A Custom Targeted Next-Generation Sequencing Gene Panel for the Diagnosis of Genetic Nephropathies (2024)

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Supplementary Materials

To the Editor:

Traditional strategies for determining the precise genetic cause of kidney disease are costly and time consuming. However, recent advances in high-throughput sequencing have dramatically decreased the cost, time, and labor involved. With new data emerging about the role of genetics in kidney disease, realizing the era of personalized and precision medicine will require a precise diagnosis and identification of the underlying molecular pathogenesis.^1,2 We describe a project to design and validate a next-generation sequencing (NGS) panel for the full spectrum of genetic nephropathies in a CLIA-approved laboratory and to test it in a series of kidney biopsies with unknown molecular pathogenesis.

A custom targeted NGS gene panel was designed covering 301 genes described in kidney disease (the full list, which may be helpful for others developing similar panels, is provided in table a of Item S1). Validation runs showed accurate results, with a mean of >99% coverage of the targeted regions at a depth greater than 20×. Performance characteristics of the assay were excellent (table b of Item S1), based on comparison to a reference genome and Sanger sequencing.³

After assay validation, we sequenced extracted DNA from kidney biopsy samples under an IRB-approved protocol (Schulman Associates IRB). We sought to interrogate cases in which the pathogenesis remained unknown after kidney biopsy. Fifty consecutive kidney biopsies with adequate DNA that met inclusion criteria were sequenced. This included patients younger than 40 years, with biopsy evidence of either idiopathic FSGS or cases without FSGS that had at least moderate chronic injury with no discernable cause either by biopsy or clinical parameters. Variants identified were classified according to relevant guidelines.⁴ We were able to identify pathogenic or likely pathogenic variants in 23 patients (Table 1). Five patients had a known family history of kidney disease, including 3 with FSGS on biopsy and 2 with CKD, NOS. Of those with a family history, 4 of 5 had an identified genetic cause for their kidney disease. All pathogenic variants were validated by a second modality such as APOL1 genotyping or Sanger sequencing, with 100% accuracy.

In both the FSGS and idiopathic CKD groups, >40% of the participants studied had evidence of a monogenic cause for their kidney disease. There was an especially large genetic disease burden among the African Americans studied, secondary to APOL1 risk alleles (table c of Item S1). We found that this is by far the largest contributor to genetic kidney disease in our patients and is responsible for nephropathy more than twice as often as all other genes combined (Table 1). Outside of African Americans, we identified only one patient (identified as Hispanic) who harbored one APOL1 risk allele. Information for patients’ clinical details and genetic diagnosis is presented in table d of Item S1.

The pattern of FSGS on biopsy in the primary FSGS group was not predictive of the presence of genetic abnormalities. Of 22 (61%) cases that showed a nonspecific NOS FSGS pattern, 9 (41%) had pathogenic variants identified by the panel, including 5 (23%) with APOL1 risk alleles. The rest included 2 cases with pathogenic variants in INF2 and 1 case each of COL4A4 and COL4A5 variants. Twelve cases showed a collapsing pattern of FSGS (33%). Seven (58%) of these cases had evidence of a genetic cause, including 6 (50%) due to APOL1 risk variants. Overall, cases with the collapsing pattern of FSGS were more likely to have a genetic cause than those with an NOS pattern, but the difference was not significant (P = 0.5).

Genetic testing enabled a specific molecular pathogenesis-based diagnosis in 46% of biopsies studied. This more specific genetic diagnosis provides important information for a number of reasons. Some data suggest that these patients might be less likely to respond to immunosuppressant medications.⁵ If verified, patients with an identified genetic cause could potentially be spared long-term intensive immunosuppressant regimens with the accompanying serious adverse side effects. Moreover, if specific treatment were to become available, the patient would not know they were eligible without knowledge of what is causing the disease. There was 1 patient with COQ2-associated FSGS in which the genetic diagnosis indicates the need for a specific treatment.⁶ Also, knowledge of the specific cause often offers peace of mind for patients, and a genetic diagnosis provides opportunities for genetic counseling and family planning decisions.

Testing this cohort provided us with important information about our kidney biopsy population and also enabled us to validate the NGS assay on DNA extracted from kidney biopsy tissue. Our results suggest that a panel-based approach using NGS is an efficient and cost-effective approach to the diagnosis of genetic nephropathies.

Supplementary Material

Acknowledgements

Support: This research was supported by NIH/NIDDK grant R43DK103432.

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