The Role Of Genetic Testing in The Diagnostic Workfow Of Pediatric Patients With Kidney Diseases: The Experience Of A Single Institution Ⅱ

CES and family segregation studies allowed the identification of causative variants in a signifcant proportion of patients 

All patients underwent CES and were analyzed for variant prioritization and annotation following the criteria described above (Fig. 1). Overall, variants were detected in 154 patients (80.6%) with 37 patients (19.4%) presenting no variants. Sanger validation of the identified variant(s) and family segregation studies have been performed so far in 90 out of the 154 patients (58.4%). This approach allowed us to (i) confirm the variants identified by CES in all cases, (ii) confirm their segregation with the phenotype, and (iii) identify de novo variants. Among the group of patients in which variants were identified by CES, a conclusive genetic report was obtained in 71 (46.1%; in 49 patients, variants were validated by Sanger sequencing and family segregation studies), while 22 (14.3%) and 61 (39.6%) patients remained with an uncertain genetic diagnosis or were classified as inconclusive, respectively (Fig. 3).

Overall, the application of CES followed by, whenever possible, family segregation studies allowed us to reach a genetic diagnosis in a signifcant proportion of cases. The diagnostic yield is heterogenous when considering the different disease macro-categories with ciliopathies showing the higher diagnostic rate (74.4% of patients diagnosed), followed by nephrolithiasis and tubulopathies (45.5% and 45%, respectively), glomerulopathies and CAKUT (24.2 and 20.6%). The macro-category “others” was the most heterogeneous one and presented the lowest diagnostic yield, with only 1 patient having a genetic diagnosis out of 14 (7.1%; Table  1). Not surprisingly, when considering a positive versus a negative family history, the former group of patients showed a higher diagnostic rate with 59.5% of patients diagnosed (Table 1).

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Variant distribution and characteristics in the diagnosed cohort of patients 

Looking at the patients with a conclusive genetic report, 96 variants in 32 genes were identified and listed in patients’ genetic reports (Fig.  4a and Additional file 1: Tables S1–S6). A few considerations can be drawn from analyzing the data: (i) Several patients presented with more than one variant either in the same or in different genes, not considering the compound heterozygous variants in recessive genes (e.g., #41, #43, #109; Fig.  4a and Additional file 1: Tables S2, S3). A signifcant proportion of these cases belong to the ciliopathies macro-category and specifically to polycystic kidney disease, posing the question of whether additional variants within the PKD1 gene may have a clinical impact, leading to an earlier diagnosis (manuscript in preparation). (ii) The most recurrently mutated genes within the cohort were COL4A5 and PKD1, in keeping with Alport and autosomal-dominant polycystic kidney disease (ADPKD) disease frequencies. (iii) Most of the identified variants are missense mutations (n=53; 55.2%), followed by frameshift (n=15; 15.6%), nonsense (n=14; 145.6%), and splicing variants (n=9; 9.4%). Moreover, 5 different CNVs were reported involving genes causative of CAKUT or glomerular diseases (5.2%; Fig. 4a and Additional fle 1: Tables S1–S6). (iv) Nephrolithiasis and tubulopathies presented the highest number of C5 variants (n=6 and n=4, respectively) and the lowest number of C3 mutations (n=1 each), which were on the contrary highly represented in ciliopathies (n=20). C4 variants represented the most mutations among all disease macro-categories (Fig. 4b).

v) Within the conclusive genetic reports, CES resulted in the identification of pathogenic C5 variants in 17 patients (24%), likely pathogenic C4 variants in 34 (47.9%), variants of unknown significance-VUS C3 in 16 subjects (22.5%) and in 4 cases variants classified as C3/C4 (5.6%). When looking at the type of variants/ frequency within patients and mode of inheritance, the majority of the C5 variants were heterozygous mutations (n=8), followed by homozygous (n=6) and compound heterozygous (n=2) variants. A C5 CNV was part of this category. A similar pattern of distribution appeared for C4 and C3 variants, with heterozygous mutations being the most represented (n=23 and n=9, respectively; Fig. 4c).

(vi) No signifcant diferences in the diagnostic rate were highlighted when considering European versus non-European subjects, even though it has to be taken in mind that the former group represented the great majority of the cohort. Finally, (vii) independently of the disease category considered, most of the identified variants were already published and associated with specific clinical phenotypes (Fig. 4d). Ciliopathies, and in particular polycystic kidney disease, was the only suspicion presenting a signifcant number of unpublished variants (13 out of 46), probably because of the higher number of variants identified compared to the other disease categories. All the identified variants are detailed in Additional file 1: Tables S1–S6.

Discussion 

Tough rare in children, CKD has a profoundly negative impact on normal growth and development, compromising quantity and quality of life. Te most recent analyses on adult and pediatric patients, who have received a kidney transplant or are included in the transplant waiting list or are present in the registries of the European Renal Association-European Dialysis and Transplant Association (ERA-EDTA) indicate that up to 27% of them are undiagnosed at the time of transplantation [7]. In line with these data, by analyzing the Transplant Registry of the Italian National Transplant Center, we recently reported that approximately 17.2% of the pediatric cohort was without a clear clinical diagnosis [6]. In addition, when considering the different disease categories, the great majority were affected by rare conditions and up to 50% by a monogenic disease [6]. These results suggest that genetic screening may be a valuable addition for increasing the diagnostic rate. It also represents a potent tool to confirm clinical diagnosis, as well as understanding the genetics underlining more complex or syndromic diseases, finally impacting on prognosis, management, and patients’ treatment.

Here, we report the results of the systematic use of a powerful genetic test, such CES in the diagnostic work fow of pediatric patients affected by nephropathies. Overall, a conclusive genetic test, based on CES followed by Sanger-based segregation studies, was obtained in 37.1% of patients, with a certain degree of heterogeneity when considering the different disease macro-categories. As expected, based on clinical presentation, the highest detection rates were obtained for ciliopathies (74.4%), followed by nephrolithiasis (45.5%) and tubular diseases (45%), while most glomerular diseases and CAKUT remained undiagnosed. In the case of glomerular diseases, a negative genetic test is important per se in that it rules out a structural cause for the disease, with signifcant implications for clinical management and transplantation outcome.

These data are in line with previously published results, even though some diferences may be registered based on the group of patients considered, especially for highly homogeneous cohorts mainly based on the same ethnic group (Table  2). It must be noted that many of the families where a VUS was identified are currently under investigation for variant segregation, most likely improving these performances.

These results underline the importance of an NGS-based genetic test together with family segregation studies and/or complementary tests (e.g., MLPA, array-CGH) as part of the routine diagnostic workflows. In addition to the relevance of having a diagnosis, these tests allow us to identify other family members that may carry the same pathogenic variants as well as estimate the risk of disease recurrence. Moreover, considering that a signifcant percentage of these patients require kidney transplantation at some point, the availability of a genetic test to screen family members carries important implications in the selection of a live donor within the family.

A second point to be discussed is the importance of distinguishing between genetic and non-genetic causes for some diseases. As an example, in the presence of a child with steroid-resistant nephrotic syndrome (SRNS), it is essential to rule out conditions caused by mutations in genes coding for structural proteins of the podocyte. This can help to refine therapy, as children carrying pathogenic variants in podocyte genes generally do not benefit from immunosuppressive therapy or predicting prognosis, as “immunologic” SRNS is more likely to recur after transplantation. Genetic diagnosis may also result in fewer kidney biopsies, particularly for patients with glomerulopathy.

A third relevant consideration in favor of genetic testing in the clinical diagnostic workflow of pediatric patients is the translational impact of the identifcation of genetic variants. Indeed, there are actionable genes meaning that the corresponding disease conditions can be treated based on the presence of pathogenic variants, as in the case of renin-angiotensin blockade for patients carrying pathogenic variants in COL4A3/COL4A4/ COL4A5 genes. On the same line, having a genetic report may avoid useless or even deleterious treatment, such as immunosuppressive therapies for patients carrying mutations in collagen-coding genes [5]. Moreover, it can be useful for patients stratification and to assess the potential risk of recurrence after a kidney transplant. As an example, patients diagnosed with atypical Hemolytic Uremic Syndrome and with a positive genetic report identifying pathogenic variants in CFH, C3 or CFB genes, are at moderate to high risk of recurrence after transplantation [28]. For them, the administration of eculizumab showed signifcant positive results with no relapse or relapse in a minority of cases, while its administration can be avoided for those patients at low risk [28–30].

A fourth point concerns family planning, as we are dealing with a pediatric population with parents who may wish to have additional children. The availability of a genetic diagnosis may be extremely useful for genetic counseling proposing to the couple all the available options for a future pregnancy. In line with this point, considering the present cohort, in 2 different cases a prenatal diagnosis was performed via Sanger sequencing, screening the fetus for the specific variant originally found by CES in the proband.

A fourth point that needs to be stressed concerns the number of C3-VUS variants identified by NGS, always posing a serious dilemma about their role in disease onset and progression and how they can be communicated during genetic counseling. In the last years, this topic has been addressed by setting up and designing novel computational methodologies that take in consideration not only nucleotide conservation, and protein impact but also gene-association networks and pathway connections. Additional hints in deciphering the real meaning of C3 variants may come from transcriptomic analyses through the detection of aberrant expression or aberrant splicing mechanisms, as well as functional validation studies [31, 32]. Lastly, it is important to stress the relevance of periodic re-analysis of negative or inconclusive genetic reports and periodic re-evaluation of C3 variants. This kind of approach relies on (i) the discovery of new gene-disease/variant-disease associations, (ii) updated information from publicly available databases, (iii) classification of genetic variants based on functional evidence, (iv) amelioration of the in silico tools used for data alignment and variants annotation [33]. Up to now, no clear indication of the time interval after which negative/inconclusive cases or C3 variants must be re-analyzed has been provided by the Italian Society of Human Genetics (SIGU) or by the American College of Medical Genetics and Genomics guidelines. However, both these institutions suggest reviewing negative cases or variant classification either based on new findings by the lab or by external sources (e.g., literature or databases) or following clinicians’ requests [34–36]. In line with this final point of discussion, it is worthy noting that in some cases, a single variant in recessive genes highly compatible with the clinical phenotype was found. While per se, these variants cannot explain clinical presentation, it is important to re-analyze and possibly to re-align original sequencing data to determine whether a second causative variant can be found.

A final point to be discussed is the financial impact of these tests on the National Healthcare system. In the Italian system, CES followed by analyses of a limited number of genes (<8) is in the range of 1200 euros, all included, while larger panels are approximately double the amount. While these costs may seem elevated, a timely diagnosis may avoid unnecessary additional tests, including biopsies and may lead to optimized patient care and to early identification of family members with the same disease or at risk of developing the same disease. Family segregation studies are in the range of 150 euros per variant tested per person. In our view, to make this diagnostic work efficient and sustainable, it is necessary to identify local/ regional “reference hubs” that can centralize these analyses, reducing costs and accumulating essential experience in variant calling and interpretation.

Overall, these results confirm the relevance of including routine genetic testing and counseling in the diagnostic workflow of pediatric patients affected by nephropathies where a monogenic condition is suspected or with a positive family history. For these patients genetic testing should be considered at the beginning of their diagnostic journey, as it may improve clinical management, spare unnecessary treatments, or diagnostic procedures, identify other family members potentially at risk of having the same genetic variants and, in case of a kidney transplant, lead to optimal live-donor selection.

References

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3. uremic syndrome advances. Cells. 2021;10(12):3580. Kagan M, Pleniceanu O, Vivante A. The genetic basis of congenital anomalies of the kidney and urinary tract. Pediatr Nephrol. 2022;37:2231. 4. Torra R, Furlano M, Ortiz A, Ars E. Genetic kidney diseases as an under-recognized cause of chronic kidney disease: the key role of international registry reports. Clin Kidney J. 2021;14(8):1879–85. 

5. Participants KC. Genetics in chronic kidney disease: conclusions from a Kidney Disease: Improving Global Outcomes (KDIGO) Controversies Conference. Kidney Int. 2022;101(6):1126–41. 

6. Vaisitti T, Peritore D, Magistroni P, Ricci A, Lombardini L, Gringeri E, Catalano S, Spada M, Sciveres M, Di Giorgio A, et al. The frequency of rare and monogenic diseases in pediatric organ transplant recipients in Italy. Orphanet J Rare Dis. 2021;16(1):374. 

7. Kramer A, Boenink R, Stel VS, Santiuste de Pablos C, Tomovic F, Golan E, Kerschbaum J, Seyahi N, Ioanou K, Beltran P, et al. The ERA-EDTA Registry Annual Report 2018: a summary. Clin Kidney J. 2021;14(1):107–23.

8. Domingo-Gallego A, Pybus M, Bullich G, Furlano M, Ejarque-Vila L, Lorente-Grandoso L, Ruiz P, Fraga G, Lopez Gonzalez M, Pinero-Fernandez JA, et al. Clinical utility of genetic testing in early-onset kidney disease: seven genes are the main players. Nephrol Dial Transpl. 2022;37(4):687–96.

9. Tullus K, Webb H, Bagga A. Management of steroid-resistant nephrotic syndrome in children and adolescents. Lancet Child Adolesc Health. 2018;2(12):880–90. 10. Kienzl-Wagner K, Waldegger S, Schneeberger S. Disease recurrence-the sword of damocles in kidney transplantation for primary focal segmental glomerulosclerosis. Front Immunol. 2019;10:1669

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