In this report, a COL4A4 heterozygous variant, not yet discussed in detail in the literature, was shown to segregate with features of AS in two generations of a single family. The reported variant, c.5007delC (p.Leu1670Ter), is classified by consensus as likely pathogenic among different clinical genetic testing laboratories and is a nonsense variant in the COL4A4 gene. These findings were disclosed to all patients in the case report. In addition, the pattern of segregation with disease in affected family members that only had one copy of this variant, suggests an AD mode of inheritance. The classification of the reported variant changed over time based on additional clinical information regarding phenotype, family history, and the role of the variant in protein translation. This case highlights the challenges that arise in the current era of genetic medicine. Clinicians should ensure they obtain a thorough family history and collaborate with geneticists and genetic counselors to verify that all variants associated with a disease of interest, not just known pathogenic variants, are evaluated. This case also demonstrates how genetic testing can lead to non-invasive, early diagnosis and improve monitoring, prevention, and/or mitigation of disease progression.
Next-generation sequencing and similar technologies allow researchers to investigate the significance of disease-associated variants cost-effectively and quickly for clinical diagnoses [20,21,22,23,24,25]. Consistency between the significance of disease-associated variants is necessary [16, 17]. Thus in 2015, the ACMG developed a framework for variant interpretation and determination of pathogenicity. Each variant classification is assigned a direction, benign or pathogenic, and the evidence criteria for classification is based on a level of strength: stand‐alone (A), very strong (VS), strong (S), moderate (M), or supporting (PP) . Despite these guidelines, nuanced classifications of variants and internal data can lead to different classifications of the same variant by separate genetic testing laboratories, such as Natera, Invitae, and GeneDx in this case report.
One of the salient points of this case is that the variant’s classification changed overtime. Initially, Natera (Natera, San Carlos, CA, USA) classified the variant as a VUS using a modified version of the ACMG criteria. The variant in this case results in the conversion of leucine to a premature termination at nucleotide 198 of 4875 of exon 48. This nonsense variant, which is located in the last exon, is not anticipated to result in nonsense mediated decay and therefore interpretation of such variants is always made with caution (PVS1_strong) [15, 26, 27]. In addition to the low frequency of the variant in gnomAD (PM2_supporting), the newly provided evidence from the co-segregation data (PP1_supporting) was sufficient to upgrade its classification to likely pathogenic . Invitae (Invitae, San Francisco, CA, USA) used the Sherloc scoring system, which cannot be directly compared with the ACMG framework for variant classification. They used evidence that it is a loss of function (LOF) variant and that LOF is a known mechanism of disease in COL4A4, similar to Natera’s PVS1, for their original VUS classification [28,29,30]. Their classification was upgraded from VUS to pathogenic due to the discovery of another patient in their laboratory that had a pathogenic variant upstream this case’s variant. The upstream variant is a nonsense variant that causes protein function loss, which did not directly correlate with evidence from the ACMG criteria. In their upgrade, they also included the variant’s absence in the population according to ExAC (ClinVar accession number: SCV001580980.2), which was similar to Natera’s PM2. GeneDx only incorporated the information that this was a nonsense variant in a gene where LOF is a known mechanism of disease and that it was predicted to disrupt the last 21–23 amino acids of the protein (PVS1). However, because the variant’s location was so close to the C-terminus or 3’ end of the gene, the evidence’s strength was decreased to strong evidence. This led GeneDx to classify this variant as a VUS and as of the date of this publication, the variant remains a VUS (ClinVar accession number: SCV001993731.1). Although the classification varied among these different clinical genetic testing laboratories, the authors agree that by consensus this variant is likely pathogenic.
According to the Online Mendelian Inheritance in Man (OMIM) data, pathogenic variants in COL4A4 are associated with ARAS or ADAS, which can be difficult to distinguish when observing the early clinical symptoms of AS . However, the COL4A4 heterozygous variant described in this case appears to have an AD mode of inheritance. In addition, patient 3, who was 20 years old and healthy, lacked the variant.
Phenotypically, age-related penetrance and the spectrum of phenotypes of a single variant are important to take into consideration when evaluating COL4A4 ADAS [11, 12]. Kidney biopsies of patients with ADAS demonstrate a predominance of thickening and thinning of the GBM, and less commonly, wrinkling of the GBM and foot process effacement . The histology observed in our patients have similarities with the kidney biopsy reports of patients heterozygous for a pathogenic variant in COL4A4. Patient 1’s biopsy was atypical regarding the numerous lamellar “zebra bodies”. However, patient 1’s biopsy had significant effacement of podocyte foot processes with wrinkling of the basement membranes and patient 2’s biopsy showed alternate thinning and thickening of the GBM without lamellations, which were consistent with kidney biopsy findings from COL4A4 heterozygotes.
The mean age of onset of kidney failure in ADAS is 52.8 years. However, the type of variant impacts the timing of kidney failure. Patients who have variants that lead to a premature termination of translation develop kidney failure at 47.1 years compared to 55.2 years in patients with missense variants . Most of the patients in this case report seem to have a disease progression consistent with patients with ADAS, except for patient 1. As compared to reports of mild disease, patient 1’s disease progression was very rapid. There are a couple of factors that likely accelerated patient 1’s disease course. There was a delay in the diagnosis and monitoring of this patient because she did not have routine follow up and care with a nephrologist. Second, she had multiple pregnancies, which in turn can accelerate CKD progression . Aside from her disease course, patient 1 exhibited the most common symptoms associated with AS, hematuria and proteinuria. The other affected patients in this family also display common AS symptoms. The older affected children (patients 2 and 4) have more clinical features, due to age-related penetrance.
Based on the co-segregation and findings, it is very convincing that this variant is contributing to pathogenicity. However, it should be noted that there were some limitations in this case report. The classifications from all these genetic testing laboratories indicated PVS1 as evidence for pathogenicity. However, more research as to the exact impact of the variant on the translated protein is still needed. In addition, patients only received gene panel testing, and not whole exome or whole genome sequencing. Thus, we cannot definitively conclude whether another variant in an untested gene may be contributing to this family’s phenotype. Patient 2 experienced neuropathic pain, not typically associated with AS, and the clinical team believes that this is caused by another clinical condition. Patient 1’s biopsy displayed numerous lamellar “zebra bodies”, which was also atypical. The clinical team is unsure as to what caused this.
Defining the pathogenicity of this COL4A4 variant has several clinically meaningful implications. All the proband’s children, except for patient 2, were spared an invasive biopsy. Follow up and monitoring by a nephrologist was started early on, prior to onset of several clinical symptoms. Patient 2 was treated with an angiotensin-converting enzyme inhibitor early on and counseled about the risk–benefit of pregnancy. Patients and parents were clinically informed about nephroprotective measures, such as avoiding NSAIDs, nephrotoxins, and maintaining a healthy diet.
In conclusion, we are the first to describe a family with this c.5007delC (p.Leu1670Ter) COL4A4 variant in detail in the literature. This is a variant classified as likely pathogenic by consensus and has an AD mode of inheritance. Nephrologists should recognize the benefit of collaborating with genetic laboratories and share the relevant clinical and family history of the patient to appropriately classify variants and determine pathogenicity. In turn, it would benefit genetic testing laboratories to regularly update their data and find centralized ways, in addition to ClinVar and ClinGen, to share their molecular data. Proper diagnosis, consistent classification of variants, and early monitoring can be beneficial for the treatment of patients with AS.