Framework From a Multidisciplinary Approach for Transitioning Variants of Unknown Significance From Clinical Genetic Testing in Kidney Disease to a Definitive Classification

Abstract

Introduction: Monogenic causes in over 300 kidney-associated genes account for approximately 12% of end stage kidney disease (ESKD) cases. Advances in sequencing and large customized panels enable the noninvasive diagnosis of monogenic kidney disease at relatively low cost, thereby allowing for more precise management for patients and their families. A major challenge is interpreting rare variants, many of which are classified as variants of unknown significance (VUS). We present a framework in which we thoroughly evaluated and provided evidence of pathogenicity for HNF1B-p.Arg303His, a VUS returned from clinical diagnostic testing for a kidney transplant candidate.

Methods: A blueprint was designed by a multidisciplinary team of clinicians, molecular biologists, and diagnostic geneticists. The blueprint included using a health system-based cohort with genetic and clinical information to perform deep phenotyping of VUS heterozygotes to identify the candidate VUS and rule out other VUS, examination of existing genetic databases, as well as functional testing.

Results: Our approach demonstrated evidence for pathogenicity for HNF1B-p.Arg303His by showing similar burden of kidney manifestations in this variant to known HNF1B pathogenic variants, and greater burden compared to noncarriers.

Conclusion: Determination of a molecular diagnosis for the example family allows for proper surveillance and management of HNF1B-related manifestations such as kidney disease, diabetes, and hypomagnesemia with important implications for safe living-related kidney donation. The candidate gene-variant pair also allows for clinical biomarker testing for aberrations of linked pathways. This working model may be applicable to other diseases of genetic etiology.

Keywords: HNF1B-MODY; autosomal dominant tubulointerstitial kidney disease (ADTKD); chronic kidney disease; genetics; hypomagnesemia; pancreatitis.

Graphical abstract

Figure 1

Imaging and histopathology of Family A proband (case AIII-1) HNF1B-p.Arg303His heterozygote showed bilateral small kidneys and oligomeganephronia. (A) Axial, (B) sagittal, and (C) coronal images from noncontrast CT scan taken at age 24 show small kidneys without cysts, and no overt abnormality of the pancreas and the liver. (D) Hematoxylin and eosin staining of renal biopsy specimen showed focal interstitial inflammation and mild tubular injury (10×10 magnification). Focal interstitial fibrosis is also present, which is highlighted by (E) Jones silver staining (4×10 magnification). (F) In the cortical areas, the number of glomeruli is reduced. Mild tubular hypertrophy associated with mildly enlarged glomeruli is observed as shown in a representative image (20×10 magnification).

Figure 2

Family A pedigree and pathogenic classification of HNF1B-p.Arg303His. (A) Male members are represented in squares, females in circles. Filled-in symbols indicate affected members with clinical features previously observed in HNF1B loss of function patients are shown, open symbols show unaffected members, and hashed symbols are members highly suspicious for the kidney disease. Positive sign indicates that the individual is heterozygous for HNF1B-p.Arg303His, and a negative sign indicates that the individual was genotyped and does not harbor HNF1B-p.Arg303His. The proband is identified with an arrow. Each generation with informative health records are labeled with Roman numerals on the left. (B) Pathogenic reclassification of HNF1B-p.Arg303His per ACMG-AMP guidelines. The rules and the explanation for meeting the rules before this study and after this study are indicated. For cosegregation evidence, see text. CKD, chronic kidney disease; HTN, hypertension, eGFR, estimated glomerular filtration rate.

Figure 3

Serum biomarkers of HNF1B-p.Arg303His cases indicate decline in kidney and pancreatic function similar to HNF1B whole gene deletion. Lifetime laboratory measures from age 18 to 60 years for individuals with HNF1B whole gene deletion (17q12 microdeletion), HNF1B-p.Arg303His (case AII-1, case AIII-1, case AIII-2, and case BI-1), and noncarriers of HNF1B are plotted against the age of measurement. Linear regression was performed for 17q12 microdeletion and noncarrier groups. For clarity, only regression lines for noncarriers and 95% confidence intervals are shown. Data for HNF1B-p.Arg303His are shown as individual measurement per case due to a smaller number of tests. Biochemical measures and number of individuals with measures are as follow: (A) Estimated GFR (n for noncarriers = 115,298; n for 17q12 microdeletion = 12), (B) Serum magnesium (n noncarriers = 21,411; n 17q12 microdeletion = 4), (C) Serum lipase (n noncarriers = 24,323, n 17q12 microdeletion = 4), (D) Serum amylase (n noncarriers = 18,116; n 17q12 microdeletion = 4). See materials and methods for details.

Figure 4

Proposed framework for transitioning a VUS from clinical genetic testing to a more definitive classification. The case presented in this study provided evidence for a framework that can be utilized to prioritize rare VUS results from clinical genetic testing for other kidney diseases. In the study, clinical genetic testing returned multiple VUS for a kidney transplant candidate and her family members. Identifying the VUS and other known pathogenic variants of that gene in the MyCode database and deep phenotyping of individuals with the VUS compared to noncarriers narrowed the VUS pool to HNF1B-p.Arg303His. Studies of variant effect on protein function, additional clinical workup (Mg wasting), and studies of variant cosegregation with disease traits provide further supporting evidence of pathogenicity. gnomAD, genome aggregation database; HGMD, human gene database; LOVD, Leiden Open Variation Database; P/LP, pathogenic/likely pathogenic, VUS, variant of unknown significance.