GABA transaminase deficiency. Case report and literature review.

GABA transaminase deficiency should be considered in the differential diagnosis of early onset epileptic encephalopathies. This case was diagnosed post-mortem, but increased vigilance to this will allow for earlier diagnoses in other infants and families. This is a case study which involved diagnosis of a rare neurometabolic disorder in one of the babies in the family and eventual genetic counselling of the family. The family has been offered pre-implantation genetic diagnosis for future pregnancies. This case reporting has been approved by the hospital research and ethical committee.

Report of a case of Raine syndrome and literature review.

We report on a case of Raine syndrome with a mutation in FAM20C and typical phenotypic features consisting of midface hypoplasia, hypoplastic nose, choanal atresia, wide fontanelle, exophthalmos, generalized osteosclerosis and intracranial calcification. New features in our patient are cerebellar hypoplasia and pachygyria. We review the literature and conclude that the triad of hypoplastic nose, exophthalmos and generalized osteosclerosis and/or intracranial calcification is consistent in all molecularly confirmed cases.

Study of the cytokine polymorphisms in correlation to rejection and graft survival in renal allograft donors and recipients from a homogenous Saudi population.

OBJECTIVES: Allograft outcome can be improved with the discovery of risk factors that influence adverse events and may allow individualization of patients' treatment. Rejection is the main hurdle to successful transplantation and the immune response is the key effecter to rejection development. Hence, the major objective of the present study was to assess the relationship between single nucleotide polymorphisms (SNPs) in 5 cytokine genes, HLA mismatch and graft outcome in a cohort of 100 Saudi kidney transplant recipients and 100 living related donors at a single transplant center. MATERIALS & METHODS: Genotyping of the following positions: TNFA (-308G/A), TGFB1 (codon 10T/C, codon 25C/G), IL-10 (-1082G/A, -819C/T, -592C/A), IL-6 (-174C/G), and IFNG (+874T/A) were performed. RESULTS: The majority of the donors whose recipients presented with either cellular or antibody mediated graft rejection (90% and 100%) respectively were found to be significantly (p=0.0351) associated with intermediate or high IL-10 producing haplotypes, compared to those with stable grafts (58.66%). Haplotypes linked with lower IL-10 production were not detected in the donors or their recipients with antibody mediated graft rejection compared to donors with stable graft (41.33%). The distribution of donor IL-10-1082 haplotypes (GG, GA, AA) showed a statistically significant association of IL-10-1082 GA genotype (p=0.0351) with rejection, when grouped according to patients' rejection status. No other statistically significant deviations were observed in the donors' genotypes. Analyses of cytokine polymorphisms in the recipients revealed no significant association. Multivariate logistic regression analyses showed that only HLA-DRB1 mismatch significantly influenced graft loss (p=0.0135). CONCLUSION: This study demonstrates that the donor IL-10 genotypes and HLA-DRB1 mismatch are key determinants in graft outcome after renal transplantation.

A novel syndrome of lethal familial hyperekplexia associated with brain malformation.

BACKGROUND: Hyperekplexia (HPX) is a rare non-epileptic disorder manifesting immediately after birth with exaggerated persistent startle reaction to unexpected auditory, somatosensory and visual stimuli, and non-habituating generalized flexor spasm in response to tapping of the nasal bridge (glabellar tap) which forms its clinical hallmark. The course of the disease is usually benign with spontaneous amelioration with age. The disorder results from aberrant glycinergic neurotransmission, and several mutations were reported in the genes encoding glycine receptor (GlyR) α1 and ß subunits, glycine transporter GlyT2 as well as two other proteins involved in glycinergic neurotransmission gephyrin and collybistin. METHODS: The phenotype of six newborns, belonging to Saudi Arabian kindred with close consanguineous marriages, who presented with hyperekplexia associated with severe brain malformation, is described. DNA samples were available from two patients, and homozygosity scan to determine overlap with known hyperkplexia genes was performed. RESULTS: The kindred consisted of two brothers married to their cousin sisters, each with three affected children who presented antenatally with excessive fetal movements. Postnatally, they were found to have microcephaly, severe hyperekplexia and gross brain malformation characterized by severe simplified gyral pattern and cerebellar underdevelopment. The EEG was normal and they responded to clonazepam. All of the six patients died within six weeks. Laboratory investigations, including metabolic screen, were unremarkable. None of the known hyperkplexia genes were present within the overlapping regions of homozygosity between the two patients for whom DNA samples were available. CONCLUSIONS: We present these cases as a novel syndrome of lethal familial autosomal recessive hyperekplexia associated with microcephaly and severe brain malformation.

Killer cell immunoglobulin-like receptor gene diversity in the Saudi population.

Killer cell immunoglobulin-like receptors (KIRs) influence the outcome of haematopoetic stem cell transplantation by modulating the cytotoxic ability of natural killer (NK) cells and a subset of T cells. KIRs are also highly polymorphic and could therefore be good population genetic markers, much like their human leukocyte antigen (HLA) ligands. This study represents the first report on distribution of 16 KIR genes in 162 unrelated healthy Saudi individuals. All the 16 KIR genes were observed in the studied population and the four framework genes (KIR2DL4, 3DL2, 3DL3 and 3DP1) were present in all individuals. Forty- one distinct KIR profiles were expressed in our population, 11 of which had not been previously described in other populations including the Middle Eastern population. AA1, the most common genotypic profile was observed at a frequency of 26.5%. The group A haplotype was more frequent (53%) in the Saudi population compared to the group B haplotype (47%). The pattern of the inhibitory KIR/HLA ligands were also analyzed and 52.3% of the Saudi population was found to express two pairs of the inhibitory KIR/HLA-C. The KIR gene frequencies suggests that the Saudi population shares common general features with the Middle Eastern and other populations, but still has its own unique frequencies of several KIR loci.

Defective gammadelta T-cell function and granzyme B gene polymorphism in a cohort of newly diagnosed breast cancer patients.

OBJECTIVE: The purpose of this study was to examine the antitumor immune function of gammadelta T cells and to scan the granzyme B gene for the known single nucleotide polymorphism in breast cancer patients and normal controls. MATERIALS AND METHODS: Levels, cytotoxicity, and functional capacity of gammadelta T cells in peripheral blood mononuclear cells were assessed by flow cytometry, (51)Cr release, and ELISpot assays, respectively. Furthermore, sequence based typing was adopted to screen for granzyme B gene polymorphism. RESULTS: We have found that the frequency and function of gammadelta T cells are reduced both in peripheral blood mononuclear cells of 30 newly diagnosed breast cancer patients (2 [1.2, 3]), compared with 38 normal controls (3.2 [2.5, 5.7]) (p=0.02). In addition, resting gammadelta T cells from breast cancer patients produced significantly more interleukin-6 and tumor necrosis factor-alpha than normal controls. Moreover, ex vivo stimulation of gammadelta T cells with zoledronic acid and interleukin-2 compensated in part for this deficiency, as it stimulated the proliferation, cytokine production, and enhanced the expression of messenger RNA of granzyme B. Interestingly, when the known granzyme B gene polymorphism was screened, we found the prevalence of the mutated genotype RAH/RAH to be significantly (p<0.017) associated with breast cancer patients (14.30%) compared with normal donors (1.40%). Cytotoxicity exerted by gammadelta T cells on Daudi and MCF-7 was significantly higher in donors with the wild-type QPY/QPY (50%) compared with donors with RAH/RAH (21%). CONCLUSIONS: Our data suggest that reduction in the proportion of gammadelta T cells and granzyme B gene polymorphism leads to defective immune function in breast cancer patients. Treatment with zoledronic acid amend partially this fault. Further studies of gammadelta T cells function and granzyme B gene polymorphism in cancers, as well as the potential therapeutic use of zoledronic acid are warranted.

Sanguinarine-dependent induction of apoptosis in primary effusion lymphoma cells.

Primary effusion lymphoma (PEL) is an incurable, aggressive B-cell malignancy that develops rapid resistance to conventional chemotherapy. In efforts to identify novel approaches to block proliferation of PEL cells, we found that sanguinarine, a natural compound isolated from the root plant Sanguinaria canadendid, inhibits cell proliferation and induces apoptosis in a dose-dependent manner in several PEL cell lines. Our data show that sanguinarine treatment of PEL cells results in up-regulation of death receptor 5 (DR5) expression via generation of reactive oxygen species (ROS) and causes activation of caspase-8 and truncation of Bid (tBid). Subsequently, tBid translocates to the mitochondria causing conformational changes in Bax, leading to loss of mitochondrial membrane potential and release of cytochrome c to the cytosol. Sanguinarine-induced release of cytochrome c results in activation of caspase-9 and caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage, leading to induction of caspase-dependent apoptosis. In addition, we show that pretreatment of PEL cells with carbobenzoxy-Val-Ala-Asp-fluoromethylketone, a universal inhibitor of caspases, abrogates caspase and PARP activation and prevents cell death induced by sanguinarine. Moreover, treatment of PEL cells with sanguinarine down-regulates expression of inhibitor of apoptosis proteins (IAP). Finally, N-acetylcysteine, an inhibitor of ROS, inhibits sanguinarine-induced generation of ROS, up-regulation of DR5, Bax conformational changes, activation of caspase-3, and down-regulation of IAPs. Taken together, our findings suggest that sanguinarine is a potent inducer of apoptosis of PEL cells via up-regulation of DR5 and raise the possibility that this agent may be of value in the development of novel therapeutic approaches for the treatment of PEL.

Skin-homing CD8+ T lymphocytes show preferential growth in vitro and suppress CD4+ T-cell proliferation in patients with early stages of cutaneous T-cell lymphoma.

A total of 27 T-lymphocyte cell strains were established from skin biopsies of 24 patients with various stages of cutaneous T-cell lymphoma (CTCL) by addition of the T-cell growth factors interleukin (IL)-2 and IL-4. Cellular proliferation and phenotypic changes were measured over 3 months in culture, and T-cell clones were studied using T-cell receptor-? re-arrangement techniques. An average outgrowth of 134 million T-lymphocytes from a 4-mm skin biopsy was observed over 2 months. Initially, most T-cells expressed the CD4+ phenotype. In 17 cell strains from patients with early CTCL a statistically significant predominance of CD8+ T-lymphocytes developed over 8-weeks' culture, indicating that CD8+ T-cells controlled the growth of CD4+ T cells, whereas CD4+ T-cells were predominant in cell strains from advanced CTCL (p <0.05). TCR-? re-arrangement studies revealed, on average, 12 T-cell clones per cell strain, which was reduced over time to 6 T-cell clones per cell strain. Lymphocytes from peripheral blood could kill lymphocytes from an autologous cell strain, suggesting the presence of autoreactive cytotoxic T-cells. Our study suggests how skin-homing CD8+ T-lymphocytes from patients with early stage CTCL can suppress the in vitro growth of skin-homing CD4+ T-lymphocytes, indicating immune surveillance.

Evaluation of cytotoxic potential of newly synthesized antiviral aminopyrazoloquinoline derivatives.

In the present study, we screened newly synthesized antiviral aminopyrazoloquinoline derivatives for cytotoxic potential in human normal and breast cancer cell lines using apoptosis as biomarker. These derivatives and the well known antiviral drug, acyclovir, were incubated with the normal (MCF-10A, MCF-12A) and cancer (MCF-7, MDA-MB-231) cell lines at 10, 50 and 100 µM for 72 h at 37°C. Both the parent compounds and their sugar derivatives were found to be differentially cytotoxic in various cell lines. MCF-7 cells were more or less completely resistant to all these compounds while MDA-MB-231 cells were significantly killed by apoptosis. The methoxy derivative of aminopyrazoloquinoline (compound 3) was found to be the most cytotoxic in the normal breast epithelial cell lines (MCF-10A and MCF-12A) and MDA-MB-231 cell lines at 100 µM killing over 90% of the cells with up to 80% apoptosis. Interestingly MCF-7 cells showed only up to 50% killing at 100 µM dose with less than 20% apoptosis. Acyclovir did not cause any cytotoxicity, apoptosis or cell cycle arrest in any of the cells lines at the doses tested. Our results suggest that the newly synthesized antiviral compounds have an associated risk of being cytotoxic compared to the acyclovir.

Evaluation of apoptosis-induction by newly synthesized phthalazine derivatives in breast cancer cell lines.

Newly synthesized phthalazine derivatives including copper and platinum complexes were evaluated for cytotoxicity in human breast cancer cell lines. The cells were incubated with the compounds (100 microM) for 72 h and cytotoxicity, apoptosis and DNA content were measured by flow cytometery. Our results suggest that the parent (H1-2), copper (C1-2)- and platinum (P1-2)-derivatized compounds were relatively more active in inducing apoptosis and cell killing in both human breast cancer cell lines, MDA-MB-231 cells being the more sensitive. Other compounds showed weak or no response towards these parameters except H-5 causing 40% apoptosis in MDA-MB-231 cells. Addition of copper or platinum in the structures generally reduced the apoptotic potential. Possible roles for structure activity relationships are discussed.

Immature B cell malignancies synthesize VEGF, VEGFR-1 (Flt-1) and VEGFR-2 (KDR).

Expression of VEGF and VEGFR support a role for angiogenic pathways in the pathogenesis of some hematological malignances. Our goal was to determine if expression of these angiogenic molecules also extend to childhood precursor B cell acute lymphoblastic leukemia (pre-B ALL). We now show that transcripts of VEGF, and its receptors VEGFR-1 and VEGFR-2 are concomitantly expressed in both ALL cell lines and primary pre-B ALL. Western blot and ELISA consistently detected VEGF protein in the supernatants of the cell lines. Similarly, VEGFR-1 and VEGFR-2 proteins are also detectable by FACS analysis. Interestingly, the expression of the receptors in immature B cells is limited to the intra-cytoplasmic compartment and may suggest either internalization of the receptors or a block in trafficking of the receptor to the surface.

Flow cytometric ploidy analysis of testicular biopsies from sperm-negative wet preparations.

BACKGROUND: The use of testicular sperm in assisted reproduction depends on the availability of sperm in wet preparations. It is not always possible to recover sperm from the testis, even with previous sperm-positive histopathological findings. The purpose of this study was to evaluate the sperm-negative wet preparation search results with flow cytometric ploidy analysis and histopathological examination. METHODS: Two pieces of testicular tissue were obtained from azoospermic patients to investigate the spermatogenic status of the testis, and to determine the presence of sperm through a wet preparation. The testicular tissue was shredded and then vortexed; the cellular suspension was then processed for a wet preparation sperm search, while the residual tissue was exposed to enzymatic digestion for flow cytometric ploidy analysis. RESULTS: A total of 38 patients had sperm-negative wet preparation results. Of those, six (16%) were shown to have haploid cells after flow cytometric analysis. Histopathological examination showed three samples with maturation arrest at the spermatid stage, and the other three at the spermatocyte stage. CONCLUSIONS: Flow cytometric ploidy analysis can be used to verify the results of a wet preparation sperm search when no sperm were detected. Flow cytometric ploidy analysis can also reveal the presence of spermatids when no sperm are available.