
tion performed with another kit (Mentype1
Argus X-8) revealed
both AMELX and AMELY fragments in sample S9 (Fig. 5).
Moreover, sample S9 showed hemizygous X-STR profile with
only one allele at each X-STR locus. These findings support the
assumption 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 needs
to be further investigated as there is only data of amplification
with Identifiler1
Plus for this sample. Absence of AMELX in
S10 can be caused by either a primer-binding site mutation at
the amelogenin locus or a deletion of the amelogenin locus of
X-chromosome.
4. Discussion
In the present study, to our knowledge, we first reported cases
of amelogenin abnormalities in Belarusian population. Herein, 11
cases of amelogenin abnormalities collected during 15-year
practice in forensic DNA analysis in Belarus were described.
Genetic mechanisms underlying AMELX or AMELY dropouts
identified in these cases fall into four categories: (1) deletion
involving AMELY and DYS458 loci (n = 4), (2) loss of the long arm of
the 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) or
AMELY (n = 2) loci.
Deletions in Yp11.2 region are the major cause of the failure
of AMELY allele amplification [5,8,12]. AMELY dropout is often
combined with deletion of the DYS458 locus [7–9,11,12]. On
the other hand, the DYS458 null allele may serve as a strong
indicator of the AMELY-negative sample. High frequency of the
AMELY-DYS458 deletion pattern may be explained by small
physical distance (1.19 Mb) between these two loci [5,7–9].
Absence of AMELY and DYS458 amplification caused by Yp11.2
deletion has been detected in various populations with different
frequency (Table 2). It can be explained by population-specific
differences and by different sizes of analyzed population groups
reported in the papers [7,21]. The high percent of AMELY and
DYS458 null alleles have been identified in Nepalese males and
Malaysian Indians (a migrant male group from India) (Table 2). At
the same time AMELY and DYS458 null alleles have not been
detected in Malaysian Chinese. In the present study, we detected
4 cases with AMELY-DYS458 allelic pattern out of 11 cases of
amelogenin-negative Belarusian males (Table 2).
Large Yp11.2 deletion patterns DYS456-AMELY-DYS458 or
AMELY-DYS458-DYS19 have been identified in AMELY-negative
males in several studies [7,8,10]. We have not identified such
deletion patterns in AMELY-negative Belarusian males. In all 9
AMELY-negative cases Y-STR markers DYS456 and DYS19 were
successfully 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], and
Indian (10/4257, 0.230%) [23], (1/100, 1%) [9], (5/270, 1.852%) [24]
populations is notably higher than that observed in some
Caucasian 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 or
AMELY-DYS458 deletion pattern in the Belarusian population
cannot be directly compared to that of above-mentioned Caucasian
population groups on the several reasons: (1) there are no data of
DYS458 marker amplification because this Y-STR marker was not
used in typing of the samples [21,25–27]; (2) different amount of
samples analyzed in the studies.
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

6. Paternity testing samples S2.1 and S2.2, S4.1 and S4.2 were
collected from related men (father–son sample pairs). It means
that the deletion AMELY-DYS458 identified in these samples is not
a de novo mutation, but was transmitted from the father to
the son. Therefore, the deletion in the short arm of the
Y-chromosome Yp11.2, containing AMELY and DYS458 loci, did
not affect fertility as paternity was proven in both cases S2
and S4. Similar observations have been done by several authors
[2,7,8,11,12,27,28]. Deletions in the long arm of the Y-chromosome
are associated with azoospermia and can cause reproduction
failure 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 identify
three 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 S7
exhibited a heterozygous profile with two alleles at 7 out of 8
X-STR loci. This finding confirms the presence of two X-
chromosomes in samples S5 and S7. Although the X-STR analysis
was not done for sample S6, we combined samples S5 and S6 in
one group because of the similar genetic findings in Y-STR typing
in the samples.
Long arm loss of Y-chromosome has been detected in the
AMELY-negative samples S5 and S6. Only four Y-STR markers,
DYS393, DYS456, DYS458, and DYS19, could be amplified in these
samples. In addition, heterozygous profile at 7 out of 8 analyzed X-
STR loci has been identified in sample S5. SRY has been detected in
both S5 and S6 samples. Cases with similar genetic findings have
been described in several studies [7,31]. It has been proposed a
putative genetic mechanism explaining the discrepant pattern
observed in the above-mentioned samples: DYS19 is transferred to
the distal IR3 element (inverted repeats region) by a paracentric
inversion, followed by the translocation of the terminal segment of
Y-chromosome, including SRY, DYS393, DYS456, DYS19, and
DYS458, onto the X-chromosome [7,31].
A case with loss of most of Y-chromosome in a man with
XX male syndrome similar to S7 (current study) was described
by Ma et al. [7]. Two out of 16 Y-STR loci, DYS393 and DYS456,
were amplified in S7. Ma and coauthors proposed a genetic
model explaining mechanism underlying deletion pattern
observed in these samples: AMELY together with the Yp markers,
DYS458, DYS19, and the whole long arm of Y-chromosome
undergo deletion, while the distal Yp markers, including SRY,
DYS393, and DYS456, are translocated onto the short arm of
X-chromosome [7].
A plausible explanation for AMELY or AMELX dropout in four
samples, S8, S9, S10, S11, can be a mutation in primer-binding
region of AMELY (samples S8 and S11) or AMELX (samples S9 and
S10).
Successful amplification of all 16 Y-STR loci in the AMELY-
negative samples S8 and S11 suggested that AMELY dropout in
these samples resulted from the point mutation in the primer-
binding site of AMELY. AMELY null allele was detected in S8 with
Identifiler1
Plus Kit. Since different kit (e.g. GlobalFiler1
or
Mentype1
Argus X-8 Kits) might use different pair of primers for
AMELY allele, this sort of amelogenin abnormalities may not
appear in other PCR kits.
AMELX locus that was not detected in S9 with Identifiler1
Plus Kit was successfully identified with Mentype1
Argus X-8
Kit. In addition, sample S9 showed a hemizygous profile at all
eight X-STR loci. These data point towards the presence of
primer-binding site mutation at AMELX locus. Zehethofer
and Rolf described similar genetic findings for a pair of samples
from paternity testing group (farther-son) [14]. AMELX allele
dropouts are believed to be mainly caused by mutations in
the primer-binding region [5]. Moreover, the loss of AMELX
Table 2
Frequency 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

7. allele is more common in some populations [5]. AMELX dropouts
have been detected in Polish population (1/5534, 0.018%) [17],
males from West Africa (10/503, 2%) [18], and African–American
males (48/144,391, 0.033%) [5].
In summary, our data indicate that the combined use of
different kits such as Identifiler1
Plus or GlobalFiler1
for
autosomal 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 overcome
problems related to the wrong gender determination and to
elucidate the genetic mechanisms underlying amelogenin abnor-
malities. A new PCR multiplex GenderPlex presented by Esteve et
al. [32] gives additional possibilities for a forensic DNA expert to
avoid sex misinterpretation.
The data on the incidence of amelogenin abnormalities in the
global population are scarce. Therefore, we believe that the data
presented in current study along with planned study on the
frequency of AMELY and AMELX dropouts in Belarusian population
would 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 State
Medical Forensic Service of the Republic of Belarus.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.fsigen.2014.10.014.
References
[1] M.A. Jobling, I.C. Lo, D.J. Turner, G.R. Bowden, A.C. Lee, Y. Xue, D. Carvalho-Silva,
M.E. Hurles, S.M. Adams, Y.M. Chang, T. Kraaijenbrink, J. Henke, G. Guanti, B.
McKeown, R.A. van Oorschot, R.J. Mitchell, P. de Knijff, C. Tyler-Smith, E.J. Parkin,
Structural variation on the short arm of the human Y chromosome: recurrent
multigene deletions encompassing Amelogenin Y, Hum. Mol. Genet. 16 (2007)
307–316, http://dx.doi.org/10.1093/hmg/ddl465.
[2] D.K. Jha, J.Pd. Rijal, N.T. Chhetri, Nepalese null AMELY males and their y-haplo-
types, Sci. World 8 (2010) 97–101, 10.3126/sw.v8 i8.3858.
[3] K.M. Sullivan, A. Mannucci, C.P. Kimpton, P. Gill, A rapid and quantitative DNA sex
test: fluorescence-based PCR analysis of XY homologous gene amelogenin, Bio-
techniques 15 (1993) 636–638, 640–641.
[4] A. Mannucci, K.M. Sullivan, P.L. Ivanov, P. Gill, Forensic application of a rapid
quantitative DNA sex test by amplification of the X–Y homologous gene amplifi-
cation, Int. J. Legal Med. 106 (1994) 190–193, http://dx.doi.org/10.1007/
BF01371335.
[5] J. Butler, Advanced Topics in Forensic DNA Typing: Methodology, Acad. Press,
2011, pp. 130–132.
[6] A.M. Cadenas, M. Regueiro, T. Gayden, N. Singh, L.A. Zhivotovsky, P.A. Underhill,
R.J. Herrera, Male amelogenin dropouts: phylogenetic context, origins and impli-
cations, Forensic Sci. Int. 166 (2007) 155–163, http://dx.doi.org/10.1016/j.
forsciint.2006.05.002.
[7] Y. Ma, J.Z. Kuang, J. Zhang, G.M. Wang, Y.J. Wang, W.M. Jin, Y.P. Hou, Y chromo-
some interstitial deletion induced Y-STR allele dropout in AMELY-negative indi-
viduals, Int. J. Legal Med. 126 (2012) 713–724, http://dx.doi.org/10.1007/s00414-
012-0720-8.
[8] W. Chen, W. Wu, J. Cheng, Y. Zhang, Y. Chen, H. Sun, Detection of the deletion
on Yp11.2 in a Chinese population, Forensic Sci. Int. Genet. 8 (2014) 73–79, http://
dx.doi.org/10.1016/j.fsigen.2013.07.003.
[9] Y.M. Chang, R. Perumal, P.Y. Keat, R.Y.Y. Yong, D.L. Kuehn, L. Burgoyne, A distinct
Y-STR haplotype for amelogenin negative males characterised by a large Yp11.2
(DYS458-MSY1-AMEL-Y) deletion, Forensic Sci. Int. 166 (2007) 115–120, http://
dx.doi.org/10.1016/j.forsciint.2006.04.013.
[10] X. Ou, W. Chen, H. Chen, F. Zhao, J. Zheng, D. Tong, Y. Chen, A. Chen, H. Sun, Null
alleles of the X and Y chromosomal amelogenin gene in a Chinese population, Int.
J. Legal Med. 126 (2012) 513–518, http://dx.doi.org/10.1007/s00414-011-0594-1.
[11] R. Kumagai, Y. Sasaki, T. Tokuta, H. Biwasaka, Y. Aoki, DNA analysis of family
members with deletion in Yp11.2 region containing amelogenin locus, Leg. Med.
10 (2008) 39–42, http://dx.doi.org/10.1016/j.legalmed.2007.05.009.
[12] S. Turrina, G. Filippini, D. De Leo, Evaluation of deleted region from Yp11.2 of two
amelogenin negative related males, Forensic Sci. Int. Genet. Suppl. Ser. 2 (2009)
240–241, http://dx.doi.org/10.1016/j.fsigss.2009.08.157.
[13] A. Sharp, K. Kusz, J. Jaruzelska, W. Tapper, M. Szarras-Czapnik, J. Wolski, P. Jacobs,
Variability of sexual phenotype in 46,XX(SRY+) patients: the influence of spread-
ing X inactivation versus position effects, J. Med. Genet. 42 (2005) 420–427,
http://dx.doi.org/10.1136/jmg.2004.022053.
[14] K. Zehethofer, B. Rolf, A molecular analysis of three amelogenin negative males
in two routine paternity tests, Forensic Sci. Int. Genet. 5 (2011) 550–551, http://
dx.doi.org/10.1016/j.fsigen.2010.04.006.
[15] A. de la Chapelle, H. Hortling, M. Niemi, J. Wennstroem, XX sex chromosomes in a
human male. First case, Acta Med. Scand. 175 (1964) 25–28.
[16] E. Vorona, M. Zitzmann, J. Gromoll, A.N. Schu¨ring, E. Nieschlag, Clinical, endo-
crinological, and epigenetic features of the 46 XX male syndrome, compared with
47,XXY Klinefelter patients, J. Clin. Endocrinol. Metab. 92 (2007) 3458–3465,
http://dx.doi.org/10.1210/jc.2007-0447.
[17] A. Maciejewska, R. Pawłowski, A rare mutation in the primer binding region of the
Amelogenin X homologue gene, Forensic Sci. Int. Genet. 3 (2009) 265–267, http://
dx.doi.org/10.1016/j.fsigen.2009.01.010.
[18] C. Alves, M. Coelho, J. Rocha, A. Amorim, The Amelogenin locus displays a high
frequency of X homologue failures in Sa˜o Tome´ Island (West Africa), Int. Congr.
Ser. 1 (2006) 271–273, http://dx.doi.org/10.1016/j.ics.2005.10.036.
[19] J.G. Shewale, S.L. Richey, S.K. Sinha, Anomalous Amplification of the Amelogenin
Locus Typed by AmpFLSTR1
Profiler PlusTM
Amplification Kit, vol. 2, FSC, 2000
Available at http://www.fbi.gov/about-us/lab/forensic-science-communications/
fsc/oct2000/index.htm/shewale.htm.
[20] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual,
second ed., Cold Spring Harbor Laboratory Press, New York, 1989.
[21] M. Steinlechner, B. Berger, H. Niederstatter, W. Parson, Rare failures in the
amelogenin sex test, Int. J. Legal Med. 116 (2002) 117–120, http://dx.doi.org/
10.1007/s00414-001-0264-9.
[22] F.R. Santos, A. Pandya, C. Tyler-Smith, Reliability of DNA-based sex tests, Nat.
Genet. 18 (1998) 103, http://dx.doi.org/10.1038/ng0298-103.
[23] V.K. Kashyap, S. Sahoo, T. Sitalaximi, R. Trivedi, Deletions in the Y-derived
amelogenin gene fragment in the Indian population, BMC Med. Genet. 7
(2006) 37–43, http://dx.doi.org/10.1186/1471-2350-7-37.
[24] K. Thangaraj, A.G. Reddy, L. Singh, Is the amelogenin gene reliable for gender
identification in forensic casework and prenatal diagnosis? Int. J. Legal Med. 116
(2002) 121–123, http://dx.doi.org/10.1007/s00414-001-0262-y.
[25] A. Michael, P. Brauner, Erroneous gender identification by the amelogenin sex
test, J. Forensic Sci. 49 (2004) 258–259.
[26] E. Bosch, A.C. Lee, F. Calafell, E. Arroyo, P. Henneman, P. de Knijff, M.A. Jobling,
High resolution Y chromosome typing: 19 STRs amplified in three multiplex
reactions, Forensic Sci. Int. 125 (2002) 42–51, http://dx.doi.org/10.1016/S0379-
0738(01)00627-2.
[27] W. Lattanzi, M. Di Giacomo, G.M. Lenato, G. Chimienti, G. Voglino, A large
interstitial deletion encompassing the amelogenin gene on the short arm of
the Y chromosome, Hum. Genet. 116 (2005) 395–401, http://dx.doi.org/
10.1007/s00439-004-1238-z.
[28] I. Ferreira, Sequence Variation of the Amelogenin Gene on the Y-chromosome,
Thesis submitted for the degree Philosophiae Doctor (Ph.D.) in Biochemistry
at the North-West University (Potchefstroom Campus), November 2010,
http://hdl.handle.net/10394/4412.
[29] P. Yen, The fragility of fertility, Nat. Genet. 29 (2001) 243–244, http://dx.doi.org/
10.1038/ng1101-243.
[30] D.J. Ballard, C. Phillips, G. Wright, C.R. Thacker, C. Robson, A.P. Revoir, D. Syn-
dercombe Court, A study of mutation rates and the characterisation of interme-
diate, null and duplicated alleles for 13 Y chromosome STRs, Forensic Sci. Int.
155 (2005) 65–70, http://dx.doi.org/10.1016/j.forsciint.2004.12.012.
[31] P. Balaresque, E.J. Parkin, L. Roewer, D.R. Carvalho-Silva, R.J. Mitchell, R.A. van
Oorschot, J. Henke, M. Stoneking, I. Nasidze, J. Wetton, P. de Knijff, C. Tyler-Smith,
M.A. Jobling, Genomic complexity of the Y-STR DYS19: inversions, deletions
and founder lineagescarrying duplications, Int. J. Leg. Med. 123 (2009) 15–23,
http://dx.doi.org/10.1007/s00414-008-0253-3.
[32] C.A. Esteve, H. Niederstatter, W. Parson, ‘‘GenderPlex’’ a PCR multiplex for reliable
gender determination of degraded human DNA samples and complex gender
constellations, Int. J. Legal Med. 123 (2009) 459–464, http://dx.doi.org/10.1007/
s00414-008-0301.
S. Borovko et al. / Forensic Science International: Genetics 15 (2015) 98–104104