SP025
ATYPICAL HEMOLYTIC UREMIC SYNDROME TARGETED RE-SEQUENCING STUDY IN A SOUTH ITALIAN COHORT OF PATIENTS

Introduction and Aims: Atypical hemolytic uremic syndrome (aHUS) is a rare disease characterized by microangiopathic hemolytic anemia, thrombocytopenia and renal dysfunction. It is a multifactorial disease due to autoimmune or genetic factors leading to deregulated alternative complement pathway activation.

At present twelve genes are associated to aHUS. However, the correlation genotype-phenotype is still mostly obscure. We reconstructed the entire clinical records of the Apulian Region in Italy, enrolling twenty patients and two relatives. This corresponds to a prevalence of 5/million people, consistent with the reported worldwide frequency of 1-9 cases/million. We performed targeted massively parallel re-sequencing of 12 known and 1 candidate gene in all patients to investigate the role of the genotype in determining the phenotypic spectrum of aHUS and identify novel mutations. The overlapping with other diseases such as Dense Deposits Disease (DDD), C3 glomerulopathy and Age-related Macular Degeneration (AMD), all sharing the alternative complement pathway deregulation, was also investigated.

Methods: Patients and their relatives were accurately selected for their history of sporadic aHUS. Target enrichment was performed using an Illumina TruSeq Custom Amplicon panel designed for our 13 genes, achieving an overall 97% coverage across all targets.

Library preparation was performed according to the TruSeq Custom Amplicon library preparation guide. Sequencing was performed on an Illumina MiSeq Desktop Sequencer. Annotation of variants was performed using Illumina VariantStudio software. All variants were evaluated for their pathogenicity according to CADD, Sift and Polyphen algorithms.

Results: Overall, 92.3% of the targets (13 genes) was sequenced at >25X coverage, required for accurate variant calling. We also were able to validate sequence data on 3 patients previously screened with Sanger technology for mutations in at least part of the genes of interest, achieving overall 100% of sensitivity across 100% variants.

We identified 30 missense and one nonsense variants fulfilling our quality criteria in known aHUS genes, plus seven missense variants in our candidate gene. Several combined variants were detected. Their presence might explain the different phenotypic nuances of our patients, as well as, in some cases, signs of other Complement-related diseases, such as AMD and DDD, although all the patients definitely express a aHUS phenotype.

Conclusions: Overall, our data suggest that (1) our candidate gene should be added to the list of genes to be screened for aHUS, (2), the overlapping with other complement-related diseases is considerable, often resulting in complex phenotypes, (4) mutations should therefore always be analyzed in combination, never singularly, (5) the high-throughput strategy is the only feasible in this scenario.

This approach would lead to a precise molecular assessment of aHUS patients which is an essential requirement in the management and treatment of the disease, in order to optimize the decision-making process towards them, especially in the perspective of renal transplant.