No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
A distinct Y-STR haplotype for Amelogenin negative males characterized by a large Y(p)11.2 (DYS458-MSY1-AMEL-Y) deletion
Abstract
The use of STR multiplexes with the incorporated gender marker Amelogenin is common practice in forensic DNA analysis. However, when a known male sample shows a dropout of the Amelogenin Y-allele, the STR system falsely genotypes it as a female. To date, our laboratory has observed 18 such cases: 12 from our Y-STR database and six from casework. A study on 980 male individuals in the Malaysian population using the AmpFlSTR Y-filer has revealed a distinct Y-chromosome haplotype associated with the Amelogenin nulls. Our results showed that whilst the Amelogenin nulls were noticeably absent among the Chinese, both the Indians and Malays exhibited such mutations at 3.2 and 0.6%, respectively. It was also found that the Amelogenin negative individuals predominantly belonged to the J2e lineage, suggesting the possibility of a common ancestor for at least some of these chromosomes. The null frequencies showed concordance with the data published in Chang et al. [Higher failures of Amelogenin sex test in an Indian population group, J. Forensic Sci. 48 (2003) 1309-1313] on a smaller Malaysian population of 338 males which used a Y-STR triplex. In the current study, apart from the absence of the Amelogenin Y-locus, a complete absence of the DYS458 locus in all the nulls was also observed. This study together with the 2003 study has indicated a similar deletion region exists on the Y(p)11.2 band in all the 18 Y-chromosomes. Using bioinformatics, this deletion has been mapped to a region of at least 1.13 Mb on the Y(p)11.2 encompassing the Amelogenin, MSY1 minisatellite and DYS458 locus. Further, the Y-filer haplotypes revealed an additional null at Y-GATA H4 in two of the Indian males presented here.
... In such a case, a male sample would falsely appear as a female. Some cases of these AMELY negative males have been reported since 2005 [3][4][5][6][7][8][9][10]. At that time, the AmpFlSTR Profiler PCR amplification kit and/or AmpFlSTR Identifiler PCR amplification kit (ID) (Applied Biosystems) was used as STR kits included AMEL for forensic casework. ...
... The cause of the AMELY allele dropout was identified in our research and reported previously [11]. Other many researchers have also reported the cause [3][4][5][6][7]. Most of the causes of AMELY deletions are large-scale deletions encompassing AMELY on Yp11.2. ...
... There were only a few reports about AMELY negative males more than 15 years ago [3][4][5][6]. They were not found in the Chinese population at that time, but have been reported after that [8,9,12,15]. ...
Gender identification in forensic DNA typing is an important tool for criminal investigation as well as STR typing. Most methods are based on a size difference of amelogenin X and Y (AMELX and AMELY). There have been some reports that the method by amelogenin (AMEL) incorrectly typed some males as females because the AMELY allele is not detected. AMELY allele dropout is often caused by deletions encompassing AMELY on Yp11.2 and accompanied with Y-STR allele dropout (especially DYS458). However, an unusual deletion was found in our laboratory. The AMELY allele could be recovered by using another commercial kit and alternative AMEL primer sets, and the Y-STR markers resulted in a complete profile. Sequencing results showed that there was an 8 bp deletion in AMELY at the position corresponding to 41–48 bp downstream from the 3′ end of the 6 bp deletion site in AMELX. This is considered a novel mutation at a primer binding region.
... Rev Odonto Cienc 2017;32(1): [1][2][3][4][5][6][7][8][9] Brazil's southeastern population Physical Anthropology | Carvalho et al. ...
... Forensic skeletal examination of remains offers several advantages: it is relatively easy to apply, results are rapidly available, costs are low, and only basic osteometric instruments are required. Disadvantages are that they cannot normally be applied to children's skeletons, and they must be population specific, as has been pointed out by many studies [1][2][3][4][5][6][7][8]. ...
... From the technical and criminalist point of view, DNA can be collected from any biologic specimen, however, the biological material recovered in forensic scenes may suffer environmental changes (temperature, soil pH, humidity), that may cause breaks and changes in the chain of nucleotides and, consequently, modify the composition and the normal structure of the DNA, making the analysis impossible [3]. In DNA analysis for sex determination, traditionally, amelogenin has been used and became a standardized method in the kits used in the human identification [8]. ...
OBJECTIVE: This study aimed to determine the sex in human craniums using methodologies of Physical Anthropology, quantitative (Forensic Data Anthropology Bank, FDB, 1986) and one qualitative (Walker, 2008) and genetic analysis by amelogenin.METHODS: The sample was composed of 66 skulls (34 males and 32 females) from the Center for Study and Research in Forensic Science, Guarulhos, SP. The methodologies were applied by two researchers who were unaware of the cranium’s sexes. For the statistical analysis, there were performed descriptive analysis, average, standard deviation, linear discriminant analysis and logistic regression.RESULTS: The qualitative methodology presented an accuracy of 89.52%. For the DNA, it was possible to determine the sex in 86.15% of the sample. Analyzing the results for each skull in three different methodologies, we reached 100% correct.CONCLUSION: As a result of this study, it is recommended that physical anthropology be the chosen method if it presents good accuracy when applied to different populations or if it is validated for the analyzed population. Otherwise, genetic analysis should be used for the determination of the sex.
... 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][7][8][9][10][11][12]. ...
... 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][8][9]11,12]. On the other hand, the DYS458 null allele may serve as a strong indicator of the AMELY-negative sample. ...
... 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][8][9]. ...
... Generally, the male's AMELX is detected while AMELY is undetectable in AMELY-null cases, which will result in the incorrect genotyping of male samples as females. According to the previous studies, the null of AMELY can be caused by the abnormality of Y chromosomes [6,7], the mutation in primer binding sites [8] and/or the deletion encompassing AMELY on Yp11.2 [3,[8][9][10][11][12][13][14][15][16][17]. In cases with abnormal Y chromosomes, Y-STRs on the q arms of Y chromosomes were not detected. ...
... Since the deletion of AMELY among populations are increasingly reported and might have a population-specific pattern, it is important to characterize the region with the deletion of AMELY [12]. During the routine paternity testing in our laboratory we observed two AMELY-null cases. ...
... In contrast, the AMELY deletion has a low frequency in O3 haplogroup. Based on reports in the literature to date, the total frequency of the AMELY deletion in Chinese population is relatively lower than that in other populations [3,12,14,16]. ...
The amelogenin (AMEL) is widely used in many multiplex PCR kits for gender determination. However, the null of amelogenin Y (AMELY) can result in the incorrect genotyping of male samples as females. In this study, we report the deletion of AMELY in two cases with a deletion frequency of 0.019% (2/10526) in our laboratory. The deletion region with AMELY was mapped by using other twelve loci, which shows the class I deletion pattern. Further, the Y chromosome short tandem repeat (Y-STR) typing shows that these two cases share the same haplotype with other two cases from previous reports. The haplogroup of the two cases was predicted as O3 haplogroup with a 100% probability. Altogether, this study will provide evidence to further demonstrate the deletion of AMELY in Chinese population.
... Both X-and Y-chromosome homologs can be amplified in a single reaction, and the amplification of the amelogenin gene has the advantage of an internal positive control since the X chromosome sequence should always be present [3,11]. Several studies have reported mutations, such as null alleles at the AMELX and AMELY loci [3,6,[12][13][14][15][16]. Most Y-specific amplification failures of the amelogenin marker are due to large deletions spanning this locus, whereas X-specific dropout is less common and is usually determined by point mutations at the primer-binding sites. ...
... Genes 2023, 14,1986 8 of 10 analyzed using the Amel-A primer or Amel-A A/G primer (A). The quantification of the amelogenin gene was subjected to real-time PCR using SYBR ® green dye with Amel-A primer or Amel-A A/G primer (B). ...
The study of gender markers is essential in forensic genetic analysis. Mutations in the X or Y homologs of the amelogenin gene can be misleading, resulting in serious mistakes in forensic genetic analysis. We recently discovered two male cases of the X homolog of the amelogenin (AMELX) allelic dropout while analyzing short tandem repeat genotypes obtained from crime scene evidence. Subsequently, we evaluated the molecular characteristics of AMELX allelic dropout in this study. We used two previously reported amelogenin primers to verify a half level of amelogenin gene amplification intensity in the two male cases, which we confirmed was caused by AMELX allelic dropout. We then characterized the point mutation using Sanger sequencing and designed mutation-specific primers that could overcome AMELX allelic dropout. Short tandem repeat genotyping analysis confirmed that the AMELX allelic dropout was recovered by the mutation-specific primer designed specifically for this case. The sequencing of the AMELX allele revealed a single-point variant from A→G at base position 7 downstream from the 3′ end in the amelogenin forward primer-binding region. This point mutation was identically found in two different male cases, resulting in AMELX allelic dropout. To our knowledge, these mutations and the X homolog amplification failure of amelogenin have not been reported in the Korean population. Our study provides a reliable approach to AMELX allelic dropout due to rare case mutations and could enable the better interpretation of gender markers for forensic samples.
... The first intron of AMELX is six base-pairs (bp) [6] shorter and primers that target both genes are included in many forensic DNA kits in order to reveal the sex of the donor. As early as 1998 [7] there were reports in the literature of 2/24 males in a Sri Lankan population that lacked AMELY (AMELYnull) and others since have reported different classes of deletions involving the gene [8], seen predominantly in South Asians with a frequency of between 1% and 6.5% in other larger studies of this population group [9][10][11][12][13]. Sometimes the deletion will encompass a larger area resulting in incomplete Y chromosome STR loci in profiles routinely used in Y chromosome forensic analysis. ...
... Rumours abounded however that Mudgett had escaped the gallows and his great-great grandson commissioned an investigation with the aim of proving that. Historical dental examination, however, matched with medical examination reports from Holmes and the Y23 chromosome haplotype from the skeleton also matched that of his descendant [13]. While the rumours of Holmes escaping his execution are now proven to be unfounded, this analysis has not resolved other conspiracy theories. ...
Originally relatively ignored in forensic investigations because its genetic analysis lacks inference of individual identification, the value of Y chromosome analysis has been proven in cases of sexual assault, particularly where the amount of material left by a male assailant is limited in comparison with female DNA. All routine analysis of autosomal DNA, however, targets a gene (AMELY) on the Y chromosome in order to identify the sex of the DNA source and this is discussed in the context of the genetic structure of this male-specific chromosome. Short-tandem repeat markers on the chromosome are tested in dedicated multiplexes that have developed over time and these are described alongside international guidance as to their use in a forensic setting. As a marker of lineage, the Y chromosome provides additional tools to assist in the inference of ancestry, both geographical and familial and the value of Y chromosome testing is illustrated through descriptions of cases of criminal and historical interest. A decision to analyse the Y chromosome has to be considered in the context, not only of the circumstances of the case, but also with regard to the ethical questions it might raise, and these are discussed in relation to the cases that have been described in more detail in the accompanying online supplementary material.
... In particular, to examine the influence of postmortem DNA modifications in ancient samples, we detect a C$G transversion polymorphism from both sense and antisense strands and confirm allelic dropout in degenerated DNA using the SNP primers for deamination analysis. Furthermore, the SRY gene, as an alternative Y-specific marker, is coamplified with the AMEL gene because largeregion deletions in the Y chromosome are frequently observed and responsible for AMELY allelic dropout, resulting in incorrect conclusions regarding the sex associated with the sample [34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50]. Thus, this novel sense-antisense AMEL PCR-APLP assay with SRY analysis incorporates "bidirectional analysis" to examine samples from both sense and antisense strands to enable the analysis of massively degraded DNA. ...
... Several reports have indicated that there are ethnic differences in the frequency of AMELY deletion. The frequency of AMELY deletion was high in male samples coming from India, Nepal, and Sri Lanka [4,34,36,[39][40][41][42] and, conversely, low in some populations, including Chinese, Italian, Australian, and Spanish ones [35, 38, 41, 42, 44-46, 49, 50]. Failure to amplify AMELY could lead to erroneous results in forensic practice and archeological work; therefore, it should be taken into careful consideration even in such populations. ...
Sex determination is important in archeology and anthropology for the study of past societies, cultures, and human activities. Sex determination is also one of the most important components of individual identification in criminal investigations. We developed a new method of sex determination by detecting a single-nucleotide polymorphism in the amelogenin gene using amplified product-length polymorphisms in combination with sex-determining region Y analysis. We particularly focused on the most common types of postmortem DNA damage in ancient and forensic samples: fragmentation and nucleotide modification resulting from deamination. Amplicon size was designed to be less than 60 bp to make the method more useful for analyzing degraded DNA samples. All DNA samples collected from eight Japanese individuals (four male, four female) were evaluated correctly using our method. The detection limit for accurate sex determination was determined to be 20 pg of DNA. We compared our new method with commercial short tandem repeat analysis kits using DNA samples artificially fragmented by ultraviolet irradiation. Our novel method was the most robust for highly fragmented DNA samples. To deal with allelic dropout resulting from deamination, we adopted “bidirectional analysis,” which analyzed samples from both sense and antisense strands. This new method was applied to 14 Jomon individuals (3500-year-old bone samples) whose sex had been identified morphologically. We could correctly identify the sex of 11 out of 14 individuals. These results show that our method is reliable for the sex determination of highly degenerated samples.
... The typical sex test included in commercial kits is based on the amelogenin described by [1] but different studies showed that is not always reliable. Deletion of Y copy amelogenin gene, AMELY-null, or the AMELX null have been reported with a frequency less to 8% [2][3][4][5]. There are other genetic markers lying on the sex-determination region Y (SRY) on the Y chromosome [2,3,5] included in dual quantification commercial kits. ...
... This first case could be related with deletions on the chromosome encompassing the AMELY. Chang et al. [4] report amelogenin negative males and absence of the some Y-STRS markers indicating a large Y deletion. In the second case, the most possible explanation is point mutations in the amelogenin-priming sites [5]. ...
Sex determination is important in forensic cases of missing persons, rape or to establish the sexual identity. There are molecular tools to determine the sex such as the simultaneous quantification of autosomal DNA and Y chromosome and the typification of amelogenin and Y chromosome markers but do not always yield accurate data on the determination of whether the individual is male or female and this can be related to mutational effects or in the possible discrepancies between the biological gender and the legal gender. Also in mixtures profiles cases, where there is a female predominant profile, may have problems in identifying the male component in the amelogenin and not always is successful the amplification of Y chromosome specific markers whose outcome can confirm the presence of a male profile. In this research were proposed different cases related to sex determination associated with mutations in the amelogenin sex marker or in theirs primers annealing regions, hermaphroditism, aneuploidies and the potential results of the forensic genetic routine markers whose interpretation should be used carefully to avoid misunderstanding in our reports.
... Cases of AMELY-negative males are more frequently detected worldwide; the genetic mechanisms underlying AMELY dropout involve deletions of different sizes, encompassing the AMELY locus, and mutations in the primerbinding region of the AMELY allele (Jobling et al., 2007;Butler, 2011). Deletions in the Yp11.2 region, a major cause of the AMELY null allele, are often combined with the absence of adjacent Y-STR loci DYS456 and/or DYS458 (Jobling et al., 2007;Cadenas et al., 2007;Ma et al., 2012;Chen et al., 2014;Chang et al., 2007;Ou et al., 2012;Kumagai et al., 2008;Turrina et al., 2009;Borovko et al., 2015). ...
Sex discordance between cell-free DNA (cfDNA) testing and ultrasound examination is rare but can cause significant patient discomfort and uncertainty. Here, we present two clinical cases where a closer examination of raw sequencing data allowed us to anticipate possible discrepancies caused by the insertion of Y-chromosome regions into the maternal genome. We used Illumina’s VeriSeq NIPT Solution v2 and a proprietary bioinformatics pipeline to analyze cfDNA in the maternal bloodstream. Paired-end sequencing data were aligned to the reference genome (hg19). Non-duplicated aligned reads were aggregated into 100-kb bins, adjusted for CG bias, and further aggregated into 5-Mb windows. Z-scores were calculated for autosomes, sex chromosomes, and 5-Mb bins. The two clinical cases were classified as low-risk male fetuses according to the primary statistics (case A: NCVx = 0.3; NCVy = 40.6; native fetal fraction (FFi) = 5.1%, and case B: NCVx = −0.3, NCVy = 40.7, FFi = 10.8%); however, the Y-chromosome-based FF (FFy) was significantly lower than the default FF estimate (FFy ≅ 2% in both cases). Plots of X and Y chromosome Z-scores for each 5-Mb bin, according to genomic position, identified bins with Z-scores significantly higher than those expected for any pregnancy with a male fetus. The genomic coordinates of these bins overlapped with the amelogenin (AMELY) and protein kinase Y-linked (PRKY) genes, respectively. Amplification of these regions in the DNA isolated from the white blood cells fraction confirmed the presence of Y-chromosome insertions in the maternal genome. This study highlights a new source of discrepancy in cfDNA testing due to maternal genomic variations. These findings suggest the need for improvements to current bioinformatics pipelines to identify and exclude possible maternal perturbations from the classification algorithms used for aneuploidy and sex calls.
... In 1998, first time sex determination based on the amelogenin marker was questioned [7]. Afterword various studies have shown AMELY deletion in many of the population studies around the globe [8,9]. AMELY is now been well characterized and its deletion mapping data is available [10,11]. ...
In forensics, DNA plays an important role to link the victim and accused to the scene of a crime. It also identifies the gender in missing person identifications, mass disaster cases, and helps in the analysis of samples of sexual assault. The presence of the Y chromosome in a sample determines that it is of a male person. Modern multiplex STR kits have amelogenin locus as a gender-determining marker. Two single-copy genes located on Xp22.1–Xp22.3 (AMELX) and Yp11.2 (AMELY) encode the amelogenin locus. Although, dropout of Amelogenin Y in a known male, may falsely be exhibited as female with these kits. In this case, confirmation of Y amelogenin is done with Y chromosome-specific kits. This type of dropout at amelogenin Y was observed globally, especially in Asian populations. In our study, out of 102 and 100 known male samples from Haryana and Rajasthan state, three and one samples showed amelogenin Y deletion with a frequency of 2.94 and 1.00%. Haryana Brahmin has the highest amelogenin Y deletion among the studied Indian populations. Hence, in this study, a comparison was discussed with other Indian and worldwide populations.
... ese samples were labeled as SQ-1-SQ-18. e frequency of AMEL-Y-null males in various populations has been studied and has been shown to vary in different populations ranging from 0.037 to 10% [38][39][40][41][42]. ...
Amelogenin is a common sex typing marker encountered in forensic case work. Phenotypically normal males have been reported in the literature who exhibit anomalous amelogenin allele. These males express only a single amelogenin peak representing AMEL-X and are called as AMEL-Y-null males. Gender misclassification of such individuals is an obvious consequence of this mutation, as a male sample would falsely appear to be a female sample. This study was aimed to attribute the AMEL-Y-null male DNA profiles encountered in forensic casework in the Pakistani population to appropriate phylogenetic clade based on shared ancestry. A total of 18 null AMEL-Y males were screened out of the sample pool of 5000 male individuals, reflecting mutational frequency of 0.36%. A common phylogenetic ancestor is suggested for 17 individuals, based on computational analysis of the Y-STR haplotypes, shown to be belonging to the J haplogroup while only one sample belonged to the R group. The samples in J groups showed homology with subclades J2b2a M241 and J2b2a PH1648, while R group individual showed 100% homology with R1a. Data are reported after haplotype network development of AMEL-Y-null Pakistani males using Network 10.0 for the study of evolutionary distances and emergence of nodes.
... Primer site mutation of both AMELX and AMELY genes was theorised as one of the reasons for sex typing failure, causing the lack of an amplicon which caused confusion while interpreting DNA mixtures, especially in sexual assault cases 1 . A study conducted by Chang et al. 6 showed a significant number of AMELY null individuals amongst Malay males (0.6%) and Indian males (3.2%), and none of Chinese male individual. A similar study also suggested that sex typing based solely on amelogenin is insufficient for use in routine forensic work 6 . ...
Sex determination is one of the basic components in victim identification. There are many available methods, namely forensic anthropology method and conventional DNA typing method. In this study, nested PCR technique was employed in sex typing of burnt teeth through amelogenin (AMEL) and sex-determining region Y (SRY) markers. In this study, 17 teeth samples were burnt at temperatures that ranged from 100°C to 500°C for 2 min-10 min. The whole tooth was used for DNA extraction by phenol-chloroform method. Accurate sex determination was achieved in 13 samples by both AMEL and SRY markers. The SRY marker achieved higher sensitivity as compared to AMEL marker. The sensitivity of both markers was improved consequent to nested PCR. Factors such as degraded DNA materials and the presence of tooth caries greatly affect sex typing results. Results showed that nested PCR proved to be a good method to amplify highly degraded DNA material as it greatly increased the DNA copy, and thus increased the possibility of sex typing.
... Several cases of deletion at the Yp11.2 region have been investigated using various genetic markers [5][6][7]. Different results in these studies indicate a variation of the size of the deletions. In our cases, the minimum size of about 2 Mb can only be estimated due to the missing Y-STRs in the Yfiler Plus kit and their positions on the Y chromosome (Table 1) and AMELY. ...
In forensic genotyping the gender specific marker Amelogenin is a valuable tool for gender determination. This marker is therefore standard in all commercial forensic genetic kits. However, gonosomale aberrations, deletions or primer binding site mutations can cause problems in the correct interpretation of the marker Amelogenin and consequently a valid sex determination. Misleading results can be obtained based on incorrect interpretation of the phenotype or genetic intersexuality even though the genotyping based on Amelogenin itself was performed and evaluated correctly. In our routine casework we observed six cases with discrepancies regarding the biological and the “legal” gender. Further investigation of these cases revealed varying reasons for these discrepancies. But not all cases could be completely resolved and clarified.
... This suggests that, AMELY-deleted individual can be correctly identified through alternative sex markers such as sex-determining region Y (SRY) marker and steroid sulfatase (STS) marker as showed in another study (Morikawa et al. 2011). The AMELY-deleted frequency as high as 3.2% was reported among Malaysian population based on race (Chang et al. 2007), however, this cannot be further investigated in this study as the information on the race of tooth owner was not collected. Furthermore, the shortest amplicon used in this study was 212 bp, which is still considered as a large PCR product in analyzing degraded DNA samples. ...
Sex determination is one of the basic components in victim identification. This study aims to ascertain the sex of an individual from burnt teeth samples exposed at different temperature and time through nested polymerase chain reaction (PCR) on the amelogenin (AMEL) sex marker, to calculate the specificity and sensitivity, and to compare with previous relevant studies. A total of 17 teeth samples was subjected to burning at different temperatures ranging from 100°C to 500°C, at 2 to 10 minutes. The whole tooth was used for deoxyribonucleic acid (DNA) extraction by phenol-chloroform method. All samples were quantified for DNA concentration and then analyzed with nested PCR using two pairs of AMEL primer and results of sex typing were recorded. Out of 17 samples, genomic DNA extracted from 6 samples have concentrations ranging from 27.3 – 130.6 ng/μL. Nested PCR could amplify 16 samples for AMEL gene. Sex typing using AMEL gene showed 76.47% accuracy. Sensitivity of AMEL primer was increased from 6.67% to 63.64% using nested PCR technique; specificity of both external and internal primer was reported at 100%. Nested PCR of AMEL gene proved to be a suitable method for unequivocal determination of sex from degraded DNA samples.
... This simultaneous deletion of loci DYS570, DYS576, DYS458, DYS449, DYS481, DYS627 and DYS448 has been previously recorded in one other British South Asian individual (final frequency of 0.0039). Large deletions encompassing the AMELY region and involving the adjacent loci DYS570, DYS576, DYS458, DYS481 as well as the distant locus DYS448 have been previously reported in individuals with South Asian ancestry [1,10,11]. Loci DYS449 and DYS627, included in the Yfiler Plus PCR kit, were not analysed in these previous studies. However, given their close proximity to the other loci involved in this extensive deletion it is of no surprise to find these loci additionally deleted when genotyping with Yfiler Plus, and indeed the typing of DYS627 further identifies the bounds of this deletion. ...
A total of 3128 Y-STR profiles from three UK and one Irish population have been analysed with the PowerPlex Y23 system and are reported here. Instances of haplotype sharing between apparently unrelated individuals were identified and further investigated with the use of the 5 additional markers within the Yfiler Plus kit, resulting in a reduction by 76% in the number of shared haplotypes. Furthermore, Yfiler Plus was also employed to verify locus deletions and duplications observed in Y23 genotypes while inconsistencies between the two kits were sequenced, revealing underlying Y23 primer binding site mutations in loci DYS392 and DYS576. Finally, the mechanism behind a previously reported population specific peak shift observed in DYS481 in South Asian samples has been evaluated and further investigated in a novel case of this phenomenon seen in a Black British individual featuring a different flanking region mutation.
... Human identification of blood sample by amplification of Cyt b (157 bp) has been successfully reported previously [15] but the amplified target of Cyt b primers were reported differently compared to this study. For sex identification of blood samples, the efficiencies of AMEL(1), AMEL(2) and SRY primers on sex determination were compared because false interpretation of AMEL amplification had been reported earlier [16,17]. In addition, contamination of sample has previously caused incorrect interpretation of SRY analysis [18]. ...
This study aimed to identify DNA of human origin and determine human sex from bloodstains by duplex PCR analysis. Two pairs of primers amplifying Amelogenin gene, AMEL(1) and AMEL(2) and a primer pair targeting Sex Determining Region Y gene [SRY] were compared at the preliminary stage to measure the accuracy of sex determination. Human specific region of Cytochrome b (Cyt b) gene sized 412 bp successfully targeted only human-origin DNA, while other animal DNAs (dog, cat, cow, chicken and pig) did not produce any PCR product. SRY primer showed 100% accuracy on male identification with 197 bp amplicon. Therefore, the SRY primer pair was chosen for sex identification. The optimal annealing temperature of duplex PCR analysis was 55C, producing the most distinctive PCR products, determining male with 197 bp and 412 bp bands and female with only 412 bp. For sensitivity, the smallest blood sample that could be detected was 20 l. For specificity, both human origin and sex determination could be detected in all ratios of mixed human and dog bloods, including using 100 fold less human blood to animal blood. Determination of male blood in mixed male and female blood samples was also investigated. The results showed that male blood could be determined in mixed female blood sample at ratios less than 10 fold. This study demonstrated that duplex PCR analysis of Cyt b and SRY is a reliable tool to investigate human DNA and sex of questioned bloodstains.
... First, we identified our cell line as HCT-8 by DNA STR profiling with a percent match higher than 80%. The only mismatch was at the AMEL Y locus, a region reported sometimes as deleted [17] a CGH analysis performed on our cell line, pointed out a large deletion at the Y(p)11.2 region encompassing the AMEL locus. ...
The major cause of failure in cancer chemotherapy is the development of multidrug resistance (MDR), and the characterization of biological factors involved in this response to therapy is particularly needed. A doxorubicin-resistant HCT-8/R clone was selected from sensitive parental cells and characterized analyzing several parameters (cell cycle phase distribution, apoptotic activity, expression, distribution and functionality of the P-gp efflux pump, the response to other chemotherapy agents, its ultrastructural features, invasiveness, and transcriptomic profile). HCT-8/R cells showed a peculiar S phase distribution, characterized by a single pulse of proliferation, resistance to drug-mediated apoptosis, increased expression and functionality of P-gp and overexpression of stem cell markers (CD44 and aldehyde dehydrogenase 1A2). At the ultrastructural level, HCT-8/R presented a greater cell volume and several intracytoplasmic vesicles respect to HCT-8. Moreover, the resistant clone was characterized by cross resistance to other cytotoxic drugs and a greater capacity for migration and invasion, compared to parental cells. Our data reinforce the concept that the MDR phenotype in HCT-8/R cells is multifactorial and involves multiple mechanisms, representing an interesting tool to understand the biological basis of MDR and to test strategies that overcome resistance to chemotherapy.
... The high correlation between 45,X0 percentage and amelogenin Y/X ratio confirms that QF-PCR is a reliable method for estimation of the Y/X ratios. Although there are reports for AMELY allelic dropout as a result of the deletion in this region of Y chromosome, the increased frequency is mainly reported in Asian populations [30,31,32]. We have not detected loss of AMELY among males included in this study as well as more than 4000 male samples analyzed in our laboratory for the presence of the most common chromosomal aneuploidies, paternity and male infertility (unpublished data). ...
Background
Although age-related loss of chromosome Y (LOY) in normal hematopoietic cells is a well-known phenomenon, the phenotypic consequences of LOY have been elusive. However, LOY has been found in association with smoking, shorter survival and higher risk of cancer. It was suggested that LOY in blood cells could become a predictive biomarker of male carcinogenesis.
Aims, Methods & Findings
To investigate the association of LOY in blood cells with the risk for development of colorectal (CC) and prostate cancers (PC), we have analyzed DNA samples from peripheral blood of 101 CC male patients (mean age 60.5±11.9 yrs), 70 PC patients (mean age 68.8±8.0 yrs) and 93 healthy control males (mean age 65.8±16.6 yrs). The methodology included co-amplification of homologous sequences on chromosome Y and other chromosomes using multiplex quantitative fluorescent (QF) PCR followed by automatic detection and analysis on ABI
... AMELY and its homolog, AMELX, are often used for gender identification [68]. Deletions of AMELY occur frequently in certain ethnic populations [69][70][71]. Research regarding AMELY function is rare, especially in human prostate cancer, but we believe that it may be a potential gender-related gene and a biomarker in human prostate cancer. In the future, more experiments and clinical samples are still needed to validate the importance of this gene in prostate cancer. ...
Prostate cancer is a type of cancer that occurs in the male prostate, a gland in the male reproductive system. Because prostate cancer cells may spread to other parts of the body and can influence human reproduction, understanding the mechanisms underlying this disease is critical for designing effective treatments. The identification of as many genes and chemicals related to prostate cancer as possible will enhance our understanding of this disease. In this study, we proposed a computational method to identify new candidate genes and chemicals based on currently known genes and chemicals related to prostate cancer by applying a shortest path approach in a hybrid network. The hybrid network was constructed according to information concerning chemical-chemical interactions, chemical-protein interactions, and protein-protein interactions. Many of the obtained genes and chemicals are associated with prostate cancer.
... The widely use of Y-STR markers in this context contributes to reveal the occurrence of rare allelic variants such as silent mutations, deletions and duplication. For instance, the dropout of the Amelogenin Y-allele has been associated with a microdeletion of at least 1.13 Mb on the Y (p) 11.2 region, also affecting MSY1 minisatellite and the DYS458 locus [1]. Also, null alleles in the Y-STR haplotype have been characterized in relation to AZFa, b and c regions, in association with male infertility [2]. ...
In the context of a post mortem paternity case with the alleged grandfather as the only available reference sample, an acceptable LR value was reached (363830) by means of 20 autosomal markers (PowerPlex21 System). The child’s YFiler haplotype showed a signal loss affecting markers DYS385a/b, DYS392 and DYS448. This finding was confirmed with the PPY23 marker set, where the loss of signal was also observed in DYS549 locus. The missing alleles map to the long arm of the Y chromosome, which encompass the AZFb + c deletion region.
Since the grandfather’s haplotype showed a complete profile, it could be assumed that the observed microdeletion is a de novo event which could have been occurred during grandfather’s or father’s meiosis. In addition, an inconsistency between Grandfather/child profile was also observed in the DYS481 marker, which could be explained by assuming a meiosis mutation (23 to 24).
... In nine samples, a large deletion was detected at Yp11.2 encompassing the AMELY region that removed four adjacent loci (DYS570, DYS576, DYS458 and DYS481). All these samples were of Asian ancestry, namely Indians from Singapore, Tamils from Southern India and British Asians with reported origins from Pakistan or India, where this type of deletion is frequent [27,28]. Furthermore, two of the nine samples also carried a null allele at DYS448 [29]. ...
... Table1: Comparisons of Y-STR haplotypes of null AMELY males.with amelogenin allele negative men, caused by large scale (>1 Mb) deletions14 . It often corresponds to specific Ychromosome binary haplogroup affiliation 12 suggestive of common Y-chromosome lineal ancestries within STR haplotype based data sets27 .A comparative Y-STR profile of null AMELY males with other study is listed in theTable 1. ...
Amelogenin is the well-liked process for sex typing in recent days. Exceptional failures to amplify AMELY accurate products can cause erroneous gender identification consequence male samples mistakenly identified as females. A total of 9 null AMELY males were noticed in 200 males of 72 Nepalese surnames by means of 4.5% frequency. No amplification of DYS458 Marker in all null AMELY males but great sharing of amplified Y STR alleles among them is a probable sign of the general phylogenetic origin. Key words: Forensic; Gender determination; Amelogenin; Null AMELY; Y-STR haplotypes etc. DOI: 10.3126/sw.v8i8.3858 Scientific World Vol.8(8) 2010 pp.97-101
... Population studies on Malaysian male individuals revealed a significant proportion of Amelogenin Y-nulls in the Indian ethnic group (3.6% frequency) and 0.88% frequency in the Malay ethnic group due to a deletion of the gene on the Y chromosome and this deletion has been mapped to a region of at least 1.13 Mb on the Yp11.2 encompassing the Amelogenin, MSY1 minisatellite and DYS458 locus [5,6]. ...
The gender marker Amelogenin is commonly incorporated in commercial multiplex STR kits such as the AmpFlSTR® Identifiler® PCR amplification kit. However, false gender determination can occur when there is dropout of the Amelogenin Y-allele, particularly in the absence of reference specimens. Failure of the Amelogenin sex test can result in misinterpretations and their forensic relevance should not be underestimated. This paper discussed the risks of failure of the Amelogenin sex test among the Indian and Malay ethnic groups in the Malaysian population. Its forensic relevance was demonstrated in a case study of an unknown Amelogenin negative male discovered in the DNA on a lady decorative hair band.
... In nine samples, a large deletion was detected at Yp11.2 encompassing the AMELY region that removed four adjacent loci (DYS570, DYS576, DYS458 and DYS481). All these samples were of Asian ancestry, namely Indians from Singapore, Tamils from Southern India and British Asians with reported origins from Pakistan or India, where this type of deletion is frequent [27,28]. Furthermore, two of the nine samples also carried a null allele at DYS448 [29]. ...
In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend towards smaller genetic distances resulting from larger numbers of markers became apparent.
The identification of species and their sex from small biological samples is of scientific interest in forensic science. Various identification techniques have been developed; however, DNA‐based PCR is the most specific and sensitive technique compared to protein‐based methods. Although PCR amplification of the amelogenin (AMEL) has been used in different species for sex determination, the reliability of the AMEL test may sometimes be challenged due to amplification failure of AMEL Y in males, resulting in incorrect gender identification. Therefore, this study aimed to establish a simple, reliable and accurate PCR protocol for the amplification of the AMEL gene from blood gDNA isolated by a single‐step DNA isolation method using primers of different GC% to ascertain the sex of ovine. This methodology may also be applicable to various biological samples for sex determination. It was concluded that the touchdown PCR was more suitable for GC‐rich primers and low GC% primers were suitable with modified conventional PCR for gender identification. The use of PCR enhancers at denaturation temperatures of 94°C and 95°C was found ineffective for the amplification of AMEL to determine the sex. In summary, all primers used showed successful amplification.
Accurate gender determination is crucial in many scientific disciplines, especially in prenatal diagnosis of X-linked diseases and forensic investigations. Today, molecular techniques, especially typing for a length variation in the X-Y homologous amelogenin gene (AMEL X and AMEL Y), are used for gender assignation. This amelogenin is an integral part of most PCR multiplex kits for gender determination marker, but in 1998 there was a report of two normal males being typed as female with this marker. Subsequently, a small number of males with amelogenin deleted genes have been reported in various populations but little data are available characterizing these deletions. This review aims to explore possible relationships among the AMEL Y deleted samples and Y-chromosome microsatellite haplotypes. Also, attempts are made to determine the frequency of males with AMEL Y deleted gene in various countries across the globe. Although some studies have shown that males with AMEL Y deleted gene are extremely rare in most populations, typing an additional gender-determining locus should be considered in forensic investigations where the reference sample is of unknown gender.
In recent years, an increasing number of abnormal DNA genotypes caused by chromosomal abnormalities have been revealed in cases of individual identification and sex-typing analysis, especially analyses of the amelogenin and short tandem repeat (STR) loci on the sex chromosomes. Here, we report a 17-year-old female with Turner syndrome typed as male due to the presence of the amelogenin Y allele. The Y-STR haplotype showed allele dropout of three Y-STR loci (DYS549, DYS392 and DYS448). Further examination showed that the proband's karyotype was 45,X/46,X,del(Y) (q11.23), and the deletion of the Yp11.2 region was confirmed to encompass the observed microdeletion of the azoospermia factor (AZF)b + c region. One challenge in forensics is inaccurate sex typing of individuals at the molecular level, particularly for individuals with chromosomal abnormalities. This case suggests that various medical evaluations, including the examination of sex-related manifestations, karyotypes, and clinical phenotypes of individuals, along with the detection of sex-typing gene markers will be beneficial to overcome the issues caused by cytogenetic disorders of the sex chromosomes.
Background:
Jeju Island is the largest island of South Korea, located southwest far from the mainland of Korea, and has a unique history and its own cultures that are distinguished from those of the other regions of the Korean mainland. However, the Jeju population has not been deeply investigated to date to understand their genetic structure, which may reflect their historical and geographical background.
Objective:
To identify the genetic characteristics and biogeographic origin of people of Jeju Island based on the statistical analysis of genetic data using lineage markers.
Methods:
17 Y-STRs data for 615 unrelated males and mitochondrial DNA haplogroup data for 799 unrelated individuals residing on Jeju Island were generated, and analyzed to investigate genetic diversity and genetic characteristics using statistical methods including pairwise Fst or Rst, Analysis of molecular variance (AMOVA) and Multidimensional scaling (MDS).
Results:
For male individuals of Jeju Island, unique genetic characteristics were observed in the analysis of Y-STRs, including low haplotype diversity, strong association with surnames, genetic difference from other regions of Korea, and common genetic variation of the Y-STR loci known to be predominant in Northern populations, such as Mongolians. Statistical analysis of the mitochondrial DNA haplogroups also revealed similar results that showed low haplogroup diversity and high frequency of haplogroup Y prevalent mostly in ethnic populations around the Sea of Okhotsk in Northeastern Asia. All these results suggest that Jeju Island is genetically distinct from other regions of Korea, possibly being a subpopulation in Korea, and related closely to Northern Asian populations.
Conclusion:
The findings in the genetic approach could support understanding of the historical background of Jeju Island that is consistent with evidence from other multidisciplinary studies.
Male DNA screening is important in forensic investigations, such as sexual assault cases. Although quantitative real-time PCR is a robust method for detection of male DNA, it is time-consuming and labor-intensive. We herein report the development of a male DNA-targeted loop-mediated isothermal amplification (LAMP) assay that can be used for both laboratory-based fluorescence analysis and on-site lateral flow detection. The two detection systems are independent, but we streamlined the reaction before the detection by introducing a fluorescence probe and biotin-labeled primer into a single reaction. This allowed the evaluation of fluorescence signal followed by lateral flow detection. Both the fluorescence and lateral flow analyses detected as low as 10 pg of male DNA. We also integrated an alkaline lysis method with our LAMP assay. The direct assay successfully detected male DNA from forensic samples without purification. The workflow requires only <40 min for fluorescence analysis and <45 min for lateral flow detection. Furthermore, when combined with a lateral flow strip, this workflow does not require any sophisticated instruments. These findings suggest that our assay is a promising strategy for on-site male DNA screening as well as laboratory-based testing.
The AGCU Expressmarker 20 + 20Y Kit is a newly devised short tandem repeat (STR) multiplex system that simultaneously analyses a set of 19 autosomal STR loci, 20 Y chromosomal STR loci and the amelogenin locus with six-dye fluorescent labelling. Here, the AGCU Expressmarker 20 + 20Y system was validated following the guidelines published by the Scientific Working Group on DNA Analysis Methods (SWGDAM), including PCR-condition, sensitivity, mixtures, species specificity, models of inhibition, precision, stutter percentage, concordance, population genetic studies, performance on three kinds of degraded DNA and a type of casework samples. The results indicated that the kit had high sensitivity when there was a small amount of DNA (0.0625 ng), more than one male (minor: major=1:19), or a mixture of males and females (male: female=1:32), models of inhibition (250 μM hematin, 1500 ng/μL humic acid and more than 100 ng/μl tannic acid) and degraded samples. The kit showed high precision level with standard deviation of allele size no more than 0.0930. Furthermore, this system was also tested in 444 random male samples of Chinese Han and Hui population, showing its high discrimination capability in Han and Hui population. Meanwhile, the system was applicable to the case of the AMELY abnormality. In short, the kit was verified and proved to be a robust, reliable and suitable tool for human identification and casework samples.
In forensics, analysis of biological samples for identification and verification is quite often and usually very challenging. Y-Short tandem repeats (Y-STRs) and associated Single Nucleotide Polymorphisms (Y-SNPs) are the most widely used markers in forensic analysis due to their typing simplicity and high level of diversity. The panels of Y-STRs presently used in forensics are a promising tool in fatherhood cases, especially when the suspected father is not present and in population genetics also to answer different questionable paternal lineages amongst many worldwide human populations. Male Specific region of Y-Chromosome or Non-recombining region (NRY) does not undergo recombination. This unique characteristic of Y-Chromosome increases its significance in field of forensics. This review illustrates the timescale development of Y-Chromosome Short Tandem Repeats (Y-STRs) marker kits applied in forensics and population genetics. The content depicts the advancements in the field of STR multiplex PCR systems with improved proficiency, reactivity and inhibitor tolerance. These Y-STR markers kits are composed of di, tri, tetra, penta and hexa nucleotide repeats. Tetra nucleotide markers are most frequently used as compared to other nucleotides. Initial kits (minimal haplotype) had very few markers but now using multiplex PCR based system kit like SureID® PathFinder Plus kit a scientist can analyze more than 40 markers in a reaction with better discriminating power and greater efficacy. In the present review, significance of Y-Chromosomal STRs in forensics and population genetics with various Y-STR markers kit is discussed.
In this study, we located eight samples with null alleles of amelogenin out of 10,750 cases, and discussed the influence in gender identification and forensic personal identification. Amelogenin was detected and retested by several autosomal STR kits and sex chromosomal STR kits, and the causes were analyzed by chromosome karyotype analysis and Y chromosome microdeletion detection if necessary. Suspected AMEL-X loss was observed in five samples, but no abnormality was detected in the X-STR loci. AMEL-X was recovered when samples were retested by other detection systems designed with different primers. One sample had AMEL-X and X-STR loci loss, and the karyotype was chimeric 45,X0[70]/46,X,+mar[13].Two male samples lost AMEL-Y fragment, and both of them lost DYS522-DYS570-DYS576 loci, but no abnormalities were found in the STS loci of SRY and AZF regions. Therefore, when carrying out gender identification by using amelogenin, it is essential to focus on null alleles of amelogenin. In especially, deal with the samples collected from the individuals who had chromosomal hereditary disorders(e.g. Turner Syndrome and Oligospermia / Azoospermia). In order to achieve this, laboratories should have various techniques to verify the null alleles of amelogenin and ensure accurate genotyping. Accurate genotyping of amelogenin and DNA database establishment are vital for personal identification.
Recently, proteomic analysis of sex-specific amelogenin peptides in tooth enamel has been proposed as a promising new method for the assignment of sex to human skeletal remains in archaeological and forensic settings. The method was initially based on the surface acid etching of tooth enamel and subsequent identification of a marker peptide that could be attributed to sex-chromosome specific amelogenins via mass spectrometry. The previous three years have brought forth several technical improvements that increased sensitivity of the method, notably development of new enamel protein extraction techniques, advanced bioinformatic data processing for the detection of multiple sex-specific amelogenin peptides and statistical framework for the estimation of both male and female sexes. This focus article shows that the proteomic method, even after these technical improvements, has the same limitation as the PCR-based amelogenin sex tests do, which directly use amelogenin alleles as markers for the sex chromosomes. The fact is that tooth enamel in some phenotypically normal males lacks Y amelogenin-specific peptides as a result of the Y amelogenin allele (AMELY) deletion. The prevalence of AMELY negative males is generally lower than 1% but may approach 10% in Indian subcontinent populations. Genetic studies indicate that the alteration might be several thousand years old. Using a proteomic approach to sex estimation, these individuals would be falsely identified as females with a potentially significant impact on forensic investigations or archaeological work. Therefore, this possibility should be taken into consideration even in populations with a low frequency of AMELY deletion and regardless of the female sex probability predicted by the existing statistical model. Unfortunately, no proteomics-based control measures exist because the genes for non-amelogenin proteins (ameloblastin, enamelin, and tuftelin) are located on autosomal chromosomes.
The Y-chromosome DNA testing is important for a number of different applications of human genetics including forensic evidence examination, paternity testing, historical investigations, studying human migration patterns throughout history, and genealogical research. In terms of forensic applications, there are both advantages and limitations to Y-chromosome testing; the primary value of the Y-chromosome in forensic DNA testing is that it is found only in males. Using ChrY-specific polymerase chain reaction (PCR) primers can improve the chances of detecting low levels of the perpetrator's DNA in a high background of a female victim's DNA. Y-chromosome tests have also been used to verify amelogenin Y-deficient males. The same feature of the Y-chromosome that gives it an advantage in forensic testing, namely maleness, is also its biggest limitation. A majority of the Y-chromosomes are transferred directly from father to son without recombination to shuffle their genes and provide greater genetic variety to future generations.
The ability to identify the sex of embryo and control of sex ratio has a great commercial importance to livestock industry. Prediction of embryonic sex could be useful in the management decisions of sex selection in breeding programs. Several methods have been attempted to determine the sex but the PCR‐based sexing method is generally favored, as it is cost effective, simple and reliable. The aim of the present study was to identify sex of sheep embryos produced in vitro through amplification of GAPDH, SRY and AMEL genes present in gDNA of embryos through PCR. To avoid false interpretation of the result by no amplification of SRY in female embryos a duplex PCR was approached to amplify combinedly SRY and GAPDH genes. Sex specific blood was used in PCR as positive control. In vitro sheep embryos were produced as per standardized protocol of laboratory. Sexing of sex specific blood and in vitro produced embryos were approached though PCR to amplify the respective genes using gDNA present in the sample without its traditional isolation. The accuracy of sex prediction for embryos was 100% by this procedure.
Here, we present work on the improved hybridisation and release of specially designed pegylated double stranded DNA strands with a pendant toehold. For this forensically relevant DNA namely amelogenin (AMEL) (a sex determination gene) and the human c-fms (macrophage colony-stimulating factor) proto-oncogene for the CSF-1 receptor (CSF1PO) short tandem repeat (STR) were employed. Magnetic beads functionalised with oligonucleotide capture probes with complementarity to the dsDNA PCR product toehold were fabricated. Characterisation of the beads was achieved through dynamic light (DLS) scattering and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy showing successful attachment of the oligonucleotides. The time frame of hybridisation between the dsPCR product toehold and the oligonucleotide modified beads was decreased from 6 h to 10 min by pre-incubation of the oligonucleotide functionalised magnetic beads in 2% aqueous sodium dodecyl sulphate (SDS) solution. Fluorescent microscopy was used to determine the time of toehold mediated strand displacement of the captured dsPCR product from the magnetic bead. Initially, displacement was achieved within 6 h. This was subsequently reduced to 3 h by the addition of a rate accelerator, PEG6000 at 10% v/v.
Sexing of biological evidence is an important aspect in forensic investigations. A routinely used molecular-genetic approach to this endeavour is the amelogenin sex test, which is integrated in most commercially available polymerase chain reaction (PCR) kits for human identification. However, this assay is not entirely effective in respect to highly degraded DNA samples. This study presents a homogeneous PCR assay for robust sex diagnosis, especially for the analysis of severely fragmented DNA. The introduced triplex for the X and Y chromosome (TriXY) is based on real-time PCR amplification of short intergenic sequences (<50bp) on both gonosomes. Subsequent PCR product examination and molecular-genetic sex-assignment rely on high-resolution melting (HRM) curve analysis. TriXY was optimized using commercially available multi-donor human DNA preparations of either male or female origin and successfully evaluated on challenging samples, including 46 ancient DNA specimens from archaeological excavations and a total of 16 DNA samples extracted from different segments of eight hair shafts of male and female donors. Additionally, sensitivity and cross-species amplification were examined to further test the assay's utility in forensic investigations. TriXY's closed-tube format avoids post-PCR sample manipulations and, therefore, distinctly reduces the risk of PCR product carry-over contamination and sample mix-up, while reducing labour and financial expenses at the same time. The method is sensitive down to the DNA content of approximately two diploid cells and has proven highly useful on severely fragmented and low quantity ancient DNA samples. Furthermore, it even allowed for sexing of proximal hair shafts with very good results. In summary, TriXY facilitates highly sensitive, rapid, and costeffective genetic sex-determination. It outperforms existing sexing methods both in terms of sensitivity and minimum required template molecule lengths. Therefore, we feel confident that TriXY will prove to be a reliable addition to the toolbox currently used for sex-typing in forensic genetics and other fields of research.
A total of 223 samples from the general population of Ladakh in Northwestern India were amplified at 17 Y-STR loci using the AmpFlSTR® Yfiler™ system. The DNA profiles generated were employed to generate allelic frequencies, gene diversity, haplotype diversity and discrimination capacity values as well as number of different haplotypes, fraction of unique haplotypes and Rst pair wise genetic distances. Multidimensional Scaling (MDS) and Correspondence Analysis (CA) were performed with Rst values and allelic frequencies, respectively. The 17-loci discrimination capacity of Ladkah was found to be 0.8093. Eleven out of the 16 loci have diversity values greater than 0.6, and 13 loci possess values greater than 0.5. Ladakh exhibits no significant genetic difference to seven of the 15 reference forensic databases after Bonferroni correction, three of which are located in South Central Asian and four are from the Himalayan region. Rst genetic distance values before and after Bonferroni corrections illustrate the capacity of the Yfiler system to discriminate among Himalayan populations. The intermediate position of the Ladakh population in the MDS and CA plots likely reflects genetic flow and admixture with neighboring populations.
Short tandem repeat (STR) sequences are named by the length of DNA regions with the repeat unit. STRs have become popular DNA repeat markers because they are easily amplified by the polymerase chain reaction (PCR) without the problems of differential amplification. This chapter discusses the types of STR markers. STR allele nomenclature, a common nomenclature, is developed to aid in interlaboratory reproducibility and comparisons of data in the forensic DNA community. An allelic ladder is an artificial mixture of the common alleles present in the human population for a particular STR marker. For DNA typing markers to be effective across a wide number of jurisdictions, a common set of standardized markers must be used. Today both Applied Biosystems and the Promega Corporation have STR kits that address the needs of the DNA typing community and cover a common set of STR loci. Each manufacturer of STR kits provides allelic ladders that may be used for accurate genotyping. Applied Biosystems introduced two new technologies with their AmpFlSTR Identifiler kit when it was released in 2001. By far the most popular method for sex typing today is the amelogenin system because it can be performed in conjunction with STR analysis. STR Base, an Internet-accessible informational database, officially launched in July 1997 in anticipation of the impact of STR markers on DNA typing and the need for a common source of information that could evolve as the process improved.
Accurate gender determination is widely used and it is crucial in many scientific disciplines, especially in profiling for DNA databasing, forensic casework (e.g., identifying the gender of biological material in stains of unknown origin), analysis of archeological specimens, preimplantation/prenatal diagnosis and post-natal diagnosis (e.g., X-linked diseases or children with ambiguous genitals). Today, molecular techniques, also based on length variation in the X-Y homologous amelogenin gene (AMELX and AMELY), are used for sex determination. In humans, the amelogenin gene is a single copy gene located on Xp22.1-Xp22.3 and Yp11.2 and it is sufficiently conserved, so the simultaneous detection of the X and Y alleles using polymerase chain reaction can lead to gender determination. There is a size difference of 6 bp between the X and the Y genes in the most widely used PCR primer set. The presence of two amplified products indicates a male genotype, while a single amplicon implies a female genotype. Several studies, published since 1998, have shown that normal males may be typed as females with this test because the amelogenin gender test may not always be concordant with true male gender: AMELY deletions may result in no amplification product and normal males being typed as female with the test (negative male). To date literature data have supported that the null allele is the result of a larger deletion on the short arm of the Y chromosome and that this occurs in different percentages in different population groups. Considering the consequences of the result obtained using only the amelogenin marker and the potential related interpretation difficulties, the gender misinterpretation may be troublesome in some cases, both in clinical practice and forensic caseworks. Different strategies have been proposed to solve this misinterpretation, such as the use of additional markers to resolve the possible occurrence of AMEY deletion. In this paper we propose a review of the incidence in failures of gender testing among different populations and the different strategies proposed in literature in case of doubt regarding the presence of deleted AME in the DNA profile.
Lineage markers include mitochondrial DNA (mtDNA) and Y-chromosome short tandem repeat (Y-STR) haplotypes that are transferred directly from generation to generation either from mother to child in the case of mtDNA, or from father to son in the case of the Y-chromosome. X-chromosome markers are another tool that can be used for genetic identity testing. Lineage markers can be helpful in missing persons investigations, disaster victim identification, forensic casework where other evidence is limited, and some complex kinship situations. X-chromosome analysis is especially helpful in assessing some kinship scenarios.
In clandestine drug laboratories, precursors, chemicals and solvents are used during the synthesis of drugs. At the scene, identification of precursors and chemicals is not sufficient and conclusive by colour tests. Using portable devices such as Attenuated Total Reflectance-Fourier Transform Infrared Spectrometer (ATR-FTIR) and Raman Spectrometer, the forensic chemist will be able to obtain actionable information during the investigation. Colour tests method is limited for identification of certain types of drugs in contrast to these
portable devices which not only cover identification of a wide range of drugs but can also provide confirmatory results. Both these techniques also help in identification of precursors and chemicals used in the synthesis of drugs at the clandestine laboratories. Further useful information such as the synthetic route employed can be obtained through the identification of the precursors and chemicals. This article presents the application of FTIR and Raman techniques as a useful tool in the identification of drugs, chemicals and solvents which can
provide confirmatory results and indication of possible synthetic route used during the synthesis of drugs at clandestine drugs laboratory.
The Y-chromosome, as with other chromosomes in the cell, is subject to mutations. However, unlike autosomal genes, the Y chromosome does not undergo recombination, and therefore individuals from different geographical regions may have differing distribution patterns with respect to Y-chromosome mutations. More detailed knowledge and information regarding Y-chromosome mutations might therefore provide insights into phylogenetic history and personal identification. Here, we describe a case study involving genotype-phenotype discrepancy in an Indian male individual. We found that the mistyping in sex determination was caused by a deletion in the amelogenin Y (AMEL Y) gene. Furthermore, on examining the short tandem repeat (Y-STR) loci using the PowerPlex® Y23 System, we found four more deleted loci on Yp11.2 (DYS576, DYS481, DYS570, and DYS458) in this sample. We performed deletion mapping for this sample, and we propose that the microdeletion on the Yp11.2 locus occurred approximately in the 6.44 Mb to 9.75 Mb region. Previous studies have reported that the AMEL Y deletion is a common mutation in the Indian population. Taking into account regional differences, we also analyzed several area-specific Y-chromosome mutations.
Assigning the gender of a DNA contributor in forensic analysis is typically achieved using the amelogenin test. Occasionally, this test produces false-positive results due to deletions occurring on the Y chromosome. Here, a four-marker “YFlag” method is presented to infer gender using single-base extension primers to flag the presence (or absence) of Y-chromosome DNA within a sample to supplement forensic STR profiling. This method offers built-in redundancy, with a single marker being sufficient to detect the presence of male DNA. In a study using 30 male and 30 female individuals, detection of male DNA was achieved with c. 0.03 ng of male DNA. All four markers were present in male/female mixture samples despite the presence of excessive female DNA. In summary, the YFlag system offers a method that is reproducible, specific, and sensitive, making it suitable for forensic use to detect male DNA.
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 3.60Mb to 8.29Mb in the Y chromosome, and the overall frequency was 0.0077%. The AMELY-DYS458 deletion pattern was broke from 6.74Mb to 9.17Mb, 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.
Short tandem repeat (STR) loci are commonly used in forensic casework, familial analysis for human identification, and for monitoring hematopoietic cell engraftment after bone marrow transplant. Unexpected genetic variation leading to sequence and length differences in STR loci can complicate STR typing, and presents challenges in casework interpretation. Copy number variation (CNV) is a relatively recently identified form of genetic variation consisting of genomic regions present at variable copy numbers within an individual compared to a reference genome. Large scale population studies have demonstrated that likely all individuals carry multiple regions with CNV of 1kb in size or greater in their genome. To date, no study correlating genomic regions containing STR loci with CNV has been conducted. In this study, we analyzed results from 32,850 samples sent for clinical array comparative genomic hybridization (CGH) analysis for the presence of CNV at regions containing the 13 CODIS (Combined DNA Index System) STR, and the Amelogenin X (AMELX) and Amelogenin Y (AMELY) loci. Thirty-two individuals with CNV involving STR loci on chromosomes 2, 4, 7, 11, 12, 13, 16, and 21, and twelve with CNV involving the AMELX/AMELY loci were identified. These results were correlated with data from publicly available databases housing information on CNV identified in normal populations and additional clinical cases. These collective results demonstrate the presence of CNV in regions containing 9 of the 13 CODIS STR and AMELX/Y loci. Further characterization of STR profiles within regions of CNV, additional cataloging of these variants in multiple populations, and contributing such examples to the public domain will provide valuable information for reliable use of these loci.
In humans, the amelogenin gene is present on both the X and the Y chromosomes: there are size differences in this gene between these chromosomes, which have been utilised for sexing in forensic casework and prenatal diagnosis. The assay typically generates a 106bp long fragment from the X chromosome and a 112bp long fragment from the Y chromosome. Several studies have shown that the amelogenin gender test may not always be concordant with true male gender. Deletions of AMELY can result in no amplification product and these null AMELY alleles can occur in different percentages in different population groups. The literature data support that the null allele is the result of a larger deletion on the short arm of the Y chromosome. Considering the consequences of the result obtained using only the amelogenin marker, and potential interpretation difficulties in the few cases where gender misinterpretation may be problematic, in this paper we propose a method for the identification of samples with deleted AMEL based on a small polyacrylamide-gel electrophoresis of a duplex PCR product of a novel marker residing in the SRY gene, which results in a 197bp long PCR product in combination with primers for AMEL. This method can be applied, as an additional assay, in case of doubt regarding the presence of deleted AME in the DNA profile.
We describe the first haploid minisatellite, the human Y chromosome-specific locus, MSY1. It consists of an array of 48-114 AT-rich 25 bp repeats of at least five different variant types. A minisatellite variant repeat PCR (MVR-PCR) system gives Y-specific DNA codes, with a virtual heterozygosity of 99.9%, making MSY1 by far the single most variable locus on the Y. African populations contain the most diverged MSY1 structures. MSY1 is the only Y-chromosomal system where the characteristics of large numbers of mutations can be studied in detail: it provides a uniquely powerful tool both for the investigation of mutation in a haploid system, and for the dating of paternal lineages.
A sample of 526 Y chromosomes representing six Middle Eastern populations (Ashkenazi, Sephardic, and Kurdish Jews from Israel; Muslim Kurds; Muslim Arabs from Israel and the Palestinian Authority Area; and Bedouin from the Negev) was analyzed for 13 binary polymorphisms and six microsatellite loci. The investigation of the genetic relationship among three Jewish communities revealed that Kurdish and Sephardic Jews were indistinguishable from one another, whereas both differed slightly, yet significantly, from Ashkenazi Jews. The differences among Ashkenazim may be a result of low-level gene flow from European populations and/or genetic drift during isolation. Admixture between Kurdish Jews and their former Muslim host population in Kurdistan appeared to be negligible. In comparison with data available from other relevant populations in the region, Jews were found to be more closely related to groups in the north of the Fertile Crescent (Kurds, Turks, and Armenians) than to their Arab neighbors. The two haplogroups Eu 9 and Eu 10 constitute a major part of the Y chromosome pool in the analyzed sample. Our data suggest that Eu 9 originated in the northern part, and Eu 10 in the southern part of the Fertile Crescent. Genetic dating yielded estimates of the expansion of both haplogroups that cover the Neolithic period in the region. Palestinian Arabs and Bedouin differed from the other Middle Eastern populations studied here, mainly in specific high-frequency Eu 10 haplotypes not found in the non-Arab groups. These chromosomes might have been introduced through migrations from the Arabian Peninsula during the last two millennia. The present study contributes to the elucidation of the complex demographic history that shaped the present-day genetic landscape in the region.
Until recently, the Y chromosome seemed to fulfil the role of juvenile delinquent among human chromosomes--rich in junk, poor in useful attributes, reluctant to socialize with its neighbours and with an inescapable tendency to degenerate. The availability of the near-complete chromosome sequence, plus many new polymorphisms, a highly resolved phylogeny and insights into its mutation processes, now provide new avenues for investigating human evolution. Y-chromosome research is growing up.
Analysis of 89 biallelic polymorphisms in 523 Turkish Y chromosomes revealed 52 distinct haplotypes with considerable haplogroup substructure, as exemplified by their respective levels of accumulated diversity at ten short tandem repeat (STR) loci. The major components (haplogroups E3b, G, J, I, L, N, K2, and R1; 94.1%) are shared with European and neighboring Near Eastern populations and contrast with only a minor share of haplogroups related to Central Asian (C, Q and O; 3.4%), Indian (H, R2; 1.5%) and African (A, E3*, E3a; 1%) affinity. The expansion times for 20 haplogroup assemblages was estimated from associated STR diversity. This comprehensive characterization of Y-chromosome heritage addresses many multifaceted aspects of Anatolian prehistory, including: (1) the most frequent haplogroup, J, splits into two sub-clades, one of which (J2) shows decreasing variances with increasing latitude, compatible with a northward expansion; (2) haplogroups G1 and L show affinities with south Caucasus populations in their geographic distribution as well as STR motifs; (3) frequency of haplogroup I, which originated in Europe, declines with increasing longitude, indicating gene flow arriving from Europe; (4) conversely, haplogroup G2 radiates towards Europe; (5) haplogroup E3b3 displays a latitudinal correlation with decreasing frequency northward; (6) haplogroup R1b3 emanates from Turkey towards Southeast Europe and Caucasia and; (7) high resolution SNP analysis provides evidence of a detectable yet weak signal (<9%) of recent paternal gene flow from Central Asia. The variety of Turkish haplotypes is witness to Turkey being both an important source and recipient of gene flow.
Sex tests based on amelogenin are part of various PCR multiplex reaction kits widely used for human gender identification and have important applications in forensic casework, prenatal diagnosis, DNA databasing and blood sample storage. The two most common sex tests based on amelogenin are represented by primer sets that delimit a 6-bp deletion on the X chromosome to produce X/Y fragments of 106/112 or 212/218 bp, respectively. Few cases of AMELY deletion, usually considered as polymorphisms, have been reported so far and a detailed characterization of the molecular alteration is still lacking. In this study, we describe a large interstitial deletion of the Y short arm encompassing the AMELY locus in two unrelated individuals. The first case was identified in an oligozoospermic, otherwise phenotypically normal, 32-year-old man during the screening for Y microdeletions performed on a sample of infertile males. The second one was found among amniotic liquid samples tested by quantitative fluorescence-polymerase chain reaction and cytogenetic analysis for prenatal diagnosis. The extent of the deletion, spanning approximately 2.5 Mb, was better characterised by pulsed-field gel electrophoresis, followed by fluorescence in situ hybridization and STS marker analysis.
Deletions of the Y chromosome are a significant cause of spermatogenic failure. Three major deletion intervals have been defined and termed AZFa, AZFb and AZFc. Here, we report an unusual case of a proximal AZFb deletion that includes the Y chromosome palindromic sequence P4 and a novel heat shock factor (HSFY). This deletion neither include the genes EIF1AY, RPS4Y2 nor copies of the RBMY1 genes. The individual presented with idiopathic azoospermia. We propose that deletions of the testis-specific HSFY gene family may be a cause of unexplained cases of idiopathic male infertility. This deletion would not have been detected using current protocols for Y chromosome microdeletion screens, therefore we recommend that current screening protocols be extended to include this region and other palindrome sequences that contain genes expressed specifically in the testis.
The Y chromosome contains the largest nonrecombining block in the human genome. By virtue of its many polymorphisms, it is now the most informative haplotyping system, with applications in evolutionary studies, forensics, medical genetics, and genealogical reconstruction. However, the emergence of several unrelated and nonsystematic nomenclatures for Y-chromosomal binary haplogroups is an increasing source of confusion. To resolve this issue, 245 markers were genotyped in a globally representative set of samples, 74 of which were males from the Y Chromosome Consortium cell line repository. A single most parsimonious phylogeny was constructed for the 153 binary haplogroups observed. A simple set of rules was developed to unambiguously label the different clades nested within this tree. This hierarchical nomenclature system supersedes and unifies past nomenclatures and allows the inclusion of additional mutations and haplogroups yet to be discovered.
[Supplementary Table 1, available as an online supplement at www.genome.org , lists all published markers included in this survey and primer information.]
Sex identification of forensic samples (bloodstains and decomposed tissue) by polymerase chain reaction (PCR) was investigated. Amplification of a segment of the amelogenin gene using a pair of primers revealed both Y- and X-specific bands at the same time. The gene has counterparts in both the X and Y chromosomes and a small deletion in the former made it possible to distinguish them. Analysis of the X-specific band is the most reliable method for sex identification. THe locus includes a single copy gene so a sample of 250 ng/tube of deoxyribonucleic acid (DNA) is required for identification. Amplification of part of the DYZ1 locus was attempted as an alternative method for analysis of infinitesimal amounts of sample. Even DNA from putrefied tissue could be analyzed by PCR because the locus consists of thousands of copies of repeating units pHY10.
Over the past few years, the Australian forensic science community has adopted a common methodology and technology in the application of DNA profiling for investigative and forensic purposes. The ultimate objective of this initiative is the establishment of a national DNA database similar to that used in the UK. An integral part of this methodology is the use of "Profiler Plus," a nonaplex of STRs combined with amelogenin, a locus utilized for sex determination. This paper reports the results from a case where a mutation in the annealing region of the amelogenin primers appears to have resulted in the failure to amplify the amelogenin Y-homolog from a phenotypically normal male. The result was confirmed using two different primer sets that amplify different regions of the amelogenin gene. This situation suggests that the genetic determination of sex based on the amelogenin sequences from specimens of unknown origin, such as crime scene samples, should not be considered infallible.
This paper delivers population genetic data on Y-chromosomal short tandem repeat (STR) polymorphisms along with reports of unusual observations and casework. Population studies were carried out on the Y-specific STR polymorphisms DYS19, DYS385 I+II, DYS389 I+II, DYS390, DYS391, DYS392, and DYS393 in population samples from North India, Turkey, and Germany. In all three populations the vast majority of haplotypes was observed only once, especially in the Turkish group. Highly unusual cases are reported. In a German individual, we observed the variant allele DYS392*11.1, whereas a Turkish haplotype revealed a duplication at locus DYS19. Application of Y-chromosomal STR markers to forensic genetics was demonstrated in two cases: 1) a deficient paternity case, and 2) a father/son pair, where the Amelogenin primers failed to amplify the Y-homolog. In forensic genetics, Y-chromosomal STR polymorphisms are highly welcomed as an additional tool.
In humans, the amelogenin gene is present on both the X and the Y chromosomes. However, there are size differences in this gene between these chromosomes, which have been utilised for sexing in forensic casework and prenatal diagnosis. Our study using the AmpFl STR Profiler Plus kit, showed a deletion of Y chromosome-specific amelogenin in five Indian males (1.85%). We propose to call them "deleted-amelogenin males" (DAMs), who but for the detection of the presence of other Y-specific markers (e.g. SRY, STR and 50f2) would have been identified as females. Considering the consequences of the result obtained only using the amelogenin marker, we suggest the use of additional Y chromosome markers for unambiguous gender identification.
Determination of sex using the amelogenin sex test is well established in the forensic field especially for casework and DNA databasing purposes. The sex test is part of commercially available PCR kits. Among 29,432 phenotypic male individuals stored in the Austrian National DNA database, 6 individuals were found to lack the amelogenin Y-specific PCR product which was confirmed using alternative amelogenin primers. The amplification of eight Y-chromosomal STR markers resulted in full profiles in five out of the six samples, one sample failed to amplify Y-STRs at all. The amplification of a fragment of the SRY gene gave positive results in all six samples, confirming the male phenotype of the individuals. The observed failure rate of the amelogenin sex test was 0.018% in this study.
We identified and characterized 14 novel short-tandem-repeats (STRs) on the Y chromosome and typed them in two samples, a globally diverse panel of 73 cell lines, and 148 individuals from a European-American population. These Y-STRs include eight tetranucleotide repeats (DYS449, DYS453, DYS454, DYS455, DYS456, DYS458, DYS459, and DYS464), five pentanucleotide repeats (DYS446, DYS447, DYS450, DYS452, and DYS463), and one hexanucleotide repeat (DYS448). Sequence data were obtained to designate a repeat number nomenclature. The gene diversities of an additional 22 Y-STRs, including the most commonly used in forensic databases, were directly compared in the cell line DNAs. Six of the 10 most polymorphic markers include the newly identified Y-STRs. Furthermore, these novel Y-STRs greatly improved the resolution of paternal lineages, above the level obtained with commonly used Y-STRs, in the European-American population.
About 30% of couple infertilities are of male origin, some of them caused by genetic abnormalities of the Y chromosome. Deletions in AZF region can cause severe spermatogenic defects ranging from non-obstructive azoospermia to oligospermia. The intracytoplasmatic sperm injection technique (ICSI) is rapidly becoming a versatile procedure for human assisted reproduction in case of male infertility. The use of ICSI allows Y chromosome defects to be passed from father. The goal of our study is to evaluate the frequency of microdeletions in the long arm of Y chromosome, within the AZF regions, in these cases of infertilities, using molecular genetics techniques. Thirty infertile men with azoospermia or oligozoospermia, determined by spermogram, were studied after exclusion of patients with endocrine or obstructive causes of infertility. Peripheral blood DNA was extracted from each patient, then amplified by multiplex PCR with STS genomic markers from the Y chromosome AZF zones. Each case was checked by multiplex PCR through coamplification with the SRY marker. Three men with microdeletions of the long arm of the Y chromosome were diagnosed among the 30 patients, corresponding to a proportion of 10%. The relatively high proportion of microdeletions found in our population suggest the need for strict patient selection to avoid unnecessary screening for long arm Y chromosome microdeletions. The molecular diagnostics was performed according to the current European Academy of Andrology laboratory guidelines for molecular diagnosis of Y chromosomal microdeletions.
The aim of the present work was to improve the discriminatory potential, and hence the probative value, of Y-STR-based testing by extending the set of Y chromosome STR loci available for forensic casework. In accordance with the requirements of a Y chromosome multiplex analytical system developed specifically for forensic casework use, we have sought to maximize the number of loci able to be co-amplified, ensure appropriate assay sensitivity (1–2 ng of input genomic DNA), balance inter-locus signals and minimize confounding female DNA artifacts.
Two Y chromosome STR systems, multiplex I (MPI) and multiplex II (MPII), have been developed which permit the robust co-amplification of 18 Y-STRs. The loci include DYS19, DYS385(a) and (b), DYS388, DYS389I and II, DYS390, DYS391, DYS392, DYS393, DYS425, DYS434, DYS437, DYS438, DYS439, Y-GATA-C4, Y-GATA-A7.1 (DYS460) and Y-GATA-H4. The two multiplex systems are robust over a wide range of primer, magnesium, and DNA polymerase concentrations and perform well under a variety of cycling conditions. Complete male haplotypes can be obtained with as little as 100–250 pg of template DNA. Although a limited number of female DNA artifacts are observed in mixed stains in which the male DNA comprises 1/100 of the total, the male profile is easily discernible. Slightly modified versions of MPI and MPII demonstrate a significant reduction in female artifacts. Thus, it may not be necessary to employ a differential extraction strategy to obtain a male haplotype (or haplotypes in the case of multiple male donors) in cases of sexual assault. The potential utility of MPI and MPII for forensic casework is exemplified by their ability to dissect out the male haplotype in post-coital vaginal swabs and to determine the number of male donors in mixed semen stains.
This study has emphasized the need for novel Y-STR multiplexes developed for forensic use to undergo a series of validation exercises that go beyond simply optimizing the PCR reaction conditions. Specifically, stringent performance checks on their efficacy need to be carried out using casework-type specimens in order to determine potential confounding effects from female DNA.
The human sex test in forensic multiplexes is based on the amelogenin gene on both the X and Y chromosomes commonly used in sex genotyping. In this study of 338 male individuals in a Malaysian population comprising Malays, Chinese and Indians, using the AmpFlSTR Profiler Plus kit, the amelogenin test gave a significant proportion of null alleles in the Indian ethnic group (3.6% frequency) and 0.88% frequency in the Malay ethnic group due to a deletion of the gene on the Y chromosome. This sex test also failed in a forensic casework sample. Failure of the amelogenin test highlights the need for more reliable sex determination than is offered by the amelogenin locus in the Malay and Indian populations. The gender of the Indian-Malay amelogenin nulls was confirmed by the presence of three Y-STR alleles (DYS438, DYS390 and DYS439). For the Indian ethnic group, one of the Y-STR forms a stable haplotype with the amelogenin null. The amelogenin-deletion individuals also showed a null with a male-specific minisatellite MSY1, indicating that a very large deletion was involved that included the amelogenin and the MSY1 loci on the short arm of the Y chromosomes (Yp).
Human gender identification, based on the amelogenin gene, has important applications in forensic casework, prenatal diagnosis, DNA databasing, and blood sample storage. However, we report on the first known case, in the Israeli population, of an amelogenin sex test failure on a phenotypically normal male. He was typed as a female by both the AmpFlSTR SGM plus and GenePrint kits. Subsequent, karyotyping of the soldier's blood sample showed no abnormalities. These results suggest that the determination of sex, based on the amelogenin test, should be interpreted cautiously.
The Samaritan community, which numbered more than a million in late Roman times and only 146 in 1917, numbers today about 640 people representing four large families. They are culturally different from both Jewish and non-Jewish populations in the Middle East and their origin remains a question of great interest. Genetic differences between the Samaritans and neighboring Jewish and non-Jewish populations are corroborated in the present study of 7,280 bp of nonrecombining Y-chromosome and 5,622 bp of coding and hypervariable segment I (HVS-I) mitochondrial DNA (mtDNA) sequences. Comparative sequence analysis was carried out on 12 Samaritan Y-chromosome, and mtDNA samples from nine male and seven female Samaritans separated by at least two generations. In addition, 18-20 male individuals were analyzed, each representing Ethiopian, Ashkenazi, Iraqi, Libyan, Moroccan, and Yemenite Jews, as well as Druze and Palestinians, all currently living in Israel. The four Samaritan families clustered to four distinct Y-chromosome haplogroups according to their patrilineal identity. Of the 16 Samaritan mtDNA samples, 14 carry either of two mitochondrial haplotypes that are rare or absent among other worldwide ethnic groups. Principal component analysis suggests a common ancestry of Samaritan and Jewish patrilineages. Most of the former may be traced back to a common ancestor in the paternally-inherited Jewish high priesthood (Cohanim) at the time of the Assyrian conquest of the kingdom of Israel.
PCR amplification of part of the X-Y homologous amelogenin gene with a single primer pair has been used as a sex identification test because it generates different length products from the X and Y chromosomes. Using a commercially available kit that contains amelogenin primers, we report a single phenotypically normal Caucasian male out of 327 males tested to date that failed to show an X chromosome-specific PCR product. Using alternative amelogenin primers external to but encompassing the initial amplicon, an X chromosome-specific product was seen. Sequence analysis of this X-specific PCR product revealed a C to G mutation at the most 3' base of the initial reverse amelogenin PCR primer. An alternative reverse PCR primer with this most 3' base deleted showed X- and Y-specific products from the case study male. Rare mutations that result in a failure to amplify sex chromosome-specific products can result in incorrect gender identification.
A mosaic karyotype consisting of a 45,X cell line and a second cell line containing a normal or an abnormal Y chromosome is relatively common and is associated with a wide spectrum of clinical phenotypes. The aim of this study was to investigate patients with such a mosaic karyotype for Y chromosome material loss and then study the possible association of the absence of these regions with the phenotype, diagnosis, and Y-chromosome instability. We studied 17 clinically well-characterized mosaic patients whose karyotype consisted of a 45,X cell line and a second cell line containing a normal or an abnormal Y chromosome. The presence of the Y chromosome centromere was verified by fluorescence in situ hybridization (FISH) and was then characterized by 44 Y-chromosome specific-sequence tagged site (STS) markers. This study identifies a high frequency of Yq chromosome deletions (47%). The deletions extend from interval 5 to 7 sharing a common deleted interval (6F), which overlaps with the azoospermia factor region (AZF) region. This study finds no association between Y-chromosome loci hosting genes other than SRY, and the phenotypic sex, the diagnosis, and the phenotype of the patients. Furthermore, this study shows a possible association of these deletions with Y-chromosome instability.
This study examined the tissue distribution, cellular localization, and developmental expression of the PEPT2 protein in rat brain. Immunoblot and immunocytochemistry analyses were performed with specific rat PEPT1 and PEPT2 antisera developed in our laboratory. Rats were examined from fetus (gestation for 17 days) to adult (day 75). On immunoblot analysis, the PEPT2 protein was detected in cerebral cortex, olfactory bulb, basal ganglia, cerebellum, and hindbrain sections of adult brain, with the strongest signals in cerebral cortex. No PEPT1 protein was found in brain. Expression levels of the PEPT2 protein in cerebral cortex were maximal in the fetus and declined rapidly with advancing age. Adult protein levels were approximately 14% of that observed in fetus. In immunofluorescence experiments, the strongest PEPT2 signals were observed in epithelial cells of the choroid plexus for both adult and neonate brains. The PEPT2 protein was exclusively expressed on the apical membrane (CSF-facing) of choroid plexus epithelia. In double labeling experiments, PEPT2 immunoreactivity in adult brain colocalized with NeuN, a neuronal marker, but not with GFAP, an astrocyte marker. In contrast, in neonatal brain, PEPT2 immunoreactivity colocalized with both GFAP and NeuN. These findings demonstrate that the PEPT2 protein is found throughout the brain. The apical expression of PEPT2 in choroid plexus suggests that it is involved in the export of neuropeptides, peptide fragments, and peptide-like drugs from cerebrospinal fluid. PEPT2 may also play a role in the regulation of neuropeptide concentrations in extracellular fluid, especially during early development.
Although considerable cultural impact on social hierarchy and language in South Asia is attributable to the arrival of nomadic Central Asian pastoralists, genetic data (mitochondrial and Y chromosomal) have yielded dramatically conflicting inferences on the genetic origins of tribes and castes of South Asia. We sought to resolve this conflict, using high-resolution data on 69 informative Y-chromosome binary markers and 10 microsatellite markers from a large set of geographically, socially, and linguistically representative ethnic groups of South Asia. We found that the influence of Central Asia on the pre-existing gene pool was minor. The ages of accumulated microsatellite variation in the majority of Indian haplogroups exceed 10,000-15,000 years, which attests to the antiquity of regional differentiation. Therefore, our data do not support models that invoke a pronounced recent genetic input from Central Asia to explain the observed genetic variation in South Asia. R1a1 and R2 haplogroups indicate demographic complexity that is inconsistent with a recent single history. Associated microsatellite analyses of the high-frequency R1a1 haplogroup chromosomes indicate independent recent histories of the Indus Valley and the peninsular Indian region. Our data are also more consistent with a peninsular origin of Dravidian speakers than a source with proximity to the Indus and with significant genetic input resulting from demic diffusion associated with agriculture. Our results underscore the importance of marker ascertainment for distinguishing phylogenetic terminal branches from basal nodes when attributing ancestral composition and temporality to either indigenous or exogenous sources. Our reappraisal indicates that pre-Holocene and Holocene-era--not Indo-European--expansions have shaped the distinctive South Asian Y-chromosome landscape.
We have analyzed 16 Y-STR loci (DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635 or Y-GATA C4, DYS392, Y-GATA H4, DYS437, DYS438 and DYS448) from the non-recombining region of the human Y-chromosome in 980 male individuals from three main ethnic populations in Malaysia (Malay, Chinese, Indian) using the AmpFlSTR((R)) Y-filertrade mark (Applied Biosystems, Foster City, CA). The observed 17-loci haplotypes and the individual allele frequencies for each locus were estimated, whilst the locus diversity, haplotype diversity and discrimination capacity were calculated in the three ethnic populations. Analysis of molecular variance indicated that 88.7% of the haplotypic variation is found within population and 11.3% is between populations (fixation index F(ST)=0.113, p=0.000). This study has revealed Y-chromosomes with null alleles at several Y-loci, namely DYS458, DYS392, DYS389I, DYS389II, DYS439, DYS448 and Y-GATA H4; and several occurrences of duplications at the highly polymorphic DYS385 loci. Some of these deleted loci were in regions of the Y(q) arm that have been implicated in the occurrence of male infertility.
- C Cinnioglu
- R King
- T Kivisild
- E Kalfoglu
- S Atasoy
- G L Cavalleri
- A S Lillie
- C C Roseman
- A A Lin
- K Prince
- P J Oefner
- P Shen
- O Semino
- L L Cavalli-Sforza
- P A Underhill
C. Cinnioglu, R. King, T. Kivisild, E. Kalfoglu, S. Atasoy, G.L. Cavalleri,
A.S. Lillie, C.C. Roseman, A.A. Lin, K. Prince, P.J. Oefner, P. Shen, O.
Semino, L.L. Cavalli-Sforza, P.A. Underhill, Excavating Y-chromosome
haplotype strata in Anatolia, Hum. Genet. 114 (2004) 127–148.
The Y-chromosome consortium, A nomenclature system for the tree of human Y-chromosomal binary haplogroups
The Y-chromosome consortium, A nomenclature system for the tree of
human Y-chromosomal binary haplogroups, Genome Res. 12 (2002) 339-348.
The human Y chromosome: an evolutionary marker comes of age
- Jobling