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American Journal of Kidney Diseases

Phenocopies, Phenotypic Expansion, and Coincidental Diagnoses: Time to Abandon Targeted Gene Panels?

Published:August 15, 2020DOI:https://doi.org/10.1053/j.ajkd.2020.07.003
      Related article, p. 460
      The main goal of precision medicine is to ascertain the correct diagnosis in an individual patient to deliver the most appropriate prognosis, counseling, and treatment. Accurate diagnosis is therefore the main prerequisite in precision medicine for clinical management of both Mendelian and non-Mendelian diseases. Precise diagnosis and medical management of human diseases, especially genetic ones, can be hampered by phenocopies, genetic and phenotypic heterogeneity, and coincidental co-occurrences. When discussing genetic findings that do not fit the original clinical suspicion, we should consider phenocopies, that is, the presence of a phenotype that is clinically indistinguishable from the one for which we conduct a specific test, but that is caused by a mutation in another gene or by environmental insults. There are other situations that might be confused as phenocopies, such as phenotypic expansion (the occurrence of additional phenotypes that were not previously appreciated in a given syndrome), coincidental diagnosis (the discovery of a mutation that causes a phenotype that is unrelated to the clinical phenotype), and simply wrong diagnosis. At times differentiating between these categories might be merely semantic and we must acknowledge that clinical tentative diagnoses are made with the clinical data available at a certain point in time. Therefore, concepts such as age-related penetrance for certain phenotypic presentations should be taken into consideration when assessing genotype-phenotype correlations for certain genes and loci. The paradigm is not different from that of other fields of medicine, in which clinicians apply a diagnostic workup to patients (with or without a genetic disease) based on clinical presentation, laboratory tests, and imaging studies at a certain time in the disease course. When a diagnosis is not achieved, often—and this is particularly frequent for many genetic diseases—the patient undergoes a diagnostic odyssey, a process particularly taxing for patients and their family members. Thus, phenomena such as phenocopies, phenotypic expansion, and coincidental diagnosis affect the ability to make a precise diagnosis and thus the best test(s) to achieve the same.
      In this issue of AJKD, Riedhammer et al
      • Riedhammer K.M.
      • Braunisch M.C.
      • Günthner R.
      • et al.
      Exome sequencing identification of phenocopies in patients with clinically presumed hereditary nephropathies.
      tackle the issue of phenocopies by conducting exome sequencing (ES) of a cohort of 174 independent patients with clinically presumed hereditary kidney diseases. The phenotypic spectrum included a variety of conditions: focal segmental glomerulosclerosis/steroid-resistant nephrotic syndrome (FSGS/SRNS; 28% of cases), Alport syndrome (AS; 20%), congenital anomalies of the kidney and urinary tract (CAKUT; 17%), ciliopathies (11%), autosomal dominant tubulointerstitial kidney disease (ADTKD; 3%), VACTERL association (5%), and an unspecified “other” group (16%). Overall, the authors established a genetic diagnosis in 30% of patients in the cohort, but with considerable variability among the different categories. A diagnosis could be established in 50% of the ADTKD group, 47% of the AS and ciliopathy groups, 27% of the CAKUT group, 16% of the FSGS/SRNS group, and 30% in the “other” group, whereas none of the VACTERL cases could be solved by using ES. This diagnostic rate is more or less in line with published data,
      • van der Ven A.T.
      • Connaughton D.M.
      • Ityel H.
      • et al.
      Whole-exome sequencing identifies causative mutations in families with congenital anomalies of the kidney and urinary tract.
      ,
      • Groopman E.E.
      • Marasa M.
      • Cameron-Christie S.
      • et al.
      Diagnostic utility of exome sequencing for kidney disease.
      although lower than expected for the AS and ciliopathy categories.
      Of note, 40% of cases had a positive family history. Knowledge of the family history coupled with detailed clinical information including the presence or absence of syndromic manifestations have been shown to improve the overall diagnostic rate in ES studies.
      • Geara A.S.
      • Kallish S.
      • Hogan J.J.
      The impact of whole-exome sequencing on kidney disease ontology: the tip of the iceberg?.
      Although the current study focuses on the investigation of a cohort of unrelated individuals with presumed hereditary disease, the identification and clinical interpretation of variants in the context of trio design (ie, the contextual sequencing and analysis of each affected index case and their parents) has been shown to be a very effective approach for diagnosing patients, particularly those affected by de novo variants.
      • Krumm N.
      • O’Roak B.J.
      • Shendure J.
      • Eichler E.E.
      A de novo convergence of autism genetics and molecular neuroscience.
      In general, highly penetrant disorders with significant effects on the affected individual’s reproductive fitness result from de novo variants or recessive inheritance. Trio testing allows for phasing of multiple variants in the same gene to discern their cis or trans configuration, which is crucial for evaluating variants associated with recessive disorders. Furthermore, the inclusion of family members such as parents and siblings can also be useful for confirming the segregation of identified variants among affected family members, thereby increasing the confidence of either classifying novel variants as pathogenic or helping to classify a variant as benign when it does not segregate with the phenotype.
      The authors next focused on phenocopies and reported them in 10 of 52 diagnosed cases (19%, or 6% of the total cohort). Again, the phenocopy rate differed among the diagnostic categories, with 6 cases involving AS and glomerular diseases (immunoglobulin A [IgA] nephropathy and SRNS/FSGS). These individuals presented with AS at biopsy but were found to conform to SRNS/FSGS by ES or had a glomerular disease diagnosed based on biopsy and clinical grounds but by using ES were found to have AS-causing mutations in COL4A genes. In contrast, no phenocopies were identified in the ADTKD or CAKUT categories. These findings confirm the increasing evidence supporting the identification of COL4A mutations in familial and sporadic forms of FSGS.
      • Gibson J.
      • Gilbert R.D.
      • Bunyan D.J.
      • Angus E.M.
      • Fowler D.J.
      • Ennis S.
      Exome analysis resolves differential diagnosis of familial kidney disease and uncovers a potential confounding variant.
      • McCarthy H.J.
      • Bierzynska A.
      • Wherlock M.
      • et al.
      Simultaneous sequencing of 24 genes associated with steroid-resistant nephrotic syndrome.
      • Gast C.
      • Pengelly R.J.
      • Lyon M.
      • et al.
      Collagen (COL4A) mutations are the most frequent mutations underlying adult focal segmental glomerulosclerosis.
      In a recently published study, COL4A genes were among the top genes with pathogenic mutations identified using ES in a cohort of 3,300 patients with chronic kidney disease, yet only 40% of such cases carried a diagnosis of AS or thin basement membrane nephropathy, and 16% presented with FSGS.
      • Groopman E.E.
      • Marasa M.
      • Cameron-Christie S.
      • et al.
      Diagnostic utility of exome sequencing for kidney disease.
      Explanations reside in the challenges associated with histopathologic diagnosis of AS and FSGS due to extensive heterogeneity in pathologic presentations and in some subjectivity in the interpretation of the kidney biopsy, which remains the primary method for diagnosing glomerular diseases.
      • Yao X.D.
      • Chen X.
      • Huang G.-Y.
      • et al.
      Challenge in pathologic diagnosis of Alport syndrome: evidence from correction of previous misdiagnosis.
      Despite comprehensive clinical and pathologic evaluation, misclassification of AS is common, with mesangial proliferative glomerulonephritis (eg, IgA nephropathy) and FSGS accounting for the most common misdiagnoses (in up to 27% and 19% of patients with AS, respectively) in one study of 52 biopsies from patients with AS.
      • Yao X.D.
      • Chen X.
      • Huang G.-Y.
      • et al.
      Challenge in pathologic diagnosis of Alport syndrome: evidence from correction of previous misdiagnosis.
      Interestingly, in a case of tubulopathy clinically diagnosed as distal renal tubular acidosis with hypercalciuria and nephrocalcinosis, the authors identified a compound heterozygous pathogenic genotype in PCSK1, a gene usually associated with a recessive syndrome characterized by obesity, diabetes, and diarrhea.
      • Ramos-Molina B.
      • Martin M.G.
      • Lindberg I.
      PCSK1 variants and human obesity.
      In the absence of more detailed clinical and laboratory information, distal renal tubular acidosis may have been wrongly diagnosed because of metabolic hyperchloremic acidosis arising from the diarrhea, rather than constituting a true phenocopy.
      The rapid evolution of next-generation sequencing has improved diagnostic accuracy for multiple genetic diseases, including hereditary nephropathies, by permitting the efficient, inexpensive, and unbiased interrogation of multiple candidate genes in parallel. Molecular analysis has proven paramount in providing accurate diagnosis for subsets of kidney diseases that have been confounded by clinical and phenotypic heterogeneity
      • van der Ven A.T.
      • Connaughton D.M.
      • Ityel H.
      • et al.
      Whole-exome sequencing identifies causative mutations in families with congenital anomalies of the kidney and urinary tract.
      ,
      • Groopman E.E.
      • Marasa M.
      • Cameron-Christie S.
      • et al.
      Diagnostic utility of exome sequencing for kidney disease.
      or simply by our limitation in clinical diagnosis purely based on signs and symptoms, laboratory testing, and imaging studies. It is increasingly recognized that only a handful of monogenic kidney diseases can be confidently diagnosed without genetic testing.
      • Armstrong M.E.
      • Thomas C.P.
      Diagnosis of monogenic chronic kidney diseases.
      Also, we are now discovering that the clinical diagnosis of many kidney diseases presenting with characteristic renal and extrarenal manifestations is complicated by expanded or overlapping presentations, making molecular diagnosis all the more important.
      • Groopman E.E.
      • Marasa M.
      • Cameron-Christie S.
      • et al.
      Diagnostic utility of exome sequencing for kidney disease.
      ,
      • Ars E.
      • Torra R.
      Rare diseases, rare presentations: recognizing atypical inherited kidney disease phenotypes in the age of genomics.
      ,
      • Gee H.-Y.
      • Otto E.A.
      • Hurd T.W.
      • et al.
      Whole-exome resequencing distinguishes cystic kidney diseases from phenocopies in renal ciliopathies.
      Improved diagnostic yield and description of phenocopies and phenotypic expansions have been achieved for a number of kidney diseases, particularly with the growing use of ES technologies, rather than limited sequencing of gene panels.
      • Gee H.-Y.
      • Otto E.A.
      • Hurd T.W.
      • et al.
      Whole-exome resequencing distinguishes cystic kidney diseases from phenocopies in renal ciliopathies.
      • Bergmann C.
      ARPKD and early manifestations of ADPKD: the original polycystic kidney disease and phenocopies.
      • Westland R.
      • Bodria M.
      • Carrea A.
      • et al.
      Phenotypic expansion of DGKE-associated diseases.
      In the current study by Riedhammer et al, strictly targeted next-generation sequencing panels (<25 kilobases) did not or would not identify any of the phenocopy cases that had a tentative clinical diagnosis of a monogenic disease. The current recommendations for targeted panels are largely based on the assumption that the superior and complete coverage of genes tested by the panels will result in a higher diagnostic rate for well-defined phenotypes.
      • Xue Y.
      • Ankala A.
      • Wilcox W.R.
      • Hegde M.R.
      Solving the molecular diagnostic testing conundrum for Mendelian disorders in the era of next-generation sequencing: single-gene, gene panel, or exome/genome sequencing.
      ,
      • Consugar M.B.
      • Navarro-Gomez D.
      • Place E.M.
      • et al.
      Panel-based genetic diagnostic testing for inherited eye diseases is highly accurate and reproducible, and more sensitive for variant detection, than exome sequencing.
      Therefore, a panel is not ideal for uncovering genetic phenocopies of disease in cases in which clinical features overlap multiple conditions or less differentiated phenotypes. Moreover, the rapid and continuous discovery of novel genes associated with human diseases makes panels rapidly obsolete, requiring frequent re-design. However, ES and genome sequencing offer the advantage of inclusively evaluating all genes collectively. Both are “durable” tests that although conducted only once, can be interrogated multiple times during a person’s life. They also pose greater potential (or risk) for identifying variants in candidate genes unsuspected of causing the patient’s kidney disorder, as well as incidental findings unrelated to the patient’s primary clinical presentation, thereby challenging the downstream decisions of clinical prognosis, management, and counseling.
      • Rasouly H.M.
      • Groopman E.E.
      • Heyman-Kantor R.
      • et al.
      The burden of candidate pathogenic variants for kidney and genitourinary disorders emerging from exome sequencing.
      ,
      • Bick D.
      • Jones M.
      • Taylor S.L.
      • Taft R.J.
      • Belmont J.
      Case for genome sequencing in infants and children with rare, undiagnosed or genetic diseases.
      This evolving situation raises the question of whether the expanded use of molecular diagnostics requires greater accuracy in clinical diagnosis to address the issue of genetic phenocopies, phenotypic expansion, and coincidental diagnosis. Alternatively, comprehensive genetic tests may increasingly replace the need for precision in clinical diagnosis before testing. We believe that the addition of genome sequencing, transcriptomics, metabolomics, and proteomics in the diagnostic armamentarium has the potential to maximize precision medicine approaches. Hence, it is not the question of if targeted panels will be abandoned, but when will this happen, and based on the most recent results, including the ones presented by Riedhammer et al, the time has likely arrived. This also implies that third-party payers should understand these nuances and embrace and support more comprehensive tests for genetic kidney diseases.

      Article Information

      Authors’ Full Names and Academic Degrees

      Dina F. Ahram, PhD, Vimla S. Aggarwal, MBBS, and Simone Sanna-Cherchi, MD.

      Support

      None.

      Financial Disclosure

      The authors declare that they have no relevant financial interests.

      Peer Review

      Received May 24, 2020, in response to an invitation from the journal. Direct editorial input from an Associate Editor and a Deputy Editor. Accepted in revised form June 26, 2020.

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