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BACKGROUND AND AIMS Alport Syndrome (AS; ORPHA 63) is one of the most frequent hereditary kidney diseases (HKD), caused by COL4A5 mutations in X-linked AS (XLAS) and COL4A3-4 mutations in the autosomal forms, dominant (ADAS) and recessive (ARAS). Definitive diagnosis of AS is essential for starting treatment, prognosis and genetic counseling. Next-generation sequencing (NGS) has been implemented in our lab and integrated in a multidisciplinary approach with nephrologists, pediatricians, and clinical and molecular geneticists from four hospitals in the Spanish Region of Murcia (1.5 million inhabitants). Our aim is to evaluate the yield of this approach, the genetic spectrum in AS and the predicting factors for a positive genetic result. METHOD During 3-year activity, 181 individuals (164 families) with diagnostic suspicion of AS have been analyzed by a coordinated clinical multidisciplinary protocol and NGS after informed consent. A customized Agilent panel was designed to capture 113 genes, including those related to AS: COL4A3-5. Interpretation of sequence variants was performed according to the American College of Medical Genetics and Genomics Guidelines. Sanger sequencing was performed to confirm variants identified by NGS and to segregate them in the families. After a negative result with a high suspicion of HKD, multiplex ligation-dependent probe amplification (MLPA), amplified NGS panel or clinical exome were performed. RESULTS Genetic variants were detected in 73 patients (40.9%). A total of 49 patients (27.1%) from 46 families showed pathogenic and probably pathogenic variants associated with AS. Additionally, nine patients (5.5%) were reclassified as other HKD (Table 1). After all, 58 patients (32.0%) had an HKD genetic diagnosis. There were 47 incidental findings. Comparing patients with final diagnosis of AS with patients with negative diagnosis, the former were 71.2% women (P = 0.013), mean age 49 ± 13 y.o. (no statistical differences). Among genetic AS patients, 88.1% had microhematuria (three patients had only persistent 0–5 RBC/HPF), 20.3% had hearing loss and 6.8% had ophthalmologic disturbances, with no statistical differences between groups. Hematuria quantification, characteristic glomerular hematuria or proteinuria did not associate with AS genetic diagnosis (P = 0.310, 0.056 and 0.598, respectively). Mean eGFR (CKD-EPI) and albumin-to-creatinine ratio at diagnostic were 71.89 ± 32.52 mL/min/1.73 m2 and 670 ± 870 mg/gr, without statistical differences. Positive family history was clearly present in 76.3% of final AS group (P = 0.009). Eleven patients had undergone a kidney biopsy before genetic test, and in 28 patients it was avoided because of NGS results. CONCLUSION NGS in a multidisciplinary approach has enabled the diagnosis of a collagen IV disease in 27.1% of patients with suspicion of AS and the identification of additional 5.5% with other HKD, leading to a total yield in molecular characterization of 32.6%. Additionally, the need of kidney biopsy has decreased. The most frequent AS gene in our cohort is COL4A4 followed by COL4A3 linked to ADAS, which is the predominant AS type. A positive family history was the only predicting factor for a positive genetic result, whereas glomerular hematuria, hearing loss or ophthalmologic disturbances did not predict the result. This multidisciplinary approach, with active participation of nephrologists and clinical geneticists, allows better interpretation of genetic variants, management of patients and families and accurate genetic counseling.

Background and Aims Autosomal Polycystic kidney Disease (ADPKD; ORPHA 730), Alport Syndrome (AS; ORPHA 63) and Familial Haematuria (FH) are the most frequent inherited kidney diseases. Next-generation Sequencing (NGS) has facilitated their molecular identification. A multidisciplinary team from four hospitals, with nephrologists, pediatricians, and clinical and molecular geneticists, has been formed in the Spanish region of Murcia (1.5 million inhabitants) with the lab implementation of NGS. Our aim is to evaluate the genetic spectrum in AS, FH and ADPKD and the clinical utility of this comprehensive approach. Method During 1-year activity, 114 individuals with diagnostic suspicion of ADPKD, AS or FH have been evaluated by a coordinated clinical protocol with periodic cases discussions. A customized Agilent panel was designed to capture 113 genes associated with several genetic diseases, including some related to PKD, AS or Familial Haematuria (FH): PKD1, PKD2, PKHD1, HNF1β, COL4A1, COL4A3, COL4A4 and COL4A5. Interpretation of sequence variants was performed according to the American College of Medical Genetics and Genomics (ACMG) Guidelines. Sanger sequencing was performed to confirm variants identified by NGS and to segregate them in the families. Exon 1 of PKD1 gene was also sequenced by Sanger method, due to the suboptimal capture of this region by NGS. Results We detected genetic variants in 63 patients (55.3%), pathogenic or probably pathogenic variants in 54 (47.8%). 31 patients had a variant in AS associated genes: 10 in COL4A3, 18 in COL4A4, 2 in COL4A1 and 1 in COL4A5. There were 13 pathogenic variants, 12 probably pathogenic variants and 6 variants of uncertain clinical significance (VUCS). Among them, 27 had an AD inheritance, 1 AR and 3 were sporadic. All the patients with any variant had microhaematuria, a 68% had also proteinuria, and mean eGFR at diagnostic was 63.79±21 ml/min/1.73m2. 61% had auditory disturbances and 11% ophthalmologic alterations. 4 of them had underwent kidney biopsy previously, but 3 were not adequately diagnosed, so they were reclassified after the molecular diagnosis. In total, 16 kidney biopsies were avoided by the genetic diagnosis. On the other hand, 29 patients had a variant in the ADPKD associated genes: 24 in PKD1 and 5 in PKD2. There were 20 pathogenic variants and 4 probably pathogenic variants, and their inheritance was confirmed AD in 27 patients, whereas new sporadic mutations were identified in 2 patients. 22 patients had big or enormous kidneys on sonography, although 7 had normal size. Hepatic cysts were present on 5%. 19 patients had hypertension, with a mean age of diagnosis of 47±14 years. Additionally, 1 patient was diagnosed on AR polycystic disease with homozygosity PKHD1 pathogenic variant. Among all the scope, familial history was clearly present in 43 patients, uncertain in 11, and not present in 9 patients. Conclusion The multidisciplinary approach to hereditary kidney diseases, with the active participation of nephrologists and clinical geneticists, has allowed a molecular diagnostic yield of 48% among patients with AS and ADPKD, employing NGS technology. This has led to a quicker diagnostic result in our region, the reclassification of some patient’s diagnosis, a decrease in invasive diagnostic procedures (such as kidney biopsy) and the correspondent adverse events and cost savings. Additionally, the AD inheritance pattern in AS has been confirmed as the most frequent in the region. The active participation of nephrologists in genomic medicine teams results in a better characterization of the hereditary kidney diseases, helping in the genetic variant interpretation and management of these patients and their families.

Nanoenabled products (NEPs) have numerous outdoor uses in construction, transportation or consumer scenarios, and there is evidence that their fragments are released in the environment at low rates. We hypothesized that the lower surface availability of NEPs fragment reduced their environmental effects with respect to pristine nanomaterials. This hypothesis was explored by testing fragments generated by intentional micronisation ("the SUN approach"; Nowack et al. Meeting the Needs for Released Nanomaterials Required for Further Testing: The SUN Approach. Environmental Science & Technology, 2016 (50), 2747). The NEPs were composed of four matrices (epoxy, polyolefin, polyoxymethylene, and cement) with up to 5% content of three nanomaterials (carbon nanotubes, iron oxide, and organic pigment). Regardless of the type of nanomaterial or matrix used, it was observed that nanomaterials were only partially exposed at the NEP fragment surface, indicating that mostly the intrinsic and extrinsic properties of the matrix drove the NEP fragment toxicity. Ecotoxicity in multiple assays was done covering relevant media from terrestrial to aquatic, including sewage treatment plant (biological activity), soil worms (Enchytraeus crypticus), and fish (zebrafish embryo and larvae and trout cell lines). We designed the studies to explore the possible modulation of ecotoxicity by nanomaterial additives in plastics/polymer/cement, finding none. The results support NEPs grouping by the matrix material regarding ecotoxicological effect during the use phase. Furthermore, control results on nanomaterial-free polymer fragments representing microplastic had no significant adverse effects up to the highest concentration tested.

Introduccion y objetivos La caracterizacion molecular de cardiopatias incluye una entidad congenita no infrecuente, el sindrome de Noonan. Presentamos el estudio de seis genes de la via RAS-MAPK en pacientes espanoles: perfil genotipico, impacto de la cardiopatia y expresividad clinica. Metodos Compusieron la poblacion en estudio 643 pacientes (y 182 familiares) diagnosticados por dismorfologos, cardiologos y endocrinopediatras de 74 hospitales (11 comunidades). Estudio primario de PTPN11 y complementario de SOS1, RAF1, BRAF, KRAS y HRAS, estratificado y orientado por signos clinicos, mediante secuenciacion de exones recurrentes (un 80-95% de mutaciones descritas). Resultados Se documento mutacion en 230 pacientes (91 mujeres, 139 varones) de 200 familias (31%), 172 PTPN11 +, 14 SOS1 +, 9 RAF1 + y 5 BRAF +, con referencia explicita a la cardiopatia padecida en 156 casos indice; 103 presentaban estenosis de la valvula pulmonar; 12, estenosis de la valvula pulmonar y miocardiopatia hipertrofica; 18, miocardiopatia hipertrofica y 14, otra cardiopatia; en solo 9 casos se encontraba ausente. En 23/30 familiares positivos no habia o no constaba cardiopatia. El rendimiento diagnostico fue superior (p = 0,016) para las muestras de algunos centros (53%; 14/32), y alcanzo el 64% (9/14; p = 0,019) en profesionales concretos. El rendimiento cayo al 18% en los pacientes sin datos clinicos facilitados. El dato genotipico reoriento el diagnostico clinico en 26 pacientes. Conclusiones El 94% de los pacientes portadores de mutacion presentaban cardiopatia, el 79% estenosis de la valvula pulmonar y el 12% miocardiopatia hipertrofica. En el 76% de los familiares positivos con rasgos clinicos compatibles, no se habia documentado la cardiopatia. El estudio molecular es una herramienta util en estos sindromes, aunque debe progresarse en la objetivacion del diagnostico clinico

Introduction and objectives Molecular characterization of congenital heart diseases now includes the not infrequent dysmorphic Noonan syndrome. A study of 6 genes of the RAS-MAPK pathway in Spanish patients is presented: the impact of heart disease, clinical expressivity, and diagnostic yield are investigated. Methods The study included 643 patients (and 182 family members) diagnosed by dysmorphologists, cardiologists, and pediatric endocrinologists from 74 tertiary hospitals. Bidirectional sequencing analysis of PTPN11, SOS1, RAF1, BRAF, KRAS and HRAS focused on exons carrying recurrent mutations accounting for 80% to 95% of previously described mutations. Results Mutations were detected in 230 patients (91 women and 139 men) in 200 (31%) families (172 PTPN11 +, 14 SOS1 +, 9 RAF1 +, 5 BRAF +). There was specific reference to the heart defect suffered in 156 index cases: 103 patients had shown pulmonary stenosis, 12 pulmonary stenosis with hyperthrophic cardiomyopathy, 18 hypertrophic cardiomiopathy, and 14 other cardiopathies; heart disease was absent in 9 index cases. Heart disease had not been documented in 23 of 30 family members with positive genotype and compatible clinical signs. Diagnostic yield was higher ( P =.016) for samples from some centers (53%; 14/32) and even from certain professionals (64%; 9/14; P =.019). Characterization rate was 18% in patients for whom clinical data were not available. Genotyping led to a more precise diagnosis in 26 patients. Conclusions Most patients (94%) with a positive genotype had known congenital heart disease, 79% pulmonary stenosis and 12% hyperthrophic cardiomyopathy. Cardiopathy had not been documented in 76% of family members carrying the mutation. Molecular study is a useful tool in these syndromes but a more rigorous clinical diagnosis should be intended as well.