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Patients with primary antibody deficiency (PAD) are predisposed to develop meningoencephalitis that is often considered to be enteroviral. However, there is a paucity of literature on this subject, and there are no studies from developing countries. We analyzed our cohort of children with PAD who developed meningoencephalitis. This complication was observed in 11/135 (8.1%) patients with PAD - 4 patients had X-linked agammaglobulinemia (XLA), and 7 had common variable immunodeficiency (CVID). The mean age at onset of neurological illness was 8.6 years (range: 2-28 years). Presenting features included seizures (n=7), neurodevelopmental delay (n=2), regression of milestones (n=1), and acute flaccid paralysis (n=1). Trough IgG levels were found to be low in 9 (81.8%) patients at the time of development of neurological symptoms. Herpes simplex virus (HSV), cytomegalovirus (CMV), and Streptococcus pneumoniae were isolated in 1 patient each. No etiological agent was identified in cerebrospinal fluid of 8 patients. Eight (72.7%) patients had altered signal hyperintensities in gray matter and deep white matter on magnetic resonance imaging (MRI), while 3 patients showed global cerebral atrophy. All patients were treated with high-dose intravenous immunoglobulin (IVIg). Fluoxetine was given to 2 patients. Eight (72.7%) patients in the present series have succumbed, while three have recovered with varying degrees of neurological sequelae. To conclude, meningoencephalitis is an uncommon complication in patients with PAD and is associated with high morbidity and mortality in our setting. Early diagnosis of immune deficiency and initiation of replacement immunoglobulin therapy may prevent the development of neurological complications.

Autosomal recessive agammaglobulinemia (ARA) is a primary immunodeficiency characterized by absent peripheral B cells, severe hypogammaglobulinemia, and absent BTK gene mutations. In ARA, mutations occur in genes encoding the pre-B cell receptor (pre-BCR) or downstream signaling proteins. In this work, we used candidate gene and whole-exome sequencing to investigate the molecular basis of ARA in 6 patients from 4 consanguineous North-African families. Sanger sequencing of candidate genes encoding the pre-BCR components (ΙGΗΜ, CD79A, CD79B, IGLL1, and VPREB1) was initially performed and determined the genetic defect in five patients. Two novel mutations in IGHM (p.Val378Alafs*1 and p.Ile184Serfs*21) were identified in three patients from two unrelated kindred and a novel nonsense mutation was identified in CD79A (p.Trp66*) in two siblings from a third kindred. Whole-exome sequencing (WES) was performed on the sixth patient who harbored a homozygous stop mutation at position 407 in the RAG2 gene (p.Glu407*). We concluded that conventional gene sequencing, especially when multiple genes are involved in the defect as is the case in ARA, is costly and time-consuming, resulting in delayed diagnosis that contributes to increased morbidity and mortality. In addition, it fails to identify the involvement of novel and unsuspected gene defects when the phenotype of the patients is atypical. WES has the potential to provide a rapid and more accurate genetic diagnosis in ARA, which is crucial for the treatment of the patients.

BackgroundWiskott Aldrich syndrome (WAS) is characterized by bleeding manifestations, recurrent infections, eczema, autoimmunity, and malignancy. Over the last decade, improved awareness and better in-house diagnostic facilities at several centers in India has resulted in increased recognition of WAS. This study reports collated data across major primary immunodeficiency diseases (PID) centers in India that are involved in care of children with WAS and highlights the varied clinical presentations, genetic profile, and outcomes of patients in India.MethodsRequest to share data was sent to multiple centers in India that are involved in care and management of patients with PID. Six centers provided requisite data that were compiled and analyzed.ResultsIn this multi-institutional cohort, clinical details of 108 patients who had a provisional diagnosis of WAS were received. Of these, 95 patients with ‘definite WAS’ were included Fourteen patients were classified as XLT and 81 patients as WAS. Median age at onset of symptoms of patients was 3 months (IQR 1.6, 6.0 months) and median age at diagnosis was 12 months (IQR 6,48 months). Clinical profile included bleeding episodes (92.6%), infections (84.2%), eczema (78.9%), various autoimmune manifestations (40%), and malignancy (2.1%). DNA analysis revealed 47 variants in 67 cases. Nonsense and missense variants were the most common (28.4% each), followed by small deletions (19.4%), and splice site defects (16.4%). We also report 24 novel variants, most of these being frameshift and nonsense mutations resulting in premature termination of protein synthesis. Prophylactic intravenous immunoglobulin (IVIg) was initiated in 52 patients (54.7%). Hematopoietic stem cell transplantation (HSCT) was carried out in 25 patients (26.3%). Of those transplanted, disease-free survival was seen in 15 patients (60%). Transplant related mortality was 36%. Outcome details were available for 89 patients. Of these, 37% had died till the time of this analysis. Median duration of follow-up was 36 months (range 2 weeks- 12 years; IQR 16.2 months- 70 months).ConclusionsWe report the first nationwide cohort of patients with WAS from India. Bleeding episodes and infections are common manifestations. Mortality continues to be high as curative therapy is not accessible to most of our patients.

BackgroundSevere Combined Immune Deficiency (SCID) is an inherited defect in lymphocyte development and function that results in life-threatening opportunistic infections in early infancy. Data on SCID from developing countries are scarce.ObjectiveTo describe clinical and laboratory features of SCID diagnosed at immunology centers across India.MethodsA detailed case proforma in an Excel format was prepared by one of the authors (PV) and was sent to centers in India that care for patients with primary immunodeficiency diseases. We collated clinical, laboratory, and molecular details of patients with clinical profile suggestive of SCID and their outcomes. Twelve (12) centers provided necessary details which were then compiled and analyzed. Diagnosis of SCID/combined immune deficiency (CID) was based on 2018 European Society for Immunodeficiencies working definition for SCID.ResultsWe obtained data on 277 children; 254 were categorized as SCID and 23 as CID. Male-female ratio was 196:81. Median (inter-quartile range) age of onset of clinical symptoms and diagnosis was 2.5 months (1, 5) and 5 months (3.5, 8), respectively. Molecular diagnosis was obtained in 162 patients - IL2RG (36), RAG1 (26), ADA (19), RAG2 (17), JAK3 (15), DCLRE1C (13), IL7RA (9), PNP (3), RFXAP (3), CIITA (2), RFXANK (2), NHEJ1 (2), CD3E (2), CD3D (2), RFX5 (2), ZAP70 (2), STK4 (1), CORO1A (1), STIM1 (1), PRKDC (1), AK2 (1), DOCK2 (1), and SP100 (1). Only 23 children (8.3%) received hematopoietic stem cell transplantation (HSCT). Of these, 11 are doing well post-HSCT. Mortality was recorded in 210 children (75.8%).ConclusionWe document an exponential rise in number of cases diagnosed to have SCID over the last 10 years, probably as a result of increasing awareness and improvement in diagnostic facilities at various centers in India. We suspect that these numbers are just the tip of the iceberg. Majority of patients with SCID in India are probably not being recognized and diagnosed at present. Newborn screening for SCID is the need of the hour. Easy access to pediatric HSCT services would ensure that these patients are offered HSCT at an early age.

BackgroundThere is paucity of literature on XLA from developing countries. Herein we report the clinical and molecular profile and outcome in a multicenter cohort of patients with XLA from India.MethodsData on XLA from all regional centers supported by the Foundation for Primary Immunodeficiency Diseases (FPID), USA and other institutions providing care to patients with PIDs were collated. Diagnosis of XLA was based on European Society for Immunodeficiencies (ESID) criteria.ResultsWe received clinical details of 195 patients with a provisional diagnosis of XLA from 12 centers. At final analysis, 145 patients were included (137 ‘definite XLA’ and eight ‘probable/possible XLA’). Median age at onset of symptoms was 12.0 (6.0, 36.0) months and median age at diagnosis was 60.0 (31.5, 108) months. Pneumonia was the commonest clinical manifestation (82.6%) followed by otitis media (50%) and diarrhea (42%). Arthritis was seen in 26% patients while 23% patients developed meningitis. Bronchiectasis was seen in 10% and encephalitis (likely viral) in 4.8% patients. Pseudomonas aeruginosa was the commonest bacterial pathogen identified followed by Streptococcus pneumoniae, Staphylococcus aureus and Klebsiella pneumoniae. Molecular analysis revealed 86 variants in 105 unrelated cases. Missense variants in BTK gene were the most common (36%) followed by frameshift (22%) and nonsense variants (21%). Most pathogenic gene variants (53%) were clustered in the distal part of gene encompassing exons 14–19 encoding for the tyrosine kinase domain. Follow-up details were available for 108 patients. Of these, 12% had died till the time of this analysis. The 5-year and 10-year survival was 89.9% and 86.9% respectively. Median duration of follow-up was 61 months and total duration of follow-up was 6083.2 patient-months. All patients received intravenous immunoglobulin (IVIg) replacement therapy. However, in many patients IVIg could not be given at recommended doses or intervals due to difficulties in accessing this therapy because of financial reasons and lack of universal health insurance in India. Hematopoietic stem cell transplant was carried out in four (2.8%) patients.ConclusionThere was a significant delay in the diagnosis and facilities for molecular diagnosis were not available at many centers. Optimal immunoglobulin replacement is still a challenge

Hyper IgE syndrome (HIES) encompasses a group of primary immunodeficiency diseases (PIDs) that is characterized by severe atopy, and recurrent infections and markedly elevated serum IgE levels. The majority of HIES cases suffer from autosomal dominant mutations in the signal transducer and activator of transcription 3 gene. A minority of cases display autosomal recessive inheritance, and one form is caused by mutations in the dedicator of cytokinesis 8 (DOCK8) gene. Here we describe the first recognized and diagnosed case of DOCK8 deficiency in the Philippines. A 14 year-old-girl was referred due to recalcitrant atopic dermatitis, recurrent sinopulmonary infections, with widespread warts on the face, trunk and extremities. She had no coarse facial features or retained primary teeth, whereas she presented with widespread viral skin infections and multiple allergic diseases. Laboratory examinations revealed elevations in eosinophil count and serum IgE. The level of T-cell receptor excision circles was undetectable. The patient was suspected to have HIES with a probable DOCK8 deficiency. Genetic analysis disclosed a large genomic deletion involving exons 2-4 in the DOCK8 gene. A combination of recalcitrant atopic dermatitis, asthma, food allergies, with viral skin infections should increase the physician's consideration of a PID. Patients with HIES accompanied by warts and T-cell deficiency can be strongly suspected to have DOCK8 deficiency.

Most of the literature on liver abscess in chronic granulomatous disease (CGD) emanates from developed countries. Data from developing countries are scarce. In this study, we report clinical features, microbiological profile, and treatment difficulties encountered while managing liver abscesses in patients with CGD at a tertiary care centre in North-West India. Case records of children with CGD and liver abscesses at Pediatric Immunodeficiency Clinic, Advanced Pediatrics Centre, Postgraduate Institute of Medical Education and Research, Chandigarh, India were analyzed. Seven of 68 patients (10.29%) with CGD presented with hepatic abscess. One patient had 2 recurrences. All were males and age-range at presentation was 7 months–22 years. Mutation analysis was carried out in all patients—3 had defects in CYBB gene; 2 in NCF1; 2 in NCF2 gene. Staphylococcus aureus was isolated from 5 patients. Duration of antimicrobial treatment ranged from 3 weeks to 7 months. Open drainage was required in 1 patient, and 1 patient was treated with a prolonged course of prednisolone. Two children succumbed to the illness. This is the largest reported experience of liver abscesses in patients with CGD from the developing world. Staphylococcus aureus was the commonest pathogen isolated. In our experience, prolonged courses of antimicrobials are usually necessary in these patients. Glucocorticoids can reduce inflammatory response and facilitate early resolution of abscesses in CGD.

Background Very-early-onset inflammatory bowel disease (VEOIBD) is a chronic inflammatory disease of the gastrointestinal tract occurring during infancy or early childhood. NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome has emerged as a crucial regulator of intestinal homeostasis; however, whether NLRP3 variants may modify VEOIBD risk is unknown. Objective We sought to investigate whether and how a rare NLRP3 variant, found in 3 patients with gastrointestinal symptoms, contributes to VEOIBD development. Methods Whole-exome sequencing and bioinformatic analysis were performed to screen disease-associated NLRP3 variants from a cohort of children with VEOIBD. Inflammasome activation was determined in reconstituted HEK293T human embryonic kidney cells with NLRP3 inflammasome components, doxycycline-inducible NLRP3 macrophages, as well as PBMCs and biopsies from patients with NLRP3 variants. Pathogenesis of the variants was determined using a dextran sulfate sodium–induced acute colitis model. Results We identified a dominant gain-of-function missense variant of NLRP3, encoded by rs772009059 (R779C), in 3 patients with gastrointestinal symptoms. Functional analysis revealed that R779C increased NLRP3 inflammasome activation and pyroptosis in macrophages. This was mediated by enhanced deubiquitination of NLRP3 via binding with deubiquitinases BRCC3 and JOSD2, which are highly expressed in myeloid cells. In a dextran sulfate sodium–induced acute colitis model, NLRP3-R779C in hematopoietic cells resulted in more severe colitis, which can be ameliorated via knockdown of BRCC3 or JOSD2. Conclusions BRCC3 and JOSD2 mediate NLRP3-R779C deubiquitination, which promotes NLRP3 inflammasome activation and the risk of developing VEOIBD.

We report on 2 Asian siblings with X-linked inhibitor of apoptosis deficiency that arose from a novel deletion that presented with Epstein-Barr virus disease and hemophagocytic lymphohistiocytosis. This disease is ascribed to dysfunction in the nucleotide binding and oligomerization domain receptor pathway, tested using a modified muramyl dipeptide–mediated assay.

Talaromyces (Penicillium) marneffei is an AIDS-defining infection in Southeast Asia and is associated with high mortality. It is rare in non-immunosuppressed individuals, especially children. Little is known about host immune response and genetic susceptibility to this endemic fungus. Genetic defects in the interferon-gamma (IFN-γ)/STAT1 signaling pathway, CD40/CD40 ligand- and IL12/IL12-receptor-mediated crosstalk between phagocytes and T-cells, and STAT3-mediated Th17 differentiation have been reported in HIV-negative children with talaromycosis and other endemic mycoses such as histoplasmosis, coccidioidomycosis, and paracoccidioidomycosis. There is a need to design a diagnostic algorithm to evaluate such patients. In this article, we review a cohort of pediatric patients with disseminated talaromycosis referred to the Asian Primary Immunodeficiency Network for genetic diagnosis of PID. Using these illustrative cases, we propose a diagnostics pipeline that begins with immunoglobulin pattern (IgG, IgA, IgM, and IgE) and enumeration of lymphocyte subpopulations (T-, B-, and NK-cells). The former could provide clues for hyper-IgM syndrome and hyper-IgE syndrome. Flow cytometric evaluation of CD40L expression should be performed for patients suspected to have X-linked hyper-IgM syndrome. Defects in interferon-mediated JAK-STAT signaling are evaluated by STAT1 phosphorylation studies by flow cytometry. STAT1 hyperphosphorylation in response to IFN-α or IFN-γ and delayed dephosphorylation is diagnostic for gain-of-function STAT1 disorder, while absent STAT1 phosphorylation in response to IFN-γ but normal response to IFN-α is suggestive of IFN-γ receptor deficiency. This simple and rapid diagnostic algorithm will be useful in guiding genetic studies for patients with disseminated talaromycosis requiring immunological investigations.

Primary B-cell immunodeficiencies are risk factors for the generation of vaccine-derived polioviruses. We report immunodeficiency-associated vaccine-derived poliovirus serotype 3 in an 11-week-old boy with X-linked agammaglobulinemia. Unique characteristics of this case include early age of presentation, high viral evolutionary rate, and the child’s perinatal exposure to human immunodeficiency virus.

This study determined the circulating levels of TGF-β1 and its association with aortic dilation and elastic properties in congenital heart patients. Forty-six patients after tetralogy of Fallot (TOF) repair, 21 patients post arterial switch and 15 patients post atrial switch for transposition of the great arteries (TGA), 27 patients post Fontan procedure, and 36 controls were studied. Aortic dimensions and elastic properties and ventricular function were assessed by echocardiography. Serum TGF-β1, metalloproteinase (MMP)-2 and MMP-9 levels were quantified. Compared with controls, all groups of patients had significantly larger ascending aortic dimensions and worse elastic properties (all p

Background X-linked agammaglobulinemia (XLA) is an X-linked genetic defect in maturation of B lymphocytes that results in the absence of B lymphocytes in the peripheral blood and profound hypogammaglobulinemia. It is caused by a mutation in the BTK gene located on the X chromosome. There are no large series describing XLA from the developing world. Objective To analyze the clinical features, immunologic and genetic characteristics, and outcomes of 36 patients with XLA diagnosed and managed for a period of 2 decades. Methods Diagnosis of XLA was made on the basis of presence of BTK gene mutation or marked reduction of B lymphocytes in peripheral blood with a family history of an affected male relative. The diagnosis was confirmed by genetic mutation studies in 28 patients with 25 unique mutations in the BTK gene. Results There was a significant delay in diagnosis in most of the patients. The mean (SD) delay in the diagnosis was 4.2 (3.5) years. Point mutations were the most common mutations detected, accounting for 68% of all mutations. Deletions and insertions were also seen in a few cases. Four of the mutations are novel mutations that have not been previously reported. Seven of the 36 patients (19%) were dead at the time of analysis in the present cohort. The mean survival was 137 months (95% confidence interval, 13–163 months). Conclusion The present study is perhaps the largest series of patients with XLA from any developing country so far.

Background X-linked agammaglobulinemia (XLA) is a primary immunodeficiency caused by Bruton’s tyrosine kinase (BTK) mutation. Patients are susceptible to severe enterovirus infections. The underlying mechanism remains unknown. BTK is involved in toll-like receptors pathway, which initiates antiviral responses including interferon (IFN) productions. Objective To demonstrate type I and III IFN productions in dendritic cells of XLA patients is decreased in response to oral poliovirus vaccine (OPV) but not H1N1 virus. Methods Monocyte-derived dendritic cells (MoDCs) were derived from nine XLA patients aged 22–32 years old and 23 buffy coats from Hong Kong Red Cross blood donors. LFM-A13 was used to inhibit BTK. OPV Sabin type 1 and H1N1 influenza virus were used to stimulate MoDCs with RPMI as mock stimulation. The antiviral cytokine productions and phenotypic maturation of MoDCs were determined 24 h post-stimulation. OPV RNA was determined at 0, 6, 12, and 24 h post-stimulation. Results Upon OPV stimulation, IFN-α2, IFN-β, and IFN-λ1 productions in MoDCs from XLA patients and BTK-inhibited MoDCs of healthy controls were significantly lower than that from healthy controls. Whereas upon H1N1 stimulation, the IFN-α2, IFN-β, and IFN-λ1 productions were similar in MoDCs from XLA patients, BTK-inhibited MoDCs of healthy controls and healthy controls. The mean fluorescent intensities (MFI) of CD83, CD86, and MHC-II in MoDCs from XLA patients in response to OPV was similar to that in response to mock stimulation, while the MFI of CD83, CD86, and MHC-II were significantly higher in response to H1N1 stimulation than that in response to mock stimulation. Whereas, the MFI of CD83, CD86, and MHC-II in MoDCs of healthy controls were significantly higher in response to both OPV and H1N1 stimulation compared to that in response to mock stimulation. Conclusion Production of type I and III IFN in response to OPV was deficient in MoDCs from XLA patients, but was normal in response to H1N1 due to deficient BTK function. Moreover, phenotypic maturation of MoDCs from XLA patients was impaired in response to OPV but not to H1N1. These selective impairments may account for the unique susceptibility of XLA patients toward severe enterovirus infections.

Background Autoimmune lymphoproliferative syndrome (ALPS) is a genetic disorder of lymphocyte homeostasis due to impaired apoptosis. It was initially regarded as a very rare disease, but recent studies show that it may be more common than previously thought. Defects in a couple of genes have been identified in a proportion of patients with ALPS, but around one-third of such patients remain undefined genetically. Objective We describe 2 siblings presenting with ALPS-like disease. This study aimed to identify the genetic cause responsible for this phenotype. Methods Whole-exome sequencing and molecular and functional analyses were used to identify and characterize the genetic defect. Clinical and immunological analysis was also performed and reported. Results The 2 patients presented with chronic lymphadenopathy, hepatosplenomegaly, autoimmune hemolytic anemia, immune thrombocytopenia, and the presence of antinuclear autoantibody and other autoantibodies, but normal double-negative T cells. They also suffered from recurrent infections. Novel compound heterozygous mutations of RASGRP1 encoding Ras guanyl nucleotide releasing protein 1 were identified in the 2 siblings. The mutations impaired T-cell receptor signaling, leading to defective T-cell activation and proliferation, as well as impaired activation-induced cell death of T cells. Conclusions This study shows for the first time that RASGRP1 mutation should be considered in patients with ALPS-like disease. We also propose to investigate the intracellular proteins involved in the T-cell receptor signaling pathway in similar patients but with unknown genetic cause.

X-linked hyper IgM Syndrome (XLHIGM), the most frequent form of the Hyper IgM syndromes is a primary immune deficiency resulting from a mutation in the CD40 ligand gene (CD40LG). We analyzed the clinical and laboratory features of ten patients with XLHIGM, who were diagnosed at a tertiary care hospital in North India. Most common infections were sinopulmonary infections (80%) and diarrhea (50%). Sclerosing cholangitis and necrotising fasciitis were noted in one patient each. Three novel mutations in CD40LG (c.429_429 delA, p. G144DfsX5; c.500 G > A, p.G167E and c.156 G > C, p.K52 N) were detected. In addition, we found one missense mutation, two splice site mutations and two large deletions, which have been previously reported. Four (4) patients had expired at the time of analysis. We report the first series of XLHIGM from North India where we have documented unique features such as pulmonary alveolar proteinosis and infections with Mycobacterium sp.

International audience; Background: Severe combined immunodeficiency (SCID) is fatal unless treated with hematopoietic stem cell transplant. Delay in diagnosis is common without newborn screening. Family history of infant death due to infection or known SCID (FH) has been associated with earlier diagnosis. Objective: The aim of this study was to identify the clinical features that affect age at diagnosis (AD) and time to the diagnosis of SCID.Methods: From 2005 to 2016, 147 SCID patients were referred to the Asian Primary Immunodeficiency Network. Patients with genetic diagnosis, age at presentation (AP), and AD were selected for study.Results: A total of 88 different SCID gene mutations were identified in 94 patients, including 49 IL2RG mutations, 12 RAG1 mutations, 8 RAG2 mutations, 7 JAK3 mutations, 4 DCLRE1C mutations, 4 IL7R mutations, 2 RFXANK mutations, and 2 ADA mutations. A total of 29 mutations were previously unreported. Eighty-three of the 94 patients fulfilled the selection criteria. Their median AD was 4 months, and the time to diagnosis was 2 months. The commonest SCID was X-linked (n = 57). A total of 29 patients had a positive FH. Candidiasis (n = 27) and bacillus CalmetteGuerin (BCG) vaccine infection (n = 19) were the commonest infections. The median age for candidiasis and BCG infection documented were 3 months and 4 months, respectively. The median absolute lymphocyte count (ALC) was 1.05 x 10(9)/L with over 88% patients below 3 x 10(9)/L. Positive FH was associated with earlier AP by 1 month (p = 0.002) and diagnosis by 2 months (p = 0.008), but not shorter time to diagnosis (p = 0.494). Candidiasis was associated with later AD by 2 months (p = 0.008) and longer time to diagnosis by 0.55 months (p = 0.003). BCG infections were not associated with age or time to diagnosis.Conclusion: FH was useful to aid earlier diagnosis but was overlooked by clinicians and not by parents. Similarly, typical clinical features of SCID were not recognized by clinicians to shorten the time to diagnosis. We suggest that lymphocyte subset should be performed for any infant with one or more of the following four clinical features: FH, candidiasis, BCG infections, and ALC below 3 x 10(9)/L.

International audience; Letter to the Editor

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.

International audience; X-linked agammagobulinemia (XLA) is a primary immunodeficiency caused by Bruton's tyrosine kinase (BTK) gene defect. XLA patients have absent or reduced number of peripheral B cells and a profound deficiency in all immunoglobulin isotypes. This multicenter study reports the clinical, immunological and molecular features of Bruton's disease in 40 North African male patients. Fifty male out of 63 (male and female) patients diagnosed with serum agammaglobulinemia and non detectable to less than 2 % peripheral B cells were enrolled. The search for BTK gene mutations was performed for all of them by genomic DNA amplification and Sanger sequencing. We identified 33 different mutations in the BTK gene in 40 patients including 12 missense mutations, 6 nonsense mutations, 6 splice-site mutations, 5 frameshift, 2 large deletions, one complex mutation and one in-frame deletion. Seventeen of these mutations are novel. This large series shows a lower frequency of XLA among male patients from North Africa with agammaglobulinemia and absent to low B cells compared with other international studies (63.5 % vs 85 %). No strong evidence for genotype-phenotype correlation was observed. This study adds to other reports from highly consanguineous North African populations, showing lower frequency of X-linked forms as compared to AR forms of the same primary immunodeficiency. Furthermore, a large number of novel BTK mutations were identified and could further help identify carriers for genetic counseling.

Aim Infantile-onset inflammatory bowel disease (IO-IBD) with the onset of disease before 12 mo of age, is a different disease entity from childhood IBD. We aimed to describe the clinical features, outcome and role of mutation in interleukin-10 (IL-10) and interleukin-10 receptors (IL-10R) in Asian children with IO-IBD. Methods All cases of IO-IBD, defined as onset of disease before 12 mo of age, seen at University Malaya Medical Center, Malaysia were reviewed. We performed mutational analysis for IL10 and IL10R genes in patients with presenting clinical features of Crohn's disease (CD). Results Six [13%; CD = 3, ulcerative colitis (UC) = 2, IBD-unclassified (IBD-U) = 1] of the 48 children (CD = 25; UC = 23) with IBD have IO-IBD. At final review [median (range) duration of follow-up: 6.5 (3.0-20) years], three patients were in remission without immunosuppression [one each for post-colostomy (IBD-U), after standard immunosuppression (CD), and after total colectomy (UC)]. Three patients were on immunosuppression: one (UC) was in remission while two (both CD) had persistent disease. As compared with later-onset disease, IO-IBD were more likely to present with bloody diarrhea (100% vs 55%, P = 0.039) but were similar in terms of an associated autoimmune liver disease (0% vs 19%, P = 0.31), requiring biologics therapy (50% vs 36%, P = 0.40), surgery (50% vs 29%, P = 0.27), or achieving remission (50% vs 64%, P = 0.40). No mutations in either IL10 or IL10R in the three patients with CD and the only patient with IBD-U were identified. Conclusion The clinical features of IO-IBD in this Asian cohort of children who were negative for IL-10 or IL-10R mutations were variable. As compared to childhood IBD with onset of disease after 12 mo of age, IO-IBD achieved remission at a similar rate.

Circulating carboxy-terminal propeptide of type I procollagen (PICP) and amino-terminal propeptide of type III procollagen (PIIINP) are biomarkers of collagen synthesis. We tested the hypothesis that circulating PICP and PIIINP are altered and may correlate with ventricular volume load and function in patients with repaired tetralogy of Fallot (TOF).Serum PICP and plasma PIIINP levels were determined in 39 patients with repaired TOF aged 17.7 ± 4.1 years and 25 healthy controls and correlated with right ventricular (RV) and left ventricular (LV) volumes, functional indices, and mechanical dyssynchrony as assessed by 3-dimensional and tissue Doppler echocardiography. Compared with controls, patients had significantly higher circulating PICP (P = .016) and PIIINP (P = .008) levels, worse RV function with intra-RV mechanical delay (all P.001), impaired LV systolic functional indices (all P.05), and greater LV systolic dyssynchrony index (SDI) (P.001). For the whole cohort, circulating PICP and PIIINP levels correlated with age (P = .001 and P.001, respectively), body mass index (P = .033 and P = .012, respectively), LV eccentricity (P = .035 and P = .046, respectively), RV end-diastolic volume (P = .029 and P = .047, respectively), and LV SDI (both P.001). In addition, PICP levels correlated negatively with RV and LV isovolumic acceleration and RV ejection fraction. Multiple linear regression analysis identified LV SDI as a significant independent correlate of circulating levels of PICP (β = .31, P = .045) and PIIINP (β = .37, P = .004).Circulating levels of PICP and PIIINP correlate positively with LV mechanical dyssynchrony in patients after TOF repair, implicating a possible role of increased collagen synthesis in its pathogenesis.

Matrix metalloproteinases (MMPs) are capable of degrading extracellular matrix proteins, which are important determinants of arterial stiffness. This study aimed to test the hypothesis that MMP-3 and MMP-9 polymorphisms may modulate aortic stiffness and magnitude of aortic root dilation in patients after surgical repair of tetralogy of Fallot (TOF). We analyzed the MMP-3 promoter and MMP-9 -1562 C>T polymorphism in 79 TOF patients aged 19.9 ± 9.5 years and determined their associations with aortic stiffness and sinotubular dimension. Genotypic and allelic frequencies of MMP-3 for the 6A6A genotype and MMP-9 for the T allele did not differ between patients and published control data (all p>0.05). For the MMP-3 locus, patients with a 6A6A genotype and those with a 6A6A/5A6A genotype had similar aortic stiffness (p=0.60), heart-femoral pulse wave velocity (p=0.63), and z score of sinotubular junction (p=0.81). For the MMP-9 locus, the -1562T allele carriers had significantly lower aortic stiffness (p=0.005), slower heart-femoral pulse wave velocity (p=0.03), and smaller z score of sinotubular junction (p=0.047). Multivariate linear regression identified MMP-9 polymorphism (β=-0.31, p=0.005) as a significant correlate of aortic stiffness after adjustments for age at study, age at operation, sex, body mass index, systolic and diastolic blood pressures, and MMP-3 polymorphism. In conclusion, MMP-9 but not MMP-3 polymorphism exerts a modulating influence on aortic stiffness and aortic root dilation in patients after TOF repair.

Severe combined immunodeficiencies (SCID) are a group of rare inherited disorders with profound defects in T cell and B cell immunity. From 2005 to 2010, our unit performed testing for IL2RG, JAK3, IL7R, RAG1, RAG2, DCLRE1C, LIG4, AK2, and ZAP70 mutations in 42 Chinese and Southeast Asian infants with SCID adopting a candidate gene approach, based on patient's gender, immune phenotype, and inheritance pattern. Mutations were identified in 26 patients, including IL2RG (n = 19), IL7R (n = 2), JAK3 (n = 2), RAG1 (n = 1), RAG2 (n = 1), and DCLRE1C (n = 1). Among 12 patients who underwent hematopoietic stem cell transplantation, eight patients survived. Complications and morbidities during transplant period were significant, especially disseminated bacillus Calmette-Guérin disease which was often difficult to control. This is the first cohort study on SCID in the Chinese and Southeast Asian population, based on a multi-centered collaborative research network. The foremost issue is service provision for early detection, diagnosis, management, and definitive treatment for patients with SCID. National management guidelines for SCID should be established, and research into an efficient platform for genetic diagnosis is needed.

Wiskott-Aldrich syndrome (WAS) is a rare primary immunodeficiency disease, with an incidence of 4/1,000,000 live male births. In China, an estimated number of 35 babies with WAS are born each year, but likely many remain undiagnosed.The objectives of study were to review the clinical and molecular characteristics of a cohort of Chinese children with WAS and to describe the long-term outcome of those who underwent hematopoietic stem cell transplant (HSCT).Records of 35 patients diagnosed with WAS during 1991-2008 were reviewed. Genetic diagnosis was established by direct gene sequencing.All patients had classical WAS phenotype. WASP mutations were identified in 33 patients from 29 families. Nine patients underwent HSCT at a mean age of 22.1 months (match-unrelated donor, n = 5; mismatched related donor, n = 2; matched-sibling donor, n = 2). Post-transplant immune hemolytic anemia and thrombocytopenia occurred in three patients with complete resolution. All patients survived without significant long-term complications and had full platelet, T and B lymphocyte recovery within 2 years post-transplant.In the past decade, there has been significant improvement in clinical and genetic diagnosis of WAS in Chinese. We demonstrated excellent long-term survival in patients who underwent HSCT. Early workup for transplant should be advocated for children with classical WAS before they suffer from major disease complications and morbidities.

OBJECTIVE. We tested the hypothesis that single-nucleotide polymorphisms of inflammatory genes C-reactive protein (CRP) and tumor necrosis factor α (TNF-α) may exert influence on susceptibility to Kawasaki disease and its arterial sequelae. METHODS. We analyzed the CRP +1444 C→T and TNF-α −308 G→A polymorphisms in 167 patients aged 8.9 ± 4.1 years with a history of Kawasaki disease (73 with and 94 without coronary aneurysms) and 124 healthy control subjects. For patients with Kawasaki disease, we further determined whether these single-nucleotide polymorphisms were associated with coronary aneurysms, carotid arterial stiffening, and intima-media thickness. RESULTS. Genotypic and allelic frequencies of CRP +1444 for T carrier and TNF-α −308 for A carrier were significantly higher in patients than in control subjects. The genotypic and allelic distributions did not differ between patients with and those without coronary aneurysms; however, patients with CRP +1444 CT/TT genotype compared with those with a CC genotype and patients with TNF-α −308 GA/AA genotype compared with those with a GG genotype had significantly greater carotid arterial stiffness and intima-media thickness. Carriers of both CRP +1444 T allele and TNF-α −308 A allele had the highest susceptibility to Kawasaki disease and a significant trend of increased arterial stiffness and intima-media thickness compared with those who carried either 1 or none of the rare alleles. Multiple linear regression analysis identified CRP +1444 allele carrier as a significant determinant of both carotid stiffness and carotid intima-media thickness and TNF-α −308 A allele carrier as a determinant of only intima-media thickness. CONCLUSIONS. Our findings suggest that CRP +1444 C→T and TNF-α −308 G→A polymorphisms are associated with predisposition to Kawasaki disease and, in patients with Kawasaki disease, increased carotid arterial stiffness and intima-media thickness in the long-term.

Chronic granulomatous disease (CGD) is a rare disorder of phagocytic oxidative bursts leading to recurrent pyogenic infections. Affected individuals are most prone to infections caused by staphylococci, Salmonella, Candida, and Aspergillus, but previously we observed a high incidence of Mycobacterium tuberculosis infection in Chinese children with CGD.To determine the spectrum of infections in patients with X-linked CGD, with an emphasis on mycobacterial infections, and to review all CYBB gene mutations identified in our center.From 1988 to 2005, 17 Chinese male children were diagnosed to have X-linked CGD. Fifteen mutations were identified, including 3 splice site defects (IVS1-1GC, 266GA, IVS3-1GA), 5 missense mutations (591TC, 627TA, 949TA, 1039TA, 1512GC), 3 nonsense mutations (882CT, 1451CA, 1569GT), 1 insertion (756_757insA), and 3 deletions (660_662delTTC, 727delT, 1341delT). Eight of these were novel mutations. Recurrent pneumonia, lymphadenitis, and bacterial skin abscess were the commonest types of infection. Seven patients had tuberculosis (TB). Seven patients had prolonged scarring or abscess formation at the Calmette-Guérin bacillus (BCG) injection site, and 1 had disseminated BCG infection. Three patients had pulmonary aspergillosis. Four patients underwent hemopoietic stem cell transplantation, but 2 died of complications.Patients with CGD are susceptible to TB and BCG complications. Our observation suggests that oxidative burst is probably important in host defense against mycobacterial infections. Because interferon-gamma is the key cytokine involved in mycobacterial immunity, there may be a stronger indication for its use in CGD patients living in areas endemic for TB.

International audience; X linked agammaglobulinemia (XLA) is the first described primary immunodeficiency and the most common form of agammaglobulinemia. It is characterized by susceptibility to recurrent infections, profound decrease of all immunoglobulin isotypes and very low level of B lymphocytes in peripheral blood. The disorder is caused by mutations in the Bruton's Tyrosine Kinase (BTK). Nine male patients suspected to have XLA from nine unrelated families were enrolled in this study. We performed sequencing of the BTK gene in all nine patients, and in the patients' relatives when possible. The XLA diagnosis was confirmed for six patients with six different mutations; we identified a novel mutation (c.1522GN A) and five known mutations. One third of nine unrelated patients do not have mutations in BTK and thus likely suffer from autosomal recessive agammaglobulinemia in the setting of consanguinity. Our results support that the autosomal recessive agammaglobulinemia can be more common in Algeria.

Our study aimed at investigating the distribution, persistence and interchange of viral strains among peripheral blood mononuclear cells (PBMC), plasma and saliva of primary Epstein-Barr virus (EBV) infection subjects. Twelve infectious mononucleosis (IM) patients and eight asymptomatic individuals (AS) with primary EBV infection were followed longitudinally at several time points for one year from the time of diagnosis, when blood and saliva samples were collected and separated into PBMC, plasma and saliva, representing circulating B cell, plasma and epithelial cell compartments, respectively. To survey the viral strains, genotyping assays for the natural polymorphisms in two latent EBV genes, EBNA2 and LMP1, were performed and consisted of real-time PCR on EBNA2 to distinguish type 1 and 2 viruses, fluorescent-based 30-bp typing assay on LMP1 to distinguish deletion and wild type LMP1, and fluorescent-based heteroduplex tracking assays on both EBNA2 and LMP1 to distinguish defined polymorphic variants. No discernible differences were observed between IM patients and AS. Multiple viral strains were acquired early at the start of infection. Stable persistence of dominant EBV strains in the same tissue compartment was observed throughout the longitudinal samples. LMP1-defined strains, China 1, China 2 and Mediterranean+, were the most common strains observed. EBNA2-defined groups 1 and 3e predominated the PBMC and saliva compartments. Concordance of EBNA2 and LMP1 strains between PBMC and saliva suggested ready interchange of viruses between circulating B cell and epithelial cell pools, whilst discordance of viral strains observed between plasma and PBMC/saliva indicated presence of viral pools in other undetermined tissue compartments. Taken together, the results indicated that the distribution, persistence and interchange of viral strains among the tissue compartments are more complex than those proposed by the current model of EBV life cycle., published_or_final_version

In our previous study, a rat homolog of human nonmuscle myosin heavy chain-B (nmMHC-B) was identified by mRNA differential display comparing of transformed against nontransformed Rat 6 cells overexpressing mutant p53val135 gene. The nmMHC-B was found to be expressed in normal Rat 6 embryo fibroblast cell line, but markedly suppressed in the mutant p53val135-transformed Rat 6 cells. To examine the possible involvement of nmMHC-B in cell transformation, we first cloned and sequenced the full length cDNA of rat nmMHC-B, which was then cloned into an ecdysone-expression vector. The resulting construct was introduced into the T2 cell line, a mutant p53val135-transformed Rat 6 cells lacking the expression of the endogenous nmMHC-B. The clonal transfectants, expressing muristerone A-induced nmMHC-B, displayed a slightly flatter morphology and reached to a lower saturation density compared to the parental transformed cells. Reconstitution of actin filamental bundles was also clearly seen in cells overexpressing the nmMHC-B. In soft agar assays, nmMHC-B transfectants formed fewer and substantially smaller colonies than the parental cells in response to muristerone A induction. Moreover, it was strikingly effective in suppressing the tumorigenicity of the T2 cells when tested in nude mice. Thus, the nmMHC-B, known as a component of the cytoskeletal network, may act as a tumor suppressor gene. Our current finding may reveal a novel role of nmMHC-B in regulating cell growth and cell signaling in nonmuscle cells. Oncogene (2001) 20, 58 - 68.

Background. Penicillium marneffei infection is indigenous to Southeast Asia. Majority of penicilliosis occurs in patients with AIDS, and less commonly with secondary immunodeficiencies. Penicilliosis is rare in otherwise healthy persons, but information on their immunological status is often lacking. Methods. From 1996 to 2009, we diagnosed penicilliosis in 5 children. Their clinical features, immunological findings, and genetic studies were analyzed. A systematic review of the English and Chinese literature was performed. Case reports/series on patients ,18 years with penicilliosis were included, and patients stated to be human immunodeficiency virus (HIV)–positive excluded. Results. All of our 5 patients were HIV negative. Presentations included fungemia (n 5 2), multifocal lymphadenopathy (n 5 2), and necrotizing pneumonia (n 5 1). Four patients had recurrent mucocutaneous candidiasis. Hyperimmunoglobin E syndrome was diagnosed in 1 patient, while another had functional defect in interleukin-12/interferon-c axis. Three patients were lymphopenic with low natural killer cell counts, but a specific immune defect was not identified. Systematic review of 509 reports on human penicilliosis identified 32 patients aged 3 months to 16 years with no known HIV infection. Twenty-four patients (75%) had disseminated disease, and 55% died of penicilliosis. Eight patients had primary immunodeficiencies or blood disorders, while 4 others had abnormal immune functions. Immune evaluations of the remaining patients were unstated. Conclusion. Penicilliosis is a severe disease causing high mortality in children. As an AIDS-defining illness, penicilliosis should be regarded as an indicator for underlying immunodeficiency in HIV-negative individuals. Immunological investigations should be performed, especially in those with recurrent infections. Multicentered collaborative studies are needed to collect information on long-term prognosis and define immune defects underlying penicilliosis. Penicillium marneffei is a pathogenic fungus indigenous to Southeast Asia. Before the human immunodeficiency virus (HIV) epidemic, P. marneffei was an extremely rare pathogen in humans. Early cases of natural infections in human were first reported in Thailand and the southern part of China, particularly Guangxi

WOS: 000283840300002, PubMed ID: 21057261, Interleukin-12 receptor beta 1 (IL-12R beta 1) deficiency is the most common form of Mendelian susceptibility to mycobacterial disease (MSMD). We undertook an international survey of 141 patients from 102 kindreds in 30 countries. Among 102 probands, the first infection occurred at a mean age of 2.4 years. In 78 patients, this infection was caused by Bacille Calmette-Guerin (BCG; n = 65), environmental mycobacteria (EM; also known as atypical or nontuberculous mycobacteria) (n = 9) or Mycobacterium tuberculosis (n = 4). Twenty-two of the remaining 24 probands initially presented with nontyphoidal, extraintestinal salmonellosis. Twenty of the 29 genetically affected sibs displayed clinical signs (69%); however 8 remained asymptomatic (27%). Nine nongenotyped sibs with symptoms died. Recurrent BCG infection was diagnosed in 15 cases, recurrent EM in 3 cases, recurrent salmonellosis in 22 patients. Ninety of the 132 symptomatic patients had infections with a single microorganism. Multiple infections were diagnosed in 40 cases, with combined mycobacteriosis and salmonellosis in 36 individuals. BCG disease strongly protected against subsequent EM disease (p = 0.00008). Various other infectious diseases occurred, albeit each rarely, yet candidiasis was reported in 33 of the patients (23%). Ninety-nine patients (70%) survived, with a mean age at last follow-up visit of 12.7 years +/- 9.8 years (range, 0.5-46.4 yr). IL-12R beta 1 deficiency is characterized by childhood-onset mycobacteriosis and salmonellosis, rare recurrences of mycobacterial disease, and more frequent recurrence of salmonellosis. The condition has higher clinical penetrance, broader susceptibility to infections, and less favorable outcome than previously thought., Rockefeller University Center for Clinical and Translational Science [5UL1RR024143-03]; Rockefeller University; ANRFrench National Research Agency (ANR); PHRC; EUEuropean Union (EU) [LHSP-CT-2005-018736]; BNP Paribas Foundation; Dana Foundation; March of DimesMarch of Dimes; Fondation pour la Recherche MedicaleFondation pour la Recherche Medicale; Howard Hughes Medical InstituteHoward Hughes Medical Institute, The St. Giles Laboratory of Human Genetics of Infectious Diseases is supported by grants from The Rockefeller University Center for Clinical and Translational Science grant number 5UL1RR024143-03, and The Rockefeller University, ANR, PHRC, EU (LHSP-CT-2005-018736), BNP Paribas Foundation, the Dana Foundation, and the March of Dimes. LDB is supported by the Fondation pour la Recherche Medicale as part of the PhD program of Pierre et Marie Curie University (Paris, France). JLC was an International Scholar of the Howard Hughes Medical Institute.

X-linked agammagobulinemia (XLA) is a primary immunodeficiency disorder caused by Bruton's tyrosine kinase (Btk) gene mutation. Recent studies suggested genotype-phenotype correlation in XLA, but a definitive association remains controversial. We examined the relationship between specific Btk gene mutations and severity of clinical presentation in 62 patients with XLA. Disease severity was assessed by the age of disease onset and the presence of severe infections, while mutations were classified into severe and mild based on structural and functional consequence by bioinformatics analysis. Fifty-six Btk mutations were identified in 62 patients from 57 kindreds. Variation in phenotypes was observed, and there was a tendency of association between genotype and age of disease onset as well as occurrence of severe infections. A critical analysis of the circumstances upon presentation also revealed that under-recognition of recurrent infections and relevant family history are important hurdles to timely diagnosis of XLA.

Jaundice is common in ethnic Chinese infants, but to our knowledge Crigler-Najjar syndrome (CN syndrome) type I has never been reported in China. A Chinese girl with severe jaundice was recently diagnosed to have CN syndrome type I by analyzing the bilirubin-uridinediphospho (UDP)-glucuronosyltransferase gene (UGT1A1). The patient was homozygous for a nonsense mutation that replaced glutamine (CAG, amino acid 239) with stop codon (TAG) at nucleotide number 715 (715C-->T) in exon 1. No mutation was found in exons 2-5. Her parents were heterozygous for the same mutant. The patient had an average bilirubin level of 300-500 mumol/L and a peak of 701 mumol/L. Daily phototherapy for 15 h was required to keep the bilirubin levels within 280-320 mumol/L. The unconjugated hyperbilirubinaemia apparently resulted from homozygous nonsense mutation of UGT1A1, which could completely abolish the UGT activity towards bilirubin (hepatic glucuronidation) and result in CN syndrome type I. Identification of the genetic defect is very useful for gene therapy, especially for DNA/RNA chimera therapy, and can be used as an antenatal screening test to identify the affected offsprings.

The Wiskott-Aldrich Syndrome (WAS) is an X-linked recessive immunodeficiency caused by mutation in the gene encoding WAS protein (WASP). The disease is characterized by eczema, thrombocytopenia and severe immunodecificency and is associated with extensive clinical heterogeneity. Mutation studies indicated that the mutated genotypes are also highly variable. In this study, we performed PCR-direct sequencing analysis of the WAS gene in six unrelated Chinese families. Five novel mutations identified, included two nonsense mutations (506C→T, 1388G→T), a small insertion (685-686insCGCA) and two single-base deletions (384delT, 984delC). All of the mutations are predicted to lead to premature translational termination of WASP. © 2002 Wiley-Liss, Inc.

Frizzled-related protein (Frp) is a new family of secreted proteins involved in tumorigenesis and Wnt-signaling pathway. Previous study has shown that rat Frp (rFrp) gene was found to be differentially expressed in Rat 6 fibroblast cell line overexpressing p53val135 (R6#13-8). The rFrp gene was otherwise silent in normal parental Rat 6 cells. To elucidate the molecular basis of the transcriptional activation of rFrp, we have isolated and analyzed a 2-kilobase pair promoter region of the rFrp gene. Mapping of transcription initiation sites of rFrp showed the existence of multiple initiation sites. Transfection studies of serial deletion constructs in both Rat 6 and CHOK1 cell lines revealed that the region from −202 to −144 contains cis-acting elements essential for the efficient transcription of rFrp. This work provides a transcriptional regulation basis for Frp and gives insight into its implication in tumorigenesis.

In mammals, growth hormone-releasing hormone (GHRH), acting via the GHRH receptor, plays an important role in the regulation of growth hormone (GH) synthesis and secretion as well as the proliferation and differentiation of somatotropes in the pituitary. In fishes, information concerning the functional role of the characterized GHRHs is limited. For that reason, a putative goldfish GHRH receptor cDNA was characterized in this study. The receptor cDNA is 2,243 bp in length, encoding a 438-amino-acid-long polypeptide with 7 putative transmembrane-spanning regions, which is a characteristic of G-protein-coupled receptors. The receptor, when expressed in COS-7 cells, showed minimal responses (2-fold cAMP responses) when stimulated with 100 nM of human GHRH, pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP). However, this receptor was found to be specific for a carp GHRH-like peptide isolated from the brain of common carp (Cyprinus carpio); there was a significant and dose-dependent increase in intracellular cAMP (a maximum response of 22-fold increase with an EC50 of 0.1 nM) when the transfected cells were stimulated with this peptide. As a preliminary study to investigate the functional role of this receptor, the tissue distribution of the mRNA was analyzed by reverse-transcription-polymerase chain reaction. The receptor mRNA was found to be present in the brain, pituitary, gut, gill, heart, liver, skeletal muscle, spleen, ovary and testis. Together with a goldfish PACAP type 1 receptor and a VIP1 receptor recently isolated in our laboratory, characterization of this putative GHRH receptor provides the molecular basis for the future understanding of the neuroendocrine control of growth and reproduction by these neuropeptides in goldfish as well as other teleosts.