Genetic diversity, evolution and selection in the major histocompatibility complex DRB and DQB loci in the family Equidae.

BACKGROUND: The mammalian Major Histocompatibility Complex (MHC) is a genetic region containing highly polymorphic genes with immunological functions. MHC class I and class II genes encode antigen-presenting molecules expressed on the cell surface. The MHC class II sub-region contains genes expressed in antigen presenting cells. The antigen binding site is encoded by the second exon of genes encoding antigen presenting molecules. The exon 2 sequences of these MHC genes have evolved under the selective pressure of pathogens. Interspecific differences can be observed in the class II sub-region. The family Equidae includes a variety of domesticated, and free-ranging species inhabiting a range of habitats exposed to different pathogens and represents a model for studying this important part of the immunogenome. While equine MHC class II DRA and DQA loci have received attention, the genetic diversity and effects of selection on DRB and DQB loci have been largely overlooked. This study aimed to provide the first in-depth analysis of the MHC class II DRB and DQB loci in the Equidae family. RESULTS: Three DRB and two DQB genes were identified in the genomes of all equids. The genes DRB2, DRB3 and DQB3 showed high sequence conservation, while polymorphisms were more frequent at DRB1 and DQB1 across all species analyzed. DQB2 was not found in the genome of the Asiatic asses Equus hemionus kulan and E. h. onager. The bioinformatic analysis of non-zero-coverage-bases of DRB and DQB genes in 14 equine individual genomes revealed differences among individual genes. Evidence for recombination was found for DRB1, DRB2, DQB1 and DQB2 genes. Trans-species allele sharing was identified in all genes except DRB1. Site-specific selection analysis predicted genes evolving under positive selection both at DRB and DQB loci. No selected amino acid sites were identified in DQB3. CONCLUSIONS: The organization of the MHC class II sub-region of equids is similar across all species of the family. Genomic sequences, along with phylogenetic trees suggesting effects of selection as well as trans-species polymorphism support the contention that pathogen-driven positive selection has shaped the MHC class II DRB/DQB sub-regions in the Equidae.

A de novo reciprocal chromosomal translocation t(3;6)(p14;q26) in the black Lucano pig.

In the past two decades, several cytogenetic screening programmes identified different chromosome rearrangements in pig, most of which represented by reciprocal translocation (rcp). This chromosome abnormality does not involve the variation in the number of chromosomes, but only the rearrangement of genetic material, resulting in phenotypically normal carriers with fertility problems. During an occasional cytogenetic screening, a new reciprocal translocation was detected in the black Lucano pig native breed. We analysed 15 animals reared by a family-run piggery in Basilicata region (Southern Italy). After karyotyping, four pigs (two boars and two sows) revealed two unpaired chromosomes. Analysis of the RBA karyotype and the dual-colour FISH technique confirmed that these pigs showed the same reciprocal translocation involving the chromosomes SSC3 and SSC6. The precise location of breakpoints was identified by RBH-FISH t(3;6)(p14;q26), whereas the analysis of the pedigree showed a case of Mendelian inheritance within a family, after the de novo occurrence of the new rcp. Considering the consequences of the rcp on the fertility, this study points out the importance of the cytogenetic screening in the native breeds for the safeguard of the genetic biodiversity and the sustainability of the rural areas.

The major histocompatibility complex in Old World camelids and low polymorphism of its class II genes.

BACKGROUND: The Major Histocompatibility Complex (MHC) is a genomic region containing genes with crucial roles in immune responses. MHC class I and class II genes encode antigen-presenting molecules expressed on the cell surface. To counteract the high variability of pathogens, the MHC evolved into a region of considerable heterogeneity in its organization, number and extent of polymorphism. Studies of MHCs in different model species contribute to our understanding of mechanisms of immunity, diseases and their evolution. Camels are economically important domestic animals and interesting biomodels. Three species of Old World camels have been recognized: the dromedary (Camelus dromedarius), Bactrian camel (Camelus bactrianus) and the wild camel (Camelus ferus). Despite their importance, little is known about the MHC genomic region, its organization and diversity in camels. The objectives of this study were to identify, map and characterize the MHC region of Old World camelids, with special attention to genetic variation at selected class MHC II loci. RESULTS: Physical mapping located the MHC region to the chromosome 20 in Camelus dromedarius. Cytogenetic and comparative analyses of whole genome sequences showed that the order of the three major sub-regions is "Centromere - Class II - Class III - Class I". DRA, DRB, DQA and DQB exon 2 sequences encoding the antigen binding site of the corresponding class II antigen presenting molecules showed high degree of sequence similarity and extensive allele sharing across the three species. Unexpectedly low extent of polymorphism with low numbers of alleles and haplotypes was observed in all species, despite different geographic origins of the camels analyzed. The DRA locus was found to be polymorphic, with three alleles shared by all three species. DRA and DQA sequences retrieved from ancient DNA samples of Camelus dromedarius suggested that additional polymorphism might exist. CONCLUSIONS: This study provided evidence that camels possess an MHC comparable to other mammalian species in terms of its genomic localization, organization and sequence similarity. We described ancient variation at the DRA locus, monomorphic in most species. The extent of molecular diversity of MHC class II genes seems to be substantially lower in Old World camels than in other mammalian species.

Illegitimate recombination between T cell receptor genes in humans and pigs (Sus scrofa domestica).

T cell receptor (TCR) genes (TRA/TRD, TRB and TRG) reside in three regions on human chromosomes (14q11.2, 7q34 and 7p14, respectively) and pig chromosomes (7q15.3-q21, 18q11.3-q12 and 9q21-22, respectively). During the maturation of T cells, TCR genes are rearranged by site-specific recombination. Occasionally, interlocus recombination of different TCR genes takes place, resulting in chromosome rearrangements. It has been suggested that the absolute number of these "innocent" trans-rearrangements correlates with the risk of lymphoma. The aims of this work were to assess the frequencies of rearrangements with breakpoints in TCR genes in domestic pig lymphocytes and to compare these with the frequencies of corresponding rearrangements in human lymphocytes by using fluorescence in situ hybridization with chromosome painting probes. We show that frequencies of trans-rearrangements involving TRA/TRD locus in pigs are significantly higher than the frequency of translocations with breakpoints in TRB and TRG genes in pigs and the frequencies of corresponding trans-rearrangements involving TRA/TRD locus in humans. Complex structure of the pig TRA/TRD locus with high number of potential V(D)J rearrangements compared to the human locus may account for the observed differences. Furthermore, we demonstrated that trans-rearrangements involving pig TRA/TRD locus occur at lower frequencies in γδ T cells than in αß T lymphocytes. The decrease of the frequencies in γδ T cells is probably caused by the absence of TRA recombination during maturation of this T cell lineage. High numbers of innocent trans-rearrangements in pigs may indicate a higher risk of T-cell lymphoma than in humans.

Impact of karyotype organization on interlocus recombination between T cell receptor genes in Equidae.

The T cell receptor (TCR) genes (TRA, TRB, TRD and TRG) reside in 3 different chromosomal regions. During the maturation of T lymphocytes, the TCR genes are rearranged by site-specific recombination, a process that also predisposes T cells to aberrant rearrangements. Illegitimate recombination between the TCR genes occurs at a low level in healthy individuals, but this frequency may correlate with the risk of lymphoma. The aim of this work was to investigate interlocus recombination in equids. Illegitimate rearrangements were studied in peripheral blood lymphocytes by FISH with painting and BAC probes and by sequencing of PCR products, and the frequencies of recombination were assessed in horses and 4 other equids. The presence of several trans-rearrangement products between the TRA and TRG genes was verified by PCR in all investigated equids. Frequencies of trans-rearrangements in horses are higher than in humans, and colocalization of the TCR genes on the same chromosome increases the incidence of trans-rearrangements between them. The orientation of the TCR genes does not impact interlocus recombination itself but does affect the viability of cells carrying its products and consequently the number of trans-rearrangements observed in lymphocytes.

The utility of chromosome microdissection in clinical cytogenetics: a new reciprocal translocation in sheep.

Local sheep breeders and scientists in Italy cooperate and conduct research on the genetic improvement of autochthonous genetic types (AGTs) by various approaches, including a cytogenetic breeding selection since 2011. The Laticauda sheep (Ovis aries, 2n = 54) breed is one of the AGTs reared in the Campania region (southern Italy). Performing cytogenetic analyses, we have detected and described a novel reciprocal translocation in a Laticauda sheep identified as 54,XX t(18;23)(q14;q26). Our data support recurring appeals that suggest the regular performance of cytogenetic analyses for monitoring genetic health of livestock species. In total, 5 cases of reciprocal translocations in sheep are known, including the new case. None of them has any phenotypic effect on the living offspring. However, affected animals are characterized by sterility or have a low fertility which can have an effect on breeding success and on economical balance. Presence and kind of the described novel chromosomal aberration were detected by performing CBA-banding and FISH mapping with telomeric probes. RBA-banding allowed the karyotyping of sheep chromosomes and the identification of aberrant chromosomes and regions involved in the new reciprocal translocation. Whole chromosome painting (WCP) probes received from equivalent chromosomes in cattle and the derivative sheep chromosome 18 confirmed the cytogenetic data. This way, our study underlined both the importance of WCP probes by chromosome microdissection and a new way to use WCP probes directly generated from derivative chromosomes.

Comprehensive meiotic segregation analysis of a 4-breakpoint t(1;3;6) complex chromosome rearrangement using single sperm array comparative genomic hybridization and FISH.

Complex chromosomal rearrangements (CCR) represent rare structural chromosome abnormalities frequently associated with infertility. In this study, meiotic segregation in spermatozoa of an infertile normospermic carrier of a 4-breakpoint t(1;3;6) CCR was analysed. A newly developed array comparative genomic hybridization protocol was used, and all chromosomes in 50 single sperm cells were simultaneously examined. Three-colour FISH was used to analyse chromosome segregation in 1557 other single sperm cells. It was also used to measure an interchromosomal effect; sperm chromatin structure assay was used to measure chromatin integrity. A high-frequency of unbalanced spermatozoa (84%) was observed, mostly arising from the 3:3 symmetrical segregation mode. Array comparative genomic hybridization was used to detect additional aneuploidies in two out of 50 spermatozoa (4%) in chromosomes not involved in the complex chromosome rearrangement. Significantly increased rates of diploidy and XY disomy were found in the CCR carrier compared with the control group (P < 0.001). Defective condensation of sperm chromatin was also found in 22.7% of spermatozoa by sperm chromatin structure assay. The results indicate that the infertility in the man with CCR and normal spermatozoa was caused by a production of chromosomally unbalanced, XY disomic and diploid spermatozoa and spermatozoa with defective chromatin condensation.

Supernumerary Marker Chromosome Identified in Asian Elephant (Elephas maximus).

We identified a small, supernumerary marker chromosome (sSMC) in two phenotypically normal Asian elephants (Elephas maximus): a female (2n = 57,XX,+mar) and her male offspring (2n = 57,XY,+mar). sSMCs are defined as structurally abnormal chromosomes that cannot be identified by conventional banding analysis since they are usually small and often lack distinct banding patterns. Although current molecular techniques can reveal their origin, the mechanism of their formation is not yet fully understood. We determined the origin of the marker using a suite of conventional and molecular cytogenetic approaches that included (a) G- and C-banding, (b) AgNOR staining, (c) preparation of a DNA clone using laser microdissection of the marker chromosome, (d) FISH with commercially available human painting and telomeric probes, and (e) FISH with centromeric DNA derived from the centromeric regions of a marker-free Asian elephant. Moreover, we present new information on the location and number of NORs in Asian and savanna elephants. We show that the metacentric marker was composed of heterochromatin with NORs at the terminal ends, originating most likely from the heterochromatic region of chromosome 27. In this context, we discuss the possible mechanism of marker formation. We also discuss the similarities between sSMCs and B chromosomes and whether the marker chromosome presented here could evolve into a B chromosome in the future.

Chromosomal damage in occupationally exposed health professionals assessed by two cytogenetic methods.

The study assessed occupationally induced chromosomal damage in hospital personnel at risk of exposure to antineoplastic drugs and/or low doses of ionizing radiation by two cytogenetic methods. Cultured peripheral blood lymphocytes of eighty-five hospital workers were examined twice over 2 to 3 years by classical chromosomal aberration analysis and fluorescence in situ hybridization. The comparison of the 1st and the 2nd sampling of hospital workers showed a significant increase in chromatid and chromosomal aberrations (all p < .05) examined by classical chromosomal aberration analysis, and in unstable aberrations (all p < .05) detected by fluorescence in situ hybridization. Both cytogenetic methods were able to detect an increase of unstable aberrations in the 2nd sampling. The raised frequency of unstable cytogenetic parameters suggested higher recent exposure to genotoxic agents.

Chromosome damage in regions with different levels of air pollution.

Air pollution is an important environmental factor influencing human health. In this study, we compared chromosome damage in city policemen from three cities in the Czech Republic: industrial Ostrava characterized by high levels of benzo[a]pyrene, Prague with heavy traffic emitting nitrogen oxides, and relatively clean Ceske Budejovice located in an area with predominantly agricultural activity. Chromosomal aberrations in lymphocytes were evaluated by fluorescence in situ hybridization with painting probes for chromosomes 1, 2, 3, and 4 in spring and autumn. An increase in the frequency of unstable chromosome aberrations, that is, dicentric chromosomes and acentric fragments, was observed in spring samples from Ostrava (p = .014 and p = .044, respectively) and Prague (p = .002 and p = .006, respectively) in comparison with Ceske Budejovice. The difference was significant only for samples taken after the winter period, when the concentration of pollutants in the air increases due to poor dispersion conditions. An increased frequency of dicentric chromosomes was observed in spring compared to autumn in both Ostrava and Prague (p = .017 and p = .023, respectively), but not in Ceske Budejovice. More breakpoints were observed on chromosome 1 than on the other chromosomes examined (p < .001). The number of breakpoints in the heterochromatin region 1p11-q12 was lower than in other parts of chromosome 1 (p < .001), suggesting a protective function of heterochromatin against damage. Our study showed, that air pollution increased the frequency of unstable chromosome aberrations, especially dicentric chromosomes. However, we did not show an effect on stable chromosome rearrangements.

Ingraft chimerism in lung transplantation--a study in a porcine model of obliterative bronchiolitis.

BACKGROUND: Bronchial epithelium is a target of the alloimmune response in lung transplantation, and intact epithelium may protect allografts from rejection and obliterative bronchiolitis (OB). Herein we study the influence of chimerism on bronchial epithelium and OB development in pigs. METHODS: A total of 54 immunosuppressed and unimmunosuppressed bronchial allografts were serially obtained 2-90 days after transplantation. Histology (H&E) was assessed and the fluorescence in situ hybridization (FISH) method for Y chromosomes using pig-specific DNA-label was used to detect recipient derived cells in graft epithelium and bronchial wall, and donor cell migration to recipient organs. Ingraft chimerism was studied by using male recipients with female donors, whereas donor cell migration to recipient organs was studied using female recipients with male donors. RESULTS: Early appearance of recipient-derived cells in the airway epithelium appeared predictive of epithelial destruction (R=0.610-0.671 and p<0.05) and of obliteration of the bronchial lumen (R=0.698 and p<0.01). All allografts with preserved epithelium showed epithelial chimerism throughout the follow-up. Antirejection medication did not prevent, but delayed the appearance of Y chromosome positive cells in the epithelium (p<0.05), or bronchial wall (p<0.05). CONCLUSIONS: In this study we demonstrate that early appearance of Y chromosomes in the airway epithelium predicts features characteristic of OB. Chimerism occurred in all allografts, including those without features of OB. Therefore we suggest that ingraft chimerism may be a mechanism involved in the repair of alloimmune-mediated tissue injury after transplantation.

Comparative chromosome painting in Chacoan peccary, Catagonus wagneri.

Chacoan peccary (Catagonus wagneri, 2n=20) is the most endangered of three extant species of Tayassuidae. Its karyotype has been studied only by differential chromosome staining methods so far. To establish a comparative cytogenetic map of the peccary, we used cross-species hybridization with porcine (Sus scrofa, 2n=38) painting probes. Painting revealed 30 evolutionary conserved autosomal segments between pig and peccary. The q-arm of the submetacentric chromosome X is homologous to the porcine X chromosome, while the p-arm is composed of heterochromatin. Nucleolar organizer regions were detected on chromosomes 8 and 9 which are homologous to pig chromosomes 8 and 4/18, respectively. Fusions of chromosomes homologous to pig chromosomes 4/7 and 4/18 and fission of chromosome 7 are synapomorphic characters shared by Catagonus wagneri and Tayassu pecari but not by Pecari tajacu. Our results confirmed a high rate of karyotype evolution in Tayassuidae and a closer relationship of Catagonus wagneri with Tayassu pecari than with Pecari tajacu.

Semen quality and sperm DNA integrity in city policemen exposed to polluted air in an urban industrial agglomeration.

BACKGROUND: We examined sperm quality in a cohort of city policemen in Ostrava at the end of a period with high concentrations of air pollutants (winter) and in the same cohort at the end of a relatively low exposure period (summer). METHODS: The study group was comprised of 54 nonsmoking city policemen living and working in Ostrava, Czech Republic. Average daily air-pollutant concentrations recorded by stationary monitoring for 90 days preceding the collection of semen samples were evaluated for different city districts of Ostrava. Standard semen parameters were assessed according to the guidelines of the World Health Organization (2010). The parameters were semen volume, sperm concentration, sperm morphology, sperm motility, acrosome reaction and sperm plasma membrane integrity. Sperm DNA damage was analysed by the sperm chromatin structure assay (SCSA). Sperm motion characteristics were determined by Computer Assisted Semen Analysis (CASA). RESULTS: The concentrations of all monitored pollutants (particulate matter, sulphur dioxide, nitrogen oxides, carbon monoxide, benzo[a]pyrene, benzene) were significantly increased during winter (p < 0.001), except for ozone, the concentration of which was significantly higher during summer. Sperm volume, concentration, % vitality, % sperm morphology (normal form) and % acrosome-intact sperm did not differ significantly between the monitoring periods. The percentages of total motility and progressive motility were significantly higher in March, i.e. at the end of winter (p = 0.001). However, CASA testing showed differences in sperm motion kinetics between spring and autumn samples. In the spring samples, we found a significantly lower % of straightness (p = 0.044) and the length of straight-line path (p = 0.01), while linearity and straight-line velocity were near the borderline value (p = 0.064; p = 0.054, respectively). As compared to summer, high exposure to air pollution during winter significantly increased the extent of sperm chromatin integrity damage (median 22.6 vs. 18.6%) (p = 0.003) and the proportion of immature spermatozoa (median 11.2 vs. 9.9%, p = 0.001). Sperm DNA damage negatively correlated with total motility and progressive motility (r = -0.611, -0.299; p < 0.001). The negative correlation with vitality, normal morphology and acrosome-intact sperm (r = -0.522, -0.550 and -0.511, respectively) was also significant (p < 0.001). CONCLUSION: The examination of the same cohort of city policemen at the end of a period of high air pollution and at the end of relatively low exposure reduced the effects of age, different lifestyles, different occupational exposures, localities and genetic polymorphism on sperm quality impairment associated with air pollution. This study did not demonstrate impaired standard semen parameters in association with exposure. It was shown that sperm chromatin damage and the percentage of immature sperm were highly sensitive to air pollution.

Chromosomal evolution in Raphicerus antelope suggests divergent X chromosomes may drive speciation through females, rather than males, contrary to Haldane's rule.

Chromosome structural change has long been considered important in the evolution of post-zygotic reproductive isolation. The premise that karyotypic variation can serve as a possible barrier to gene flow is founded on the expectation that heterozygotes for structurally distinct chromosomal forms would be partially sterile (negatively heterotic) or show reduced recombination. We report the outcome of a detailed comparative molecular cytogenetic study of three antelope species, genus Raphicerus, that have undergone a rapid radiation. The species are largely conserved with respect to their euchromatic regions but the X chromosomes, in marked contrast, show distinct patterns of heterochromatic amplification and localization of repeats that have occurred independently in each lineage. We argue a novel hypothesis that postulates that the expansion of heterochromatic blocks in the homogametic sex can, with certain conditions, contribute to post-zygotic isolation. i.e., female hybrid incompatibility, the converse of Haldane's rule. This is based on the expectation that hybrids incur a selective disadvantage due to impaired meiosis resulting from the meiotic checkpoint network's surveillance of the asymmetric expansions of heterochromatic blocks in the homogametic sex. Asynapsis of these heterochromatic regions would result in meiotic silencing of unsynapsed chromatin and, if this persists, germline apoptosis and female infertility.

The effects of age on DNA fragmentation, the condensation of chromatin and conventional semen parameters in healthy nonsmoking men exposed to traffic air pollution.

BACKGROUND: Numerous studies have investigated age-based declines in semen traits, but the impact of paternal age on semen parameter values remains inconclusive. OBJECTIVES: The aim of this study was to detect an impact of age on semen quality was studied in healthy nonsmoking men exposed to traffic air pollution. METHODS: Semen samples from 150 Prague City policemen aged 23 to 63 years were examined for standard semen parameters, sperm DNA fragmentation and high DNA stainability. RESULTS: A significant positive correlation was found between age and %DFI (r = .359, P < .001), and negative correlations were found between age and sperm vitality (r = -.247, P < .001), the % acrosome-intact sperm (r = -.202, P = .013) and the % normal sperm heads (r = -.204, P = .012). A weak but significant negative correlation was found for high DNA stainability (% HDS) vs age (r = -.161, P = .050). No significant correlation was detected between male age and the other investigated semen quality parameters. At ages of 23 to 30, 31 to 40, 41 to 50, and 51 to 63 years, the mean %DFI values were 12.7 ± 7.18, 14.7 ± 7.42, 19.6 ± 11.25, and 34.2 ± 15.08, respectively. CONCLUSION: Our study shows a strong relationship (P < .001) between the age of men and sperm DNA fragmentation in an occupational cohort at risk of exposure to heavy traffic-related air pollution in a large city center.

Satellite DNA in Neotropical Deer Species.

The taxonomy and phylogenetics of Neotropical deer have been mostly based on morphological criteria and needs a critical revision on the basis of new molecular and cytogenetic markers. In this study, we used the variation in the sequence, copy number, and chromosome localization of satellite I-IV DNA to evaluate evolutionary relationships among eight Neotropical deer species. Using FISH with satI-IV probes derived from Mazama gouazoubira, we proved the presence of satellite DNA blocks in peri/centromeric regions of all analyzed deer. Satellite DNA was also detected in the interstitial chromosome regions of species of the genus Mazama with highly reduced chromosome numbers. In contrast to Blastocerus dichotomus, Ozotoceros bezoarticus, and Odocoileus virginianus, Mazama species showed high abundance of satIV DNA by FISH. The phylogenetic analysis of the satellite DNA showed close relationships between O. bezoarticus and B. dichotomus. Furthermore, the Neotropical and Nearctic populations of O. virginianus formed a single clade. However, the satellite DNA phylogeny did not allow resolving the relationships within the genus Mazama. The high abundance of the satellite DNA in centromeres probably contributes to the formation of chromosomal rearrangements, thus leading to a fast and ongoing speciation in this genus, which has not yet been reflected in the satellite DNA sequence diversification.

Karyotypic relationships in Asiatic asses (kulan and kiang) as defined using horse chromosome arm-specific and region-specific probes.

Cross-species chromosome painting has been applied to most of the species making up the numerically small family Equidae. However, comparative mapping data were still lacking in Asiatic asses kulan (Equus hemionus kulan) and kiang (E. kiang). The set of horse arm-specific probes generated by laser microdissection was hybridized onto kulan (E. hemionus kulan) and kiang (E. kiang) chromosomes in order to establish a genome-wide chromosomal correspondence between these Asiatic asses and the horse. Moreover, region-specific probes were generated to determine fusion configuration and orientation of conserved syntenic blocks. The kulan karyotype (2n = 54) was ascertained to be almost identical to the previously investigated karyotype of onager E. h. onager (2n = 56). The only difference is in fusion/fission of chromosomes homologous to horse 2q/3q, which are involved in chromosome number polymorphism in many Equidae species. E. kiang karyotype differs from the karyotype of E. hemionus by two additional fusions 8q/15 and 7/25. Chromosomes equivalent to 2q and 3q are not fused in kiang individuals with 2n = 52. Several discrepancies in centromere positions among kulan, kiang and horse chromosomes have been described. Most of the chromosome fusions in Asiatic asses are of centromere-centromere type. Comparative chromosome painting in kiang completed the efforts to establish chromosomal homologies in all representatives of the family Equidae. Application of region-specific probes allows refinement comparative maps of Asiatic asses.

Monozygotic twins with discordant karyotypes following preimplantation genetic screening and single embryo transfer: case report.

PURPOSE: to report a case of monozygotic monochorial diamniotic twins with discordant karyotypes. METHODS AND RESULTS: the pregnancy was achieved following a treatment cycle with intracytoplasmic sperm injection (ICSI) and preimplantation genetic screening (PGS) for chromosomes X, Y, 13, 16, 18, 21, 22. One embryo euploid for studied chromosomes was transferred. Prenatal ultrasonography revealed monozygotic twins. One fetus had growth retardation, multiple organ abnormalities and polyhydramnion. The other twin had normal ultrasound appearance. Delivery on week 29 of gestation resulted in the birth of two females, a stillborn twin with karyotype 45,XX,-13[12]/46,XX,r(13)[3] and a healthy twin with normal karyotype. CONCLUSIONS: the discordance in the twins' karyotypes originated from a mosaic embryo. Structural chromosomal abnormality of the affected twin could not be revealed using standard PGS investigation. Embryo splitting occurred probably due to apoptotic process in an early stage of embryo development. Apoptosis represents one of the possible mechanisms which can explain the embryo twinning process globally.

Complex variation in the KLRA (LY49) immunity-related genomic region in horses.

Natural killer (NK) cells play important roles in innate and adaptive immunity, as well as in the reproduction of placental mammals. Ly49 (KLRA) molecules represent a lectin-like type of NK cell receptor encoded within a complex genomic region, the NK cell complex. In rodents and horses, an expansion of the genes encoding Ly49 receptors leading to the formation of a gene family was observed. High sequence similarities and frequent high polymorphism of multiple family members represent an obstacle both for their individual identification and for annotation in the reference genomes of their respective species. Here, we focused on resolving complex variation of the KLRA gene family observed in domestic and Przewalski's horses. The KLRA (LY49) genomic region contains six genes (KLRA2-KLRA7) and one putative pseudogene, KLRA1. Two types of polymorphism were observed in the horses analyzed. Copy number variation between haplotypes was documented for the gene KLRA7 by polymerase chain reaction. As expected, the major source of variation of all KLRA genes, including KLRA7, is because of single nucleotide polymorphisms, many of them being nonsynonymous substitutions. Extensive allelic variability of the expanded KLRA (LY49) genes was observed. For four out of the six functional KLRA, high numbers of novel allelic amino acid sequence variants were identified in the genes studied, suggesting that this variation might be of functional importance, especially in the context of high polymorphism of their presumed ligands encoded by major histocompatibility complex class I genes. In fact, polymorphic amino acid sites were mostly found in the ligand-binding C-type lectin-like domain of the putative receptor molecule.

Hybridization of the 18 alpha-satellite probe to chromosome 1 revealed in PGD.

Although the chromosome 18 alpha-satellite probe is considered to have a very low polymorphism rate, the routine use of this probe in prenatal diagnosis revealed rare variants in size and copy number of these sequences. A polymorphic signal was detected in preimplantation genetic diagnosis (PGD) for aneuploidy, in a patient with repeated early miscarriages. A third small signal of chromosome 18 alpha-satellite probe was observed in two of four evaluated embryos. Hybridization to the woman's metaphasic lymphocytes revealed that the small signal was localized in the pericentromeric region of chromosome 1. Reanalysis of blastomeres with telomeric probes for chromosome 18q confirmed the presence of only two copies of chromosome 18. Options for verifying PGD analysis results, to prevent misdiagnosis in cases of suspected polymorphism, are discussed. Although some authors speculate about a possible role of heterochromatin polymorphism in infertility, this rare polymorphism of 18 alpha-satellite sequences is in itself probably a normal variant. This is the third report of a cross-hybridization of the chromosome 18 alpha-satellite probe and the first report of the localization of the polymorphic 18 alpha-satellite signal to chromosome 1.