A New Method for Sex Determination Based on Detection of

A New Method for Sex Determination Based on Detection of SRY, STS and Amelogenin Gene Regions with Simultaneous

Amplification of Their Homologous Sequences by a Multiplex PCR

Toshio Morikawaa,b, Yuji Yamamotoa＊, and Satoru Miyaishia

aDepartment of Legal Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan, and

bForensic Science Laboratory, Hiroshima Prefectural Police Headquaters, Hiroshima 730-0825, Japan

We have developed a new method for sex determination based on simultaneous detection of the SRY (sex-determining region Y), STS (steroid sulfatase) and amelogenin (AMELX and AMELY) gene regions and their homologous sequences. The sex of 246 blood samples was correctly determined by this method. An AMELY-deleted male sample, which would have been erroneously considered female based solely on analysis of the amelogenin locus, was successfully identified as male by the present method. The detection limit of this method was 63 pg of genomic DNA, and the male DNA component could be detected from mixed samples having a male : female ratio as low as 1 : 10. This method was useful for degraded DNA and possessed the human specificity. Practical application to 35 autopsy cases is described.

Key words: sex determination, SRY (sex-determining region Y), multiplex PCR, forensic casework

ex determination from the biological specimens gathered from crime scenes plays an important

role in criminal investigations [1]. A molecular bio-logical method for sex determination based on poly-merase chain reaction (PCR) has been applied to forensic cases; in this method, the region of DYZ1 repeats or the centromeric alphoid repeats in DYZ3 on the Y chromosome must be amplified separately from the region of DXZ1 repeats on the X chromo-some in a singleplex manner [2-5].　 Methods for sex determination using a singleplex PCR for a sequence in the SRY (sex-determining

region Y) gene on the Y chromosome (Yp) have also been reported [6-9]. These methods can indicate a male genotype by the presence of the amplified product from the SRY gene, but can not accurately indicate a female genotype. One of the reasons for this limitation is that the amplicon may be absent due to lack/degen-eration of DNA in the examined specimen. The other reason is that a technical error during the examination process might cause a false negative result.　 Recently, various PCR kits for short tandem repeats (STRs) have been marketed, and the sex test included in these kits has become a standardized method. In the test, homologous sequences in amelo-genin genes on the X and Y chromosomes (AMELX and AMELY) are simultaneously amplified [10], and the sample of female origin can be discriminated from

S

Acta Med. Okayama, 2011Vol. 65, No. 2, pp. 113ﾝ122CopyrightⒸ 2011 by Okayama University Medical School.

Original Article http ://escholarship.lib.okayama-u.ac.jp/amo/

Received November 19, 2010 ; accepted December 9, 2010.＊Corresponding author. Phone : ＋81ﾝ86ﾝ235ﾝ7198; Fax : ＋81ﾝ86ﾝ235ﾝ7201E-mail : [email protected] (Y. Yamamoto)

a false negative by the single peak derived from the X chromosome on the electropherogram [11]. However, misidentification of a male as female may occur when the male subject has an interstitial deletion in the Yp involving AMELY, which has been found in some populations [6-8, 12-20].　 In this study, we developed a new method for sex determination using a multiplex PCR. A major advan-tage of our method is its ability to detect both upstream and downstream sequences of the SRY gene simultaneously, which assures a remarkable consis-tency between the test result and the genotypic sex. Another advantage is that the presence of the X and 7th chromosome-specific fragments acts as an internal positive control, which by itself can assure successful amplification and positively indicate a female sample. There has been no previous attempt to simultaneously amplify the SRY region and the internal positive control sequence.　 Finally, experiments were performed to evaluate the accuracy, sensitivity, species specificity, and applicability of the new sex determination method to male/female mixed samples, degraded DNA samples or to specimens in forensic casework.

Materials and Methods

　 Principles of sex determination and primer design. Two target sequences that could be used to identify the male origin of samples were defined in both the downstream and upstream regions of the SRY gene, and primer sets to amplify these regions (named SRY-a and SRY-b, respectively) were designed. There are sequences homologous to these target

sequences in the X or 7th chromosome, respectively (the homology of each region was 71.1ｵ and 58.5ｵ), and products with different lengths can be simultane-ously amplified by PCR using each primer set. Such products can act as internal positive controls. A primer set for another target sequence, an alternative male marker, was designed to amplify a sequence in the first intron of the STS (steroid sulfatase) gene on the X chromosome (STS-1) and its homologous sequence on the Y chromosome (the homology of the region was 84.5ｵ).　 The sequences of the three primer sets described above were determined by using a BLAT search (http://www. genome. ucsc. edu/cgi-bin/hgBlat, accessed April, 2006) to amplify products with different sizes, and these primer sets were used in the present multi-plex PCR together with that for the amplification of the amelogenin locus described by Sullivan et al. [11]. To aid non-template adenylation in the PCR process, a 5ʼ tail consisting of the 7-base sequence GTTTCTT [21] was added to the forward primers for the STS-1 and amelogenin (Table 1).　 Sample preparation. Human control DNAs of both sexes (male: 9948 Male DNA; female: 9947A DNA) were purchased from Promega Corporation (Madison, WI, USA). A DNA sample obtained from a male subject with the interstitial deletion of Yp involving AMELY (CM1) [19] was also used. Peripheral blood samples were collected from unre-lated Japanese males (n＝204) and females (n＝42) after receipt of informed consent. The concentration of these samples was adjusted to 0.1ng/µl (0.5ng/tube).　 DNA samples extracted from blood or muscle of

114 Acta Med. Okayama　Vol. 65, No. 2Morikawa et al.

Table 1　 Primer sequences, dye labelling, concentrations and PCR product sizes of SRY, STS and amelogenin loci

Gene Primername 5ʼ-3ʼ Sequence [Dye] Final

conc.Product size(Chromosome)

SRY

SRY-a F AGCCCCTGTGGAAAAAATTAGTTTT 0.80μM 77bp 83bpSRY-a R AAAACACCATAGTTTTATATGGAGTAAAGGAAC [NED] 0.50μM (Y) (X)

SRY-b F TTGTGCAGCCATCACCTCT 0.04μM 130bp 123bpSRY-b R AAATCAGATTAATGGTTGCT [NED] 0.04μM (Y) (7)

STS STS-1 F GTTTCTTCAGGCACATTTTGGAGCTAT 0.05μM 166bp 158bpSTS-1 R GGTTAGTTCAGGTGTAGACC [NED] 0.05μM (Y) (X)

Amelo-genin

AMEL F GTTTCTTCCCTGGGCTCTGTAAAGAATAGTG 0.04μM 120bp 114bpAMEL R ATCAGAGCTTAAACTGGGAAGCTG [6-FAM] 0.04μM (Y) (X)

male nonhuman primates (chimpanzee, gorilla and Japanese macaque), non-primate mammals (cat, dog, raccoon dog, cow, sheep, goat, pig, wild boar, horse, rabbit, guinea pig, mouse, rat, hamster, weasel and whale), birds (cock, pigeon and crane), a reptile (crocodile), an amphibian (frog), a fish (tuna) and bacteria (Escherichia coli and Bacillus subtilis) were subjected to PCR amplification. The concentration of these samples was adjusted to 0.4ng/µl (2.0ng/tube).　 Male/female mixed DNA samples were prepared using human control DNA with male : female ratios of 30 : 1, 20 : 1, 5 : 1, 1 : 1, 1 : 5, 1 : 10, 1 : 20 or 1 : 30. In each mixture the total amount of DNA was adjusted at 0.5ng per reaction. To create samples of degraded DNA, a cotton cloth was stained with 5µl of peripheral blood from each of 2 males and 2 females and heated in an oven at 150℃ for 0.5, 1, 1.5, 2, 2.5, 3, 6, 8, 10, 15 or 20h. To create samples like those used in forensic casework, blood, skeletal muscle, kidney, costal cartilage, tooth (molar) and bone (femur) samples were collected from 35 cadavers at autopsy. The DNA concentration from heated bloodstains or autopsy materials was adjusted to 0.1ng/µl. When the total amount of DNA extracted was under 0.5ng, all of the extracted DNA was used for the PCR.　 An EZ1 DNA Investigator Kit (Qiagen, Hilden, Germany) was used to extract DNA from bloodstains and casework samples. The bacterial DNA samples were extracted using a QIAamp DNA Mini Kit (Qiagen). The phenol-chloroform method was also used for peripheral blood and animal samples. The DNA was quantified using a Human Genome DNA Quantification Kit (Takara Bio, Otsu, Japan) on a Smart Cycler II (Cepheid, Sunnyvale, CA, USA).　 The use of human materials in this study was approved by the Ethics Committee on Genetic Studies of the Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences.　 Condition of PCR and detection of the ampli-fied products. The PCR mixture contained 2 units of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA), 1×PCR Gold Buffer, 2.0mM MgCl2, 200µM of each dNTP, 3µg BSA, each primer set in the appropriate concentra-tion as shown in Table 1, and 5µl of DNA template in a final volume of 15µl. The multiplex amplification conditions were optimized in a GeneAmp PCR System

9700 Thermal Cycler (Applied Biosystems) and con-sisted of enzyme activation at 95℃ for 10min, fol-lowed by 34 cycles of denaturation at 94℃ for 30sec, annealing at 54℃ for 1min, and extension at 72℃ for 1min. A final extension was performed at 60℃ for 60min.　 After PCR, a 1µl aliquot of the product was added to 9µl of Hi-Di formamide (Applied Biosystems) con-taining 0.3µl of size standard solution (GeneScan 600 LIZ; Applied Biosystems) and used as the sample for the capillary electrophoresis using an Applied Biosys-tems 3130xl Genetic Analyzer (Applied Biosystems). The sample was injected at 3kV for 10sec and sepa-rated at 15kV for 28min in the POP 4 polymer (Applied Biosystems). The electropherogram was analyzed using Gene Mapper ID software ver. 3.2 (Applied Biosystems) with a peak height threshold of 50 relative fluorescence units (RFU).　 Sequence analysis. PCR products amplified with unlabeled primers were subjected to DNA sequencing on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems) using a DYEnamic ET Terminator Cycle Sequencing Kit (GE Healthcare, Little Chalfont, UK) or a BigDye Terminators ver. 1.1 Cycle Sequencing Kit (Applied Biosystems).

Results

　 Amplification of human control DNA and DNA from peripheral blood samples. The electropherograms for the human control DNA of both sexes amplified by the present multiplex PCR are shown in Fig. 1. When the male DNA was amplified, 8 peaks (4 pairs) were observed. Those derived from 4 Y chromosome-specific sequences and their homolo-gous sequences on the X or 7th chromosome served as internal positive controls. Sequence analysis revealed that all fragments were concordant with the reported sequences. In the analysis of the female DNA, only the 4 peaks serving as internal positive controls were observed. When we attempted to determine the sex of 246 blood samples donated from sex-known individuals (204 males and 42 females) by the present method, all of them were correctly determined.　 Amplification of DNA obtained from an AMELY-deleted male. In the PCR using the DNA sample obtained from an AMELY-deleted male (CM1), 7 fragments were detected. Lacking the

115A New Method for Sex DeterminationApril 2011

Y-specific fragment, only the X-specific fragment was detected in the amelogenin locus. In contrast, the Y-specific male marker fragments of the SRY-a, SRY-b and STS-1 loci were detected with the internal positive control fragments (Fig. 2).　 Species specificity. Electropherograms obtained by PCR using the DNA of male primates, including a human, are shown in Fig. 3. From the amelogenin locus, 2 fragments were amplified in all primates as well as in the human sample. The frag-ments derived from the other three loci (SRY-a, SRY-b and STS-1) showed distinct differences in both size and number between the human and the other primates. There was no amplified fragment that was found to be common to one or more loci of all investi-gated mammals. From the DNA samples of the birds, a reptile, an amphibian, a fish and bacteria, nothing was amplified.　 Sensitivity. All of the expected products could be amplified using 63 pg or more of human control DNA in both males and females. Off-scale signals (i.e.,

fluorescent signals exceeding the dynamic range) caused by using more than 2 ng DNA did not affect the interpretation of the electropherogram. Amplification from less than 31 pg DNA resulted in fragment drop-outs (Fig. 4).　 Amplification of male/female mixed sam-ples. When the mixtures of male and female con-trol DNA in various ratios were examined, the peak height changed in accordance with the male/female ratio, as shown in Fig. 5. All 8 of the expected frag-ments were amplified when the ratio ranged from 1 : 0 to 1 : 10. The samples with a male: female ratio of 1 : 20 or lower resulted in partial dropouts of the male component.　 Amplification of degraded DNA. When the degraded DNA prepared by heating the bloodstains was amplified by the present multiplex PCR, all frag-ments were detected and the sex could be correctly determined from all of the bloodstains heated for up to 10h, but not from those heated for longer than 15h, as shown in Fig. 6.


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Fig. 1　 Electropherograms obtained from human control DNA. Male and female control DNA samples (0.5ng each) and a negative control sample (sterile water) were amplified. The male sample represents 8 fragments and the female sample represents 4 fragments. No peaks were observed in the negative control. The target region is shown above the peak. The X-axis indicates the length of the DNA frag-ment. The Y-axis indicates relative fluorescence units (RFU).


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Fig. 2　 Electropherograms obtained from an AMELY-deleted male sample (CM1). In the amelogenin locus, only one fragment derived from AMELX was observed. The boxes over the panel show the target region. Target fragments are expected to appear in shaded areas in the electropherograms. The X-axis indicates the length of the DNA fragment. The Y-axis indicates relative fluorescence units (RFU).

Japanese Macaque

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Fig. 3　 Electropherograms obtained from male primates. The boxes over the panel show the target region. Target fragments are expected to appear in shaded areas in the electropherograms. The X-axis indicates the length of the DNA fragment. The Y-axis indicates relative fluorescence units (RFU).

　 Forensic casework samples. When we applied our method to 35 forensic cases in which DNA degradation was expected, the determined sex was concordant with the morphological features or circum-stantial evidences in all cases (Table 2). In Case 35, human remains were found under the sea in a harbor. The postmortem interval was estimated to be about 5 years and the soft tissues were completely lost. As shown in Fig. 7, eight peaks were detected without a nonspecific product using the DNA extracted from a femur and the victim was determined to be male.

Discussion

　 In recent years, sex determination using the amelogenin locus has become a routine method in forensic casework. However, it has been pointed out that sex determination based on this locus may mis-judge male samples as female samples [6]. In this study, we have developed a new multiplex PCR

method for sex determination amplifying the SRY, STS and amelogenin sequences simultaneously with the internal positive controls, and validated its useful-ness for forensic casework.　 Using our new multiplex PCR method, all 8 and 4 expected fragments respectively, were detected from male and female control DNAs. In the examination of 246 blood samples by our method, there was no con-tradiction between the results and the known sex of the donors. When the AMELY-deleted male sample was analyzed, only an X chromosome-specific fragment was amplified from the amelogenin locus and, based on solely this locus, the sample would have been consid-ered female. However, considering the results obtained from additional markers, i.e., SRY-a, SRY-b and STS-1, the sample was determined to be male. This suggests that our method is applicable even to specimens from AMELY-deleted males. Moreover, samples of female origin can be accurately determined by this method, since the additional three markers


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Fig. 4　 Sensitivity of the present multiplex PCR for sex determination using serially diluted control DNA. The target region is shown above the peak. The X-axis indicates the length of the DNA fragment. The Y-axis indicates relative fluorescence units (RFU).

include internal positive controls.　 Next, we examined the species specificity of our method using 27 nonhuman samples. This is important because, in practice, it is sometimes unknown whether the specimen to be analyzed is human. The results showed that all of the nonhuman samples could be readily discriminated from human samples, and thus this method is thought to be useful for forensic case-work.　 Any new method to be applied to modern forensic

cases must achieve a detection limit of subnanogram quantities of DNA, because many samples contain such minute quantities. In our method, the sex could be correctly determined using 63 pg of template DNA, a quantity equivalent to approximately 10 cells. In the examination using male/female mixed samples, a decrease of peak height in the male components was observed concomitant with the reduction of male DNA. However, all male components could be identified from the mixture having a male : female ratio of 1 : 10, and


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Fig. 5　 Sex determination from male/female mixed samples by the present methods. The target region is shown above the peak. The X-axis indicates the length of the DNA fragment. The Y-axis indicates relative fluorescence units (RFU).


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Fig. 7　 Sex determination from human remains undersea for 5 years (Case 35). The DNA extracted from a femur was examined. The sample was determined to be male. The boxes over the panel show the target region. Target fragments are expected to appear in shaded areas in the electropherograms. The X-axis indicates the length of the DNA fragment. The Y-axis indicates relative fluorescence units (RFU).

thus our method was thought to be applicable to the DNA samples in cases of sexual assault. Sex determi-nation from samples containing only degraded DNA is also required in forensic casework. In our investiga-tion using heated bloodstains as a model of DNA deg-radation, the sex was correctly determined from the sample heated at 150℃ for 10h. This finding also indicates that our method would be satisfactory for practical application, since, in the routinely used ABO genotyping method, the typing has been reported to be uncertain for samples heated at 150℃ longer

than 2h [22].　 In 35 forensic cases, we successfully determined the sex from all of the specimens of blood, muscle, organ, cartilage, tooth and bone, in which DNA degradation might have occurred due to environmental insults. In the Case 35, DNA extracted from a femur bone that had been undersea for 5 years was subjected to our method and the sex was determined to be male, a result concordant with the morphological findings and police investigation. This and the other cases demon-strated that our multiplex PCR method was useful for


Table 2　 Sex determination of various forensic casework samples

Case Specimen Postmortem environment

Postmortem interval

Sex determination

Present methodAutopsy findings

and policeinvestigations

1 Muscle House 2 days Male Male 2 Blood House 2-3 days Male Male 3 Blood Sewage pond 2-3 days Female Female 4 Muscle Drain 2-3 days Male Male 5 Blood Pond 3-5 days Female Female 6 Blood House 4-5 days Male Male 7 Blood River 1 week Male Male 8 Blood Car 1 week Male Male 9 Muscle Well 1 week Female Female10 Kidney Sea 1 week Male Male11 Kidney Sea 1 week Male Male12 Kidney Sea 1 week Female Female13 Cartilage House 1 week Male Male14 Blood Yard 1-2 weeks Male Male15 Muscle Sea 1-2 weeks Male Male16 Kidney House 1-2 weeks Male Male17 Kidney House 2 weeks Female Female18 Muscle House 2-3 weeks Male Male19 Kidney House 2-3 weeks Male Male20 Muscle Yard 3 weeks Male Male21 Kidney Sea 2-4 weeks Male Male22 Muscle House 3-4 weeks Female Female23 Kidney House 3-4 weeks Female Female24 Blood River 1-2 months Female Female25 Muscle House 2-3 months Male Male26 Cartilage Sea 2-3 months Male Male27 Kidney House 3-6 months Male Male28 Cartilage Car 3-6 months Male Male29 Cartilage Mountain 9 months Male Male30 Cartilage House 9 months Male Male31 Cartilage Mountain 0.5-1 year Male Male32 Tooth House 1 year Male Male33 Tooth Drain 1 year Male Male34 Tooth Mountain 2.5 years Male Male35 Femur Sea 5 years Male Male

forensic casework.　 Conclusion. The present method of multiplex PCR targeting SRY, STS, and amelogenin gene regions was proven to be an accurate, reliable and robust means of determining the sex of origin of vari-ous forensic biological samples. A major advantage of our method is its applicability to samples from AMELY-deleted males. Another advantage is that this method includes internal positive controls deriving from the X and 7th chromosomes, which assure the accurate determination of female origin.

Acknowledgments.　The authors wish to thank Dr. Mark Jobling, University of Leicester, for providing the AMELY-deleted male sample (CM1). This work was partially supported by a Grant-in-Aid for Scientific Research (No.20932018) from the Japan Society for the Promotion of Science.

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