MO039: Multidisciplinary approach improves genetic diagnosis of Alport syndrome in the next-generation sequencing ERA
Authors: Galan Carrillo Isabel , Galbis Liliana , Martínez Jiménez Víctor , Pablo Ortuño Pedro , Roca Susana , David Castro Juan , Ramos Fernanda , Rodríguez Lidia , Guillén Encarnación 
Published: May 2022

Abstract

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.