The Evolving Role Of Diagnostic Genomics in Kidney Transplantation Ⅱ

Considerations Around Testing Interpretation of Results 

There are caveats to genetic testing that are applicable to testing in anticipation of a kidney transplant. The first consideration is the way in which genetic test results are reported. A positive test result occurs when (i) a variant has been identified that is classified as pathogenic or likely pathogenic according to the American College of Medical Genetics variant classification criteria56 and is within a gene that has a clear evidence-based relationship to a kidney phenotype and (ii) the number of variants identified in the gene matches the mode of inheritance of the condition (i.e., 2 variants on separate alleles for autosomal recessive disorders) (Figure 2). If the patient’s clinical phenotype matches the kidney phenotype associated with the gene in which the pathogenic or likely pathogenic variant has been identified, the condition associated with the gene is the likely diagnosis. A negative test result means no reportable gene variants have been identified. Importantly, this does not mean that the patient’s kidney disease is not genetic, and one should not provide reassurance that a form of GKD has been excluded in the patient. This outcome could be the result of a number of factors. First, owing to limitations in the testing technology’s ability to detect the disease-causing variant, for example, intronic or regulatory region variants. Second, there may be an undiscovered gene or mechanism involved in the kidney disease. Third, the disease-causing gene may not have been analyzed due to incorrect panel selection and/or misphenotyping. This reinforces the importance of the initial and thorough clinical assessment. Further genetic investigations, potentially in the research context, may need to be pursued, where available, to identify the underlying cause of the GKD.

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A VUS is a variant in a gene where there is insufficient evidence to classify it as either pathogenic, likely pathogenic, likely benign, or benign (Table 1). This is an uninformative result for the patient and cannot be interpreted as the cause of the patient’s kidney disease. A VUS may be highly suspicious if the patient’s clinical phenotype matches the kidney phenotype associated with the gene in which a pathogenic or likely pathogenic variant is usually identified and the number of variants identified in the gene matches the mode of inheritance of the condition. In this situation, further clarification might be obtained by first re-reviewing the patient’s clinical phenotype, sometimes with nongenetic investigations (e.g., imaging, biopsy), but caution must be taken not to overcall the implication of a VUS in the disease. Second, clarification can be obtained with segregation studies with targeted testing of the VUS in affected and unaffected family members. Third, diagnostic genomics staff can review the VUS in population databases, such as gnomAD or ClinVar, to determine population frequency in healthy controls. Last, diagnostic genomics staff can perform in silico predictions for pathogenicity or arrange functional studies of the variant in model systems through research collaborations. In many instances, these additional steps may not be sufficient to change the variant’s classification, but reappraisal of the VUS every 2 to 3 years is recommended with the benefit of additional knowledge and tools. It is important to note that predictive testing for a VUS cannot be offered in potential live-related donors as it cannot be confidently linked with the disease.

In addition, most KF cases are related to diabetes, hypertension, or autoimmune conditions, and it is unlikely an underlying monogenic cause be identified in these situations. Similarly, many kidney diseases are polygenic, and so a single pathogenic gene variant may not carry the same weight with regard to diagnosis, and Mendelian inheritance patterns may not apply. Recently, genome-wide association studies have discovered thousands of genetic variants associated with the disease, with polygenic risk scores aggregating the individual effects of these variants and correlating them with disease risk. Unfortunately, this is not well established in KF and yet to enter mainstream clinical practice.

Predictive Testing of Potential Related Living Donors 

After a potential renal transplant recipient has undergone genetic testing with a causative gene variant identified, apparently unaffected relatives who are considering kidney donation can be offered testing for the same gene variant. The process of offering testing for a familial gene variant in an apparently unaffected individual is called predictive genetic testing and aims to clarify their risk of kidney disease, which can be a significant health revelation, and potentially determine their suitability to proceed with kidney donation. Many studies have indicated that predictive testing carries significant anxiety for many people,57-59 and as such it is important that they undergo proper pretest and post-test counseling.

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A positive predictive test outcome means that the individual has the identified familial gene variant, and although previously considered a potential donor, it can now be referred early for surveillance or started on therapies that may change the disease course. In the same instance, this individual no longer has the opportunity to donate to their relative which may have a potentially negative psychological impact on them, the recipient, and their relationship.

A negative predictive test outcome means that the patient does not carry the familial gene variant and can be considered for donation. It also carries complex psychological impacts, with survivor guilt a common experience in family members with a negative test result. This describes the situation in which a patient tests negative for the familial variant associated with a disease in their family, and they feel guilt toward those who have tested positive or are affected. There is limited information about predictive testing in kidney disease, but studies performed with patients being tested for Huntington’s disease reveal that 10% of those who receive a negative predictive result have difficulty coping with their gene status based on assessments of psychological well-being.60

There are other nonmedical implications to predictive genetic testing, such as the impact on the ability to obtain income or life insurance. It is important that patients are aware of these issues and have the opportunity to make informed decisions. Therefore, predictive genetic testing should only be performed with appropriate pretest and post-test genetic counseling by a clinical geneticist or genetic counselor.

Common GKDs ADPKD

ADPKD is the most common genetic condition that results in KF. In approximately 78% of phenotypical cases, there is a pathogenic variant in the PKD1 gene, with approximately 15% having a pathogenic variant in the PKD2 gene.61 Direct PKD gene sequencing has a higher cost and longer turnaround time. Pseudogenes are also problematic in genetic testing of ADPKD as PKD1 bears 97.7% similarity in sequence to 6 pseudogenes.62 GS has been found to efficiently circumvent these challenges,62 with this now being revealed in clinical contexts.63

The genotype of patients with ADPKD is predictive of the clinical course. Compared with patients with PKD2, those with PKD1 gene mutations progress to KF on average 20 years earlier and die at a younger age.64 Importantly, up to 18% of affected individuals might experience significant intrafamilial disease variability65 compared with that anticipated by their genotype and/ or family history.

The diagnosis of ADPKD requires an age-specific renal phenotype and a 50% risk of inheritance based on a positive family history. Currently, ultrasound is used first line to assess for the renal phenotype. In all patients (both PKD1 and PKD2), the overall sensitivity, specificity, and accuracy of ultrasound for diagnosis is 97%, 100%, and 98%, respectively.66 In families with a PKD1 or PKD2 mutation, there is variable emergence of renal cysts by age 40 years, constituting a clinical diagnosis of ADPKD, though phenocopy and atypical (non-PKD1/PKD2) forms of ADPKD can confound this along with a sometimes erroneous reassurance of excluding ADPKD at younger ages based on an apparent absence of renal cysts. Consequently, the current recommendation for ultrasound-based exclusion of a diagnosis of ADPKD in an at-risk individual with an affected first-degree family member is the absence of kidney cysts at 40 years of age or older.66 More recently, diagnostic criteria for magnetic resonance imaging have been established for those aged 16 to 40 years. The presence of >10 renal cysts in patients in this age group is sufficient for diagnosis of ADPKD in an at-risk individual, with 100% positive predictive value and sensitivity.67 Conversely, a total of <10 renal cysts in the same patient population can be considered sufficient for disease exclusion, with a negative pre-dictive value of 100% and a specificity of 98.3%.67 For potential living kidney donors in this age range, a more conservative criterion of <5 renal cysts on magnetic resonance imaging for disease exclusion has been suggested.67 Diagnostic criteria are not yet established for CT.68

Failure to confirm or exclude a diagnosis of ADPKD and atypical forms of cystic kidney disease69,70 has implications for both donors and recipients in the context of kidney transplantation. First, genetic testing in the recipient can be performed to confirm the type of

cystic kidney disease. In such situations, the affected recipient would undergo genetic testing of typical and atypical ADPKD genes, and if a mutation is found, then a related donor would undergo targeted testing for the identified causative familial variant. If that variant is identified, the potential donor would be excluded from proceeding to kidney donation.71 The primary application of genetic testing for ADPKD in at-risk but seemingly unaffected relatives is not recommended unless an established disease-causative variant has previously been identified in an affected family member. It is recommended that if there are no other suitable donor options, related donors over the age of 40 years undergo renal ultrasound, and those between 18 and 40 years of age undergo magnetic resonance imaging screening, and if cystic kidney disease is unable to be confidently excluded, the potential donors are screened for an identified familial ADPKD-causative variant to determine their eligibility to donate.

SRNS 

SRNS accounts for 15% of childhood cases of nephrotic syndrome and 40% of adult-onset cases of nephrotic syndrome.72 In the last 20 years, >39 genes have been identified as involved in the pathogenesis of SRNS,73 with both autosomal dominant and recessive inheritance patterns. Furthermore, 85% of SRNS presenting clinically before 3 months of age and 66% of cases presenting before 1 year of age can be explained by biallelic/recessive mutations in 1 of the following 4 genes: NPHS1, NPHS2, LAMB2, or WT1. 74 Other studies have reflected the high incidence of monogenetic causes for SRNS,75-77 with 29.5% of all allcomers in a worldwide cohort of patients who pre-sented with SRNS before age 25 years having an identifiable mutation.78 It is recommended that those who have presented with SRNS or phenotypes compatible with such a diagnosis, particularly at younger ages of onset, are offered genetic testing before renal transplantation.

Familial Hematuria and COL4A-Related Nephropathy 

Benign familial hematuria, now most often known as thin basement membrane disease, is characterized by the presence of recurrent and/or persistent microhematuria, often though not exclusively first detected in childhood or adolescence. The diagnosis of this condition has been best traditionally defined by histopathology revealing a thinned glomerular basement membrane on electron microscopy examination of a kidney biopsy. Clinically, it is also inferred as a diagnosis by the presence of isolated recurrent or persistent microhematuria without other lower urinary tract pathology or significant proteinuria. Both thin basement membrane disease and Alport syndrome develop as a result of pathogenic variants in the COL4A3, COL4A4, and COL4A5 genes. Most cases (85%) of Alport syndrome are inherited in an X-linked pattern and are as a result of COL4A5 gene variants.79 The autosomal dominant and autosomal recessive cases are due to monoallelic or biallelic mutations in COL4A3 and COL4A4, respectively.79 In the case of X-linked Alport syndrome, the previous belief that females are more mildly affected than males is being appropriately challenged, and there is a broad and broadening phenotype spectrum in affected females, which includes KF.80

Genetic testing for a potential transplant recipient with familial hematuria should be considered if there are extrarenal manifestations making a diagnosis of Alport syndrome more likely. This would be useful for the affected recipient in confirming the diagnosis and for potential at-risk related family members to identify suitable potential kidney donors. Such testing can also ensure early intervention and follow-up for both kidney and other organ involvement. We would recommend genetic testing for any potential kidney transplant recipients with a diagnosis of thin basement membrane disease or Alport syndrome and cascade testing for any related donor deemed to be at risk for the familial gene variant to determine diagnostic clarity and donor suitability. In addition, in some parts of the world, such as French Polynesia, the population prevalence of Alport syndrome has been noted to be higher than anticipated. Genetic and clinical studies have provided an understanding of the basis of this,81 and such information is important to consider as part of both recipient and donor assessment within a kidney transplantation setting, particularly in a globalized community.

Fabry Disease

Fabry disease, an X-linked lysosomal storage disorder, is caused by the deficiency of alpha-galactosidase A enzyme and the progressive intracellular accumulation of globotriaosylceramide. It has multisystemic manifestations, including hypertrophic cardiomyopathy, dysrhythmias, valvular insufficiency, kidney disease, gastrointestinal dysmotility, hypohidrosis, acropar asthesias, and cerebrovascular accidents. The Fabry Registry has revealed that KF occurs in 14% of males and 2% of females with the condition, with a median age of commencement of kidney replacement therapy at 38 years in both groups.82

Those with the clinical phenotype usually undergo testing of alpha-galactosidase A levels as a first-line diagnostic test, with deficiency being definitive in the diagnosis of hemizygous males. Enzyme levels may not be as reliable in diagnosing heterozygous females.

This is mainly driven by random X inactivation and is the reason many females require genotyping to confirm the diagnosis.83 Other emerging nongenetic tests, such as Lyso-GB3 testing, can also assist among suspected female cases and in circumstances of diagnostic uncertainty. We recommend genetic testing in both males and females who have an unclear etiology of kidney disease and have features concerning Fabry disease before transplantation.

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