Microsatellite Polymorphism Analysis Research Articles

Pulmonary Aspergillosis in Humboldt Penguins-Susceptibility Patterns and Molecular Epidemiology of Clinical and Environmental Aspergillus fumigatus Isolates from a Belgian Zoo, 2017-2022.

Aspergillus fumigatus is the main causative agent of avian aspergillosis and results in significant health problems in birds, especially those living in captivity. The fungal contamination by A. fumigatus in the environment of Humboldt penguins (Spheniscus humboldti), located in a Belgian zoo, was assessed through the analysis of air, water, sand and nest samples during four non-consecutive days in 2021-2022. From these samples, potential azole-resistant A. fumigatus (ARAF) isolates were detected using a selective culture medium. A total of 28 veterinary isolates obtained after necropsy of Humboldt penguins and other avian species from the zoo were also included. All veterinary and suspected ARAF isolates from the environment were characterized for their azole-resistance profile by broth microdilution. Isolates displaying phenotypic resistance against at least one medical azole were systematically screened for mutations in the cyp51A gene. A total of 14 (13.6%) ARAF isolates were identified from the environment (n = 8) and from Humboldt penguins (n = 6). The TR34/L98H mutation was observed in all resistant environmental strains, and in two resistant veterinary strains. To the best of our knowledge, this is the first description of this mutation in A. fumigatus isolates from Humboldt penguins. During the period 2017-2022, pulmonary aspergillosis was confirmed in 51 necropsied penguins, which reflects a death rate due to aspergillosis of 68.0%, mostly affecting adults. Microsatellite polymorphism analysis revealed a high level of diversity among environmental and veterinary A. fumigatus isolates. However, a cluster was observed between one veterinary isolate and six environmental strains, all resistant to medical azoles. In conclusion, the environment of the Humboldt penguins is a potential contamination source of ARAF, making their management even more complex.

Identification of novel variations of oculocutaneous albinism type 2 with Prader-Willi syndrome/Angelman syndrome in two Chinese families.

Objective: Oculocutaneous albinism (OCA) is an autosomal recessive disorder caused by a variety of genomic variations. Our aim is to identify the molecular basis of OCA in two families and lay the foundation for prenatal diagnosis. Methods: Four types of OCA-causing mutations in the TYR, p, TYRP1, or SLC45A2 genes were screened. Linkage analysis was performed because the mutations found in the p gene violated the laws of classical Mendelian heredity. Primer-walking sequencing combined with microsatellite and single-nucleotide polymorphism analysis was used to ascertain deletion ranges. Bioinformatics methods were used to assess the pathogenicity of the new mutations. Results: Proband 1 was diagnosed as OCA2 with Prader-Willi syndrome (PWS) due to a novel atypical paternal deletion (chromosome 15: 22330347-26089649) and a pathogenic mutation, c.1327G>A (Val443Ile), in the p gene of the maternal chromosome. The prenatal diagnosis results for family 1 indicated the fetus was a heterozygous carrier (c.1327G>A in the p gene) with a normal phenotype. Proband 2 was diagnosed as OCA2 with Angelman syndrome (AS) due to a typical maternal deletion of chromosome 15q11-q13 and a novel mutation, c.1514T>C (Phe505Ser), in the p gene of the paternal chromosome. This novel mutation c.1514T>C (Phe505Ser) in the p gene was predicted as a pathogenic mutation. Conclusion: Our study has shown clear genotype-phenotype correlations in patients affected by distinct deletions of the PWS or AS region and missense mutations in the p gene. Our results have enriched the mutation spectrum of albinism diseases and provided insights for more accurate diagnosis and genetic counseling.

P468 Aspergillosis in a colony of Humboldt penguins (Spheniscus humboldti) under managed care: a clinical and environmental investigation in a French zoological park

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesAspergillosis is an opportunistic fungal infection due to Aspergillus spp., and primarily A. fumigatus. The disease is pervasive in avian populations, especially those under managed care. The incidence and the management of avian aspergillosis can be critically impacted by exposure to high levels of spores via environmental contamination and by strains resistant to azole drugs. The present study aimed at assessing the impact of environmental contamination on the clinical incidence of aspergillosis in penguins managed under human care. ZooParc de Beauval was chosen for the investigations, because this zoological facility hosts a large colony of Humboldt penguin (Spheniscus humboldti) and it is geographically located in the rural countryside of the Loir-et-Cher district, France, surrounded by numerous large crop fieldsMaterial and methodsThe fungal contamination in the environment was assessed through three serial sessions of surface sampling in bird nests: all isolates were counted, identified by DNA sequencing, and then systematically screened for resistance mutations and MICs (minimal inhibitory concentrations) elevation for the A. fumigatus strains. In addition, the clinical incidence of aspergillosis was evaluated in the penguin population over a 3-year period. A microsatellite-based analysis tracked A. fumigatus isolates circulation.ResultsEnvironmental investigations highlighted a substantial increase in the fungal load during the Autumn season (>12-fold vs. the other timepoints) and a large overrepresentation of species belonging to the Aspergillus section Fumigati (ranging from 22.7 to 94.6%) (Fig. 1). Only one cryptic species (A. nishimurae) and one isolate exhibiting resistance mutation (G138S in the cyp51A gene; MIC itraconazole >4 μg/ml) were detected. The overall incidence of aspergillosis was measured at ∼3.4% case-years and was observed mostly in juvenile penguins (Fig. 1). The analysis of microsatellite polymorphism revealed a high level of genetic diversity among A. fumigatus isolates, except for one strain that was largely over-represented during the Autumn sampling session. A limited number of isolate genotypes was collected from clinical cases and from the environment.ConclusionsFungal environmental contamination and incidence of aspergillosis in penguins appeared variable depending on seasons, but the rural location of the penguin habitat did not seem to influence the emergence of resistant strains.

Comprehensive analysis of microsatellite polymorphisms in human populations.

Microsatellites (MS) are tandem repeats of short units, and have been used for population genetics, individual identification, and medical genetics. However, studies of MS on a whole-genome level are limited, and genotyping methods for MS have yet to be established. Here, we analyzed approximately 8.5 million MS regions using a previously developed MS caller for short reads (MIVcall method) for three large publicly available human genome sequencing data sets: the Korean Personal Genome Project, Simons Genome Diversity Project, and Human Genome Diversity Project. Our analysis identified 253,114 polymorphic MS. A comparison among different populations suggests that MS in the coding region evolved by random genetic drift and natural selection. In an analysis of genetic structures, MS clearly revealed population structures as SNPs and detected clusters that were not found by SNPs in African and Oceanian populations. Based on the MS polymorphisms, we selected MS marker candidates for individual identification. Finally, we applied our method to a deep sequenced ancient DNA sample. This study provides a comprehensive picture of MS polymorphisms and application to human population studies.

Aspergillosis in a colony of Humboldt penguins (Spheniscus humboldti) under managed care: a clinical and environmental investigation in a French zoological park.

Abstract Aspergillosis is pervasive in bird populations, especially those under human care. Its management can be critically impacted by exposure to high levels of conidia and by resistance to azole drugs. The fungal contamination in the environment of a Humboldt penguin (Spheniscus humboldti) group, housed in a French zoological park next to numerous large crop fields, was assessed through three serial sessions of surface sampling in nests, in 2018–20: all isolates were counted and characterized by sequencing. When identified as Aspergillus fumigatus, they were systematically screened for resistance mutations in the cyp51A gene and tested for minimal inhibitory concentrations (MICs) determination. At the same time, the clinical incidence of aspergillosis was evaluated in the penguin population by the means of systematic necropsy and mycological investigations. A microsatellite-based analysis tracked the circulation of A. fumigatus strains. Environmental investigations highlighted the substantial increase of the fungal load during the summer season (>12-fold vs. the other timepoints) and a large overrepresentation of species belonging to the Aspergillus section Fumigati, ranging from 22.7 to 94.6% relative prevalence. Only one cryptic species was detected (A. nishimurae), and one isolate exhibited G138S resistance mutation with elevated MICs. The overall incidence of aspergillosis was measured at ∼3.4% case-years, and mostly in juveniles. The analysis of microsatellite polymorphism revealed a high level of genetic diversity among A. fumigatus clinical isolates. In contrast, one environmental strain appeared largely overrepresented during the summer sampling session. In all, the rural location of the zoo did not influence the emergence of resistant strains. Lay summary

Genetic relations among wild populations of Saccharina japonica in the western North Pacific

We performed microsatellite polymorphism analysis for wild populations of Saccharina japonica, a kelp species that is, among wild production areas, mainly produced in Hokkaido, is indispensable as an ingredient of Japanese food that differs in application and value depending on the origin (variety), growing in southern Sakhalin, Primorsky Krai, and Hokkaido to investigate the genetic relations among the populations. A total of 230 alleles (Polymorphic Information Content: 0.08–0.69) were detected from 18 loci. In the analysis by region, the differentiation index (Fst) and genetic distance (DS) were lower between the Sakhalin southwestern-end populations or the Sakhalin southern coast populations and the Hokkaido populations. However, gene structure analysis showed that the clusters dominant in the kelp in the Sakhalin or Hokkaido population group also existed in the other population group, suggesting that genes are exchanged between these regions. When analyses were performed by origin, one of the Sakhalin southwestern-end populations formed a clade with the Hokkaido populations in the phylogenetic tree, both having similar genetic structure, and the Fst and DS values were particularly low with the Hokkaido East populations (S. japonica var. diabolica. The Fst and DS values were also low between the five Sakhalin southern populations – a sister group with the Hokkaido East populations in the phylogenetic tree – and the North Hokkaido populations (S. japonica var. ochotensis), indicating that these Sakhalin southwestern-end and southern populations may be used as alternatives to Hokkaido populations for culinary purposes.

222 Effective donor selection before superovulation treatment for Japanese Black beef cattle

Japanese Black beef cattle (JBBC) are a leading Wagyu breed in Japan. Embryo transfers are being used to improve JBBC, a sturdy, fast-maturing breed prized for its marbled beef; however, relevant donor selection techniques have not been fully established. Accordingly, in this study, in order to produce embryos efficiently, we aimed to investigate donor selection techniques based on follicle counts at different time points (Experiment 1) and an associated genetic marker (Experiment 2). Statistical differences were evaluated with Student's t-test or Welch's t-test, regarding P<0.05 as significant. In Experiment 1, we initially targeted 57 JBBCs for evaluation. Their follicle counts were determined by ultrasound 3 days before the start of superovulation (pre), close to the time of follicular wave emergence, and they were grouped according to number of follicles (0-19, 20-29, 30-39, and ≥40). Each cow was administered a tapered dose of FSH-R (20 AU) for superovulation, and then subjected to AI with cryopreserved semen from bulls of proven fecundity. Embryos were collected 7 days after AI by non-surgical intrauterine reflux. Post-AI follicle counts were done before ovulation and did not differ significantly from pre-superovulation follicle count in any group (range: −1.09 to 0.61). We then targeted a further 12 JBBC undergoing superovulation and AI with the same procedures, for counting of follicles at 30 days before (Day −30) embryo recovery (ER), pre-superovulation, and AI, and counting of corpora lutea and large follicles at ER, to investigate correlations between counts. We used decision coefficient (R2), regarding R2 ≥ 0.5 as significant. We found positive correlations (R2) at Day −30 with pre-superovulation (0.91), AI (0.63), and ER (0.63), and at pre-superovulation with AI (0.69) and ER (0.74). In Experiment 2, we targeted 69 JBBCs for detection of mutations in the GRIA1 gene, which encodes inotropic glutamate receptor AMPA1 with a known association with ovulation rate, and embryo yield measurements. DNA obtained from blood was subject to microsatellite polymorphism analysis, for mutations at base 917 of GRIA1 exon 7. We identified 33 cows (48%) as bearers of the wild-type allele (GG), 29 cows (42%) as bearers of the heterozygous mutation (GA), and seven cows (10%) as bearers of the homozygous mutation (AA). Average embryo yield was highest in wild-type cows, followed by heterozygous mutants and then homozygous mutants, and was significantly lower for homozygous mutants than for the other two types. Our study results demonstrated that follicle count before superovulation and GRIA1 gene analysis have utility for selecting donors for embryo transfer in JBBC. Furthermore, follicle counts at the early time point of 30 days before ER can be a useful indicator for this selection.

An historical and biogeographical assessment of European Merino sheep breeds by microsatellite markers

Although the Merino breed originated in the Iberian Peninsula, the Merino and Merino-derived sheep breeds have nowadays been widely distributed across the world both as purebreds and as admixed populations. In Europe, the Merino trunk has both high phenotypic and genetic diversity which originated from several genetic types adapted to different economic and farming environments. For these reasons, Merino and Merino-derived breeds must be presently considered cosmopolitan breeds that have an important role worldwide in the production of high-quality wool. This paper presents the results of the genetic characterization by 35 STR markers of 539 animals which are the most important 16 Merino and Merino-derived sheep breeds in Europe. The results highlighted a remarkable level of genetic variability in all breeds. Overall, microsatellite polymorphism analysis revealed the Merino Pozo Blanco as the most homogeneous breed.The subdivision between the studied breeds was shown by the STRUCTURE analysis even though a genetic admixture is particularly evident in the three Italian Merino derived breeds.In this study, the historical background of the Merino breed has been assessed from the genetic point of view, and the Spanish origin of the Merinos branch seems to be confirmed. In general, despite their common ancestral origin, nowadays the investigated Merino and Merino-derived breeds present a genetic identity in accordance with their geographical location probably due to different selection strategies.

Establishment and characterization of a prostate cancer cell line from a prostatectomy specimen for the study of cellular interaction.

Though human prostate cancer (PCa) heterogeneity can best be studied using multiple cell types isolated from clinical specimens, the difficulty of establishing cell lines from clinical tumors has hampered this approach. In this proof-of-concept study, we established a human PCa cell line from a prostatectomy surgical specimen without the need for retroviral transduction. In a previous report, we characterized the stromal cells derived from PCa specimens. Here, we characterized the epithelial cells isolated from the same tumors. Compared to the ease of establishing prostate stromal cell lines, prostatic epithelial cell lines are challenging. From three matched pairs of normal and tumor tissues, we established one new PCa cell line, HPE-15. We confirmed the origin of HPE-15 cells by short tandem repeat microsatellite polymorphism analysis. HPE-15 cells are androgen-insensitive and express marginal androgen receptor, prostate-specific antigen and prostate-specific membrane antigen proteins. HPE-15 expresses luminal epithelial markers of E-cadherin and cytokeratin 18, basal cell markers of cytokeratin 5 and p63 and neuroendocrine marker of chromogranin A. Interestingly, HPE-15 Cells exhibited no tumorigenicity in different strains of immune-deficient mice but can become tumorigenic through interaction with aggressive cancer cell types. HPE-15 cells can thus serve as an experimental model for the study of PCa progression, metastasis and tumor cell dormancy.

An Analysis of Microsatellite Polymorphism in the Population of the Arctic Rainbow Smelt Osmerus dentex from Eastern and Western Kamchatka

The genetic diversity of ten microsatellite loci is examined in samples of Arctic rainbow smelt. The expected heterozygosity estimates vary in the range of 12.6–83.9% (mean 55.3%). The degree of genetic differentiation is statistically significant: θ = 2.8%, 95% CI (1.4–4.9%). The greatest differences are found between datasets of smelt in Eastern and Western Kamchatka. The samples from Lake Nerpich’e demonstrate the maximum estimates of divergence from the other datasets from both the western and eastern coasts.

The role of ecological factors in determining phylogeographic and population genetic structure of two sympatric island skinks (Plestiodon kishinouyei and P. stimpsonii).

We conducted comparative phylogeographic and population genetic analyses of Plestiodon kishinouyei and P. stimpsonii, two sympatric skinks endemic to islands in the southern Ryukyus, to explore different factors that have influenced population structure. Previous phylogenetic studies using partial mitochondrial DNA indicate similar divergence times from their respective closest relatives, suggesting that differences in population structure are driven by intrinsic attributes of either species rather than the common set of extrinsic factors that both presumably have been exposed to throughout their history. In this study, analysis of mtDNA sequences and microsatellite polymorphism demonstrate contrasting patterns of phylogeography and population structure: P. kishinouyei exhibits a lower genetic variability and lower genetic differentiation among islands than P. stimpsonii, consistent with recent population expansion. However, historical demographic analyses indicate that the relatively high genetic uniformity in P. kishinouyei is not attributable to recent expansion. We detected significant isolation-by-distance patterns among P. kishinouyei populations on the land bridge islands, but not among P. stimpsonii populations occurring on those same islands. Our results suggest that P. kishinouyei populations have maintained gene flows across islands until recently, probably via ephemeral Quaternary land bridges. The lower genetic variability in P. kishinouyei may also indicate smaller effective population sizes on average than that of P. stimpsonii. We interpret these differences as a consequence of ecological divergence between the two species, primarily in trophic level and habitat preference.

Strong genetic differentiation but not local adaptation toward the range limit of a coastal dune plant

All species have limited geographic distributions; but the ecological and evolutionary mechanisms causing range limits are largely unknown. That many species' geographic range limits are coincident with niche limits suggests limited evolutionary potential of marginal populations to adapt to conditions experienced beyond the range. We provide a test of range limit theory by combining population genetic analysis of microsatellite polymorphisms with a transplant experiment within, at the edge of, and 60 km beyond the northern range of a coastal dune plant. Contrary to expectations, lifetime fitness increased toward the range limit with highest fitness achieved by most populations at and beyond the range edge. Genetic differentiation among populations was strong, with very low, nondirectional gene flow suggesting range limitation via constraints to dispersal. In contrast, however, local adaptation was negligible, and a distance-dependent decline in fitness only occurred for those populations furthest from home when planted beyond the range limit. These results challenge a commonly held assumption that stable range limits match niche limits, but also raise questions about the unique value of peripheral populations in expanding species' geographical ranges.

Specific features of microsatellite polymorphism in two species of Arabidopsis

The results of an analysis of microsatellite polymorphism in two closely related Arabidopsis species that differing by their crossing types are presented. Considerable genetic diversity was revealed in Arabidopsis thaliana populations located at the northern periphery of the species range (Karelia), which was not typical of self-pollinating species. Populations from the northern boundary of the species range in the basin of Lake Onega were found to be more polymorphic (P99% = 0.43; Hexp = 0.17) as compared to populations of the islands of Lake Ladoga, which are located approximately 300 km to the south (P99% = 0.39; Hexp = 0.15). It is suggested that the high population polymorphism of A. thaliana in the northern part of its range is most likely associated with hostile growing conditions and is the basis for the adaptation processes. A. thaliana populations are highly differentiated (G(ST) = 0.783), which is typical of inbred species. However, such a high interpopulation differentiation is probably due to the low level of gene flow. Specifically, five populations out of seven examined are located on the islands, i.e., are to some extent isolated. These great differences between A. thaliana populations in terms of polymorphism, as well as the high haplotype diversity, suggests postglacial colonization of the Karelian territory by many different ancestral forms. The participation of unique alleles of microsatellite loci with many dinucleotide repeats in the adaptive evolution of A. thaliana is discussed. A species population that is rare for Karelia, Arabidopsis lyrata ssp. petraea, is in complete isolation and, due to specific features of the microevolutionary processes in such populations, is characterized by the polymorphism level, which is low for outbred species (P99% = 0.64; Hexp = 0.16). Nevertheless, this population exists in Karelia for a long period of time, pointing to its successful adaptation to these conditions.

Trypanosoma rangeli displays a clonal population structure, revealing a subdivision of KP1(−) strains and the ancestry of the Amazonian group

Assessment of the genetic variability and population structure of Trypanosoma rangeli, a non-pathogenic American trypanosome, was carried out through microsatellite and single-nucleotide polymorphism analyses. Two approaches were used for microsatellite typing: data mining in expressed sequence tag /open reading frame expressed sequence tags libraries and PCR-based Isolation of Microsatellite Arrays from genomic libraries. All microsatellites found were evaluated for their abundance, frequency and usefulness as markers. Genotyping of T. rangeli strains and clones was performed for 18 loci amplified by PCR from expressed sequence tag/open reading frame expressed sequence tags libraries. The presence of single-nucleotide polymorphisms in the nuclear, multi-copy, spliced leader gene was assessed in 18 T. rangeli strains, and the results show that T. rangeli has a predominantly clonal population structure, allowing a robust phylogenetic analysis. Microsatellite typing revealed a subdivision of the KP1(−) genetic group, which may be influenced by geographical location and/or by the co-evolution of parasite and vectors occurring within the same geographical areas. The hypothesis of parasite–vector co-evolution was corroborated by single-nucleotide polymorphism analysis of the spliced leader gene. Taken together, the results suggest three T. rangeli groups: (i) the T. rangeli Amazonian group; (ii) the T. rangeli KP1(−) group; and (iii) the T. rangeli KP1(+) group. The latter two groups possibly evolved from the Amazonian group to produce KP1(+) and KP1(−) strains.

Analysis of microsatellite polymorphism in Candida albicans strains isolated from patients with vulvo-vaginal candidiasis in Guizhou region

Objective To investigate the genotype distribution of microsatellite locus CAI among Candida albicans （ C.albicans ） strains and to evaluate its relationship with the epidemic of vulvovaginal candidiasis （ VVC） in Guizhou region .Methods Ninety independent C.albicans strains isolated from patients with VVC in Guizhou were investigated based on single-strand conformation polymorphisms （ SSCP ） and GeneScan analysis .The genotypes of C.albicans strains were identified by microsatellite locus CAI polymorphism analysis .The gene polymorphism and the cluster of C.albicans strains were analyzed by using software SPSS 19.0.A logistic regression model was used to analyze the relationship between genotype distribution of CAI microsatellite among C.albicans strains and VVC infection .Results Twenty-seven distinct CAI genotypes with various patterns were identified from 90 C.albicans strains by GeneScan analysis .Cluster analysis showed that the C.albicans strains were classified into three clusters （ ClusterⅠto Cluster Ⅲ） . Three predominant genotypes including 30-45, 32-46 and 30-46 and other 7 highly similar genotypes belonged to clusterⅡthat accounted for 70.0%（63 strains） in all strains.The odds ratio for the predominant genotypes associated with VVC infection was 4.3.Conclusion The predominant distribution of genotypes was observed among the isolated C.albicans strains.The predominant genotypes of C.albicans were highly associated with the occurrence of VVC . Key words: Single-strand conformation polymorphism; Candida albicans; Genotype

Molecular Characterization of Herpes Simplex Virus 2 Strains by Analysis of Microsatellite Polymorphism

The complete 154-kbp linear double-stranded genomic DNA sequence of herpes simplex virus 2 (HSV-2), consisting of two extended regions of unique sequences bounded by a pair of inverted repeat elements, was published in 1998 and since then has been widely employed in a wide range of studies. Throughout the HSV-2 genome are scattered 150 microsatellites (also referred to as short tandem repeats) of 1- to 6-nucleotide motifs, mainly distributed in noncoding regions. Microsatellites are considered reliable markers for genetic mapping to differentiate herpesvirus strains, as shown for cytomegalovirus and HSV-1. The aim of this work was to characterize 12 polymorphic microsatellites within the HSV-2 genome by use of 3 multiplex PCR assays in combination with length polymorphism analysis for the rapid genetic differentiation of 56 HSV-2 clinical isolates and 2 HSV-2 laboratory strains (gHSV-2 and MS). This new system was applied to a specific new HSV-2 variant recently identified in HIV-1-infected patients originating from West Africa. Our results confirm that microsatellite polymorphism analysis is an accurate tool for studying the epidemiology of HSV-2 infections.

Matching Maternal Isodisomy in Mucinous Carcinomas and Associated Ovarian Teratomas Provides Evidence of Germ Cell Derivation for Some Mucinous Ovarian Tumors

The tissue derivation of mucinous ovarian carcinoma remains a mystery; however, rare tumors are associated with mature teratoma. Two decades ago, studies of chromosomal heteromorphisms and DNA polymorphisms proved that ovarian teratomas arise during female gametogenesis. We sought to exploit the relationship between mucinous carcinoma and associated teratoma to provide molecular evidence for tissue of origin. Seventeen cases of mucinous ovarian carcinoma were studied, 6 of which had associated mature teratoma. DNA was extracted from the mucinous carcinoma, teratoma, and normal dissected tissue from formalin-fixed, paraffin-embedded sections. Twelve polymorphic microsatellite markers were used to allelotype each sample. Alleles from the teratomas and carcinomas were scored as homozygous (1 allele present in the tumor when normal tissue was heterozygous), heterozygous (2 alleles present matching normal tissue), or noninformative (normal tissue was homozygous). Of the 6 carcinoma/teratoma pairs, 2 showed complete matching homozygosity for informative markers (isodisomy), whereas 2 showed matching heterozygosity. One case did not have the corresponding teratoma available for comparison but demonstrated complete homozygosity and was presumed to be isodisomic. The remaining case had a teratoma homozygous for 7 of 10 informative markers, whereas the matching carcinoma was homozygous for only 2 of these markers. Carcinomas without associated teratoma demonstrated variable zygosity. Microsatellite polymorphism analysis demonstrates that mucinous ovarian carcinomas usually clonally match associated teratomas when present and often show evidence of complete isodisomy, indicating that at least some mucinous carcinomas arise from female gametes and thus are of germ cell origin. The zygosity patterns in mucinous carcinomas without teratoma suggest that these tumors may arise through a different mechanism.

Analysis of Microsatellite Polymorphisms in South Indian Patients with Non Syndromic Cleft Lip and Palate

Non syndromic cleft lip and/or palate (NSCLP) is a complex congenital anomaly with varying incidence among patients of different geographical origins. Multiple contributing factors are known to trigger the cleft formation. There are several genes involved in the aetiology of NSCLP and they are different in different populations. The genetic components of clefts that underlie the susceptibility to respond to the environment still remain unclear. In this study, five microsatellite polymorphisms from five candidate genes were employed to analyze the association between these genes and NSCLP in 83 patients and 90 controls. Genotyping was performed by separating and visualizing the fluorescently-labeled polymerase chain reaction (PCR) products. The association of the five microsatellite polymorphisms with NSCLP was tested by using the CLUMP v1.9 program that uses the Monte Carlo method. The genotypic distribution is in Hardy-Weinberg equilibrium in the control group for only the MSX1 and DLX3 genes. The RARA microsatellite was significantly associated with NSCLP. Our results suggest that the RARA gene is involved in pathogenesis of cleft lip and palate in South Indians.

Fetal Membrane Cells for Treatment of Steroid-Refractory Acute Graft-Versus-Host Disease

The placenta protects the fetus from the mother's immune system. We have previously found that fetal membrane cells (FMCs) isolated from term placenta prevent alloreactivity in vitro. FMCs share many features with bone marrow-derived mesenchymal stromal cells (MSCs), which we previously introduced to treat severe acute graft-versus-host disease (GVHD). Here, we tested FMCs for treatment of steroid-refractory acute GVHD. After two passages in culture, approximately 10(9) FMCs were obtained from one single placenta, although not all cells from passage 0 and passage 1 were used for expansion. The FMCs were positive for CD29, CD44, CD73, CD90, CD105, and CD49d but were negative for hematopoietic, endothelial, and epithelial markers. Microsatellite polymorphism analysis showed that FMCs were of maternal origin. All FMCs used showed normal karyotype. Nine patients who had undergone hematopoietic stem cell transplantation (HSCT) and who had developed steroid-refractory grade III-IV acute GVHD were given 0.9-2.8 × 10(6) FMCs per kg at 15 infusions. Median age was 57 years. There was no toxicity from infusion of FMCs in eight patients. One patient had seizures after infusion. Two of eight evaluable patients had a complete response and four had a partial response, giving an overall response rate of 75%. Two patients showed no response at all. Three patients are alive from 6 to 21 months after HSCT. One patient is well and two have chronic GVHD. Thus, FMCs may be successfully used for immune modulation and tissue repair.

Fetal Membrane Cells for Treatment of Steroid-Refractory Acute Graft-Versus-Host Disease

The placenta protects the fetus from the mother's immune system. We have previously found that fetal membrane cells (FMCs) isolated from term placenta prevent alloreactivity in vitro. FMCs share many features with bone marrow-derived mesenchymal stromal cells (MSCs), which we previously introduced to treat severe acute graft-versus-host disease (GVHD). Here, we tested FMCs for treatment of steroid-refractory acute GVHD. After two passages in culture, approximately 10(9) FMCs were obtained from one single placenta, although not all cells from passage 0 and passage 1 were used for expansion. The FMCs were positive for CD29, CD44, CD73, CD90, CD105, and CD49d but were negative for hematopoietic, endothelial, and epithelial markers. Microsatellite polymorphism analysis showed that FMCs were of maternal origin. All FMCs used showed normal karyotype. Nine patients who had undergone hematopoietic stem cell transplantation (HSCT) and who had developed steroid-refractory grade III-IV acute GVHD were given 0.9-2.8 × 10(6) FMCs per kg at 15 infusions. Median age was 57 years. There was no toxicity from infusion of FMCs in eight patients. One patient had seizures after infusion. Two of eight evaluable patients had a complete response and four had a partial response, giving an overall response rate of 75%. Two patients showed no response at all. Three patients are alive from 6 to 21 months after HSCT. One patient is well and two have chronic GVHD. Thus, FMCs may be successfully used for immune modulation and tissue repair.