High risk APOL1 genotypes and kidney disease among treatment naïve HIV patients at Kano, Nigeria.

INTRODUCTION: Racial disparities are known in the occurrence of kidney disease with excess risks found among people of African descent. Apolipoprotein L1 (APOL1) gene variants G1 and G2 are associated with kidney disease among HIV infected individuals of African descent in the USA as well as among black population in South Africa. We set out to investigate the prevalence of these high-risk variants and their effects on kidney disease among HIV infected patients in Northern Nigeria with hitherto limited information despite earlier reports of high population frequencies of these alleles from the Southern part of the country.
METHODS: DNA samples obtained from the whole blood of 142 participants were genotyped for APOL1 G1 and G2 variants after initial baseline investigations including assessment of kidney function. Participants comprised 50 HIV positive patients with no evidence of kidney disease, 52 HIV negative individuals with no kidney disease and 40 HIV positive patients with chronic kidney disease (CKD) evidenced by persistent proteinuria and/or reduced eGFR, who also had a kidney biopsy. All the HIV positive patients were newly diagnosed and treatment naïve.
RESULTS: The distribution of the APOL1 genotypes among the study participants revealed that 24.6% had a G1 risk allele and 19.0% a G2. The frequency of the High Risk Genotype (HRG) was 12.5% among those with CKD compared to 5.8% in the HIV negative group and zero in the HIV positive no CKD group. Having the HRG was associated with a higher odds for developing HIV Associated Nephropathy (HIVAN) (2 vs 0 risk alleles: OR 10.83, 95% CI 1.38-84.52; P = 0.023; 2 vs 0 or 1 risk alleles: OR 5.5, 95% CI 0.83-36.29; P = 0.07). The HRG was also associated with higher odds for Focal Segmental Glomerulosclerosis (FSGS) (2 vs 0 risk alleles: OR 13.0, 95% CI 2.06-81.91; P = 0.006 and 2 vs 0 or 1 risk alleles: OR 9.0, 95%CI 1.62-50.12; P = 0.01) when compared to the control group.
CONCLUSION: This study showed a high population frequency of the individual risk alleles of the APOL1 gene with higher frequencies noted among HIV positive patients with kidney disease. There is high association with the presence of kidney disease and especially FSGS and HIVAN among treatment naive HIV patients carrying two copies of the HRG.

Introduction

There is global disparity in the occurrence of kidney disease with an excess risk for persons of African Ancestry as they were shown to have four times higher incidence of chronic kidney disease (CKD) compared to people of European descent [1], together with a more rapidly progressive course of the disease, as reported in the Multi-Ethnic Study of Atherosclerosis (MESA) during 5 years of follow-up [2]. Data analysis from the US revealed that people of African descent showed a consistent yearly increase in the incidence of End Stage Kidney Disease (ESKD) that is 3.5 to 5-fold higher than people of European descent [3]. This racial/ ethnic disparity in the occurrence of kidney disease is also noted among HIV infected patients. Eggers et al reported that the possibility of developing kidney disease among HIV infected people of African descent is at least 50 times higher than that of people of European descent [4]. Nearly 90% of ESKD attributed to HIV-Associated Nephropathy (HIVAN) was likely to occur in persons of African ancestry [5].

The excess risk of kidney disease among people of African descent could not entirely be explained by factors such as access to care and other socioeconomic factors [6]. The possibility of genetic factors being significant in the development of kidney disease among this population group was supported by findings from many studies [7, 8]. Variants in the gene located on chromosome 22 that encode for Apolipoprotein L1 (APOL1) have been found to be strongly associated with increased risk of various forms of kidney diseases among people of African descent, with three times greater risk of developing lupus nephritis, seven times greater risk of developing hypertensive nephrosclerosis, seventeen times greater risk of developing primary Focal Segmental Glomerulosclerosis (FSGS) and twenty nine times greater risk to develop HIVAN [9, 10]. There were reports of even stronger associations with the development of HIVAN [11, 12]. Kasembeli et al, in a study among black people in South Africa, showed that the presence of these high risk variants was associated with an 89-fold odds for developing HIVAN [13].

These alleles include the APOL1 G1 variant comprising two non-synonymous coding variants rs73885319 and rs60910145 and the G2 allele with a 6 base pair deletion that removes two amino acids (rs71785313).

APOL1 high-risk genotypes (HRGs) are defined as the occurrence of two risk alleles in any of the combinations (homozygous G1/G1, homozygous G2/G2 and compound heterozygous G1/G2). Patients with the HRG carry an increased risk of kidney disease. The heterozygous forms (G1/G0 or G2/G0), with only one risk allele, are known as low risk genotypes and are associated with a lesser risk of kidney disease. These kidney disease risk variants occur in higher frequency among individuals of West African descent [14]. This was later found to have evolutionary origins by way of natural selection for a trait that offers protection against infection with Trypanosoma brucei, a protozoan that causes sleeping sickness, which is endemic in parts of Sub-Saharan Africa. These traits encode for circulating APOL1 that functions as a trypanolytic factor leading to death of the trypanosome parasites in human serum [15]. However this positive selective advantage against sleeping sickness has associated increased risk of developing CKD among the carriers of the HRG.

Genetic predilection to HIVAN has been described among people of African descent but has not been widely studied among native Africans with the study among the South African black population by Kasembeli et al and few other studies from Nigeria as well as a study among children in the Democratic Republic of Congo (DRC) [13, 16–18]. We hypothesised that the frequency of APOL 1 high risk genotypes will be high, with higher odds for kidney disease among HIV infected treatment naive patients in Kano. We therefore set out to investigate the frequencies of these variants and their association with kidney disease among HIV infected persons from this part of Northern Nigeria, a region with scarce information regarding these risk alleles despite previous reports of high population frequencies of these alleles from the South Western and South Eastern parts of the country which were predominantly inhabited by the Yoruba and Igbo population groups respectively [14, 19].

Methods

Participants in this cross-sectional study were recruited consecutively from the dedicated HIV clinic of Aminu Kano Teaching Hospital (AKTH), one of the approved United States President’s Emergency Plan for AIDS Relief (PEPFAR) centers in Northern Nigeria. One hundred and fifty five participants comprising 50 treatment naive HIV positive patients with no evidence of kidney disease (HIV positive no CKD), 52 HIV negative participants with no kidney disease (HIV negative no CKD) and 53 HIV positive patients with kidney disease evidenced by persistent proteinuria and/or reduced estmated glomerular filtration rate (eGFR), who had a kidney biopsy (HIV positive CKD) also. The HIV positive CKD patients had no obvious other known causes of kidney disease, such as diabetes and hypertension, and were not being treated for any kidney disease prior to enrolment and had no contraindication to kidney biopsy. The HIV negative with no kidney disease were recruited from among volunteer hospital workers and patients at general out patients who presented with minor ailments. Patients were recruited into the study between April 2016 and March 2018.

A spring-loaded automatic biopsy gun (BARD 16/18 G, 22 mm edge) was used to perform percutaneous kidney biopsy under real-time ultrasound guidance as a daytime procedure. Before the biopsy, the patients were counseled on the proceedure, had a complete blood count including platelet measurement. The coagulation profile was also checked to rule out any bleeding tendency, and an informed consent for the proceedure was obtained. The patients were monitored before and after the biopsy according to the established unit protocol and were discharged after at least 8 hours of observation and no obvious complications observed.

All participants gave a written informed consent to participate and underwent an initial assessment with a focused clinical examination and baseline investigations including serum creatinine, CD4 count, complete blood count, Hepatitis B surface antigen and Hepatitis C antibody tests. The IDMS traceable Jaffe method was used to determine creatinine levels in both the blood and urine samples using the Cobas C 311 Clinical Chemistry Analyser (Roche Diagnostics Rotkreuz, Switzerland) at Aminu Kano Teaching Hospital (AKTH). The eGFR was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation without use of the ethnicity factor.

Genomic DNA was extracted from whole blood using the Maxwell DNA purification kit (Promega AS1010, USA) as per the manufacturer’s protocol. DNA concentrations were determined by the NanoDropTM 2000 spectrophotometer (Thermo Scientific, USA) and the quality was assessed by means of the A260/280 ratios. A 260/280 ratio of 1.8 to 2.0 was considered as indicative of a pure DNA preparation free of contamination. DNA samples were genotyped for SNPs rs73885319, rs60910145 and rs71785313 using a PCR-restriction fragment length polymorphism assay as described by Nqebelele et al. [20] Genomic studies were carried out in the Department of Internal Medicine Research Laboratory at the University of Witwatersrand, Johannesburg.

Ethical approval to conduct the study was granted by the Research Ethics Committee of AKTH (NHREC/21/08/AKTH/1090) and University of Witwatersrand Human Research Ethics Committee (M140116).

Statistics

Data was analysed using STATA v14.2 (Stata Corp, College Station, TX). Demographic and clinical characteristics were expressed as mean ± SD. Comparisons of continuous variables were made using a Kruskal-Wallis test or ANOVA test, as appropriate. Categorical variables were expressed as frequencies and compared using a chi-squared test. Associations between the risk of kidney disease and APOL1 risk alleles were explored using logistic regression models. A two-tailed p-value <0.05 was considered significant. The SNP data was tested for any departure from Hardy-Weinberg equilibrium.

Results

A total of 155 participants were studied with a mean age of 36.1 ± 9.3years and 55% were males. Complete data on genotyping was available for 142 participants (40 HIV positive CKD patients, 50 HIV positive controls and 52 HIV negative controls). Table 1 shows the characteristics of the participants. The HIV positive CKD patients were older than the HIV positive no-CKD patients (38.8 ± 9.3 vs. 31.2 ± 7.4 years, P<0.001); however the age was similar to the HIV negative no-CKD controls (38.8 ± 9.3 vs 38.0 ± 9.3, P = 0.648). The HIV positive CKD patients also had higher blood pressure (systolic: 120 vs 110 mm Hg, P = 0.001) when compared with the no-CKD controls, both HIV positive and negative. All the HIV positive CKD patients had a kidney biopsy. The histology results revealed that HIVAN (characterised by collapsing FSGS, cystic tubular dilatation, interstitial infiltrates), was reported in 17 (32%) of the patients. Majority of the patients had FSGS [20 (37.7%)] and HIVAN [32% (17)] as their histologic diagnosis while 6 (11.3%) had no significant pathological finding, 7(13.2%) had chronic interstitial nephritis, and acute tubular necrosis and membranoproliferative glomerulonephritis were reported in one patient each. The majority of the FSGS seen in this study were of the NOS sub type with only a few being of the tipped sub type. Among the patients that had immunofluorescence evaluation of their tissues (n = 10), none showed features of immune complex disease.

Table 1

Characteristics of the study participants.

Characteristic	HIV CKD (n = 53)	HIV positive controls(n = 50)	HIV negative controls (n = 52)	P-value
Male, n (%)	34 (64.2%)	20 (40.0%)	32 (61.5%)	0.027
Age (years)	38.8 ± 9.3	31.2 ± 7.4	38.0 ± 9.3	0.083
Serum creatinine (μmol/L)	210 (143–302)	82 (70–90)	82 (73–87.5)	<0.001
eGFR (ml/min/1.73m2)	34.4 (26.4–48.9)	107 (87–128)	110.5 (100.5–120.5)	<0.001
Systolic BP (mmHg)	120.0 (110–132)	110.0 (110–120)	110.0 (110–120)	0.001
Diastolic BP (mmHg)	77.0 (65–84)	60.0 (60–70)	69.5 (60–70)	<0.001
Body mass index (kg/m2)	22.26 ± 2.6	22.5 ± 7.7	21.9 ± 6.6	0.770
CD4 (cells/mm3)	115 (74–304)	265 (179–402)	-	0.005

HIV = Human Immune deficiency Virus, CKD = Chronic Kidney Disease, eGFR = estimated glomerular filtration rate, BP = blood pressure; n = number

The frequency of APOL 1 genotypes showed that 24.6% (35/142) of the study participants had at least 1 G1 risk allele and 19.0% (27/142) had a G2 risk allele. The haplotype G-G-I (G1GM), comprising the derived allele at both rs73885319 (p.S342G) and rs60910145 (p.I384M) and the ancestral insertion (I) allele at rs71785313, was the most frequent G1 configuration seen in 20.4% of all the study participants. Table 2 depicts the distribution of the various haplotypes across different groups of study participants.

Table 2

Distribution of APOL1 SNP genotypes and haplotypes in the study participants with HIV positive CKD, HIV positive no-CKD and HIV negative no-CKD.

APOL 1 variants	HIV Positive CKD (n = 40)	HIV Positive no-CKD (n = 50)	HIV Negative no-CKD controls (n = 52)	P-value	P-value HIV+ CKD vs HIV+ no-CKD	P-value HIV+ CKD vs HIV- no-CKD
rs73885319
AA, n (%)	32 (80.0%)	43 (89.0%)	36 (69.2%)	0.008	0.143	0.023
AG, n (%)	5 (12.5%)	7 (14.0%)	16 (30.8%)
GG, n (%)	3 (7.5%)	0	0
rs60910145
TT, n (%)	30 (75.0%)	43 (86.0%)	36 (69.2%)	0.153	0.289	0.373
TG, n (%)	9 (22.5%)	7 (14.0%)	16 (30.8%)
GG, n (%)	1 (2.5%)	0	0
rs71785313
Insertion/Insertion	28 (70.0%)	46 (92.0%)	41 (78.8%)	0.027	0.007	0.331
Insertion/Deletion	12 (30.0%)	4 (8.0%)	11 (21.2%)
Deletion/Deletion	0	0	0
Haplotypes
G0	35	50	49
G1GM	8	6	15
G1G+	2	1	1
G1+M	2	1	1
G2	12	4	11

HIV = Human Immune deficiency Virus, CKD = Chronic Kidney Disease.

The alleles of the ancestral haplotype G0 include; The G1GM haplotype with two missense alleles; the G1G+ haplotype with one missense risk allele at rs73885319; the G1+M has one missense variant at rs60910145. The G2 haplotype has the 6 base pair deletion risk allele at rs71785313.

The frequency of the HRG was 5.6% (8/142) among all the study participants. Three participants had the homozygous G1/G1 genotype and five had the compound heterozygote G1/G2 form; none of the study participants had the homozygous G2/G2 form. However the frequency of the HRG was 12.5% among those with HIV CKD compared to 5.8% in the HIV negative controls and zero in HIV positive no-CKD group; this difference was statistically significant (p = 0.025). Table 3 compares the distribution of the risk alleles among the various study groups.

Table 3

APOL1 genotype distribution among HIV positive CKD patients and control patients (HIV positive no-CKD and HIV-negative no-CKD).

Characteristic	HIV positive CKD (n = 40)	HIV positive no-CKD controls (n = 50)	HIV-negative no-CKD controls (n = 52)	p-value	p-value HIV+ CKD vs HIV+ no-CKD	p-value HIV+ CKD vs HIV- no-CKD
0 risk alleles
G0/G0	20 (50.0%)	38 (76.0%)	27 (51.9%)	0.013	0.015	1.000
1 risk allele
G0/G1	5 (12.5%)	8 (16.0%)	14 (26.9%)	0.13	0.175	0.673
G0/G2	10 (25.0%)	4 (8.0%)	8 (15.4%)
2 risk alleles
G1/G1	3 (7.5%)	0	0	0.025	0.015	0.288
G1/G2	2 (5.0%)	0	3 (5.8%)
G2/G2	0	0	0

Data is represented as n (%) HIV = Human Immune deficiency Virus, CKD = Chronic Kidney Disease,

We found a 10.8 fold higher odds for HIVAN among those with 2 risk alleles vs no risk alleles (OR 10.83, 95%CI 1.38–84.52; P = 0.023) and a 5.5 fold higher odds for those with 2 vs 1or 0 risk alleles (OR 5.50, 95%CI 0.83–36.29; P = 0.07) compared to controls. Associations were stronger among those with FSGS (2 vs 0 risk alleles: OR 13.0, 95%CI 2.06–81.91; P = 0.006 and 2 vs 0 or 1 risk alleles: OR 9.0, 95% CI 1.62–50.12; P = 0.012) and remained significant after adjusting for age and gender. Table 4 depicts the OR in the various groups, both unadjusted and after adjustments for age and gender.

Table 4

Odds ratios for associations of APOL1 risk alleles with HIVAN and FSGS versus controls.

	Unadjusted OR (95% CI)	P-value	Adjusted OR* (95% CI)	P-value
HIVAN
No of Risk Alleles
1 vs 0	3.82 (1.07–13.61)	0.038	2.51 (0.63–9.92)	0.189
2 vs 0	10.83 (1.38–84.52)	0.023	4.81 (0.52–44.90)	0.168
2 vs 1	2.83 (0.40–19.87)	0.295	2.58 (0.36–18.63)	0.347
2 vs 1 or 0	5.50 (0.83–36.29)	0.077	3.76 (0.54–26.35)	0.182
FSGS
No of Risk Alleles
1 vs 0	2.29 (0.65–8.07)	0.196	1.55 (0.40–6.00)	0.526
2 vs 0	13.0 (2.06–81.91)	0.006	14.00 (1.77–110.67)	0.012
2 vs 1	5.67 (0.92–34.99)	0.062	5.30 (0.83–33.95)	0.079
2 vs 1 or 0	9.0 (1.62–50.12)	0.012	8.85 (1.55–50.74)	0.014

*for age, gender. HIV = Human Immune deficiency Virus, CKD = Chronic Kidney Disease,

Discussion

This study conducted in Northern Nigeria determined the frequencies of APOL1 genotypes among treatment naïve HIV infected patients with biopsy-proven kidney disease and those with no evidence of kidney disease.

The prevalence of the haplotypes G1 (24.6%) and G2 (19.0%) in this population was lower than that reported in previous studies involving Nigerian populations; 45 to 50% for G1 among Yoruba population in the South Western parts of Nigeria and 30% among the Igbo population in the South Eastern parts of the country [12, 14, 19, 21]. This finding is also lower than that of our previous report, which comprised of a larger cohort with a multi-ethnic population of treatment experienced HIV patients [17]. It is also lower than the 40% reported for the Ashanti population and higher than the 11.4% reported among the Bulsa population, both from Ghana [20]. The G1 prevalence in our study participants was similar to the 20–22% reported from 3 surveys among populations of African descent [10, 22]. The prevalence was however higher than the 7.3% for G1 and 11.1% for G2 reported in a South African black population [13]. The G2 prevalence of 19% in this study was higher than the 7.5% and 16.6% reported in the Yoruba population [14, 22]. The G2 frequency in this study was lower than the 24.4% in the Igbo and 21.4% in the Bulsa in Ghana. It was however similar to the 13–15% among people of African descents in the US and the 12.9% among the Asante population in Ghana [10, 21]. Variations in the frequencies of these haplotypes among diverse African populations were also reported by Tzur et al when they studied 676 samples from 12 African populations that included Cameroon (2 population groups), Congo, Ethiopia (4 population groups), Ghana (2 population groups), Malawi, Mozambique and Sudan. The haplotype distribution differed between groups from the same country and also across the continent; for example in the Bulsa and Asante populations of Ghana, the frequencies were 11% and 41% respectively [21]. The findings in this study also differ from that of an Ethiopian population where they reported zero frequencies of these APOL 1 risk alleles among HIV infected patients with associated absence of HIVAN [23].

From the lower frequencies of these haplotypes in our study compared to those from the southern part, it may be possible to speculate on a possible relaxation of the natural selection push exerted by the Trypanosome infection in these regions by ancestors of the ethnic populations in this Northern part of Nigeria as previously suggested [17]. The study area is some distance from the Trypanosome infection belt and the Atlantic coastal areas of West Africa, the hub for the trans-Atlantic slave trade from which most people of African descent trace their ancestral journeys to the Caribbean and the Americas [24]. The fact that this is a hospital-based study as against a community based survey may be associated with recruitment bias and may also explain the discrepancies observed. There is a need for a multicenter comparative study with a large sample size to explain the differences in the frequencies of these alleles in different geographic locations within the same country. The frequencies of the HRG defined by the presence of 2 risk alleles (G1/G1, G2/G2, or G1/G2) among all participants in this study was 5.6% and it was 12.5% among the HIV positive CKD group. This overall lower frequency was similar to a previous report from this center among ART experienced cohort, although lower than that reported from the southern part of the country [18] and lower than the 11% reported for people of African descent [7] and 7% reported among DRC children [9]. However the higher frequency of the HRG among those with kidney disease was also reported by Kasembeli et al in which the frequency of the HRG was 17% among all participants but was as high as 89% among those with HIVAN [13] and the report by Ekrikpo et al that showed overall higher frequencies of the HRG of more than 70% in the study population with even much higher frequencies among HIV patients with kidney disease [18].

This study found that having the 2 high risk allele genotype was a predictor of FSGS compared to no risk alleles. The association was maintained in the recessive form (2 vs 1 or 0 risk alleles:). The association was also strong with HIVAN (2 vs 0 risk alleles) although the association did not reach statistical significance in the recessive form (2 vs 1 or 0 risk alleles). Kopp et al also reported this association in a cohort of FSGS and HIVAN; a greater association was observed among HIVAN compared to FSGS [10] Kasembeli et al reported an even greater association with HIVAN among a black South African cohort but no significant association with FSGS [13]. The lower strength of the association among our patients may be due to a smaller sample size compared to both the Kopp and Kasembeli studies. This calls for further study with a larger sample size on the effect of this HRG and other APOL1 nephropathies, not only among HIV infected patients but also in the general population.

This study provides information on the high frequencies of the APOL1 genotypes among treatment naïve HIV infected patients from this region, though the frequencies were not as high as those reported among population groups in the western and eastern parts of Nigeria. The study also confirmed the association between the occurrence of these HRGs and kidney disease, and specifically FSGS and HIVAN histological types among HIV infected patients despite the relatively lower frequencies; this was hitherto not reported from this part of the country This calls for the inclusion of evaluation of kidney function among newly diagnosed HIV patients as recommended [25], to allow for early detection and subsequent initiation of ART as extrapolation from a previous study showed that the life time risk of developing HIVAN among HIV patients carrying 2 high risk alleles may reach as high as 50% [10]. This finding also may be of significance considering the multiple risk factors for kidney disease among HIV infected individuals. The strong association with APOL1 HRG may provide an additional strategy in the evaluation and management of kidney disease risk among these patients. This study is limited by a small sample size and cross sectional design that did not allow for the assessment of the impact of the HRG on the progression of the kidney disease. However the patients being ART naïve and having a definitive diagnosis of CKD with kidney histology adds to the strength of the association of HRG of APOL1 with specific forms of kidney disease among the study patients.

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9 Jul 2021

PONE-D-21-10394

HIGH RISK APOL1 GENOTYPES AND KIDNEY DISEASE AMONG TREATMENT NAÏVE HIV PATIENTS AT KANO, NIGERIA

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: General Comments:

The study was performed to determine the frequency of APOL1 gene frequencies in HIV-1 infected- ART naïve patients at KANO, the northern part of Nigeria. 142 samples having HIV negative and HIV positive (with or without chronic kidney disease)) were analyzed for risk alleles by PCR-RFLP assay. The authors found an association between risk alleles (G1 and G2) frequencies and CKD. While these observations in part confirm previous studies, the APOL1 gene frequencies at the KANO region

remain to be determined. Simultaneously, the obtained results with respect to published results do not give any clear picture of the association between the tested variables. Consider increasing the sample size, which might help draw definitive conclusions, especially when a broad spectrum of frequency associations is reported. Overall, the data appear to be essential to the complex area of HIV-1 therapy.

Specific Comments:

1. I would ask the authors to include line and page numbers in all future submissions as it makes reviewing difficult without these.

2. Consider building a hypothesis in the introduction

3. Table 2 was mentioned two times and did not come complete on paper when printed.

4. Consider describing Table legends.

Reviewer #2: The authors have presented data on a limited sample size on the prevalence of APOL-1 genotypes in HIV positive patients (with and without kidney disease) in Northern Nigeria, West Africa. The describe the prevalence of high-risk and low-risk genotypes on kidney disease in the region.

I have a number of concerns about the study:

Major concerns:

1. The aim of the study was to “investigate the prevalence of high-risk variants and their effects on kidney disease among HIV infected patients in Northern Nigeria with hitherto limited information despite earlier reports of high population frequencies of these alleles from the Southern part of the country.” There are a number of prevalence studies from the same country region including this one from the same centre with a much larger sample size:

(Apolipoprotein-1 risk variants and associated kidney phenotypes in an adult HIV cohort in Nigeria. Wudil UJ, Aliyu MH, Prigmore HL, Ingles DJ, Ahonkhai AA, Musa BM, Muhammad H, Sani MU, Nalado AM, Abdu A, Abdussalam K, Shepherd BE, Dankishiya FS, Burgner AM, Ikizler TA, Wyatt CM, Kopp JB, Kimmel PL, Winkler CA, Wester CW.Kidney Int. 2021 Apr 23:S0085-2538(21)00430-0. doi: 10.1016/j.kint.2021.03.038. Online ahead of print.).

So, it is unclear what added value this study has provided. Rather, I think their results are conflicting with recent results from the same centre.

2. Leading on to the study rationale was this statement “Genetic predilection to HIVAN has been described among African-Americans but has not been widely studied among native Africans except for a few studies such as the study among the South African black population by Kasembeli et al and another study among children in the DRC.” This is not correct as there have been several other studies e.g.:

- Association of Genetic Polymorphisms of TGF-β1, HMOX1, and APOL1 With CKD in Nigerian Patients With and Without HIV. Ekrikpo UE, Mnika K, Effa EE, Ajayi SO, Okwuonu C, Waziri B, Bello A, Dandara C, Kengne AP, Wonkam A, Okpechi I.Am J Kidney Dis. 2020 Jul;76(1):100-108. doi: 10.1053/j.ajkd.2020.01.006. Epub 2020 Apr 27.

- APOL1 risk alleles among individuals with CKD in Northern Tanzania: A pilot study. Stanifer JW, Karia F, Maro V, Kilonzo K, Qin X, Patel UD, Hauser ER.PLoS One. 2017 Jul 21;12(7):e0181811. doi: 10.1371/journal.pone.0181811.

- APOL1 genetic variants, chronic kidney diseases and hypertension in mixed ancestry South Africans. Matsha TE, Kengne AP, Masconi KL, Yako YY, Erasmus RT.BMC Genet. 2015 Jun 26;16:69. doi: 10.1186/s12863-015-0228-6.

3. The method section is not very detailed. For instance, where were the HIV negative patients recruited from and how?, little information is provided about the methods of the kidney biopsy and how it was reported, statistical methods are not sufficiently reported, etc The methods section needs to have more details to ensure clarity.

4. The results section is also lacking in detail:

- The tables do not have a legend to describe abbreviations within each Table

- Some variables do not have units (e.g. what is the unit of measurement of CD4 count?)

- Why were there two Tables labelled as “Table 3”?

- What do the empty cells in the Tables mean – “no data”, “zero”, or “not reported”?

- Why are there inconsistencies with the decimals – most of the blood pressure reported have no decimals?

- In reporting the histologies, this was stated in the results section: “…and the commonest histological diagnosis was FSGS seen in 20 (37.7%) patients followed by HIVAN seen in 19 (35.9%) …” Is this primary FSGS? How did you differentiate primary FSGS in HIV positive patients from HIVAN which also has an FSGS pattern on histology?

5. The discussion needs to probe more into the implications of your study findings, especially relative to other recent studies in the region and in Nigeria as well as implications related to kidney disease prevalence and outcomes in Nigeria.

Minor comment:

I am not sure why most of the references are very old despite there being more recent citations from the region and country.

Reviewer #3: The study investigates the frequency of high risk alleles of the APOL1 gene in HIV infected patients of Northern Nigeria and establishes their effects on kidney disease. A High Risk Genotype (HRG) is associated with an increased risk to develope HIVAN and FSGS in HIV positive group respect to the control group. In particular, carriers of 2 vs 0 risk alleles have the highest odds.

The manuscript is interesting and, despite limitations on sample size and study design that can be improved with further investigations, provides new findings on the frequencies of APOL1 genotypes in a region with limited information so far. I have some comments to clarify and better explain some matters. The PDF file doesn’t include the number of lines or the number of pages, so it was difficult to report my comments, I hope I was clear.

In the ABSTRACT, please explain the acronyms FSGS and HIVAN

In the INTRODUCTION:

1. In the phrase “these variants include the APOL1 G1 allele…..and the G2 allele” the verb “include” is not the most appropriate, the allele includes the variants and not vice-versa: please explain better the concept

2. the correct nomenclature for a protein variation is p.Ser342Gly and p.Ile384Met: please correct

3. in the phrase “such as the study among the South African black population by Kasembeli et al and another study among children in the DRC” please explain the acronym DRC

In the METHODS:

1. Which is the treatment of HIV patients (both HIV positive no CKD and HIV positive CKD)? Is there any difference between the two groups? Please explain better this subject

2. eGFR is Estimated Glomerular Filtration Rate: please correct

3. It is not specified if it has been tested the Hardy-Weinberg equilibrium: please clarify

In the RESULTS:

1. In the table 1 “Characteristics of the study participants” the unit of measure of eGFR is ml/min/1.73m2 and the unit of measure of CD4 is missing, please correct. Furthermore in my opinion it would be important to have other information such as the possible presence of coinfection (hepatitis B/C), the type of ART and the HIV viral load: please indicate whether or not you have these data, and eventually why you do not have it.

2. In the table 2 please explain what haplotypes G0, G1GM, G1G+, G1+M, G2 mean

3. In the text after table 3 you state “these associations however were not significant after adjusting for age and gender”, but the association with 2 vs 1 or 0 risk alleles is already not significant (p=0.07): please explain better this concept.

4. Why do you adjust only for age and gender? There are other risk factors that could be included as covariates in the model, such as CD4 count, viral load, ART type and blood pressure

5. There are two table 3

6. It is not specified how you determined that G1 and G2 are on two different chromosomes, therefore in compound heterozygosity (G1/G2), and not on the same: is it based on literature data or have you tested it? Please clarify this concept.

In the DISCUSSION:

1. In the phrase “variations in the frequencies of these alleles among diverse African ethnic populations…” was should be were, please correct and add the number of reference of the cited study by Tzur et al.

2. Please change the sentence “the association was also strong with HIVAN…..with a less strong association in the recessive form”, a p=0.07 is not a less strong association, is not an association at all.

3. What does it mean the expression “from this environment” in the sentence “this study provides the first information on the high frequencies of the APOL1 genotypes….”? Please explain better.

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Reviewer #1: Yes: Himanshu Batra

Reviewer #2: No

Reviewer #3: No

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23 Feb 2022

Editorial concerns raised have all been addressed sequentially

Reviewer 1 All the issues raised have been addressed we are grateful to you for the valuable contributions

Reviewer 2 We have responded to all the issues raised and we thank you

Reviewer 3. We have addressed the observations made and we thank you very much

Submitted filename: Response to Reviewer (2).doc

Click here for additional data file.

8 Apr 2022

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Weijing He, M.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I thank authors for addressing all the concerns which I raised. Therefore, I recommend the article to be published.

Reviewer #3: Reviewing again the manuscript, I realized that there is a serious inaccuracy in the calculation of allelic frequencies, that I have not considered before.

The 142 subjects participating in the study have 284 chromosomes and consequently alleles, so the G0 alleles in each group are not 35, 50 and 49, but 55, 88 and 76 respectively (20 individuals with the G0/G0 genotype have 40 G0 alleles; 38 G0/G0 have 76 G0 alleles; 27 G0/G0 have 54 G0 alleles); the G1 alleles are 38 and the G2 alleles are 27; in total there are 284 alleles and not 198 alleles resulting from the sum of the haplotypes listed in table 2 "Frequencies of the APOL1 risk alleles". Therefore, the sentence "The frequency of the individual risk allele among all study participants was 26.1% (37/142) for G1 and 19.0% (27/142) for G2" on page 9 is incorrect, the exact G1 prevalence is 38/284=13.4% and the G2 prevalence is 27/284=9.5%. It would be correct to consider the 142 subjects, if you are talking about the frequency of the genotype or of the individuals with a number of risk alleles of 0,1,2. If you are talking about allelic frequencies, you must consider all 284 alleles. In my opinion in a study dealing with genetics, this concept needs to be revised.

Another important issue, already included in my revision, concerns the concept on page 10, where you state "five had the compound heterozygote G1/G2 form". I would like to know how you say that, are you based on literature data or have you tested it and know that G1 and G2 are on two different chromosomes? You didn't answer to my previous question.

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Reviewer #1: Yes: Himanshu Batra

Reviewer #3: No

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21 Aug 2022

1.The statement ‘the individual risk allele frequency among all study participants was’ has been changed to ‘The distribution of the APOL1 genotypes among the study participants revealed that 24.6% had a G1 risk allele and 19.0% a G2’ Page 2 line 28

2.‘The statement ‘the individual risk allele frequency among all study participants was’ has been changed to ‘The frequency of APOL 1 genotypes showed that 24.6% (35/142) of the study participants had at least 1 G1 risk allele and 19.0% (27/142) had a G2 risk allele.’ Page 9 line 164

3.The title of table 2 was changed from APOL1 risk alleles frequencies to

‘Distribution of APOL1 SNP genotypes and haplotypes in the study participants with HIV positive CKD, HIV positive no-CKD and HIV negative no-CKD’ Page 9 line 170

4.The title of table 3 was changed from APOL 1 risk alleles among the study participants to

‘APOL1 genotype distribution among HIV positive CKD patients and control patients ( HIV positive no-CKD and HIV-negative no-CKD)’ page 10 line 183

5.The statement ‘The prevalence of the individual risk alleles’ has been changed to

‘The prevalence of the haplotypes page 12 line 201

6.The statement ‘From the lower frequencies of these individual alleles has been changed to.

‘From the lower frequencies of these individual haplotypes’ page 13 line 214

Our response to reviewer's comment 2

This was based on the observation of the authors and published in the Kasembeli et al (2015). Because of the close proximity of the SNPs recombination is not possible/rare. The individuals showing this novel haplotype underwent several rounds of genotyping and sanger sequencing to confirm the presence of this haplotype

28 Sep 2022

HIGH RISK APOL1 GENOTYPES AND KIDNEY DISEASE AMONG TREATMENT NAÏVE HIV PATIENTS AT KANO, NIGERIA

PONE-D-21-10394R2

Dear Dr. Aliyu Abdu ,

My apologies for the long review process, we’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Weijing He, M.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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Comments to the Author

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Reviewer #1: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I thank the authors for making the necessary changes.

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Reviewer #1: Yes: Himanshu Batra

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3 Oct 2022

PONE-D-21-10394R2

HIGH RISK APOL1 GENOTYPES AND KIDNEY DISEASE AMONG TREATMENT NAÏVE HIV PATIENTS AT KANO, NIGERIA

Dear Dr. Abdu:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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