Detection of the deletion on Yp11.2 in a Chinese population

Forensic Science International: Genetics 8 (2014) 73–79

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Detection of the deletion on Yp11.2 in a Chinese population Wenjing Chen a,1, Weiwei Wu b,1, Jianding Cheng a, Yinming Zhang a, Yong Chen a, Hongyu Sun a,* a b

Department of Forensic Medicine, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510089, PR China Department of Criminal Investigation of Zhejiang Provincial Public Security Bureau, Hangzhou 310009, PR China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 9 March 2013 Received in revised form 27 June 2013 Accepted 4 July 2013

Sex determination tests based on Amelogenin gene as part of commercial PCR multiplex reaction kits have been widely applied in forensic DNA analysis. Mutations that cause dropout of Y chromosomal Amelogenin gene (AMELY) could lead to errors in gender determination and mixture interpretation. To infer the mechanism and estimate the dropout frequency of AMELY and adjacent Y-STRs, we studied 3 samples with AMELY dropout combined with DYS458 and/or DYS456 and 37 samples with DYS456 dropout. DYS456, DYS458 and AMELY are located in the Yp11.2 region. The singleplex amplification system showed the null alleles could be caused by fragment deletion in Yp11.2 rather than a point mutation in the primer binding region. After detection of the 17 Y-STR and 77 STS markers, the deletion map showed different patterns. The DYS456-AMELY-DYS458 deletion pattern was the largest, breaking from 3.60 Mb to 8.29 Mb in the Y chromosome, and the overall frequency was 0.0077%. The AMELYDYS458 deletion pattern was broke from 6.74 Mb to 9.17 Mb, with a 0.0155% frequency. The DYS456 negative pattern was concentrated in two main deletion regions, with a 0.8220% frequency. The frequency of all negative pattern was 0.0155%. All the AMELY-DYS458 and DYS456-AMELY-DYS458, and 92% of the DYS456 deletion patterns belonged to Hg O3, the rest belonged to Hg Q. The DYS456 deletion pattern was first reported in Chinese population. The current and previous findings suggest additional gender test for ambiguous sex determination may be required. ß 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: AMELY Y-STR Deletion Yp11.2 Chinese population

1. Introduction Male-specific region of the human Y chromosome (MSY), also called non-recombining region of human Y chromosome (NRY), accounts for 95% of the Y chromosome which does not recombine with the X chromosome during meiosis [1–3]. Due to the unique characteristics of the Y chromosome, Y-chromosome specific short tandem repeat loci (Y-STR) and the Amelogenin genes (AMELX and AMELY) have been widely used in paternity testing, personal identification, prenatal testing, preimplantation genetic diagnosis, and deoxyribonucleic acid (DNA) databasing [3–10]. The Y chromosome bears a particularly large proportion of submicroscopic structural rearrangement, including deletions, insertions, duplications, large-scale copy number variants and inversions [3,7,9]. Abnormal phenomena have been identified during routine AMELY and/or Y-STR genotyping casework. AMELY dropouts will cause the false genotyping of male samples as female. Base variation in the primer binding region is one of the

* Corresponding author at: No. 74 Zhongshan Road II, Guangzhou 510089, PR China. Tel.: +86 20 87330557; fax: +86 20 87334353. E-mail addresses: [email protected], [email protected] (H. Sun). 1 Wenjing Chen and Weiwei Wu contributed equally to the article. 1872-4973/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsigen.2013.07.003

causes for the dropout of the AMELX and AMELY, but the AMELY dropout is predominantly due to fragment deletion on Yp11.2 region, and is often combined with the absence of adjacent Y-STR locus DYS458 [1,3,4,11–14]. In our study, one case showed the DYS456-AMELY-DYS458 deletion pattern and 37 cases showed the DYS456 deletion pattern. DYS456 is 2.4 Mb upper than the AMELY loci and DYS458 is 1.1 Mb lower than the AMELY loci on the Yp11.2 region [4,12], a possible hotspot of Y chromosome deletion [4,11,12]. Yp11.2 deletion mapping data has been reported in different populations [5,14–17]. Although cases with AMELY and YSTR nulls have been reported in the Chinese population, no systematic analysis of the deletion region has been performed [3,18]. The present study described the four deletion patterns and determined the deletion map in the Yp11.2 region that causes the absence of AMELY, DYS458 and DYS456, some of which has been reported in a previously published article [14]. 2. Materials and methods 2.1. Samples Buccal swab samples 1 to 38, obtained from DNA database of 4501 unrelated Chinese Han males in Zhejiang provinces [19],

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indicated null-alleles in DYS456 or DYS458 when genotyping using an AmpF/STR1 Yfiler Kit (Applied Biosystems, USA), in which the samples 1 to 37 showed null alleles at the locus DYS456 and the sample 38 showed null alleles at the locus DYS458. Samples 39–42 were collected from 8414 unrelated phenotypically normal males in Guangdong province with informed consents, indicated nullallele for AMELY when genotyping using Powerplex1 16 system (Promega Corporation, Madison, USA). Genomic DNA from 42 blood stain samples was extracted by the Chelex-100 method.

STS primers and PCR conditions were obtained from the National Institute of Health website (http://www.ncbi.nlm.nih.gov, as accessed on 15/06/2012). Special care was taken to ensure the STS markers’ effectiveness. Only the markers with a single copy, or the PCR products that were wide enough to be separated from the homologous X chromosome, were used to show the deletion map of the Yp11.2 region.

2.2. Genotyping of AMELY nulls with 17 Y-STR markers

3.1. Deletion patterns

Four samples (39, 40, 41 and 42) with AMELY allele dropout were genotyped using the AmpF/STR1 Yfiler system.

After using the alternative single primer sets, all samples continued to show the same results as before, which suggests a fragment deletion of the Y chromosome rather than a base mutation within the annealing region of the primers. This is consistent with previous studies [1,4,11,12,14,24–28]. According to Y-STR and STS genotyping, four patterns of deletion were detected. These were the DYS456 deletion pattern for samples 1–37, the AMELY-DYS458 deletion pattern for samples 38 and 39, and the DYS456-AMELY-DYS458 deletion pattern for sample 40. Samples 41 and 42 were negative for all 17 Y-STR markers and AMELY (Fig. 1), therefore no further investigation was performed.

2.3. Genotyping with singleplex amplification systems for AMELY, DYS456 and DYS458 To verify the reason for allele dropout, tests using a singleplex amplification system were carried out using primer sequences from published data, binding to alternative sites (Table 1) [14,20– 23]. Polymerase chain reaction (PCR) amplification was carried out with 12.5 mL 2  GoTaq1 Green Master Mix (Promega, USA), 1 mL of each of the primers (10 mmol/L) and 5–7 ng DNA template. Nuclease-free water was added to a total volume of 25 mL. The thermo cycling conditions were: 95 8C for 2 min, 32 cycles at 94 8C for 1 min, 60 8C for 1 min, and 72 8C for 1 min, with a final extension at 72 8C for 15 min. PCR products were separated by non-denaturing polyacrylamide gel electrophoresis (T = 6%, C = 3.3%) and subsequently visualized with silver staining.

3. Results

3.2. Hg prediction based on Y-STRs haplotypes Twenty-five different Haplotypes were identified in 40 samples (1–40). Two Hg, O3 and Q, were predicted based on Y-STRs haplotypes (Table 3). Samples 7, 12 and 36 of the DYS456 deletion pattern belonged to Hg Q. All other samples belonged to Hg O3.

2.4. Y chromosome haplogroup prediction

3.3. Deletion map of DYS456 deletion pattern

Based on Y-STR genotyping, the haplogroup (Hg) of Y chromosome was predicted using the Whit Athey’s Haplogroup Predictor 27-haplogroup program (http://www.hprg.com/hapest5/hapest5b/hapest5.htm, as updated on 10/12/2012), selecting ‘‘Equal Priors’’ in the Area Selection menu.

Samples 1–37 showed the DYS456 deletion pattern and exhibited similar fragment deletion around the DYS456. As shown in Fig. 1, the deletion regions were found on two main non-consecutive sites. The upper breakpoint was varied from upper marker sY3020 (No. 2 at 3.22 Mb) to lower marker DXYS112 (No. 9 at 3.90 Mb). The most notable breakpoint was sY3024 (No. 7 at 3.76 Mb), where seventeen (46%) samples were breaking from this marker, and thirty-five (95%) samples were negative at this marker. Twenty-six (70%) samples began to show positive results after DXYS6 (No. 10 at 4.05 Mb). The first main deletion region was ranged from 3.76 Mb to 4.05 Mb at the Y chromosome, where twenty-nine (78%) samples showed negative results. After thirty-two (86%) samples indicated positive results in sY221 (No. 11 at 4.08 Mb), all the samples indicated no results from sY3040 or sY3041 (No. 13 or No. 14 at 4.13 Mb), where the second main deletion region began from. And this deletion was extended to sY3026 (No. 27 at 4.87 Mb). Some positive parts appeared between the second deletion region. The most notable one was eighteen (49%) samples showed positive in sY3103 (No. 16 at 4.20 Mb), and majority (61%, 11/18) of them were prolonged to sY3104 (No. 17 at 4.21 Mb). Ignoring the interspersed positive results, the second main deletion region was from 4.13 Mb to 4.87 Mb, where thirty-five (95%) samples showed negative results. A negative result was considered more important than a positive result, as negative results could indicate either a true deletion or a variation at the markers. The longest breakpoint boundary surrounding DYS456 extended from 3.22 Mb to 4.89 Mb (sample 8), while the shortest breakpoint extended from 3.76 Mb to 4.23 Mb (sample 17). Ignoring the sporadic positive results, the lengths of the deletion region extended from 0.47 Mb to 1.67 Mb (Fig. 1).

2.5. Deletion mapping using STS markers in Yp11.2 region Seventy-seven sequence-tagged site (STS) markers, spaced at approximately 20–600 kb, were selected to detect the deletion region. These STS markers were selected from DYS393 (located in 3.13 Mb segment of the Y chromosome) to DYS19 (located in 9.52 Mb segment of the Y chromosome), containing the DYS456, AMELY and DYS458 null alleles (Table 2). Table 1 Primer pairs for AMELY, DYS456 and DYS458. Markers

Primer sequence (50 –30 )

Product size (Repeats)

AMEL

F-ACCTCATCCTGGGCACCCTGG R-AGGCTTGAGGCCAACCATCAG

AMELX: 212 bp AMELY: 218 bp

DYS456

1: F-GGACCTTGTGATAATGTAAGATA R-CCCATCAACTCAGCCCAAAAC 2: F-ACTCGGACTGGCTCATCTTG R-CCCATCAACTCAGCCCAAAAC 3: F-TCAGCCTGCAGATGGTCT R-TTTTGAACTCTTGGCCTCAA

141–161 bp (13–18)

1: F-GCAACAGGAATGAAACTCCAAT R-GTTCTGGCATTACAAGCATGAG 2: F-GGGTGGTGGAGGTTACTGTG R-CTAGAGGTTCCTGCCACCAC 3: F-GGTGGTGGAGGTTACTGTGA R-TTCCTGACCTTGTGATCCAG

132–160 bp (13–20)

DYS458

189–209 bp (13–18) 318–338 bp (13–18)

308–336 bp (13–20) 221–249 bp (13–20)

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Table 2 All the markers used and their location on the Y-chromosome. No.

Markers

Location (Mb)

No.

Markers

Location

No.

Markers

Location

* 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 * 22 23 24 25 26 27 28 29

DYS393 sY3020 sY3063 sY3066 sY1254 sY3023 DYS661 sY3024 sY3025 DXYS112 DXYS6 sY211 DYS253 sY3040 sY3041 sY3042 sY3103 sY3104 sY3105 sY3106 sY3107 sY3114 DYS456 sY3043 sY3044 sY3055 G65998 sY3019 sY3026 sY3027 sY3134

3.13 3.16 3.22 3.33 3.54 3.60 3.64 3.76 3.85 3.90 4.05 4.08 4.08 4.13 4.13 4.17 4.20 4.21 4.21 4.22 4.22 4.23 4.31 4.23 4.28 4.33 4.57 4.59 4.87 4.89 4.99

30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 * 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59

sY3056 sY1240 sY716 sY1241 TSPY DYS665 sY605 DYS666 sY74 DYS667 G66289 AMELY DYS455 sY1320 DYS463 DYS458 sY881 sY1090 DYS449 DYS656 GDB:187576 SHGC102071 DYS454 G66129 G65898 G65901 DYS688 DYS689 DYS690 G66264 DYS691

5.16 5.31 5.42 6.10 6.17 6.59 6.61 6.62 6.62 6.63 6.66 6.74 6.97 7.03 7.68 7.93 7.97 8.16 8.27 8.22 8.23 8.26 8.28 8.29 8.31 8.32 8.33 8.37 8.43 8.46 8.47

60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 * * * * * * * * * * * *

G65900 DYS693 G66104 DYS257 DYS695 G66267 G66266 G66265 DYS697 SHGC80640 sY1209 DYS702 DYS245 sY1293 sY1249 GDB187630 G66114 sY1243 DYS19 DYS391 DYS635 DYS437 DYS439 DYS389 DYS438 DYS390I/II Y GATA H4 DYS385a/b DYS392 DYS448

8.48 8.56 8.57 8.62 8.65 8.67 8.71 8.71 8.82 8.93 8.96 9.03 9.12 9.17 9.17 9.38 9.40 9.47 9.52 14.10 14.38 14.47 14.52 14.61 14.94 17.27 18.74 20.83 22.63 24.36

The markers with * are STR loci from the AmpF/STR1 Yfiler Kit, others are STS markers.

3.4. Deletion map of AMELY-DYS458 deletion pattern Samples 38 and 39 presented simultaneous deletions in both AMELY and DYS458. Two samples showed two parts of the deletion, from AMELY (No. 41 at 6.74 Mb) to sY1293 or sY1249 (No. 73 or No. 74 at 9.17 Mb), separated by a 0.26 Mb normal region (from G66267 numbered as 65 at 8.67 Mb to SHGC80640 numbered as 69 at 8.93 Mb) which showed positive amplified results (Fig. 1). Comparing the Yp11.2 deletion region among different population, the breakpoint boundaries were approximately on the pericentromeric region of the short arm of the Y-chromosome [11], around the TSPY array (located on the 6.17 Mb of the Y chromosome), which is immediately downstream of the IR3 inverted repeat. Parts of the deletion region boundary were prolonged to TSPY4 array. 3.5. Deletion map of DYS456-AMELY-DYS458 deletion pattern The deletion region of sample 40 was from sY3023 (No. 5 at 3.60 Mb) to G66129 (No. 52 at 8.29 Mb), including parts of constant positive region. The upper deletion part of this pattern was similar with DYS456 deletion pattern, while the lower part presented different deletion region with AMELY-DYS458 deletion pattern, which was only partial overlapped around DYS458. This is the longest deletion observed in Yp11.2 region. 3.6. Frequencies of different deletion patterns Thirty-seven samples from Zhejiang province showed the DYS456 deletion pattern, with a 0.822% (37/4501) frequency. Two samples showed the AMELY-DYS458 deletion pattern, including sample 38 from Zhejiang Province, with a frequency of 0.0222% (1/ 4501), and sample 39 from Guangdong province, with a 0.0119%

(1/8414) frequency, overall prevalence was 0.0155% (2/12915). As only sample 40 showed the DYS456-AMELY-DYS458 deletion, the frequency was 0.0077% (1/12915). The total prevalence of AMELYDYS568 deletion pattern and DYS456-AMELY-DYS458 deletion pattern caused by Yp11.2 deletion was 0.0232% (3/12915). The all negative pattern (samples 41 and 42) was observed twice in Guangdong, but not in Zhejiang, the prevalence was 0.0155% (2/ 12915).

4. Discussion Y-STR dropout has been reported in various populations. Null alleles of DYS456, DYS458 [29], DYS448 [30,31], DYS385 [32], DYS392, DYS389I, DYS389II, DYS439, DYS448 and Y-GATA H4 [33] have been observed, and caused by different reasons such as mutations in the primer binding sites and fragment deletion of Y chromosome. Deletions of DYS385, DYS392 and DYS460 within the region termed azoospermia factor b (AZFb) suggest an area of at least 1.8 Mb deleted from the Y chromosome which may be a possible cause of infertility in males [34]. Most of the reports showed that AMELY nulls caused by deletion of Yp11.2 were combined with a dropout of DYS458 instead of DYS456 [3,4,17,24,35]. The large deletion in DYS456-AMELYDYS458 was screened in our study and also reported in another Chinese population [17]. A deletion on Yp11.2 resulted in 37 individuals with a single DYS456 dropout should be noted as it indicates diversity between Chinese and other population groups. AMELY and DYS458 null alleles caused by Yp11.2 deletion have been reported in various populations, with a higher frequency in Sri Lankan (8.3%) [36], Nepalese (6.5%) [37] and Indian populations (0.23–3.2%) [4,24,35]. The frequency of AMELY and DYS458 null alleles seems lower in Chinese population (0–0.0155%). AMELY and DYS458 null alleles have not been detected in Chinese from Malay

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W. Chen et al. / Forensic Science International: Genetics 8 (2014) 73–79

Fig. 1. The deletion map of Yp11.2 for the samples with Amelogenin and (or) DYS456, DYS458 allelic dropouts. Approximate locations of 77 STSs (including Amelogenin) and 17 Y-STR were determined by database searching. The yellow region (+) indicates the positive result and the blue region () indicates the negative result performed by PCR. The samples were displayed in order of the deletion region and haplogroup.

Table 3 Haplogroup (Hg) prediction based on Y-STRs haplotypes. Hg

Prob. (%)

DYS456

DYS389I

DYS390

DYS389II

DYS458

DYS19

DYS385a/b

DYS393

DYS391

DYS439

DYS635

DYS392

Y GATA H4

DYS437

DYS438

DYS448

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

O3 O3 O3 O3 O3 O3 Q O3 O3 O3 O3 Q O3 O3 O3 O3 O3 O3 O3 O3 O3 O3 O3 O3 O3 O3 O3 O3 O3 O3 O3 O3 O3 O3 O3 Q O3 O3 O3 O3

88.9 91.1 96.6 98.8 68.3 83.7 97.5 59.4 59.1 57.4 87.1 70.2 58.3 78.1 78.1 78.1 78.1 78.1 78.1 78.1 99.1 72.3 72.3 72.3 72.3 72.3 72.3 72.3 72.3 72.3 72.3 83.6 70.6 86.4 64.3 73.6 79.4 99.0 99.5 96.8

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – 15 17 –

13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 12 12 14

24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 23 25

29 29 29 29 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 31 28 30 30

16 17 17 16 16 16 16 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 – – –

14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 15 14

13–19 13–19 13–19 13–19 13–19 13–19 13–19 13–18 13–18 13–18 13–19 13–19 13–19 13–19 13–19 13–19 13–19 13–19 13–19 13–19 13–19 13–19 13–19 13–19 13–19 13–19 13–19 13–19 13–19 13–19 13–19 13–19 13–19 13–19 13–20 14–20 13–19 13–14 13 13–18

14 14 15 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 15 14 14 14 12 13 12

10 10 10 10 10 10 10 11 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 11 11 10

12 12 12 12 12 12 12 12 12 12 11 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 13 14 12 12 12 12 12 12 12

21 21 21 21 21 21 22 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 20 19 22

14 15 14 14 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 14 14 13

12 12 13 13 13 13 13 12 13 12 12 12 12 12 12 12 12 12 12 12 13 13 13 13 13 13 13 13 13 13 13 12 13 12 12 13 13 12 12 11

15 16 16 15 15 16 17 15 15 16 16 14 15 16 16 16 16 16 16 16 15 15 15 15 15 15 15 15 15 15 15 16 15 16 16 15 15 15 14 15

11 11 12 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 10 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 10 10

21 20 22 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 18 20

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77

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Table 4 Summary of contemporary AMELY and DYS458 null alleles reported. Only those with evidence of Yp11.2 fragment deletion but not point mutation were included, arranged based on the frequency from high to low. Population

No. of null/no. of individuals

Freq. (%)

Proximate Deletion region (markers)

Haplogroup (null/individuals)

Ref.

Sri Lankan Nepalese Malaysian Indians Indian (general) Singaporean Indian Israel Malaysian Malays Singaporean Malays Indian (whole) Japanese

2/24 5/77 10/315 5/270 3/175 1/96 2/334 1/182 10/4 257 1/500

8.3 6.5 3.2 1.85 1.76 1.0 0.6 0.6 0.23 0.2

J2e1 J2b2-M241 J2 No data J2e1 (2/175); F* (1/175) No data J2 J2e1 J2 No data

[36] [37] [4] [35] [3] [42] [4] [3,4] [24] [12]

Chinese

3/12 891

0.0232

O3 (3/12915)

Current study

Chinese

8/79 304

0.010

Located around TSPYB (about 6.2 Mb) 8.75 Mb (sY2180)–10.1 Mb (DYS19) At least 1.13 Mb (DYS458-MSY1-AMELY) 1 Mb around AMELY 6.15 Mb (sY1241)–9.97 Mb (sY59) No data At least 1.13 Mb (DYS458-MSY1-AMELY) 6.15 Mb (sY1241)–9.97 Mb (sY59) No data 6.44 Mb (sY1242)–8.95 Mb (DYS487); 9.23 Mb (sY1293); 9.98 Mb (sY59)–10.01 Mb (AC025819U) 6.74 Mb (AMELY)–8.65 Mb (DYS695); 8.96 Mb (sY1209)–9.17 Mb (sY1293). 3.60 Mb (sY3023)–8.29 Mb (G66129) No data

J2 (5/79304); I1b1b, N, L (1/79304)

[17]

Malaysian Chinese Singaporean Chinese Austrian (Caucasian) Italy Italy Italy

0/331 0/210 5/28 182 1/13 000 No data No data

0 0 0.018 0.008 No data No data

No data 2.5 Mb (sY1242-TSPY) 4.91 Mb (G65933)–7.97 Mb (G66004) 4.71–8.06 Mb; 6.44–7.97 Mb

and Singapore, although this might be caused by the low sample size (331 and 210, respectively) [3,17]. Y-SNP typing could not be performed in current study due to the limited amount of samples. Y-STRs haplotype was performed to predict the Hg using the Whit Athey’s Haplogroup Predictor. Thirty-four (24/37, 92%) samples out of the DYS456 deletion pattern, all of the AMELY-DYS458 deletion pattern and the DYS456AMELY-DYS458 deletion pattern were assigned to Hg O3, which is a subclade of Hg O. Hg O is a Southeast Asian and East Asian lineage dating back 28,000–41,000 years ago. Three samples (7, 12 and 36) of DYS456 deletion pattern belonged to Hg Q, which is believed to have arisen in Central Asia or South Asia approximately 17,000– 22,000 years ago [38]. As shown in Table 4, the samples with AMELY nulls caused by Yp11.2 deletion showed a high frequency of Hg J2. The Hg L, Hg N, and Hg I1b1b were also present in Chinese populations according to Ma et al. [17]. The Hg R1b is more frequent among the Italian population, whereas AMELY nulls combined with DYS458 and/or DYS456 in the current study belonged to Hg O3. The similar Hg origin within a population indicated diversity [3,4,17,37]. Their different haplotypes were irrelevant to the deletion regions, suggesting that the deletions occurred independently [11]. Based on the distribution and frequency, it is unlikely that this deletion has arisen as a consequence of natural selection on Y chromosomes, but indicates a founding event in some haplotypes [4]. Majority of the deletion regions were within the TSPY proximity, consequently, the deletions may have been caused by an intrachromosomal non-homologous recombination between the TSPY array [1,3,11]. Determining the DNA sequence deletions would be potentially valuable to further investigate the actual mutational mechanism. Paternity testing samples 39 and 40 were collected from phenotypically normal fathers who had conceived their sons naturally. The abnormal genotype was passed down to the offspring which means that the deletion type did not cause reproductive failure. The infertility was presumed to have other origins [4,11,39]. The absence of Y-STR products from certain loci in the long arm of the Y-chromosome can cause infertility in male populations. AZFa, AZFb, and AZFc deletions are the azoospermia

No data R1b3 No data R1b

[4] [3] [28] [11] [27] [13]

factor related regions [17,40,41]. This evidence suggests that Yp11.2 deletion does not interfere with reproductive ability [11]. Even though the frequency of the Amelogenin deletion is low in some populations, considering its relevance to gender identification, the sex determination should not be based on the Amelogenin typing alone, as AMELY null could lead to the wrong conclusion [42]. For the single sample that showed AMELY null, it should not be assumed to be female, and the Y-STR genotyping should be performed to confirm whether there is any other allele dropout. For the mixture samples, the AMELY dropout could also mislead the results. It is necessary that Y-STR nulls should be investigated carefully in forensic interpretation to avoid false exclusion. Recently, DYS391 was commended as one of the Combined DNA Index System (CODIS) Core Loci as a complementary marker of the sex determination by the CODIS Core Loci Working Group [43]. DYS391 is located in the pericentromere of the Y chromosome and its polymorphism is relative low, so it can be more stable with low mutation rate and it is easier to be added into a multiplex system as a short amplicon [43]. All samples with Yp11.2 deletion in the current study showed normal genotypes in DYS391. To elucidate the mechanism of this deletion, further studies should focus on the diversity of the deletion map between different ethnic populations and geographical positions [3]. Acknowledgments This study was supported by the National Natural Science Foundation of China (81273347) and the Fundamental Research Funds for the Central Universities (09YKPY78).

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