Edna Sadayo Miazato Iwamura

Human identification and analysis of DNA in bones

The introduction of molecular biology techniques, especially of DNA analysis, for human identification is a recent advance in legal medicine. Substantial effort has continuously been made in an attempt to identify cadavers and human remains after wars, socio-political problems and mass disasters. In addition, because of the social dynamics of large cities, there are always cases of missing people, as well as unidentified cadavers and human remains that are found. In the last few years, there has also been an increase in requests for exhumation of human remains in order to determine genetic relationships in civil suits and court action. The authors provide an extensive review of the literature regarding the use of this new methodology for human identification of ancient or recent bones.

A qualitative study of compact bone microstructure and nuclear short tandem repeat obtained from femur of human remains found on the ground and exhumed 3 years after death.

Forensic identification of human remains is composed of anthropological study of race, sex, age, etc. By using these traditional methods, inconclusive or nonidentified cases could be subjected to DNA analysis. However, in spite of advances in human identification techniques, especially by PCR-amplified DNA, some limitations that affect the ability of obtaining DNA from human remains still persist. Light microscope sections of postmortem compact bones from human remains are presented here for the purpose of increasing a forensic examiners prediction of successful nuclear DNA typing. Femoral compact bones were obtained from 7 human remains found on the ground, in different degrees of decomposition, and were cleaned by boiling to remove soft tissues, 8 collections of bones having undergone natural decomposition, not boiled (as no soft tissue was adhered), and 5 cadavers 12 to 16 hours postmortem. The histologic sections were stained by hematoxylin and eosin, the loci CSF1PO, TPOX, TH01, F13A01, FESFPS, vWA, D16S539, D7S820, D13S317, and amelogenin were amplified by PCR, and the polyacrylamide gel was stained with silver. The results presented here clarify questions concerning the viability of DNA for identification analysis, and they also may help to establish better strategies for optimization of DNA extraction and analysis in compact bones of human remains.

Potential definition of the time of death from autolytic myocardial cells:: a morphometric study

Morphometric methods and transmission electron microscopy were used to quantify modifications occurring in the mitochondria of dog myocardium during the first four hours of autolysis. Myocardial fragments were obtained from the outer free wall of the left ventricle, during anesthesia (control-zero) and at 15, 45, 120, and 240 min after cardiac arrest, maintaining the heart in situ at 22 degrees C. During the 240 min of autolysis, the main parameters evaluated showed: (a) a decrease in the number of mitochondria from 0.31 to 0.12 per micron 3 of cytoplasm. The decrease over the first 45 min reached 50% of the initial value; (b) an increase in mitochondrial volume, three times greater after the first 45 min (from 0.92 to 2.68 micron 3) and four times greater after 240 min (from 0.92 to 3.79 micron 3); (c) an increase in mitochondrial outer membrane surface area from 5.51 to 12.54 micron 2; (d) an increase in the surface area of individual mitochondria inner membrane and cristae from 27.60 to 56.96 micron 2. The progressive nature of the alterations and the difference in the numerically expressed values allow correlation with the time of somatic death. The authors emphasize the need for further studies in order to complement the present study.

Y-STRs in forensic medicine: DNA analysis in semen samples of azoospermic individuals.

ABSTRACT: The incidence of rape has increased, especially in metropolitan areas, such as the city of São Paulo. In Brazil, studies about it have shown that the majority of this type of crime is committed by the relatives and persons close to the victim. This has made the crime more difficult to be denounced, as only 10% of the cases are reported to competent police authorities. Usually, cytological exams are carried out in sex crime investigations. The difficulty in showing the presence of spermatozoa is frequent, but it does not exclude the presence of male DNA. The absence of spermatozoa in material collected from rape victims can be due to several factors, including the fact that the agressor suffers from azoospermia. This condition can be the result of a successful vasectomy. As the majority of DNA in the ejaculation sample is from spermatozoa, there is much less DNA to be analyzed. This study presents the application of Y‐STRs (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, and DYS393) in DNA analysis of sperm samples from 105 vasectomized men. The study demonstrated a great variation in DNA concentration. DNA extraction and amplification was possible in all sperm samples even in the absence of spermatozoa. The same profile was observed, for each individual, from DNA extracted from blood, pre‐ and postvasectomy semen samples. The use of markers specific for Y chromosome in sex crime cases, especially in the absence of spermatozoa, is very important, mainly because in most situations there is a small quantity of the agressors DNA in the medium and a large quantity of the victims DNA.

Population Genetics of Nine Short Tandem Repeat Loci Allele Frequency Distribution in a Brazilian Population Sample

Gene and genotype frequencies in relation to the D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317, and D7S820 loci were determined in a sample of 290 unrelated individuals (204 Caucasians and 86 mulattoes) living in the city of São Paulo, Brazil. The sex test Amelogenin was also performed in all subjects from our sample, revealing the expected sex in all instances. Allele frequency data obtained from the analysis of these samples were in the usual range of other population groups with similar racial background. In the sample of Caucasian individuals, panmictic proportions were ruled out in relation to TPOX and CSF1PO loci, but only in the latter was the overall frequency of heterozygotes significantly less than expected. In the sample of mulattoes, Hardy-Weinberg proportions were rejected in relation to FGA and CSF1PO loci, but in no instance were the overall numbers of heterozygotes different from the corresponding expected ones under panmixia. Taking into account all this and also the number of tests performed, the degree of genetic heterogeneity of Brazilian populations, and the critical level reached by the significant results (1% < &agr;<5%), the departures from panmixia here observed can be considered to be negligible in altering significantly biologic relationship odds calculated under the assumption of random matings.

Post-mortem forensic identity testing: application of PCR to the identification of fire victim

CONTEXTnDNA analysis has been used with success in the identification of carbonized corpses and victims of large accidents. The analysis requires relatives of crash victims to donate blood for analysis. The relatives are generally willing contribute to the identification by giving a blood sample.nnnOBJECTIVEnTo describe the use of the polymerase chain reaction (PCR) for genetic characterization of one victim extensively burned by fire.nnnDESIGNnCase report.nnnCASE REPORTnDNA was extracted from blood of the cardiac chamber, and 15 different loci (D1S80, ApoB, D17S30, D3S1744, D18S849, D12S1090, FGA, D7S820, D1S533, D9S304, HUMCSF1PO, HUMTPOX, HUMTHO1, amelogenin and HLA-DQA1) were analyzed using the PCR technique. Results from all loci typing of the corpse were then compared to that of his alleged biological parents, revealing a genetic compatibility.

Population and mutation analysis of Y-STR loci in a sample from the city of São Paulo (Brazil)

The haplotypes of seven Y-chromosome STR loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, and DYS393) were determined in a sample of 634 healthy Brazilian males (190 adult individuals and 222 father-son pairs). The 412 adults were unrelated, and the 222 father-son pairs had their biological relationship confirmed using autosomal STRs (LR > 10,000). Among the 412 adults, a total of 264 different 7-loci haplotypes were identified, 210 of which were unique. The most frequent haplotype was detected in 31 instances, occurring with a frequency of 7.52%. The haplotype diversity index was calculated as 98.83%. Upon transmission of the 1,554 alleles, in 222 father-son pairs, six mutations were observed, with an average overall rate of 3.86 x 10-3 per locus. A haplotype with a duplicated DYS389I locus, and another with duplicated DYS389I, DYS389II, and DYS439 loci were detected in both fathers and their respective sons.

Bioethics, Intellectual Property and Genomics

RHCFAP/3043SEGRE M et al. - Bioethics, Intellectual Property and Genomics. Rev. Hosp. Clin. Fac. Med. S. Paulo 56(4):97-102, 2001.Scientific and technological advances will never be anti-ethical. For mankind, “a search for new ways ” means better conditionsfor achieving autonomy. Everything that has been discovered in the last few decades, from how to control the birth rate, to the recentprobability of being able to clone human beings, and, within these latest discoveries, the extraordinary possibility of manipul atingthe human gene itself, empowers us to assume control of what until now was unknown, and thus we will be able to attain a betterquality of life.With respect to the ideal , it is unquestionable that the knowledge that could substantially change our lives on this planet shouldbe within reach of the world society; society itself should be responsible for monitoring the applications of this knowledge.The present article discusses the ethics and the patenting of the human genome, arguments for and against gene patenting,patents and research into human embryos, legal aspects, and intellectual property in the field of the human genome.DESCRIPTORS: Bioethics. Genomics. Human Rights. Patenting. Intellectual Property.Scientific and technological ad-vances will never be anti-ethical. Formankind, “a search for new ways”means better conditions for achievingautonomy. Everything that has beendiscovered in the last few decades,from how to control the birth rate, tothe recent probability of being able toclone human beings, and, within theselatest discoveries, the extraordinarypossibility of manipulating the humangene itself, empowers us to assumecontrol of what until now, was un-known, and thus we will be able to at-tain a better quality of life.The fear, which is understandable,regarding what is going to happen themoment we modify our habitat andourselves is founded on the fear of theunknown. We live with tragedies, suchas wars, oppressions, inequalities ofevery type, affronts to human dignityat all levels, diseases, etc., but the mo-ment we are told “Man is playing ofGod”, many of us belittle ourselveswithout debating the issue. We forgetthat man has played this game for allof recorded history, and that if it werenot for this game, we would not havewon the battle against so many dis-eases— a victory that has prolongedlife expectation for decades, reducedinfant mortality, etc .What could be frightening is theway in which we apply the new knowl-edge, which could be used for dis-crimination, oppression, and extermi-nation, as so many other advances inknowledge have been used. It could besaid to those who condemn and abhorthe discovery of nuclear energy that,considered on its own merit, this ad-vancement in knowledge was an enor-mous success for science; what wecondemn was its use in killing millionsof human beings. We must rememberthat a treaty of non-proliferation ofnuclear weapons was drawn up andsigned and that, up to the present time,it has been respected; therefore, it isclear that we are (or, if not, should be),capable of responsibly administeringthe application of new techniques withthe intent of applying them in waysthat are compatible to our sense of eth-ics. Therefore, since we accept thatknowledge will never be ethical oranti-ethical a priori, we will not raise

GENE AND GENOTYPE FREQUENCIES FOR HLA-DQA1 IN CAUCASIANS AND MULATTOES IN BRAZIL

Gene and genotype frequencies of the HLA-DQA1 locus were determined in a sample of 197 unrelated individuals (144 Caucasians and 53 Mulattoes), living in the city of São Paulo, Brazil. The Mulatto group consisted of mixed individuals who presented at least one negroid physical characteristic or declared themselves to be of mixed ancestry. A total of six different alleles were identified with frequencies ranging from 0.087 to 0.316 in the Caucasian population and from 0.066 to 0.330 in the Mulatto population. We observed an increased frequency of allele 1.2 among Mulattoes in relation to Caucasians. The sample heterozygote frequency was 0.722 among Caucasians and 0.736 among Mullatoes. No significant deviations from Hardy-Weinberg equilibrium were found either in the Caucasian or in the Brazilian Mullato population samples.

Allele Frequency Distribution of Three STR Loci (CSF1PO, TPOX, and TH01) in a Brazilian Population Sample*

After informing 331 unrelated Brazilian individuals (240 Caucasians and 91 Mulattoes) and getting their consent, blood samples were collected. DNA was extracted from 5 mL of peripheral blood obtained from each of 331 volunteers by the salting-out procedure (1). PCR analysis was performed using the GenePrint™. STR Multiplex System (CTT Multiplex, Promega Corporation, Madison, WI) under conditions recommended by the manufacturer. The amplified fragments were submitted to electrophoresis on denatured polyacrylamide gels and visualized after silver staining. Allele identification was achieved by comparison of the amplified fragments with the allelic ladder included in the reagent set. Statistical analysis: gene and genotype frequencies were estimated using standard counting procedures; for comparing gene counts between samples and for testing Hardy-Weinberg proportions within each sample, Chisquared tests were used throughout. All these procedures are described in detail by Weir (2). In order to locate the categories responsible for significant values in contingency tables, the method of adjusted standardized residuals described by Haberman was applied (3,4). Tables 1, 2, and 3 summarize the frequencies and Table 4 describes the observed and expected heterozygosities. The complete data set is available to any interested researcher upon request.