Clinical utility of genetic testing in early-onset kidney disease: seven genes are the main players.

BACKGROUND: Inherited kidney diseases are one of the leading causes of chronic kidney disease (CKD) that manifests before the age of 30 years. Precise clinical diagnosis of early-onset CKD is complicated due to the high phenotypic overlap, but genetic testing is a powerful diagnostic tool. We aimed to develop a genetic testing strategy to maximize the diagnostic yield for patients presenting with early-onset CKD and to determine the prevalence of the main causative genes. METHODS: We performed genetic testing of 460 patients with early-onset CKD of suspected monogenic cause using next-generation sequencing of a custom-designed kidney disease gene panel in addition to targeted screening for c.428dupC MUC1. RESULTS: We achieved a global diagnostic yield of 65% (300/460), which varied depending on the clinical diagnostic group: 77% in cystic kidney diseases, 76% in tubulopathies, 67% in autosomal dominant tubulointerstitial kidney disease, 61% in glomerulopathies and 38% in congenital anomalies of the kidney and urinary tract. Among the 300 genetically diagnosed patients, the clinical diagnosis was confirmed in 77%, a specific diagnosis within a clinical diagnostic group was identified in 15%, and 7% of cases were reclassified. Of the 64 causative genes identified in our cohort, 7 (COL4A3, COL4A4, COL4A5, HNF1B, PKD1, PKD2 and PKHD1) accounted for 66% (198/300) of the genetically diagnosed patients. CONCLUSIONS: Two-thirds of patients with early-onset CKD in this cohort had a genetic cause. Just seven genes were responsible for the majority of diagnoses. Establishing a genetic diagnosis is crucial to define the precise aetiology of CKD, which allows accurate genetic counselling and improved patient management.

Description of Two Families with New Mutations in Familial Cerebral Cavernous Malformations Genes.

Cerebral cavernous malformations (CCMs) are dilated aberrant leaky capillaries located in the Central Nervous System. Familial CCM is an autosomal dominant inherited disorder related to mutations in KRIT1, Malcavernin or PDCD10. We show two unrelated families presenting familial CCM due to two new mutations in KRIT1 and PDCD10, producing truncated proteins. Clinical phenotype was highly variable among patients from asymptomatic individuals to diplopia, seizures or severe intracranial hemorrhage. PDCD10 patients usually show a more aggressive course and they frequently showed multiple meningiomas. This work provides evidence for the pathogenicity of two new mutations in CCM genes and supports previous findings regarding familial CCM and multiple meningiomas.

New cis-Acting Variants in PI*S Background Produce Null Phenotypes Causing Alpha-1 Antitrypsin Deficiency.

Alpha-1 antitrypsin deficiency (AATD) is an inherited condition characterized by reduced levels of serum AAT due to mutations in the SERPINA1 (Serpin family A member 1) gene. The Pi*S (Glu264Val) is one of the most frequent deficient alleles of AATD, showing high incidence in the Iberian Peninsula. Herein, we describe two new alleles carrying an S mutation but producing a null phenotype: QOVigo and QOAachen. The new alleles were identified by sequencing the SERPINA1 gene in three patients who had lower AAT serum levels than expected for the initial genotype. These alleles are the result of combined mutations in cis in a PI*S allele. Sequencing detected the S mutation in cis with Tyr138Cys (S+Tyr138Cys) in two patients, whereas a third one had the S mutation in cis with Pro391Thr variant (S+Pro391Thr). When expressed in a cellular model, these variants caused strong AAT polymerization and very low AAT secretion to almost undetectable levels. The isoelectric focusing method for plasma AAT phenotyping did not show AAT protein encoded by the novel mutant alleles, behaving as null. We called these alleles PI*S-plus because the S variant was phased with another variant conferring more aggressive characteristics to the allele. The current data demonstrate that the clinical variability observed in AATD can be explained by additional genetic variation, such as dual cis-acting variants in the SERPINA1 gene. The possible existence of other unrevealed variants combined in the PI*S alleles should be considered to improve the genetic diagnosis of the patients.

Characterization of Novel Missense Variants of SERPINA1 Gene Causing Alpha-1 Antitrypsin Deficiency.

The SERPINA1 gene is highly polymorphic, with more than 100 variants described in databases. SERPINA1 encodes the alpha-1 antitrypsin (AAT) protein, and severe deficiency of AAT is a major contributor to pulmonary emphysema and liver diseases. In Spanish patients with AAT deficiency, we identified seven new variants of the SERPINA1 gene involving amino acid substitutions in different exons: PiSDonosti (S+Ser14Phe), PiTijarafe (Ile50Asn), PiSevilla (Ala58Asp), PiCadiz (Glu151Lys), PiTarragona (Phe227Cys), PiPuerto Real (Thr249Ala), and PiValencia (Lys328Glu). We examined the characteristics of these variants and the putative association with the disease. Mutant proteins were overexpressed in HEK293T cells, and AAT expression, polymerization, degradation, and secretion, as well as antielastase activity, were analyzed by periodic acid-Schiff staining, Western blotting, pulse-chase, and elastase inhibition assays. When overexpressed, S+S14F, I50N, A58D, F227C, and T249A variants formed intracellular polymers and did not secrete AAT protein. Both the E151K and K328E variants secreted AAT protein and did not form polymers, although K328E showed intracellular retention and reduced antielastase activity. We conclude that deficient variants may be more frequent than previously thought and that their discovery is possible only by the complete sequencing of the gene and subsequent functional characterization. Better knowledge of SERPINA1 variants would improve diagnosis and management of individuals with AAT deficiency.

The NER-related gene GTF2H5 predicts survival in high-grade serous ovarian cancer patients.

OBJECTIVE: We aimed to evaluate the prognostic and predictive value of the nucleotide excision repair-related gene GTF2H5, which is localized at the 6q24.2-26 deletion previously reported by our group to predict longer survival of high-grade serous ovarian cancer patients. METHODS: In order to test if protein levels of GTF2H5 are associated with patients' outcome, we performed GTF2H5 immunohistochemical staining in 139 high-grade serous ovarian carcinomas included in tissue microarrays. Upon stratification of cases into high- and low-GTF2H5 staining categories (> and ≤ median staining, respectively) Kaplan-Meier and log-rank test were used to estimate patients' survival and assess statistical differences. We also evaluated the association of GTF2H5 with survival at the transcriptional level by using the on-line Kaplan-Meier plotter tool, which includes gene expression and survival data of 855 high-grade serous ovarian cancer patients from 13 different datasets. Finally, we determined whether stable short hairpin RNA-mediated GTF2H5 downregulation modulates cisplatin sensitivity in the SKOV3 and COV504 cell lines by using cytotoxicity assays. RESULTS: Low expression of GTF2H5 was associated with longer 5-year survival of patients at the protein (hazard ratio [HR], 0.52; 95% CI, 0.29 to 0.93; p=0.024) and transcriptional level (HR, 0.80; 95% CI, 0.65 to 0.97; p=0.023) in high-grade serous ovarian cancer patients. We confirmed the association with 5-year overall survival (HR, 0.55; 95% CI, 0.38 to 0.78; p=0.0007) and also found an association with progression-free survival (HR, 0.72; 95% CI, 0.54 to 0.96; p=0.026) in a homogenous group of 388 high-stage (stages III-IV using the International Federation of Gynecology and Obstetrics staging system), optimally debulked high-grade serous ovarian cancer patients. GTF2H5-silencing induced a decrease of the half maximal inhibitory concentration upon cisplatin treatment in GTF2H5-silenced ovarian cancer cells. CONCLUSION: Low levels of GTF2H5 are associated with enhanced prognosis in high-grade serous ovarian cancer patients and may contribute to cisplatin sensitization.

Deletion at 6q24.2-26 predicts longer survival of high-grade serous epithelial ovarian cancer patients.

Standard treatments for advanced high-grade serous ovarian carcinomas (HGSOCs) show significant side-effects and provide only short-term survival benefits due to disease recurrence. Thus, identification of novel prognostic and predictive biomarkers is urgently needed. We have used 42 paraffin-embedded HGSOCs, to evaluate the utility of DNA copy number alterations, as potential predictors of clinical outcome. Copy number-based unsupervised clustering stratified HGSOCs into two clusters of different immunohistopathological features and survival outcome (HR = 0.15, 95%CI = 0.03-0.81; Padj = 0.03). We found that loss at 6q24.2-26 was significantly associated with the cluster of longer survival independently from other confounding factors (HR = 0.06, 95%CI = 0.01-0.43, Padj = 0.005). The prognostic value of this deletion was validated in two independent series, one consisting of 36 HGSOCs analyzed by fluorescent in situ hybridization (P = 0.04) and another comprised of 411 HGSOCs from the Cancer Genome Atlas study (TCGA) (HR = 0.67, 95%CI = 0.48-0.93, Padj = 0.019). In addition, we confirmed the association of low expression of the genes from the region with longer survival in 799 HGSOCs (HR = 0.74, 95%CI = 0.61-0.90, log-rank P = 0.002) and 675 high-FIGO stage HGSOCs (HR = 0.76, 95%CI = 0.61-0.96, log-rank P = 0.02) available from the online tool KM-plotter. Finally, by integrating copy number, RNAseq and survival data of 296 HGSOCs from TCGA we propose a few candidate genes that can potentially explain the association. Altogether our findings indicate that the 6q24.2-26 deletion is an independent marker of favorable outcome in HGSOCs with potential clinical value as it can be analyzed by FISH on tumor sections and guide the selection of patients towards more conservative therapeutic strategies in order to reduce side-effects and improve quality of life.

Assessing fishing and marine biodiversity changes using fishers' perceptions: the Spanish Mediterranean and Gulf of Cadiz case study.

BACKGROUND: The expansion of fishing activities has intensively transformed marine ecosystems worldwide. However, available time series do not frequently cover historical periods. METHODOLOGY: Fishers' perceptions were used to complement data and characterise changes in fishing activity and exploited ecosystems in the Spanish Mediterranean Sea and Gulf of Cadiz. Fishers' interviews were conducted in 27 fishing harbours of the area, and included 64 fishers from ages between 20 to >70 years old to capture the experiences and memories of various generations. Results are discussed in comparison with available independent information using stock assessments and international convention lists. PRINCIPAL FINDINGS: According to fishers, fishing activity substantially evolved in the area with time, expanding towards deeper grounds and towards areas more distant from the coast. The maximum amount of catch ever caught and the weight of the largest species ever captured inversely declined with time. Fishers (70%) cited specific fishing grounds where depletion occurred. They documented ecological changes of marine biodiversity during the last half of the century: 94% reported the decline of commercially important fish and invertebrates and 61% listed species that could have been extirpated, with frequent mentions to cartilaginous fish. Declines and extirpations were in line with available quantitative evaluations from stock assessments and international conventions, and were likely linked to fishing impacts. Conversely, half of interviewed fishers claimed that several species had proliferated, such as cephalopods, jellyfish, and small-sized fish. These changes were likely related to trophic cascades due to fishing and due to climate change effects. The species composition of depletions, local extinctions and proliferations showed differences by region suggesting that regional dynamics are important when analysing biodiversity changes. CONCLUSIONS/SIGNIFICANCE: Using fishers' perceptions, fishing and ecological changes in the study area were documented. The recovery of local ecological knowledge provides valuable information complementing quantitative monitoring and evaluation surveys.

Mutaciones de Splicing / Splicing Mutations

El splicing o maduración del pre-mRNA es un mecanismo que está adquiriendo gran relevancia no sólo por las posibilidades que ofrece de expansión del proteoma, sino también por su implicación en la patofisiología de las enfermedades humanas. Las mutaciones de splicing alteran el procesamiento del mRNA al afectar a las secuencias del pre-mRNA (mutaciones en cis) o a las proteínas que intervienen en el splicing (mutaciones en trans). Las secuencias que pueden verse alteradas son: las limitantes de exones e intrones, la zona de ramificación, la zona rica en pirimidinas y también otros elementos reguladores que incrementan o suprimen la selección de un exón. La comprensión en profundidad del efecto de las mutaciones sobre el splicing puede abrir la puerta a su tratamiento mediante terapia moléculas (AU)

Splicing or maturation of pre-mRNA is a mechanism that is becoming more relevant not only for its potential expansion of the proteome, but also for its involvement in the physiopathology of human diseases. Splicing mutations after the processing of mRNA by affecting pre-mRNA sequences (mutations in cis) or proteins involved in splicing (mutations in trans). The sequences that can be altered are: the limiting areas between exons and interns, the branch site, the polypirimidine tract and, finally, other regulatory elements that enhance or suppress the selection of an exon. The whole understanding of the effect of mutations on the splicing processes will help to design molecular therapy targets to correct these defects (AU)

Large Evaluation of Anti-Cardiolipin and anti-Beta2Glycoprotein I Assays: Results from the AutoimmunityWorkshop of the Spanish Society of Immunology

Despite their clinical utility and the importance that laboratory testshave in APS diagnosis, probably the most important drawback of suchtests is the elevated intra- and inter-laboratory variation. The aim of thepresent work was to assess the multilaboratory performance of aCL (..) (AU)

A pesar de la indudable utilidad clínica y de la importancia de laspruebas de laboratorio en el diagnóstico del síndrome antifosfolípido(APS), probablemente el mayor defecto de dichas pruebas es su elevadavariabilidad intra- e inter-laboratorio. El objetivo del presente trabajo fueevaluar el comportamiento de los ensayos (..) (AU)

Carcinoma micropapilar infiltrante de la mama en un varón. A propósito de un caso / Invasive micropapillary carcinoma of the male breast. A case report

Introducción: El carcinoma micropapilar infiltrante de la glándula mamaria representa una variante histológica con una alta incidencia de metástasis ganglionares y un alto índice de recidivas locorregionales, que se ha descrito de forma muy ocasional en la mama masculina. Caso clínico: Se presenta un caso de un varón de 75 años con un carcinoma micropapilar infiltrante de la mama derecha de 2 cm y con metástasis en uno de 11 ganglios axilares. Los receptores hormonales fueron positivos y el c-erbB 2 determinado inmunohistoquímicamente fue negativo. La evolución a los 17 meses del diagnóstico fue satisfactoria. Conclusión: El carcinoma micropapilar infiltrante también puede presentarse en la mama masculina con las mismas características clínicas y morfológicas que en la mama femenina

Introduction: Invasive micropapillary carcinoma of the breast is a histological subtype of carcinoma with a high incidence of lymph node metastasis and frequent recurrences that has only been described occasionally in the male breast. Clinico-pathological report: A case of a 75 year old male with a 2 cm invasive micropapillary carcinoma and metastasis in one out of 11 axillary lymph nodes is presented. Hormone receptors were positive and c-erbB 2 detected immunohistochemically was negative. The follow-up was successful after 17 months of diagnosis. Conclusion: Invasive micropapillary carcinoma can also occur in the male breast with the same clinic and morphologic characteristics than in the female breast (AU)

The impaired response of NK cells from HIV-infected progressor patients to A-class CpG oligodeoxynucleotides is largely dependent of a decreased production of IL-12.

We have reported that NK cells from HIV-infected progressors showed a markedly lower IFN-gamma production in response to an A-class CpG oligodeoxynucleotide as compared to LTNP subjects and healthy HIV-negative individuals. This functional defect was not related to the number of circulating plasmacytoid dendritic cells, nor to the alpha-interferon secreted. In contrast, defective response correlated negatively with the frequency of myeloid dendritic cells. Furthermore, peripheral blood mononuclear cells from LTNPs as well as those from some healthy HIV-negative donors secreted large amounts of IL-12 in unstimulated cultures and in response to the CpG-ODN whereas, HIV-progressor cells showed impaired responses and low level of spontaneous secretion. The addition of a monoclonal anti-IL-12 reduced the response to the CpG-ODN in a dose-dependent manner. These results suggest that the impaired response of NK cells to the CpG-ODN in HIV-progressors is largely dependent on a decreased production of IL-12.

HIV-infected progressors and long-term non-progressors differ in their capacity to respond to an A-class CpG oligodeoxynucleotide.

We used an A-class CpG oligodeoxynucleotide to explore innate immunity in HIV infection and observed that natural killer cells from progressors showed a markedly lower IFN-gamma production in response to the oligonuclotide as compared with long-term non-progressing subjects and healthy HIV-negative individuals. This functional defect was found in patients who showed a long immunological reduction and in those who had had a recent reduction in their CD4 cell counts.