Molecular Challenges in the Diagnosis of X-Linked Chronic Granulomatous Disease: CNVs, Intronic Variants, Skewed X-Chromosome Inactivation, and Gonosomal Mosaicism.

Chronic granulomatous disease (CGD) is a prototypical inborn error of immunity affecting phagocytes, in which these cells are unable to produce reactive oxygen species. CGD is caused by defects in genes encoding subunits of the NADPH oxidase enzyme complex (CYBA, CYBB, CYBC1, NCF1, NCF2, NCF4); inflammatory responses are dysregulated, and patients are highly susceptible to recurrent severe bacterial and fungal infections. X-linked CGD (XL-CGD), caused by mutations in the CYBB gene, is the most common and severe form of CGD. In this study, we describe the analytical processes undertaken in 3 families affected with XL-CGD to illustrate several molecular challenges in the genetic diagnosis of this condition: in family 1, a girl with a heterozygous deletion of CYBB exon 13 and skewed X-chromosome inactivation (XCI); in family 2, a boy with a hemizygous deletion of CYBB exon 7, defining its consequences at the mRNA level; and in family 3, 2 boys with the same novel intronic variant in CYBB (c.1151 + 6 T > A). The variant affected the splicing process, although a small fraction of wild-type mRNA was produced. Their mother was a heterozygous carrier, while their maternal grandmother was a carrier in form of gonosomal mosaicism. In summary, using a variety of techniques, including an NGS-based targeted gene panel and deep amplicon sequencing, copy number variation calling strategies, microarray-based comparative genomic hybridization, and cDNA analysis to define splicing defects and skewed XCI, we show how to face and solve some uncommon genetic mechanisms in the diagnosis of XL-CGD.

Detection and evolutionary dynamics of somatic FAS variants in autoimmune lymphoproliferative syndrome: Diagnostic implications.

Autoimmune lymphoproliferative syndrome (ALPS) is a rare primary immune disorder characterized by impaired apoptotic homeostasis. The clinical characteristics include lymphoproliferation, autoimmunity (mainly cytopenia), and an increased risk of lymphoma. A distinctive biological feature is accumulation (>2.5%) of an abnormal cell subset composed of TCRαß+ CD4-CD8- T cells (DNTs). The most common genetic causes of ALPS are monoallelic pathogenic variants in the FAS gene followed by somatic FAS variants, mainly restricted to DNTs. Identification of somatic FAS variants has been typically addressed by Sanger sequencing in isolated DNTs. However, this approach can be costly and technically challenging, and may not be successful in patients with normal DNT counts receiving immunosuppressive treatment. In this study, we identified a novel somatic mutation in FAS (c.718_719insGTCG) by Sanger sequencing on purified CD3+ cells. We then followed the evolutionary dynamics of the variant along time with an NGS-based approach involving deep amplicon sequencing (DAS) at high coverage (20,000-30,000x). Over five years of clinical follow-up, we obtained six blood samples for molecular study from the pre-treatment (DNTs>7%) and treatment (DNTs<2%) periods. DAS enabled detection of the somatic variant in all samples, even the one obtained after five years of immunosuppressive treatment (DNTs: 0.89%). The variant allele frequency (VAF) range was 4%-5% in pre-treatment samples and <1.5% in treatment samples, and there was a strong positive correlation between DNT counts and VAF (Pearson's R: 0.98, p=0.0003). We then explored whether the same approach could be used in a discovery setting. In the last follow-up sample (DNT: 0.89%) we performed somatic variant calling on the FAS exon 9 DAS data from whole blood and purified CD3+ cells using VarScan 2. The c.718_719insGTCG variant was identified in both samples and showed the highest VAF (0.67% blood, 1.58% CD3+ cells) among >400 variants called. In summary, our study illustrates the evolutionary dynamics of a somatic FAS mutation before and during immunosuppressive treatment. The results show that pathogenic somatic FAS variants can be identified with the use of DAS in whole blood of ALPS patients regardless of their DNT counts.

Impact of cytomegalovirus infection prior to hematopoietic stem cell transplantation in children with inborn errors of immunity.

The presence of active viral infections has an impact on the prognosis of patients undergoing hematopoietic stem cell transplantation (HSCT). Nevertheless, the number of reports of cytomegalovirus infection in patients with inborn errors of immunity (IEI) who undergo HSCT is relatively low. To analyze the effect of cytomegalovirus infection acquired prior to curative treatment on patient survival in 123 children with IEI. An observational and retrospective study was performed with patients younger than 18 years diagnosed with IEI who were candidates for HSCT, gene therapy, or thymus transplantation at five hospitals in Spain between 2008 and 2019. We included 123 children, 25 infected by cytomegalovirus prior to undergoing curative treatment (20.3%). At IEI diagnosis, 24 of the patients were already infected, 21 of whom had symptomatic cytomegalovirus disease (87%), while the other three patients developed disease before undergoing curative treatment. The patients with cytomegalovirus infection had higher mortality than those without (p = 0.006). Fourteen patients developed refractory cytomegalovirus infection (56%), all of whom died, while no patients with non-refractory infection died (p = 0.001) All deaths that occurred before curative treatment and three of the five after the treatment were attributed to cytomegalovirus. Patients with refractory cytomegalovirus disease had the highest pre-HSCT mortality rate (64.3%), compared with the non-infected children and those with non-refractory cytomegalovirus disease (10.1%) (p < 0.0001). CONCLUSION: Prevention and prompt control of cytomegalovirus infection, together with early HSCT/gene therapy, are crucial for improving the prognosis in children with IEI. WHAT IS KNOWN: • Cytomegalovirus is the most frequent viral infection in children with inborn errors of immunity who are candidates to hematopoietic stem cell transplantation (HSCT). • Active viral infections at the time of HSCT lead to worse prognosis. WHAT IS NEW: • In children with inborn errors of immunity and indication of HSCT, refractory cytomegalovirus disease is associated with a very high mortality rate, compared with non-infected children and those with non-refractory cytomegalovirus disease. • In patients with novel transplantation indications, the presence and treatment response of CMV infection should be considered to decide the best possible moment for HSCT.

Inborn errors of TLR3- or MDA5-dependent type I IFN immunity in children with enterovirus rhombencephalitis.

Enterovirus (EV) infection rarely results in life-threatening infection of the central nervous system. We report two unrelated children with EV30 and EV71 rhombencephalitis. One patient carries compound heterozygous TLR3 variants (loss-of-function F322fs2* and hypomorphic D280N), and the other is homozygous for an IFIH1 variant (loss-of-function c.1641+1G>C). Their fibroblasts respond poorly to extracellular (TLR3) or intracellular (MDA5) poly(I:C) stimulation. The baseline (TLR3) and EV-responsive (MDA5) levels of IFN-ß in the patients' fibroblasts are low. EV growth is enhanced at early and late time points of infection in TLR3- and MDA5-deficient fibroblasts, respectively. Treatment with exogenous IFN-α2b before infection renders both cell lines resistant to EV30 and EV71, whereas post-infection treatment with IFN-α2b rescues viral susceptibility fully only in MDA5-deficient fibroblasts. Finally, the poly(I:C) and viral phenotypes of fibroblasts are rescued by the expression of WT TLR3 or MDA5. Human TLR3 and MDA5 are critical for cell-intrinsic immunity to EV, via the control of baseline and virus-induced type I IFN production, respectively.

Newborn Screening for SCID. Experience in Spain (Catalonia).

Newborn screening (NBS) for severe combined immunodeficiency (SCID) started in Catalonia in January-2017, being the first Spanish and European region to universally include this testing. In Spain, a pilot study with 5000 samples was carried out in Seville in 2014; also, a research project with about 35,000 newborns will be carried out in 2021-2022 in the NBS laboratory of Eastern Andalusia. At present, the inclusion of SCID is being evaluated in Spain. The results obtained in the first three and a half years of experience in Catalonia are presented here. All babies born between January-2017 and June-2020 were screened through TREC-quantification in DBS with the Enlite Neonatal TREC-kit from PerkinElmer. A total of 222,857 newborns were screened, of which 48 tested positive. During the study period, three patients were diagnosed with SCID: an incidence of 1 in 74,187 newborns; 17 patients had clinically significant T-cell lymphopenia (non-SCID) with an incidence of 1 in 13,109 newborns who also benefited from the NBS program. The results obtained provide further evidence of the benefits of early diagnosis and curative treatment to justify the inclusion of this disease in NBS programs. A national NBS program is needed, also to define the exact SCID incidence in Spain.

Epigenome-wide association study of COVID-19 severity with respiratory failure.

BACKGROUND: Patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the coronavirus disease 2019 (COVID-19), exhibit a wide spectrum of disease behaviour. Since DNA methylation has been implicated in the regulation of viral infections and the immune system, we performed an epigenome-wide association study (EWAS) to identify candidate loci regulated by this epigenetic mark that could be involved in the onset of COVID-19 in patients without comorbidities. METHODS: Peripheral blood samples were obtained from 407 confirmed COVID-19 patients ≤ 61 years of age and without comorbidities, 194 (47.7%) of whom had mild symptomatology that did not involve hospitalization and 213 (52.3%) had a severe clinical course that required respiratory support. The set of cases was divided into discovery (n = 207) and validation (n = 200) cohorts, balanced for age and sex of individuals. We analysed the DNA methylation status of 850,000 CpG sites in these patients. FINDINGS: The DNA methylation status of 44 CpG sites was associated with the clinical severity of COVID-19. Of these loci, 23 (52.3%) were located in 20 annotated coding genes. These genes, such as the inflammasome component Absent in Melanoma 2 (AIM2) and the Major Histocompatibility Complex, class I C (HLA-C) candidates, were mainly involved in the response of interferon to viral infection. We used the EWAS-identified sites to establish a DNA methylation signature (EPICOVID) that is associated with the severity of the disease. INTERPRETATION: We identified DNA methylation sites as epigenetic susceptibility loci for respiratory failure in COVID-19 patients. These candidate biomarkers, combined with other clinical, cellular and genetic factors, could be useful in the clinical stratification and management of patients infected with the SARS-CoV-2. FUNDING: The Unstoppable campaign of the Josep Carreras Leukaemia Foundation, the Cellex Foundation and the CERCA Programme/Generalitat de Catalunya.

[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.

First Universal Newborn Screening Program for Severe Combined Immunodeficiency in Europe. Two-Years' Experience in Catalonia (Spain).

Severe combined immunodeficiency (SCID), the most severe form of T-cell immunodeficiency, can be screened at birth by quantifying T-cell receptor excision circles (TRECs) in dried blood spot (DBS) samples. Early detection of this condition speeds up the establishment of appropriate treatment and increases the patient's life expectancy. Newborn screening for SCID started in January 2017 in Catalonia, the first Spanish and European region to universally include this testing. The results obtained in the first 2 years of experience are evaluated here. All babies born between January 2017 and December 2018 were screened. TREC quantification in DBS (1.5 mm diameter) was performed with the Enlite Neonatal TREC kit from PerkinElmer (Turku, Finland). In 2018, the retest cutoff in the detection algorithm was updated based on the experience gained in the first year, and changed from 34 to 24 copies/µL. This decreased the retest rate from 3.34 to 1.4% (global retest rate, 2.4%), with a requested second sample rate of 0.23% and a positive detection rate of 0.02%. Lymphocyte phenotype (T, B, NK populations), expression of CD45RA/RO isoforms, percentage and intensity of TCR αß and TCR γδ, presence of HLA-DR+ T lymphocytes, and in vitro lymphocyte proliferation were studied in all patients by flow cytometry. Of 130,903 newborns screened, 30 tested positive, 15 of which were male. During the study period, one patient was diagnosed with SCID: incidence, 1 in 130,903 births in Catalonia. Thirteen patients had clinically significant T-cell lymphopenia (non-SCID) with an incidence of 1 in 10,069 newborns (43% of positive detections). Nine patients were considered false-positive cases because of an initially normal lymphocyte count with normalization of TRECs between 3 and 6 months of life, four infants had transient lymphopenia due to an initially low lymphocyte count with recovery in the following months, and three patients are still under study. The results obtained provide further evidence of the benefits of including this disease in newborn screening programs. Longer follow-up is needed to define the exact incidence of SCID in Catalonia.

Epidemiology of congenital Chagas disease 6 years after implementation of a public health surveillance system, Catalonia, 2010 to 2015.

BackgroundChagas disease is endemic in Latin America and affects 8 million people worldwide. In 2010, Catalonia introduced systematic public health surveillance to detect and treat congenital Chagas disease.AimThe objective was to evaluate the health outcomes of the congenital Chagas disease screening programme during the first 6 years (2010-2015) after its introduction in Catalonia.MethodsIn a surveillance system, we screened pregnant women and newborns and other children of positive mothers, and treated Chagas-positive newborns and children. Diagnosis was confirmed for pregnant women and children with two positive serological tests and for newborns with microhaematocrit and/or PCR at birth or serology at age 9 months.ResultsFrom 2010 to 2015, the estimated screening coverage rate increased from 68.4% to 88.6%. In this period, 33,469 pregnant women were tested for Trypanosoma cruzi and 937 positive cases were diagnosed. The overall prevalence was 2.8 cases per 100 pregnancies per year (15.8 in Bolivian women). We followed 82.8% of newborns until serological testing at age 9-12 months and 28 were diagnosed with Chagas disease (congenital transmission rate: 4.17%). Of 518 siblings, 178 (34.3%) were tested and 14 (7.8%) were positive for T. cruzi. Having other children with Chagas disease and the heart clinical form of Chagas disease were maternal risk factors associated with congenital T. cruzi infection (p < 0.05).ConclusionThe increased screening coverage rate indicates consolidation of the programme in Catalonia. The rate of Chagas disease congenital transmission in Catalonia is in accordance with the range in non-endemic countries.

Extended immunophenotyping reference values in a healthy pediatric population.

BACKGROUND: For the accurate diagnosis of immunodeficiencies is crucial to compare patients' immunology laboratory values with age-sex matched controls, yet there is a paucity of normal values for most populations. OBJECTIVES: To define appropriate reference values of extended lymphocyte subpopulations and T-cell receptor excision circle (TRECs) levels in healthy pediatric donors between 1 month and 18 years of age. METHODS: Extended immunophenotyping values were obtained by analysis of multiparameter flow cytometry panels for the following subpopulations: CD4+ and CD8+ Naive, Effector, Effector Memory and Central Memory, T helper subpopulations and their degrees of activation, T Regulatory cells, Recent Thymic Emigrants (RTE), B Lymphocyte subpopulations (Transitional, Naive, Preswitch-Memory, Switch-Memory, Plasmablasts, CD21low, and Exhausted), and subpopulations for Monocytes, NK cells and Dendritic Cells. RESULTS: Median values and the 10th and 90th percentiles were obtained for 32 lymphocyte and monocyte subpopulations, and for TRECs levels in each age group of children. Naive CD4+ and CD8+ T-cell populations tended to decrease with age, with significant difference between the groups, in parallel with the reduction in thymic function assessed by TRECs counts and the recent thymic emigrant population. Relative numbers of Th cell populations tended to increase with age. The percentage of class-switched B cell populations showed a significant increase between the youngest group and the others. CONCLUSION: This study provides essential data for interpreting extended immunophenotyping profiles in the pediatric and young adult populations, which could be of value for the diagnosis of PIDs and immune-mediated diseases, particularly those associated with subtle immunological abnormalities. © 2018 International Clinical Cytometry Society.

Evaluating the Genetics of Common Variable Immunodeficiency: Monogenetic Model and Beyond.

Common variable immunodeficiency (CVID) is the most frequent symptomatic primary immunodeficiency characterized by recurrent infections, hypogammaglobulinemia and poor response to vaccines. Its diagnosis is made based on clinical and immunological criteria, after exclusion of other diseases that can cause similar phenotypes. Currently, less than 20% of cases of CVID have a known underlying genetic cause. We have analyzed whole-exome sequencing and copy number variants data of 36 children and adolescents diagnosed with CVID and healthy relatives to estimate the proportion of monogenic cases. We have replicated an association of CVID to p.C104R in TNFRSF13B and reported the second case of homozygous patient to date. Our results also identify five causative genetic variants in LRBA, CTLA4, NFKB1, and PIK3R1, as well as other very likely causative variants in PRKCD, MAPK8, or DOCK8 among others. We experimentally validate the effect of the LRBA stop-gain mutation which abolishes protein production and downregulates the expression of CTLA4, and of the frameshift indel in CTLA4 producing expression downregulation of the protein. Our results indicate a monogenic origin of at least 15-24% of the CVID cases included in the study. The proportion of monogenic patients seems to be lower in CVID than in other PID that have also been analyzed by whole exome or targeted gene panels sequencing. Regardless of the exact proportion of CVID monogenic cases, other genetic models have to be considered for CVID. We propose that because of its prevalence and other features as intermediate penetrancies and phenotypic variation within families, CVID could fit with other more complex genetic scenarios. In particular, in this work, we explore the possibility of CVID being originated by an oligogenic model with the presence of heterozygous mutations in interacting proteins or by the accumulation of detrimental variants in particular immunological pathways, as well as perform association tests to detect association with rare genetic functional variation in the CVID cohort compared to healthy controls.

TNFAIP3 haploinsufficiency is the cause of autoinflammatory manifestations in a patient with a deletion of 13Mb on chromosome 6.

There is scarce literature about autoinflammation in syndromic patients. We describe a patient who, in addition to psychomotor and growth delay, presented with fevers, neutrophilic dermatosis, and recurrent orogenital ulcers. Comparative Genomic Hybridization (CGH) array permitted to identify a 13.13Mb deletion on chromosome 6, encompassing 53 genes, and including TNFAIP3 gene (A20). A20 is a potent inhibitor of the NF-kB signalling pathway and restricts inflammation via its deubiquitinase activity. Western blotting and immunoprecipitation assays showed decreased A20 expression and increased phosphorylation of p65 and IkBa. Patient's cells displayed increased levels of total K63-linked ubiquitin and increased levels of ubiquitinated RIP and NEMO after stimulation with TNF. We describe the molecular characterization of an autoinflammatory disease due to a large chromosomal deletion and review the phenotypes of patients with A20 haploinsufficiency. CGH arrays should be the first diagnostic method for comprehensive analysis of patients with syndromic features and immune dysregulation.

Chronic Recurrent Multifocal Osteomyelitis and Thalidomide in Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency that leads to severe recurrent infection and inflammatory complications that are usually difficult to diagnose and treat. Several hyperinflammation mechanisms, such as decreased neutrophil apoptosis, toll-like receptor activation imbalance, Th17 cell induction, Nrf2 activity deficiency, and inflammasome activation, have been described in CGD patients However, there have been no reports of chronic recurrent multifocal osteomyelitis as an inflammatory complication in CGD, and the differential diagnosis of this condition with infectious osteomyelitis is challenging. Thalidomide has been used to treat several inflammatory manifestations in CGD patients with good clinical results. Here, we report the case of a previously asymptomatic 11-year-old boy who consulted for difficulty walking and pain at the back of the right thigh, with increased inflammatory markers. Multifocal bone involvement was seen on bone scintigraphy, and acute-phase reactants were elevated. On the basis of a suspected diagnosis of infectious osteomyelitis, broad-spectrum antibiotic therapy was started, with no clinical response. Bone biopsy and microbiological tests yielded negative results; at that point, chronic recurrent multifocal osteomyelitis was suspected. The patient was unresponsive to nonsteroidal antiinflammatory drugs and corticosteroids. Thalidomide was started, and within 6 months, clinical and radiologic resolution of the condition was achieved with no adverse effects. More than 1 year after stopping thalidomide, the patient remained free of symptoms and inflammatory parameters are within normal levels. Thalidomide has a favorable safety profile compared with other alternatives and could be considered a feasible therapeutic option for this type of condition in selected patients.

Clinical and structural impact of mutations affecting the residue Phe367 of FOXP3 in patients with IPEX syndrome.

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a monogenic autoimmune disease characterized by early-onset life-threatening multisystemic autoimmunity. This rare hereditary disorder is caused by loss-of-function mutations in the gene encoding the forkhead box P3 (FOXP3) transcription factor, which plays a key role in the differentiation and function of CD4(+)CD25(+) natural regulatory T cells (Tregs), essential for the establishment and maintenance of natural tolerance. We identified a novel mutation in the FOXP3 gene affecting the Phe367 residue of the protein (F367V) in a family with three male siblings affected by IPEX. Two other mutations affecting the FOXP3 Phe367 residue (F367L and F367C) have been described previously. This unique situation of three mutations affecting the same residue in FOXP3 led us to study the molecular impact of these mutations on the structure of FOXP3 protein. Structure analysis showed that Phe367 is involved in a rich interaction network related to both monomer and dimer structure stabilization, and is crucial for FOXP3 regulatory activity. The relevance of this location is confirmed by the results of SIFT and PolyPhen-2 pathogenicity predictions for F367V mutation. In summary, as assessment of the pathogenicity of a novel mutation is crucial to achieve a proper molecular diagnosis, we analysed the impact of mutations affecting the Phe367 residue using a combined approach that provides a mechanistic view of their pathogenic effect.

Cystatin C: A Marker for Inflammation and Renal Function Among HIV-infected Children and Adolescents.

BACKGROUND: Renal disease is a leading cause of morbidity in HIV-infected adults in the highly active antiretroviral therapy (HAART) era. Cystatin C has been proposed as a more sensitive marker of renal function, but it may be affected by ongoing inflammation. We aimed to study the cystatin C levels in a cohort of HIV-infected pediatric patients at 3 Spanish centers. METHODS: This is a multicenter cross-sectional observational study. Renal function was assessed by means of first morning urine protein/creatinine and albumin/creatinine ratios and creatinine-estimated glomerular filtration rates (GFRs), together with the following inflammation markers: cystatin C, reactive C protein, ß-2-microglobulin and 25(OH)-vitamin D levels. A control group of healthy children and adolescents was used. RESULTS: Eighty-three patients (51 females, median age: 13.3 years; 32 males, median age: 13.6 years) and 44 controls (24 females, median age: 12.2 years; 20 males, median age: 10.9 years) were included. Among the former, mean CD4 cell count was 860/mm, 29(35%) patients had a previous AIDS diagnosis, 73(88%) were on HAART and HIV viremia was undetectable in 61(73%). No differences in cystatin C levels were observed between the 2 groups. In HIV-infected patients, cystatin C levels correlated with GFR (r = -0.27; P = 0.01), age at first HAART (r = -0.21; P = 0.05), and ß-2-microglobulin (r = 0.569; P < 0.01). In multivariate analysis, lower GFR (P = 0.014) and higher ß-2-microglobulin levels (P = 0.001) remained as independent risk factors for higher cystatin C values. CONCLUSIONS: Cystatin C values were associated with GFR and ß-2-microglobulin. Cystatin C may be useful as a marker of renal function in HIV-infected pediatric patients, independently of ongoing inflammation or viremia.

From Severe Combined Immunodeficiency to Omenn syndrome after hematopoietic stem cell transplantation in a RAG1 deficient family.

BACKGROUND: Mutations in RAG genes cause a spectrum of severe immunodeficiencies ranging from Severe Combined Immunodeficiency (SCID) T-B-NK+ to Omenn syndrome (OS) through intermediate phenotypes, even for the same alteration. Nowadays, hematopoietic stem cell transplantation (HSCT) is the unique curative treatment available. METHODS: We describe three related patients from a Moroccan consanguineous family. Patient 1 developed at 1 month of age moderate eczematous dermatitis with eosinophilia, followed by infections and enteritis. He was transplanted and received reduced intensity conditioning regimen previous to HSCT. His brother, patient 2, was born preterm with a severe neonatal erythroderma, hepatosplenomegaly and lymphadenopathy. Patient 3, cousin of the two siblings, was also born preterm and fulfilled all criteria for classical OS. Immunological evaluation was performed and RAG genes were sequenced. RESULTS: Immunological data from all three patients were very diversed, from T lymphopenia to marked lymphocytosis, and different degrees of eosinophilia and IgE levels. Non-responder T cells and absent B cells were constant. All patients presented the same homozygous mutation in RAG1 gene (c.631delT). Patient 1 fully recovered both clinically and immunologically after HSCT. Two years later, he lost the accomplished lymphoid chimera and the disease relapsed as a classical OS, leading to patient's death. CONCLUSIONS: This is the first report of a RAG1 deficient patient with a changed clinical and immunological phenotype from SCID to OS after HSCT. The use of a myeloablative conditioning regimen that eliminates reminiscent T cells might have improved patient's outcome and it should be considered in similar cases.

Hyperlactatemia and in utero exposure to antiretrovirals: is the control group the clue?

Perinatal antiretroviral (ARV) exposure has been related to hyperlactatemia and lactic acidosis in infants born to HIV-infected mothers. Our objective was to determine the incidence of these conditions during the first year of life in uninfected infants born to HIV-infected mothers and compare the data with infants born to mothers with hepatitis C virus (HCV) infection. We investigated the relationships between hyperlactatemia and neurological and neurodevelopmental disorders by conducting a prospective, comparative cohort study (October 2004 to October 2007) consecutively including children of HIV- and HCV-infected mothers. Liver enzymes, pH, lactic acid, and plasma amino acids were determined at 1.5, 3, 6, and 12 months of life. Pathological hyperlactatemia was defined as lactate >2.1 mmol/liter together with alanine >475 µmol/liter. Seventy-nine patients (39 HIV-exposed patients and 40 unexposed patients) were included. Baseline maternal characteristics in the two groups were similar. Almost 90% of HIV-infected mothers received HAART during gestation, while 10.3% were given AZT monotherapy. Eight newborns received combined therapy and 31 received AZT-based monotherapy. Twelve patients (five exposed and seven nonexposed) had some neurological disorder, and four other patients (one vs. three) showed signs of neurodevelopmental delay, with no significant differences between the groups (p=0.34). Pathological hyperlactatemia was detected in 56.4% (95% CI 39.6-72.2) and 57.5% (95% CI 40.9-73.0) of patients, respectively (p=0.92), and this condition was more frequent in preterm children (p<0.05). ARV use during pregnancy and the neonatal period was not associated with pathological hyperlactatemia. The presence of hyperlactatemia was not associated with neurological or neurodevelopmental disorders. No association was established between the use of ARV agents and the development of hyperlactatemia or neurological disorders in HIV-exposed children during their first year of life.

Voriconazole drug monitoring in the management of invasive fungal infection in immunocompromised children: a prospective study.

OBJECTIVES: To evaluate voriconazole plasma level monitoring in immunocompromised children and determine the relationship of plasma levels with dose, safety and efficacy. METHODS: We used a prospective study including all consecutive children with invasive fungal infection (IFI) treated with voriconazole between August 2008 and May 2010. IFI diagnosis and clinical outcome evaluation were based on European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group ('EORTC/MSG') definitions. RESULTS: A total of 196 voriconazole plasma trough measurements from 30 patients (median age 10 years) obtained during 2135 days of voriconazole therapy were analysed. Nineteen patients (63%) presented with proven or probable IFI. Voriconazole plasma levels varied widely and 73% of patients required dose adjustment. The median voriconazole dose was 20 mg/kg/day and the median duration of therapy was 6 weeks. Age 5 was the smallest value defining two groups on which the correlation between dose and plasma levels had a different behaviour, and this relationship was especially significant for patients <5 years old (Spearman's rank correlation coefficient=0.38213, P=0.008). For patients <5 years old the median dose to achieve therapeutic levels was 38.0 mg/kg/day (12-40.0) and for those ≥5 years old it was 15 mg/kg (4-52). Voriconazole plasma levels showed a significant relationship with early outcome (P=0.0268), but not late outcome (P=0.2015). Overall mortality was 42% and a significant relationship with voriconazole therapeutic plasma levels was not demonstrated. A significant relationship was established between plasma levels above normal range and skin and neurological toxicity (P=0.0001), but this could not be demonstrated for liver toxicity. CONCLUSIONS: Our study confirms the large variability in voriconazole trough plasma levels in children and a trend to non-linear pharmacokinetics in older patients. In addition, doses significantly higher than those recommended in younger children seem warranted and a significant relationship between plasma voriconazole above the normal range and some adverse events is confirmed.