Nine-year experience in Gaucher disease diagnosis at the Spanish reference center Fundación Jiménez Díaz.

BACKGROUND: Fundación Jiménez Díaz (FJD) is a reference center for genetic diagnosis of Gaucher disease (GD) in Spain. Genetic analyses of acid ß-glucosidase (GBA) gene using different techniques were performed to search for new mutations, in addition to those previously and most frequently found in the Spanish population. Additionally, the study of the chitotriosidase (CHIT1) gene was used to assess the inflammatory status of patients in the follow-up of enzyme replacement therapy (ERT). We present the genetic data gathered during the last nine years at FJD. METHODS: Blood samples from patients with suspected GD were collected for enzymatic and genetic analyses. The genetic analysis was performed on DNA from 124 unrelated suspected cases and 57 relatives from 2007 to 2015, starting with a mutational screening kit, followed by Sanger sequencing of the entire gene and other techniques to look for deletions. CHIT1 was also studied to assess the reliability of this biomarker. RESULTS: In 46 out of 93 GD patients (49.5%) the two mutant alleles were found. We detected 21 different mutations. The most common mutation was N370S (c.126A > G; p.Asp409Ser current nomenclature) (in 50.5% of patients), followed by L444P (c.1448T > C; p.Leu483Pro current nomenclature) (in 24.7%). The most common heterozygous compound genotype observed (18.3%) was c.1226A > G/c.1448T > C (N370S/L444P). Two novel mutations were found (del. Ex.4-11 and c.1296G > T; pW432C), as well as p.S146L, only once previously reported. Two patients showed the homozygous state for the duplication of CHIT1. CONCLUSION: N370S and L444P are the most common mutations and other mutations associated to Parkinson's disease have been observed. This should be taken into account in the genetic counseling of GD patients.

Urinary excretion of platelet-activating factor in human and experimental nephrosis.

BACKGROUND: Platelet-activating factor (PAF) is a phospholipid that has been implicated in the pathogenesis of glomerulonephritis and can be synthesized by glomerular cells in response to different stimuli. PAF increases glomerular permeability to proteins and urinary PAF has been determined to be of renal origin. In order to assess whether urinary PAF can be found augmented in situations of glomerular damage without glomerular leukocyte infiltration, urinary PAF was quantified in human and experimental nephrosis. METHODS: Urinary PAF was quantified by platelet bioassay and glomerular PAF by incorporation of 3H-acetate into PAF. PAF was characterized by its behaviour on thin-layer chromatography and high performance liquid chromatography and the blockade of its bioactivity by receptor antagonists. RESULTS: Urinary PAF excretion was significantly higher in patients with active idiopathic nephrotic syndrome than in controls (5.8+/-1.5 versus 1.7+/-0.75 mg/24 h; P<0.05) and patients in remission (1.63+/-0.75 ng/24 h; P<0.02). In rats with nephrosis induced by puromycin aminonucleoside there was an early increase in urinary PAF excretion (138+/-19 versus 49+/-22 pg/24 h in controls; P<0.035) that coincided with the augmented glomerular PAF synthesis (67+/-3.4 versus 36+/-1.2 DPM/mg protein in controls; P<0.003). CONCLUSIONS: These results suggest that the synthesis of PAF in the kidney may be involved in the pathogenesis of the proteinuria in idiopathic nephrotic syndrome and that urinary PAF excretion may be a good marker of disease activity.

Long-term effect of nonsteroidal anti-inflammatory drugs on the production of cytokines and other inflammatory mediators by blood cells of patients with osteoarthritis.

Most of the previous studies dealing with the effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on the synthesis of inflammatory mediators involved in joint damage have been done in cells cultured in vitro or in blood cells from patients treated for short periods of time. In this work we have evaluated the long-term effect of aceclofenac, a new NSAID, and diclofenac on the production of a series of inflammatory mediators by blood cells from 30 patients with severe knee osteoarthritis. Both aceclofenac and diclofenac significantly inhibited prostaglandin E2 (PGE2) synthesis by blood mononuclear and polymorphonuclear cells after 180 days of treatment. However, no clear effect was noted on leukotriene B4 (LTB4) and platelet activating factor (PAF) production. The generation of O-2 by polymorphonuclear cells, stimulated with FMLP, was decreased after 15 days of treatment with both drugs, but reached normal values after 180 days. Interleukin-1 beta (IL-1 beta) production decreased significantly at 180 days with both drugs in the group of high producer patients. In a few (n = 3) patients with high basal mononuclear cell tumor necrosis factor alpha (TNF alpha) production, this also decreased on treatment for 180 days with the NSAIDs. In the remaining low TNF alpha-producing patients, TNF alpha production tended to increase. Interleukin-6 (IL-6) synthesis was not affected by aceclofenac while it was diminished by diclofenac. The decrease in IL-6 in all treated patients was significantly correlated with a worsening of the clinical condition. On the whole, these data could afford a pathogenetic basis for the long-term employment of these drugs in patients with inflammatory conditions.

Presence of circulating antibodies against brush border antigens (Fx1A) in a patient with membranous nephropathy and bilateral pyeloureteral stenosis. Comparison with idiopathic membranous nephropathy.

In a patient with membranous nephropathy and bilateral pyeloureteral stenosis with hydronephrosis, we examined the possibility that an increase in the intratubular pressure could facilitate the passage of the Fx1A antigens to the circulation. Elevated serum anti-Fx1A antibodies were detected in this particular patient by ELISA on three occasions during the disease follow-up, even though he was in clinical remission. These antibodies reacted in vitro with the tubular brush border of a normal human kidney. The anti-Fx1A antibodies isolated from the patient's sera by affinity chromatography competed with the rabbit anti-Fx1A antisera binding to plates coated with human Fx1A antigen. In immunoblotting studies the isolated specific IgG antibodies from that patient reacted with a 180 kDa antigen of the human Fx1A and with less intensity with 75 kDa and 50-55 kDa polypeptides. In none of 12 patients with idiopathic membranous nephropathy could the circulating anti-Fx1A antibodies be demonstrated. On the whole, this particular case suggests that on some occasions increased intratubular pressure could cause the release of Fx1A antigens, facilitating an autologous immunocomplex nephritis. These antigens, by contrast, do not seem to play any role in most cases of membranous nephropathy in man.

IgA immune aggregates stimulate platelet-activating factor and superoxide anion production by human neutrophils. A comparison with IgG aggregates.

We have examined the possibility that human polymorphonuclear cells exposed to IgA immune complexes can mediate the production of platelet-activating factor (PAF) and oxygen radicals. We found that human IgA and IgG immune aggregates stimulated, to a similar extent, PAF and O2- production by human polymorphonuclear cells (PMN) in a concentration and time dependent manner. The PAF, that was largely associated with cells, was shown to be identical to synthetic PAF, as determined by physicochemical, chromatographic and enzymatic assay. Furthermore, de novo synthesis of PAF by PMN was shown to occur by incorporation of radioactive precursors, such as [3H]acetate. The addition of normal human serum to PMN incubated with IgG aggregates resulted in a significant amount of PAF formation which was not observed with IgA aggregates. By contrast, no change was seen in PMN O2- with either aggregates. The preincubation of PMN with cytochalasin B, an inhibitor of phagocytosis, did not affect PAF and O2- production by both aggregates. The results suggest that the interaction of PMN with the IgA complexes in blood vessel walls of different tissues can result in the release of lipid mediators, such as PAF and oxygen radicals that could contribute to the inflammatory response.

Expression and synthesis of cross-reactive idiotypic antibodies by peripheral blood lymphocytes and their suppression by anti-idiotypes in patients with IgA nephropathy.

We have recently described that patients with IgA nephropathy present high serum levels of anti-BSA idiotypic antibodies that were well correlated with the existence of hematuria. Furthermore, these Id were found in circulating and renal deposited immune complexes. In the present work, we examined the expression of surface idiotypic determinants on PBL by flow cytometry and their in vitro production, using as reagent anti-idiotypic antibodies previously well characterized. The presence of cross-reactive Id-bearing cells was observed in 5 out of 6 patients studied, with frequencies ranging from 3 to 12% of lymphocytes. After 7 days of culture, the spontaneous synthesis of idiotypic antibodies by PBL was found elevated in 6 out of 13 (46%) patients. A major Id cell expression and production was noted in patients with active disease as defined by hematuria. The preincubation of PBL with 20 and 50 micrograms of anti-idiotypic antibodies/2 x 10(6) cells for 3 days induced a significant inhibition of cross-reactive Id production in a dose-dependent fashion, with a degree of suppression between 12 and 50% in five out of six patients studied. In the above assays, as negative controls, we used the anti-Id antibodies previously adsorbed on an Id-Sepharose column. On the whole, these results suggest that patients with IgA nephropathy present dysfunctions in the Id-Anti-Id network that could play an important role in the pathogenesis of this disease.

Clinical significance of polymeric and monomeric IgA complexes in patients with IgA nephropathy.

IgA nephropathy is currently considered an immune complex (IC) disease. However, though several groups have demonstrated the presence of IgA-IC in the sera of patients by various techniques, a correlation with clinical activity of the nephropathy has not always been found. Since these assays detect (simultaneously) polymeric and monomeric IgA-IC, the pathogenicity of these two classes of complexes could not be established. In this work, we have studied in 66 patients with IgA nephropathy the existence and significance of such IC, by means of a technique described in our laboratory, based on the specific binding of secretory component for polymeric IgA. Furthermore, IgG-ICs were also determined by the standard Raji cell assay in ELISA. The prevalence of these complexes was as follows: Multimeric (polymeric and monomeric) IgA-ICs were detected in 55% of 66 patients studied, polymeric IgA-ICs in 30%, monomeric IgA-ICs in 39%, and IgG-ICs in 46%. The intermittency of all these complexes was clearly noted in sequential examinations. A significant correlation (P less than .025) with hematuria was only found with polymeric IgA-IC, but not with multimeric IgA-IC, monomeric IgA-IC, or IgG-IC. Polymeric IgA-ICs were more frequently observed at the initial phases of the disease. Analytical ultracentrifugation showed that polymeric IgA-IC was of larger size than monomeric IgA-IC. The major pathogenicity of polymeric IgA-IC is in agreement with the finding of this immunoglobulin at the mesangial level in patients and animals with IgA nephropathy.

[Mitral prolapse syndrome and hypothyroidism (author's transl)]. / Síndrome del prolapso mitral en hipotiroidismo.

Authors observe a case of hypothyroidism associated with advanced development in weight and height and mitral valve prolapse syndrome (MPS), with rupture of cordae tendinae. The sudden death of a sister promotes the hypothesis of hypothyroidism with a complication of MPS not diagnosed during life and in turn gives rise to comments about this rare morbid association. Hereditary implications, evolutionary risks and necessary reserves in the classification of infantile cardiac murmurs apparently not significant are commented.