#2446 Collagen IV diseases—genetic testing yield of a single-center cohort

Background and Aims Knowledge about genetic causes of chronic kidney disease (CKD) is one of the key gaps in global kidney research and recent International Society of Nephrology recommendations encourage the adoption of genetic testing to enable a goal of providing precision medicine based on individual risk (1). A recent whole-exome sequencing study showed that genetic inheritance may be responsible for up to 10% of CKD diagnoses, many of which may be previously undiagnosed or mis-diagnosed (2). Continued advances in DNA sequencing technology have made genetic testing, even whole-exome sequencing, applicable to routine clinical diagnoses. In order to test the hypothesis that genetic testing can provide valuable information to increase the accuracy and precision of diagnosis in CKD, we designed a gene panel to prospectively provide genetic testing in a subset of patients with CKD defined by a specific set of inclusion criteria. Method Reata Pharmaceuticals is partnering with Invitae on a program called KidneyCode, which provides no-charge genetic testing to enable diagnosis of three specific rare monogenic causes of CKD: Alport syndrome (AS), autosomal dominant polycystic kidney disease (ADPKD) due to PKD2 mutations, and focal segmental glomerulosclerosis (FSGS), as well as detection of variants in one of the autosomal recessive polycystic kidney disease gene, PKHD1. Invitae’s renal disease panel includes 17 genes (ACTN4, ANLN, CD2AP, COL4A3, COL4A4, COL4A5, CRB2, HNF1A, INF2, LMX1B, MYO1E, NPHS1, NPHS2, PAX2, PKD2, PKHD1, and TRPC6), and its assay includes both full-gene sequencing and intragenic deletion/duplication analysis using next-generation sequencing (NGS). The assay targets the coding exons and flanking 10bp of intronic sequences. Invitae’s method of variant classification uses a systematic process for assessing evidence based on guidelines published by the American College of Medical Genetics (3). Patients in the US at risk for hereditary CKD (eGFR ≤ 90 mL/min/1.73m2 plus hematuria or a family history of CKD) or with a known diagnosis of AS or FSGS are eligible. Family members of those with suspected or known AS or FSGS are also eligible. All participants in the KidneyCode program have access to genetic counseling follow-up at no additional charge. Results In the first five months of the KidneyCode program, 152 genetic tests have been completed. A genetic variant was reported in 87 patients. Of those 87 patients, 67 patients had 75 variants in COL4A3, 4, or 5 genes (34 Pathogenic/Likely Pathogenic (P/LP), 41 Variants of Uncertain Significance (VUS)), 20 patients had 24 variants in genes associated with FSGS (3 P/LP, 21 VUS), 15 patients had 20 variants in PKHD1 (1 P/LP, 19 VUS), and 2 patients had variants in PKD2 (1 P/LP, 1 VUS). Of the 34 patients with Pathogenic or Likely Pathogenic COL4A variants, 19 reported a previous diagnosis of Alport syndrome. Other diagnoses in patients with COL4A mutations included FSGS, thin basement membrane disease, and familial hematuria. Extra-renal manifestations such as hearing loss and eye disease were reported in 7 of the 34 patients with COL4A variants. Conclusion Initial results with the KidneyCode panel demonstrate the utility of NGS and support the hypothesis that combining genetic testing with clinical presentation and medical history can significantly improve accuracy and precision of diagnosis in patients with hereditary CKD.

DNA-based screening and population health: a points to consider statement for programs and sponsoring organizations from the American College of Medical Genetics and Genomics (ACMG)

Should We Diagnose Autosomal Dominant Alport Syndrome When There Is a Pathogenic Heterozygous COL4A3 or COL4A4 Variant?

Background and Aims Familial hematuria diseases are a heterogeneous group of monogenic conditions caused by mutations in one of the collagen IV genes: COL4A3 (2q36.3), COL4A4 (2q36.3), and COL4A5 (Xq22.3) that are expressed in the glomerular basement membranes (GBM) and are responsible for the most frequent forms of microscopic hematuria (MH), Alport syndrome, and thin basement membrane nephropathy (TBMN). Recent data suggest that about 1% (1 in 106 individuals) of the world population may have heterozygous predicted pathogenic COL4A3 or COL4A4 variants, a frequency that leads to the occasional superimposition of TBMN with other glomerulopathies. Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a major genetic disorder affecting up to 12.5 million individuals worldwide and it is the fourth most common global cause of renal replacement therapy. Two are the principal causative genes: PKD1(16p13.3) and PKD2 (4q22.1). The aim of the study was to describe a cohort of clinically ADPKD patients studying collagen IV genes COL4A3, COL4A4, and COL4A5. Method We performed NGS with Sophia Genetic “Nephropathies Solution” Panel on clinically ADPKD patients (pts). This panel includes 44 genes (target region 105.8 kb) involved in different types of nephropathies. We considered variants that are categorized as Variant of Uncertain Significance (Vus), likely pathogenic, or pathogenic (Class 3,4, or 5 of American College of Medical Genetics (ACMG)) in COL4A3, COL4A4, and COL4A5 genes. Results We analyzed 250 consecutive clinically ADPKD pts (consecutive in the outpatient clinic). We found 13 (6.3%) clinically potentially interesting (Class 3,4 or 5 of ACMG) variants in collagen genes. We found 2 pts that were negative for PKD1, PKD2, PKHD1 analysis: 1 pt carrier COL4A4 NM_000092.5: c.2908C>T (p.Gln970Ter), Pathogenic and c.2756A>G (p.Glu919Gly) Vus; 1 male pt carrier COL4A3 NM_000091.5: c.609+3_609+6del, Vus and COL4A5 NM_000495:c.169G>A p.(Gly57Arg), likely pathogenic. We also found 4 pts with 4 Vus variants in collagens genes combined with at least 1 pathogenic or likely pathogenic variant in PKD1 or PKD2 genes. We found 3 pts with likely pathogenic variants in COL4A4 or COL4A3 genes combined with Vus or likely benign variants in PKD1 or PKD2 genes and 1 patient with 1 likely pathogenic variant in COL4A3 and 1 likely pathogenic variant in PKD1. Conclusion Variants in collagen genes are very frequent and the associated phenotype is varied, sometimes very mild such as microhematuria. Furthermore, penetrance has to be considered. The study outlines the importance of considering also collagen genes, even if we have a clear clinical manifestation of ADPKD. An additional variant in a kidney already compromised can have an impact on the phenotype. Analysis of genes involved in these 2 pathologies could lead to a better understanding of the phenotype-genotype correlation at least in patients with both conditions.

Type IV Collagen Mutations in Familial IgA Nephropathy

Eligibility Criteria and Genetic Testing Results from a High-Risk Cohort for Hereditary Breast and Ovarian Cancer Syndrome in Southeastern Ontario

Background and aims Alport syndrome (AS) and thin basement membrane nephropathy (TBMN) are genetically heterogeneous kidney diseases that manifest with kidney involvement, AS also with hearing and visual impairment. Electron microscopic examination shows characteristic ultrastructural changes in the glomerular basement membrane (GBM) in AS and TBMN, but cannot reliably distinguish between the two entities. Therefore, molecular genetic studies and the identification of individual pathogenic variants in the target genes are crucial to confirm the diagnosis. The aim of our study is to determine the significance of the genetic variants associated with the ultrastructural changes of GBM found in the renal biopsy. Method We retrospectively analyzed records of patients from the University Medical Centre Ljubljana in which ultrastructural GBM changes characteristic for AS and TBMN were found in the renal biopsy, including their clinical, laboratory and histological features. Molecular genetic investigations were made using the new generation sequencing method (NGS) to determine germline pathogenic variants in genes directly related to AS and TBMN (COL4A3, COL4A4 and COL4A5) and in genes that are important in syndromes with a related clinical picture (COL4A6, CD151, LMX1B, MYH9, FN1 and MYO1E). The pathogenicity of variants was determined according to the Clinical Molecular Genetic Society best practice guidelines. Results The study included 73 patients with ultrastructurally confirmed thin GBM. Indications for kidney biopsy were chronic nephritis syndrome in 33 patients (45%), acute worsening of renal function in 16 (22%), nephrotic syndrome in 9 (12%), nephrotic proteinuria and chronic kidney disease in 6 (8%), isolated proteinuria in 5 (7%) and suspected vasculitis in 4 patients (6%). At the time of kidney biopsy average age was 50 ± 16 years, average creatinine 112 ± 70 µmol/L and average estimated glomerular filtration rate (eGFR) 65 ± 24 ml/min/1.73 m2, 7 patients had eGFR below 30 ml/min/1.73 m2. Average daily proteinuria was 2.4 ± 3.3 gr and 88% of patients had hematuria. 38% of them have hearing impairment and 43% had positive familiar history of kidney disease. We recorded 33 patients (45%) with genetic variants associated with AS or TBMN: 22 patients (67%) with pathogenic variants, 2 patients (6%) with likely pathogenic and 9 patients (27%) with variants of uncertain significance. The presence of a positive genetic analysis was associated with a higher level of hematuria (p = 0.003), a lower level of proteinuria (p = 0.004) and a positive family history of kidney disease (p < 0.001). It was also associated with hearing loss, but the association was not statistically significant (p = 0.099). Patients with genetic variants had similar eGFR to those with negative genetics (63.9 ml/min/1.73 m2 (58.3-69.4) vs 66.2 ml/min/1.73 m2 (60.0-72.3)), p = 0.698. We also found no differences in GBM thickness in patients without compared to those with identified gene variants (213 nm (160-315) vs. 209 nm (140-410)), p = 0.761. 32 patients (44%) had only focal chronic changes and ultrastructural changes of thin GBM present on kidney biopsy and 41 patients (56%) had also been diagnosed with other renal pathology. When comparing these two groups, patients without associated pathology had lower daily proteinuria (p = 0.048), higher level of hematuria (p = 0.021), frequent hearing impairment (p = 0.008) and also positive genetics analysis (p = 0.016). Conclusion We have shown that in patients with proven ultrastructural alterations in GBM, the presence of so far recognized genetic variants associated with AS and TBMN is 45%, and corresponds to the frequency of other phenotypic characteristics of AS, including hearing impairment and positive family history. In the future, additional clinical tools will be required to accurately determine the role of coexistent thin GBM on the prognosis of patients with other relevant kidney pathology and other genetic tools beyond NGS should be employed to explore other pathologic genetic variants associated with the AS spectrum.

The use of fetal exome sequencing in prenatal diagnosis: a points to consider document of the American College of Medical Genetics and Genomics (ACMG)

Background: The role of germline ALK and PHOX2B variants in genetic predisposition to neuroblastoma (NB) is well established; however, alterations in genes associated with other syndromes, including RASopathies, Fanconi anemia, and Li-Fraumeni, have also been detected. Emerging data suggest potential roles for DNA damage repair (DDR) pathway genes in NB predisposition. The use of next-generation sequencing (NGS) technologies facilitates the unbiased detection of both known and novel germline variants. Methods: Patients (pts) with newly diagnosed and relapsed malignancies, including 43 NB pts, were referred to our institutional pediatric oncology NGS study to sequence germline DNA, tumor DNA, and RNA. Germline DNA was sequenced using a custom pediatric cancer panel (Agilent Sure Select capture technology) targeting 15,000 exons across 864 genes (1000x coverage). Variant pathogenicity was classified according to ACMG (American College of Medical Genetics and Genomics) criteria and evaluated at multidisciplinary molecular tumor boards. Rare variants of uncertain significance (VUS) with supportive corresponding somatic data, patient phenotypes, literature, and/or in-silico functional analyses were termed “Variants of uncertain significance with limited evidence for pathogenicity” (VUS-LEP). Results: Analyses have been completed for 41/43 enrolled NB pts. Patient history was retrospectively categorized as “high genetic predisposition risk” (HGPR) in 14/41 pts based on ≥1 of the following criteria: (1) family history of NB and/or significant family history of other cancer(s); (2) patient with NB and another metachronous or synchronous malignancy; (3) NB with congenital abnormalities; (4) multifocal NB. Germline pathogenic (P), likely pathogenic (LP) variants, or VUS-LEP in a known cancer predisposition gene were identified in 6/14 HGPR pts (43%), predominantly in DDR-related genes (PALB2, BRCA1/CHEK2, CHEK2/PALB2, NF1, DICER1, MITF). Of note, no ALK or PHOX2B germline variants were identified for the five pts with NB family history. For the 27 non-HGPR pts, germline P, LP variants or VUS-LEP in DDR genes were most prevalent and detected in 8/27 pts (30%), in genes such as BAP1, BARD1 (n=2), BLM, BRCA2, CHEK2, RAD51, RAD51D/NBN. In this non-HGPR population, we also identified a germline P TP53 variant (n=1), a LP FH variant (n=1) as well as additional germline VUS-LEPs in EZH2 (n=1) and ERCC2 (n=1). Conclusion: Prospective sequencing identified frequent germline variants in NB pts, predominantly in genes involved in DDR and homologous recombination, regardless of classification as HGPR. We intend to complete and report somatic characterization of the tumors, with a focus on mutational signatures and 11q loss encompassing ATM. Our findings may have implications for future targeted treatment recommendations (e.g., PARP inhibitors) as well as for appropriate genetic counseling for pts and families. Citation Format: Sarah Cohen-Gogo, Karin Langenberg-Ververgaert, Anita Villani, Winnie Lo, Ted Young, Nisha Kanwar, Scott Davidson, Nathaniel Anderson, Bailey Gallinger, Mehdi Layeghifard, Larissa Waldman, Daniel Morgenstern, Ledia Brunga, Stephen Meyn, David Malkin, Adam Shlien, Meredith Irwin. Prospective germline next-generation sequencing in pediatric patients with neuroblastoma identifies frequent alterations in genes involved in DNA damage repair [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B07.

Refinement of pathogenicity classification of variants associated with familial hypercholesterolemia: Implications for clinical diagnosis

The etiology of 46, XY disorders of sex development (46, XY DSD) is complex, and studies have shown that different series of patients with 46, XY DSD has different genetic spectrum. In this study, we aimed to investigate the underlying genetic etiology in a Chinese series of patients with 46, XY DSD by whole exome sequencing (WES). Seventy patients with 46, XY DSD were enrolled from the Peking Union Medical College Hospital (Beijing, China). The detailed clinical characteristics were evaluated, and peripheral blood was collected for WES to find the patients' rare variants (RVs) of genes related to 46, XY DSD. The clinical significance of the RVs was annotated according to American College of Medical Genetics and Genomics (ACMG) guidelines. A total of 57 RVs from nine genes were identified in 56 patients with 46, XY DSD, which include 21 novel RVs and 36 recurrent RVs. Based on the American ACMG guidelines, 43 variants were classified as pathogenic(P) or likely pathogenic (LP) variants and 14 variants were defined as variants of uncertain significance (VUS). P or LP variants were identified in 64.3% (45/70) patients of the series. Thirty-nine, 14, and 4 RVs were involved in the process of androgen synthesis and action, testicular determination and developmental process, and syndromic 46, XY DSD, respectively. The top three genes most frequently affected to cause 46, XY DSD were AR, SRD5A2, and NR5A1. Seven patients were found harboring RVs of the 46, XY DSD pathogenic genes identified in recent years, namely DHX37 in four patients, MYRF in two patients, and PPP2R3C in one patient. We identified 21 novel RVs of nine genes, which extended the genetic spectrum of 46, XY DSD pathogenic variants. Our study showed that 60% of the patients were caused by AR, SRD5A2 or NR5A1 P/LP variants. Therefore, polymerase chain reaction (PCR) amplification and Sanger sequencing of these three genes could be performed first to identify the pathogeny of the patients. For those patients whose pathogenic variants had not been found, whole-exome sequencing could be helpful in determining the etiology.

<h3>Background</h3> This study was undertaken to compare the proportions of conditions referred/detected in probands of at-risk families at a single Inherited Cardiac Conditions (ICC) clinic and to assess the uptake of genetic testing and the yield of actionable genetic testing results in these individuals. <h3>Methods</h3> In this retrospective study, we interrogated the clinic’s proband database (n=1,817; representing a total of 3,316 condition-affected persons) in July 2020. We extracted data about those whose primary diagnosis was one of six ICCs: Hypertrophic Cardiomyopathy (HCM; n=444), Dilated Cardiomyopathy (DCM; n=235), Long QT Syndrome (LQTS; n=220), Arrhythmogenic Cardiomyopathy (ACM; n=30), Brugada Syndrome (BrS; n=25) and Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT; n=9). We also included Sudden Adult Death Syndrome (SADS; n=165) as an additional category. Genetic variants which were classified as Pathogenic (P), Likely Pathogenic (LP) and Variant of Uncertain Significance (VUS) by American College of Medical Genetics were grouped together as ‘not negative’. P and LP variants are directly clinically actionable while VUSs are not. <h3>Conclusion</h3> The overall utilisation of genetic testing was relatively low at 32.4% in this cohort. The overall not negative (P, LP or VUS) rate for genetic testing across the ICCs probands in whom genetic testing was pursued was 60.7%. Combined P/LP variant rates within the not negative group in the larger cohorts of HCM (n=86) and LQTS (n=88) were 82.5% and 84.7% respectively. VUS rates vary from 0% to 30% within the not negative groups (excluding SADS where it was 100%). Factors contributing to low rates of genetic testing and phenotypic predictors of positive genotype warrant further investigation.

Background and Aims Alport syndrome (AS) is an inherited nephropathy caused by pathogenic variants in COL4A3 (autosomal dominant -AD- and autosomal recessive -AR- inheritance), COL4A4 (AR) and COL4A5 (X-linked dominant -XLD). It is characterized by glomerular nephropathy with hematuria progressing to end-stage renal disease, frequently associated with sensorineural deafness and ocular anomalies. In thin basement membrane disease (TBMD) most patients are asymptomatic and are incidentally noted to have microhematuria, mild proteinuria, and occasionally gross hematuria, with normal renal function. This condition is caused by pathogenic variants in COL4A3 and COL4A4 (AD). We have systematically applied genetic testing to pediatric and adult patients with clinical features suggestive of AS (Table 1). Method Clinical exome sequencing (CES) was performed on a cohort of 95 patients referred to our center from 2019 to 2022. Analyses were performed on an in-silico designed panel including 523 renal genes. Whenever possible, identified variants were segregated by Sanger sequencing. Results We identified causative (C4/C5) variants in 43 (45.3%) patients. Among them, genetic diagnosis of AS was obtained in 25 patients (58.1%): one (4.0%) with biallelic variants in COL4A4, 13 (52.0%) with COL4A5 variants (6 females and 7 males), 5 (20.0%) with COL4A3 variants (4 heterozygous and 1 homozygous) and 6 (24.0%) cases with coexisting variants in one or more other collagen genes. In 16 (37.2%) patients, we found causative monoallelic variants compatible with TBMD: 12 (75.0%) patients with variants in COL4A4 and 4 (25.0%) in COL4A3. Lastly, in 2 cases, we found variants in COL4A1 (#120130) and MYH9 (#160775), both genes associated with other renal diseases with a clinical presentation partially overlapping with AS. In additional 16 cases (16.8%), we identified C3 variants in collagen genes. In the remaining patients, genetic testing was negative (14 patients, 14.7%) or inconclusive (22, 23.2%). Of note, we identified 6 families with digenic AS. In 4 of them - 3 with COL4A5/COL4A4 variants and one with COL4A3/COL4A4 variants – the co-existence of two variants was associated to earlier renal failure, compared to family members bearing a single variant. In contrast, in the remaining 2 families (33.3%), members with COL4A5/COL4A3 missense variants had a milder phenotype compared to male family members with the single variant COL4A5 variant. In families with digenic AS, at least one variant involved the substitution of a glycine with another amino acid in the triple helix domain, frameshift indel variants in the triple helix domain or inframe indel in the collagen IV domain non-collagenous. To better understand the impact of multiple variants on collagen structure, computational studies are currently ongoing. Conclusion CES is a powerful tool to clearly define the diagnosis when AS or TBMD are suspected, as witnessed by a detection rate of 43.2%. Genetic diagnosis allows to identify the causative gene offering the possibility of extending diagnosis to other family members, also in the prenatal setting. In addition, it offers the possibility to identify multiple variants in collagen genes associated with digenic AS, as well as to find variants in other genes implicated in kidney disease. Notably, the presence of digenic variants is not necessarily predictive of a worse disease outcome. For all these reasons, molecular diagnosis can be useful to improve clinical management, to calculate recurrence risk and to better define the prognosis of the patient.

Points to consider: is there evidence to support BRCA1/2 and other inherited breast cancer genetic testing for all breast cancer patients? A statement of the American College of Medical Genetics and Genomics (ACMG)

A Multimodality Approach to Assessing Factor I Genetic Variants in Atypical Hemolytic Uremic Syndrome