1 INTRODUCTION

Alport syndrome (AS), a type IV collagen disease, is the second most common monogenic cause for end-stage renal failure (ESRF).^1-3 AS is caused by pathogenic variants in the genesCOL4A3, COL4A4, and COL4A5, each encoding an alpha-chain of type IV collagen, an important component of the basal membrane in kidney, cochlear, and eyes.^{1, 2} The α(IV)-chain consists of a long collagen tail with an arrangement of repeating Gly-X-Y amino acid molecules, which is important for the structure of the triple helix, its flexibility and its unique mechanical properties.⁴ AS can be inherited in an X-linked (XLAS) or autosomal recessive (ARAS) form. Pathogenic variants in the COL4A5 (Xq22.3) gene cause XLAS, which accounts for 80–85% of all patients with AS.^{5, 6} Therefore, mostly males are affected and a distinct genotype–phenotype correlation could be detected.⁷ In female patients with a heterozygous variant in COL4A5, a variable intrafamilial and interfamilial penetrance exists resulting in a broad spectrum of clinical symptoms ranging from mild microscopic hematuria to severe AS.^8-10 Autosomal recessive AS results from biallelicpathogenic variants in COL4A3 (2q36.3) and COL4A4 (2q36.3) in about 15%.^{3, 7} In some cases a digenic inheritance was proposed.¹¹ The existence of an autosomal dominant inheritance (ADAS) (monoallelic pathogenic variants in COL4A3 and COL4A4), which is also described in the literature, is contested.¹² Past literature mostly described it as a very rare form of AS, only one recent study postulated a frequency of 31%.¹³ The current recommendation is to describe these patients as carrier of AS or as patients with a thin basement membrane nephropathy (TBMN).¹² Heterozygous carriers of pathogenic variants in COL4A3 or COL4A4 also have an increased risk for glomerular pathological changes presenting as focal segmental glomerular sclerosis (FSGS) and ESRF.^{8, 14}

The disruption of type IV collagen structure is the pathophysiological hallmark in AS and leads to a dysfunction of the glomerular filter with hematuria, proteinuria, and progressive renal failure. Patients with AS can also develop (high-frequency-) hearing impairment and ocular findings, which include maculopathy and anterior lenticonus, which is pathognomonic for AS.^15-18 Diagnostic criteria for patients with AS also include a positive family history of microscopichematuria or renal failure, renal biopsy, and genetic testing.¹⁹ Genetic testing ofCOL4A3, COL4A4, and COL4A5 is broadly available in most countries and has emerged as gold standard for the accurate diagnosis of AS.²⁰ To evaluate renal biopsy, electron microscopy (EM) should be performed. EM shows pathognomonic changes including thinning, areas of thinning and thickening and/or a complex splitting of the glomerular basement membrane (GBM).¹⁹ The age of ESRF differs among patients with different types of variants. Better genotype–phenotype correlations exist for patients with XLAS than for patients with ARAS, but as of the current literature there seems to be no significant difference in clinical course of male patients with XLAS and patients (both sexes) with ARAS.²⁰ The probability of ESRF for an untreated 30-year-old male patient with XLAS varies between 50% for missense variants, 70% for splice-site variants, and up to 90% for large rearrangements, nonsense variants, and frame shift variants.⁷

Registry data have shown that the progress of the renal manifestation in A scan be delayed by an early start of angiotensin-converting enzyme inhibitor (ACEi)treatment in proteinuric patients with chronic kidney disease (CKD) stage 2.²¹ To clarify whether an even earlier start (CKD stage 0 or 1) is safe and effective, the EARLY PRO-TECT Alport trial (NCT01485978) was initiated in 2012.²² In the present study, we address the question, which arose during and after primary data analysis of the EARLY PRO-TECT Alport trial: can the classifications according to Gross et al.⁴ and Bekheirnia et al.⁶ be helpful in judging the clinical course and the response to therapy in genetically solved patients of the EARLY PRO-TECT Alport trial and a (likely) pathogenic variant in COL4A5?

Our data shall provide evidence for genetic counseling of families with AS in everyday clinical practice and shall underline the important duty of the clinician to provide complete assessment of renal and extra renal symptoms and, if available, biopsy results to allow the geneticist a precise and accurate assessment of identified variants.

2 MATERIAL AND METHODS

3 STATISTICAL ANALYSIS

Group comparisons were made by a two-tailed Fisher's exact test with a significance level (α) of 0.05. For the analysis, patients were divided in those who did receive treatment and those who received placebo. After that each subset was classified according to Gross et al.⁴ and Bekheirniaet al.⁶ and each group was tested for a possible association between disease progress and risk classification by Fisher's exact test. Descriptive statistics are displayed as median and inter-quartile range (IQR). Statistics were calculated by STATA version 14.2 (STATA, College Station, TX).

4 RESULTS

Patients were evaluated and analyzed according to a flow chart (Figure 1). Out of 66 children who entered the treatment phase of the EARLY PRO-TECT Alport trial, five had to be excluded because of protocol violation or withdrawn consent. Twenty children qualified for randomization (all male) and 42 children (two girls and 40 boys) were openly treated. The patients' median age was 8 years (IQR 8). In 98% (60/61), molecular genetic testing was performed. One boy was not tested, because of his typical AS symptoms and two relatives with genetic testing, in which the variant causing AS could not be found. Therefore, the following results are based on these 60 patients (Table 1). See Table 1 for a complete account of the phenotypic and genotypic data including applied ACMG criteria for each variant.

5 DISCUSSION

Early genetic diagnosis in children with AS is crucial for early therapy to delay renal failure and improve life-expectancy. The present study is a genotype–phenotype correlation of patients of the EARLY PRO-TECT Alport trial. Although the study size is small, the patients are phenotypically very well ascertained and variant interpretation was transparent and reproducible using ACMG criteria with latest amendments.^{29, 31}

In the present study, we address the question whether the classifications according to Gross et al.⁴ and Bekheirnia et al.⁶are helpful in discriminating the clinical course and the response to therapy in genetically solved patients with AS and a causative variant in COL4A5.

First of all, in 8/60 (13%) patients of this study causative variants (or a fitting genotype) could not be identified leading to a genetically unsolved case. This could be due to several factors: (i) these patients carry their causative variant(s) within non-analyzed regions of the genes COL4A3, COL4A4 or COL4A5 (e.g., deep-intronic variants, which are usually not covered by sequencing methods capturing protein-coding and adjacent regions), (ii) they have a different monogenic disease mimicking AS which they were not tested for (e.g., hereditary FSGS) or they do not have a monogenic but a multifactorial disease resembling AS (“phenocopy”). To approach these facts, comprehensive genetic testing including genes associated with phenotypically related hereditary kidney disorders, for example, by comprehensive panel or ES testing, should be performed in patients with suspicion of AS and negative COL4A3-5 genetic testing. Unfortunately, this was not included as part of the original trial and only two cases were analyzed by ES.

Of the remaining 52 patients, a genetic diagnosis could be made. Forty-five of these patients carried a causative variant in COL4A5, and 41/45(91%) patients passed through the complete trial. Using the classification system of Gross et al.⁴ a statistically significant discrimination concerning the allocation to severity groups and disease progress of the 41 patients who passed through the complete trial was not possible. On the other hand, using the classification system of Bekheirnia et al.⁶ at least a borderline significance (p = 0.05) could be found when associating the severity groups with disease progress. The non-significant discrimination of clinical course by variant using Gross et al.⁴ in contrast to Bekheirnia et al.⁶ might be due to small sample size and/or the fact that Bekheirnia et al.⁶ classifies all missense variants in the less-severe group compared to Gross et al.⁴ Generally speaking, in both classification systems there was a trend in the group of treated patients that those with a less-severe or intermediate variant had less likely a progress compared to patients with a severe variant. Whether this trend concerning reduced disease progress in the less-severe or intermediate group is attributable to genotype combined with treatment or the genotype alone cannot be discerned by these data as the placebo group had too few patients to compare the outcome stratified according to the variant classifications. Unfortunately, the number of patients in this study was limited and maybe therefore significant results concerning the classification according to Gross et al.⁴ cannot be seen. Concerning other limitations, for most of the patients, no genetic report with specific information on testing method, quality parameters, and genes studied were available as the genetic analysis was performed before the patients were part of the trial.

For the VUS in our cohort, one can acknowledge that a further investigation of splicing effect on RNA level (synonymous variant NM_000495.4:c.384G > A, p.(Lys128=), near splice variant NM_000495.4:c.81 + 4A > C,p.(?) in COL4A5and canonical splice variantNM_000092.4 c.[4810-1G > A];[4810-1G > A] affecting the acceptor splice site of the last exon of COL4A4) and segregation analysis (in-frame indel NM_000495.4 c.453_455del p.[Pro152del] in COL4A5) could lead to an upgrade of variant rating to “likely pathogenic.” This was not possible in this cohort, as index patients and families were not accessible due to the study design of the EARLY PRO-TECT Alport trial.

The results of this study indicate, as other genotype–phenotype correlations showed before, that genetic testing is not only prime to diagnose AS but also for knowing the exact genotype to assess clinical course. Our data indicate that there is a possible better prognosis for missense (less-severe) variants, which is comparable with the literature data that patients with a severe variant develop ESRF earlier than patients with a less-severe variant.⁷ Of note, in our study population, the most common co-morbidities were allergies, asthma, and skin diseases, all of which can lead to recurrent infections, which also possibly could contribute to disease progression in children with AS.³² One can speculate that this individual effect can be explained by the distinct impact of missense variants on type IV collagen assembly, mechanical stability, and collagen degradation. For example, the less vulnerable the GBM is to hyperfiltration and mechanical stress, which is the case in glycine-missense variants compared to splice-site or truncating variants, the later AS progresses to (irreversible) loss of glomeruli and renal fibrosis and the better ACEis' effect on lowering filtration pressure will delay progression.⁴ Nevertheless, currently a precise prediction of the therapeutic response to nephro protective therapy in patients with specific variants is difficult just on the basis of the present study results.

Up to now, there are hundreds of different and individual variants in the huge COL4A3-5 genes with a size between 150 and 250 kb. Furthermore, the complexity of the type IV collagen triple helix extracellular suprastructure formation, function, and degradation is too little understood. Both facts make it difficult to judge the clinical course of an AS patient from a genetic test result alone. Therefore, this study should highlight the importance of interdisciplinary collaboration between clinicians and human geneticists in order to comprehensively evaluate AS patients. The workup of these patients should include repeated comparison between genetic findings and clinical symptoms including family history to also identify phenocopies (Figure 2).

In addition, it should be pointed out that the most common variant in the trial wasp. (Gly624Asp) in COL4A5, identified in a hemizygous state in eight male XLAS patients. 2/8 (25%) children with this variant showed a progress of the disease during the trial. This variant is known in the literature to lead to a rather mild clinical phenotype (“hypomorphic” variant) with late onset of renal failure (median age of 50 years at ESRF).³³ In contrast, a recent case report described a more severe phenotype in patients carrying this variant.³⁴ The data of this clinical trial further support the doubt that this variant is “hypomorphic” in all carriers. Despite good response to ramipril therapy, according to our data, 2/8 patients with this variant also showed a disease progress. This is in contrast to the literature as there are only adult patients described.

6 CONCLUSION

The complexity of genetic testing in AS make it imperative for clinicians and geneticists to work closely together. In the future, a patient's individual genotype might be helpful to predict response to therapy with a possible advantage for missense (less-severe) variants to be more responsive, but more data is currently still needed to confirm this hypothesis. We recommend to use a stringent workflow between geneticist and referring clinician. The early diagnosis by an experienced geneticist allows prompt surveillance and swift initiation of therapy once treatable symptoms of AS become apparent (proteinuria, hypertension), which has the potential to delay renal failure by decades and improve life-expectancy in our young patients. Furthermore, larger studies should be initiated on an international collaborative basis to further improve genotype–phenotype correlations in AS, especially concerning response to treatment.

CONFLICT OF INTEREST

The authors declare no potential conflict of interest.