Prevalence of Kidney Health Genetic Variants in Adults with Sickle Cell Nephropathy

Abstract

The pathophysiology and genetic risk for sickle cell disease (SCD)-related chronic kidney disease (CKD) are not well understood. In 70 adults with SCD-related CKD and without APOL1 inherited in a high-risk pattern, 24 (34%) had pathogenic variants in candidate genes using KidneySeq^™. A moderate impact INF2 variant was observed in 20 (29%) patients and those with 3 versus 0–2 pathogenic or moderate impact glomerular genetic variants had higher albuminuria and lower estimated glomerular filtration rate (adjusted P≤0.015). Using a panel of pre-selected genes implicated in kidney health, we observed several variants in people with sickle cell nephropathy.

Graphical abstract

The pathophysiology of sickle cell nephropathy is complex with damage to the vasculature, glomerulus, and tubules implicated in sickle cell disease-related chronic kidney disease. Using a panel of pre-selected genes linked to kidney health, we observed variants in 34% (24/70) of adults with sickle cell nephropathy and without APOL1 G1 and G2 variants inherited in a high-risk pattern. The presence of 3 or more gene variants impacting glomerular function was associated with lower estimated glomerular filtration rate (eGFR) and higher urine albumin-to-creatinine ratios.

Introduction

Sickle cell disease (SCD) is an inherited red blood cell disorder that leads to chronic kidney disease (CKD) in approximately half of adults.(Guasch, et al 2006) Although SCD is classically considered a monogenic disorder, other genetic modifiers affect the risk of acute and chronic kidney damage. For example, when the APOL1 G1 and G2 risk variants are inherited in a homozygous or compound heterozygous pattern, individuals with SCD have an approximately 7-fold greater risk of CKD progression.(Saraf, et al 2017) Approximately 11% of patients with SCD co-inherit the APOL1 variants in this high-risk pattern suggesting variants in other genes implicated in kidney function and health may impact CKD risk in this patient population.

Several genetic disorders have been implicated in glomerular or tubulointerstitial disease processes in the general population. For example, a gene implicated in proximal tubular health, LRP2, encodes the megalin receptor.(Christensen, et al 2012) In conjunction with cubulin, megalin is responsible for endocytic reabsorption of all filtered proteins by the proximal tubule. Mutations in LRP2 have been associated with CKD in patients and with progression of kidney disease in C57BL/6JTac transgenic mice.(Charlton, et al 2020) Identifying the prevalence of these types of inherited genetic disorders implicated in kidney health in individuals with SCD may point to pathophysiologic mechanisms and stratify those at risk for the development and progression of kidney disease.

We investigated the prevalence of variants in candidate genes involved in kidney health using the KidneySeq^™ gene panel in SCD adults with CKD who did not co-inherit the APOL1 kidney risk variants in a high-risk pattern.

Methods:

Institutional review board approval and written informed consent were obtained at the time of recruitment and before clinical data and biosample collection. We identified 70 unrelated patients with SCD from our longitudinal registry of 439 patients at the University of Illinois at Chicago (UIC) who were diagnosed with CKD before age 45 years without high-risk APOL1 status and with sufficient DNA samples, based upon a DNA concentration of ≥100ng/μL using the Qubit fluoremeter and absence of non-fragmented or degraded DNA as detected by TapeStation Analysis. Chronic kidney disease was defined as having urine albumin-to-creatinine ratio (ACR) ≥30mg/g creatinine or estimated glomerular filtration rate (eGFR) <60mL/min/1.73 m² on two repeat measurements over at least three months of follow up during an outpatient clinic visit. Of 439 SCD patients, 51% (225/439) met this definition of CKD and 36% (160/439) had evidence of CKD before age 45 years. High-risk APOL1 status was observed in 14% (59/432) of genotyped SCD patients and defined as being either homozygous (G1/G1, G2/G2) or compound heterozygous (G1/G2) for the G1 (S342G and I384M substitutions) and/or G2 (N388 and Y389 deletions) variants determined by polymerase chain reaction.(Saraf, et al 2017) Thirty-six patients with only one APOL1 risk allele were included in this study. Hypertension was defined as a systolic blood pressure ≥130mmHg or diastolic blood pressure ≥80mmHg on at least two outpatient visits.

DNA samples from these 70 SCD patients were processed by the University of Iowa Institute of Human Genetics for analysis by the 330-gene kidney panel, KidneySeq^™ Version 5.(Mansilla, et al 2021) Pathogenic and moderate impact variants were identified using criteria developed by the American College of Medical Genetics and Genomics.(Richards, et al 2015, Thomas, et al 2020) These genetic variants were further classified as involving glomerular, tubular, renin-angiotensin-aldosterone system (RAAS), or other functions based upon literature searches for the respective genes. The association between 0–2 versus 3 genetic variants involved in glomerular function with eGFR and urine ACR was performed by linear regression analysis adjusting for age, sex, hypertension and diabetes status using Systat 13 (Systat Software Corporation; Chicago, IL).

Results & Discussion:

The median age of the cohort when CKD was confirmed was 32 (interquartile range [IQR] 25–38) years, 35 (50%) were female, 67 (96%) were hemoglobin SS or Sβ⁰-thalassemia genotype, 23 (33%) had hypertension, and 3 (4%) were diabetic. At the time of sample collection, 39 (56%) patients were on hydroxyurea, 18 (26%) were on an angiotensin converting enzyme-inhibitor or angiotensin receptor blocker and the median eGFR and urine ACR were 122 (IQR, 88–132) mL/min/1.73m² and 248 (IQR, 103–641) mg/g creatinine, respectively (Supplementary Table 1).

Pathogenic variants involved in glomerular, tubular, renin-angiotensin-aldosterone system (RAAS) or other genes implicated in CKD risk were identified in 24 (34%) patients (Figure 1A; Supplementary Table 2). This includes pathogenic variants in NPHP4, NPHS1, GLA, SLC4A1, and AGT observed in ≥2 patients with CKD. We identified 13 genes with moderate impact variants involved in glomerular (n=7), tubular (n=4) and RAAS-related (n=2) genes in ≥ 2 patients (Figure 1B; Supplementary Table 2). SCD patients with 3 pathogenic or moderate impact genes impacting glomerular function had significantly lower eGFR (β −33.3±12.8; P=0.015) and higher urine ACR (natural log β 1.7±0.6; P=0.0096) compared to those with 0 – 2 pathogenic or moderate impact genes, adjusting for age, sex, hypertension, and diabetes status (Figure 1C, 1D).

Figure 1:

(A) Pathogenic variants identified in patients with SCD. (B) Moderate impact genetic variants involved in glomerular, tubular, and RAAS function observed in ≥ 2 patients with SCD. (C, D) Association of kidney function in SCD patients with 0 – 2 (n = 66) versus 3 (n = 4) pathogenic or moderate impact glomerular genes. P-values adjusted for age, sex, hypertension and diabetes status.

KidneySeq^™ is a targeted genetic risk panel of 330 candidate genes causally linked to renal disease in the general population. In this cohort of SCD adults with non-APOL1 related CKD, the KidneySeq^™ panel detected the presence of several pathogenic and moderate impact variants involved in glomerular, tubular, and RAAS function. The prevalence of pathogenic variants in our SCD cohort (24/70) was relatively similar to what was observed in a cohort of patients with suspected genetic renal disease (54/127) (P=0.25) referred to the University of Iowa Institute of Human Genetics supporting the need to evaluate additional genetic modifiers for sickle cell nephropathy.

Glomerulopathy is a common renal complication observed in up to 68% of adults with SCD.(Guasch, et al 2006) Renal biopsies from patients with SCD reveal several pathologic features in the glomerulus, including focal and segmental glomerulosclerosis, membranoproliferative glomerulopathy, and glomerular hypertrophy.(Bhathena and Sondheimer 1991) We observed several candidate gene variants implicated in glomerular health in our cohort of SCD patients with CKD. NPHS1 is a candidate gene that encodes nephrin, a protein which is a critical component of the glomerular filtration barrier and is involved in signal transduction pathways regulating podocyte cell adhesion and survival.(Martin and Jones 2018) Initial reports identified NPHS1 mutations that led to a truncated protein and nephrotic syndrome in children from Finland.(Patrakka, et al 2000) In a cohort of African Americans, NPHS1 variants were associated with a 2.8-fold greater risk of all-cause end stage kidney disease.(Bonomo, et al 2014) Three patients from our SCD cohort had pathogenic variants in NPHS1.

Invertin-formin 2 (INF2) modulates actin assembly and nephrin localization to the slit diaphragm thereby playing an essential role in glomerular health.(Ueda, et al 2023) Autosomal dominant mutations in INF2 have been identified in 12 – 17% of inherited cases of focal segmental glomerulosclerosis, a common pathologic feature observed in sickle cell nephropathy.(Boyer, et al 2011, Ueda, et al 2023). Mutations in INF2 have been linked to up to 17% of familial and 1% of sporadic cases of focal segmental glomerulosclerosis.(Barua, et al 2013) We identified moderate impact INF2 variants in 20 (29%) patients with sickle cell nephropathy, which is substantially higher than the allele frequency of 0.04 reported in the background African American population, pointing to this gene as a strong candidate for future investigation.

In addition to identifying several pathologic or moderate impact variants in genes involved in glomerular health, we observed an association between the presence of three of these variants with lower eGFR and higher urine ACR. This panel of glomerular genes should be validated in larger cohorts of patients with SCD, particularly those that did not co-inherit high-risk APOL1. Several other genes implicated in tubular health (LRP2, ATP6V1C2, NPHP4, SLC4A1, EHHADH, FOXI1) and RAAS (CACNA1H, CACNA1D, AGT) were also identified and their association with tubular function, blood pressure, and CKD risk should be investigated in future studies.

Our study is limited by the small sample size and selection of patients with SCD that already developed CKD. The association of these genes with the development and progression of CKD will need to be validated in larger, longitudinal studies including comparing frequencies of these gene variants in SCD populations with versus without evidence of CKD and to the general African American population. Future studies will also be needed to investigate whether the pathogenic variants predict earlier development of CKD and for gene interactions between identified genes and with APOL1 risk variants. In conclusion, using a genetic risk panel of pre-selected genes implicated in kidney disease, we found that several pathogenic variants are enriched in people with sickle cell nephropathy. Further evaluation of these candidate genes may elucidate mechanisms and greater risk for the development of sickle cell nephropathy.

Data Availability:

The data that support the findings of this study are available upon reasonable request from the corresponding author.