Guidelines for Genetic Testing and Management of Alport Syndrome

Abstract

Genetic testing for pathogenic COL4A3–5 variants is usually undertaken to investigate the cause of persistent hematuria, especially with a family history of hematuria or kidney function impairment. Alport syndrome experts now advocate genetic testing for persistent hematuria, even when a heterozygous pathogenic COL4A3 or COL4A4 is suspected, and cascade testing of their first-degree family members because of their risk of impaired kidney function. The experts recommend too that COL4A3 or COL4A4 heterozygotes do not act as kidney donors. Testing for variants in the COL4A3–COL4A5 genes should also be performed for persistent proteinuria and steroid-resistant nephrotic syndrome due to suspected inherited FSGS and for familial IgA glomerulonephritis and kidney failure of unknown cause.

Introduction

With the trend toward “mainstreaming” genetic testing, nephrologists may need to: explain the implications of finding a COL4A3–COL4A5 variant to patients and their families; obtain consent (for testing, using the results in studying family members, submitting variants to databases, and participating in related research); indicate that pathogenic variants are not always identified and that assertions sometimes change with time; describe the likely kidney outcomes; and start treatment to slow disease progression.

Other developments in genetic testing include the reporting of “variants of uncertain significance” (VUS), which are not clearly pathogenic or benign and may be reclassified over time; the recognition of “hypomorphic” COL4A5 variants, which are associated with hematuria only, late-onset kidney failure, or a thinned glomerular membrane; and the finding of “digenic” COL4A3–COL4A5 variants.

Background

Alport syndrome is the most common inherited kidney disease, even more prevalent than autosomal dominant polycystic kidney disease (¹). The autosomal dominant form affects about one in 100 of the population (²), and the more severe X-linked form affects about one in 2000 (³). Clinical features range from isolated hematuria to kidney failure, often with hearing loss, lenticonus, and fleck retinopathy (⁴). X-linked disease is caused by pathogenic variants in COL4A5 and autosomal recessive inheritance by biallelic pathogenic variants in COL4A3 or COL4A4 (^5–7). Digenic Alport syndrome occurs with variants in two of the COL4A3–COL4A5 genes and may have worse clinical features than a single variant (^8–10).

Individuals with a pathogenic heterozygous COL4A3 or COL4A4 variant are diagnosed with “thin basement membrane nephropathy” or “autosomal dominant Alport syndrome” (²,¹¹) and are considered carriers of autosomal recessive Alport syndrome. There is currently no consensus on the terminology, and these guidelines use “COL4A3 and COL4A4 heterozygotes” (¹¹). Heterozygotes with a pathogenic variant have hematuria and sometimes proteinuria, hypertension, and kidney impairment, but they rarely have the hearing loss or ocular abnormalities typical of X-linked Alport syndrome (²,¹²,¹³).

The genes affected in Alport syndrome, COL4A5, COL4A3, and COL4A4, code for the collagen IV α5-, α3-, and α4-chains (⁶,⁷,¹⁴), which form the α3α4α5 heterotrimer that is the major constituent of the basement membranes of the glomerulus, ear, and eye and explains the associated clinical features (¹⁵,¹⁶).

X-linked and autosomal recessive Alport syndrome have the highest risk of kidney failure. Most men and 15%–30% of women with X-linked inheritance, and most of those with autosomal recessive disease, develop kidney failure (¹⁷,¹⁸). Risk factors for progressive kidney impairment include the genetic variants’ location and type (¹⁹,²⁰). There is little correlation between variant features and kidney failure in women with X-linked disease (²¹), but their identification is important because of their own and their offspring’s risk of kidney failure (²¹). The risk for COL4A3 and COL4A4 heterozygotes has been estimated to be 10%–20% (^22–24), but genetic studies in unbiased cohorts suggest that it is much less (²⁵).

Making the diagnosis of Alport syndrome is critical because effective inexpensive treatment with renin-angiotensin-aldosterone system (RAAS) blockade delays the development of kidney failure (²⁶,²⁷). In addition, new agents will soon be available. However, making the diagnosis is often difficult on the basis of clinical features, family history, and even kidney biopsy (²⁸). In contrast, genetic testing is sensitive and accurate. It also indicates the mode of inheritance and, sometimes, the likelihood of early-onset kidney failure and extrarenal features (¹⁹).

What Is New Since the Last Guidelines?

The strategy to develop these guidelines is described in Box 1, and new developments are in Box 2. Major changes include the recommendations to identify all individuals with pathogenic heterozygous COL4A3 and COL4A4 variants as early as possible in order to enable monitoring and treatment and avoid unnecessary investigations, and to consider that the risk of kidney donation for COL4A3 and COL4A4 heterozygotes is in most cases unacceptable (²⁸,²⁹). Even minimizing the risk by only accepting donors aged more than 40 years with normal kidney function and normal urinary protein levels (³⁰) was not considered appropriate by the panel of Alport experts.

Box 1.

Strategy for developing the guidelines

The past 5 years have seen a great increase in the availability of massively parallel sequencing. The aim of these guidelines is to provide the most up-to-date advice for nephrologists and clinical geneticists on genetic testing and management of the diseases associated with pathogenic COL4A3–COL4A5 variants. This is important with the trend to “mainstreaming” genetic testing, where nephrologists who request testing must also explain the implications of finding pathogenic variants to their patients; obtain consent (for testing, using the results for other family members, submitting variants to databases, and participating in related research); indicate that pathogenic variants are not always identified and that variant interpretations sometimes change with time; describe the likely kidney outcomes; and implement the treatments to slow disease progression. These guidelines are also for clinical geneticists who need to stay up to date with developments in many different specialties. The guidelines are not evidence based because there are still too few prospective randomized controlled trials in Alport syndrome, but rather, they depend on expert opinion using the Reporting Tool for Practice Guidelines in Health Care: The Reporting Items for Practice Guidelines in Healthcare statement (⁷⁰).

In February 2020, a group of 47 clinical and nonclinical experts in Alport syndrome from Europe, North America, and Australia all involved in genetic testing for Alport syndrome or the care of these patients met at Chandos House, London, United Kingdom, to review the current recommendations for genetic testing for COL4A3–COL4A5 variants and to further refine the American College of Medical Genetics and Genomics/Association for Molecular Pathology criteria (⁵⁴) for evaluating COL4A3–COL4A5 variants. The recommendations on asserting pathogenicity are published separately. The group comprised adult nephrologists (n=12), pediatric nephrologists (n=6), histopathologists (n=2), geneticists (n=17), laboratory scientists (n=4), researchers (n=2), industry scientists (n=3), and a patient representative of the Alport UK Patient Support group (n=1). A subgroup spent the first afternoon reviewing previous recommendations and deciding on the topics to be updated and the gaps in our current knowledge. They presented their work that evening and were provided with feedback. This group spent most of the following day discussing their recommendations, which they again presented to the whole group; after the meeting, five smaller groups refined the recommendations, and the resulting document was amended and circulated to all participants for review.

Two major issues arose from these discussions (the risks of kidney failure and of kidney donation for COL4A3 or COL4A4 heterozygotes), and therefore, background information (“What is new since the last guidelines?”) was circulated for further opinion (Box 2). Recommendations were incorporated into the final document when there was >70% agreement. Nineteen attendees, 17 of whom were clinicians from three continents, contributed to these recommendations.

The draft manuscript was then reviewed by an independent Alport expert, the document was further amended on the basis of her suggestions, and it was again circulated for approval prior to submission for publication.

Box 2.

What is new since the last guidelines?

The indications for testing for COL4A3–COL4A5 variants include further phenotypes. These guidelines also include major changes to the recommendations for genetic testing for persistent hematuria where a heterozygous pathogenic COL4A3 or COL4A4 variant is suspected (²⁸); the recommendations for testing first-degree family members of COL4A3 or COL4A4 heterozygotes; and the recommendation that COL4A3 and COL4A4 heterozygotes should not act as kidney donors. Additional issues include consent, the finding of “variants of uncertain significance” as well as “hypomorphic” and “digenic” variants, and the clinician’s role in managing these.

The following background was provided to inform decision making on the two main issues.

The risks of kidney failure with pathogenic heterozygous COL4A3 or COL4A4 variants.

Individuals with pathogenic heterozygous COL4A3 or COL4A4 variants have a higher risk of proteinuria, hypertension, and kidney impairment and have a higher risk of kidney failure (²,¹²). Some studies suggest that 10%–20% of COL4A3 or COL4A4 heterozygotes have kidney failure by the age of 70 (^22–24), but most reports have been in hospital-based series with more severe disease. Although COL4A5 variants are much less common than COL4A3 and COL4A4 variants (1:20) in any population, they occur approximately as often in series with kidney failure, suggesting that the risk of kidney failure with pathogenic heterozygous variants is much less than with COL4A5 variants (⁷¹).

COL4A3 or COL4A4 heterozygotes acting as kidney donors.

The second issue was whether COL4A3 or COL4A4 heterozygotes should be allowed to act as kidney donors (Table 1).

There are already guidelines that address using kidney donors with hematuria (⁷²). Hematuria occurs in 3% of potential kidney donors (⁷³) and is often due to COL4A3–COL4A5 variant–associated disease (⁷⁴). Recent Kidney Disease Improving Global Outcomes guidelines cite a study by one of the authors of this document that suggests that heterozygous COL4A3 or COL4A4 variants are uncommonly associated with kidney failure (²) and that, on the basis of a small series with short follow-up periods, these individuals may act as donors (⁷²). Other recommendations have used an association with kidney failure for a particular mutation to exclude a kidney donor (³⁰).

However, after 15 years, the observed risk of kidney failure in kidney donors is already 3.5 to 5.3 times greater than the projected risk in the absence of donation (⁷⁵). The risk of proteinuria, hypertension, and kidney impairment is also likely to be higher with kidney donation even in the short term. There is increasing evidence from patient reports and series that kidney donation from individuals with hematuria or heterozygous pathogenic COL4A3 or COL4A4 variants is followed by worsening proteinuria or kidney function over even a brief period of review (^76–78). Thus, in a cohort of 20 individuals with persistent hematuria followed for a median of 2.3 years after kidney donation, 8% developed persistent proteinuria, and many had a deterioration in kidney function (⁷⁹).

Recommendation 1: Genetic Testing for a Pathogenic Variant in COL4A3–COL4A5

Genetic testing for a pathogenic variant in COL4A3–COL4A5 is typically recommended where there is persistent dysmorphic hematuria >6 months in duration where there is no other obvious cause (Box 3). Hematuria is highly penetrant with pathogenic COL4A3–COL4A5 variants, occurring in nearly all men and 95% of women with a COL4A5 variant (³¹), and at least two thirds of those with a heterozygous pathogenic COL4A3 or COL4A4 variant (²). Episodes of macroscopic hematuria are common in children and adults and are often coincident with mucosal infections, but they also occur in other forms of glomerulonephritis (³²,³³).

Box 3.

Genetic testing for a pathogenic variant in COL4A3–COL4A5

Genetic testing is recommended where there is

• • persistent dysmorphic hematuria >6 months;
• • persistent proteinuria >0.5 g/d, steroid-resistant nephrotic syndrome, or biopsy-proven FSGS;
• • sensorineural hearing loss with hematuria and a family history of hematuria or kidney impairment;
• • lenticonus, fleck retinopathy, or temporal retinal thinning;
• • GBM lamellation or thinning or an abnormal collagen IV α-chain composition;
• • kidney failure without another obvious cause, especially with hematuria and a family history of hematuria or kidney impairment; and
• • familial IgA glomerulonephritis (that is, IgA glomerulonephritis in one person together with a family history of hematuria or kidney impairment).

The COL4A3–COL4A5 genes should be included in gene panels for persistent hematuria, glomerulonephritis, FSGS, and kidney failure where the cause is unknown but suspected to be genetic.

The likelihood of identifying a pathogenic COL4A3–COL4A5 variant is higher where hematuria and a family history of hematuria or kidney impairment are present: that is, where the disease is familial. The likelihood of detecting a pathogenic COL4A3–COL4A5 variant appears to be the same for children and adults.

Genetic testing is also performed

• • to determine the mode of inheritance with certainty; and
• • to avoid performing a kidney biopsy.

Genetic testing is recommended in the first-degree relatives (parent, sibling, and offspring) of a person with a known pathogenic variant in COL4A3–COL4A5 as follows:

• • To establish the genetic status of the first-degree relatives of a person with a pathogenic variant in COL4A5. Hematuria is unreliable in detecting whether an individual is affected (⁸⁰).
• • To demonstrate that a person has autosomal recessive Alport syndrome due to compound heterozygous variants with one variant inherited from each parent and that the variants are thus in trans (on opposite chromosomes). In trans variants in COL4A3 or COL4A4 have more severe disease than those in cis (on the same chromosome) variants.
• • To test for the presence of a pathogenic variant in the first-degree relatives of a person with a heterozygous COL4A3 or COL4A4 variant.
• • To establish whether the partner of a person who is heterozygous for a pathogenic COL4A3 or COL4A4 variant has a pathogenic variant in one of the COL4A3–COL4A5 genes where the partner has hematuria, has a family member with hematuria or kidney impairment, or is consanguineous, because of the risk of autosomal recessive or digenic Alport syndrome in their offspring.
• • To confirm that a family member who wishes to be a kidney donor does not have the pathogenic variant.

The likelihood of detecting a pathogenic COL4A3–COL4A5 variant is higher where there is also kidney impairment or a family history of hematuria or kidney impairment. In 101 individuals with hematuria and a family history of hematuria or kidney impairment, 80% had a pathogenic COL4A3–COL4A5 variant (³⁴). Nine percent of those in another series of 3315 adults with suspected inherited kidney disease had a pathogenic COL4A3–COL4A5 variant (²⁴). The likelihood of finding a pathogenic variant in COL4A5 is also higher when there is hearing loss, lenticonus or fleck retinopathy (³⁵), or an abnormal glomerular basement membrane (GBM) (³⁶). A sensorineural hearing loss is very common in X-linked and autosomal recessive Alport syndrome (³⁷), but also occurs with other forms of inherited kidney disease (³⁸). There are very few other causes of diffuse GBM lamellation (²⁸) or thinning, and these findings alone are an indication for genetic testing.

However, COL4A3–COL4A5 pathogenic variants are also found when there is persistent proteinuria >500 mg/d or steroid-resistant nephrotic syndrome due to FSGS in both children and adults (²³,³²,³⁹). The proteinuria probably results from podocyte loss (⁴⁰,⁴¹) and secondary glomerulosclerosis. COL4A3–COL4A5 pathogenic variants are the most common cause of FSGS in many series (³⁹).

Hearing loss, lenticonus, fleck retinopathy, and FSGS with COL4A3–COL4A5 variants are all more common in adults than children (¹⁷,⁴⁰,⁴²,⁴³).

In addition, a COL4A3–COL4A5 pathogenic variant is found in 10%–35% of those with kidney failure of unknown cause (⁴⁴). In general, pathogenic variants are detected more often where there is familial disease and where there are extrarenal features, but they are not detected more often with a younger age at presentation (⁴⁵).

IgA glomerulonephritis commonly occurs together with thin basement membrane nephropathy (⁴⁶,⁴⁷) or with pathogenic COL4A3–COL4A5 variants (⁴⁸,⁴⁹). This is often when there are also other family members with hematuria, which is attributed to familial IgA disease but is more likely due to an undetected pathogenic COL4A3–COL4A5 variant in family members. The coexistence is currently unexplained.

There have now been several reports of kidney cysts associated with thin basement membrane nephropathy or COL4A4 or COL4A5 variants (⁴³,⁵⁰) after autosomal dominant polycystic kidney disease has been excluded. The cysts are evident from the age of 35, and they are often found with proteinuria, FSGS, and kidney impairment; however, they are not themselves large enough to affect kidney function. Cysts may be bilateral, but kidneys are normal size. The Alport experts believed that there was currently insufficient evidence to recommend testing for COL4A3–COL4A5 variants in cystic kidney panels but that the presence of kidney cysts should not discourage testing for pathogenic COL4A3–COL4A5 variants when these are suspected. The finding of hematuria or a family history of hematuria or kidney function is further encouragement for testing for COL4A3–COL4A5 variants in cystic kidney disease.

The Alport experts considered that it was important to undertake genetic testing even when a pathogenic heterozygous COL4A3 or COL4A4 variant is suspected. It is often not possible to distinguish clinically between a pathogenic variant in the COL4A5 gene and one in COL4A3 or COL4A4 with confidence, but the implications are very different. Although COL4A3 or COL4A4 heterozygotes have a very low risk of kidney failure, they may develop proteinuria, hypertension, or kidney impairment, and their first-degree family members should also undergo genetic testing. It is also not possible to predict the risk of kidney failure from the nature of the COL4A3 and COL4A4 variants, but proteinuria, hypertension, and kidney impairment all suggest risk factors for disease progression. COL4A3 and COL4A4 heterozygotes have a small (<1%) risk of offspring with autosomal recessive or digenic Alport syndrome. Recognizing that a person has hematuria from a COL4A3 or COL4A4 variant means that they will not require further unnecessary investigations such as kidney biopsy or cystoscopy.

Recommendation 2: Clinical Assessment and Genetic Testing

Investigations for clinical manifestations may be performed before genetic testing primarily to confirm the suspicion of a COL4A3–COL4A5–related disorder, and afterward, to determine extrarenal features. The finding of the typical clinical manifestations encourages genetic testing. Clinical features should also be assessed if genetic testing is not available (Box 4). There have been no changes to the clinical assessment guidelines except to extend the indications for testing.

Box 4.

Clinical assessment where COL4A3–COL4A5 variants are suspected

The individual who is to undergo genetic testing may

• • be tested for serum creatinine/eGFR, urinary red blood cell count, and urinary albumin-creatinine (proteinuria and kidney impairment as well as hypertension are all indications for starting treatment);
• • undergo audiometry, retinal photography (including peripheral views), and retinal optical coherence tomography (OCT); and
• • ask their family members for any history of hematuria, kidney failure, or other features consistent with a pathogenic COL4A3–COL4A5 variant.

Appropriate tests include audiometry for hearing loss (³⁷), retinal photography for fleck retinopathy (⁸¹), and OCT for temporal retinal thinning (⁸²). Hearing loss and fleck retinopathy are found in men and women with X-linked or autosomal recessive disease (⁸¹). Temporal retinal thinning is found in men with X-linked disease and men and women with autosomal recessive disease (⁸³). The peripheral retinopathy is more common than the central fleck retinopathy in men and women with X-linked or autosomal recessive Alport syndrome (⁸¹). The peripheral retina is at least two disk diameters from the central macula and is seen on nonstandard but easily obtainable views. The demonstration of temporal retinal thinning requires a dilated optic fundus. Audiometry, retinal photography, and retinal OCT are all more likely to be abnormal in adults than children, and they may also be performed as pregenetic testing in an informative adult family member, such as the mother of a boy with a suspected COL4A5 variant, if the tests are unhelpful or not available in a child.

Recommendation 3: Consent for Genetic Testing

The international trend toward “mainstreaming” (⁵¹) genetic testing means that a nephrologist who suspects a COL4A3–COL4A5 variant may request testing for the patient without review by a clinical geneticist. Sometimes, such an assessment is required. Geneticists and genetic counselors can also provide the patient with information on the risks and benefits of genetic testing, reproductive risks, and pregnancy planning, and they can help identify other family members for cascade testing. They may also be involved in the multidisciplinary meetings that decide on variant classification.

• • The patient should be offered the opportunity to discuss the risks and benefits of genetic testing, including with an independent professional if they wish. The benefits of genetic testing are that, in many cases, it confirms or excludes a diagnosis and, where appropriate, allows the commencement of monitoring and treatment; that other family members may be identified and treated; and that the whole family may make informed reproductive decisions. The risks are that it may not result in a diagnosis or that the result is inconclusive or incorrect. There is also possibly anxiety associated with the testing and uncertainty.
• • When obtaining consent, the clinician should explain and record whether targeted testing for variants in the COL4A3–COL4A5 genes or a more comprehensive approach is being undertaken. The clinician should explain that the causative variant(s) may not be identified and that sometimes variant classification, typically a VUS, changes with time as new information becomes available.
• • The clinician may explain that undergoing genetic testing affects the ability to obtain medical insurance or life insurance in some jurisdictions.
• • The clinician may request permission to use the genetic information to evaluate tests from other family members. Increasingly, it is believed that this anonymized information should be available to inform genetic testing in other family members, especially because treatment for Alport syndrome with RAAS blockade delays kidney failure onset (⁵²,⁵³).
• • The clinician should indicate that the laboratory will submit the genetic variant or variants and their assessments of pathogenicity in an anonymized form to variant databases and disease registries in order to help others with the same condition.

Recommendation 4: Genetic Testing and Reporting Results

Guidelines for testing and reporting are well established (⁴⁶,⁴⁷,⁵⁴,⁵⁵); however, practices vary (⁵⁶), and variant interpretations from different laboratories may differ. International efforts to harmonize both testing and reporting procedures are underway (⁵⁷).

• • Genetic testing for clinical use should be performed in an accredited laboratory. The results from research studies should be validated in an accredited laboratory before they are used clinically.
• • Where Alport syndrome is suspected, all three COL4A3–COL4A5 genes should be examined for pathogenic variants. Even when a pathogenic variant is identified, the other genes should be examined and all pathogenic or likely pathogenic variants reported because each may contribute to, or modify, the phenotype.
• • Gene variants should be described using the Reference Sequence, HGVS nomenclature (⁵⁸), and American College of Medical Genetics and Genomics/Association for Molecular Pathology criteria (⁵⁴). This may include an in-house subclassification for a VUS that indicates that it is more likely to be pathogenic or benign (⁵⁴).
• • The laboratory should report a variant that is pathogenic, likely pathogenic, or a VUS that is suspected to be pathogenic.
• • The patient should also be offered a copy of the laboratory results with the diagnosis, the gene name, the variant type, the American College of Medical Genetics and Genomics/Association for Molecular Pathology classification, and interpretation.

Recommendation 5: Actions When Digenic Variants Are Identified

Digenic Alport syndrome occurs where there are pathogenic variants in two of the three COL4A3, COL4A4, and COL4A5 genes (^8–10). Most of the Alport experts considered that variants that occurred in another podocyte or basement membrane gene (⁹,¹⁰,^59–62) together with one of the COL4A3–COL4A5 genes were examples of modifier genes rather than of digenic disease (⁸,⁶¹).

Digenic Alport syndrome is not common but is the reason for the recommendation to examine all three COL4A3–COL4A5 genes in at-risk individuals. The most common form of digenic Alport syndrome has both a pathogenic COL4A3 and COL4A4 variant and may demonstrate a more severe clinical phenotype than found with a single variant (⁹). Thus, affected individuals are more likely to develop proteinuria, hypertension, and kidney failure than heterozygotes.

Because the COL4A3 and COL4A4 variants are located head to head on chromosome 2, both variants may be inherited together on the same chromosome (in cis) when inheritance is autosomal dominant and occurs in successive generations, or on opposite chromosomes (in trans) (¹⁰) where the combination of variants occurs in only a single generation.

• • The term digenic Alport syndrome is used when there are two pathogenic variants in different COL4A3–COL4A5 genes. Each variant that is pathogenic, likely pathogenic, or a VUS that is likely to be pathogenic, and hence, clinically significant should be reported.
• • When individuals have a COL4A5 and a COL4A3 or COL4A4 variant, they should be treated with RAAS blockade from the time of diagnosis because of their risk of kidney failure.
• • For COL4A3 and COL4A4 variants, the cis or trans nature of digenic variants should be identified because this determines whether the offspring inherit one or both variants and the risk of kidney impairment in future generations of the family.
• • When individuals have digenic pathogenic variants in COL4A3 and COL4A4, they should be treated with RAAS blockade from the time of diagnosis because of their risk of kidney failure.
• • First-degree family members of individuals with digenic Alport syndrome should undergo cascade testing to identify affected family members.
• • Individuals with digenic Alport syndrome should not act as kidney donors.

Recommendation 6: Actions When a Pathogenic or Likely Pathogenic Variant Is Identified

• • All men with X-linked Alport syndrome should be treated with RAAS blockade from the time of diagnosis. This is because 90% of men with a pathogenic COL4A5 variant develop kidney failure by the age of 40 (¹⁷). Women with X-linked Alport syndrome should be treated from the onset of microalbuminuria, hypertension, or kidney impairment (²⁸) because these represent risk factors for kidney failure, which occurs in 15%–30% of affected women by the age of 60 (¹⁸).
• • All men and women with autosomal recessive Alport syndrome should be treated with RAAS blockade from the time of diagnosis (²⁸). The onset of kidney failure with autosomal recessive Alport syndrome is possibly earlier than even in men with X-linked disease (⁶³).
• • Where individuals have digenic pathogenic COL4A3 and COL4A4 variants, they should be treated with RAAS blockade from the time of diagnosis because of their risk of kidney failure (those with a COL4A5 together with a COL4A3 or COL4A4 variant, which is much less common, will already be treated according to the guidelines for those with a COL4A5 variant).
• • Individuals with a heterozygous pathogenic COL4A3 or COL4A4 variant should be treated with RAAS blockade from the onset of microalbuminuria, hypertension, or kidney impairment.
• • These recommendations are the same for children and for adults (⁶⁴).

“Hypomorphic” pathogenic variants in COL4A5 are identified increasingly, but it is uncertain how common they are. Hypomorphic variants in COL4A5 are typically associated with milder disease: for example, hematuria only, late-onset kidney failure, or a thinned rather than lamellated GBM (⁶⁵,⁶⁶). The most common hypomorphic variant in COL4A5 is p.(Gly624Asp), which accounts for up to half of all pathogenic variants in some European populations (⁶⁶). It is important for clinicians to be aware of hypomorphic variants and that, despite being common, they are “actionable” because they may eventually cause kidney failure and thus require monitoring and treatment. In general, actionability depends on the balance of burden, tolerance of risk, and acceptability of treatment (⁶⁷).

Recommendation 7: Actions When a Variant of Uncertain Significance Is Detected

The clinical significance of variants detected with massively parallel sequencing may be difficult to ascertain with current protocols and knowledge.

• • It is preferable for a VUS to be discussed with the referring clinician or a multidisciplinary team prior to reporting, especially if the clinical phenotype is inconsistent with the variant.
• • A VUS in a COL4A3–COL4A5 gene that is more likely to be pathogenic should be reported as such. That is, a VUS should, if possible, be further classified by a laboratory in a way that indicates that it is “more likely to be benign” or “more likely to be pathogenic” (⁵⁴).
• • The classification of a VUS may change to pathogenic or benign after further examination of the family, additional independent reports, or expert review. A VUS may be further investigated with segregation studies in the family to confirm that it is only found in clinically affected family members and is absent from those who are unaffected.
• • The laboratory may re-examine the variant’s classification if requested to do so by a nephrologist or geneticist, usually because the diagnosis is still not known and new information has become available.
• • The report form indicates the laboratory’s policy on reporting and reviewing a VUS, such as, that reviews may occur at the clinician’s request, at 18-month intervals, on two further occasions.
• • Clinical advice should be provided for people with a VUS on the basis of their personal characteristics, such as age and sex and family history. A VUS should not be used for medical decision making or predictive cascade testing in families (⁵⁴). Prenatal and preimplantation genetic testing should not be performed on the basis of the presence of a VUS.
• • A VUS in a gene that is not relevant to the clinical diagnosis is not reported.

Recommendation 8: Actions When No Pathogenic or Likely Pathogenic Variant Is Identified

The Alport experts did not have a uniform approach, but these were some of their strategies.

• • The quality of the sequencing should be reviewed to confirm adequate gene coverage. Massively parallel sequencing is less sensitive for deep intronic variants, large deletions, and copy number variation. Synonymous variants resulting in a downstream stop codon through activation of a downstream cryptic splice site, insertions, and duplications should be considered.
• • Another technique for detecting variants, such as mRNA sequencing (⁶⁸), multiplex ligation-dependent probe amplification, or whole-genome sequencing, may be used.
• • The search strategies should be indicated in the report.
• • The diagnosis and clinical and histologic features should be re-evaluated preferably in consultation with the clinician or a multidisciplinary team, taking into account the extrarenal features, family history, and GBM ultrastructure.
• • A kidney biopsy should be performed or re-evaluated (in particular, light microscopic and immunostaining examination for an additional form of glomerulonephritis and ultrastructural examination for GBM lamellation or thinning).
• • Hearing loss suggests the diagnosis of Alport syndrome but also occurs with other diseases (³⁸). Lenticonus, fleck retinopathy, and temporal retinal thinning strongly suggest Alport syndrome. Lenticonus is only found in men with X-linked Alport syndrome and men and women with recessive disease (⁶⁹). The fleck retinopathy is common in men and women with X-linked or autosomal recessive Alport syndrome, but it does not occur with heterozygous pathogenic COL4A3 or COL4A4 variants.
• • Another inherited kidney disease may be considered, including FSGS (due to one of many genes); MYH9-related nephropathy (OMIM 155100, MYH9); dense deposit disease (OMIM 613913, multiple complement pathway genes); CFHR5 disease (OMIM 604819, CFHR5); hereditary angiopathy, nephropathy, aneurysms, and muscle cramps (OMIM 611773, COL4A1); nail patella syndrome (OMIM 161200, LMX1β); and mitochondrial disease (where massively parallel sequencing is less sensitive). Apart from FSGS, these conditions are much less common than pathogenic COL4A3–COL4A5 variants.
• • The individual and their family may be referred to a specialized service or research laboratory to be investigated for a novel genetic cause. This is difficult where there are few family members, autosomal dominant inheritance, or suspected incomplete penetrance.

Recommendation 9: Genetic Testing of Family Members

• • In general, first-degree family members of individuals with X-linked Alport syndrome (parents, siblings, and offspring) should undergo cascade genetic testing regardless of the results of other investigations. Genetic testing is more sensitive than hematuria in identifying affected family members (³¹).
• • First-degree family members of individuals with autosomal recessive Alport syndrome should undergo cascade testing.
• • First-degree family members of individuals with digenic Alport syndrome should undergo cascade testing.
• • First-degree family members of individuals with heterozygous COL4A3 or COL4A4 variants should undergo cascade testing.

Recommendation 10: Kidney Transplantation and Donation

Women who have COL4A5 variants should not act as kidney donors because of their inherent risk of kidney failure and the additional burden of donation on kidney function (²¹). The situation has been less clear for individuals with pathogenic heterozygous COL4A3 or COL4A4 variants, but most demonstrate a deterioration in kidney function over the years following donation that is greater than occurs in other donors (Table 1).

• • Potential donors suspected of having COL4A3–COL4A5 variants should undergo genetic testing prior to kidney transplantation to confirm the diagnosis and determine the mode of inheritance and any variant characteristics associated with a poor prognosis.
• • The results of genetic testing may exclude a family member as a potential kidney donor.
• • Women who have a COL4A5 variant should not act as kidney donors because of their own risk of kidney impairment and of further deterioration after donation.
• • Individuals with digenic variants in COL4A3–COL4A5 should not act as kidney donors.
• • Individuals with a heterozygous COL4A3 or COL4A4 variant should also not act as kidney donors because of their own risk of kidney impairment and of a further deterioration after donation.

Table 1. - The outcomes of kidney donors with pathogenic heterozygous COL4A3 or COL4A4 variants

Age of Donor, yr	Sex	Diagnosis of Pathogenic Heterozygous COL4A3 or COL4A4 Variant	Initial eGFR of Donor, ml/min per 1.73 m2	Follow-Up Period, mo	Last eGFR, ml/min per 1.73 m2	Reference
49±8	1 M, 10 F	Thinned GBM on biopsy	90±3	56.8±32.0 (6.4–120.1)	59±6 (P=0.40)	(78)
>46	1 M	COL4A3 variant (Gly637Arg)	90	84	60	(76)
50	1 F	Thinned GBM on biopsy	90 (presumed)	48	Decreased by 30%	(77)

M, male; F, female; GBM, glomerular basement membrane.

Limitations of These Recommendations

There are still gaps in our knowledge upon which guidelines such as these are based (Box 5).

Box 5.

Remaining gaps in our knowledge and the opportunities for future research

• • The risks of COL4A3 and COL4A4 variants (in particular, the likelihood of and age at onset of proteinuria, hypertension, and kidney impairment).
• • The risks in digenic Alport syndrome of developing proteinuria, kidney failure, hearing loss, retinal abnormalities, and a lamellated GBM.
• • The risks and characteristics of hypomorphic variants, how they can be identified, whether they result in more severe disease when combined with a variant in a modifying gene, whether they are actionable, and the indications for treatment.
• • The significance of pathogenic COL4A3–COL4A5 variants in cystic kidney disease and whether COL4A3–COL4A5 genes should also be included in gene panels for cystic kidney disease.
• • The optimal approach where a pathogenic variant in COL4A3–COL4A5 is expected but not found.

The most relevant treatment study is EARLY-PROTECT, in which children diagnosed with Alport syndrome underwent treatment with an ACE inhibitor that reduced proteinuria and delayed kidney impairment (²⁷); however, there are no studies yet in adults, and there are no published assessments of cost effectiveness or quality of life evaluations.

In conclusion, pathogenic COL4A3–COL4A5 variants are the most common cause of inherited kidney disease and manifest as a spectrum of disease that includes hematuria, proteinuria, FSGS, familial IgA glomerulonephritis, kidney cysts, and kidney failure. Early diagnosis is important in part because progression to kidney failure can be delayed with RAAS blockade.

Disclosures

L. al-Rabadi reports receiving honoraria from Atheneum, Guidepoint, and the National Institutes of Health and serving as a scientific advisor or member of the Alport Syndrome Foundation Medical Advisory Board. K. Claes reports consultancy agreements with Astellas and Sanofi, serving as a scientific advisor or member of Alexion and Astellas, receiving a speaker's fee from Menarini, and other interests/relationships with Fresenius Medical Care. C. Deltas has a research collaboration agreement with Regeneron. S. Gear reports other interests/relationships with Alport UK, a UK patient organization for patients living with Alport syndrome. M. Gregory reports serving as a scientific advisor or member of the Alport Syndrome Foundation Medical Advisory Board, the Idaho Medical Advisory Board, and the National Kidney Foundation of Utah. P. Hilbert is an employee of the Institute of Pathology and Genetics. S. Landini reports serving as a member of the ERKNet Pediatric Unit, Institution Florence Meyer Children's Hospital. R. Lennon reports consultancy agreements with Travere Therapeutics, serving as a scientific advisor or member of the Kidney Research UK grants panel and the Scientific Advisory Research Network for the Alport Syndrome Foundation, funding from The Wellcome Trust, other interests/relationships with Kidney Research UK, and serving as a trustee for Alport UK and a trustee for Kidneys for Life. B.S. Lipska-Zietkiewicz has received honoraria from Takeda and Travere for giving lecutres. B.S. Lipska-Zietkiewicz reports serving as a section editor of experimental nephrology and genetics for Nephron, a scientific journal by Karger. B.S. Lipska-Zietkiewicz reports serving as a Molecular Diagnostics Task Force member; a Hereditary Glomerulopathies WG cochair, European Reference Network for Rare Kidney Diseases; a member of the European Society of Pediatric Nephrology; and a member of the International Pediatric Neprhology Association. A. Lungu reports employment with the Fundeni Clinical Institute, serving as a scientific advisor or member of Chiesi, and serving on the speakers bureau for Alexion. F. Mari reports serving as a scientific advisor or member of SienaGenTest s.r.l. and serving as a member of the Scientific Committee of the Italian Alport Syndrome Patients’ Association. L. Massella reports serving on the editorial board of Nephron for the section “Case Studies in Genetics.” A. Renieri reports receiving research funding from Travere Therapeutics, Inc. H.M. Rothe reports receiving honoraria from VIFOR Pharma and serving on the speakers bureau for VIFOR Pharma. J. Savige reports employment with Northern Health and other interests/relationships with Alport Foundation Australia and the PKD Australia Scientific Board. A. van Eerde reports serving on the European Renal Association—European Dialysis and Transplant Association Working Group Inherited Kidney Diseases Board, serving as an European Rare Kidney Disease Reference Network Working Group chair, and receiving funding from the Dutch Kidney Foundation. E. Watson reports employment with the National Health Service (United Kingdom). All remaining authors have nothing to disclose.

Funding

None.