Short Tandem Repeat Sequences Research Articles

High-Throughput Sequencing of Core STR Loci for Forensic Genetic Investigations using the Roche Genome Sequencer FLX Platform

The analysis and profiling of short tandem repeat (STR) loci is routinely used in forensic genetics. Current methods to investigate STR loci, including PCR-based standard fragment analyses and capillary electrophoresis, only provide amplicon lengths that are used to estimate the number of STR repeat units. These methods do not allow for the full resolution of STR base composition that sequencing approaches could provide. Here we present an STR profiling method based on the use of the Roche Genome Sequencer (GS) FLX to simultaneously sequence multiple core STR loci. Using this method in combination with a bioinformatic tool designed specifically to analyze sequence lengths and frequencies, we found that GS FLX STR sequence data are comparable to conventional capillary electrophoresis-based STR typing. Furthermore, we found DNA base substitutions and repeat sequence variations that would not have been identified using conventional STR typing.

Statistical model for degraded DNA samples and adjusted probabilities for allelic drop-out

Abstract DNA samples found at a scene of crime or obtained from the debris of a mass disaster accident are often subject to degradation. When using the STR DNA technology, the DNA profile is observed via a so-called electropherogram (EPG), where the alleles are identified as signal peaks above a certain level or above a signal to noise threshold. Degradation implies that these peak intensities decrease in strength for longer short tandem repeat (STR) sequences. Consequently, long STR loci may fail to produce peak heights above the limit of detection resulting in allelic or locus drop-outs. In this paper, we present a method for measuring the degree of degradation of a sample and demonstrate how to incorporate this in estimating the probability of allelic drop-out. This is done by extending an existing method derived for non-degraded samples. The performance of the methodology is evaluated using data from degraded DNA, where cases with varying amounts of DNA and levels of degradation are investigated.

Irradiation-Based Myeloablative Cord Blood Transplantation for Hematologic Malignancies In Adults.

AbstractAbstract 4583We retrospectively analyzed of the engraftment, Transplant-related complications and survival after unrelated cord blood transplantation (UCBT) using irradiation-based myeloablative conditioning in adult with hematologic malignancies. Between September 2006 and June 2010, 29 consecutive adult patients with hematological malignancies were treated with UCBT, Thirteen of them were advanced-stage disease and 11 of them were high risk or refractory disease. All patients received four fractionated 12 Gy TBI, total dose 12 g/m2 cytarabine and total dose 120 mg/kg cyclophosphamide as myeloablative conditioning. The median age was 21 years; the median weight was 57 kg. Double or multiple UCB grafts were used for 17 patients, while single UCB graft for 12 patients. the median number of nucleated cells was 3.83×107/kg and 5.25×107/kg. All patients were given a combination of cyclosporine A (CsA) and mycophenolate mofetil (MMF) for graft-versus-host disease (GVHD) prophylaxis. All patients obtained engraftment. Median time to neutrophil≥0.5×109/L was 20 days (range 14–37) and platelet engraftment in 25 patients (≥20×109/L) was 35 days (range 25–49). Chimerism was assessed by PCR analysis of short tandem repeat (STR) sequences on whole blood or bone marrow weekly in cases who were fully donor chimeric from 7 days to 21 days after transplantation. 16 cases developed acute GVHD, more than grade II in three cases. Six of twenty-four patients who survived more than 100 days developed limited chronic GVHD.17 cases were alive and in hematologic remission at a median follow-up of 504 days (range 49 ~ 1245). The probability of over survival at 3 years was 58.6%. Five cases relapsed. Nine of twelve cases died of transplant related complications and infection. These results suggest that UCBT after TBI-based myeloablative conditioning could be safely and effectively used for adult patients with hematologic malignancies. Disclosures:Sun:the Fund of the Key Medical Project of Anhui Provincial healthy department (2010A005): Research Funding; the Clinical Technology foundation of Anhui Provincial healthy department (2008A011): Research Funding; the Fund of Anhui Provincial “115” Industrial Innovation Program: Research Funding.

STR sequence analysis for characterizing normal, variant, and null alleles

DNA sequence variation is known to exist in and around the repeat region of short tandem repeat (STR) loci used in human identity testing. While the vast majority of STR alleles measured in forensic DNA laboratories worldwide type as “normal” alleles compared with STR kit allelic ladders, a number of variant alleles have been reported. In addition, a sequence difference at a polymerase chain reaction (PCR) primer binding site in the DNA template can cause allele drop-out (i.e., a “null” or “silent” allele) with one set of primers and not with another. Our group at the National Institute of Standards and Technology (NIST) has been sequencing variant and null alleles supplied by forensic labs and cataloging this information on the NIST STRBase website for the past decade. The PCR primer sequences and strategy used for our STR allele sequencing work involving 23 autosomal STRs and 17 Y-chromosome STRs are described along with the results from 111 variant and 17 null alleles.

Outcome of Umbilical Cord Blood Transplantation in 51 Patients with Hematologic Malignancies.

Abstract 4342 Background and ObjectivesUmbilical cord blood transplantation (UCBT) has recently emerged as a curative approach for patients with hematologic malignancies who lack a suitable HLA-matched donor. We report the outcome of UCBT for 51 hematologic malignancies from a single hospital in Southeast China. Design and MethodsFifty-one cases of UCBT for hematologic malignancies at Anhui Provincial Hospital from March 2000 to May 2009 were retrospectively characterized, including 20 cases of acute lymphoblastic leukemia (ALL), 12 acute myeloid leukemia (AML), 12 chronic myeloid leukemia (CML) (accelerated phase 5, blast crisis 4), 2 mixed acute leukemia(MIX-AL),4 myelodysplastic syndrome (MDS), and one case of non-Hodgkin lymphoma (NHL). Double UCB grafts were used for 27 patients, while single UCB graft was used for 24 patients. All but one UCB grafts were from unrelated donors. The median cell doses infused were 4.37×107TNC/kg (range, 2.41?16.24), 2.96×105 CD34/kg (range, 0.67?29.28) in single UCBT or 4.44×107TNC/kg (range, 2.76?7.70), 2.67×105 CD34/kg (range, 0.8?9.35) in double UCBT. Myeloablative conditioning was given to 46 patients and nonmyeloablative regimen was given to 5 patients according to the standard protocols. Myeloablative regimen consisted of fractionated total body irradiation (TBI) 1,200 cGy, cyclophosphamide 120 mg/kg, cytarabine 8g/m2 (n=26) or of modified BUCY2 consisting of busulfan 16mg/kg orally or 12.8 mg/kg intravenously, cyclophosphamide 120 mg/kg, and rabbit antithymocyte globulin (ATG, Fresenius, Germany) 7.5mg/kg (n=20). Reduced intensity conditioning consisted of fludarabine 160 mg/m2, BU6.4m/kg, ATG 7.5mg/kg and TBI 3Gy. All patients were given a combination of cyclosporin A (CsA) and mycophenolate mofetil (MMF) for graft-versus-host disease (GVHD) prophylaxis. ResultsOf the 51 patients, 34 had advanced stage of disease. The patients' age ranged from 3 to 48 years (median 19 yr) and the body weight ranged from 15 to 84 kg (median 53 kg), respectively. Although one patient with advance-staged chronic myeloid leukemia (CML) died 16 days after UCBT and 6 patients showed primary graft failure, 44 of the 50 patients (88%) achieved engraftment, with neutrophil recovery (≥0.5×109/L) occurring at a median of 19 days (range, 12-39) and platelet recovery (≥20×109/L) occurring at a median of 34 days (range, 21-73), respectively. All of the 44 patients gained complete donor chimerism by PCR analysis of short tandem repeat (STR) sequences on whole blood or bone marrow from day 7 to day 100 after transplantation. Half of the patients (22/44, 50%) developed acute GVHD with 19 cases of grade I/II disease (43.2%) and 3 cases of grade III disease (6.8%). Only five patients (16.1%) developed chronic GVHD, with 2 in skin, 1 in intestinal, 1 in liver, and 1 patient with refractory chronic GVHD with thrombocytopenia. Three patients had relapse (2 year relapse rate of 5.9%) with one in central nervous system, who achieved remission after intrathecal injection of chemotherapy regimen.,one with CML blast crisis achieved remission after oral imatinib,and the third died of relapsed disease. The median follow-up duration of surviving patients was 19 months (range, 3-113) with a 2 year overall survival rate of 54.5%. A total of 20 patients died during the study, with a 2 year transplant related mortality rate of 37.3%. The most common cause of death was severe infection (12) due to bacteria, fungi and/or viruses, and other causes included acute GVHD (1), disease progression (1), veno-occlusive disease of the liver (1), sudden cardiac death (1), cerebral hemorrhage (1), fulminant viral hepatitis (1), early rejection (1),and systemic failure (1). Interpretation and ConclusionsUCBT could be safely and effectively used for patients with hematologic malignancies. The use of double UCB as a strategy extends the availability of transplantation. Prevention and control of infection are essential for long-term survival after UCBT. Disclosures:No relevant conflicts of interest to declare.

Donor cell-derived acute lymphocytic leukemia after allogeneic stem cell transplantation for multiple myeloma

Donor cell leukemia (DCL) is a rare, but well-known, complication after allogeneic hematopoietic cell transplantation. We report a case of donor cell-derived acute lymphocytic leukemia (ALL) occurring in a 55-year-old man after allogeneic bone marrow transplantation (allo-BMT) from an HLA-matched unrelated donor for refractory multiple myeloma (MM). Molecular analysis using short tandem repeat sequences proved the ALL to be of donor origin. He underwent combination chemotherapy and second allo-BMT from an alternative donor. After second allo-BMT, extramedullary myeloma relapsed as tumor, but was successfully treated with proteasome inhibitor, bortezomib. However, he died from severe graft-versus-host disease four months after the second transplantation. Although more than 50 cases of DCL have been reported, there have been only two reports of DCL developed in MM patients including our case. This rare complication may give some insights into leukemogenesis.

Genotyping of IRGM Tetranucleotide Promoter Oligorepeats by Fluorescence Resonance Energy Transfer

Fluorescence resonance energy transfer (FRET) genotyping has been well established for the rapid assessment of single nucleotide polymorphisms (SNPs) and deletions. A design is presented that allows the typing of short tandem oligo repeat sequences using the LightTyper/LightCycler system. The protocol was evaluated and applied to the typing of a tetranucleotide promoter repeat of the human gene encoding the immunity-related GTPase family, M (IRGM) molecule in >2000 individuals from Ghana, West Africa.

Use of Short Tandem Repeat Sequences to Study Mycobacterium leprae in Leprosy Patients in Malawi and India

BackgroundInadequate understanding of the transmission of Mycobacterium leprae makes it difficult to predict the impact of leprosy control interventions. Genotypic tests that allow tracking of individual bacterial strains would strengthen epidemiological studies and contribute to our understanding of the disease.Methodology/Principal FindingsGenotyping assays based on variation in the copy number of short tandem repeat sequences were applied to biopsies collected in population-based epidemiological studies of leprosy in northern Malawi, and from members of multi-case households in Hyderabad, India. In the Malawi series, considerable genotypic variability was observed between patients, and also within patients, when isolates were collected at different times or from different tissues. Less within-patient variability was observed when isolates were collected from similar tissues at the same time. Less genotypic variability was noted amongst the closely related Indian patients than in the Malawi series.Conclusions/SignificanceLineages of M. leprae undergo changes in their pattern of short tandem repeat sequences over time. Genetic divergence is particularly likely between bacilli inhabiting different (e.g., skin and nerve) tissues. Such variability makes short tandem repeat sequences unsuitable as a general tool for population-based strain typing of M. leprae, or for distinguishing relapse from reinfection. Careful use of these markers may provide insights into the development of disease within individuals and for tracking of short transmission chains.

Structural Diversity and Extreme Stability of Unimolecular Oxytricha nova Telomeric G-Quadruplex

Oxytricha nova telomeric DNA contains guanine-rich short-tandem repeat sequences (GGGGTTTT) n and terminates as a single strand at the 3'-end. This single-stranded overhang forms a novel DNA structure, namely, G-quadruplex, comprising four quartets. In this study, we investigated the structures and dynamics of unimolecular Oxytricha nova ( O. nova) telomeric G-quadruplexes by performing single molecule fluorescence resonance energy transfer (FRET) spectroscopy and bulk circular dichroism (CD) measurements. We observed that unimolecular O. nova G-quadruplexes exhibit structural polymorphism according to monovalent cations. In the presence of Na (+), only antiparallel conformation is detected, which was demonstrated in previous studies; however, in the presence of K (+), they fold into two different conformations, a parallel conformation and an antiparallel one different from that induced by Na (+). Furthermore, these G-quadruplexes show extremely high stability in their dynamics when compared with human G-quadruplexes. While human telomeric G-quadruplexes that possess three quartets display fast dynamic behavior (<100 s) at low K (+) concentrations or high temperatures, O. nova G-quadruplexes maintain their conformational state for a long time (>1000 s), even at the lowest K (+) concentration and the highest temperature investigated. This high stability is primarily due to an extra quartet that results in additional cation coordination. In addition to cation coordination, we propose that other factors such as base stacking and the size of the thymine loop may contribute to the stability of O. nova G-quadruplexes; this is based on the fact that the O. nova G-quadruplexes were observed to be more stable than the human ones in the presence of Li (+), which is known to greatly destabilize G-quadruplexes because of imprecise coordination. This extreme stability of four-quartet G-quadruplexes enables telomere protection even in the absence of protective proteins or in the case of abrupt environmental changes, although only a single G-quadruplex structure can be derived from the short single-stranded overhang.

Short tandem repeat sequences in the Mycoplasma genitalium genome and their use in a multilocus genotyping system

BackgroundSeveral methods have been reported for strain typing of Mycoplasma genitalium. The value of these methods has never been comparatively assessed. The aims of this study were: 1) to identify new potential genetic markers based on an analysis of short tandem repeat (STR) sequences in the published M. genitalium genome sequence; 2) to apply previously and newly identified markers to a panel of clinical strains in order to determine the optimal combination for an efficient multi-locus genotyping system; 3) to further confirm sexual transmission of M. genitalium using the newly developed system.ResultsWe performed a comprehensive analysis of STRs in the genome of the M. genitalium type strain G37 and identified 18 loci containing STRs. In addition to one previously studied locus, MG309, we chose two others, MG307 and MG338, for further study. Based on an analysis of 74 unrelated patient specimens from New Orleans and Scandinavia, the discriminatory indices (DIs) for these three markers were 0.9153, 0.7381 and 0.8730, respectively. Two other previously described markers, including single nucleotide polymorphisms (SNPs) in the rRNA genes (rRNA-SNPs) and SNPs in the MG191 gene (MG191-SNPs) were found to have DIs of 0.5820 and 0.9392, respectively. A combination of MG309-STRs and MG191-SNPs yielded almost perfect discrimination (DI = 0.9894). An additional finding was that the rRNA-SNPs distribution pattern differed significantly between Scandinavia and New Orleans. Finally we applied multi-locus typing to further confirm sexual transmission using specimens from 74 unrelated patients and 31 concurrently infected couples. Analysis of multi-locus genotype profiles using the five variable loci described above revealed 27 of the couples had concordant genotype profiles compared to only four examples of concordance among the 74 unrelated randomly selected patients.ConclusionWe propose that a combination of the MG309-STRs and MG191-SNPs is efficient for general epidemiological studies and addition of MG307-STRs and MG338-STRs is potentially useful for sexual network studies of M. genitalium infection. The multi-locus typing analysis of 74 unrelated M. genitalium-infected individuals and 31 infected couples adds to the evidence that M. genitalium is sexually transmitted.

In-Situ Proliferation May Explain the Persistence of Host Derived Langerhans Cells Following Allogeneic Hematopoietic Stem Cell Transplantation with Reduced Intensity Conditioning.

Langerhans cells (LCs) are antigen presenting cells found in the epidermis. They are thought to originate from bone marrow precursors although studies in mice suggest they have the ability to self renew in situ. Murine models have also shown that recipient LCs are required for the development of acute graft versus host disease (GVHD) following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Engraftment of LCs following human allo-HSCT has previously been assessed using migration from isolated epidermal sheets, which may be inaccurate because of the potential for differential migration of donor and recipient cells. We therefore studied LC engraftment using fluorescence immunophenotyping and simultaneous in-situ hybridisation for X/Y chromosomes in sex mismatched transplants. Skin biopsies were performed at days 28, 56 and 100, 6 months and 1 year post transplant on 8 patients receiving alemtuzumab based RIC allo-HSCT regimens. Ten micron cryosections were taken, LCs labelled with anti-CD1a and X/Y chromosomes detected with the CEP X/Y probe kit. Slides were examined on a Zeiss LSM510 Meta confocal microscope and Z-stack images were collected to cover a volume of 100μm3. Chimerism of purified CD15+ and CD3+ peripheral blood cells was determined in parallel by polymerase chain reaction amplification of short tandem repeat sequences. Results are tabulated below and show that compared to CD15+ myeloid blood cells, donor LC engraftment is markedly delayed (day 28, 56 and 100 paired t-tests give P<0.0001, P=0.001 and P=0.0302 respectively). Of the 8 patients studied, 1 went on to develop biopsy proven GVHD. In all cases studied, recipient LCs could still be detected albeit in small numbers at the 1-year time point. Since these findings suggested that epidermal LCs are either very persistent or replicate in-situ, we next stained samples of normal and post transplant GVHD skin with both CD1a and the proliferation marker Ki67. In both cases a proportion of CD1a+ cells also expressed Ki67 (13.5% of normals, n=8; 18.6% of GVHD n=9 p=0.33). Both recipient and donor derived LCs appear to be capable of proliferating since both male and female Ki67+ CD1a+ cells were identified in 2 cases for which appropriate samples were available. In conclusion, our results provide independent confirmation that recipient LCs may persist for a prolonged period following allo-HSCT and show that at least in part, this may be due to self renewal within the epidermis.Post transplant chimerism analysisCell TypeDay 28Day 56Day 1006 Months1 YearLC18.5 (n=8)73 (n=7)90 (n=7)93.5 (n=6)97 (n=3)CD3+99 (n=8)80.5 (n=6)66.5 (n=6)79.5 (n=4)100 (n=3)CD15+100 (n=8)100 (n=6)100 (n=6)100 (n=4)100 (n=2)Median % donor chimerism at specific time points following allo-HSCT.

Monoclonal B Cell Lymphocytosis as a Marker of Inherited Predisposition to Chronic Lymphocytic Leukemia.

Background: Segregation analyses suggest that CLL has one of the highest inherited risks among all types of cancer. However, no common germline mutations that confer an inherited predisposition to CLL have been found. Identification of a CLL predisposition gene should lead to improved prevention and treatment strategies. Gene identification studies to date have been limited by few affected patients per family, late age of disease onset, and probable genetic heterogeneity. Identification of a phenotypic marker for carriers of a CLL predisposition gene would dramatically improve the likelihood of finding a causative gene. Monoclonal B-cells with typical CLL immunophenotype (monoclonal B-cell lymphocytosis, MBL) can be identified in normal relatives of patients with overt CLL. We hypothesize that MBL identify unaffected carriers of a CLL predisposition gene.Methods: We used 6-color flow cytometric methods to analyze peripheral blood samples for MBL. We defined MBL as a population of CD5+, CD20lo cells that were kappa/lambda restricted and comprised at least 2% of the CD19+ peripheral B cell compartment. We identified extended families segregating 2 or more members with CLL from patients receiving care at Duke and the Durham VA. We collected peripheral blood from both affected and unaffected family members for complete blood count, flow cytometry for MBL detection, and DNA purification. Genotyping studies were performed using PCR to amplify highly polymorphic short tandem repeat sequences (STR) at candidate loci.Results: We ascertained 16 extended families with CLL and obtained samples from 138 family members. Twelve of the 113 (11%) unaffected participating family members had MBL. In one family, evaluation of 32 members revealed 3 with overt CLL and 5 with MBL. Two distinct inheritance patterns emerge upon review of the families ascertained and analyzed to date. Five families suggested a dominant mode of inheritance of the combined CLL/MBL phenotype: multiple family members with MBL, vertical transmission, and an approximate 50% affection rate for among persons over age 40. In these 5 families, 11 of 64 (17%) unaffected family members had MBL while in the other 11 families no unaffected family members had MBL (0 of 48). In some of the latter 11 families, analysis of obligate carriers of a CLL predisposition gene showed no MBL. We pooled the 5 families with MBL for screening of candidate loci associated with an inherited predisposition to CLL from previously published linkage studies. Using markers at these candidate loci, we excluded linkage to 5q22.1, 6p22, 9q21.33, 10q24.1, 11q13.1, 13q22.2, and 14q32.2.Conclusions: A combined MBL/CLL phenotype appears to be inherited as a dominant trait in some cases of familial CLL. This supports the hypothesis of genetic heterogeneity in the development of CLL. A familial analysis of both CLL and MBL greatly increases the statistical power for genetic analysis, and may allow for families to be pooled for genetic analysis based on the MBL phenotype. Gene identification studies are ongoing.

Spontaneous deletions and reciprocal translocations in Saccharomyces cerevisiae: influence of ploidy

Studying spontaneous chromosomal rearrangements throws light on the rules underlying the genome reshaping events occurring in eukaryotic cells, which are part of the evolutionary process. In Saccharomyces cerevisiae, translocation and deletion processes have been frequently described in haploids, but little is known so far about these processes at the diploid level. Here we investigated the nature and the frequency of the chromosomal rearrangements occurring at this ploidy level. Using a positive selection screen based on a particular mutated allele of the URA2 gene, spontaneous diploid revertants were selected and analysed. Surprisingly, the diploid state was found to be correlated with a decrease in chromosome rearrangement frequency, along with an increase in the complexity of the rearrangements occurring in the target gene. The presence of short DNA tandem repeat sequences seems to be a key requirement for deletion and reciprocal translocation processes to occur in diploids. After discussing the differences between the haploid and diploid levels, some mechanisms possibly involved in chromosome shortening and arm exchange are suggested.

Donor Cell-derived Acute Myeloblastic Leukemia After Allogeneic Peripheral Blood Hematopoietic Stem Cell Transplantation for Juvenile Myelomonocytic Leukemia

Despite its rarity, donor cell leukemia (DCL) is a most intriguing entity. We report here the case of a 5 year-old girl with juvenile myelomonocytic leukemia and normal female karyotype who developed acute myeloblastic leukemia with a karyotype of 46, X, t(X; 7) (p21; p11.2), der(7) t(3; 7) (q13.3; q22) 5 months after peripheral blood hematopoietic stem cell transplantation from her HLA-matched sister. We performed the analysis of short tandem repeat sequence markers to DNA obtained from donor peripheral blood, patient's peripheral blood including leukemic blasts and patient's hair root. This analysis showed that the leukemic blood DNA matched the donor blood DNA and not the patient's DNA, thus confirming DCL. To our knowledge, this is the first case of DCL after peripheral blood SCT for juvenile myelomonocytic leukemia.

STR sequence variants revealed by Pyrosequencing technology

Pyrosequencing is a fast, real-time, non-electrophoretic sequencing-by-synthesis method that can be used to genotype short tandem repeat (STRs) markers. In this study, a total of 18 Y-chromosome and autosomal STRs have been successfully analyzed using Pyrosequencing technology. Several sequence variants were detected, demonstrating that additional information besides the fragment length can be provided in a forensic DNA investigation by these assays.

Simultaneous analysis of microsatellite instability and loss of heterozygosity by capillary electrophoresis with a homemade kit

Microsatellite instability (MSI) and loss of heterozygosity (LOH), the alteration in length and strength of short tandem repeat sequences are an important molecular characteristic of many human tumors. MSI and LOH analysis has become an attractive method for diagnostic and tumor research purposes. A method for the simultaneous analysis of MSI and LOH at the five microsatellite loci (BAT-26, D17S261, D3S1283, D2S123 and D3S1611) was developed employing a cheap homemade kit to replace the expensive commercial kit on ABI 310 capillary genetic analyzer. After studying the effect of temperature and urea denaturant on microsatellite analysis, 8 mol/L urea and 60 degrees C were selected for assessing accurately fragment size of microsatellite alleles. Based on this method, 52 sporadic gastric cancers were screened, and MSI and LOH, at least one locus was observed in 15 of 52 (28.8%) patients. Moreover, it is found that a statistically significant association exists between MSI and LOH and tumor-differentiated level.

Distribution of the alleles at loci D16S539, D7S820, and D13S317 in hydatidiform mole genome from Chinese women and its relationship with clinical prognosis

Using polymerase chain reaction and denaturating polyacrylamide gel electrophoretic techniques, we studied 53 cases of hydatidiform moles. Of these, 41 cases were genetically complete hydatidiform moles (g-CHM) whose genome were totally paternally derived. We investigated the distribution of the alleles in the short tandem repeat sequences at loci D16S539, D7S820, and D13S317 in these cases. In particular, we analyzed the allelic distribution and potential significance in cases with traceable benign and invasive moles (i.e., persistent trophoblastic tumor [PTT]). Among 41 g-CHM cases, there were six alleles at D16S539, five alleles at D7S820 (the frequencies of alleles 9 and 10 were respectively lower and higher than those in Beijing population), and seven alleles at D13S317; the heterozygosity of loci D16S539, D7S820, and D13S317 was 0.0732, 0.0976, and 0.0732, respectively. Among 23 benign cases, there were six alleles at D16S539, four at D7S820, and six at D13S317; among 11 PTT cases, there were five alleles at D7S820 and four alleles each at D16S539 and D13S317. The frequencies of allele 9 at D16S539 and allele 10 at D7S820 were higher than in benign cases ( P < 0.05). There were significant differences in frequencies of alleles 9 and 10 at D7S820 between the cases and the Beijing population, and heterozygosity at the three loci was lower in the cases than in the population. In addition, invasiveness of hydatidiform mole correlated to the frequency of allele 9 at loci D16S539 and allele 10 at D7S820.

Short Tandem Repeat Polymorphism in the Flanking Region of the Human Phosphoglycerate Kinase Gene in a Japanese Population

The human phosphoglycerate kinase (PGK1) gene is located within Xqll-Xql3 and is closely linked to the androgen receptor gene within a region implicated in a number of X-chromosome-linked urologic disorders. A polymorphism of a TATC short tandem repeat (STR) is present downstream from the PGK1 3' nuclease-sensitive site. We present the PGK1 flanking STR sequence and population genetic data for 190 Japanese males and 83 Japanese females. Ten STR alleles and 29 genotypes were identified in the population. Five alleles--*10, *11, *12, *13, and *14--were common in the Japanese with frequencies greater than 10%. No significant deviations from Hardy-Weinberg equilibrium were established. The power of discrimination was 0.993 for females and 0.819 for males; heterozygosity was 0.759 for females; and the polymorphic information content was 0.936. These data indicate that this STR locus shows a high degree of polymorphism in this Japanese population and may prove to be a useful genetic marker in forensic medicine, in determining the clonality of neoplasms, and potentially in studying predisposition to prostate cancer and other urologic diseases.

Microsatellite typing of the rhesus macaque MHC region

To improve the results gained by serotyping rhesus macaque major histocompatibility complex (MHC) antigens, molecular typing techniques have been established for class I and II genes. Like the rhesus macaque Mamu-DRB loci, the Mamu-A and -B are not only polymorphic but also polygenic. As a consequence, sequence-based typing of these genes is time-consuming. Therefore, eight MHC-linked microsatellites, or short tandem repeats (STRs), were evaluated for their use in haplotype characterization. Polymorphism analyses in rhesus macaques of Indian and Chinese origin showed high STR allelic diversity in both populations but different patterns of allele frequency distribution between the groups. Pedigree data for class I and II loci and the eight STRs allowed us to determine extended MHC haplotypes in rhesus macaque breeding groups. STR sequencing and comparisons with the complete rhesus macaque MHC genomic map allowed the exact positioning of the markers. Strong linkage disequilibria were observed between Mamu-DR and -DQ loci and adjacent STRs. Microsatellite typing provides an efficient, robust, and quick method of genotyping and deriving MHC haplotypes for rhesus macaques regardless of their geographical origin. The incorporation of MHC-linked STRs into routine genetic tests will contribute to efforts to improve the genetic characterization of the rhesus macaque for biomedical research and can provide comparative information about the evolution of the MHC region.

Successful Unrelated Cord Blood Transplantation in a Child with β-Thalassemia Major

We report here the first successful transplant of unrelated umbilical cord blood (UCB) for a child with beta-thalassemia major in Taiwan. A total of 2.48 x 10(5)/kg CD34 cells were infused into a 3(1/2)-year-old girl following conditioning with a pre-transplant cytoreductive/immunosuppressive regimen of busulfan and cyclophosphamide. The absolute neutrophil count first exceeded 0.5 x 10(9)/l on day 17, and the patient became red cell- and platelet-independent on days 34 and 49, respectively. Engraftment of donor hematopoietic populations was documented by cytogenetic analysis, hemoglobin electrophoresis, Southern blot analysis for thalassemia markers, and molecular analysis of short tandem repeats sequences. The patient was ex-thalassemic for more than 15 months post transplant. From this experience, it is postulated that UCB will indeed allow us to expand the available donor pool for recipients with an HLA DRB1 mismatched at one locus and reduce the risk of graft-versus-host disease.