Analysis of mutations in the IL2RG gene in 2 asian infants with X-linked severe combined immunodeficiency

No disponible

Chronic granulomatous disease: two decades of experience from a tertiary care centre in North West India.

Chronic granulomatous disease (CGD) results from an inherited defect in the phagocytic cells of the immune system. It is a genetically heterogenous disease caused by defects in one of the five major subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. There is a paucity of data from India on CGD. We herein describe the clinical features in 17 children with CGD from a single tertiary referral center in India. A detailed analysis of the clinical features, laboratory investigations and outcome of 17 children 7 with X-linked (XL) and 10 with autosomal recessive (AR) form was performed. Diagnosis of CGD was based on an abnormal granulocyte oxidative burst evaluated by either Nitroblue Tetrazolium (NBT) test or flow cytometry based Dihyrorhodamine 123 assay or both. The molecular diagnosis was confirmed by genetic mutation analysis in 13 cases. The mean age at diagnosis and the age at onset of symptoms was significantly lower in children diagnosed with XL- CGD compared those with AR disease. Mutations were detected in CYBB gene in 6 patients with XL-CGD and NCF-1 gene mutations were observed in 7 cases of AR- CGD. The course and outcome of the disease was much worse in children diagnosed with X-linked form of disease compared to AR forms of the disease; 4/7 (57%) children with X-CGD were dead at the time of data analysis. This is one of the largest series on chronic granulomatous disease from any developing country.

Novel Mutation of IL2RG Gene in a Korean Boy With X-linked Severe Combined Immunodeficiency

No disponible

XPS and NEXAFS studies of VUV/O3-treated aromatic polyurea and its application to microchip electrophoresis.

In this study, the authors performed X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure (NEXAFS) studies of vacuum ultraviolet (VUV)/O3-treated aromatic polyurea films to investigate their treatment effects. XPS and NEXAFS spectra indicate that the benzene ring was cleaved after treatment and that carboxyl, hydroxyl, ketone and aldehyde groups were formed at the cleaved sites. The VUV/O3-treated polyurea film was applied to a polymethylmethacrylate (PMMA) microchip for microchip electrophoresis (MCE) of bovine serum albumin (BSA). Fast electro-osmotic mobility of 4.6×10(-4) cm²/V/s as well as reduction of the BSA adhesion was achieved. This functional surface is useful for high-speed MCE analysis.

Ataxia-telangiectasia in a patient presenting with hyper-immunoglobulin M syndrome.

Ataxia-telangiectasia (AT) and hyper-immunoglobulin M (HIGM) syndrome are both primary immunodeficiency diseases caused by different genetic defects. While a small proportion of AT patients have increased serum immunoglobulin (Ig) M concentrations during the course of a disease, a high level of IgM at onset is rare. We report the case of an 8-year-old girl who had experienced recurrent respiratory infection, cutaneous abscesses, and hepatosplenomegaly since the age of 2 years. She was diagnosed with HIGM based on the results of immunological studies, including low IgG and IgA levels and raised serum IgM concentrations. However, at the age of 4 years, a neurological examination revealed gait disturbance and telangiectatic lesions on the conjunctiva; therefore, a diagnosis of AT was suggested. In spite of regular intravenous immunoglobulin infusions and antimicrobial prophylaxis, the patient experienced several episodes of respiratory infection and eventually died of respiratory failure at the age of 8 years. Further molecular analysis revealed a novel homozygous missense mutation in exon 53 (c.8250C>T, p.2622Ala>Val) of the ATM gene. Patients with AT and the HIGM phenotype may not develop clinical characteristics of AT for some time. While patients with AT and increased serum IgM levels could have a considerably more severe disease course and a shorter survival, IgM levels could be considered a prognostic factor.

Hyper IgM syndrome and complement Clq deficiency in an individual with systemic lupus erythematosus-like disease.

Many immunedeficiency syndromes are associated with autoimmune disorders. We here report on a girl with a systemic lupus erythematosus-like disease who suffered from both hyperimmunoglobulin M syndrome (HIGMS) and C1q deficiency. Despite severe central nervous system-lupus like disease, probably due to C1q deficiency, kidney function was relatively spared. IgM autoantibody might play a protective role against lupus-glomerulonephritis.

Unique activation status of peripheral blood mononuclear cells at acute phase of Kawasaki disease.

Although Kawasaki disease (KD) is characterized by a marked activation of the immune system with elevations of serum proinflammatory cytokines and chemokines at acute phase, the major sources for these chemical mediators remain controversial. We analysed the activation status of peripheral blood mononuclear cells (PBMCs) by flow cytometry, DNA microarray and quantitative reverse transcription-polymerase chain reaction. The proportions of CD69+ cells in both natural killer cells and gammadeltaT cells at acute-phase KD were significantly higher than those at convalescent-phase KD. Microarray analysis revealed that five genes such as NAIP, IPAF, S100A9, FCGR1A and GCA up-regulated in acute-phase KD and the pathways involved in acute phase KD were related closely to the innate immune system. The relative expression levels of damage-associated molecular pattern molecule (DAMP) (S100A9 and S100A12) genes in PBMCs at acute-phase KD were significantly higher than those at convalescent-phase KD, while those of TNFA, IL1B and IL6 genes were not significantly different between KD patients and healthy controls. Intracellular production of tumour necrosis factor-alpha, interleukin-10 and interferon-gamma in PBMCs was not observed in KD patients. The present data have indicated that PBMCs showed a unique activation status with high expression of DAMP genes but low expression of proinflammatory cytokine genes, and that the innate immune system appears to play a role in the pathogenesis and pathophysiology of KD.

Severe developmental delay and epilepsy in a Japanese patient with severe congenital neutropenia due to HAX1 deficiency.

HAX1 deficiency has recently been identified as a cause of severe congenital neutropenia (SCN), but little is known about the phenotype. We described an SCN patient with a homozygous 256C-to-T transition causing an R86X mutation in the HAX1 gene. Notably, the patient has been complicated by epilepsy and severe delay of motor, cognitive, and intellectual development; each developmental quotient was 21-26 at 7 years old. Growth failure and dental development delay were also noted. Neurodevelopmental delay in this patient expands the clinical phenotype of HAX1 deficiency and suggests an important role of HAX1 on neural development as well as myelopoiesis.

Expression profile of mRNAs from human pancreatic islet tumors.

In order to understand the tIssue specificity of the endocrine pancreas, it is important to clarify the expression profile of mRNAs in various states of the tIssue. A total of approximately 9000 non-redundant expressed genes from human pancreatic islets and insulinoma have so far been determined as expressed sequence tags (ESTs) and deposited in public databases. In the present study towards the identification of a complete set of genes expressed in human pancreatic islets, we have determined 3'-ESTs of 21267 clones randomly selected from a cDNA library of human pancreatic islet tumors. Clustering analysis generated 6157 non-redundant sequences comprising 2323 groups and 3834 singletons. Nucleotide and peptide database searches show that 3103 of them represent known human sequences or homologs of genes identified in other species and 58 are new members of structurally related families. The sequences were classified on the basis of the putative protein functions encoded, and were assigned to the respective chromosome by database analysis. The sequences were also compared with the EST databases (dbEST and EPConDB) including ESTs from normal pancreatic islet, insulinoma, and fetal pancreas. Since 3384 genes were newly found to be expressed in human pancreatic islets and 587 of them were unique to the islets, this study has considerably expanded the catalog of genes expressed in the endocrine pancreas. The larger collection of pancreatic islet-related ESTs should provide a better genome source for molecular studies of differentiation, tIssue-specific functions, and tumorigenesis of the endocrine pancreas as well as for genetic studies of diabetes mellitus.

A new simplified method for preparation of a synthetic phage antibody with practically acceptable detection sensitivity on immunoblots.

We produced a library of phage clones displaying a synthetic repertoire of 1x10(8) scFv antibody fragments by combinatorial mutagenesis of several amino acid residues in complementarity determining region 3 (CDR3) of a single antibody sequence used as a template. Phage antibodies specific to a cerebellum-specific protein, MEGF1/fat2, were successfully isolated from clones in this library, but their affinity in binding to MEGF1/fat2 was too low for use in immunoblotting applications. In an effort to obtain a practically useful phage antibody from these primary phage antibodies, a new simplified method based on PCR using unequally degenerate oligonucleotides was devised and applied. A secondary phage antibody showing 5-fold slower dissociation from the targeted antigen than the parental one was successfully obtained from a mutant library consisting of about 10(7) clones through only two rounds of panning. This affinity maturation followed by fusion with bacterial alkaline phosphatase improved sensitivity in immunoblot assays, with the detection limit lowered to one nanogram level. The results indicate that this method offers a straightforward route to generate a recombinant phage antibody with practically acceptable immunoblot sensitivity from a medium-size single-pot library, from which specific and high-affinity primary phage antibodies are difficult to isolate directly.

Identification of three novel non-classical cadherin genes through comprehensive analysis of large cDNAs.

The terminal sequences of long cDNAs from human brains were subjected to an improved method of motif-trap screening. This process resulted in the identification of three novel genes that encode proteins with 27, 27, and six cadherin domains that we denoted as KIAA1773, KIAA1774 and KIAA1775, respectively. Sequence analysis indicated that the products of these genes were non-classical cadherins. KIAA1773 was found to be a mammalian homologue of the Drosophila dachsous gene but the remaining two genes did not have any likely homologues in public databases. Assessment of their expression in rat tissues indicated that these genes are expressed in highly distinct and tissue-specific patterns. Notably, KIAA1775 is expressed almost exclusively in the olfactory bulb in the rat brain. In situ hybridization further showed that KIAA1775 is strongly expressed by the mitral and tufted cells in the main and accessory olfactory bulbs, suggesting that KIAA1775 may be important in the formation and maintenance of neuronal networks, particularly those in the olfactory bulb. This study clearly shows the importance and usefulness of our cDNA project in search for genes encoding large proteins, as this project has allowed us to identify several novel non-classical cadherin genes that have thus far not been detected by conventional methods.

Prediction of the coding sequences of unidentified human genes. XXI. The complete sequences of 60 new cDNA clones from brain which code for large proteins.

As an extension of a sequencing project of human cDNA clones which encode large proteins of unidentified genes, we herein present the entire sequences of 60 cDNA clones for the genes named KIAA1879-KIAA1938. The cDNA clones were isolated from size-fractionated cDNA libraries derived from human fetal brain, adult whole brain and amygdala, and their protein-coding sequences were predicted. Thirty-seven cDNA clones entirely sequenced in this study were selected as cDNAs which have coding potentiality by in vitro transcription/translation experiments, and the remaining 23 cDNA clones were chosen by computer-assisted analysis of terminal sequences of cDNAs. The average sizes of the inserts and corresponding open reading frames of cDNA clones analyzed here were 4.5 kb and 2.2 kb (733 amino acid residues), respectively. Sequence analyses against the public databases enabled us to annotate the functions of the predicted products of the 25 genes; 84% of these predicted gene products (21 gene products) were classified into proteins related to cell signaling/communication, nucleic acid management, and cell structure/motility. In addition to the sequence information about these 60 genes, their expression profiles were also studied in some human tissues including brain regions by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.

Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase.

Helical filaments driven by linear molecular motors are anticipated to rotate around their axis, but rotation consistent with the helical pitch has not been observed. 14S dynein and non-claret disjunctional protein (ncd) rotated a microtubule more efficiently than expected for its helical pitch, and myosin rotated an actin filament only poorly. For DNA-based motors such as RNA polymerase, transcription-induced supercoiling of DNA supports the general picture of tracking along the DNA helix. Here we report direct and real-time optical microscopy measurements of rotation rate that are consistent with high-fidelity tracking. Single RNA polymerase molecules attached to a glass surface rotated DNA for >100 revolutions around the right-handed screw axis of the double helix with a rotary torque of >5 pN nm. This real-time observation of rotation opens the possibility of resolving individual transcription steps.

Prediction of the coding sequences of unidentified human genes. XX. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro.

To accumulate information on the coding sequences of unidentified genes, we have carried out a sequencing project of human cDNA clones which encode large proteins. We herein present the entire sequences of 100 cDNA clones of unidentified human genes, named KIAA1776 and KIAA1780-KIAA1878, from size-fractionated cDNA libraries derived from human fetal brain, adult whole brain, hippocampus and amygdala. Most of the cDNA clones to be entirely sequenced were selected as cDNAs which were shown to have coding potentiality by in vitro transcription/translation experiments, and some clones were chosen by using computer-assisted analysis of terminal sequences of cDNAs. Three of these clones (fibrillin2/KIAA1776, MEGF10/KIAA1780 and MEGF11/KIAA1781) were isolated as genes encoding proteins with multiple EGF-like domains by motif-trap screening. The average sizes of the inserts and corresponding open reading frames of eDNA clones analyzed here reached 4.7 kb and 2.4 kb (785 amino acid residues), respectively. From the results of homology and motif searches against the public databases, the functional categories of the predicted gene products of 54 genes were determined; 93% of these predicted gene products (50 gene products) were classified as proteins related to cell signaling/communication, nucleic acid management, or cell structure/motility. To collect additional information on these genes, their expression profiles were also studied in 10 human tissues, 8 brain regions, spinal cord, fetal brain and fetal liver by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.

Association of structural polymorphisms in the human period3 gene with delayed sleep phase syndrome.

Recent progress in biological clock research has facilitated genetic analysis of circadian rhythm sleep disorders, such as delayed sleep phase syndrome (DSPS) and non-24-h sleep-wake syndrome (N-24). We analyzed the human period3 (hPer3) gene, one of the human homologs of the Drosophila clock-gene period (Per), as a possible candidate for rhythm disorder susceptibility. All of the coding exons in the hPer3 gene were screened for polymorphisms by a PCR-based strategy using genomic DNA samples from sleep disorder patients and control subjects. We identified six sequence variations with amino acid changes, of which five were common and predicted four haplotypes of the hPer3 gene. One of the haplotypes was significantly associated with DSPS (Bonferroni's corrected P = 0.037; odds ratio = 7.79; 95% CI 1.59-38.3) in our study population. Our results suggest that structural polymorphisms in the hPer3 gene may be implicated in the pathogenesis of DSPS.