Framework from a multidisciplinary approach for transitioning variants of unknown significance from clinical genetic testing in kidney disease to a definitive classification

IntroductionMonogenic causes account for up to 40% of patients in cohorts enriched with end stage kidney disease (ESKD) or familial kidney disease (Westland R. Renkema K.Y. Knoers N. Clinical Integration of Genome Diagnostics for Congenital Anomalies of the Kidney and Urinary Tract., Groopman E.E. Marasa M. Cameron-Christie S. Petrovski S. Aggarwal V.S. Milo-Rasouly H. et al.Diagnostic Utility of Exome Sequencing for Kidney Disease., Connaughton D.M. Kennedy C. Shril S. Mann N. Murray S.L. Williams P.A. et al.Monogenic causes of chronic kidney disease in adults., Mann N. Braun D.A. Amann K. Tan W. Shril S. Connaughton D.M. et al.Whole-Exome Sequencing Enables a Precision Medicine Approach for Kidney Transplant Recipients.). Genetic testing has become an invaluable tool in the diagnosis of various types of chronic kidney disease (CKD), including but not limited to autosomal dominant tubulointerstitial kidney disease (ADTKD), autosomal dominant polycystic kidney disease (ADPKD), congenital anomalies of the kidney and urinary tract (CAKUT), focal segmental glomerulosclerosis (FSGS), and CKD of unknown cause (Groopman E.E. Marasa M. Cameron-Christie S. Petrovski S. Aggarwal V.S. Milo-Rasouly H. et al.Diagnostic Utility of Exome Sequencing for Kidney Disease., Knoers N. Antignac C. Bergmann C. Dahan K. Giglio S. Heidet L. et al.Genetic testing in the diagnosis of chronic kidney disease: recommendations for clinical practice.). Genetic testing allows for more precise molecular diagnosis—in some cases diagnostic reclassification. Benefits of establishing a molecular diagnosis include earlier treatment with disease-modifying therapies, screening for extrarenal manifestations, avoidance of invasive procedures such as kidney biopsy, and important implications for family planning and living kidney donation (Connaughton D.M. Kennedy C. Shril S. Mann N. Murray S.L. Williams P.A. et al.Monogenic causes of chronic kidney disease in adults., Knoers N. Antignac C. Bergmann C. Dahan K. Giglio S. Heidet L. et al.Genetic testing in the diagnosis of chronic kidney disease: recommendations for clinical practice., Jayasinghe K. Stark Z. Kerr P.G. Gaff C. Martyn M. Whitlam J. et al.Clinical impact of genomic testing in patients with suspected monogenic kidney disease.).HNF1B encodes the hepatocyte nuclear factor 1β (HNF1B) and is a member of the homeodomain-containing superfamily of transcription factors involved in the development of kidney, urogenital tract, pancreas, liver, brain, and parathyroid gland (Groopman E.E. Marasa M. Cameron-Christie S. Petrovski S. Aggarwal V.S. Milo-Rasouly H. et al.Diagnostic Utility of Exome Sequencing for Kidney Disease.). Pathogenic HNF1B variants lead to a wide spectrum of phenotypic expression ranging from non-insulin dependent, maturity onset diabetes of the young (MODY), pancreatic hypoplasia, liver cholestasis, and several renal phenotypes. Renal phenotypes include an ADPKD phenocopy spectrum (renal cysts and diabetes [RCAD] syndrome), an ADTKD phenocopy spectrum (ADTKD-HNF1B), CAKUT, and biochemical anomalies across all (e.g. hypomagnesemia, hyperuricemia, hyperparathyroidism) (

Edghill EL, Bingham C, Ellard S, Hattersley AT. Mutations in hepatocyte nuclear factor-1beta and their related phenotypes. J Med Genet. 432006. p. 84-90.

, Ulinski T. Lescure S. Beaufils S. Guigonis V. Decramer S. Morin D. et al.Renal phenotypes related to hepatocyte nuclear factor-1beta (TCF2) mutations in a pediatric cohort., Clissold R.L. Hamilton A.J. Hattersley A.T. Ellard S. Bingham C. HNF1B-associated renal and extra-renal disease-an expanding clinical spectrum., Adalat S. Woolf A.S. Johnstone K.A. Wirsing A. Harries L.W. Long D.A. et al.HNF1B mutations associate with hypomagnesemia and renal magnesium wasting., van der Made C.I. Hoorn E.J. de la Faille R. Karaaslan H. Knoers N.V. Hoenderop J.G. et al.Hypomagnesemia as First Clinical Manifestation of ADTKD-HNF1B: A Case Series and Literature Review., Raaijmakers A. Corveleyn A. Devriendt K. van Tienoven T.P. Allegaert K. Van Dyck M. et al.Criteria for HNF1B analysis in patients with congenital abnormalities of kidney and urinary tract.). The phenotypic expression of HNF1B heterozygotes varies even between individuals with the same mutation within families, possibly as a result of temporal stochastic variations in HNF1B expression during nephrogenesis(Clissold R.L. Hamilton A.J. Hattersley A.T. Ellard S. Bingham C. HNF1B-associated renal and extra-renal disease-an expanding clinical spectrum.). As there are a large number of genes associated with HNF1B renal phenotypes, the use of exome sequencing or massively parallel sequencing (MPS) can be very helpful (Westland R. Renkema K.Y. Knoers N. Clinical Integration of Genome Diagnostics for Congenital Anomalies of the Kidney and Urinary Tract., Groopman E.E. Marasa M. Cameron-Christie S. Petrovski S. Aggarwal V.S. Milo-Rasouly H. et al.Diagnostic Utility of Exome Sequencing for Kidney Disease., Mann N. Braun D.A. Amann K. Tan W. Shril S. Connaughton D.M. et al.Whole-Exome Sequencing Enables a Precision Medicine Approach for Kidney Transplant Recipients., Knoers N. Antignac C. Bergmann C. Dahan K. Giglio S. Heidet L. et al.Genetic testing in the diagnosis of chronic kidney disease: recommendations for clinical practice., Mallett A.J. McCarthy H.J. Ho G. Holman K. Farnsworth E. Patel C. et al.Massively parallel sequencing and targeted exomes in familial kidney disease can diagnose underlying genetic disorders., Rao J. Liu X. Mao J. Tang X. Shen Q. Li G. et al.Genetic spectrum of renal disease for 1001 Chinese children based on a multicenter registration system., Eckardt K.U. Alper S.L. Antignac C. Bleyer A.J. Chauveau D. Dahan K. et al.Autosomal dominant tubulointerstitial kidney disease: diagnosis, classification, and management--A KDIGO consensus report.).MPS has become increasingly available and more affordable in clinical practice(Cocchi E. Nestor J.G. Gharavi A.G. Clinical Genetic Screening in Adult Patients with Kidney Disease., Phillips K.A. Deverka P.A. Hooker G.W. Douglas M.P. Genetic Test Availability And Spending: Where Are We Now? Where Are We Going?.). MPS allows the evaluation of multiple genes and thus can be particularly useful for monogenic kidney disorders with broad differential genetic causes. A consequence of testing dozens if not hundreds of genes linked to kidney disease is that many variants of unknown significance (VUS) are often detected. In various cohorts, up to 10-100% of genetic results may be VUS, classified according to the American College of Medical Genetics-Association for Molecular Pathology (ACMG-AMP) criteria (Connaughton D.M. Kennedy C. Shril S. Mann N. Murray S.L. Williams P.A. et al.Monogenic causes of chronic kidney disease in adults., Elhassan E.A.E. Murray S.L. Connaughton D.M. Kennedy C. Cormican S. Cowhig C. et al.The utility of a genetic kidney disease clinic employing a broad range of genomic testing platforms: experience of the Irish Kidney Gene Project., Thomas C.P. Freese M.E. Ounda A. Jetton J.G. Holida M. Noureddine L. et al.Initial experience from a renal genetics clinic demonstrates a distinct role in patient management., Lieberman K.V. Chang A.R. Block G.A. Robinson K. Bristow S.L. Devarajan P. et al.The KIDNEYCODE program: Diagnostic yield and clinical features of individuals with chronic kidney disease.). VUS present a diagnostic and ethical challenge in genetic testing and lack of resolution may result in delays in treatment and management (Hay E. Cullup T. Barnicoat A. A practical approach to the genomics of kidney disorders.). Variant reclassification from VUS to (likely) benign, or (likely) pathogenic improves as data sharing and variant curation efforts from expert panels (e.g. ClinGen and Genomics England) expand (Knoers N. Antignac C. Bergmann C. Dahan K. Giglio S. Heidet L. et al.Genetic testing in the diagnosis of chronic kidney disease: recommendations for clinical practice.). Apart from case studies, implementation and strategies to efficiently transition kidney gene VUS to a more definitive classification are lacking.Clinically unselected research population databases present a unique resource that can be used to triage VUS. The MyCode DiscovEHR database, currently comprised of ∼173,000 individuals who have exome sequencing and linked-electronic health records (EHR) (Carey D.J. Fetterolf S.N. Davis F.D. Faucett W.A. Kirchner H.L. Mirshahi U. et al.The Geisinger MyCode community health initiative: an electronic health record-linked biobank for precision medicine research.), includes rich, longitudinal in-patient and out-patient data on multiple generations spanning over 20 years that could be leveraged for studying clinical features of heterozygotes of rare VUS that are returned from clinical genetic testing.

In this exemplar study, we present a framework of efforts from a multidisciplinary team with expertise in nephrology, endocrinology, molecular biology, and diagnostic genetics to gather evidence for a diagnostic reclassification of HNF1B c.907C>T p.Arg303His from a VUS to a likely pathogenic variant, identified through a clinical MPS panel for a patient with CKD of unknown cause.

DISCUSSIONIn an effort to streamline the genetic diagnosis for a kidney transplant candidate, we present a framework to prioritize rare VUS results from clinical genetic testing for kidney disease developed through efforts from a multidisciplinary team (Figure 4). The standard workflow following receipt of a rare VUS result consistent with the clinical phenotype should include literature review, examination of reference databases (e.g. ClinVar, LOVD, gnomAD), and if other affected family members exist, additional deep phenotyping (e.g. FEmg)(van der Made C.I. Hoorn E.J. de la Faille R. Karaaslan H. Knoers N.V. Hoenderop J.G. et al.Hypomagnesemia as First Clinical Manifestation of ADTKD-HNF1B: A Case Series and Literature Review., Raaijmakers A. Corveleyn A. Devriendt K. van Tienoven T.P. Allegaert K. Van Dyck M. et al.Criteria for HNF1B analysis in patients with congenital abnormalities of kidney and urinary tract.), and family testing to test for co-segregation. Consultation with a Clingen curation panel or experts in the gene of interest should be considered. If resources allow, additional research from other sources may be helpful to provide supportive evidence. In this case, we collaborated with colleagues who performed in vitro functional testing, albeit with inconclusive results. We also interrogated a large research cohort (MyCode) to compare phenotypic traits of HNF1B Arg303His heterozygotes with individuals with 17q12 microdeletion and noncarriers to demonstrate consistency in supporting pathogenicity. It should be noted that these types of associative analyses do not explicitly fulfill ACMG criteria. Regardless, demonstration of lower magnesium levels and lower eGFR in the heterozygotes compared to non-carriers provided additional supportive evidence to fulfill the ACMG PP1 criteria, hence, reclassification of the variant from VUS to LP.Figure thumbnail gr4

Figure 4Proposed 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 co-segregation with disease traits provide further supporting evidence of pathogenicity. Abbreviations: P/LP Abbreviations: P/LP pathogenic/likely pathogenic, VUS variant of unknown significance, LOVD Leiden Open Variation Database, gnomAD Genome Aggregation Database, HGMD Human Gene Database

Using data from our research population cohort, we show that HNF1B-p.Arg303His heterozygotes had eGFR and serum magnesium levels that were comparable or lower than those seen in individuals with 17q12 microdeletion. Even though the proband’s sister does not presently have CKD as defined as eGFR 2, her eGFR measured twice at age 21 (less than the 1st percentile for her age and sex), and her serum magnesium level at age 21 were significantly lower than noncarriers of pathogenic HNF1B variants (mean [95% CI] 1.8 mg/dL vs. 2.3 [2.1, 2.5], n = 174 individuals measured at age 21 years old in noncarriers). Notably, we observed that 3 of the 4 HNF1B-p.Arg303His cases had elevated serum lipase compared to individuals with17q12 microdeletion and noncarriers of pathogenic HNF1B variants. With HNF1B as the candidate gene, we further confirmed that the proband and her mother had hypermagnesuria (FEMg) as seen with HNF1B extrarenal abnormalities(van der Made C.I. Hoorn E.J. de la Faille R. Karaaslan H. Knoers N.V. Hoenderop J.G. et al.Hypomagnesemia as First Clinical Manifestation of ADTKD-HNF1B: A Case Series and Literature Review., Adalat S. Hayes W.N. Bryant W.A. Booth J. Woolf A.S. Kleta R. et al.HNF1B Mutations Are Associated With a Gitelman-like Tubulopathy That Develops During Childhood.).Our index case and four other family members had multiple VUS returned from clinical genetic testing. There were only two VUS that were shared amongst the family members: PTH1R-p.Ala72Val and HNF1B-p.Arg303His, but the phenotype of tubulointerstitial kidney disease, hypomagnesemia and pancreas dysfunction was consistent only with HNF1B. Typical signs of PTH1R mutations such as Murk Jansen type of metaphyseal chondrodysplasia, characterized by abnormal height, hypercalcemia, bone deformities, and renal calcification, were absent (Saito H. Noda H. Gatault P. Bockenhauer D. Loke K.Y. Hiort O. et al.Progression of Mineral Ion Abnormalities in Patients With Jansen Metaphyseal Chondrodysplasia.), and the PTH1R p.Ala72Val was also harbored by the proband’s asymptomatic cousin (Case AIII-3).Our study exemplifies the important role of large, unselected cohorts with robust EHR data to provide corroborating evidence of clinical traits for the gene-disease pair associated with rare VUS. It is important to note that variable penetrance and clinical phenotype of monogenic disorders (e.g. HNF1B) can make determination of pathogenicity more challenging, and large cohorts can be very useful to provide confidence on pathogenicity (Mirshahi2022-medrxiv). In a disease with high heterogeneity even within the same family, the presence of similar clinical spectrum between the proband, her family members, the MyCode participant with HNF1B-p.Arg303His, and an individual in ClinVar is highly supportive of this variant being causal for her CKD. Indeed, we observed renal and extra-renal features in all 7 individuals from clinical data (Family A and Family C, ClinVar individual with scant data) as well as the participant from the MyCode research study (Family B) who all had HNF1B-p.Arg303His in common. Reports of other renal abnormalities in individuals with HNF1B-p.Arg303Ser and p.Arg303Cys lend support that the arginine residue at this locus is important in HNF1B function. The Grantham’s distance which predicts the dissimilarity of amino acid substitutions by composition, polarity, and molecular volume for arg to his (Kompatscher A. de Baaij J.H.F. Aboudehen K. Hoefnagels A. Igarashi P. Bindels R.J.M. et al.Loss of transcriptional activation of the potassium channel Kir5.1 by HNF1β drives autosomal dominant tubulointerstitial kidney disease.) Amino acid difference formula to help explain protein evolution.). Further, the arginine 303 is in the POUH domain and is conserved in multiple species including human, mouse, rat, frog, and zebrafish (Supplement Figure S2).PKHD1 and FXYD2 are known transcriptional targets of HNF1B, and disturbed transcription of these genes may cause kidney malformation and hypomagnesemia, respectively (Ferrè S. de Baaij J.H. Ferreira P. Germann R. de Klerk J.B. Lavrijsen M. et al.Mutations in PCBD1 cause hypomagnesemia and renal magnesium wasting., Hiesberger T. Shao X. Gourley E. Reimann A. Pontoglio M. Igarashi P. Role of the hepatocyte nuclear factor-1beta (HNF-1beta) C-terminal domain in Pkhd1 (ARPKD) gene transcription and renal cystogenesis., Ferrè S. Veenstra G.J. Bouwmeester R. Hoenderop J.G. Bindels R.J. HNF-1B specifically regulates the transcription of the γa-subunit of the Na+/K+-ATPase.). Our luciferase reporter experiments using wild-type HNF1B and HNF1B-p.Arg303His showed similar transactivation of the PKHD1 and FXYD2 promoters. However, absence of an effect by the mutant on transactivation effect does not exclude pathogenicity. For example, HNF1B-p.Val61Gly showed comparable transactivation potential to wildtype HNF1B in a luciferase reporter assay(Granberg C.F. Harrison S.M. Dajusta D. Zhang S. Hajarnis S. Igarashi P. et al.Genetic basis of prune belly syndrome: screening for HNF1β gene.), even though this variant was observed in 3 children with HNF1B-related disorders: a child with a single ovary, a single kidney, and a hemi-uterus(

Edghill EL, Bingham C, Ellard S, Hattersley AT. Mutations in hepatocyte nuclear factor-1beta and their related phenotypes. J Med Genet. 432006. p. 84-90.

), and a child with prune belly syndrome and congenital genitourinary malformation(Granberg C.F. Harrison S.M. Dajusta D. Zhang S. Hajarnis S. Igarashi P. et al.Genetic basis of prune belly syndrome: screening for HNF1β gene.), and a child with multicystic dysplastic kidney(

Hoskins BE, Cramer CH, 2nd, Tasic V, Kehinde EO, Ashraf S, Bogdanovic R, et al. Missense mutations in EYA1 and TCF2 are a rare cause of urinary tract malformations. Nephrol Dial Transplant. 23. England2008. p. 777-779.

). Mild to no alterations in in vitro studies were also observed in another homeodomain variant, HNF1B-p.Arg295His; this variant was observed in a family of multiple kidney and pancreatic anomalies(Bohn S. Thomas H. Turan G. Ellard S. Bingham C. Hattersley A.T. et al.Distinct molecular and morphogenetic properties of mutations in the human HNF1beta gene that lead to defective kidney development., Barbacci E. Chalkiadaki A. Masdeu C. Haumaitre C. Lokmane L. Loirat C. et al.HNF1beta/TCF2 mutations impair transactivation potential through altered co-regulator recruitment.). It was previously shown that the C-terminal domain of HNF1B and coactivators modify histone acetylase activity(Hiesberger T. Shao X. Gourley E. Reimann A. Pontoglio M. Igarashi P. Role of the hepatocyte nuclear factor-1beta (HNF-1beta) C-terminal domain in Pkhd1 (ARPKD) gene transcription and renal cystogenesis.), hence mutations of HNF1B may reduce histone acetylation on target promoters(Gong Y. Ma Z. Patel V. Fischer E. Hiesberger T. Pontoglio M. et al.HNF-1beta regulates transcription of the PKD modifier gene Kif12.). The absent or mild impact of mutants on luciferase activity may be due to transient nature of the expression of HNF1B in the assay as well as differences in chromatin state compared to the more complex in vivo situation. (Inoue F. Kircher M. Martin B. Cooper G.M. Witten D.M. McManus M.T. et al.A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity.).Our study is not without limitations. While clinical features of HNF1B were observed in the proband’s grandmother and grand-aunt, we lacked genetic testing on these 2 individuals(Clissold R.L. Hamilton A.J. Hattersley A.T. Ellard S. Bingham C. HNF1B-associated renal and extra-renal disease-an expanding clinical spectrum.). In addition, we cannot exclude the presence of unknown pathogenic structural variants in the intronic regions not covered on the gene panel. Regardless, the clinical spectrum observed in the four Arg303His families, the Arg303Ser family, and the Arg303Cys patient provides robust evidence that this locus does not tolerate these amino acid changes. Given the rarity of the variant (only 1 unrelated individual in MyCode), we could not conduct formal case-control analyses that are used in ACMG classifications. Future large-scale collaborations are needed to improve the power to conduct formal case-control statistical comparisons for rare variants.Despite the high utility of genetic testing in CKD, upwards of 10-100% of tests returned a VUS in part because the painstaking work of gathering and adjudicating evidence to determine the pathogenicity (Connaughton D.M. Kennedy C. Shril S. Mann N. Murray S.L. Williams P.A. et al.Monogenic causes of chronic kidney disease in adults., Elhassan E.A.E. Murray S.L. Connaughton D.M. Kennedy C. Cormican S. Cowhig C. et al.The utility of a genetic kidney disease clinic employing a broad range of genomic testing platforms: experience of the Irish Kidney Gene Project., Thomas C.P. Freese M.E. Ounda A. Jetton J.G. Holida M. Noureddine L. et al.Initial experience from a renal genetics clinic demonstrates a distinct role in pat

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