Amelogenin Gene Research Articles

Sex determination by simultaneous amplification of equine SRY and amelogenin genes.

A quick method for sex determination of horses was developed. Simultaneous amplification of the equine sex-determining region of the Y chromosome gene (SRY) and amelogenin gene (AMEL) accomplished the determination of the presence of both the Y chromosome and SRY gene. In agarose gel electrophoresis, a normal stallion showed 1 SRY band and 3 AMEL (AMELX, AMELY, and AMELX/AMELY heteroduplex) bands, and a normal mare showed a single AMELX band. In XY-mares, 3 AMEL bands were detected as in a normal stallion, but no SRY band. The present method enables a quick diagnosis for XY-mare prior to cytogenetic analysis.

Sex Identification Using PCR with Non-invasive Samples in Chimpanzee, Bonobos and Japanese macaques

The sex was identified by the polymerase chain reaction (PCR) using DNA extracted from noninvasive samples, such as urine, feces, food wadges, and hair samples, of Pan troglodytes, Pan paniscus, and Macaca fuscata. The Amelogenin gene was successfully amplified by PCR, and X- and Y-specific DNA regions had different lengths. The ZFX and ZFY genes were also amplified by PCR, and restriction fragment length polymorphism (RFLP) depicted different patterns between the sexes. Sex identification using DNA from noninvasive samples obtained from unidentified wild animals will help to study the range and social structure of these species.

Personal Identification by DNA Analysis of Samples from Dental Prostheses Made of Acrylic Resin.

The procedures and possibilities for personal identification by DNA analysis of samples from dental prostheses made of acrylic resin were investigated. Acrylic resin pieces dipped in whole saliva for a moment, dried in air and left for at least two months at room temperature retained saliva stains that could be used as materials for DNA analysis for personal identification. The amounts of DNA extracted from 0.5×0.5×0.1cm resin pieces prepared from 55 resin prostheses used by the patients ranged from 35.7ng to 1.52μg and were significantly different among the samples. The amounts of extractable DNA did not correlate with the period of time the prostheses were left at room temperature or the period of time they were used in the oral cavity. Sex determination by amplification of segments of the amelogenin gene and typing of the 184bp fragment in the D4S43 locus was possible from all of the extracted DNA. Resin prostheses used in the oral cavity and left at room temperature for as long as approximately 200 days could be used for DNA extraction followed by DNA analysis. The present results provide a procedure that can be used from sampling of DNA from resin prostheses used in the oral cavity to typing by DNA analysis.

Sex determination based on fecal DNA analysis of the amelogenin gene in sika deer (Cervus nippon).

A sex determination method using DNA extracted from feces has been developed for sika deer (Cervus nippon). We determined a partial sequence of the amelogenin gene of sika deer, which exists on both X and Y chromosomes with a deletion region on the Y chromosome. Based on the sexually dimorphic sequences, we designed a pair of primers which could amplify DNA fragments the lengths of which are different between males and females. PCR products were detected in 34 out of 37 fecal samples collected from captured deer and the sexes estimated by the present method were perfectly matched with the actual sexes.

Mutational analysis of X-linked amelogenesis imperfecta in multiple families

Seven mutations in the amelogenin gene are associated with X-linked amelogenesis imperfecta. These mutations can produce reductions in the amount of enamel and the degree of mineralization. Two families have been identified from western North Carolina exhibiting features of amelogenesis imperfecta, characterized by brown enamel in affected males and interposed vertical bands of normal appearing and brown enamel in presumably heterozygous females. Mutational analysis reveals a C–A mutation in exon 6 at codon 41 of the X-chromosomal amelogenin gene, resulting in a pro–thr change in all individuals having the amelogenesis imperfecta phenotype. This mutation was previously reported in a family with X-linked hypomaturation amelogenesis imperfecta. There is no known relationship between any of the three families but the presence of similar phenotypes and common mutations suggests they may be distantly related. For individuals from all three families, the haplotype for six highly polymorphic loci flanking the amelogenin gene was determined. A common haplotype was demonstrated among two of the three families, suggesting that the mutation may have been inherited from a common ancestor. The finding that the third family had a distinct haplotype may indicate that the C–A mutation at codon 41 represents a mutational hotspot that occurs with greater frequency than other known amelogenin gene mutations. The phenotype resulting from this mutation was highly consistent in affected male members of the same family and between families.

Gender determination in single bovine blastomeres by polymerase chain reaction amplification of sex-specific polymorphic fragments in the amelogenin gene.

A sensitive technique for the sexing of bovine embryos was developed using polymerase chain reaction (PCR) amplification of the bovine amelogenin (bAML) gene on the X- and Y-chromosomes of Holstein dairy cattle. Cloning and DNA sequencing showed a 45.1% homology between the fifth intron of the bAML-X and bAML-Y gene with multiple deletions. A pair of sex-specific primers was designed to allow amplification of a single fragment of 467-bp from the X-chromosome of female cattle and two fragments of 467-bp and 341-bp from the X- and Y-chromosomes of male cattle. The primers were successfully applied to bovine sexing from single blastomeres isolated from day-6 to day-7 cow embryos by direct cell lysis and PCR. Our protocol of embryo sexing should be applicable to the diagnosis of defective genes in vitro in human embryos and in other domestic or recreational animals.

Gender determination in single bovine blastomeres by polymerase chain reaction amplification of sex‐specific polymorphic fragments in the amelogenin gene

Gender determination in single bovine blastomeres by polymerase chain reaction amplification of sex‐specific polymorphic fragments in the amelogenin gene

First-trimester fetal sex prediction by deoxyribonucleic acid analysis of maternal peripheral blood

Objectives: We investigated whether the number of weeks of gestation influences the accuracy of first-trimester fetal sex prediction by analysis of deoxyribonucleic acid extracted from whole maternal blood. A comparison was also made to determine whether a difference exists between this approach and the deoxyribonucleic acid analysis of transcervical cells performed on the same group of subjects. Study Design: Deoxyribonucleic acid was isolated from 50 maternal blood samples taken between gestational weeks 7 and 11. The sex of the fetus was assessed by nested polymerase chain reaction specific for the amelogenin gene. A receiver-operating characteristic curve analysis was used to correlate the accuracy of fetal gender prediction with the gestational age and also to compare the goodness of the 2 methods under investigation. Results: Analysis of the receiver-operating characteristic curve provided a cutoff value of 9 weeks 4 days of gestation for both tests, indicating that a higher degree of accuracy in the sex assignment was obtained in those samples taken before or at this time. However, this difference was statistically significant only for analysis of deoxyribonucleic acid from maternal blood. The comparison between tests of deoxyribonucleic acid from maternal blood and from transcervical cells showed that the first approach is better, although a statistically significant difference was not found. Conclusion: Analysis of maternal blood deoxyribonucleic acid is a better approach than analysis of trans-cervical cell deoxyribonucleic acid in fetal sex prediction. The highest degree of accuracy is obtained when blood is drawn before 10 weeks of gestation. This can be important when sampling of chorionic villi should be avoided because of the risk of an X-linked disease when the fetal sex is female. (Am J Obstet Gynecol 1999;181:675-80.)

Characterization of dentin matrix protein 1 gene in crocodilia.

Several clones containing DMP1 cDNA were isolated from a caiman tooth library by screening with a platypus DMP1 probe. The caiman DMP1 shows little amino acid sequence similarity to mammalian DMP1s for much of its length. A few highly conserved regions can, however, be identified that correspond to the slowly evolving parts of the corresponding mammalian genes. Southern blot analysis using probes comprising either conserved regions or longer segments of the gene indicates that only a single DMP1 locus exists. In coding regions, exon-intron boundaries and reading frames are shared by caiman and mammalian genes with the exception of exons 1 and 5, which are longer in the caiman. The repetitive sequence of the last exon is shared by mammals and caiman as are the high Ser content and acidity due to a high proportion of Asp and Glu residues. The conserved mammalian cell-attachment signal Arg-Gly-Asp is absent in the caiman DMP1. In contrast to the amelogenin gene, the DMP1 gene appears to evolve rapidly in vertebrates.

Gonadoblastoma, Testicular and Prostate Cancers, and the TSPY Gene

The role of the Y chromosome in oncogenesis of human cancers has been somewhat controversial. Both gain and loss of the Y chromosome in different leukemia, lymphoma, and solid tumors have been reported (Abeliovich et al. 1994; Brothman 1997; Dave et al. 1996; Geburek et al. 1997; Jackson-Cook et al. 1996; Kirk et al. 1994; Konig et al. 1994; Mertens et al. 1997; Riske et al. 1994; Watanabe et al. 1996). Conceivably, both oncogenes and tumor-suppressor genes exist on this chromosome, and they may act at different points during tumorigenesis, particularly for cancers of male-specific organs, such as the testis and the prostate.

Analysis of ancient DNA from a prehistoric Amerindian cemetery.

The Norris Farms No. 36 cemetery in central Illinois has been the subject of considerable archaeological and genetic research. Both mitochondrial DNA (mtDNA) and nuclear DNA have been examined in this 700-year-old population. DNA preservation at the site was good, with about 70% of the samples producing mtDNA results and approximately 15% yielding nuclear DNA data. All four of the major Amerindian mtDNA haplogroups were found, in addition to a fifth haplogroup. Sequences of the first hypervariable region of the mtDNA control region revealed a high level of diversity in the Norris Farms population and confirmed that the fifth haplogroup associates with Mongolian sequences and hence is probably authentic. Other than a possible reduction in the number of rare mtDNA lineages in many populations, it does not appear as if European contact significantly altered patterns of Amerindian mtDNA variation, despite the large decrease in population size that occurred. For nuclear DNA analysis, a novel method for DNA-based sex identification that uses nucleotide differences between the X and Y copies of the amelogenin gene was developed and applied successfully in approximately 20 individuals. Despite the well-known problems of poor DNA preservation and the ever-present possibility of contamination with modern DNA, genetic analysis of the Norris Farms No. 36 population demonstrates that ancient DNA can be a fruitful source of new insights into prehistoric populations.

Regulation of Amelogenin Gene Expression

The X-chromosomal amelogenin gene is expressed at a high level by ameloblast cells within the enamel organ for a short time during tooth development. Therefore, expression is both tooth specific and developmentally regulated. A Y-chromosomal amelogenin gene is also active in human and cow, but has not been detected in mouse. Genes and/or cDNAs have been cloned for mouse, human, cow, rat, pig, opossum, and hamster, and analyses have indicated that coding and upstream regions are conserved across species. Alternative splicing is extensive and produces as many as 9 mRNAs from the 7 exon murine gene, resulting from single and multiple exon skipping and alternate 3' site selection within exon 6. The pattern of alternative splicing varies both between species and during development, which is expected to result in some diversity through varying complements of amelogenin proteins associated with this highly conserved gene.

Validation of Capillary Electrophoresis for Analysis of the X-Y Homologous Amelogenin Gene

Capillary electrophoresis (CE) is a versatile technology with tremendous potential applications in forensic science. A refinement of the traditional slab-gel, separation occurs in a liquid polymer contained within a capillary. The capillary electrophoresis unit is computer-driven, semi-automating the loading and analysis of samples. As with any new technology, a full performance assessment is required before it may be implemented with confidence. This paper focuses on validation of CE technology for analysis of a portion of the X-Y homologous amelogenin gene used in gender determination. Typical Y/X ratios of peak heights for male samples were determined. Instrument linearity, sample resolution and reproducibility were examined. Samples subjected to contamination, extreme environmental conditions or extracted from a variety of substrates were also tested by CE. All samples typed correctly. Genetic material from a number of common non-primate animals was amplified with amelogenin primers. Some species yielded no product. Products derived from the animal samples that did amplify produced peaks on CE analysis readily distinguishable from those of human origin.

Identification and characterization of amelogenin genes in monotremes, reptiles, and amphibians.

Two features make the tooth an excellent model in the study of evolutionary innovations: the relative simplicity of its structure and the fact that the major tooth-forming genes have been identified in eutherian mammals. To understand the nature of the innovation at the molecular level, it is necessary to identify the homologs of tooth-forming genes in other vertebrates. As a first step toward this goal, homologs of the eutherian amelogenin gene have been cloned and characterized in selected species of monotremes (platypus and echidna), reptiles (caiman), and amphibians (African clawed toad). Comparisons of the homologs reveal that the amelogenin gene evolves quickly in the repeat region, in which numerous insertions and deletions have obliterated any similarity among the genes, and slowly in other regions. The gene organization, the distribution of hydrophobic and hydrophilic segments in the encoded protein, and several other features have been conserved throughout the evolution of the tetrapod amelogenin gene. Clones corresponding to one locus only were found in caiman, whereas the clawed toad possesses at least two amelogenin-encoding loci.

Molecular phylogenetics of the hominoid Y chromosome.

The Human Y-chromosome plays a central role in sex determination, and is composed of DNA sequences homologous to the Y-chromosome, families of Y-specific repetitive DNA sequences, and single copy sequences. We investigated the chromosomal location of Y-specific DNA sequences, in the chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), and orangutan (Pongo pygmaeus) by the fluorescence in situ hybridization (FISH) technique. The Yq subtelomeric DNA sequences (DYS427) have been observed to be intact at the presumed loci. Also, the amelogenin gene (AMELY, Yp11.2) revealed sequence homology and positional conservation in the higher primates, except in gorilla where positional divergence was observed.

Amelogenin dosage compensation in carcinoma of colon, lung, liver and kidney, is not a marker of clonality in males

The analysis of patterns of X-chromosome inactivation is becoming increasingly utilized as a marker of clonal composition of tissues from women. To date, however, no analogous system has been found for the study of clonality in tissue from men. In the current study, the methylation patterns for portions of the amelogenin genes are tested, which are encoded on both the X- and Y-chromosome (AMGX and AMGY). The polymerase chain reaction (PCR) was used to amplify portions of AMGX and AMGY from genomic DNA of carcinomas of the colon, lung, liver and kidney, as well as from matched normal somatic tissues. The amplification target includedAlu Imethylation sensitive restriction endonuclease sites as well as a 189 bp sequence which is present in AMGX but is absent in AMGY. Polymerase chain reaction amplification of AMGX and AMGY was successful using genomic DNA from both tumour and normal control tissue in 24 of the 26 cases. Pretreatment of genomic DNA withAlu Iblocked amplification of AMGX in all cases from both normal tissue and tumour. This indicates that AMGX and AMGY undergo a non-random pattern of methylation in both normal tissues and in tumours, precluding their use as a marker of clonality. Methylation ofAlu Isites in AMGY suggests that the amelogenin genes undergo dosage compensation, which raises the possibility that the expression of amelogenin is not restricted to the development of the tooth bud but may also play some other role in various tissues of the body.

Identification of two additional exons at the 3′ end of the amelogenin gene

Alternative splicing of the amelogenin gene generates a family of proteins secreted by ameloblasts that are primarily responsible for tooth enamel formation. Here the presence of two additional exons, downstream of exon 7, which are followed by an alternate polyadenylation site, is reported. Long polymerase chain reaction was used with a rat genomic amelogenin template to amplify sequences downstream from the primary polyadenylation site. Within the amplified sequences, two exons of 45 bp (exon 8) and 110 bp (exon 9) were identified. The presence of these additional exons in the human and mouse genome was indicated by Southern blot analysis. Antibodies raised against a synthetic peptide corresponding to a sequence encoded by exon 9 positively stained ameloblasts in sections of developing murine and porcine unerupted teeth. These results suggest that an amelogenin protein(s) that includes sequences derived from exons 8 and 9 is synthesized by the ameloblasts.

Molecular genetic sex determination of Medieval human remains from North Russia: Comparison with archaeological and anthropological criteria

Sex determination presents a difficult problem in archaeology and anthropology in cases of fragmentary or juvenile remains, and where grave goods are absent. Here, a molecular genetic analysis of the sex of human remain from the Early Medieval cemetery at Nefedievo, North Russia, was carried out and the results were compared with archaeological and anthropological data. Teeth without cavities (15 samples) and bones (9 samples) were used as the ancient DNA source. The repetitive sequences in DYZ1, DYZ3, DXZ3 loci, and a unique sequence in the first intron of the X-Y homologous gene amelogenin, were amplified. Sex was determined in 87.5% of the samples by archaeological criteria, in 95.8% of the samples by anthropological methods, and in 79.2% of the samples by DNA analysis. PCR allowed the sex of infant's remains to be identified in individual where the sex could not be determined by anthropological methods and in three remains where sex could not be inferred from archaeological data. Uneven preservation of nuclear DNA loci was evident.

An Amelogenin Gene Mutation Associated with X-linked Amelogenesis Imperfecta

A kindred was described with X-linked hypo-maturation Amelogenesis Imperfecta, in which the teeth of the propositus had mottled yellowish white enamel and his mother had vertical bands of opaque white and translucent enamel (Sauk et al., Am. J. Hum. Genet., 24, 267, 1972). Members of this family were contacted to obtain blood samples, in order to search for a mutation within the coding region of the X-chromosomal amelogenin gene. Primers were designed to amplify by the polymerase chain reaction (PCR) the coding regions: PCR products were sequenced directly, or cloned and sequenced. A point mutation was identified in the sequence encoding the 8th amino acid (pro > thr) of exon-6 of three affected family members. Two unaffected sisters of the propositus each had the normal DNA sequence as described by Salido et al. (Am. J. Hum. Genet., 50, 303, 1992). This proline is conserved in human X and Y chromosomal amelogenin genes, as well as in cow, pig, mouse, rat and opossum amelogenins, and is located within a highly conserved region of the protein. This mutation is significant in that it localizes a domain in the amelogenin protein predicted to be required for normal enamel formation. This study was supported by NIH grants DE10149 (CWG) and DE08239 (JR).

Sex determination by PCR analysis of the X-Y amelogenin gene.

PCR-based sex determination was first accomplished by amplifying multiple-copy sequences in the Y-chromosomal DYZ1 locus (,). These methods are quite sensitive (amplifiable from trace samples) due to the multiplicity, but it is impossible to tell whether the template DNA is from a female or whether the analysis has ended in failure when no fragment is amplified by PCR. Simultaneous amplification of X- and Y-chromosomal genes is thus necessary for a reliable sex test, in which the X-specific product may act as an internal control for PCR. From this point of view, centromeric alphoid repeats on the X and Y chromosomes have been amplified separately () or in the same reaction mixture () using two pairs of primers. These methods are also sensitive. However, a difference in the copy numbers of the repeated sequences (i.e., 5000 and 100 copies in X and Y centromeres, respectively) is likely to cause a difference in the sensitivity of PCR amplification so that the X product does not act strictly as an internal control. Sex determination by PCR analysis of X-Y homologous genes is thus most trustworthy, because X and Y sequences are of equal (single) copy number and because they can be amplified using one set of primers.