Mutations disrupting the Kennedy phosphatidylcholine pathway in humans with congenital lipodystrophy and fatty liver disease

Phosphatidylcholine (PC) is the major glycerophospholipid in eukaryotic cells and is an essential component in all cellular membranes. The biochemistry of de novo PC synthesis by the Kennedy pathway is well established, but less is known about the physiological functions of PC. We identified two unrelated patients with defects in the Kennedy pathway due to biallellic loss-of-function mutations in phosphate cytidylyltransferase 1 alpha (PCYT1A), the rate-limiting enzyme in this pathway. The mutations lead to a marked reduction in PCYT1A expression and PC synthesis. The phenotypic consequences include some features, such as severe fatty liver and low HDL cholesterol levels, that are predicted by the results of previously reported liver-specific deletion of murine Pcyt1a. Both patients also had lipodystrophy, severe insulin resistance, and diabetes, providing evidence for an additional and essential role for PCYT1A-generated PC in the normal function of white adipose tissue and insulin action.All living cells are surrounded by a lipid membrane. Eukaryotic cells also contain several internal membrane-bound organelles, which enable them to compartmentalize related biological functions and thereby to enhance the efficiency of these processes. Phospholipids are the predominant component of these membranes. Their hydrophilic head groups interact with the cytosol, whereas their hydrophobic side chains are either buried within the hydrophobic interior of a typical membrane bilayer or interact with the hydrophobic neutral lipid core of lipoproteins and lipid droplets (LDs). Phospholipids are generally defined by their organic head group with phosphatidylcholine (PC) constituting over 50% of all membrane phospholipids. PC was first isolated in the 19th century and the major enzymatic pathway involved in its synthesis was revealed by Kennedy and Weiss (1) in the 1950s. Cells synthesize PC in three consecutive steps (Fig. 1A): choline kinase phosphorylates choline before choline phosphate cytidylyltransferase 1 α (encoded by the PCYT1A gene) generates the high-energy donor CDP-choline in the rate-limiting step of the pathway. In the last step, DAG:CDP-choline cholinephosphotransferase (CPT) uses CDP-choline and diacylglycerol (DAG) to form PC (2, 3).Open in a separate windowFig. 1.Cosegregation of biallelic PCYT1A mutations with fatty liver, low HDL cholesterol levels, lipodystrophy, insulin-resistant diabetes, and short stature. (A) Schematic illustration of the Kennedy PC synthesis pathway. CK, choline kinase; CPT, CDP-choline:1,2-diacylglycerol cholinephosphotransferase; PCYT1A, choline-phosphate cytidylyltransferase A, CTP:phosphocholine-cytidylyltransferase. (B) Family pedigrees of both probands demonstrating that only compound heterozygous carriers of PCYT1A mutations manifest fatty liver (red), low HDL cholesterol (blue), lipodystrophy (yellow), and insulin resistance/type 2 diabetes (T2DM) (green). PCYT1A mutation status, height (Ht.), and body mass index (BMI) are indicated below each individual’s symbol. ND, not determined; WT, wild type. (C) The location of PCYT1A mutations E280del, V142M, and 333fs in relation to known functional domains of PCYT1A. Domain M, membrane binding domain; domain P, phosphorylated region. (D) Conservation around the V142(red*) and E280(red*) mutation sites. Sequence alignment of representative metazoan sequences in the region surrounding the mutated residues. Hydrophobic (blue) and polar (green) residues interacting with V142 are highlighted. Only residues different from the human sequence are shown. Sequence IDs: human (Homo sapiens) {"type":"entrez-protein","attrs":{"text":"P49585","term_id":"166214967"}}P49585, zebrafish (Danio rerio) F1QEN6, sea squirt (Ciona intestinalis) {"type":"entrez-protein","attrs":{"text":"XP_002130773.1","term_id":"198437270"}}XP_002130773.1, sea urchin (Strongylocentrotus purpuratus) H3I3V9, water flee (Daphnia pulex) E9G1P5, Drosophila (D. melanogaster) Q9W0D9, Caenorhabditis (C. elegans) {"type":"entrez-protein","attrs":{"text":"P49583","term_id":"32172439"}}P49583, Trichoplax (T. adherens) B3RI62. (E and F) Structure of the catalytic domain of PCYT1A highlighting the role of V142M in the core packing. The two chains in the dimer are shown in yellow and gray; the residues and the secondary structure units are highlighted in color in the yellow monomer A: loop L3 with V142, red; α-helix, green; and the interacting β-sheet, blue. The residues packing with V142 are shown in ball-and-stick and space-filling representations, the dimer stabilizing R140 is shown in ball-and-stick colored according to the atom type. E is a global view, and F is a zoomed-in view of the catalytic core.Membrane phospholipids are a defining feature of advanced life-forms so it is perhaps not surprising that the pathways involved in their synthesis are ancient, and mutations affecting them are rarely tolerated in evolution. Here, we describe the identification and characterization of pathogenic human loss-of-function mutations affecting the eponymous Kennedy pathway.     

Early worsening of diabetic retinopathy after simultaneous pancreas and kidney transplantation—Myth or reality?

Early worsening of diabetic retinopathy due to sudden glucose normalization is a feared complication of pancreas transplantation; however, its rate or severity has not been studied prospectively. We followed up 43 pancreas and kidney recipients for a composite endpoint comprising new need for laser therapy, newly diagnosed proliferation, macular edema, visual acuity worsening, and blindness over 12 months. Although 37% of patients met this primary endpoint, its severity was rather low. Mean central retinal thickness and proportion of patients with subclinical macular edema increased significantly, with spontaneous resolution in half of them. Visual acuity did not change. There was no significant difference in the absolute glycated hemoglobin (HbA1c) drop, age, and diabetes duration between the patients who met and those who did not meet the primary endpoint, but a higher proportion of patients with worsening had a recent history of laser treatment. Retinopathy remained stable in 62.8% of patients. In 26%, the visual acuity significantly improved. Although retinopathy worsening was documented in more than one‐third of patients, its evolution was not related to the magnitude of metabolic change; rather, it corresponded to the expected natural course of retinopathy. Nonetheless, comprehensive ophthalmologic care should be a substantial component of the recipient management.     

Idiopathic CD4 Lymphocytopenia: Clinical and Immunologic Characteristics and Follow-Up of 40 Patients

Idiopathic CD4 T lymphocytopenia (ICL) is a rare and severe condition with limited available data. We conducted a French multicenter study to analyze the clinical and immunologic characteristics of a cohort of patients with ICL according to the Centers for Disease Control criteria.We recruited 40 patients (24 female) of mean age 44.2 ± 12.2 (19–70) years. Patients underwent T-lymphocyte phenotyping and lymphoproliferation assay at diagnosis, and experiments related to thymic function and interferon (IFN)-γ release by natural killer (NK) cell were performed. Mean follow-up was 6.9 ± 6.7 (0.14–24.3) years. Infectious, autoimmune, and neoplastic events were recorded, as were outcomes of interleukin 2 therapy.In all, 25 patients had opportunistic infections (12 with human papillomavirus infection), 14 had autoimmune symptoms, 5 had malignancies, and 8 had mild or no symptoms. At the time of diagnosis, the mean cell counts were as follows: mean CD4 cell count: 127/mm³ (range, 4–294); mean CD8: 236/mm³ (range, 1–1293); mean CD19: 113/mm³ (range, 3–547); and mean NK cell count: 122/mm³ (range, 5–416). Most patients had deficiency in CD8, CD19, and/or NK cells. Cytotoxic function of NK cells was normal, and patients with infections had a significantly lower NK cell count than those without (p = 0.01). Patients with autoimmune manifestations had increased CD8 T-cell count. Proliferation of thymic precursors, as assessed by T-cell rearrangement excision circles, was increased. Six patients died (15%). CD4 T-cell count <150/mm³ and NK cell count <100/mm³ were predictors of death.In conclusion, ICL is a heterogeneous disorder often associated with deficiencies in CD8, CD19, and/or NK cells. Long-term prognosis may be related to initial CD4 and NK cell deficiency.Abbreviations: AIHA = autoimmune hemolytic anemia, CDC = Centers for Disease Control, CMV = cytomegalovirus, cpm = count per minute, CVID = common variable immunodeficiency, CXCR4 = C-X-C chemokine receptor type 4, HIV = human immunodeficiency virus, HLA = human leukocyte antigen, HPV = human papillomavirus, HTLV-1/2 = human T-cell lymphotropic 1/2, ICL = idiopathic CD4 T lymphocytopenia, IFN-γ = interferon-γ, IL = interleukin, JC virus = John Cunningham virus, LPA = lymphocyte proliferation assay, NK = natural killer, P = patient, PBMC = peripheral blood mononuclear cell, Pwd = pokeweed, SI = stimulation index, sj = signal joint, TREC = T-cell rearrangement excision circle     

Peripheral T cell lymphoma: immunologic and cell-kinetic observations associated with morphological progression

Peripheral T cell lymphomas (PTCLs) form a morphologically heterogeneous group of non-Hodgkin's lymphomas that are generally considered to have immunophenotypes associated with mature T cells, usually those of helper T cells. We now describe and correlate the clinical, morphological, immunologic, and cell-kinetic findings based on the evaluation of eight tissue samples obtained at various times from a 13-year-old girl with PTCL. The early morphological expressions of this patient's PTCL were those of diffuse mixed-cell lymphoma and focal large-cell lymphoma (LCL) evolving from the histologic picture of an atypical immune response (AIR). These morphological findings were associated with an immature T cell immunophenotype associated with cortical thymocytes--namely, sheep erythrocyte rosette (sER)+, T11+, Leu-2a+, Leu-3a+, HLA-DR+, OKT6-, OKT9+, OKT10+--and with cell-kinetic findings that showed no evidence of aneuploidy and few cells in S phase. Diffuse pleomorphic LCL developed, which was associated with further dedifferentiation of the neoplastic T cells to the immunophenotype sER-, T11+, Leu-2a-, Leu-3a-, HLA-DR+, OKT6-, OKT9+, OKT10- and with cell-kinetic findings that demonstrated a distinct aneuploid population and a dramatic increase in the percentage of cells in the S phase. The immunophenotype of the PTCL at the time of the patient's death was T11-, Leu-2a-, Leu-3a-, HLA-DR+, OKT6-, OKT9+, OKT10-, an immunophenotype indistinguishable from that of a non-B non-T cell lymphoma. The immunologic findings in this case also suggest that an AIR in some cases may represent a prelymphomatous state or may be a morphological expression of PTCL. These observations indicate that PTCLs may be characterized by rapidly changing clinical, morphological, immunologic, and cell kinetic findings which are best evaluated by multidisciplinary studies.     

Case Report: Congenital Radioulnar Synostosis and Its Embryological Correlation and Functional Assessment

Congenital radioulnar synostosis(CRS) is a rare anomaly and approximately 400 cases were reported worldwide so far. CRS is the failure of the longitudinal segmentation and the persistence of the cartilaginous anlage between the radius and ulna during the seventh week of development that results in a persistent bridge of tissue. Here we are discussing on a case of 25yrs old, male patient with bilateral congenital synostosis. On the left hand the pronation and supination movements are restricted completely where as on right side 10 degree of supination and 20 degree of pronation is possible. Radiologically in our patient synostosis is classified as type II variety by Wilkie(1914)1 classification and type IV by the Cleary and Omer classification(1985). The position of forearm was not found to be related to subjective functional limitation, or employment status. Main line of treatment is surgical mainly rotational osteotomy but is rarely indicated. Our patient is not able to rotate his forearm especially on the left side still he has no functional limitation so he has refused for the operative treatment. No study has objectively compared the preoperative functional limitation of the patients with their postoperative functional improvement in order to justify surgical intervention In the authors opinion the only major factor that is to be taken into consideration of operative treatment is functional limitation to the patient.     

Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization

The development and application of fluorescence in-situ hybridization (FISH) has opened the way for comprehensive studies on numerical chromosome abnormalities in human spermatozoa. FISH can be rapidly applied to large numbers of spermatozoa and thus overcomes the major limitation of karyotyping spermatozoa after penetration of zona-free hamster oocytes. The simultaneous hybridization of two or more chromosome-specific probes to spermatozoa and subsequent detection of the bound probes using different fluorescent detection systems enables two or more chromosomes to be localized simultaneously in the same spermatozoon and provides a technique for undertaking reasonable estimates of aneuploidy. The most commonly used probes are those which bind to the centromeric region of specific chromosomes. Most studies to date have concentrated on estimating aneuploidy in spermatozoa from normospermic men, although reports are beginning to appear on aneuploidy in spermatozoa from subfertile and infertile men. Multi- probe FISH studies have generally reported disomy (hyperhaploidy) estimates of 0.05-0.2% per chromosome. There is preliminary evidence that some chromosomes such as X, Y and 21 are predisposed towards higher rates of non-disjunction during spermatogenesis. There are also suggestions of inter-donor variability in aneuploidy frequencies for specific chromosomes, although this requires confirmation in larger studies. While FISH is clearly a powerful technique that has many applications in reproductive medicine, it must also be realized that it does have limitations and the technology itself is still evolving and has yet to be fully validated on spermatozoa.      

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography

In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.      

Overview and update on molecular testing in non-small cell lung carcinoma utilizing endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) samples

Lung carcinoma remains one of the most frequent and aggressive human neoplasms. Fortunately, in the last decades, the increasing knowledge of the molecular mechanisms leading to cancer development has allowed the use of targeted therapies with improvement of prognosis in many patients. Clinical management has also changed after the introduction of endobronchialultrasonographic bronchoscopy that allows a conservative staging of lung tumors, avoiding the need of mediastinoscopy for lymph node staging. Lung pathologists and cytopathologists are facing the challenge of giving the more comprehensive prognostic and predictive information with ever smaller tissue or cytological samples. The aim of this review is to summarize the molecular testing for non-small cell lung carcinoma and how pathologists can contribute to the patient's outcome with a conscious management of biological samples.