Human Sex Determination Research Articles

Role of nucleobase-specific interactions in the binding and bending of DNA by human male sex determination factor SRY

Y-chromosome-encoded master transcription factor SRY functions in the embryogenesis of therian mammals to initiate male development. Through interactions of its conserved high mobility-group (HMG) box within a widened DNA minor groove, SRY and related Sox factors induce sharp bends at specific DNA target sites. Here, we present the crystal structure of the SRY HMG domain bound to a DNA site containing consensus element 5’-ATTGTT. The structure contains three complexes in the asymmetric unit; in each complex, SRY forms 10 hydrogen bonds with minor-groove base atoms in 5’-CATTGT/ACAATG-3’, shifting the recognition sequence by one base pair (italics). These nucleobase interactions involve conserved residues Arg7, Asn10, and Tyr74 on one side of intercalated Ile13 (the cantilever side chain), and Arg20, Asn32 and Ser36 on the other. Unlike the less-bent NMR structure, DNA bend angles of the distinct box-DNA complexes range from 69-84°, similar to those observed in homologous Sox domain-DNA structures. Electrophoretic studies indicate that respective substitutions of Asn32, Ser36 or Tyr74 by Ala exhibit slightly attenuated specific DNA-binding affinity and bend angles (70-73°) relative to WT (79°). By contrast, respective substitutions of Arg7, Asn10 or Arg20 by Ala markedly impaired DNA-binding affinity in association with much smaller DNA bend angles (53-65°). In a rodent cell-based model of the embryonic gonadal ridge, full-length SRY variants bearing these respective, Ala substitutions exhibited significantly decreased transcriptional activation of SRY’s principal target gene (Sox9). Together, our findings suggest that nucleobase-specific hydrogen bonds by SRY are critical for specific DNA binding, bending, and transcriptional activation.

Forensic dentistry- A deep dive in the drop

Teeth can be put to a lot more use than just understanding and evolving dentistry. Teeth can withstand tremendous pressure and extreme conditions providing for an incomparable piece of evidence. The comparison of the ante-mortem and the post-mortem evidences can be used to establish an identity of the required personnel. The oral cavity provides an excellent field to work on when it comes to human identification, age estimation and sex determination. The Bichat’s fat pad, lips, maxillary bones and the teeth protect the palatal rugae hence making them resistant to decomposition and incineration. The palatal rugae are characteristic to every individual. Just like the fingerprints, lip prints are permanent and constant for every individual. Bite marks can be found either on the attacker or the perpetrator. Both lip prints and bite marks account for important evidence for human identification found at the site of crime in the cases of sexual abuse. As the oral cavity serves as a useful source of DNA, latter can be extracted from saliva, the oral mucosal cells and the teeth. This DNA is used for PCR analysis. Dental structures can prove as useful indictors in determining the individual’s chronological age by analysing the tooth development. The sexual dimorphism elicited by various teeth of the human dentition play a major role in sex determination of the individual. The various recent advances like- facial reconstruction, dental identification methods, tongue prints, ameloglyphics make forensic dentistry a convenient and an effective tool in human identification. Forensic dentistry should not only be restricted to the dentists but various team involved in investigations and justice should also be exposed to this arena to avoid any biased and negligent decision making.

Anomalies in Human Sex Determination: Usefulness of a Combined Cytogenetic Approach to Characterize an Additional Case with Xp Functional Disomy Associated with 46,XY Gonadal Dysgenesis

Disorders of sexual development (DSD) are a heterogeneous group of genital defects affecting chromosomal, gonadal and anatomical sex. 46,XY DSD is a subset of DSD which covers a wide range of phenotypes in which 46,XY gonadal dysgenesis (GD) is the most severe form. In this study, we report on the clinical and molecular cytogenetic findings of a study on a Tunisian girl with the syndromic form of 46,XY DSD. This case was a phenotypic female patient having several congenital anomalies including growth retardation. Karyotype, fluorescence in situ hybridization and array Comparative Genome Hybridization (array CGH) were performed. The proband exhibited a de-novo 46,X,der(Y) karyotype. Array CGH revealed a pathogenic 27.5Mb gain of an Xp21.2 chromosome segment leading to Xp functional disomy. No deletion was observed in the Y-chromosome. The duplicated region encompassed the NR0B1 (DAX1) and MAGEB genes, located within the dosage sensitive sex (DSS) reversal locus, known as promote genes responsible for human sex reversal and testis repression. The extra-dosage and interactions of these genes with different specific genes could result in the impairment of the male sex pathway. Over-dosage of KAL1 and IL1RAPL1 genes fall within the somatic features observed in the patient. To the best of our knowledge, we report on the fourth case of Xp21.2-pter duplication within Xp;Yp translocation associated with XY GD. Our findings suggest that when duplicated, the NR0B1 and MAGEB genes could be a major cause of XY GD. Therefore, we emphasize the usefulness of a combined cytogenetic approach in order to provide an accurate genetic diagnosis for those patients having syndromic XY DSD in a clinical setting.

Sex Determination and the Human Person

Abstract: For many species that reproduce sexually, how sex is expressed at different points across lifespan is highly contingent and dependent on various environmental factors. For example, in many species of fish, environmental cues can trigger a natural process of sex transition where a female transitions to male. For many species of turtle, incubation temperature influences the likelihood that turtle eggs will hatch males or females. What is the case for Homo sapiens? Is human sex expression influenced by contingent environmental factors like we see in fish and turtles, with whom we share common ancestry and DNA? Our paper explores the current biological science of sex determination and how it applies to philosophical and theological accounts of the human person. We argue that while human sex determination is not susceptible to environmental cues to the same degree we see in other species, there is sufficient variability among the pathways of human sex development to complicate simplistic biological categories of male and female.

The role of genetics and epigenetics in sex differences in human survival

Sex differences in human survival have been extensively investigated in many studies that have in part uncovered the biological determinants that promote a longer life in females with respect to males. Moreover, researches performed in the past years have prompted increased awareness about the biological effects of environmental factors that can modulate the magnitude of the sex gap in survival. Besides the genetic background, epigenetic modifications like DNA methylation, that can modulate cell function, have been particularly studied in this framework. In this review, we aim to summarize the role of the genetic and epigenetic mechanisms in promoting female advantage from the early in life (“INNATE” features), and in influencing the magnitude of the gap in sex differences in survival and ageing (“VARIABLE” features). After briefly discussing the biological bases of sex determination in humans, we will provide much evidence showing that (i) “innate” mechanisms common to all males and to all females (both genetic and epigenetic) play a major role in sex differences in lifespan; (ii) “variable” genetic and epigenetic patterns, that vary according to context, populations and exposures to different environments, can affect the magnitude of the gap in sex differences in survival. Then we will describe recent findings in the use of epigenetic clocks to uncover sex differences in biological age and thus potentially in mortality. In conclusion, we will discuss how environmental factors cannot be kept apart from the biological factors providing evidence from the field of human ecology.

Human Sex determination from the dental pulp tissue using Polymerase Chain Reaction (PCR)

Human Sex determination from the dental pulp tissue using Polymerase Chain Reaction (PCR)

Tenuous transcriptional threshold of human sex determination. II. SRY exploits water-mediated clamp at the edge of ambiguity.

Y-encoded transcription factor SRY initiates male differentiation in therian mammals. This factor contains a high-mobility-group (HMG) box, which mediates sequence-specific DNA binding with sharp DNA bending. A companion article in this issue described sex-reversal mutations at box position 72 (residue 127 in human SRY), invariant as Tyr among mammalian orthologs. Although not contacting DNA, the aromatic ring seals the domain's minor wing at a solvent-exposed junction with a basic tail. A seeming paradox was posed by the native-like biochemical properties of inherited Swyer variant Y72F: its near-native gene-regulatory activity is consistent with the father's male development, but at odds with the daughter's XY female somatic phenotype. Surprisingly, aromatic rings (Y72, F72 or W72) confer higher transcriptional activity than do basic or polar side chains generally observed at solvated DNA interfaces (Arg, Lys, His or Gln). Whereas biophysical studies (time-resolved fluorescence resonance energy transfer and heteronuclear NMR spectroscopy) uncovered only subtle perturbations, dissociation of the Y72F complex was markedly accelerated relative to wild-type. Studies of protein-DNA solvation by molecular-dynamics (MD) simulations of an homologous high-resolution crystal structure (SOX18) suggest that Y72 para-OH anchors a network of water molecules at the tail-DNA interface, perturbed in the variant in association with nonlocal conformational fluctuations. Loss of the Y72 anchor among SRY variants presumably "unclamps" its basic tail, leading to (a) rapid DNA dissociation despite native affinity and (b) attenuated transcriptional activity at the edge of sexual ambiguity. Conservation of Y72 suggests that this water-mediated clamp operates generally among SRY and metazoan SOX domains.

Tenuous Transcriptional Threshold of Human Sex Determination. I. SRY and Swyer Syndrome at the Edge of Ambiguity.

Male sex determination in mammals is initiated by SRY, a Y-encoded transcription factor. The protein contains a high-mobility-group (HMG) box mediating sequence-specific DNA bending. Mutations causing XY gonadal dysgenesis (Swyer syndrome) cluster in the box and ordinarily arise de novo. Rare inherited variants lead to male development in one genetic background (the father) but not another (his sterile XY daughter). De novo and inherited mutations occur at an invariant Tyr adjoining the motif’s basic tail (box position 72; Y127 in SRY). In SRY-responsive cell lines CH34 and LNCaP, de novo mutations Y127H and Y127C reduced SRY activity (as assessed by transcriptional activation of principal target gene Sox9) by 5- and 8-fold, respectively. Whereas Y127H impaired testis-specific enhancer assembly, Y127C caused accelerated proteasomal proteolysis; activity was in part rescued by proteasome inhibition. Inherited variant Y127F was better tolerated: its expression was unperturbed, and activity was reduced by only twofold, a threshold similar to other inherited variants. Biochemical studies of wild-type (WT) and variant HMG boxes demonstrated similar specific DNA affinities (within a twofold range), with only subtle differences in sharp DNA bending as probed by permutation gel electrophoresis and fluorescence resonance-energy transfer (FRET); thermodynamic stabilities of the free boxes were essentially identical. Such modest perturbations are within the range of species variation. Whereas our cell-based findings rationalize the de novo genotype-phenotype relationships, a molecular understanding of inherited mutation Y127F remains elusive. Our companion study uncovers cryptic biophysical perturbations suggesting that the para-OH group of Y127 anchors a novel water-mediated DNA clamp.

P-793 The effect of Nuclear Receptor Subfamily 5 Group A Member 1 (NR5A1) in human ovarian somatic cell development

Abstract Study question Can an inducible NR5A1 (encoding Steroidogenic Factor 1) cell line be used to study the functional role of NR5A1 in human female gonadal differentiation? Summary answer Conditional induction of NR5A1 in a human pluripotent stem cell (hPSC)-based model of gonadal differentiation allows us to study its effect on the differentiating cells. What is known already According to animal studies, NR5A1 is a critical factor in gonadal development. Previous research in our lab showed that CRISPR/Cas9 induced upregulation of NR5A1 during gonadal differentiation in male (XY) hPSCs increases expression of early Sertoli cell markers such as Inhibin A and Anti-Müllerian hormone (AMH) and thus pushes the cells into a more mature gonadal cell type. However, the role of NR5A1 in female gonadal differentiation has yet to be determined. Study design, size, duration The female (XX) human embryonic stem cell (hESC) line, H9, was edited using CRISPR/Cas9 to conditionally induce NR5A1. These cells were subjected to a 10/20-day gonadal differentiation protocol where NR5A1 was induced through addition of Doxycycline (DOX) and Trimethoprim (TMP) from day 4 until day 10/20. Starting from day 4 technical duplicates/triplicates from every second day were subjected to qRT-PCR. Technical triplicates from day 4, 6, 8 and 10 were subjected to bulk RNA-sequencing. Participants/materials, setting, methods The cell line that was used in this project is a female hESC line, H9 (WiCell). A previously established protocol for gonadal differentiation (Sepponen et al., 2017) was followed wherein different cell culture media and matrices were supplemented with small molecules, inhibitors and growth factors. The CRISPR/Cas9 genome-editing system was used to generate the NR5A1 inducible cell line. Cell characterisation was performed using qRT-PCR and bulk RNA sequencing (NovaSeq S4) methods. Main results and the role of chance qRT-PCR results showed that without NR5A1 induction there was almost no expression of this gene, but its expression increased drastically upon addition of DOX and TMP to the culture media. NR5A1 induction seemed to upregulate FOXL2 and WNT4, which are crucial genes for female gonadal differentiation, although the increase was not statistically significant. AMH showed a significant increase on day 8 of differentiation after NR5A1 induction. We also saw an increase in the expression of steroidogenic genes such as STAR, CYP11A1, CYP17A1 and CYP19A1 after NR5A1 induction. Subjecting the cells to NR5A1 induction for an additional 10 days resulted in the upregulation of bipotential gonadal markers GATA4, WT1 and EMX2 after NR5A1 induction. Similarly, the female gonadal markers RSPO1 and WNT4 were upregulated over time and WNT4 expression further increased upon NR5A1 induction. NovaSeq S4 data showed that on day 8, after induction of NR5A1, genes related to reproductive system development and female pregnancy were upregulated. These included DHH, NR5A1, CYP19A1, AR, INHA and NR0B1. On day 10 genes related to reproductive system development such as FOXL2, NR5A1, INHA, LHCGR, and NR0B1 were also upregulated after NR5A1 induction. Limitations, reasons for caution The effect of NR5A1 induction on expression levels of bipotential gonadal markers GATA4, LHX9, WT1, EMX2 and female gonadal markers FOXL2, RSPO1, WNT4 was small in comparison to differentiation without additional NR5A1 induction. Moreover, we lack a protocol to generate bipotential gonadal cells expressing NR5A1 without inducing NR5A1 through CRISPR/Cas9. Wider implications of the findings Taken together the results will provide valuable insight and novel information on the function of NR5A1 in early female gonadal development and more in general on human ovarian development and sex determination. This knowledge will increase our understanding of pathological gonadal development, which often underlies disorders of sex development. Trial registration number Not applicable

493 The use of visual arts to teach complex ideas on non-binary sex/gender traits and identities.

OBJECTIVES/GOALS: Recent scientific discoveries show that human sex determination and differentiation is a spectrum of developmental processes and that sex/gender traits and identities not always fit binarity. This study aims to determine whether the visual arts can effectively transmit these complex scientific ideas on sex and gender variance. METHODS/STUDY POPULATION: A one-hour lecture for undergraduate students enrolled in a behavioral neuroendocrinology course included 17th century Spanish paintings and representative work from contemporary LGBTQIA+ artists in photography. Pre and post self-evaluation was conducted through five multiple choice questions. Chi Square test was employed for statistical analysis, which required elimination of no responses from item analysis. Statistical significance was defined as p value < 0.05. Three artistic images and two scientific images were included in the questions. Acquisition of content-specific knowledge on diverse body configurations, differences of sex development (DSD), transsexuality, histopathology of the gonad and neuroanatomy of the hypothalamus was evaluated. RESULTS/ANTICIPATED RESULTS: Twenty students completed the online pre- and post-test. We found that a 17th century Spanish painting was effective at making the distinction between transsexuality and intersexuality (p < 0.0005). Similarly, an artistic image that reinterprets the Vitruvius Man was effective for presenting the argument that diverse body configurations does not imply clinical pathology (p < 0.0001). Last, a scientific image showing the histopathology of an ovotestis was effective for introducing undergraduate students to diagnostic criteria for DSD (p < 0.02). No significant differences were found in the use of an artistic photograph depicting a young female to male transsexual individual nor in the use of a scientific image showing the neuroanatomical localization of the hypothalamus. DISCUSSION/SIGNIFICANCE: The learning of complex scientific concepts on human sex determination and differentiation can be affected by preconceived ideas, values, and attitudes towards sex and gender variance. The visual arts can provide a familiar ground of understanding between teachers and learners to transform such preconceptions.

Sex-Related Effects on Cardiac Development and Disease.

Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality. Interestingly, male and female patients with CVD exhibit distinct epidemiological and pathophysiological characteristics, implying a potentially important role for primary and secondary sex determination factors in heart development, aging, disease and therapeutic responses. Here, we provide a concise review of the field and discuss current gaps in knowledge as a step towards elucidating the “sex determination–heart axis”. We specifically focus on cardiovascular manifestations of abnormal sex determination in humans, such as in Turner and Klinefelter syndromes, as well as on the differences in cardiac regenerative potential between species with plastic and non-plastic sexual phenotypes. Sex-biased cardiac repair mechanisms are also discussed with a focus on the role of the steroid hormone 17β-estradiol. Understanding the “sex determination–heart axis” may offer new therapeutic possibilities for enhanced cardiac regeneration and/or repair post-injury.

Research Article MQS-PCR: a simple and quick multiplex PCR molecular method for human chromosomal sex determination in DNA samples

Research Article MQS-PCR: a simple and quick multiplex PCR molecular method for human chromosomal sex determination in DNA samples

Raman spectroscopy of fingerprints and chemometric analysis for forensic sex determination in humans

From the late 90 s until recently, some forensic research has been dedicated to the development of analytical techniques to explore the chemical components present in fingerprints, in order to find other information besides authorship. Raman spectroscopy is a technique of nondestructive analysis of a wide variety of forensic evidence, including fingerprints, at the crime scene. In this context, the aim of this work is to explore Raman spectroscopy and the supervised methods, Partial Least Squares and Support Vector Machine for Discriminant Analysis (PLSDA and SVMDA, respectively), as means to determine sex based on fingerprints obtained from male and female donors and submitted to different conditions (dark and light). Considering a period up to seven days from the collection of the fingerprint, the results showed correct discrimination rates ranging from approximately 80–93%.

Expanding the spectrum of syndromic PPP2R3C-related XY gonadal dysgenesis to XX gonadal dysgenesis.

Homozygous variants in PPP2R3C have been reported to cause a syndromic 46,XY complete gonadal dysgenesis phenotype with extragonadal manifestations (GDRM, MIM# 618419) in patients from four unrelated families, whereas heterozygous variants have been linked to reduced fertility with teratozoospermia (SPGF36, MIM# 618420) in male carriers. We present eight patients from four unrelated families of Turkish and Indian descent with three different germline homozygous PPP2R3C variants including a novel in-frame duplication (c.639_647dupTTTCTACTC, p.Ser216_Tyr218dup). All patients exhibit recognizable facial dysmorphisms allowing gestalt diagnosis. In two 46,XX patients with hypergonadotropic hypogonadism and nonvisualized gonads, primary amenorrhea along with absence of secondary sexual characteristics and/or unique facial gestalt led to the diagnosis. 46,XY affected individuals displayed a spectrum of external genital phenotypes from ambiguous genitalia to complete female. We expand the spectrum of syndromic PPP2R3C-related XY gonadal dysgenesis to both XY and XX gonadal dysgenesis. Our findings supported neither ocular nor muscular involvement as major criteria of the syndrome. We also did not encounter infertility problems in the carriers. Since both XX and XY individuals were affected, we hypothesize that PPP2R3C is essential in the early signaling cascades controlling sex determination in humans.

Identification and functional analysis of fourteen NR5A1 variants in patients with the 46 XY disorders of sex development

Human sex determination and differentiation is a complex process, during which NR5A1 plays a central role via the transcriptional regulation of key modulators involved in steroidogenesis. Approximately 8-15% of 46,XY DSD are caused by variants in the NR5A1 gene. Therefore, screening for variants in the NR5A1 gene was performed in a Chinese cohort of sixty-two 46,XY DSD patients with no AR or SRD5A2 variants via next-generation sequencing (NGS). Fourteen variants in the NR5A1 gene were identified in 16 patients from 14 unrelated families, including nine novel variants. These variants included eight heterozygote missense variants, two heterozygote frameshift variants, two heterozygote nonsense variants, one heterozygote nonframeshift deletion-insertion variant, and one homozygous missense variant. Functional assays showed that the transcriptional activity of the 11 variants was significantly reduced. In this study, 11 NR5A1 pathogenic variants were identified. These novel variants further expand the existing spectrum of the NR5A1 variants associated with 46,XY DSD, which will, in turn, assist in the molecular diagnosis of DSD.

SUN-034 Combined CNV, Haplotyping and Whole Exome Sequencing Implicates Inherited Novel SRY Mutations Not Account for Familial 46,XY Sex Reversal

SRY is one of the important genes involved in the process of human sex determination. The disturbed sex determination caused by SRY mutation accounts for 10-15% cases with complete gonadal dysplasia (CGD), also known as 46, XY sex reversal. Recently, three distal enhancers are disclosed in the upstream of SOX9 gene. In an inherited 46, XY sex reversal pedigree with 5 patients, p.Arg76Leu mutation of SRY and p.G212S mutation of NR5A1were identified from the proband who present with primary amenorrhea and lack of puberty development. The missense mutation of NR5A1was found to be derived from the mother. Interestingly, the paternal inherited p.Arg76Leu mutation of SRY was revealed from other 2 CGD patients, as well as from apparent normal male family members with fertility. P.Arg76Leu variation was found have no effect to the transcriptional activity of target gene SOX9, neither alteration of the nuclear translocation of SRY. Whole exome sequencing also found SRY mutation, FGF10 mutation, GJB4 gene mutation, etc. with no segregation in the family, which suggested SNVs are not main cause of the disease in this pedigree. By copy number variation and SNP haplotype analysis, SOX9 gene far upstream deletion of 68kb was disclosed from the 3 patients in this family, containing one of the enhancers of SOX9. Real-time PCR confirmed that the heterozygous deletion of the region result in loss of SR-XY, but not eSR-B and eALDI. Therefore, single nucleotide variation (SNV) of SRY and NR5A1 are not main causes of severe phenotype of CGD, the enhancers of SOX9 should be investigated carefully in such patients.

Optimization of Method for Human Sex Determination Using Peptidome Analysis of Teeth Enamel from Teeth of Different Biological Generation, Archeological Age, and Degrees of Taphonomic Preservation.

Determination of biological sex to human remains is a fundamental requirement in anthropological, archeological, and forensic anthropological studies. Sex determination based on morphological criteria is significantly limited in the cases of juvenile remains and adult skeletons in a poor state of preservation. Regular attempts have been made to use alternative techniques to resolve this issue, including analysis of tooth enamel peptides by liquid chromatography/mass spectrometry. Optimization of this method involving acid etching of tooth enamel for 10 min followed by desalting of the products of etching on SDB-RPS StageTips microcolumns and analysis of desalted sample (1/3) by liquid chromatography/mass spectrometry allowed reliable sex determination to fossil remains within a wide range of archeological and biological ages without destructing analyzed teeth. Increasing the duration of enamel etching ensured a 2 to 3-fold increase in the total number of identified peptides and, more importantly, in the number of identified fragments of amelogenin Y isoform specific for male teeth, which facilitated reliable sex determination of fossil remains. The suggested technique was tested with 8 permanent and 15 deciduous teeth of different archaeological age and different degree of preservation. Two amelogenin Y-specific peptide sequences were identified. One of these peptides [SM(+15.99)IRPPYS)] was found in all male-derived samples without exception; the other peptide [IRPPYSS(+79.97)], which contained phosphorylated Ser66 residue, was found only in the enamel from deciduous teeth, which suggests that phosphorylation of Ser66 plays a role in the enamel formation in deciduous teeth.

Accurate Sex Identification of Ancient Elephant and Other Animal Remains Using Low-Coverage DNA Shotgun Sequencing Data.

Sex identification of ancient animal biological remains can benefit our understanding of historical population structure, demography and social behavior. Traditional methods for sex identification (e.g., osteological and morphometric comparisons) may be ineffective when animal remains are not well preserved, when sex distinguishing characteristics have not yet developed, or where organisms do not exhibit sex-associated phenotypic dimorphisms. Here we adapt a method developed for human sex determination so that it can be used to identify the sex of ancient and modern animal taxa. The method identifies sex by calculating the ratio of DNA reads aligning to the X chromosome to DNA reads aligning to autosomes (termed the Rx ratio). We tested the accuracy of this method using low coverage genomes from 15 modern elephants (Loxodonta africana) for which sex was known. We then applied this method to ancient elephant ivory samples for which sex was unknown, and describe how this method can be further adapted to the genomes of other taxa. This method may be especially useful when only low-coverage genomic data are obtainable. Furthermore, because this method relies on only the X and not the Y chromosome, it can be used to determine the sex of organisms for which a reference genome was obtained from a female or for which only the X chromosome is reported. Such taxa include the domestic cat, sheep, goat, and horse; and non-domesticated animals such as the Sumatran orangutan, western lowland gorilla and meerkat.

Alternatives to amelogenin markers for sex determination in humans and their forensic relevance.

Forensic DNA typing and subsequent molecular methods of sex determination in humans have been proven to be an imperious tool to criminal justice system. In current practice, most of the short tandem repeat (STR) based commercial kits contain amelogenin as the sexing marker. Amelogenin gene which contributes to the tooth enamel formation is present on both X and Y chromosome with a variation in base pair size. However, huge discrepancies have been observed with amelogenin based sex determination mostly due to X and Y deletion in the population and mutation in primer binding sites. Some ethnicities such as those in Indian population are affected badly with inappropriate sex determination by amelogenin marker due to the presence of high frequency of Y deletion in the population. Presence of PCR inhibitors, degradation in the DNA samples and presence of mixed DNA also contribute to the discrepancy in results obtained by amelogenin analysis. To overcome this problem, many alternative markers/techniques such as STS, SRY, TSPY, DXYS156, SNPs, DYZ1 and Next generation sequencing have been discussed in much detail with their respective pros and cons. In this regard, inclusion of one or more alternative markers along with amelogenin will decrease the anomalies in sex determination observed while using the amelogenin marker alone in forensic sample analysis.

Application of tpsDig2 Software in Nasal Angle Measurements.

Nose is considered to be a conspicuous feature of human face. Its different parameters like shape, size, nasal angles etc may indicate one's gender, age, race, adapted climatic conditions, and the environment to which one belongs to. Furthermore, it is an important component and determinant of an individual's physical outlook and appearance. The current study provides a new approach for the assistance of anthropologists and forensic experts in human identification and sex determination from the nasal angles. The study was performed on a total of 500 subjects (250 males and 250 females) belonging to north India (Mandi, Himachal Pradesh State). In comparison to the classical and manual methods for nasal angle measurements, an automated approach was followed in the present study. A Windows-based software called tpsDig2 was used for nasal angle measurements by utilization of the landmarks (nasion, pronasale, subnasale, and alare). Three nasal angles (nasal prominence angle, inter-alar angle, and nasal tip angle) were measured in all the subjects and each measurement was repeated thrice to reduce the measurement error and increase the specificity and efficiency of the results. Discriminant function statistics was used for determination of sex from different nasal angles. The results of the statistical analysis (ANOVA) performed using SPSS reveal the significant difference between males and females in all the 3 repeated measurements of nasal angles. The final discriminant classification statistics show that 64% of original grouped cases have been correctly classified and the designed statistical model can be used in several forensic scenarios for the sex determination on the basis of the available nasal angles. The study may be further useful in the identification cases pertaining to facial reconstruction. The investigation may also be helpful in providing specific correlation of the nasal angles with the other parts of the face in the cases of remodelling and reformation of craniofacial alterations and reconstructions in plastic surgery and aesthetics.