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Atypical short tandem repeat allelic patterns in a sexual assault case involving hematopoietic stem cell transplantation.

Short tandem repeat (STR) profiling is a cornerstone of forensic DNA analysis, particularly during criminal investigations. However, certain clinical conditions, such as bone marrow transplantation, can complicate interpretation. To illustrate the impact of allogeneic bone marrow transplantation on forensic STR analysis, this case report details a sexual assault investigation involving a female victim who had previously received a bone marrow transplant from a female donor. A female sexual assault victim underwent forensic examination, during which multiple biological swabs were collected. STR profiling was conducted on the victim's blood, fingernail, buccal, breast, hip, vulvar, vaginal, cervical samples, panty, and brassiere. As conflicting profiles were found, a detailed medical history was collected, and hair follicle analysis was performed to confirm the origin of the STR profiles. Blood STR profiling revealed a single female genotype, while multiple swabs, including vaginal and cervical samples, showed a second female STR profile alongside the first. Notably, the consistency and distribution of the mixed profile across samples reduced the likelihood of laboratory contamination. The medical history of the victim revealed a prior history of allogeneic bone marrow transplant. Hair follicle analysis identified the recipient's original genotype, confirming that the secondary STR profile originated from the donor. Bone marrow transplantation may result in a mixed STR profile, potentially leading to misidentification or misinterpretation of forensic evidence. Awareness of transplant history is crucial during forensic evaluations. However, such clinical history is currently not included in standard sexual-assault evidence forms, underscoring the need for procedural updates.

Female↗

Multiplex DNA short tandem repeat analysis. A useful method for determining the provenance of minute fragments of formalin-fixed, paraffin-embedded tissue.

A tiny fragment of high-grade carcinoma was found in histologic sections and in the paraffin block of a benign cervical polyp from a patient with no clinical evidence of malignancy. Thus, it raised the suspicion of block contamination. No malignant tumor was processed the same day as the polyp; however, a similar tumor had been processed 6 days earlier. Multiplex DNA short tandem repeat analysis was applied to paraffin-extracted tissue samples obtained from the polyp, the suspected contaminant, the patient's additional cervical biopsy specimen, and the putative source of contamination. The results demonstrated that the suspected contaminant and the patient's cervical tissue could not have come from the same patient and that the suspected contaminant derived from the tumor processed earlier, without reasonable doubt. We hypothesize that this friable tumor escaped from cassettes into the processor and contaminated the polyp specimen. Multiplex DNA short tandem repeat analysis can be applied to determine the provenance of minute tissue samples in surgical pathology.

Adult↗

Tandem-repeated Zebrafish zp3 genes possess oocyte-specific promoters and are insensitive to estrogen induction.

Zona pellucida (Zp) proteins are glycoproteins in fish chorion and are encoded by multiple gene families, including zp1, zp2, zp3, and potentially other zp genes. Expression of zp genes in teleosts is either in the liver under the induction of estrogen or in the ovaries. In the present study, we isolated and characterized a zebrafish zp3 genomic clone and found three tandem-repeated zp3 genes with high sequence identities. We estimated that there were 10-15 zp2 and 17-21 zp3 genes in a haploid genome. We also found some variant zp2 and zp3 subfamilies, and each subfamily may have multiple members. These zp2 and zp3 genes are distributed in several different chromosomes. Ontogenetic expression of zp2 and zp3 mRNAs was first detected at 3 wk postfertilization, which was about 5 wk earlier than initial vtg1 expression, indicating that ovary development was earlier than vitellogenesis. Both zp2 and zp3 mRNAs were expressed specifically in early-growing oocytes and are insensitive to estrogen induction. Because zp3 genes are organized in tandem repeats, to investigate whether an individual zp3 promoter is capable of driving oocyte-specific expression, green fluorescence protein (gfp)-transgenic zebrafish were developed by using a 3.8-kb zp3 5' upstream region, and we found that the gfp reporter gene was faithfully expressed in developing oocytes in zp3:gfp transgenic females. Thus, the new transgenic line not only provided a convenient living marker for monitoring female gonad development, but also demonstrated that a single zp3 gene promoter is sufficient for oocyte-specific transcription.

Amino Acid Sequence↗

Extrachromosomal circular DNA of tandemly repeated genomic sequences in Drosophila.

One characteristic of genomic plasticity is the presence of extrachromosomal circular DNA (eccDNA). This DNA is found in various eukaryotes from yeast to humans, and its levels are elevated by exposure to carcinogens. eccDNA is heterogeneous in size and composed of chromosomal sequences. In this study we used two-dimensional gel electrophoresis to detect and characterize eccDNA in Drosophila. We found eccDNA throughout the fly's life cycle. These molecules comprise up to 10% of the total repetitive DNA content, and their size ranges from <1 kb to >20 kb. The eccDNA population contains circular multimers of tandemly repeated genes such as histones, rDNA, Stellate, and the Suppressor of Stellate. Multimers of centromeric heterochromatin sequences are included in eccDNA as well. Our findings are consistent with the hypothesis that intramolecular homologous recombination between direct tandem repeats is a favorite mechanism for eccDNA formation. The level of eccDNA increased following MMS treatment of wild-type larvae, consistent with phenomena observed in cultured mammalian cells. This shows mutagen-induced eccDNA formation in the context of the whole organism for the first time. Mutations in the genes okra, mus309, and mei41 did not affect eccDNA under normal conditions or following mutagen treatment, implying that eccDNA formation is different from known pathways of DNA repair.

Animals↗

Tandem repeat structure of rhamnose-binding lectin from catfish (Silurus asotus) eggs.

The primary structure of catfish (Silurus asotus) egg lectin (SAL) was determined. SAL cDNA contained 1448-bp nucleotides and 308 amino acid residues, deduced from open reading frame. The SAL mature protein composed of 285-amino acid residues was followed by a predicted signal sequence having 23 residues. The mRNA of SAL was found to be expressed in eggs, but not in liver. SAL is composed of three tandem repeat domain structures divided into exactly 95 amino acid residues each, and all cysteine positions of each domain were completely conserved. Sequence homologies between the three domains, termed D1 (1-95), D2 (96-190) and D3 (191-285), were as follows; D1-D2, 28%; D2-D3, 33%; D1-D3, 43%. Two conserved peptide motifs, -(AN)YGR(TD)S(T)XCS(TGR)P- and -DPCX(G)T(Y)KY(L)-, appear to exist at the N- and C-terminal regions of each domain, respectively. The kinetic parameters of SAL obtained by measuring surface plasmon resonance were as follows: K(a) (M(-1)) for neohesperidosyl-BSA, 7. 1 x 10(6); for melibiosyl-BSA, 4.9 x 10(6); and for lactosyl-BSA, 5. 2 x 10(5). These results show that RBLs including SAL comprise a family of alpha-galactosyl binding lectins having characteristic tandem repeat domain structures.

Amino Acid Sequence↗

EmsB, a tandem repeated multi-loci microsatellite, new tool to investigate the genetic diversity of Echinococcus multilocularis.

In order to explore the genetic diversity within Echinococcus multilocularis (E. multilocularis), the cestode responsible for the alveolar echinococcosis (AE) in humans, a microsatellite, composed of (CA) and (GA) repeats and designated EmsB, was isolated and characterized in view of its nature and potential field application. PCR-amplification with specific primers exhibited a high degree of size polymorphism between E. multilocularis and Echinococcus granulosus sheep (G1) and camel (G6) strains. Fluorescent-PCR was subsequently performed on a panel of E. multilocularis isolates to assess intra-species polymorphism level. EmsB provided a multi-peak profile, characterized by tandemly repeated microsatellite sequences in the E. multilocularis genome. This "repetition of repeats" feature provided to EmsB a high discriminatory power in that eight clusters, supported by bootstrap p-values larger than 95%, could be defined among the tested E. multilocularis samples. We were able to differentiate not only the Alaskan from the European samples, but also to detect different European isolate clusters. In total, 25 genotypes were defined within 37 E. multilocularis samples. Despite its complexity, this tandem repeated multi-loci microsatellite possesses the three important features for a molecular marker, i.e. sensitivity, repetitiveness and discriminatory power. It will permit assessing the genetic polymorphism of E. multilocularis and to investigate its spatial distribution in detail.

Animals↗

Identification of Leishmania genes encoding proteins containing tandemly repeating peptides.

A genomic Leishmania major DNA expression library was screened using antibodies raised against L. major membranes. Two different clones were identified that encoded proteins containing regions of tandemly repeated peptides. Clone 20 encodes a repetitive peptide of 14 amino acids, while clone 39 encodes a repetitive peptide of 10 amino acids. DNA from clone 20 hybridized with two RNA species of 9,500 and 5,200 nucleotides in length, while DNA from clone 39 hybridized to a single RNA species of 7,500 nucleotides. Antibodies against clone 20 fusion protein recognized a series of L. major proteins of apparent mol wt 250,000. Regions of repetitive peptides is a characteristic shared by many malarial protein antigens and this feature has been implicated in immune evasion. Intracellular parasites such as Leishmania and Plasmodia, therefore, may have evolved similar mechanisms consisting of the expression of proteins containing tandemly repeating peptides that are involved in immune evasion.

Amino Acid Sequence↗

The organization of two related subfamilies of a human tandemly repeated DNA is chromosome specific.

Several clones containing clusters of repetitive elements were isolated from a human chromosome 22 specific library. An EcoRI-XhoI fragment of 860bp was subcloned and was shown to belong to a family of tandemly repeated DNA linked to the Y-specific 3.4 kb HaeIII band. This probe hybridizes to several sets of sequences or subfamilies. The most abundant subfamily is a 1.8kb long sequence containing one EcoRV site, and in most repeats, one AvaII and one KpnI site. Using human-rodent somatic cell hybrid DNA, we have shown that this cluster is present on human chromosome 9 although presence on chromosome 15 is not excluded. Another subfamily, 6.1kb long, appears to be exclusive of chromosome 16. By in situ hybridization with metaphasic chromosomes, these sets of repeats were mapped to the constitutive heterochromatin of a few chromosomes. Coexistence in one genome of long tandem repeats of distinct organization but similar length may represent the outcome of a continuous process of fixation of variant sequences. Homologous repeats are also abundant in four higher primate genomes (Orangutan, gorilla, chimpanzee, and man) but absent in other primates (African green monkey, rhesus monkey, baboon, and mouse lemur).

Animals↗

Pericentromeric regions of soybean (Glycine max L. Merr.) chromosomes consist of retroelements and tandemly repeated DNA and are structurally and evolutionarily labile.

Little is known about the physical makeup of heterochromatin in the soybean (Glycine max L. Merr.) genome. Using DNA sequencing and molecular cytogenetics, an initial analysis of the repetitive fraction of the soybean genome is presented. BAC 076J21, derived from linkage group L, has sequences conserved in the pericentromeric heterochromatin of all 20 chromosomes. FISH analysis of this BAC and three subclones on pachytene chromosomes revealed relatively strict partitioning of the heterochromatic and euchromatic regions. Sequence analysis showed that this BAC consists primarily of repetitive sequences such as a 102-bp tandem repeat with sequence identity to a previously characterized approximately 120-bp repeat (STR120). Fragments of Calypso-like retroelements, a recently inserted SIRE1 element, and a SIRE1 solo LTR were present within this BAC. Some of these sequences are methylated and are not conserved outside of G. max and G. soja, a close relative of soybean, except for STR102, which hybridized to a restriction fragment from G. latifolia. These data present a picture of the repetitive fraction of the soybean genome that is highly concentrated in the pericentromeric regions, consisting of rapidly evolving tandem repeats with interspersed retroelements.

Base Sequence↗

Characterization of a third simple tandem repeat polymorphism in the human glucokinase gene.

Recent studies have implicated mutations in the glucokinase gene as a cause of early-onset Type 2 (non-insulin-dependent) diabetes mellitus. Two simple tandem repeat DNA polymorphisms have been identified in this gene and used for genetic studies. However, their heterozygosity is relatively low and not all families are informative for linkage with these markers. Here we report the characterization of a third simple tandem repeat polymorphism that can be used for genetic studies.

Base Sequence↗

Tandem repeat polymers of a critical region of the human interferon-beta promoter exhibit a marked constitutive activity and enhanced responsiveness to transcriptional regulators in transfected HeLa cells.

Multiple copy tandem repeats polymers of an authentic 30-bp region of the human interferon-beta (IFN-beta) promoter between positions-91 to -62 relative to the cap site or the hexanucleotide GAAAGT derived from this region, both acted as strong constitutive regulatory elements in transfected HeLa cells. Such polymers were unresponsive to treatment with IFN-alpha despite their considerable homology with the IFN-responsive elements of other genes but were highly responsive to treatment of HeLa cells with IFN-gamma. Virus induction of HeLa cells transfected with polymers of the 30-bp region linked to a CAT gene increased the activity of the reporter gene 500- to 2,000-fold over baseline levels. Treatment with IFN-alpha prior to virus induction did not increase further CAT activity. Cotransfection of HeLa cells with the CAT gene under the control of a 12-element tandem repeat polymer of the human IFN-beta promoter and an expression vector for the IRF-1 transcriptional activator markedly increased CAT activity while cotransfection of HeLa cells with the IFN-beta construct together with an expression vector for the transcriptional regulator IRF-2 markedly decreased CAT activity relative to cells transfected with the IFN-beta polymer alone.

Base Sequence↗

Production of novel recombinant single-domain antibodies against tandem repeat region of MUC1 mucin.

Recently, the existence of "heavy-chain" antibody in Camelidae has been described. However, as yet there is no data on the binding of this type of antibody to peptides. In addition, there was not any report of production of single-domain antibodies in two-humped camels (Camelus bactrianus). In the present study, these questions are addressed. We showed the feasibility of immunizing old world camels, cloning the repertoire of the variable domain of their heavy-chain antibodies, panning and selection, leading to the successful identification of minimum-sized antigen binders. Antigen-specific fragments of the heavy-chain IgGs (V(HH)) are of great interest in biotechnology because they are very stable, highly soluble, and react specifically and with high affinity to the antigens. In this study, we immunized two camels (Camelus dromedarius and Camelus bactrianus) with homogenized cancerous tissues, synthetic peptide, and human milk fat globule membrane (HMFG), and generated two V(HH) libraries displayed on phage particles. Some single-domain antibody fragments have been isolated that specifically recognize the tandem repeat region of MUC1. The camels' single-domain V(HH) harbor the original, intact antigen binding site and reacted specifically and with high affinity to the tandem repeat region of MUC1. Indeed soluble, specific antigen binders and good affinities (in the range of 0.2 x 10(9) M(-1) to 0.6 x 10(9) M(-1)) were identified from these libraries. This is the first example of the isolation of camel anti-peptide V(HH) domains.

Animals↗

The prognostic significance of genetic polymorphisms (Methylenetetrahydrofolate Reductase C677T, Methionine Synthase A2756G, Thymidilate Synthase tandem repeat polymorphism) in multimodally treated oesophageal squamous cell carcinoma.

The present study retrospectively examined the correlation between the outcome of patients with locally advanced oesophageal squamous cell carcinoma (cT3-4 cN0-1 cM0) after multimodal treatment (radiochemotherapy+/-surgical resection), and the presence of genetic polymorphisms in genes involved in folate metabolism. In total, 68 patients who took part in a prospective multicentric trial received 5-fluorouracil (FU)-based radiochemotherapy, optionally followed by surgery. DNA was extracted from pretherapeutic tumour biopsies and was subsequently genotyped for common genetic polymorphisms of three genes (MTHFR C677T, MTR A2756G, TS tandem repeat polymorphism) involved in folate metabolism and potentially in sensitivity to 5-FU-based chemotherapy. The genotypes were correlated with tumour response to polychemotherapy, radiochemotherapy and with overall survival. Tumours with the MTR wild-type genotype (2756AA) showed a median survival time of 16 months, whereas tumours with an MTR variant genotype (2756AG/2756GG) showed a median survival time of 42 months (P=0.0463). No prognostic impact could be verified for the genotypes of the MTHFR genes and the TS gene. Among tumours treated with radiochemotherapy and subsequent resection, MTR variant genotype showed higher histopathological response rate than tumours with MTR wild-type genotype (P=0.0442). In contrast, no significant relationship between clinically determined tumour regression after polychemotherapy and polymorphisms of the three genes under analysis was observed. In conclusion, pretherapeutic determination of the MTR A2756G polymorphism may predict survival of multimodally treated oesophageal squamous cell carcinomas. Determination of MTHFR C677T and TS tandem repeat polymorphism has no predictive value.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

Analysis of nine short tandem repeat (STR) loci in the Slovenian population.

A polymerase chain reaction (PCR)-based short tandem repeat (STR) system consisting of nine loci has recently been introduced in Slovenia for use in routine forensic identity testing. Fluorescently labelled PCR products were analysed using an ABI PRISM 310 Genetic Analyzer. The STR loci analysed exhibit between 6 and 14 observed alleles per locus and have a combined matching probability of 2.3 x 10(-10).

Alleles↗

Cloning and characterisation of tandem-repeat type galectin in rainbow trout (Oncorhynchus mykiss).

Fish beta-galactoside binding lectin (galectin) cDNA was cloned from the cDNA library of rainbow trout (Oncorhynchus mykiss) head kidney. The clone contained a single open reading frame encoding 341 amino acids (aa) (38 kDa protein), including the initiator methionine. Significant sequence homology to mammalian galectin-9 (40-55% identity) was observed. Its amino acid sequence showed two distinct N- and C-terminal domains (148 and 130 aa, respectively) connected by a peptide linker (63 aa). The galectin contains two consensus WG-E-R/K motifs thought to play an essential role in sugar-binding, indicating that this lectin is a member of the tandem-repeat type galectins which have not been identified in fish. The 1.6 kDa mRNA of the lectin was found by Northern blot analyses to be widely expressed in the spleen, head kidney, thymus, peritoneal exudate cells, ovary, gills and heart. Southern blot analyses with the probe for C-terminal of the lectin showed the existence of two hybridising genes. These results suggest that rainbow trout has at least one tandem-repeat type galectin as well as proto-type galectin.

Amino Acid Sequence↗

Tibetan population data on the multiplex short tandem repeat loci--D16S539, D7S820, and D13S317.

The multiplex amplification system of three tetrameric short tandem repeats loci (D16S539, D7S820, and D13S317) have been analysed extensively in various populations for forensic application. Population genetic studies were carried out for these three loci in a population sample of 129 unrelated Tibetan individuals by using a multiplex polymerase chain reaction (PCR) followed by 4% polyacrylamide gel electrophoresis (PAGE) and silver staining. All loci were in accordance with the Hardy-Weinberg expectations. The observed heterozygosities of three loci-D16S539, D7S820, and D13S317 were 73.3%, 81.4% and 80.6%, respectively. The polymorphism information contents (PIC) were 0.84, 0.80 and 0.83, respectively. Comparing with Han population, there was no statistically significant difference except for D16S539 locus.

Chromosome Mapping↗

Analysis of 15 short tandem repeats reveals significant differences between the Arabian populations from Morocco and Syria.

The short tandem repeat (STR) systems D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX and FGA were studied in Arabian population samples from Morocco and Syria. No significant deviation from Hardy-Weinberg equilibrium could be observed in either preparation. Comparing the Moroccan and Syrian populations using the program RxC, no similarity could be observed at all 15 loci. In the Moroccan and Syrian populations the matching probability is 1 in 1.4 x 10(17) and 1 in 2.6 x 10(17), respectively. Thus, the combination of these 15 STR loci is powerful tool for forensic identification in Arabian populations.

DNA Fingerprinting↗

Typing of eight short tandem repeat (STR) loci in a Saudi Arabian population.

Allele frequency data for eight short tandem repeat (STR) loci, HUMF13A01, HUMFESFPS, HUMF13B, HUMLPL, HUMCSF1PO, HUMTPOX, HUMTHO1 and HUMvWA, were obtained for unrelated individuals in a Saudi Arabian population. All loci, except F13B (P = 0.037) and LPL (P = 0.035), meet Hardy-Weinberg expectations, based on the exact test. The most informative locus is HUMvWA (PD = 0.936) and the least discriminating is the HUMTPOX locus (PD = 0.820). There was only one observation of a departure from expectation from pairwise locus comparisons. These data can be used for estimating the frequency of STR profiles in a Saudi Arabian population.

Alleles↗