Clinical exome sequencing as a tool for genetic diagnosis of alport syndrome and thin basement membrane disease

Abstract Background and Aims Alport syndrome (AS) is an inherited nephropathy caused by pathogenic variants in COL4A3 (autosomal dominant -AD- and autosomal recessive -AR- inheritance), COL4A4 (AR) and COL4A5 (X-linked dominant -XLD). It is characterized by glomerular nephropathy with hematuria progressing to end-stage renal disease, frequently associated with sensorineural deafness and ocular anomalies. In thin basement membrane disease (TBMD) most patients are asymptomatic and are incidentally noted to have microhematuria, mild proteinuria, and occasionally gross hematuria, with normal renal function. This condition is caused by pathogenic variants in COL4A3 and COL4A4 (AD). We have systematically applied genetic testing to pediatric and adult patients with clinical features suggestive of AS (Table 1). Method Clinical exome sequencing (CES) was performed on a cohort of 95 patients referred to our center from 2019 to 2022. Analyses were performed on an in-silico designed panel including 523 renal genes. Whenever possible, identified variants were segregated by Sanger sequencing. Results We identified causative (C4/C5) variants in 43 (45.3%) patients. Among them, genetic diagnosis of AS was obtained in 25 patients (58.1%): one (4.0%) with biallelic variants in COL4A4, 13 (52.0%) with COL4A5 variants (6 females and 7 males), 5 (20.0%) with COL4A3 variants (4 heterozygous and 1 homozygous) and 6 (24.0%) cases with coexisting variants in one or more other collagen genes. In 16 (37.2%) patients, we found causative monoallelic variants compatible with TBMD: 12 (75.0%) patients with variants in COL4A4 and 4 (25.0%) in COL4A3. Lastly, in 2 cases, we found variants in COL4A1 (#120130) and MYH9 (#160775), both genes associated with other renal diseases with a clinical presentation partially overlapping with AS. In additional 16 cases (16.8%), we identified C3 variants in collagen genes. In the remaining patients, genetic testing was negative (14 patients, 14.7%) or inconclusive (22, 23.2%). Of note, we identified 6 families with digenic AS. In 4 of them - 3 with COL4A5/COL4A4 variants and one with COL4A3/COL4A4 variants – the co-existence of two variants was associated to earlier renal failure, compared to family members bearing a single variant. In contrast, in the remaining 2 families (33.3%), members with COL4A5/COL4A3 missense variants had a milder phenotype compared to male family members with the single variant COL4A5 variant. In families with digenic AS, at least one variant involved the substitution of a glycine with another amino acid in the triple helix domain, frameshift indel variants in the triple helix domain or inframe indel in the collagen IV domain non-collagenous. To better understand the impact of multiple variants on collagen structure, computational studies are currently ongoing. Conclusion CES is a powerful tool to clearly define the diagnosis when AS or TBMD are suspected, as witnessed by a detection rate of 43.2%. Genetic diagnosis allows to identify the causative gene offering the possibility of extending diagnosis to other family members, also in the prenatal setting. In addition, it offers the possibility to identify multiple variants in collagen genes associated with digenic AS, as well as to find variants in other genes implicated in kidney disease. Notably, the presence of digenic variants is not necessarily predictive of a worse disease outcome. For all these reasons, molecular diagnosis can be useful to improve clinical management, to calculate recurrence risk and to better define the prognosis of the patient.