Influence of initial clinical suspicion on the diagnostic yield of laboratory enzymatic testing in lysosomal storage disorders. Experience from a multispecialty hospital.

Lysosomal storage disorders (LSD) are a group of inherited metabolic diseases mainly caused by a deficiency of lysosomal hydrolases, resulting in a gradual accumulation of non-degraded substrates in different tissues causing the characteristic clinical manifestations of such disorders. Confirmatory tests of suspected LSD individuals include enzymatic and genetic testing. A well-oriented clinical suspicion can improve the cost-effectiveness of confirmatory tests and reduce the time expended to achieve the diagnosis. Thus, this work aims to retrospectively study the influence of clinical orientation on the diagnostic yield of enzymatic tests in LSD by retrieving clinical, biochemical, and genetic data obtained from subjects with suspicion of LSD. Our results suggest that the clinical manifestations at the time of diagnosis and the initial clinical suspicion can have a great impact on the diagnostic yield of enzymatic tests, and that clinical orientation performed in specialized clinical departments can contribute to improve it. In addition, the analysis of enzymatic tests as the first step in the diagnostic algorithm can correctly guide subsequent confirmatory genetic tests, in turn increasing their diagnostic yield. In summary, our results suggest that initial clinical suspicion plays a crucial role on the diagnostic yield of confirmatory enzymatic tests in LSD.

Lysosomal lipid alterations caused by glucocerebrosidase deficiency promote lysosomal dysfunction, chaperone-mediated-autophagy deficiency, and alpha-synuclein pathology.

Mutations in the GBA gene that encodes the lysosomal enzyme ß-glucocerebrosidase (GCase) are a major genetic risk factor for Parkinson's disease (PD). In this study, we generated a set of differentiated and stable human dopaminergic cell lines that express the two most prevalent GBA mutations as well as GBA knockout cell lines as a in vitro disease modeling system to study the relationship between mutant GBA and the abnormal accumulation of α-synuclein. We performed a deep analysis of the consequences triggered by the presence of mutant GBA protein and the loss of GCase activity in different cellular compartments, focusing primarily on the lysosomal compartment, and analyzed in detail the lysosomal activity, composition, and integrity. The loss of GCase activity generates extensive lysosomal dysfunction, promoting the loss of activity of other lysosomal enzymes, affecting lysosomal membrane stability, promoting intralysosomal pH changes, and favoring the intralysosomal accumulation of sphingolipids and cholesterol. These local events, occurring only at a subcellular level, lead to an impairment of autophagy pathways, particularly chaperone-mediated autophagy, the main α-synuclein degradative pathway. The findings of this study highlighted the role of lysosomal function and lipid metabolism in PD and allowed us to describe a molecular mechanism to understand how mutations in GBA can contribute to an abnormal accumulation of different α-synuclein neurotoxic species in PD pathology.

Biomarkers in Fabry Disease. Implications for Clinical Diagnosis and Follow-up.

Fabry disease (FD) is a lysosomal storage disorder caused by deficient alpha-galactosidase A activity in the lysosome due to mutations in the GLA gene, resulting in gradual accumulation of globotriaosylceramide and other derivatives in different tissues. Substrate accumulation promotes different pathogenic mechanisms in which several mediators could be implicated, inducing multiorgan lesions, mainly in the kidney, heart and nervous system, resulting in clinical manifestations of the disease. Enzyme replacement therapy was shown to delay disease progression, mainly if initiated early. However, a diagnosis in the early stages represents a clinical challenge, especially in patients with a non-classic phenotype, which prompts the search for biomarkers that help detect and predict the evolution of the disease. We have reviewed the mediators involved in different pathogenic mechanisms that were studied as potential biomarkers and can be easily incorporated into clinical practice. Some accumulation biomarkers seem to be useful to detect non-classic forms of the disease and could even improve diagnosis of female patients. The combination of such biomarkers with some response biomarkers, may be useful for early detection of organ injury. The incorporation of some biomarkers into clinical practice may increase the capacity of detection compared to that currently obtained with the established diagnostic markers and provide more information on the progression and prognosis of the disease.

[50 years of the Neonatal Screening Program in Catalonia.] / 50 años del Programa de Cribado Neonatal en Cataluña.

The Catalonian Newborn Screening Program (CNSP) began in 1969, in Barcelona. It was promoted by Dr. Juan Sabater Tobella and supported by Barcelona Provincial Council and Juan March Foundation. That is how the Institute of Clinical Biochemistry was born, whose aims were diagnosis, research and teaching, along with the spirit of contributing to the prevention of mental retardation. The CNSP began with the detection of phenylketonuria (PKU), and, in 1982, the Program was expanded with the inclusion of congenital hypothyroidism detection. Towards 1990, the Program covered almost 100% of all newborns (NB) in Catalonia. In 1999, the CNSP was expanded with the incorporation of cystic fibrosis. It took fourteen years, until 2013, to make the largest expansion so far, with the incorporation of 19 metabolic diseases to the screening panel. The detection of sickle cell disease began in 2015 and in 2017 the detection of severe combined immunodeficiency was included. Currently, the CNSP includes 24 diseases in its main panel. Since 1969, 2,787,807 NBs have been screened, of whom 1,724 have been diagnosed with any of these diseases, and 252 of other disorders by differential diagnosis with those included in the main panel. The global prevalence is 1: 1,617 NBs affected by any of the diseases included in the CNSP and 1: 1,140 NBs if incidental findings diagnosed through the CNSP are included.

El Programa de Cribado Neonatal de Cataluña (PCNC) se inició en el año 1969, en Barcelona, impulsado por el Dr. Juan Sabater Tobella y apoyado por la Diputación de Barcelona y la Fundación Juan March. Así nació el Instituto de Bioquímica Clínica para acometer funciones asistenciales, de investigación y docencia, con el espíritu de contribuir a la prevención del retraso mental. El PCNC se inició con la detección de la fenilcetonuria (PKU) y en el año 1982 se amplió con la detección del hipotiroidismo congénito. Hacia el año 1990 la cobertura territorial llegó casi al 100% de todos los recién nacidos en Cataluña. En 1999 se amplió el PCNC con la incorporación de la fibrosis quística y tras catorce años, en 2013, se realizó la ampliación más numerosa hasta ahora, con la incorporación de la detección de 19 enfermedades metabólicas hereditarias. En el año 2015 comenzó la detección de la enfermedad de células falciformes y en el 2017 la detección de la inmunodeficiencia combinada grave. Actualmente, el PCNC incluye la detección de 24 enfermedades. Desde su inicio en el año 1969, se han cribado 2.787.807 recién nacidos, de los cuales 1.724 han sido diagnosticados de alguna de las 24 enfermedades que componen nuestro panel principal y 252 por diagnóstico diferencial de las primeras. En total la prevalencia global es de 1:1.617 RN afectos de alguna de las enfermedades incluidas en el PCNC y de 1:1.140 RN si se incluyen los hallazgos incidentales encontrados.

Identification and characterization of the novel m.8305C>T MTTK and m.4440G>A MTTM gene mutations causing mitochondrial myopathies.

We report on two novel mtDNA mutations in patients affected with mitochondrial myopathy. The first patient, a 44-year-old woman, had bilateral eyelid ptosis and the m.8305C>T mutation in the MTTK gene. The second patient, a 56-year-old man, had four-limb muscle weakness and the MTTM gene m.4440G>A mutation. Muscle biopsies in both patients showed ragged red fibers and numerous COX-negative fibers as well as a combined defect of complex I, III and IV activities. The two mutations were heteroplasmic and detected only in muscle tissue, with a higher mutation load in COX-negative fibers. Additionally, both mutations occurred in highly conserved mt-tRNA sites, and were not found by an in silico search in 30,589 human mtDNA sequences. Our report further expands the mutational and phenotypic spectrum of diseases associated with mutations in mitochondrial tRNA genes and reinforces the notion that mutations in mitochondrial tRNAs represent hot spots for mitochondrial myopathies in adults.

Value of urinary levels of interleukin-6, epidermal growth factor, monocyte chemoattractant protein type1 and transforming growth factor ß1 in predicting the extent of fibrosis lesions in kidney biopsies of patients with IgA nephropathy. / Valor de los niveles urinarios de interleucina 6, factor de crecimiento epidérmico, proteína quimioatractante de monocitos de tipo 1 y factor de crecimiento transformante ß1 para la predicción de la extensión de las lesiones de fibrosis en biopsias de enfermos con nefropatía IgA.

OBJECTIVE: To analyse the associations between urinary levels of IL-6 EGF, MCP-1 and TGFß1 and clinical, biochemical and histopathological characteristics in patients with primary IgA nephropathy and their ability to predict the extent of lesions of glomerular and/or interstitial sclerosis. PATIENTS AND METHODS: A total of 58 patients with IgA nephropathy were studied. We determined the urine levels of IL-6, EGF, MCP-1, and TGFß1 at the time of diagnosis. The extent of glomerular and interstitial fibrosis was analyzed by quantitative morphometry and kidney biopsies were classified according to the Oxford criteria. We analysed the ability of these molecules to predict the extent of glomerular and interstitial fibrosis lesions. RESULTS: IL-6, TGFß1 and MCP-1 were associated with focal glomerulosclerosis and interstitial fibrosis extension but not with the presence of mesangial, extracapillary or endocapillary proliferation. EGF showed a negative association with interstitial fibrosis. By categorising patients according to the Oxford classification, patients with T1 and T2 scores had significantly higher levels of IL-6, MCP-1, TGF-ß1 and significantly lower levels of EGF than patients with T0 scores. By multiple regression and logistic regression analyses, the levels of MCP-1, IL-6 and EGF were independent predictors of the fibrosis surface, after adjusting for age and eGFR. CONCLUSION: The urinary concentration of IL-6, EGF and MCP-1 provides additional information that significantly improves the estimation of the surface of interstitial fibrosis in patients with IgA nephropathy.

Study of the variables associated with local complement activation in IgA nephropathy. / Estudio de las variables asociadas a la activación local del complemento en la nefropatía IgA idiopática.

OBJECTIVES: 1. To identify the variables that are associated with urinary levels of properdin, MBL, C4d, and C5b-9 in patients with idiopathic IgA nephropathy. 2. To analyse whether urinary levels of MBL and/or C4d are useful for identifying the presence of mesangial deposits of C4d/MBL. PATIENTS AND METHOD: A total of 96 patients with IgA nephropathy were studied. Demographic, clinical and biochemical variables were recorded at the time of diagnosis. Renal lesions were quantified using the Oxford classification. Immunohistochemical staining for MBL, MASP-2, properdin, C4d, and C5b-9 was performed in kidney biopsies, and in urine, the levels of properdin, MBL, C4d and C5b-9 were determined. RESULTS: In multivariate analysis, the independent predictors of C4d and MBL levels in urine were the mesangial deposits of each protein and, to a lesser extent, the urinary protein excretion. The independent predictors of urinary levels of C5b-9 were MBL properdin and proteinuria. Urinary excretion of C4d had a sensitivity of 90% (95% CI: 58,7 to 99) and a specificity of 73% (95% CI: 54-87) for detecting mesangial C4d deposits, and the level of MBL had a sensitivity of 83.9% (95% CI: 62-95) and a specificity of 81.6% (95% CI: 65-92) for identifying mesangial deposits of MBL. CONCLUSION: The main predictor of urinary concentration of C4d and MBL was the presence of their respective mesangial deposits. Urine MBL may contribute to complement activation in the tubular luz through the lectin pathway. Urinary levels of MBL and C4d could be sensitive and specific biomarkers for the identification of patients with mesangial deposits of MBL and C4d.

Clinical features, course and prognosis of idiopathic membranous nephropathy depending on the presence of antibodies against M-type phospholipase A2 receptor.

UNLABELLED: In membranous nephropathy, the presence of antibodies against M-type phospholipase A2 receptor is considered highly specific for idiopathic forms. However, no specific association to a particular clinical profile has been found for such antibodies. OBJECTIVE: To assess potential differences in initial clinical profile, course and prognosis of idiopathic membranous nephropathy depending on the presence of anti-PLA2R antibodies. METHODS: Eighty-five patients with idiopathic membranous nephropathy were included (55 anti-PLA2R-positive and 30 anti-PLA2R-negative). Clinical, biochemical and pathological variables were recorded at the time of diagnosis. Frequency of spontaneous remission, incidence of response to first-line therapy, frequency and number of recurrences, survival of renal function free from renal replacement therapy, survival of renal function free from chronic renal insufficiency and frequency of occurrence of malignant, infectious or autoimmune diseases during follow-up were recorded. RESULTS: At the time of diagnosis, anti-PLA2R-negative patients were significantly older and had a higher frequency of spontaneous remission. No differences were noted in the response to first-line treatment, frequency and number of recurrences, survival of renal function free from renal replacement therapy, or survival of renal function free from chronic renal insufficiency. CONCLUSIONS: Anti-PLA2R-negative patients with idiopathic membranous nephropathy were older and experienced spontaneous remission more often than anti-PLA2R-positive patients. No differences in terms of treatment response, recurrences, and final prognosis were observed between both groups of patients.

Evolution of antibody titre against the M-type phospholipase A2 receptor and clinical response in idiopathic membranous nephropathy patients treated with tacrolimus.

INTRODUCTION AND OBJECTIVES: The level of circulating antibodies against M-type phospolipase A2 receptor has been reported as having a significant correlation with clinical activity in idiopathic membranous nephropathy. However, the usefulness of monitoring antibody titre as a predictor of clinical response following the onset of treatment has not been formally analysed. The predictive value of the evolution of anti-PLA2R antibody titre on the clinical response of idiopathic membranous nephropathy patients treated with tacrolimus is analysed in the following study. PATIENTS AND METHOD: 36 patients with nephrotic syndrome secondary to idiopathic membranous nephropathy with immunosuppressive treatment indication criteria were treated with tacrolimus in monotherapy. The level of anti-PLA2R antibodies was determined before treatment and at 3, 6, 9 and 12 months after the onset of treatment. The study analysed the predictive value of the reduction in antibody titre and the relative and absolute reduction in antibody titre at 3 and 6 months over the period until remission and on the probability of remission at 6, 9 and 12 months. RESULTS: The relative reduction in the anti-PLA2R antibody titre was significantly greater in those patients with remission and it preceded the clinical response. No association was observed between the antibody titre prior to treatment and the mean response time or the response at 12 months. Reduction in antibody titre is significantly associated with the time until signs of remission. Relative reduction in anti-PLA2R antibody titre at 3 months had a high sensitivity and specificity to predict the response at 6 and 9 months, but not at 12 months; however the relative reduction in the antibody titre at 6 months had a high sensitivity and specificity for predicting the response at 12 months. CONCLUSION: In patients with IMN associated with anti-PLA2R antibodies, the monitoring of antibody titre following the onset of treatment is useful for estimating the time period until remission and predicting the probability of remission at 12 months.

Prevalence, diagnostic value and clinical characteristics associated with the presence of circulating levels and renal deposits of antibodies against the M-type phospholipase A2 receptor in idiopathic membranous nephropathy.

UNLABELLED: The M-type phospholipase A2 receptor (PLA2R) has been identified as one of the target antigens of the autoimmune response in idiopathic membranous nephropathy (MN). The prevalence of anti-PLA2R antibodies in patients with idiopathic MN is around 70% but this varies in accordance with geographic region, and until present, anti-PLA2R has not been shown to be associated with any particular clinical profile of the disease. METHODS: We studied 64 adults with nephrotic syndrome who were diagnosed with MN, confirmed by renal biopsy. Forty-seven patients had idiopathic MN and 17 had secondary MN. We determined the presence of circulating anti-PLA2R antibodies by indirect immunofluorescence (IIF) and their titre by ELISA, and we analysed the presence of anti-PLA2R antibody renal deposits by immunohistochemical techniques. We calculated the sensitivity and specificity of the IIF and ELISA techniques for the identification of patients with renal deposits and for the identification of those with idiopathic MN and we tested whether there were differences in the clinical profile of the disease at the time of diagnosis according to the presence or absence of anti-PLA2R antibodies. RESULTS: We did not observe significant differences in the clinical-demographic variables between patients with idiopathic and secondary MN. The prevalence of anti-PLA2R glomerular deposits by IHC was 76.6%. The IIF and ELISA techniques had a similar sensitivity (IIF 94.4% and ELISA 97.2%) and specificity (100%) for the identification of patients with anti-PLA2R renal deposits and the detection of circulating anti-PLA2R antibodies. The determination of anti-PLA2R by IIF identified patients with idiopathic MN with a sensitivity of 72.3% and a specificity of 94.2%. A titre of antibodies >15RU/ml measured by ELISA had a sensitivity of 74.45% and a specificity of 94.2% for the identification of patients with idiopathic MN. Patients with idiopathic MN and anti-PLA2R had significantly higher proteinuria figures (13.25 [P25-P75: 9.05-15.87] compared to 9.43 [P25-P75: 6.30-15] g/day, P:.018). No statistical correlation was observed between the antibody titre measured by ELISA and age, glomerular filtration rate or 24-hour proteinuria or albuminaemia. CONCLUSIONS: The techniques employed to determine anti-PLA2R in patients with MN are highly specific for the diagnosis of idiopathic forms of the glomerular disease. The frequency with which patients with MN and anti-PLA2R were identified is similar to that reported in previous studies. Staining by immunohistochemistry is the most sensitive method for detecting cases of MN associated with the presence of anti-PLA2R antibodies. The IIF and ELISA techniques allow circulating anti-PLA2R antibodies to be detected in most patients with renal deposits, but they may very infrequently have false negative results. The concordance of these tests is high. Patients with idiopathic MN and anti-PLA2R antibody renal deposits have higher proteinuria than patients that are anti-PLA2R negative, but the differences have little clinical importance.

Treatment of idiopathic focal segmental glomerulosclerosis: options in the event of resistance to corticosteroids and calcineurin inhibitors.

Half of patients with nephrotic syndrome caused by primary focal segmental glomerulosclerosis (FSGS) have resistance to treatment with steroids. In the case of corticosteroid resistance,  the best evidence-based option has classically been treatment with calcineurin inhibitors,  although recent studies indicate that mycophenolate may have similar efficacy. In patients with resistance to calcineurin inhibitors,  there is no option that allows the clinical course of the disease to be modified, and this is supported by appropriately designed clinical trials, although observational studies have suggested the potential usefulness of mycophenolate, sirolimus, rituximab, apheresis or high galactose doses as treatment options. In FSGS of idiopathic origin, resistant to steroids and calcineurin inhibitors, before taking the decision whether or not to test other immunosuppressive drugs, it might be appropriate to conduct a systematic analysis that considers: 1) evaluating whether the dose and duration of treatment with steroids and calcineurin inhibitors were suitable, 2) analysing the level of P-glycoprotein expression in lymphocytes, 3) performing a new renal biopsy if there is no electron microscopic study available for the first, 4) in young patients,  considering a genetic study to rule out the presence of the podocin variant pR229Q in combination with heterozygous mutations in NPHS2,  and 5) evaluating the seriousness and difficulty of managing the nephrotic syndrome and the likelihood of progressive loss of renal function. Currently, there are multiple study avenues that attempt to identify the pathogenic mechanisms that cause podocyte injury and there are also several studies underway to analyse the efficacy of drugs such as adalimumab, fresolimumab, rosiglitazone, ACTH (corticotropin) or galactose at high doses, whose preliminary results have generated expectations that require confirmation in larger-scale clinical studies.  In the future, it is possible that a better understanding of the pathogenic pathway or pathways that cause FSGS may allow differentiation between immunomodulable and non-immunomodulable forms,  however, this continues to be a challenge currently.

Biological markers of nephrotic syndrome: a few steps forward in the long way.

One of the major challenges modern nephrology should face is the identification of biomarkers that are associated with histopathological patterns or defined pathogenic mechanisms that might aid in the non-invasive diagnosis of the causes of nephrotic syndrome, or in establishing prognosis sub-groups based on each type of disease, thus predicting response to treatment and/or recurrence. Advancements in the understanding of the pathogenesis of the different diseases that cause nephrotic syndrome, along with the progressive development and standardisation of plasma and urine proteomics techniques, have facilitated the identification of a growing number of molecules that might be useful for these objectives. Currently, the available information for many of the possible candidates identified to date, above all those discovered using proteomics, are still very preliminary. In this review, we summarise the available evidence for the different molecules that have been best assessed using clinical studies.