ABSTRACT
The sex-determining gene SRY has undergone rapid evolution in rodents. Curiously, a new study by Miyawaki et al. reveals that a recently evolved SRY gene sequence antagonizes SRY protein stability, necessitating splicing of a novel intron. Other data suggest that this troublesome gene region has noncoding RNA functions, possibly related to conflict between sex chromosomes.
Subject(s)
Genes, sry/genetics , Genome/genetics , RNA, Untranslated/genetics , Rodentia/genetics , Sex Determination Processes/genetics , Amino Acid Sequence , Animals , Evolution, Molecular , Introns/genetics , Phylogeny , Sex Chromosomes/geneticsABSTRACT
The sex-determining region on the Y chromosome (SRY) is thought to be the central genetic element of male sex development in mammals. Pathogenic modifications within the SRY gene are associated with a male-to-female sex reversal syndrome in humans and other mammalian species, including rabbits and mice. However, the underlying mechanisms are largely unknown. To understand the biological function of the SRY gene, a site-directed mutational analysis is required to investigate associated phenotypic changes at the molecular, cellular, and morphological level. Here, we successfully generated a knockout of the porcine SRY gene by microinjection of two CRISPR-Cas ribonucleoproteins, targeting the centrally located "high mobility group" (HMG), followed by a frameshift mutation of the downstream SRY sequence. This resulted in the development of genetically male (XY) pigs with complete external and internal female genitalia, which, however, were significantly smaller than in 9-mo-old age-matched control females. Quantitative digital PCR analysis revealed a duplication of the SRY locus in Landrace pigs similar to the known palindromic duplication in Duroc breeds. Our study demonstrates the central role of the HMG domain in the SRY gene in male porcine sex determination. This proof-of-principle study could assist in solving the problem of sex preference in agriculture to improve animal welfare. Moreover, it establishes a large animal model that is more comparable to humans with regard to genetics, physiology, and anatomy, which is pivotal for longitudinal studies to unravel mammalian sex determination and relevant for the development of new interventions for human sex development disorders.
Subject(s)
Sex Determination Processes/genetics , Sex-Determining Region Y Protein/genetics , Sex-Determining Region Y Protein/metabolism , Amino Acid Sequence/genetics , Animals , DNA-Binding Proteins/genetics , Disorders of Sex Development/genetics , Frameshift Mutation/genetics , Genes, sry/genetics , HMG-Box Domains/genetics , Male , Mutation/genetics , Nuclear Proteins/genetics , Proof of Concept Study , Protein Domains/genetics , Swine/genetics , Transcription Factors/genetics , Y Chromosome/geneticsABSTRACT
The SRY initiates cascade of gene expression that transforms the undifferentiated gonad, genital ridge into testis. Mutations of the SRY gene is associated with complete gonadal dysgenesis in females with 46,XY karyotype. Primary amenorrhea is one of the clinical findings to express the genetic cause in 46,XY sex reversal. Here, we report a 26-year-old married woman presenting with primary amenorhea and complete gonadal dysgenesis. The clinical phenotypes were hypoplastic uterus with streak gonad and underdeveloped secondary sexual characters. The cytogenetic analysis confirmed 46,XY sex reversal karyotype of a female. Using molecular approach, we screened open reading frame of the SRY gene by PCR and targeted DNA Sanger sequencing. The patient was confirmed with nucleotide substitution (c.226C>A; p.Arg76Ser) at in HMG box domain of SRY gene that causes 46,XY sex reversal female. Mutation prediction algorithms suggest that alteration might be disease causing mutation and mutated (p.Arg76Ser) amino acid deleteriously affects HMG box nNLS region of SRY protein. Clinical phenotypes and in silico analysis confirmed that missense substitution (p.Arg76Ser) impaired nNLS binding Calmodulin-mediated nuclear transport of SRY from cytoplasm to nucleus. The mutation affects down regulation of male sex differentiation pathway and is responsible for 46,XY sex reversal female with gonadal dysgenesis.
Subject(s)
Gonadal Dysgenesis, 46,XY , Gonadal Dysgenesis , Adult , Base Sequence , Female , Genes, sry/genetics , Gonadal Dysgenesis, 46,XY/genetics , Humans , Male , Mutation , Mutation, Missense , Sex-Determining Region Y Protein/geneticsABSTRACT
Y chromosomes underlie sex determination in mammals, but their repeat-rich nature has hampered sequencing and associated evolutionary studies. Here we trace Y evolution across 15 representative mammals on the basis of high-throughput genome and transcriptome sequencing. We uncover three independent sex chromosome originations in mammals and birds (the outgroup). The original placental and marsupial (therian) Y, containing the sex-determining gene SRY, emerged in the therian ancestor approximately 180 million years ago, in parallel with the first of five monotreme Y chromosomes, carrying the probable sex-determining gene AMH. The avian W chromosome arose approximately 140 million years ago in the bird ancestor. The small Y/W gene repertoires, enriched in regulatory functions, were rapidly defined following stratification (recombination arrest) and erosion events and have remained considerably stable. Despite expression decreases in therians, Y/W genes show notable conservation of proto-sex chromosome expression patterns, although various Y genes evolved testis-specificities through differential regulatory decay. Thus, although some genes evolved novel functions through spatial/temporal expression shifts, most Y genes probably endured, at least initially, because of dosage constraints.
Subject(s)
Evolution, Molecular , Mammals/genetics , Y Chromosome/genetics , Animals , Birds/genetics , Conserved Sequence/genetics , Female , Gene Dosage/genetics , Genes, sry/genetics , Genomics , High-Throughput Nucleotide Sequencing , Male , Marsupialia/genetics , Receptors, Peptide/genetics , Receptors, Transforming Growth Factor beta/genetics , Selection, Genetic/genetics , Sex Chromosomes/genetics , Spatio-Temporal Analysis , Spermatogenesis/genetics , Testis/metabolism , Transcriptome/geneticsABSTRACT
46,XX testicular disorder of sex development (46,XX TDSD) is a relatively rare condition characterised by the presence of testicular tissue with 46,XX karyotype. The present study aims to reveal the phenotype to genotype correlation in a series of sex-determining region Y (SRY)-positive 46,XX TDSD cases. We present the clinical findings, hormone profiles and genetic test results of six patients with SRY-positive 46,XX TDSD and give the details and follow-up findings of our three of previously published patients. All patients presented common characteristics such as azoospermia, hypergonadotropic hypogonadism and an SRY gene translocated on the terminal part of the short arm of one of the X chromosomes. Mean ± standard deviation (SD) height of the patients was 164.78 ± 8.0 cm. Five patients had decreased secondary sexual characteristics, and three patients had gynaecomastia with varying degrees. Five of the seven patients revealed a translocation between protein kinase X (PRKX) and inverted protein kinase Y (PRKY) genes, and the remaining two patients showed a translocation between the pseudoautosomal region 1 (PAR1) of X chromosome and the differential region of Y chromosome. X chromosome inactivation (XCI) analysis results demonstrated random and skewed XCI in 5 cases and 1 case, respectively. In brief, we delineate the phenotypic spectrum of patients with SRY-positive 46,XX TDSD and the underlying mechanisms of Xp;Yp translocations.
Subject(s)
Genes, sry , Testicular Diseases , Genes, sry/genetics , Humans , Karyotyping , Male , Phenotype , Translocation, GeneticABSTRACT
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 polymerase chain reaction (PCR)-based sexing method is generally favoured, 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 glyceraldehyde 3-phosphate dehydrogenase (GAPDH), sex-determining region Y (SRY) and amelogenin genes present in genomic DNA (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.
Subject(s)
Polymerase Chain Reaction/veterinary , Sex Determination Analysis/veterinary , Sheep/embryology , Amelogenin/genetics , Amelogenin/metabolism , Animals , DNA/blood , DNA/genetics , Embryo, Mammalian , Female , Genes, sry/genetics , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Male , Polymerase Chain Reaction/methods , Sex Determination Analysis/methodsABSTRACT
Aim To highlight the complexity of infertility causes by describing the rare case of a man with a testicular disorder of sexual differentiation. Diagnosis A 33 years old Caucasian male presented with a 3-year-old history of primary infertility. His investigations revealed a low testosterone and a raised LH and FSH levels. A sample sent for sperm analysis revealed azoospermia. Chromosomal analysis and karyotyping revealed a 46 XX SRY positive karyotype. Treatment The patient was initiated on testosterone replacement and on calcium/vitamin D supplements. Conclusion Fertility evaluation requires complex assessments and a broad knowledge of possible causes.
Subject(s)
Abnormal Karyotype , Disorders of Sex Development/complications , Disorders of Sex Development/genetics , Genes, sry/genetics , Infertility, Male/etiology , Infertility, Male/genetics , Sex Differentiation/genetics , Translocation, Genetic/genetics , Adult , Azoospermia/etiology , Azoospermia/genetics , Follicle Stimulating Hormone/metabolism , Humans , Karyotyping , Luteinizing Hormone/metabolism , Male , Semen Analysis , Testosterone/deficiencyABSTRACT
Unbalanced translocations of Y-chromosomal fragments harboring the sex-determining region Y gene (SRY) to the X chromosome or an autosome result in 46,XX and 45,X testicular disorders of sex development (DSD), respectively. Of these, Y;autosome translocation is an extremely rare condition. Here, we identified a 20-year-old man with a 45,X,t(Y;7)(q11.21;q35) karyotype, who exhibited unilateral cryptorchidism, small testis, intellectual disability, and various congenital anomalies. The fusion junction of the translocation was blunt, and the breakpoint-flanking regions shared only 50% similarity. These results indicate that Y;autosome translocations can occur between 2 low-similarity sequences, probably via nonhomologous end joining. Furthermore, translocations of a Ypterq11.21 fragment to 7q35 likely result in normal or only mildly impaired male-type sexual development, along with various clinical features of 7q deletion syndrome, although their effects on adult testicular function remain to be studied.
Subject(s)
Chromosomes, Human, Pair 7/genetics , Chromosomes, Human, Y/genetics , Disorders of Sex Development/genetics , Genes, sry/genetics , Testicular Diseases/genetics , Translocation, Genetic/genetics , Adult , Chromosome Breakpoints , Female , Humans , In Situ Hybridization, Fluorescence , Infant, Newborn , Karyotype , Male , Young AdultABSTRACT
BACKGROUND: Sex reversal syndrome (SRS) is a human chromosomal abnormality disease with gender dysplasia, which is characterized by inconsistency between social sexuality and genetic sexuality. METHODS: We report a case of sex reversal syndrome with 46, XX. Chemiluminescence was used to detect serum sex hormones, including testosterone (T), luteinizing hormone (LH), and follicular stimulation (FSH), and 15 karyotype analysis. RESULTS: The levels of FSH and LH in serum were high, and the level of T in serum was low. The karyotype analysis showed that the nuclear type of the patient was 46, XX. The examination of the sex-determining region Y (SRY) gene showed positive results. CONCLUSIONS: The main principle of diagnosing the 46, XX male SRS is early determination of chromosome, gonad, and genitalia gender. When the prenatal ultrasound diagnosis of pregnant women is inconsistent with the results of cytogenetics, caution should be taken to avoid the birth of children with 46, XX male SRS.
Subject(s)
46, XX Testicular Disorders of Sex Development/genetics , Genes, sry/genetics , Sex Chromosome Aberrations , 46, XX Testicular Disorders of Sex Development/blood , Adult , Follicle Stimulating Hormone/blood , Humans , Infertility, Male/genetics , Luteinizing Hormone/blood , Male , Testosterone/bloodABSTRACT
BACKGROUND: The knowledge of the individual genetic "status" in the prenatal era is particularly relevant in the case of positive family history for genetic diseases, in advanced maternal age and in the general screening for foetal abnormalities. In this context, here, we report an innovative molecular assay which utilizes the cell-free foetal DNA (cffDNA) as a source for the early and fast detection of the foetal sex. The study involved 132 pregnant women in their first 3 months of pregnancy, who agreed to give a blood sample. All the collected samples were immediately subjected to the separation of the plasma, which was utilized for the extraction of the cffDNA. Successively, the extracted cffDNA was analysed by a quantitative PCR (qPCR) method based on Plexor-HY chemistry, which is able to simultaneously identify, quantify and discriminate the autosomal DNA from the sex-linked DNA. RESULTS: Overall, the Plexor-HY assay demonstrated to be sensitive and specific for the determination of low-template DNA, such as the cffDNA. In fact, the Plexor-HY assay has been successfully performed in all the samples, identifying 70 males and 62 females. As the foetal sex can be provided in 120 min just by utilizing a maternal blood sample as cffDNA source, the assay represents a very fast, safe and non-invasive prenatal method. CONCLUSIONS: The possibility of determining the foetal sex in the early prenatal life consents the application of our assay as a helpful screening test for subjects and families at risk of sex-linked disorders. Moreover, the early knowledge of the foetal sex may be of great help even for the specialist, who might promptly advise the patients concerning the foetal risk of inheriting sex-linked disorders and the clinical utility of performing an invasive prenatal diagnosis.
Subject(s)
DNA/genetics , Prenatal Diagnosis/methods , Sex Determination Analysis/methods , Sex Determination Processes , Adult , Female , Fetus , Genes, sry/genetics , Humans , Male , PregnancySubject(s)
Disorders of Sex Development , Dissent and Disputes , Sexual Development , Animals , Athletes/legislation & jurisprudence , Child , Chromosomes, Human, X/genetics , Chromosomes, Human, X/metabolism , Disorders of Sex Development/genetics , Disorders of Sex Development/metabolism , Disorders of Sex Development/psychology , Disorders of Sex Development/surgery , Evidence-Based Medicine/standards , Female , Genes, sry/genetics , History, 20th Century , History, 21st Century , Homosexuality, Male/genetics , Humans , Infant , Longitudinal Studies , Male , Mice , Mothers , Patient Advocacy/legislation & jurisprudence , Practice Guidelines as Topic/standards , Sexual Behavior , Sexual Development/genetics , Social Support , Testosterone/blood , United Nations , United StatesABSTRACT
Sex determining region Y gene (SRY) is located on Y chromosome and encodes a protein with 229 amino acids. In this study, ORF region of SRY with a length of 690 bp was synthesized using PCR and ligated to pET28a (+), then transformed in E.coli DH5α. E.coli BL21 (DE3) strain was chosen to express recombinant bovine SRY protein. A set of optimization steps was taken including different concentrations of IPTG, glucose, and temperatures at differed incubation times after the induction. Results showed that temperature points and different concentrations of IPTG and glucose had a significant effect (p < 0.01) on total protein and recombinant bovine SRY. After purification, various temperatures and concentrations of IPTG showed meaningful effects (p < 0.01) on the solubility of expressed recombinant SRY. Highest soluble rSRY protein amount was achieved where 0.5 mM IPTG and 0.5% glucose was used at 20°C during induction. In the absence of glucose, the highest amount of soluble recombinant SRY levels were achieved at the concentrations of 0.8 mM of IPTG at 28°C, 20°C, and 1.5 mM IPTG at 37°C during induction for 16, 24, and 8 hours, respectively. Regarding the results obtained in this study, it could be stated that by decreasing temperature and inducer concentration, soluble bovine SRY protein expression increases.
Subject(s)
Cattle/genetics , Genes, sry/genetics , Sex-Determining Region Y Protein/genetics , Y Chromosome/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Male , Open Reading Frames/genetics , Protein Domains , Recombinant Proteins , Sequence Analysis, DNA/veterinary , Sex-Determining Region Y Protein/isolation & purification , Sex-Determining Region Y Protein/metabolism , Solubility , TemperatureABSTRACT
OBJECTIVE: To explore the genetic cause of a female case with intellectual development disorder. METHODS: G banding karyotyping was performed for the patient. Following DNA extraction, the coding sequence of SRY gene was amplified with PCR and subjected to Sanger sequencing. qPCR was used to detect the copy numbers of the SRY gene. RESULTS: The karyotype of the patient was 47,XXY. PCR and qPCR analyses of the SRY gene showed a large deletion with null copy number. CONCLUSION: The female phenotype of the patient is probably due to deletion of the SRY gene on the Y chromosome. This is the first report of 47,XXY female case with deletion of the SRY gene in China.
Subject(s)
Chromosomes, Human, Y/genetics , Genes, sry/genetics , Intellectual Disability/genetics , Klinefelter Syndrome/genetics , Sequence Deletion , Base Sequence , Chromosome Banding , Female , Humans , Karyotype , Karyotyping , Male , Polymerase Chain Reaction , Review Literature as Topic , Sequence Analysis, DNA/methods , Sequence Homology, Nucleic AcidABSTRACT
Sex determination is a complex and dynamic process with multiple genetic and environmental causes, in which germ and somatic cells receive various sex-specific features. During the fifth week of fetal life, the bipotential embryonic gonad starts to develop in humans. In the bipotential gonadal tissue, certain cell groups start to differentiate to form the ovaries or testes. Despite considerable efforts and advances in identifying the mechanisms playing a role in sex determination and differentiation, the underlying mechanisms of the exact functions of many genes, gene-gene interactions, and epigenetic modifications that are involved in different stages of this cascade are not completely understood. This review aims at discussing current data on the genetic effects via genes and epigenetic mechanisms that affect the regulation of sex determination. Birth Defects Research (Part C) 108:321-336, 2016. © 2016 Wiley Periodicals, Inc.
Subject(s)
Sex Determination Processes/genetics , Sex Differentiation/genetics , Epigenomics , Female , Genes, sry/genetics , Gonads/physiology , Humans , Male , SOXE Transcription Factors/genetics , Sex Determination Analysis/methods , Transcription Factors/geneticsSubject(s)
Disorders of Sex Development/genetics , Disorders of Sex Development/physiopathology , Gonadal Steroid Hormones/metabolism , Sex Determination Processes/genetics , Sex Determination Processes/physiology , Sex , Androgen-Insensitivity Syndrome/genetics , Androgen-Insensitivity Syndrome/physiopathology , Animals , Chimera/genetics , Chimera/physiology , Chromosomes, Human, X/genetics , Chromosomes, Human, Y/genetics , Female , Genes, sry/genetics , Genotype , Gonads/physiology , Humans , Male , Mice , Mosaicism , Phenotype , Sex Characteristics , Sex Determination AnalysisABSTRACT
INTRODUCTION: The present case report describes a 6-year old subfertile pony mare, which became pregnant after the eleventh artificial insemination. The examination of the ovaries and the uterus did not reveal any abnormal clinical findings and the mare showed a regular oestrous cycle. Based on cytogenetic and molecular genetic analyses it became possible to elucidate the observed subfertility. The mosaic karyotype of the mare consisted of 63,X (20%) and 64,XX (80%) cells. A PCR analysis failed to amplify sequences from the equine SRY gene. The observed classic 63,X/64,XX mosaicism is a plausible explanation for the subfertility of the mare.
Subject(s)
Horses/genetics , Infertility, Female/veterinary , Mosaicism/veterinary , X Chromosome/genetics , Animals , Female , Genes, sry/genetics , Infertility, Female/genetics , Insemination, Artificial/veterinary , Karyotype , Polymerase Chain Reaction/veterinary , PregnancyABSTRACT
Sex chromosome genes directly influence sex differences in behavior. The discovery of the Sry gene on the Y chromosome (Gubbay et al., 1990; Koopman et al., 1990) substantiated the sex chromosome mechanistic link to sex differences. Moreover, the pronounced connection between X chromosome gene mutations and mental illness produces a strong sex bias in these diseases. Yet, the dominant explanation for sex differences continues to be the gonadal hormones. Here we review progress made on behavioral differences in mouse models that uncouple sex chromosome complement from gonadal sex. We conclude that many social and cognitive behaviors are modified by sex chromosome complement, and discuss the implications for human research. Future directions need to include identification of the genes involved and interactions with these genes and gonadal hormones.
Subject(s)
Behavior, Animal , Genes, sry/genetics , Sex Characteristics , Sex Chromosomes/genetics , Y Chromosome/genetics , Animals , Behavior, Animal/physiology , Disease Models, Animal , Humans , MiceSubject(s)
Cell-Free Nucleic Acids/analysis , Disorders of Sex Development/diagnostic imaging , Noninvasive Prenatal Testing , Patient Education as Topic , Ultrasonography, Prenatal , Adrenal Hyperplasia, Congenital/diagnosis , Adrenal Insufficiency/diagnosis , Amniocentesis , Androgens , Disorders of Sex Development/genetics , Female , Gender Identity , Genes, sry/genetics , Gonadal Dysgenesis, 46,XY/diagnosis , Humans , Male , Patient-Centered Care , Pregnancy , Pregnancy Trimester, Second , Receptors, Androgen/genetics , Translocation, Genetic , Virilism/diagnosisABSTRACT
BACKGROUND: 46,XX testicular disorder of sex development is a rare genetic syndrome, characterized by a complete or partial mismatch between genetic sex and phenotypic sex, which results in infertility because of the absence of the azoospermia factor region in the long arm of Y chromosome. CASE PRESENTATION: We report a case of a 14-year-old male with microorchidism and mild bilateral gynecomastia who referred to our hospital because of abnormal gender characteristics. The patient was treated for congenital scrotal type hypospadias at the age of 4 years. Semen analysis indicated azoospermia by centrifugation of ejaculate. Levels of follicle-stimulating hormone and luteinizing hormone were elevated, while that of testosterone was low and those of estradiol and prolactin were normal. The results of gonadal biopsy showed hyalinization of the seminiferous tubules, but there was no evidence of spermatogenic cells. Karyotype analysis of the patient confirmed 46,XX karyotype and fluorescent in situ hybridization analysis of the sex-determining region Y (SRY) gene was negative. Molecular analysis revealed that the SRY gene and the AZFa, AZFb and AZFc regions were absent. No mutation was detected in the coding region and exon/intron boundaries of the RSPO1, DAX1, SOX9, SOX3, SOX10, ROCK1, and DMRT genes, and no copy number variation in the whole genome sequence was found. CONCLUSION: This study adds a new case of SRY-negative 46,XX testicular disorder of sex development and further verifies the view that the absence of major regions from the Y chromosome leads to an incomplete masculine phenotype, abnormal hormone levels and infertility. To date, the mechanisms for induction of testicular tissue in 46,XX SRY-negative patients remain unknown, although other genetic or environmental factors play a significant role in the regulation of sex determination and differentiation.
Subject(s)
46, XX Testicular Disorders of Sex Development/genetics , Genes, sry/genetics , 46, XX Testicular Disorders of Sex Development/pathology , Adolescent , Gene Deletion , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Infertility, Male/genetics , Infertility, Male/pathology , Inhibins/analysis , Karyotyping , Male , Phenotype , Testis/pathology , Vimentin/analysisABSTRACT
Personal identification in forensics is possible with gender determination using DNA (deoxyribonucleic acid) analysis. DNA isolation from teeth samples subjected to extreme temperatures has been shown to predict the gender of the deceased. However, the literature lacks studies on DNA extracted from tooth samples exposed to freezing temperatures. This study aimed to isolate the SRY gene from the extirpated pulp of teeth that were subjected to varying temperatures for gender identification. Thirty teeth with vital pulps, divided into 3 groups were included in the study. Each group consisted of 5 male and 5 female tooth samples. The groups were exposed to diverse environmental factors for three weeks. Group 1: room temperature (R group); Group 2: high temperature (H group) and Group 3: freezing temperature (F group). Later, DNA was isolated from the pulp tissue, and the SRY gene was amplified using PCR (Polymerase Chain Reaction). The Sensitivity and Specificity of the results were analyzed. SRY gene detected in the study samples identified accurate gender with a 46.70% Sensitivity and 93.30% Specificity. Significant difference was found in the correlation between gene expression and gender among the three groups (p = 1.000). The study validates that dental pulp tissue can be a reliable source for DNA extraction. And SRY gene amplification from teeth exposed to diverse environmental conditions. Further investigations are required to validate its application in forensics.