Practical considerations for APOL1 genotyping in the living kidney donor evaluation

Abstract

Background:

Association between the apolipoprotein L1 gene (APOL1) and nephropathy has altered the epidemiology of chronic kidney disease (CKD). In addition, donor APOL1 genotypes play important roles in the time to allograft failure in kidneys transplanted from deceased donors and the safety of living kidney donation.

Methods:

This manuscript reviews genetic testing for inherited kidney disease in living kidney donors to improve donor safety. APOL1 genotyping in donors with recent African ancestry is considered.

Results:

Based on current data, transplant physicians should discuss APOL1 genotyping with potential living kidney donors self-reporting recent African ancestry. Until results from APOL1 Long-term Kidney Transplant Outcomes Network (APOLLO) ancillary studies are available, we present practical approaches from our experience for considering APOL1 genotyping in the living donor evaluation.

Conclusions:

Transplant physicians should inform potential living kidney donors at-risk for APOL1-associated nephropathy about the gene and possibility of genetic testing early in the donor evaluation, well before scheduling the donor nephrectomy. Transplant programs must weigh risks of performing a donor nephrectomy in those with two APOL1 renal-risk variants (high-risk genotypes), particularly younger individuals. Our program counsels kidney donors with APOL1 high-risk genotypes in the same fashion as with risk genotypes in other nephropathy genes. Because most African American kidney donor candidates lacking hypertension, proteinuria and reduced kidney function after workup will not possess APOL1 high-risk genotypes, genetic testing is unlikely to markedly increase donor declines and may reassure donors with regard to their long-term kidney outcomes, potentially increasing the number of African American donors.

Introduction

There is no more powerful gift to a patient with end-stage kidney disease (ESKD) than a kidney transplant. Compared to recipients of a kidney from a deceased donor, those fortunate enough to have a living donor are more likely to remain dialysis-free with functioning allografts for longer durations. Transplant physicians are obligated to provide for the safety and long-term well-being of the living kidney donor. Evaluation of medically complex donors is an inherently challenging process for transplant physicians and can be stressful for donor candidates, recipients and families. The donor nephrectomy is not medically necessary, although it may provide donors with personal and emotional benefits. Donors do not obtain medical benefit and they assume short-term perioperative and long-term medical risk. Although risks are quite low, they are present.¹ Often, there is no perfect donor candidate. Most have minor (or more) considerations. Transplant programs are familiar with the populations they treat, and each program must determine the acceptable level of risk for their living donors.

The current era of genomic medicine has benefitted organ transplantation. It is now relatively simple to screen asymptomatic potential living kidney donors to determine whether they possess risk variants in genes that have known associations with chronic kidney disease (CKD) and consequently avoid donation. This can benefit not only the donor, by preserving their renal mass and hopefully delaying the onset of nephropathy, but also the recipient who might have received a kidney predisposed to more rapid allograft failure. Risks to living kidney donors accrue over many decades. Appreciation of the risk for developing ESKD among former living kidney donors has improved by contrasting their outcomes with those from healthy populations, not the general population where a significant percentage have CKD.² Although long-term absolute risk of post donation ESKD are low among donors, relative risk of ESKD is higher in donors and attributable risk is highest in African American donors compared to donors of other ancestries.³

The apolipoprotein L1 gene (APOL1) association with nondiabetic CKD has transformed nephrology.^4,5 APOL1 is associated with ESKD in Africans, African Americans, Brazilians and Afro-Caribbeans.^6–8 APOL1 high-risk genotypes in deceased organ donors (defined as two copies of the G1 and/or G2 renal-risk variants: G1G1, G2G2, or G1G2) also contribute to more rapid failure of kidneys transplanted from African American donors.^9–11 In addition, higher risk for developing post donation CKD in living kidney donors with recent African ancestry, defined as having similar genetic make-up to those currently residing in Africa (e.g., African Americans, Hispanic blacks, Afro-Caribbeans and Africans), has been demonstrated in donors with APOL1 high-risk genotypes.¹²

There has been increasing awareness among transplant physicians, nephrologists, patients with ESKD and potential living kidney donors about the effects of APOL1 in kidney transplantation.^13–16 Despite reaching the milestone of equitable proportional rates of deceased donor kidney transplants for African American recipients, significant disparities remain in the incidence of living kidney donor transplants in this population.¹⁷ Rising concerns about higher risks for ESKD in African American donors are likely a major driver of this disparity. The present manuscript reviews the living donor evaluation from the standpoints of screening for inherited kidney disorders and consideration of APOL1 genetic testing in individuals with recent African ancestry, including African Americans. Intended for clinicians, it considers associated ethical, social and practical considerations of performing APOL1 genetic testing.

Screening for inherited kidney diseases in the living donor evaluation

Consideration of genetic counseling and/or testing in living kidney donors is important, underscored by the fact that 40 to 80% of such donors are related to the recipient.^18,19 Genetic susceptibility to kidney disease in potential donors affects their health and well-being and constitutes a risk factor for nephropathy in transplanted kidneys.²⁰ Cohort studies demonstrate that kidney donation increases the risk for ESKD; however, the absolute risk is low and varies based on ancestry and gender.^1,21,22 Risk for ESKD is generally higher in related, compared to unrelated kidney donors.^2,18

Transplant teams should inform donor candidates about the potential for genetic predisposition to kidney disease and efforts undertaken to exclude this possibility. This can be challenging because the cause of ESKD in transplant recipients is often unclear; nearly 60% carry a presumptive diagnosis of diabetes or hypertension without histopathological confirmation.²³ Many patients have an alternative (or superimposed) cause of ESKD and lack of an accurate etiology of nephropathy complicates genetic screening of related living donors.²⁴ A careful family history is critical, but this can also be challenging due to de novo mutations.

Genetic screening techniques often involve sequencing a single suspected gene for pathogenic variants or high throughput methods such as next-generation-sequencing (NGS) to sequence several genes at once. Some laboratories have developed NGS panels to evaluate a given clinical phenotype, such as focal segmental glomerulosclerosis (FSGS).^18,25,26 Genetic testing is simplest in monogenic disorders, but can be challenging for genes with heterogeneous pathology, variable penetrance, or when multiple genes are implicated.²⁷ Testing for multiple genes can also be a financial burden for transplant programs.

Genetic testing should be considered for kidney donors with a family history of potentially inherited forms of kidney disease in two or more members, whether in the same or different generations. Testing can be considered with one affected first-degree relative, if the extended family history is uncertain. When in doubt, we recommend consultation with a Molecular Genetics Service. Potential donors (and recipients) should be informed that presence of a genetic mutation may not be an absolute contraindication to donation. Factors such as heterozygous versus homozygous gene expression, donor age, absence of signs, symptoms or lack of an abnormal urinalysis could lead to favorable decisions if the estimated risk of kidney disease after donor nephrectomy is deemed acceptable.

Risk of kidney disease in donors whose families have a Mendelian disorder, such as Autosomal Dominant Polycystic Kidney disease (ADPKD) can often be determined based on genetic testing. Mutations in the PKD1 and PKD2 genes cause the majority of ADPKD and approximately 50% of affected patients develop ESKD by the age of 60 years.^28–30 Renal ultrasonography is the most common accepted screening test and can exclude presence of polycystic kidney disease in individuals older than 30 years; however, it has limitations in younger individuals. Magnetic resonance imaging has higher sensitivity and specificity and can be used as a screening test in younger individuals.³¹

Rare forms of Autosomal Dominant Tubulointerstitial Kidney Disease due to uromodulin (UMOD), mucin-1 (MUC1), renin (REN) and hepatocyte nuclear factor-1β (HNF1B) gene mutations cause ESKD and can be assessed in potential living-related donors.³² Podocytopathies such as hereditary FSGS, congenital nephrotic syndrome and steroid resistant nephrotic syndrome are also potential targets for mutation screening in prospective donors whose families have mutations in the nephrin (NPHS1), podocin (NPHS2), alpha actinin-4 (ACTN4), inverted formin-2 (INF2) and Wilms tumor-1 (WT1) genes, among others.

Genetic screening should be considered in donors whose relatives have atypical hemolytic uremic syndrome associated with mutations in genes that regulate the complement cascade (Table 1).^33,34 Screening can also be performed in donors from families with Alport syndrome (for mutations in the COL4A3, COL4A4, and COL4A5 collagen genes)³⁵ and Fabry disease (an X-linked lysosomal storage disease that results from deficient α-galactosidase A [α-Gal A] activity due to mutations in the GLA gene). Table 1 displays inherited kidney diseases that can be assessed with gene-based techniques in the living donor evaluation.

Table 1.

Inherited kidney disorders suitable for genetic screening

Disorder	Mode of inheritance	Implicated genes
Polycystic Kidney Disease	Autosomal dominant	PKD1, PKD2
Atypical Hemolytic Uremic Syndrome	Autosomal dominant*	CFB, CFH, MCP, CFI, C3
Alport syndrome	Autosomal recessive X-linked for COLA5	COL4A3, COL4A4, COL4A5
Fabry disease	X-linked	GLA
Tubulointerstitial kidney disease	Autosomal dominant	UMOD, MUC1, REN, HNF1B
Focal segmental glomerulosclerosis (FSGS)/steroid resistant nephrotic syndrome	Autosomal dominant	ACTN4, INF2, TRPC6, NPHS1, NPHS2**
Solidified glomerulosclerosis/FSGS (if recent African ancestry present)	Autosomal recessive	APOL1

^*Autosomal recessive transmission with specific mutations have been identified.

^**Additional genes implicated

APOL1 testing in living kidney donor candidates with recent African ancestry

As in native kidney disease, post donation CKD and ESKD are three times higher in African Americans, particularly in younger males, compared to European-derived populations.^19,22,36,37 Living kidney donors often have a family history of ESKD; another established risk factor for future nephropathy.³⁸ Finally, among donor candidates with risk factors for surgical nephrectomy (e.g., smokers and those with high blood pressure, mildly reduced kidney function or proteinuria), African Americans and Hispanics are less likely to have long-term health insurance necessary to allow for optimal post donation healthcare.³⁹ APOL1 plays a key role in the biologic basis for this disparity; however, a minority of individuals with high-risk genotypes will develop CKD. This suggests that a second hit is necessary to develop APOL1-associated kidney disease.⁴⁰

Long-term outcomes were assessed in 136 African American living kidney donors from Detroit.^12,41 As in the general population, 14% (19/136) had APOL1 high-risk genotypes (with 2 renal-risk variants) and 86% low-risk genotypes (0/1 renal-risk variant). Baseline characteristics were similar regardless of APOL1 genotype. However, mean predonation estimated glomerular filtration rate (eGFR) was significantly lower, albeit not markedly reduced, in those with high-risk genotypes (98±17 ml/min/1.73 m² vs. 108±20 in low-risk donors; p=0.03). Of concern, 11% (2/19) of previously healthy kidney donors with APOL1 high-risk genotypes developed ESKD and 67% developed an eGFR <60 ml/min/1.73 m² after 12 year median follow-up. None of the 117 APOL1 low-risk genotype donors developed ESKD and only 36% had a follow-up eGFR <60 ml/min/1.73 m² (p=0.01). Mean±SD serum creatinine concentrations at latest follow-up were 1.71±1.2 in APOL1 high-risk and 1.26+0.3mg/dl in low-risk donors (p=0.003). Other reports reveal APOL1 high-risk kidney donors can develop post transplant focal segmental glomerulosclerosis progressing to ESKD, along with earlier failure of the transplanted kidney.^8,42,43 These findings raise concern about the safety of live donation from individuals with two APOL1 renal-risk variants. Based on these case reports, we hypothesize premature transplant failure could also occur in some recipients of APOL1 high-risk living donor kidneys.

Similar outcomes exist in recipients of deceased donor kidney transplants. Kidneys transplanted from APOL1 high-risk donors fail more quickly.^11,44 The current Kidney Allocation System downgrades quality in all African American deceased donor kidneys regardless of APOL1 genotype, although recent data suggest that those with two APOL1 renal-risk variants (approximately 13%) appear to be at highest risk for early allograft failure.⁴⁴ These findings contributed to the need for the prospective National Institutes of Health-supported “APOL1 Long-term Kidney Transplant Outcomes Network (APOLLO)” and the APOLLO Ancillary Study, “Living Donor Extended Time Outcomes”.⁴⁵ The American Society of Transplantation and National Kidney Foundation “Kidney Disease Improving Global Outcomes” support additional studies; APOLLO and its ancillary studies will provide these data.³

Counseling the living kidney donor candidate about APOL1

Until APOLLO results become available, we believe that transplant programs should develop local guidelines to inform potential living kidney donors who self-report recent African ancestry about the role of APOL1 in kidney disease and outcomes after donation and transplantation (Figure 1). Currently, U.S. transplant programs do not universally test at-risk living donors for APOL1, nor do they consider these genotypes in the donor evaluation. For ethical reasons, we feel potential living donors reporting recent African ancestry should be informed that APOL1 gene testing is commercially available prior to decisions about candidacy.^46,47 Results can be available within 4 hours (in our lab), at low cost and typically paid for by transplant programs. We, and others, consider education about APOL1 and availability of genetic testing to be the current standard of care.^15,16,36 Kidney donors are not required to undergo genetic testing for APOL1 (or other genes). Genetic counseling is required when genetic testing is performed; this can provided by the transplant nephrologist or genetic counselor.

Figure 1.

Sample flow chart for evaluation of African American living kidney-donor candidates.

Compared to non-African Americans, young African Americans have higher rates of ESKD after donor nephrectomy. We feel it is in the best interest of younger potential donors <50 years of age with APOL1 high-risk genotypes not to proceed with donation.^12,43 Other programs may choose different age cut-offs. It may prove safer for APOL1 high-risk individuals to donate a kidney at a later age, when they would more likely have avoided the effects of modifying factors for nephropathy.⁴⁰ After age 60, similar long-term renal outcomes exist in European American and African American living kidney donors.³⁷ This supports that 60 years of age may be a reasonable cut-off for use of APOL1 risk stratification in medically eligible donors (education about APOL1 and testing should still be provided). It remains uncertain how to counsel potential donors 50 to 60 years of age. We suggest donor candidates and their physician have a frank discussion carefully weighing all elements of donor risk and benefit. Some programs may be comfortable accepting donors in their 50’s if they are otherwise medically acceptable, but data are lacking. Extrapolating retrospective results from Doshi et al.,¹² a 51-year old African American donor with two APOL1 renal-risk variants might face up to an 11% risk for ESKD and 67% chance of eGFR <60 ml/min/1.73 m² by 63 years of age. Based on this, we do not currently advocate that these individuals donate a kidney.

Medically acceptable donors who refuse to undergo APOL1 testing but wish to donate regardless of their own inherent risk for developing ESKD could proceed to nephrectomy, as long as their transplant program provided clear information about APOL1, kidney disease and transplant outcomes. We suggest documenting these discussions in the medical record.

APOL1 and sickle cell testing is performed at our program and others, to assist with risk-stratification of potential living kidney donors who self-report recent African ancestry.⁴⁸ We recognize that a small percentage of donors who report as non-African could still possess APOL1 renal-risk variants due to African ancestry they were unaware of or did not report. However, we do not feel it is cost-effective to screen them. In chromosomes from healthy European Americans, 0.3% possessed a single APOL1 renal-risk variant; two renal-risk variants were extremely rare.⁴⁹ The APOL1 association with nephropathy has much in common with the hemoglobin S gene variant (HbS) association with sickle cell disease. A single risk HbS or APOL1 variant (autosomal dominant) protects from malarial and trypanosomal infection, respectively. Risk variants are common and more often present in certain ancestral groups. Inheriting two copies of a risk variant (autosomal recessive inheritance) is required to develop sickle cell disease or CKD. In contrast to HbS, where individuals with risk genotypes will develop sickle cell disease, approximately 20% with APOL1 high-risk genotypes develop nephropathy (personal communication, Dr. Martin Pollak). Prior to the donor evaluation, most individuals with sickle cell trait presenting to our center are aware of their status because testing is performed at birth in North Carolina.

To date, our experience with APOL1 testing in living kidney donors has been positive. Potential donors first submit screening packets highlighting their medical history and listing medications. Prior to the in-person medical evaluation, our program records height, weight and blood pressure and performs a urinalysis and urine protein testing. Donors from distant locations have laboratory studies and a 24-hour urine prior to medical evaluations. We typically do not proceed with the evaluation of African American living donors who have hypertension (systolic blood pressures above 140 mmHg or diastolic pressures above 90 mmHg), an eGFR that is abnormal for their age, proteinuria (spot urine protein:creatinine ratio >200 mg/g) or other concerning features during work-up. In this fashion, exclusions for kidney disease typically occur prior to discussions about APOL1 testing as many individuals with APOL1 high-risk genotypes screen out due to low eGFR or proteinuria.

Performing the donor workup in this sequence excludes many individuals with recent African ancestry likely to have APOL1 high-risk genotypes without formal genetic testing. In turn, individuals who proceed to the full clinical evaluation with discussion of genetic testing are at lower risk for APOL1-nephropathy compared to the general population. The percentage of donor candidates screened at our program and found to have two APOL1 renal-risk variants is very low. Although we cannot fully mitigate the long-term risks from hypertension and diabetes in African American donors, they are counseled about long-term medical risks, 24-hour ambulatory blood pressure monitoring is performed when indicated, and glucose tolerance testing is currently performed on all potential donors.

Ethical considerations

Ethical concerns involving genetic testing for APOL1 have been reported.^50–52 Candidates who present for living donor evaluations do not all face the same long-term risk for CKD and it is the transplant program’s responsibility to evaluate each donor to the best of their ability. It is evident that APOL1 renal-risk variants are virtually limited to individuals with recent African ancestry. We contend that offering APOL1 testing to these individuals and considering results in donor selection should not differ from other genetic tests employed in the evaluation of living donors. In our opinion, claims of discrimination do a disservice to the African American community. Individuals who request information about APOL1 and elect to have testing performed should have been aware (prior to ordering the test) that those with high-risk genotypes are at increased risk for CKD. In our practice, they are informed that factors beyond genetic make-up contribute to risk for kidney disease, including dietary factors, smoking, lack of exercise and failure to maintain ideal body weight. Prior to the test being ordered they should be aware that no specific therapies for APOL1-kidney disease currently exist.

APOL1 testing is a scientific breakthrough; this gene displays the most powerful genetic association in common complex disease. In our view, transplant programs should provide information on APOL1 and offer testing to those with self-reported recent African ancestry. Advisory councils comprised of African Americans with kidney disease, former kidney donors, relatives of patients with ESKD and general populations espouse similar views about genetic testing and return of results, even when treatments are not available.^16,53 The number of African American living kidney donors has been declining in recent years.⁵⁴ Although future rates of living kidney donation are unknown, informing potential kidney donors that they lack APOL1 high-risk genotypes might provide them reassurance. We believe this information could increase the numbers of African American living kidney donors in the future. In addition, it has the potential to reduce the excess risk for post donation ESKD in African American living kidney donors, relative to European American. As new treatments for native APOL1-nephropathy are developed, they may prove useful in the transplant setting.⁵⁵

Conclusions

Kidney transplant programs evaluating African American living donor candidates should become familiar with the effects of APOL1 and consider offering APOL1 and non-APOL1 genetic testing. If programs lack individuals comfortable with these processes, other transplant programs and/or Clinical Laboratory Improvement Amendments-certified APOL1 labs could assist. Prospective results from the APOLLO Study will ideally clarify best practices. Until then, we recommend education about APOL1 in kidney disease and the offer of genetic testing in kidney donor candidates who self-report recent African ancestry. Our program does not typically proceed with living kidney donation from individuals with two APOL1 renal-risk variants under the age of 60 years. In addition, we treat APOL1 genetic data in a similar fashion as genotypes for other inherited kidney diseases. We provide education, offer genetic testing and counseling. In our opinion, these practices provide potential kidney donors with the ability to make the most informed and safest decision.