Bioinformatics analysis of candidate genes for milk production traits in water buffalo (Bubalus bubalis).

Water buffalo (Bubalus bubalis) is an important livestock species in developing countries due to its contribution to meat, milk production, and a certain form of labor. However, the genetic potential of buffalo milk production traits has not been fully exploited. To date, 516 candidate genes associated with milk production traits of buffalo have been identified. The present study aimed to explore the possible molecular mechanisms underlying milk production traits of this species through functional genomics analysis of these candidate genes by using different bioinformatics tools. Gene ontology (GO) analysis indicated that these candidate genes were associated with complex biological processes, such as cell proliferation and mitotic nuclear division. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis demonstrated that these candidate genes were enriched in multiple signaling pathways, such as AMPK, ErbB, Toll-like receptor, and Jak-STAT. In addition, one function module consisting of 57 nodes and 139 edges were identified from the protein-protein interaction (PPI) network. GO analysis showed that the 57 candidate genes in this function module were enriched in three main biological processes, including homeostasis, metabolism, and cell response. These three distinct biological processes are well known for regulating mammary gland activities, which explained clearly the mechanism underlying milk production traits. This study provides a novel perspective for better understanding of the biological processes linked with milk production traits. This knowledge is conducive to the improvement of milk yield and composition of this species.

Integrated analysis of SNP, CNV and gene expression data in genetic association studies.

Integrative approaches that combine multiple forms of data can more accurately capture pathway associations and so provide a comprehensive understanding of the molecular mechanisms that cause complex diseases. Association analyses based on single nucleotide polymorphism (SNP) genotypes, copy number variant (CNV) genotypes, and gene expression profiles are the 3 most common paradigms used for gene set/pathway enrichment analyses. Many work has been done to leverage information from 2 types of data from these 3 paradigms. However, to the best of our knowledge, there is no work done before to integrate the 3 paradigms all together. In this article, we present an integrated analysis that combine SNP, CNV, and gene expression data to generate a single gene list. We present different methods to compare this gene list with the other 3 possible lists that result from the combinations of the following pairs of data: SNP genotype with gene expression, CNV genotype with gene expression, and SNP genotype with CNV genotype. The comparison is done using 3 different cancer datasets and 2 different methods of comparison. Our results show that integrating SNP, CNV, and gene expression data give better association results than integrating any pair of 3 data.

The Anti-Müllerian Hormone Profile is Linked with the In Vitro Embryo Production Capacity and Embryo Viability after Transfer but Cannot Predict Pregnancy Outcome.

The current study investigated the possibility of using the AMH concentration as a predictor of the ability of Korean Hanwoo cows to produce cumulus-oocyte complexes, embryos that survive after transfer as well as the pregnancy outcome of surrogates. Eight sessions of ovum pick-up (OPU) were performed with 19 donor cows at an interval of 3-4 days. Antral follicle count (AFC), oocyte quality and in vitro embryo development were recorded for each cow. Embryos produced from cows with different AMH profiles were transferred into recipients (n = 96). Cows in the high (≥0.25 ng/ml) and intermediate (0.1≥ to <0.25 ng/ml) AMH groups had a significantly higher AFC per OPU session (20.40 ± 1.36 and 16.91 ± 1.52, respectively; mean ± standard deviation) than cows in the low AMH group (<0.1 ng/ml; 12.19 ± 2.14). In addition, more cumulus-oocyte complexes per donor were recovered in the high (11.46 ± 1.22) and intermediate (7.38 ± 0.83) AMH groups than in the low AMH group (4.77 ± 0.44). The percentage of oocytes reached blastocyst stage was significantly higher in the intermediate (47.0%) and high (38.5%) AMH groups than in the low AMH group (32.3%). The number of embryos produced per cow was higher in the high (3.9 ± 0.2) and intermediate (6.9 ± 0.6) AMH groups than in the low AMH group (2.2 ± 0.3). The percentage of embryos that gave birth to viable calves when transferred into recipients was higher for those derived from cows in the intermediate AMH group (50.7%) than for those derived from cows in the low (35.7%) and high (36.4%) AMH groups. In conclusion, a single measurement of AMH concentration predicted the in vitro embryo production potential of donor Korean native cows before OPU and is linked with embryo viability after transfer into recipients.

Effects of flunixin meglumine and prostaglandin F2 α treatments on the development and quality of bovine embryos in vitro.

Assisted reproduction procedures, such as embryo transfer (ET) and artificial insemination (AI), in cattle could induce the secretion of prostaglandin F2 -alpha (PGF2 α) from uterine horns which may in turn interrupt embryo development and implantation. This study investigated the effect of flunixin meglumine (FM), prostaglandin F2 alpha (PGF2α) and FM combined with PGF2α supplementation in culture medium (IVC-II) on the development and quality of in vitro produced bovine embryos. The development rate of embryos was significantly higher in the FM group (33.3%) than in control (24.3%), PGF2 α (23.9%) and FM + PGF2 α groups (24.5%). The percentage of hatched blastocysts was also higher (p < 0.05) in the FM group (41.2%) than in the control (27.8%) and PGF2 α groups (19.8%). While, there was no significant difference in total cell number in all experimental groups, the number of apoptotic cells was significantly higher in the PGF2 α group (8.2 ± 6.6) than in the control (4.7 ± 3.2), FM (4.7 ± 2.5) and FM + PGF2 α (4.9 ± 3.4) groups. Detected by real-time PCR, secreted vesicle seminal protein 1 (SSLP1) and prostaglandin G/H synthase 2 (PTGS2) gene expression decreased (p < 0.05) in the PGF2 α group. However, SSLP1 and PTGS2 gene expression in the FM + PGF2 α group returned to their baseline levels, similar to the control and FM groups. Caspase 3 (CAPS3) gene expression increased in the PGF2 α group compared with other groups (p < 0.05). In conclusion, addition of FM in vitro culture significantly improved embryo development as well as alleviated the negative impact of PGF2 α.

Effect of reproductive tract environment following controlled ovarian hyperstimulation treatment on embryo development and global transcriptome profile of blastocysts: implications for animal breeding and human assisted reproduction.

BACKGROUND: In mammals, the reproductive tract plays a crucial role in the success of early reproductive events and provides an optimal microenvironment for early embryonic development. However, changes in the reproductive tract environment associated with controlled ovarian hyperstimulation and the influence on the embryo transcriptome profile have not been investigated. Therefore, we investigated differences in the development rate and the transcriptome profile of bovine blastocysts developing in the reproductive tract of unstimulated or superovulated heifers. METHODS: Nineteen Simmental heifers were synchronized, superovulated and artificially inseminated; nine heifers were flushed on Day 2 after insemination and 2-4-cell stage embryos were recovered and endoscopicaly transferred to the ipsilateral oviduct of unstimulated (i.e. single-ovulating) synchronized recipients (n= 4 recipients; 25-50 embryos per recipient). The remaining 10 superovulated heifers and the unstimulated recipients were then non-surgically flushed on Day 7 to collect embryos. The blastocyst transcriptome profile was examined using the Affymetrix GeneChip Bovine Genome Array. RESULTS: The proportion of embryos, which developed to the blastocyst stage, was lower in superovulated heifers than unstimulated heifers (P< 0.05). Blastocysts that developed under the abnormal endocrine conditions associated with ovulation induction showed higher cellular and metabolic activities, as genes involved in the oxidative phosphorylation pathway, different metabolic processes and translation and transcription processes, in addition to genes expressed in response to stress, were highly expressed compared with embryos that developed in the oviduct of unstimulated animals. CONCLUSIONS: The environment in which the embryo develops in the oviduct/uterus significantly alters gene expression patterns, especially those genes that regulate metabolic activity in the embryo.

Bovine blastocysts with developmental competence to term share similar expression of developmentally important genes although derived from different culture environments.

This study was conducted to investigate the gene expression profile of in vivo-derived bovine embryo biopsies based on pregnancy outcomes after transferring to recipients. For this, biopsies of 30-40% embryos were taken from grade I blastocysts (International Embryo Transfer Society Manual) and the remaining 60-70% of the intact embryos were transferred to recipients. Frozen biopsies were pooled into three distinct groups based on the pregnancy outcome after transferring the corresponding parts, namely those resulting in no pregnancy (NP), pregnancy loss (PL), and calf delivery (CD). Array analysis revealed a total of 41 and 43 genes to be differentially expressed between biopsies derived from blastocysts resulting in NP versus CD and PL versus CD respectively. Genes regulating placental development and embryo maternal interaction (PLAC8) were found to be upregulated in embryo biopsies that ended up with CD. Embryo biopsies that failed to induce pregnancy were enriched with mitochondrial transcripts (Fl405) and stress-related genes (HSPD1). Overall, gene expression profiles of blastocysts resulting in NP and CD shared similar expression profiles with respect to genes playing significant roles in preimplantation development of embryo. Finally, comparing the transcript signatures of in vivo- and in vitro-derived embryos with developmental competence to term revealed a similarity in the relative abundance of 18 genes. Therefore, we were able to present a genetic signature associated with term developmental competence independent of the environmental origin of the transferred blastocysts.

Suppression of the transcription factor MSX1 gene delays bovine preimplantation embryo development in vitro.

This study was conducted to investigate the effect of suppressing transcription factor gene MSX1 on the development of in vitro produced bovine oocytes and embryos, and identify its potential target genes regulated by this gene. Injection of long double-stranded RNA (LdsRNA) and small interfering RNA (siRNA) at germinal vesicle stage oocyte reduced MSX1 mRNA expression by 73 and 37% respectively at metaphase II stage compared with non-injected controls. Similarly, injection of the same anti-sense oligomers at zygote stage reduced MSX1 mRNA expression by 52 and 33% at 8-cell stage compared with non-injected controls. Protein expression was also reduced in LdsRNA- and siRNA-injected groups compared with non-injected controls at both stages. Blastocysts rates were 33, 28, 20 and 18% in non-injected control, scrambled RNA (scRNA), LdsRNA- and siRNA-injected groups respectively. Cleavage rates were also significantly reduced in Smartpool siRNA (SpsiRNA)-injected group (53.76%) compared with scRNA-injected group (57.76%) and non-injected control group (61%). Large-scale gene expression analysis showed that 135 genes were differentially regulated in SpsiRNA-injected group compared with non-injected controls, of which 54 and 81 were down- and up-regulated respectively due to suppression of MSX1. Additionally, sequence homology mapping and gene enrichment analysis with known human pathway information identified several functional modules that were affected due to suppression of MSX1. In conclusion, suppression of MSX1 affects oocyte maturation, embryo cleavage rate and the expression of several genes, suggesting its potential role in the development of bovine preimplantation embryos.

Transcriptional analysis of buffalo (Bubalus bubalis) oocytes during in vitro maturation using bovine cDNA microarray.

The need for improving in vitro production of buffalo embryos necessitates a better understanding of the molecular mechanisms regulating early development including oocyte maturation. Here, we used bovine cDNA microarray platform to investigate mRNA abundance of buffalo oocytes before and after in vitro maturation. For this, a total of six pools each contains 50 immature or in vitro matured buffalo oocytes were used for mRNA isolation and subsequent cDNA synthesis. The BlueChip bovine cDNA microarray (with approximately 2000 clones) was used to analyse gene expression profiles between immature and matured oocytes. Statistical analysis of microarray data revealed a total of 104 transcripts to be differentially expressed between the two oocyte groups. Among these, transcription factors (ZFP91), M-phase mitotic cell cycle (MPHOSPH9), growth factor (BMP15) and DNA binding (HMGN2) were found to be up-regulated in immature oocytes. Similarly, matured oocytes were found to be enriched with genes involved in cytoskeleton (ACTB), hydrogen ion transporting (ATP6V1C2) and structural constituent of ribosome (RPS27A). Quantitative real-time polymerase chain reaction validated the expression profile of some selected transcripts during array analysis. In conclusion, to our knowledge, this is the first large-scale expression study to identify candidate genes differentially abundant and with potential role during buffalo oocyte maturation.

Sliding multislice MRI for abdominal staging of rectal gastrointestinal stromal tumours.

Anorectal gastrointestinal stromal tumours (GISTs) are uncommon mesenchymal neoplasms. The objective of this report was to demonstrate the value of sliding multislice (SMS) as an upcoming method of continuously moving table MRI, providing detailed abdominal staging of rectal GISTs. Integration of SMS into a high-resolution pelvic MR imaging protocol allows for both detailed assessment of rectal GISTs and depiction of the entire abdomen with high image quality. The staging of liver, malignant lymph nodes and bone metastases is now possible, prolonging pelvic MRI for only one minute.

Whole-body MRI in comparison to skeletal scintigraphy in detection of skeletal metastases in patients with solid tumors.

AIM: To evaluate the diagnostic value of whole-body magnetic resonance imaging (MRI) and skeletal scintigraphy in the detection of skeletal metastases in patients with solid tumors. MATERIALS AND METHODS: One hundred and twenty-nine tumor patients were examined with whole-body MRI using coronal TIRM sequences for the different anatomical regions. Skeletal scintigraphy was performed with 99mTc-DPD. RESULTS: In 105/129 (81%) patients, the whole-body MRI and skeletal scintigraphy findings were concordant. In 56/129 (43%) patients, both imaging modalities excluded skeletal metastases. In 49/129 (38%) patients, whole-body MRI and skeletal scintigraphy revealed metastases, however whole-body MRI demonstrated more extensive disease in 22/49 (45%) cases. In 6/49 (12%) cases, skeletal scintigraphy was superior to whole-body MRI in detecting more skeletal metastases. In 24/129 (19%) cases, the imaging findings were discordant. In 15 cases, skeletal scintigraphy was negative, whereas whole-body MRI revealed skeletal metastases. In 9 cases, skeletal scintigraphy was positive, whereas whole-body MRI failed to detect these metastases. In 77/129 (60%) patients, whole-body MRI revealed additional tumor-related findings. CONCLUSION: Whole-body MRI, as a new staging method, is superior to skeletal scintigraphy with respect to the detection of skeletal metastases and the extent of metastastic disease. Furthermore, whole-body MRI yields additional tumor-related findings. Therefore, whole-body MRI should be performed as an alternative to skeletal scintigraphy for the assessment of skeletal metastases.

Clinical and radiological findings after different treatment of odontoid fractures type Anderson II and III.

PURPOSE OF THE STUDY: In the period from 06/00 to 08/02, 31 patients with odontoid fractures type Anderson II and III were treated and stastically recorded. 25 patients were followed up; the progess of 24, documented in detail radiographically, were evaluated independently by a traumatologist and by a radiologist. The usual time of immobilization when treating odontoid fractures type Anderson type II and III with the halo-fixator is 12 weeks. For this 12 weeks that it is worn, objective assessment of bone healing is performed radiographically and the results critically considered in terms of the length of time that the halo-fixator should be worn and whether this duration should be altered on the basis of the clinical and radiological results obtained. MATERIALS AND METHODS: 16 patients with an odontoid fracture type Anderson type II were treated partly with a halo-fixator and partly by additional operative stabilization. 15 patients with a type III fracture were treated in a halo over 12 weeks. At the time of the accident the patients to be treated had to have conventional radiographic examination and a CT scan as well as a position check following reduction. After 4, 8 and 12 weeks radiographic and CT investigation was repeated. These findings were evaluated independently by a surgeon and a radiologist. The clinical follow-up was carried out using the VAS Score and, in addition, the general activity level before and after the accident was recorded in a similar way on the Tegner/Lysholm subjective activity score. RESULTS: In most cases, according to the CT scan, the osseous bridging decreased again between the 8th and 12th weeks, as defined by resorption zones seen during the fracture healing period. Radiological evidence of complete osseous bridging was only seen after 12 weeks in three cases. CONCLUSION: Conventional radiography does not seem to us to be the most suitable technical means to evaluate osseous healing in odontoid fractures. The CT is more reliable for this. According to our radiological results, osseous healing of different types of odontoid fractures takes more than 12 weeks. Despite of its known complications, the halo fixator is still a good instrument for the treatment of odontoid fractures.

Analysis of the B-cell compartment in plasma cell leukemia and multiple myeloma: immunoglobulin gene rearrangement of EBV-infected B-cell lines.

Multiple myeloma (MM) is defined as a tumoral expansion of plasma cells occurring in the bone marrow and sometimes in the peripheral blood (plasma-cell leukemia, PCL). Many reports have demonstrated a clonal expansion of B cells bearing the same idiotypic determinants as the myeloma protein (idiotypic B cells) in MM, suggesting that they could belong to the malignant clone. In order to investigate whether the B-cell population is a malignant component or not, either in the peripheral blood of patients with PCL or in the bone marrow of patients with MM, we derived B-cell lines by infecting, with the Epstein-Barr virus (EBV), cultures in limiting dilution of mononuclear cells from six patients. A limiting dilution culture was used to prevent the elimination of slowly proliferating clones by the more rapidly dividing ones, and thus to get the most exact representation of the B-cell repertoire of these patients. The cloning efficiency of the EBV-infected cells was similar in patients and healthy individuals (range: 1 in 100 to 1 in 1650 B cells). All of the clones obtained from a single patient exhibited different clonal immunoglobulin gene rearrangements (IGR), proving the validity of our cloning technique. No tumoral clones (61 clones analysed) showed the IGR pattern specific of autologous myeloma cells. These results indicate that malignant plasma cells cannot be immortalized with EBV. These results show that, if malignant B cells (pre-switch or post-switch) exist, they could be present only in a minor population, and the corollary of this is that there is a major population of non-malignant B cells in the sites of tumoral proliferation of patients with MM. This is remarkable in view of numerous reports showing a profound defect of the polyclonal B lymphopoiesis in these patients, and even an absence of B lymphocytes. Thus, these results challenge the existence of a major compartment of malignant idiotypic B cells and favor the hypothesis of non-malignant B cells sharing cross-reactive idiotypes with the autologous myeloma protein.

Rearrangements of immunoglobulin light and heavy chain genes and correlation with phenotypic markers in B-cell chronic lymphocytic leukemia.

Twenty-five patients with B-cell chronic lymphocytic leukemia (B-CLL) were investigated to correlate the immunological phenotype with the description of the Ig gene rearrangements of the B-cell clone. All patients were positive for the CD19 antigen and one pan B-antigen, markers of late cells (CD20, CD37 or Y2955). Twenty-four of the 25 patients tested expressed monoclonal cell surface immunoglobulin (SIg). The CD5 antigen was present in 21 of the 25 tested patients. Immunoglobulin gene rearrangements were detected by hybridization of the BamHI, EcoRI, BgIII, and HindIII digested genomic DNAs to the IGHJ, IGKC, IGLC, and IGLJ2 probes. Twenty-four of 25 patients had two rearranged IGH loci. The IGKC rearrangements were observed in 20 patients. In four patients, the IGK loci were deleted on both chromosomes. One patient without SIg displayed a germline pattern. All six patients with lambda producing B-CLL showed a lambda gene rearranged band, although the use of IGL polymorphism to investigate IGL rearrangements must be noted. These clonal rearrangements of IGL genes, together with the detection of either the kappa or lambda light chain of SIg, confirm that patients with B-CLL meet the developmental scheme of ordered light chain gene rearrangements.

Diagnostic value of MRI in comparison to scintigraphy, PET, MS-CT and PET/CT for the detection of metastases of bone.

The initial localization of metastases in the bone in patients with solid tumors has a relatively good prognosis in comparison with visceral metastasization. The early detection of bone marrow metastases allows for a rapid initiation of therapy and a subsequent reduction in the morbidity rate. Modern MRI is superior to the 30-year-old skeletal scintigraphy and bone marrow scintigraphy with respect to sensitivity, specificity, as well as the extent of osteal metastasis. MRI provides substantial, therapy-relevant additional information. MSCT plays an important role in the management of cancer patients in clinical routine and gives an excellent survey of the axial skeleton by demonstrating osteolytic and osteoblastic metastases. Extensive comparative studies of MRI with 18F-FDG-PET and 18F-fluoride-PET have not yet been carried out. Whole body MRI is a very promising new staging method for the oncological diagnosis of solid tumors and the detection of osteal metastases. The adoption of 18F-FDG-PET and 18F-fluoride-PET FDG as well as the side by side PET-CT image fusion and the two in one PET/CT examinations appears to be slightly less sensitive to whole body MRI in the detection of osteal metastases. Larger, prospective multicenter studies are necessary to establish these as new, promising methods for the detection of osteal metastases.

High grade cardiac lymphoma vitality monitoring by gadolinium-enhanced magnetic resonance imaging (MRI).

Primary cardiac lymphoma (PCL) is a rare disorder with a poor prognosis and response monitoring is often difficult. Delay in the diagnosis and infiltration of cardiac structures contribute to the unfavorable prognosis. We report on a 76-year-old woman who was diagnosed as having an immunoblastic B-cell PCL according to a histology attained by catheter-guided biopsy. Systemic chemotherapy with six cycles of CHOP (Cyclophosphamide, Doxorubicine, Vincristine = Oncovine, Prednisone), combined with the monoclonal anti-CD20 antibody Rituximab induced only a partial remission, based solely on monitoring of tumor size. However, cardiac gadolinium-enhanced magnetic resonance imaging (CMR) disclosed a reduced lymphoma perfusion and, therefore, indicated decreased tumor vitality. Nine months after the final treatment, the cardiac tumor further decreased to 10% of the initial size, and the patient is in sustained remission as monitored by CMR and validated by florine-18 fluorodeoxyglucose positron emission tomography (PET). Determination of PCL perfusion was, in our case, beneficial for clinical decision making on additional therapy.

Diagnostic accuracy of MRI for preoperative staging of pancreatic carcinoma: tendency for understaging.

OBJECTIVE: To evaluate the diagnostic accuracy of magnetic resonance imaging (MRI) for preoperative staging in pancreatic carcinoma. MATERIALS AND METHODS: MRI investigations, including MR-angio and MR-cholangiopancreatography (MRCP) of 19 patients who underwent surgery for pancreatic carcinoma were retrospectively evaluated by two radiologists. The size, localization of the tumor and possible infiltration of neighboring organs, as well as the presence of enlarged lymph nodes, were determined to define a preoperative, radiological TN stage. Lymph node metastasis was defined as peripancreatic lymphoma greater than 10 mm. Our findings were correlated to postoperative diagnosis. RESULTS: The T-stage was correctly evaluated in 52.6% of the cases (10/19). Understaging took place in 31.6% (6/19) and overstaging in 15.8% (3/19). In three cases of understaging, a micro-infiltration of the peripancreatic tissue was not visible in MRI. Pathologically enlarged lymph nodes were correctly found in 63.2% of the cases (12/19). Overstaging took place in 21.1% of the cases (4/19) and understaging in 15.8% (3/19). CONCLUSION: MRl for preoperative staging of pancreatic carcinoma showed a tendency to understage tumor size in this study population. Especially in cases of small tumor size, micro-infiltration of peripancreatic tissue or the common bile duct may not be detected by MRI. Concerning N-stage, the 95% confidence interval reveals a distribution of over- and understaged.

Quality improvement of transgenic cloned bovine embryos using an aggregation method: Effects on cell number, cell ratio, embryo perimeter, mitochondrial distribution, and gene expression profile.

The production of cloned embryos using conventional methods has extremely low success rates owing to low embryo quality. To improve the quality of cloned bovine embryos expressing enhanced green fluorescent protein (EGFP), we applied an aggregation culture method. The EGFP gene was transfected into bovine fetal fibroblasts using a retroviral vector system. Somatic cell nuclear transfer was performed using these cells, and the resulting embryos were cultured in aggregates or individually. Gene expression was analyzed by a microarray, and differentially expressed genes were validated by quantitative real-time polymerase chain reaction. The total number of cells per blastocyst and the ratio of inner cell mass cells to trophectoderm cells were higher in aggregated transgenic cloned blastocysts (agBL; 368.7 ± 109.6 and 1:4.8, respectively) than in in vitro-fertilized blastocysts (ivfBL; 189.8 ± 65.8 and 1:2.6, respectively) and nonaggregated transgenic cloned blastocysts (sBL; 113.1 ± 36.3 and 1:1.5, respectively; P < 0.05 and P < 0.01, respectively). Moreover, the blastocyst perimeter was larger in the agBL group than in the ivfBL and sBL groups (1168.8 ± 200.23 vs. 887.33 ± 187.62 and 678 ± 226.1 µm; P < 0.05). In addition, mitochondrial fluorescence intensity was higher in the agBL group than in the ivfBL and sBL groups (P < 0.05). The number of apoptotic cells per blastocyst was lower in the ivfBL and agBL groups than in the sBL group (3.7 ± 2.2 and 3.4 ± 2.1 vs. 6.7 ± 6.8; P < 0.05). The genes identified in the microarray belonged to 18 categories. Expression of the Krüppel-like factor 4 gene, which is associated with cell proliferation, development, and transcription, was 7.2-fold higher in the agBL group than in the ivfBL group (P < 0.05) but did not differ between the sBL and ivfBL groups (P > 0.05). Expression of the heat shock 70-kDa protein 1A gene, which is associated with apoptosis, was 12-fold higher in the sBL group than in the ivfBL and agBL groups (P < 0.05). Expression of a stemness-related gene (octamer-binding transcription factor 4) and trophectoderm-specific genes (homeobox protein CDX2 and keratin 18) was higher in the agBL group than in the sBL group (P < 0.05). However, expression of the stemness gene homeobox protein NANOG did not differ among the groups (P > 0.05). Taken together, these data suggest that the aggregation method improves the quality of cloned embryos expressing EGFP and might be helpful in animal cloning.

Twenty-four novel hemophilia B mutations revealed by rapid scanning of the whole factor IX gene in a French population sample.

Full scanning of the factor IX gene by means of denaturing gradient gel electrophoresis enabled us to determine the molecular defects in 48 out of 49 hemophiliacs and to evaluate the spectrum of factor IX mutations in the French population. Our results further document the high molecular heterogeneity of the disease and the efficiency of this rapid screening method for disease-causing mutations. This direct approach, which is based on computer-aided analysis of the whole coding, promoter and exon-flanking factor IX gene sequences, proved to be helpful for carrier detection and prenatal diagnosis in most hemophilia B families, including sporadic cases. Moreover, we were able to identify 24 novel molecular defects of various natures in the factor IX gene.

Frequent occurrence of the CFTR intron 8 (TG)n 5T allele in men with congenital bilateral absence of the vas deferens.

Isolated congenital bilateral absence of the vas deferens (CBAVD) is an autosomal recessive disorder which has recently been shown to be associated with cystic fibrosis (CF) mutations. As part of an effort to understanding the genetic basis of this disorder, we have analysed the entire coding sequence and all the intron/exon boundaries of the cystic fibrosis transmembrane conductance regulator (CFTR) gene from 45 azoospermic individuals with this phenotype. We were able to detect a CFTR gene defect in 86% of chromosomes from these subjects. In addition to identifying 9 novel CFTR gene mutations, we found that a surprisingly high proportion (84%) of men with CBAVD who are heterozygous for a CF mutation carry the intron 8 polypyrimidine 5T CFTR allele on one chromosome. We hypothesise that this tight and significant (p < 10(-6)) linkage reflects the very mild impact of this mutation on CFTR gene expression. Although genetic heterogeneity cannot be excluded, CBAVD patients in whom no CFTR mutation has been detected are likely to harbour additional unidentified mild mutations. These observations have implications for the genetic counselling of CBAVD patients and CF families, and couples undergoing in vitro fertilisation procedures.

CFTR gene mutations in adults with disseminated bronchiectasis.

The severity and type of clinical manifestations are variable in patients with cystic fibrosis (CF). The respiratory syndromes in these patients consist of lung infections associated with disseminated bronchiectasis (DB), asthma, and chronic obstructive pulmonary disease. To investigate the possible involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in chronic pulmonary disease in adults, we studied 32 DB patients with a clinically isolated respiratory syndrome. Careful analysis of all the CFTR gene exons and their flanking regions revealed a significantly increased frequency of CFTR gene mutations in these patients. Thirteen CFTR gene mutations were identified in sixteen different alleles. Six of these mutations, which have previously been reported as CF defects, were found on nine alleles. A further four, two of which had not previously been described (D192N and 406-2 AdeltaC), are potentially disease-causing mutations. We also identified three rare substitutions (R31C, L997F, T1220I), which could be involved in mild CFTR gene disease. Four patients were compound heterozygotes, one carried two CFTR gene mutations (possibly allelic) and six were heterozygous for a mutation. These results indicate that CFTR gene mutations may play a role in bronchiectatic lung disease, possibly in a multifactorial context. These findings have implications for genetic counselling of DB patients and their families.