Identification of war victims from mass graves in Croatia, Bosnia, and Herzegovina by use of standard forensic methods and DNA typing

The postmortem remains of sixty-one war victims were excavated from 6 mass graves in Bosnia and Herzegovina one and a half years after interment Using standard identification methods, including the matching of medical and dental records, the recognition of distinguishing characteristics such as the use of clothing and belongings, and video superimposition, 35 persons were identified. For the remaining 26 persons identification efforts continue. DNA typing was performed at the HLA DQA1 locus and five PM system loci. Results from DNA typing were confirmed by other methods. DNA profiles of family members of 150 missing persons are now being developed using the 6 loci. These DNA profiles will then be compared with those generated from the bone and teeth remains of the unidentified victims.     

Genetic typing of HLA class II genes in Swedish populations: application to forensic analysis

In an attempt to determine the value of DNA based typing of HLA class II loci to forensic analysis, allele and genotype frequencies at DQA1, DQB1, DPB1, and DRB1 were determined in samples from two Swedish populations using hybridization with sequence specific oligonucleotides to PCR amplified DNA. Significant allele frequency differences were observed at the DQB1 and DRB1 loci between the two populations, as well as between one of the Swedish and a Norwegian population. The average heterozygosity varies between 0.74 to 0.91 and the power of discrimination between 0.90 to 0.98, with the highest values obtained for the DRB1 locus. The probability of genotype identity by chance differs on average 2% between the populations. When applied to a paternity case with one parent deceased and a criminal case, typing of class II loci proved in both cases informative. Analyses of DR and DQ genes does not increase the power of discrimination, due to strong linkage, but offers through the reconstruction of putative haplotypes an internal control for the consistency of the typing results at several loci. Typing of the DRB1 and DPB1 loci was found to result in an approximate combined average probability of genotype identity by chance of one in a thousand.     

DNA typing in forensic medicine and in criminal investigations: a current survey

Since 1985 DNA typing of biological material has become one of the most powerful tools for personal identification in forensic medicine and in criminal investigations [1-6]. Classical DNA "fingerprinting" is increasingly being replaced by polymerase chain reaction (PCR) based technology which detects very short polymorphic stretches of DNA [7-15]. DNA loci which forensic scientists study do not code for proteins, and they are spread over the whole genome [16, 17]. These loci are neutral, and few provide any information about individuals except for their identity. Minute amounts of biological material are sufficient for DNA typing. Many European countries are beginning to establish databases to store DNA profiles of crime scenes and known offenders. A brief overview is given of past and present DNA typing and the establishment of forensic DNA databases in Europe.     

Polymorphisms of twelve short tandem repeat loci in a Taiwanese population and their application in parentage testing

With the advancement of techniques in molecular biology, rapid, sensitive, and reliable methods of DNA typing for parentage testing have become available. In this study, we evaluated the usefulness of multiplex polymerase chain reaction (PCR) with 12 unlinked short tandem repeat (STR) loci for paternity testing in Taiwan. The genetic informativeness of this test was then compared with that of conventional human leukocyte antigen (HLA) analysis in 167 parentage studies. The 12 STR loci alone provided a cumulative power of exclusion of up to 0.9998. Paternity was excluded in 59 (35.3%) cases, including 40 of 112 paternity trios and 19 of 55 paternity duos. In the 40 trios in which paternity was excluded, a mean of 6 (range, 3-9) incompatible STR markers were in the 19 duos in which paternity was excluded, a mean of 4 (range, 1-8) incompatible STR markers were noted. In the 72 trios in which the alleged paternity could not be excluded, the mean probabilities of paternity (PP) were 90.6863% with HLA testing alone, 99.9847% with STR analysis alone, and 99.9972% with combined HLA and STR analysis. In the 36 duos in which the alleged paternity could not be excluded, the mean PPs were 81.4768% with HLA testing alone, 99.6124% with STR analysis alone, and 99.9145% with combined HLA and STR analysis. These results suggest that STR analysis is very powerful when used alone for paternity trio testing and when combined with conventional serologic HLA typing for duo parentage testing in the Taiwan population.     

Deduction of the order of sexual assaults by DNA analysis of two condoms

Differential DNA extraction procedures were performed on two condoms found at a rape scene. One of the condoms was recovered intact (A), while the second condom (B) had apparently ruptured during the alleged attack. Two related suspects (cousins 1 & 2) were identified as the potential semen donors. Condom B contained DNA from the female and from one of the suspects. Condom A contained DNA from the suspect identified on condom B and from an unidentified individual. The presence of DNA from suspect 2 on both condoms led to the deduction that his sexual activity preceded that of the unidentified suspect. The ability to determine such a sequence of events using DNA typing is unusual.     

Additional approaches to DNA typing of skeletal remains: the search for "missing" persons killed during the last dictatorship in Argentina

DNA typing techniques are among the most advanced tools for human identification and can contribute to the identification of poorly preserved skeletal remains. Ten thousand people are thought to have been killed during the last dictatorship in Argentina (1976-1983) and there are few official records on the identity of the victims or the location of burials. A mass grave containing 340 skeletons was excavated using archeological methods. A small number of individuals was identified by traditional forensic methods and one family group by mitochondrial DNA (mtDNA) analysis. Due to the lack of antemortem physical information on many of the victims, the application of molecular methods is imperative to speed up the identification process. We have tested two molecular screening methods, Y chromosome-specific short tandem repeats (DYS19, DYS385, DYS389 I, DYS389 II, DYS390, DYS391, DYS392, DYS393) and amplification of autosomal microsatellites using nested primers. These methods can complement solely matrilineal mtDNA sequence data in the identification of "missing" persons.     

Outcomes of disabling cervical spine disorders in compensation injuries. A prospective comparison to tertiary rehabilitation response for chronic lumbar spinal disorders

BACKGROUND: Genotyping based on short tandem repeat (STR) regions is widely used in human identification and parentage testing, in gene mapping studies, and as an approach to studies on the etiopathogenesis and diagnosis of hereditary diseases. We wished to study a new analytical approach that uses capillary electrophoresis and multicolor fluorescence in place of slab gel electrophoresis. METHODS: We evaluated the efficiency for parentage and forensic purposes of the AmpFLSTR Profiler PlusTM typing kit that is used with the ABI Prism 310 Genetic Analyzer (System-2 STR), and that of a widely used panel of nine STRs analyzed with conventional slab-gel electrophoresis followed by radioactive detection (System-1 STR). System-2 STR, based on automated capillary electrophoresis and automated sizing of the alleles by Genotyper 2.0 software, was used to determine the allele frequency of the nine loci in 157 Caucasian subjects from southern Italy. On the basis of the data obtained, we submitted 40 trios to parentage testing. RESULTS: A higher median probability of paternity attribution and power of exclusion were obtained with System-2 STR vs System-1 STR: respectively, 99.99% and 99.95% (P <0.05) for attribution; and five and four excluding loci (P <0.05) for exclusion. The most informative and highly discriminating loci were D18S51, D21S11, and FGA. The combined probability of matching-by-chance for all nine STRs was 1.36 x 10(-12) for System-2 compared with 1.11 x 10(-7) obtained with the other system. The internal standard and allelic ladder of the System-2 STR facilitated accurate and precise genotyping; furthermore, System-2 STR and was faster than the conventional System-1 STR. CONCLUSIONS: The System-2 STR allows rapid testing with higher probabilities of attribution and a higher power of exclusion than with the comparison method with slab-gel electrophoresis.     

An outbreak of hepatitis A during a military field training exercise

Current practice for the selection of unrelated donors involves serological typing of HLA-A, -B and -DR antigens, DNA analysis of the class II region and the MLR. However, even after matching for the class II loci at the DNA level, a significant proportion of matched unrelated pairs remain MLR reactive. Ideal matching for BMT would be a match for the whole MHC haplotype rather than individual HLA loci. In the present study, we have evaluated the complementary role of class III typing in determining MHC identity. A group of 86 donor/recipient pairs, of which 14 were unrelated, was investigated using C4, Bf, HSP70 and TNF DNA probes. Phenotypically HLA-matched siblings were always identical at the C4 locus which is the most polymorphic of all the loci examined. Nine of the 14 HLA serologically matched MLR non-reactive (RRI < 20%) unrelated pairs had class III mismatching. Four of these pairs with class III mismatching were matched at the DRB and DQB loci by RFLP analysis. These results demonstrate that serological identity, DRB/DQB RFLP-matching and a negative MLR do not always match the whole haplotype in unrelated pairs. It can be concluded that the linkage of the class III loci to both HLA regions makes this region a reliable marker of the whole MHC haplotype.     

Novel primers designed for microsatellite loci in Eucalyptus and identification by PCR fingerprints

We found a novel PCR-primer which can be used for the identification on "elite-tree-selection". This primer was designed for selective hybridization at the both ends of microsatellite loci, which is well known as one of the most highpervariable region of DNA. After PCR-fingerprinting on five Eucalyptus species (E. globulus, E.citriodora, E.grandis, E. maidenii, E.bicostata), with our primer, DNA-polymorphism was observed all over the cases.     

Sex identification of archaeological human remains based on amplification of the X and Y amelogenin alleles

Sex identification of archaeological human remains is essential for the exploration of gender differences in past populations. Traditional morphometric analyses fail to identify the gender of incomplete skeletal remains and that of immature individuals. In the present work, we have established a sensitive and reliable method, based on amplification of the single-copy amelogenin-encoding gene (AMG). The Y allele carries a small deletion in the first intron, facilitating the design of distinct X- and Y-specific polymerase chain reactions. Amplification with three primers, two of which are allele-specific, allows unambiguous identification of both X and Y chromosome signals in a single reaction, providing an internal control. For added confidence, the reaction may be performed in separate tubes for each allele. Using this method, the sex was determined from the skeletal remains of 18 individuals, including young children, out of 22 examined from periods ranging from 200 to around 8000 years ago. The state of skeletal preservation ranged from poor to good. Cortical and cranial bones, as well as teeth, were found to provide sufficiently preserved DNA. The success of retrieval of amplifiable DNA was not related either to the period or to the burial site. On the other hand, the method of DNA purification was critical. In our hands, direct DNA purification by Chelex from minute samples of bone/tooth powder gave the best results. This study demonstrates the applicability of the method for gender determination in skeletal remains from different periods.     

The expert identification of the remains of the imperial family by means of molecular genetic verification of genealogical relations

The paper presents the results of international complex molecular genetic expert identification of skeletal remains of 9 subjects buried near the Koptiaki road in Ekaterinburg region, presumably belonging to the Romanov Royal Family and persons in their attendance. The armory of methods based on analysis of the least permissible amounts of DNA by the polymerase chain reaction and direct fluorescent sequencing of amplified DNA fragments included the latest scientific technologies. In addition, new methods were developed and used, which have no analogs in the world expert practice. The strategy included identification of biological gender of skeletons, of familial group among exhumed individuals, and of ties of relationship of this family with two independent maternal branches of the Romanov genealogical tree using tracing kindred markers based on mitochondrial DNA (mtDNA). The study was carried out in two stages: in 1992-1993 at Aldermaston Criminology Center of Home Office of the UK with participation of British specialists and in 1995 at Military Medical Institute of Ministry of Defence of the USA in Washington with participation of American specialists. In 1993 five skeletons were identified as Emperor Nicholas II, Empress Alexandra Fedorovna, and their three daughters with 99.6% certainty. From modern criminological viewpoint, the result could not be considered as sufficiently certain for such an extraordinary case, and therefore in 1995 molecular genetic studies of presumable remains of Nicholas II and his brother Prince Georgi? Romanov were carried out again. The results showed absolute positional identity of mtDNA genetic code of these two men due to an extremely rare genetic abnormality (heteroplasmia), and thus the problem of appurtenance of the remains to the Romanov royal family was solved.     

A simple method for extracting DNA from old skeletal material

Extraction of DNA from old skeletal material is of great importance in the identification of human remains, but is particularly difficult because the methods currently employed, especially those using phenol/chloroform, are not always satisfactory. A simple technique based on the removal of non-nucleic acid material by salting out (precipitation) with saturated sodium acetate is described; the presence of DNA in the extract being confirmed by amplification of selected sequences of the HLA-DRB1 gene using the polymerase chain reaction (PCR). The method was applied to fresh bone (five femoral heads and six vertebral bodies) and to bone from two forensic cases, 3 and 9 months post-mortem, respectively. Parallel extractions using the phenol/chloroform technique were performed on all samples in order to compare the efficiency of the two methods. Using sodium acetate precipitation, amplifiable DNA was consistently extracted from fresh bone, as well as from the two forensic cases. With the phenol/chloroform method, amplification was successful in only 7 out of 11 instances with the fresh bone samples and failed in both forensic cases. The studies also showed that an effective way of removing PCR inhibitors is to subject the extract to agarose gel electrophoresis, isolate the high molecular weight area and re-extract the DNA from the gel by boiling. It was concluded that the sodium acetate method is a valid alternative to established techniques for extracting DNA from bone and that it offers the advantages of being simple, quick, inexpensive and avoids using hazardous reagents.     

Childhood-onset psychosis: evolution and comorbidity

High resolution cytogenetics, microsatellite marker analyses, and fluorescence in situ hybridization were used to define Xq deletions encompassing the fragile X gene, FMR1, detected in individuals from two unrelated families. In Family 1, a 19-year-old male had facial features consistent with fragile X syndrome; however, his profound mental and growth retardation, small testes, and lover limb skeletal defects and contractures demonstrated a more severe phenotype, suggestive of a contiguous gene syndrome. A cytogenetic deletion including Xq26.3-q27.3 was observed in the proband, his phenotypically normal mother, and his learning-disabled non-dysmorphic sister. Methylation analyses at the FMR1 and androgen receptor loci indicated that the deleted X was inactive in > 95% of his mother's white blood cells and 80-85% of the sister's leukocytes. The proximal breakpoint for the deletion was approximately 10 Mb centromeric to FMR1, and the distal breakpoint mapped 1 Mb distal to FMR1. This deletion, encompassing approximately 13 Mb of DNA, is the largest deletion including FMR1 reported to date. In the second family, a slightly smaller deletion was detected. A female with moderate to severe mental retardation, seizures, and hypothyroidism, had a de novo cytogenetic deletion extending from Xq26.3 to q27.3, which removed approximately 12 Mb of DNA around the FMR1 gene. Cytogenetic, and molecular data revealed that approximately 50% of her white blood cells contained an active deleted X. These findings indicate that males with deletions including Xq26.3-q27.3 may exhibit a more severe phenotype than typical fragile X males, and females with similar deletions may have an abnormal phenotype if the deleted X remains active in a significant proportion of the cells. Thus, important genes for intellectual and neurological development, in addition to FMR1, may reside in Xq26.3-q27.3. One candidate gene in this region, SOX3, is thought to be involved in neuronal development and its loss may partly explain the more severe phenotypes of our patients.     

Right laparoscopic lumbar sympathectomy]

In three separate shooting incidents involving multiple gunshots, two FMJ bullets and one bullet fragment found at the scene (one from each case) were investigated for the presence of biological material from the victim after perforation. The surface of the missiles, which did not show obvious tissue traces when examined under a macroscope, was swabbed. PCR typing of up to five STR loci was performed on the small amounts of DNA extracted, which were seen below the detection limit of the slot blot quantification in one case. Nevertheless, individualisation of cellular material from the perforating projectiles was successful in each of the three cases presented. Consequently, identification of the victim wounded by a perforating bullet can reliably be achieved if contamination or removal of evidentiary material by improper handling is prevented. This technique is especially useful in cases where more than one person has fired a gun because the bullet carrying DNA can be linked to the firearm by investigation with a comparison microscope. As a by-product of this investigation, a variant allele 14 (14+4) at the VWA locus was detected.     

Lack of association between urinary creatinine and ethanol concentrations and urine/blood ratio of ethanol in two successive voids from drinking drivers

Primer extension preamplification (PEP), which can amplify sequence-independently a limited quantity of DNA as a whole, allows multiple DNA analyses by polymerase chain reaction (PCR). This technique may be applicable to forensic cases, especially in cases where only small amounts of DNA are available. To define the accuracy and sensitivity of PEP, the DNA typing results of nine loci (TH01, MCT118, HLA-DQ alpha, amelogenin, LDLR, GYPA, HBGG, D7S8, GC) by PCR with PEP (PEP-PCR) were compared with those by PCR without PEP. Both results were identical to each other for each sample tested. Furthermore, amplification of an initial genomic DNA by PEP was found to range from 15 to 600 times of the initial quantity and the efficiency of PEP may depend on the sequences flanking the loci tested. These results indicate that PEP can increase typing potential of PCR from forensic samples.     

Maternal language in infancy.

Observations of 36 preterm infants and their English-speaking mothers suggest that maternal language to the infant varies as a function of the age and ordinal position of the infant and maternal education. The relationship between maternal language at 1 mo of age and the mother's verbal style to her older infant allows for early identification of maternal language input styles and, therefore, has important implications for mother–infant intervention programs. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Direct detection and isolation of restriction landmark genomic scanning (RLGS) spot DNA markers tightly linked to a specific trait by using the RLGS spot-bombing method

We have developed a technique for isolating DNA markers tightly linked to a target region that is based on RLGS, named RLGS spot-bombing (RLGS-SB). RLGS-SB allows us to scan the genome of higher organisms quickly and efficiently to identify loci that are linked to either a target region or gene of interest. The method was initially tested by analyzing a C57BL/6-GusS mouse congenic strain. We identified 33 variant markers out of 10,565 total loci in a 4.2-centimorgan (cM) interval surrounding the Gus locus in 4 days of laboratory work. The validity of RLGS-SB to find DNA markers linked to a target locus was also tested on pooled DNA from segregating backcross progeny by analyzing the spot intensity of already mapped RLGS loci. Finally, we used RLGS-SB to identify DNA markers closely linked to the mouse reeler (rl) locus on chromosome 5 by phenotypic pooling. A total of 31 RLGS loci were identified and mapped to the target region after screening 8856 loci. These 31 loci were mapped within 11.7 cM surrounding rl. The average density of RLGS loci located in the rl region was 0.38 cM. Three loci were closely linked to rl showing a recombination frequency of 0/340, which is < 1 cM from rl. Thus, RLGS-SB provides an efficient and rapid method for the detection and isolation of polymorphic DNA markers linked to a trait or gene of interest.     

Microsatellite instability analysis: a multicenter study for reliability and quality control

The molecular biology section of the Hereditary Non-Polyposis Colorectal Cancer study group-Germany, instituted a multicenter study to test the reliability and quality of microsatellite instability (MSI) analysis. Eight laboratories compared MSI analyses performed on 10 matched pairs of normal and tumor DNA from patients with colorectal carcinomas. A variety of techniques were applied to the detection of microsatellite changes: (a) silver and ethidium bromide staining of polyacrylamide gels; (b) radioactive labeling; and (c) automated fluorescence detection. The identification of highly unstable tumors and tumors without MSI was achieved in high concordance. However, the interpretation of the band patterns resulted in divergent classifications at several microsatellite marker loci for a large fraction of this tumor/normal panel. The data on more than 30 primers per case suggest that the enlargement of the microsatellite panel to more than 10 loci does not influence the results. In this study, cases with MSI in less than 10% of loci were classified as microsatellite stable, whereas MSI was diagnosed in cases with more than 40% of all markers unstable. We propose that a panel of five microsatellite loci consisting of repeats with different lengths should be analyzed in an initial analysis. When less than two marker loci display shifts in the microsatellite bands from tumor DNA, the panel should be enlarged to include an additional set of five marker loci. The number of marker loci analyzed as well as the number of unstable marker loci found should always be identified. These criteria should result in reports of MSI that are more comparable between studies.     

Assessment of danger to themselves and their infants by rhesus macaque (Macaca mulatta) mothers.

This study investigated whether rhesus macaque (Macaca mulatta) mothers distinguish between what is dangerous to themselves and what is dangerous to their infants. The behavioral interactions between 11 mother–infant pairs and other females living in their group were analyzed in the 1st 2 mo of infant life. Mothers behaved as if they perceived higher ranking females as dangerous to both themselves and their infants, lower ranking females as dangerous to their infants but not to themselves, and their young daughters as relatively harmless to both themselves and their infants. Changes in maternal intolerance of infant handling between the 1st and 2nd mo covaried with changes in the probability of infant harassment rather than with the temporal pattern of aggression received by mothers. The possible cognitive mechanisms underlying parental recognition of infant's special needs are discussed in the light of comparative evidence from other mammalian species. (PsycINFO Database Record (c) 2010 APA, all rights reserved)