Autosomal dominant kidney disease phenocopying hypertensive nephropathy in Turkish Cypriot Families

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Deren Oygar, Ahmet Behlul, Salahi Ataç, Simge Bardak, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2844330/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Background In Cyprus, chronic kidney disease (CKD) is very common and often presents as a haematuric nephropathy caused by autosomal dominant pathogenic variants in the COL4A3 or COL4A4 genes. We investigated 57 Turkish Cypriots (TCs) with familial CKD for pathogenic variants in the COL4A3 and COL4A4 genes. Methods Probands from 53 families underwent massive parallel DNA sequencing using a glomerular gene panel for familial haematuria ( COL4A3, COL4A4, COL4A5, CFHR5 , and FN1 ), and whole exome sequencing (WES) was performed for 24 families. Twenty families were subjected to both procedures. Variants of interest were validated and tested in other family members by Sanger DNA sequencing or polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) and agarose gel electrophoresis. Results The diagnostic yield from these families was disappointing, and likely pathogenic variants were identified in only 12 of the 57 patients (genes, including COL4A3 (3), COL4A4 (2) , and COL4A5 (2)), leaving 45 unsolved families. Among the latter, a common missense variant ( COL4A4 :p. G545A), was present in four of the 45 unsolved and one of the solved families. Subsequently, we examined at least one member from a total of 85 families with evidence of familial kidney disease and a probable glomerular phenotype (at least one person with hematuria or proteinuria) and found 12 families (14%) with the p.G545A variant, which seemed to cosegregate with renal disease more often than would be expected by chance. All these families demonstrate an autosomal dominant (AD) inherited susceptibility to kidney disease associated with hypertension, variable and intermittent microscopic hematuria, and minimal proteinuria that remains at < 1 g/day until the estimated glomerular filtration rate (eGFR) falls below 30 ml/min, after which it may increase. Conclusions We suggest that COL4A4 :p. G545A may play a permissive polygenic role in a novel renal condition that phenocopies ‘hypertensive nephropathy’. This variant may be a common contributor to renal failure in the eastern Mediterranean region, thus justifying further investigation in appropriate families. Alport syndrome autosomal dominant COL4A4 tubulointerstitial disease hypertensive nephropathy Figures Figure 1 Figure 2 Figure 3 Background The Eastern Mediterranean has the highest incidence of kidney failure, as reported by the European Renal Association (ERA-EDTA) in Greece, Israel and Greek Cypriots [ 1 ]. We have previously reported a high incidence of renal failure and familial kidney disease in northern Cyprus among Turkish Cypriots, where the incidence was 2–3 times greater than that reported in northwestern Europe by the UK, Norway, Finland, and Denmark [ 2 ]. Investigation of Greek Cypriots with familial kidney disease has shown that the majority of patients have either haematuric nephropathy, often accompanied by significant proteinuria (termed here the ‘glomerular phenotype’) caused by heterozygous mutations of the COL4A3 , COL4A4 or CFHR5 genes [ 3 ] , [ 4 ] , [ 5 ] , [ 6 ], or a tubulointerstitial phenotype (that is, CKD in the absence of haematuria or glomerular proteinuria) typically associated with mutations in the PKD1, PKD2 , MUC1 or UMOD genes [ 7 ]. We are investigating more than 100 Turkish Cypriot families with evidence of CKD in at least two generations. The majority of families exhibit autosomal dominant (AD) inheritance. Our work has been aided by the number of large families with 6–10 siblings in up to four generations. Nonetheless, investigations of these families via massive parallel sequencing, either involving whole-exome sequencing or targeting of a specific panel of glomerular genes, were disappointing and yielded diagnostic findings in only 12 of the 57 families studied (genes were UMOD (4 cases), COL4A1 (1), COL4A3 (3), COL4A4 (2), and COL4A5 (2)), leaving 45 unsolved families. Among the latter, we observed that a common missense variant (COL4A4:p. G545A), which was hitherto considered to be benign or hypomorphic, was present in four of the 45 unsolved and one of the solved families. Further investigation via PCR-RFLP analysis of DNA from the families revealed that this variant cosegregated with clinical evidence of kidney disease in the affected family members. The renal disease in these families is characterized by mild hypertension, variable hematuria and minimal proteinuria, more suggestive of tubulointerstitial kidney disease and consistent with patients described as having hypertensive nephropathy. Methods Study population Cyprus is the third largest island in the Mediterranean inhabited by the majority of the Greek Cypriot and the minority of the Turkish Cypriot. Genetic studies indicate that Greek and Turkish Cypriots have similar origins with close affinities to southeastern Anatolia and the Near East and genetic continuity with the earliest inhabitants that first colonized the island approximately 10,000 years ago[ 8 ] , [ 9 ] , [ 10 ]. Turkish Cypriots are currently centred on northern Cyprus in the Turkish-administered Northern Cyprus (TRNC). A 2006 census of the TRNC population revealed that of 178,031 de jure citizens, 120,007 were born to parents who were both born in Cyprus[ 11 ]. The study was conducted at Nicosia State Hospital in northern Cyprus, a multispecialty tertiary care hospital that provides renal services to all of northern Cyprus. Families All patients were referred to the Nephrology Department of the Nicosia State Hospital for investigation of kidney disease. Family trees were created for families with 2 or more members with evidence of CKD [ 12 ] [see Figs. 1 (a) − 1(e)]. Families were contacted and were either visited in their villages or at the hospital. After providing written informed consent, all volunteer family members underwent urinalysis and renal function evaluation, and DNA was donated. The strict requirement for the use of family names (surnames) in northern Cyprus was introduced only in 1974, which made tracking families before that date more difficult. On the other hand, families of 10 children were common until the 1970s. Methods DNA Isolation and PCR-RFLP DNA extractions were carried out using the PureLinkTM Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s procedure. All DNA samples were quantified using a Qubit 2.0 fluorometer (Invitrogen, Thermo Fisher Scientific) and a Qubit dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA, USA). The index patients from a total of 53 families underwent NGS, which included parallel analysis of a 5-gene panel containing COL4A3, COL4A4, COL4A5, CFHR5, and FN1 as previously described [ 6 ],. Twenty-four patients had previously undergone whole-exome sequencing (WES), 20 of whom also underwent next-generation sequencing (NGS) via a 5-gene panel. WES analysis was performed as previously described, and patients with a minor allele frequency (MAF) > 0.005 in the 1000 Genomes Project database were excluded [ 13 ]. The nucleotide sequence around the variant COL4A4:p. G545A (rs1800516) was obtained from the NCBI database. The novel primers for PCR amplification were designed using the Primer-Blast software tool (NCBI). The sequences of primers used were as follows: forward primer: 5’-GAGACCAAGAATGCCAAGCT-3’, and reverse primer: 5’-AATAAACACCAGCCAAGCCA-3’. PCR was carried out in a volume of 25 µl containing 1x standard buffer (Amplicon, Denmark; containing 15 mM MgCl 2 ), 12.5 mM MgCl 2 , 16 mM dNTPs, 7.5 pmol from each primer, 0.75 U of Taq DNA polymerase (Amplicon, Denmark), approximately 25 ng of genomic DNA and distilled water. The primers used to amplify 491 bp fragments of the COL4A4 gene were generated by using the following PCR cycling conditions: initial denaturation at 95°C for 2 min; 30 cycles of denaturation at 95°C for 30 sec, annealing for 30 sec at 52°C and elongation at 72°C for 3 sec; and a final extension for 5 min at 72°C. The amplification products (491 bp) were visualized and photographed with a BioDoc-It Imaging System (Cole-Parmer™, Hanwell, London), an ultraviolet transilluminator, on a 1% agarose gel that was stained with ethidium bromide. Restriction fragment length polymorphism (RFLP) analysis was performed in a 20 µl total reaction volume containing 1x restriction enzyme tango buffer (33 mM Tris-acetate [pH 7.9 at 37°C], 10 mM Mg-acetate, 66 mM K-acetate and 0.1 mg/mL BSA), 10 U of XceI (NspI) (Thermo Fisher Scientific, USA), 10 µl of PCR product and nuclease-free water at 37°C for six hours. The digested products were electrophoresed for 45 min at 100 V on a 2.5% agarose gel and visualized under ultraviolet light after staining with ethidium bromide. The G allele was identified as an undigested 491 bp product, as opposed to the 349 and 142 bp products of XceI ( Nsp I) digestion yielded by the C allele. PCR products of heterozygous and mutant genotypes were verified by direct sequencing using the same set of primers (Applied Biosystems 3500xl DNA Analyser, USA) to assess the reliability of the presented assay. A schematic illustration of the COL4A4 p.G545A PCR-RFLP assay is given in Fig. 2 . A 491 bp PCR product was digested with XceI (NspI) to determine the presence of the G-to-C transition (Fig. 2 ). The genotyping results of individuals with CC and heterozygous genotypes were confirmed by Sanger sequencing, and the data obtained were consistent with the RFLP findings and precisely verified the reliability of the PCR restriction digestion assay. Datasets and mutation predictions Mutation predictions for COL4A4 :p. G545A was obtained from the gnomAD browser for exome data (GRCh37). https://gnomad.broadinstitute.org/variant/2-227946893-C-G?dataset=gnomad_r2_1 and for genomic data (GRCh38) https://gnomad.broadinstitute.org/variant/2-227082177-C-G?dataset=gnomad_r3 Information regarding previous reports of clinical significance was obtained from ClinVar https://www.ncbi.nlm.nih.gov/clinvar/variation/255015/?new_evidence=false and from the Leiden Open Variation Database (LOVD3) https://databases.lovd.nl/shared/genes/COL4A4 The mutation was also reviewed in Mutation Taster https://www.genecascade.org/MutationTaster2021/#example1 Clinical investigations Creatinine was the upper limit of normal for men (97 µmol/l), for women (80 µmol/l), and for blood urea nitrogen (BUN) (7.1 mmol/l). Kidney function (eGFR) was estimated by the MDR method. Proteinuria was initially measured via 24-hr urine collection, and a normal status was defined as a protein concentration < 140 mg/day and an albumin concentration < 30 mg/day. More recently, proteinuria has also been measured as the protein and albumin/creatinine ratio (Uacr < 30 mg/mg and Upcr < 200 mg/mg). Hematuria was investigated historically by urine microscopy and more recently by urine dipstick tests. Haematuria was deemed positive and equivalent to ‘a trace’ on a dipstick if there were 3 or more red blood cells per high-power field. Kidney biopsies were performed in two families. Biopsy specimens were routinely examined by light microscopy and immunofluorescence, and in one case electron microscopy was available. CKD staging as shown in the family tree figures: stage 0, no evidence of kidney disease; stage 1, microscopic haematuria only; stage 2, microscopic haematuria and/or proteinuria; stage 3, eGFR in the range of 59 − 30 ml/min; stage 4, eGFR 29 − 15; and stage 5, kidney failure. The diagnostic criteria were based on KDIGO guidelines[ 12 ]. Study groups investigated After we observed the p.G545A variant in our sequencing results, a member from all the families with some evidence of a glomerular phenotype (at least one person with hematuria or proteinuria) whose DNA sample was donated was tested for p.G545A (n = 85 families). Twelve families (14%) had a member with this variant. Statistical analysis Genotype and allele frequencies for both p.G545A and the wild type were calculated via the genotype counting method. The observed and expected genotype frequencies for both alleles were compared according to the Hardy‒Weinberg equilibrium by using controls and the chi-square test (2-tailed test). The analysis of cosegregation of the disease with the variant in families was tested with cosegregation analysis to evaluate whether there is evidence that a current variant underlies the distribution of a given phenotypic trait. Results Families with COL4A4:p. G545A variant COL4A4: p. G545A was initially found in 5 families investigated by DNA sequencing. Subsequently, we examined 85 families with evidence of familial kidney disease most likely with a glomerular phenotype (at least one person with hematuria or proteinuria) and found 12 families (14%) with the G545A variant. We investigated the 5 largest families and showed that the variant G545A cosegregated with kidney disease [Table 1 ]. The clinical data from these 5 families are summarized in Table 2 and by family trees [See Figs. 1 (a) – (e)]. Table 1 Analysis of observed cosegregation under the null hypothesis of no association of phenotype with the variant (i.e., assuming 50% chance of transmission of the allele in each informative meiosis) in families with p.G545A variation. Family (Fig) Informative meioses Concordant Discordant Probability of N or more concordant under null 1 (a) 16 15 1 0.0002594 1 (b) 15 9 6 0.30361938 1 (c) 15 12 3 0.01757813 1 (d) 18 14 4 0.01544189 1 (e) 4 3 1 0.3125 Legend : Binomial probability of observed cosegregation under the null hypothesis of random transmission of COL4A4 :p. G545A at each informative meiosis was calculated using the binomial cumulative distribution function, and a single affected founder was assumed for each pedigree. Combined p = 3.5 x10 − 6 . Table 2 Clinical information from family members shown on five family trees (FTs) Family Number (N) of family members on FT a All clinically affected (CKD stages 1–5) Clinically affected (CKD stages 3–5) Clinically unaffected (CKD stage 0) c No clinical Info d Age at first CKD Stage 3/KRT e Affected and age > 50 year KRT % N b N (variant present) N (variant present) N (variant present) N 1 (a) 32 20 (13/13) 9 (5/5) 7 (1/5) 5 60/65 1/11 (9%) 1 (b) 24 11 (8/9) 8 (7/7) 8 (6/6) 5 63/0 0/7 1 (c) 33 18 (8/9) 10 (6/6) 6 (2/3) 9 67/67 4/14 (29%) 1 (d) 44 20 (6/9) 10 (4/5) 8 (1/6) 16 61/60 3/15 (20%) 1 (e) 20 5 (2/3) 3 (2/2) 9 (0) 6 57/61 1/4 (25%) LEGEND : Family trees (FTs) – see Supplementary Figs. 1a-1e). The first generation is omitted from Table 1 because there are no clinical data. a Clinically affected: evidence of renal disease (stages 1–5), including all reported cases of ‘kidney disease’ but no survival data. b Variant present (x/y): y is the number of family members tested, and x is the number showing the heterozygous mutation. c No clinical information: no information available because of death or diaspora. d Median age at CKD stage 3/KRT. e Affected and aged > 50 years. KRT%: percentage who received KRT after the age of 50 years. Hypertension Mildly raised blood pressure was universal and was easily controlled with 1–2 drugs. There was no case of accelerated hypertension considered relevant to deteriorating kidney function. Microscopic hematuria Hematuria can be intermittent or absent. Among the 53 people whose urine was tested on one or more occasions, 40 had a positive result at least once (75%). Proteinuria We found that the proteinuria test was negative according to a dipstick test until the eGFR was less than 30 ml/min. Figure 3 shows proteinuria versus eGFR, documenting the observation that proteinuria was not greater than 1 g/d until the eGFR was less than 30 ml/min. We noted that for proteinuria (more than 0.2 g/d) in patients with an eGFR > 50 ml/min, approximately 50% of the urine protein was albumin, and this percentage increased to 80–90% when the proteinuria exceeded 2.0 g/d with eGFR values less than 30 ml/min. Onset of CKD Stage 3 and end-stage kidney disease (ESKD) Table 2 shows the age of onset of CKD and ESKD in the 5 families. The median age of the 38 patients with a GFR < 60 ml/min at first detection was 61 years (range 34–84). Among family members with clinical evidence of renal disease and aged older than 50 years, 19.6% had progressed to ESKD at a median age of 66 years (range 48–80 years). Renal biopsy Histology was performed for 2 families and revealed tubulointerstitial disease with interstitial fibrosis, tubular atrophy and extensive global glomerular sclerosis, with some glomeruli exhibiting segmental sclerosis (FSGS). On one of the biopsies, electron microscopy was performed and showed patchy thinning of the glomerular basement membrane (GBM) measured at 159 nm (a value less than 260 nm is diagnostic for thin glomerular basement membranes). In one family (see Fig. 1 . C), a 52-year-old female developed nephrotic syndrome, and a renal biopsy showed membranous nephropathy. She is now in remission following a course of immunosuppression. Her data are not included in Table 2 . Other clinical features In Cyprus, diabetes mellitus is very common, and we have not specifically evaluated this topic in these families. The data recorded show that of the 65 family members over the age of 50 years with clinical information available, 40% with no renal disease (stage 0) had diabetes, and 40% with stage 1–5 disease were diabetic. No specific routine investigation of hearing loss or eye problems was performed, but we are not aware of any issue with these features of Alport’s syndrome. Digenic family In one of the five families [see Fig. 1 (a)], DNA sequencing identified a second novel variant, COL4A3 :p. G877R (c.2629G > A) that is classified as likely pathogenic by Franklin and Varsome. CKD stages 3–4 were present in 4 patients with both variants, and CKD stage 3 was present once in a patient with only the p.G545A variant. In the latter case, there was no proteinuria despite an eGFR of 50 ml/min. In the former, all had proteinuria, but the maximum was 1.2 g/day, with an eGFR of 20 ml/min. Comparison of three different groups Three different groups were studied: Group 1: Family members with the p.G545A variant (see Figs. 1 a ) – e) for family trees). In total, 63 patients were tested for the p.G545A variant, and the minor allele frequency was 38% ( X 2 = 10, p = 0.001). Affected individuals were assigned to CKD Stages 1–5. All clinical information about the families is given in Table 1 . Group 2: Other Patients with Kidney Disease This group consisted of three different populations (n = 309): The primary renal diagnosis of 223 patients who started kidney replacement therapy (KRT) was either unknown or had diabetes or hypertension. Eighteen individuals (8%) had the p.G545A variant, so the variant allele frequency was 4% ( X 2 = 0.39, p = 0.53). Sixty-nine patients with clinical evidence of a haematuric glomerular disease or a renal biopsy showed secondary FSGS but no family history; 2 (3%) of these patients had p.G545A. The variant allele frequency was 1% ( X 2 = 0.01, p = 0.90). Seventeen individuals from 17 different families with kidney disease were considered to have a tubulointerstitial phenotype, but none had the p.G545A variant (0%). Group 3: Healthy Controls A total of 172 unrelated healthy volunteers, with a mean age of 56 years (range 22–103), were studied. The stages of kidney disease (CKD) in the 172 individuals in the control group ranged from 99 individuals with CKD stage 0 to 63 individuals with CKD1, 8 with CKD2 and 2 with CKD3. The minor allele frequency was 2% for the control group ( X 2 = 0.07, p = 0.78). There were 7 individuals with the variant allele; the median age was 51 years (range 33–80); 5 had CKD stage 0, one had CKD2, and one had CKD3. Discussion The large number of people affected in our five families has allowed us to perform a detailed analysis of the natural history of this kidney disease, which is indolent and asymptomatic until the disease is advanced and it constitutes an important cause of ESKD, usually in the seventh decade of life. From our 5 families, with 73 members with clinical evidence of renal disease, we observed the following: Mild hypertension is easily treated with 1–2 drugs. Proteinuria does not exceed 1 g/d until the eGFR is less than 30 ml/min (Fig. 3 ). Microscopic haematuria is variable, not always present and was found at least once in 75% of those tested. ESKD occurs in 19.6% of those affected who are aged > 50 years and has a median age of 66 years. Until CKD stage 4 is reached, patients can present with a tubulointerstitial phenotype (i.e., renal impairment with minimal or no proteinuria). The majority of families thus far have been reviewed retrospectively, and late presentation of an asymptomatic condition might be expected. Histology revealed tubulointerstitial disease with interstitial fibrosis and tubular atrophy and extensive global glomerular sclerosis, with some glomeruli exhibiting segmental sclerosis. Most likely, more important than focal and segmental glomerular sclerosis (FSGS) is the large percentage of global glomerular sclerosis evident at an early stage when the eGFR is still in excess of 50 ml/min [ 14 ]. A review of renal diseases leading to ESKD in the Middle East and North Africa revealed that in all countries, the sum of those with ‘ unknown aetiology’ and ‘ hypertensive nephropathy’ was approximately 50%[ 15 ]. This finding implies that the cause of renal failure is unknown in at least half of all patients. This finding contrasts with the European view that approximately 17% of patients with ESKD do not have a known renal diagnosis [ 1 ]. In the past 20 years, there have been an increasing number of reports of AD kidney failure associated with heterozygous pathogenic variants of COL4A3 or COL4A4 [ 4 ] , [ 16 ] , [ 17 ] , [ 18 ] , [ 19 ] [ 20 ] , [ 21 ]. Although initially considered to be a benign condition (benign familial haematuria), end-stage renal failure was first reported in 1985 in elderly patients [ 22 ]. We observed significant enrichment in the frequency of COL4A4 :p. G545A among the 85 families studied here compared with controls, and renal disease in the 5 families reported here cosegregated substantially more than would be expected by chance with this variant. It is common, and in gnomAD (v4.0), which is a register of whole-genome sequencing data, the variant is most commonly found in people from the ‘Middle East’, with an allele frequency of 0.082. In silico variant predictions suggest that COL4A4 :p. G545A is pathogenic based on the use of Polyphen as a ‘probably damaging’, SIFT as a ‘deleterious’ variant and a Grantham score of 60. The evolutionary model of variant effects yielded a high EVE score of 0.966[ 23 ]. The other predictor scores were as follows: REVEL: 0.776; CADD: 23.5; and PrimateAI: 0.575. There are also reports that it is a variant of unknown significance (VUS) [ 24 ]. Franklin and Varsome tools classify the variant as “benign” with the use of the ACMG criteria [ 25 ]. Because the variant p.G545A is so common, it cannot be regarded as a monogenic cause of renal disease, but it is better considered a predisposing or risk factor rather than a pathogenic variant. The absence of other genetic causes of kidney disease identified in all but one of the families studied here argues against the p.G545A variant acting only as a genetic modifier of disease. Our investigation of Greek Cypriots with familial kidney disease has shown that the majority of patients have haematuric nephropathy, often accompanied by proteinuria (termed here the ‘glomerular phenotype’) caused by heterozygous mutations of the COL4A3 , COL4A4 or CFHR5 genes[ 3 ] , [ 4 ] , [ 6 ]. In the Greek Cypriot population, the p.G545A variant was significantly more common in patients referred for testing because of haematuria (n = 468) than in controls in the general population (n = 368), p = 0.037. On the other hand, COL4A4 :p.G545A, either demonstrates incomplete penetrance or it acts as a genetic factor conferring higher predisposition to hematuria and CKD rather than as a causative mutation following a Mendelian inheritance. Our previous work (Deltas C and colleagues, unpublished results), revealed this variant in cohorts as follows: in people of the general population, where there was no known evidence for microscopic haematuria; in patients referred to our lab for genetic testing of microscopic haematuria but there was no clearcut family segregation when it was possible to test; and in patients who had the COL4A4 :p.G545A variant co-inherited with another clearly pathogenic variant 3 . Members of our five families fulfilled the clinical KDIGO criteria for ADTKD, which include CKD with bland urinary sediment, no to moderate proteinuria, and normal or decreased kidneys, with their pedigree consisting of at least 2 affected family members in 2 successive generations[ 26 ]. Both AD and X-linked Alport syndrome have recently been reported as causes of autosomal dominant tubulointerstitial disease (ADTKD)[ 27 ] , [ 28 ], and several mild pathogenic variants in the COL4A5 gene lead to a much attenuated form of Alport syndrome with late-onset kidney failure[ 29 ]. Conclusions Our findings do not provide a genetic explanation for kidney disease but suggest that p.G545A may, like APOL1[ 30 ], play a permissive polygenic role in this disease. Although many individuals in our 5 families fulfil the KDIGO criteria for ADTKD, we believe that further in-depth genetic investigation will reveal evidence of pathogenic variants in COL4A3 and COL4A4 , as well as a polygenic role for common variants such as p.G545A. In summary, we highlight a novel renal condition that phenocopies ‘hypertensive nephropathy’ and is likely to be a common cause of renal failure in elderly people in the eastern Mediterranean region. List of abbreviations AD Autosomal dominant ADTKD AD tubulointerstitial disease BUN Blood urea nitrogen CKD Chronic kidney disease eGFR Estimated glomerular filtration rate ERA-EDTA European Renal Association ESKD End stage kidney disease FSGS Focal and segmental sclerosis GBM Glomerular basement membrane KDIGO Kidney Disease: Improving Global Outcomes KRT Kidney replacement therapy MDRD Modification of Diet in Renal Disease NGS Next generation seuqencing PCR-RFLP Polymerase chain reaction-restriction fragment length polymorphism TC Turkish Cypriot TRNC Turkish administered Northern Cyprus WES Whole exome sequencing Declarations Ethics Approval All samples were collected in accordance with the ethical principles for medical research involving human subjects and according to the Declaration of Helsinki of the World Medical Association. This study was approved by the TRNC Ministry of Health Ethics Committee [Decision Number YTK.1.01 (Ek 002/19)]. All the research involving human participants was performed with written informed consent. and was approved by the ethics committee of Dr. Burhan Nalbantoğlu State Hospital, Nicosia. Data availability The datasets generated during and/or analysed during the current study are available from the corresponding author upon reasonable request. Competing Interests The following authors declare no competing interests: FO, DDO, AB, SA, SB, MY, GP, AM, CD, DPG, CG, or GHN. Funding Funding for FO was provided by Eastern Mediterranean University. This work was partly funded by EMU. DPG is supported by St. Peter’s Trust for Kidney, Bladder, & Prostate Research. This work was partly funded by the Cyprus Research and Innovation Foundation, program RESTART 2016-2020/INTEGRATED/0918/0043, to CD. Additionally, CD was funded by the CY -Biobank, an EU Horizon 2020 Research and Innovation Programme, under Grant Agreement No. 857122, the Republic of Cyprus, and the University of Cyprus. Author contributions Research idea and study design: DDO, GHN; clinical care, data and sample collection: DDO, AB, SA, SB, MY, FO; laboratory studies: FO, CG; data analysis/interpretation: FO, GHN, DPG, CG; statistical analysis: AM, DPG, CD. ; manuscript writing: GHN, CD, DPG, CG, FO. All the authors have read and agreed to the published version of the manuscript. Acknowledgements We would like to thank the families included in the study. We also thank Dr. Huseyin Sevay for his help with the pedigree drawings. References ERA-EDTA Registry Annual Report 2019. Amsterdam UMC, Department of Medical Informatics, Amsterdam, the Netherlands, 2021. Connor TMF, Oygar DD, Gale DP, Steenkamp R, Nitsch D, Neild GH, et al. Incidence of End-Stage Renal Disease in the Turkish-Cypriot Population of Northern Cyprus: A Population Based Study. PLoS One. 2013;8:e54394. Athanasiou Y, Voskarides K, Gale DP, Damianou L, Patsias C, Zavros M, et al. 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Frequency of COL4A3/COL4A4 Mutations amongst families segregating glomerular microscopic hematuria and evidence for activation of the unfolded protein response. Focal and segmental glomerulosclerosis is a frequent development during ageing. PLoS One. 2014;9:1–25. Stavrou C, Koptides M, Tombazos C, Psara E, Patsias C, Zouvani I, et al. Autosomal-dominant medullary cystic kidney disease type 1: Clinical and molecular findings in six large Cypriot families. Kidney Int. 2002;62:1385–94. Voskarides K, Mazières S, Hadjipanagi D, Di Cristofaro J, Ignatiou A, Stefanou C, et al. Y-chromosome phylogeographic analysis of the Greek-Cypriot population reveals elements consistent with Neolithic and Bronze Age settlements. Investig Genet. 2016;7:1. Gurkan C, Sevay H, Demirdov DK, Hossoz S, Ceker D, Teralı K, et al. Turkish Cypriot paternal lineages bear an autochthonous character and closest resemblance to those from neighbouring Near Eastern populations. Ann Hum Biol. 2017;44:164–74. 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A revised coding system for Middle East and North Africa. Saudi J Kidney Dis Transpl. 2011;22:651–61. Jefferson JA, Lemmink HH, Hughes AE, Hill CM, Smeets HJM, Doherty CC, et al. Nephrology Dialysis Transplantation Autosomal dominant Alport syndrome linked to the type IV collage a3 and a4 genes ( COL4A3 and COL4A4 ). Nephrol Dial Transplant. 1997;12:1595–9. Pescucci C, Mari F, Longo I, Vogiatzi P, Caselli R, Scala E, et al. Autosomal-dominant Alport syndrome: Natural history of a disease due to COL4A3 or COL4A4 gene. Kidney Int. 2004;65:1598–603. Furlano M, Martínez V, Pybus M, Arce Y, Crespí J, Venegas MDP, et al. Clinical and Genetic Features of Autosomal Dominant Alport Syndrome: A Cohort Study. Am J Kidney Dis. 2021;78:560-570.e1. Savige J. Heterozygous Pathogenic COL4A3 or COL4A4 Variants (Autosomal Dominant Alport Syndrome) Is Common, and Not Typically Associated With End-Stage Kidney Failure, Hearing Loss, or Ocular Abnormalities. Kidney Int Reports. 2022;7:1933–8. Matthaiou A, Poulli T, Deltas C. Prevalence of clinical, pathological and molecular features of glomerular basement membrane nephropathy caused by COL4A3 or COL4A4 mutations: a systematic review. Clin Kidney J. 2020;13:1025–36. García-Aznar JM, De la Higuera L, Besada Cerecedo L, Gandiaga NP, Vega AI, Fernández-Fresnedo G, et al. New Insights into Renal Failure in a Cohort of 317 Patients with Autosomal Dominant Forms of Alport Syndrome: Report of Two Novel Heterozygous Mutations in COL4A3. J Clin Med. 2022;11:4883. Dische FE, Weston MJ, Parsons V. Abnormally thin glomerular basement membranes associated with hematuria, proteinuria or renal failure in adults. Am J Nephrol. 1985;5:103–9. Frazer J, Notin P, Dias M, Gomez A, Min JK, Brock K, et al. Disease variant prediction with deep generative models of evolutionary data. Nature. 2021;599:91–5. Gibson J, Fieldhouse R, Chan MMY, Sadeghi-Alavijeh O, Burnett L, Izzi V, et al. Prevalence estimates of predicted pathogenic COL4A3-COL4A5 variants in a population sequencing database and their implications for alport syndrome. J Am Soc Nephrol. 2021;32:2273–90. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405–24. Eckardt KU, Alper SL, Antignac C, Bleyer AJ, Chauveau D, Dahan K, et al. Autosomal dominant tubulointerstitial kidney disease: Diagnosis, classification, and management - A KDIGO consensus report. Kidney Int. 2015;88:676–83. Wopperer FJ, Knaup KX, Stanzick KJ, Schneider K, Jobst-Schwan T, Ekici AB, et al. Diverse molecular causes of unsolved autosomal dominant tubulointerstitial kidney diseases. Kidney Int. 2022;102:405–20. Popp B, Ekici AB, Knaup KX, Schneider K, Uebe S, Park J, et al. Prevalence of hereditary tubulointerstitial kidney diseases in the German Chronic Kidney Disease study. Eur J Hum Genet. 2022;30:1413–22. Pierides A, Voskarides K, Kkolou M, Hadjigavriel M, Deltas C. X-linked, COL4A5 hypomorphic Alport mutations such as G624D and P628L may only exhibit thin basement membrane nephropathy with microhematuria and late onset kidney failure. Hippokratia. 2013;17:207–13. Daneshpajouhnejad P, Kopp JB, Winkler CA, Rosenberg AZ. The evolving story of apolipoprotein L1 nephropathy: the end of the beginning. Nat Rev Nephrol. 2022;18:307–20. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2844330","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":268883707,"identity":"dfd598b0-1973-4ac9-b933-eadf42685883","order_by":0,"name":"Fezile Ozdemir","email":"","orcid":"","institution":"Eastern Mediterranean University, North Cyprus.","correspondingAuthor":false,"prefix":"","firstName":"Fezile","middleName":"","lastName":"Ozdemir","suffix":""},{"id":268883708,"identity":"2634b6ed-8765-4333-9e4f-84f9902075f7","order_by":1,"name":"D. Deren Oygar","email":"","orcid":"","institution":"Nicosia State Hospital, Burhan Nalbantoglu General Hospital","correspondingAuthor":false,"prefix":"","firstName":"D.","middleName":"Deren","lastName":"Oygar","suffix":""},{"id":268883709,"identity":"3da9718f-f713-4c3c-b5c3-f3db3f9ba15d","order_by":2,"name":"Ahmet Behlul","email":"","orcid":"","institution":"Nicosia State Hospital, Burhan Nalbantoglu General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ahmet","middleName":"","lastName":"Behlul","suffix":""},{"id":268883710,"identity":"706b66d8-86c3-46b9-9766-c03ada9bd1df","order_by":3,"name":"Salahi Ataç","email":"","orcid":"","institution":"Nicosia State Hospital, Burhan Nalbantoglu General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Salahi","middleName":"","lastName":"Ataç","suffix":""},{"id":268883711,"identity":"d941fbfa-5d82-471b-973e-fe07b8d598d8","order_by":4,"name":"Simge Bardak","email":"","orcid":"","institution":"Nicosia State Hospital, Burhan Nalbantoglu General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Simge","middleName":"","lastName":"Bardak","suffix":""},{"id":268883712,"identity":"64e50563-7551-4471-a63c-5dd0f972490c","order_by":5,"name":"Meral Yükseliş","email":"","orcid":"","institution":"Nicosia State Hospital, Burhan Nalbantoglu General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Meral","middleName":"","lastName":"Yükseliş","suffix":""},{"id":268883713,"identity":"1e5715d4-1947-421b-8795-8c3684d9e074","order_by":6,"name":"Gregory Papagregoriou","email":"","orcid":"","institution":"School of Medicine, University of Cyprus","correspondingAuthor":false,"prefix":"","firstName":"Gregory","middleName":"","lastName":"Papagregoriou","suffix":""},{"id":268883714,"identity":"c5d2a64a-a15d-4ea7-a23c-ae10a7252118","order_by":7,"name":"Apostolos Malatras","email":"","orcid":"","institution":"biobank.cy Center of Excellence in Biobanking and Biomedical Research, University of Cyprus,","correspondingAuthor":false,"prefix":"","firstName":"Apostolos","middleName":"","lastName":"Malatras","suffix":""},{"id":268883715,"identity":"48a9558e-6ed0-4c1b-95f1-12b5d5b9313a","order_by":8,"name":"Daniel P. Gale","email":"","orcid":"","institution":"UCL Centre for Nephrology, University College London","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"P.","lastName":"Gale","suffix":""},{"id":268883716,"identity":"8037d9d7-a103-4074-ad8e-797e28dcafe5","order_by":9,"name":"Guy H. Neild","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYLCChAJmOQYG5gYwR4I4LQbMxgwMjKRoYTBgTmwgWou8A+/BBw8MrNM3HD/YwPCjhiFxZgMBLYYH+JINEgzSczecSWxg7DnGkDibkC2GDTxmEgkGh3M33AA6jLeBIXEeEVrMfwC1pBsAtTD+JUaLPAOPGTDEDieAtDCDbCHoMANmHmOgw9INZwL9cljmmIQxQe/Lt/cYfvxRYS3Pd/zwwYdvamxkZxwgZMthJM4BoiJSnpAzRsEoGAWjYBQwAACCdDyxw9mj2QAAAABJRU5ErkJggg==","orcid":"","institution":"UCL Centre for Nephrology, University College London","correspondingAuthor":true,"prefix":"","firstName":"Guy","middleName":"H.","lastName":"Neild","suffix":""},{"id":268883717,"identity":"67440923-efe6-41c1-aeae-cd4b26f47ca8","order_by":10,"name":"Constantinos Deltas","email":"","orcid":"","institution":"School of Medicine, University of Cyprus","correspondingAuthor":false,"prefix":"","firstName":"Constantinos","middleName":"","lastName":"Deltas","suffix":""},{"id":268883718,"identity":"c3f7e951-cd66-437c-9b2c-0de2cdb180fc","order_by":11,"name":"Cemal Gurkan","email":"","orcid":"","institution":"Turkish Cypriot DNA Laboratory, Committee on Missing Persons in Cyprus Turkish Cypriot Member Office, North Cyprus","correspondingAuthor":false,"prefix":"","firstName":"Cemal","middleName":"","lastName":"Gurkan","suffix":""}],"badges":[],"createdAt":"2023-04-21 08:14:27","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-2844330/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-2844330/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50063554,"identity":"0b4246cd-518f-4f95-bf50-f48bfafacdcb","added_by":"auto","created_at":"2024-01-16 20:44:53","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":618033,"visible":true,"origin":"","legend":"\u003cp\u003eFive\u003cstrong\u003e \u003c/strong\u003efamily pedigrees showing cosegregation with p.G545A.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLegend:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e**\u003c/em\u003e \u003cem\u003eFamily 1 (a) also shows cosegregation with the p.G877R variant.\u003c/em\u003eThe \u003cem\u003eCKD stage is shown as a superscript, and solid fill symbols indicate clinical evidence of CKD stages 1-5, while the half-fill symbols indicate CKD 0 (null CKD). * Red fill or red half-fill indicate the G545A mutation; green indicates the normal gene (wild type.) and clear is ‘no PCR performed’. The G877R mutation is shown in blue or half-filled lines; the wild type is shown in yellow. *CKD 0 means information available but no evidence of CKD. * CKD? - means no information was available. *CKD+ - means known to have CKD but no information available. * Diagonal lines through symbols represent premature deaths.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3859986/v1/6c2b67792b0f24377336d8cc.jpg"},{"id":50063558,"identity":"64468b13-238c-40ab-8a03-a6f30947a3f2","added_by":"auto","created_at":"2024-01-16 20:44:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":91451,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration and gel image of PCR-RFLP for \u003cem\u003eCOL4A4\u003c/em\u003e G545A (CC:349 bp and 142 bp, GC: 491 bp, 349 bp and 142 bp, GG:491 bp).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3859986/v1/e16c4f6ce63c2e2117b6830b.png"},{"id":50063560,"identity":"5f85923a-101b-41e6-86d0-e3ca02413618","added_by":"auto","created_at":"2024-01-16 20:44:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":18604,"visible":true,"origin":"","legend":"\u003cp\u003eProteinuria (up) g/day versus estimated glomerular filtration rate (eGFR)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLegend: \u003c/strong\u003eeGFR\u003cstrong\u003e (\u003c/strong\u003eml/min) versus maximum Upg/d in 53 patients in our 5 families.\u003c/p\u003e\n\u003cp\u003eProteinuria increases substantially after the eGFR becomes less than 30 ml/min.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3859986/v1/8613a80ff3711e7cc47d4bf7.png"},{"id":55265624,"identity":"194ff073-2a42-4d98-9d4b-1b5a1c6d2033","added_by":"auto","created_at":"2024-04-25 02:10:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":917205,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2844330/v2/9d27db94-6513-4d7c-9e7c-152a05ae1983.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Autosomal dominant kidney disease phenocopying hypertensive nephropathy in Turkish Cypriot Families","fulltext":[{"header":"Background","content":"\u003cp\u003eThe Eastern Mediterranean has the highest incidence of kidney failure, as reported by the European Renal Association (ERA-EDTA) in Greece, Israel and Greek Cypriots [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. We have previously reported a high incidence of renal failure and familial kidney disease in northern Cyprus among Turkish Cypriots, where the incidence was 2\u0026ndash;3 times greater than that reported in northwestern Europe by the UK, Norway, Finland, and Denmark [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInvestigation of Greek Cypriots with familial kidney disease has shown that the majority of patients have either haematuric nephropathy, often accompanied by significant proteinuria (termed here the \u0026lsquo;glomerular phenotype\u0026rsquo;) caused by heterozygous mutations of the \u003cem\u003eCOL4A3\u003c/em\u003e, \u003cem\u003eCOL4A4\u003c/em\u003e or \u003cem\u003eCFHR5\u003c/em\u003e genes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], or a tubulointerstitial phenotype (that is, CKD in the absence of haematuria or glomerular proteinuria) typically associated with mutations in the \u003cem\u003ePKD1, PKD2\u003c/em\u003e, \u003cem\u003eMUC1\u003c/em\u003e or \u003cem\u003eUMOD\u003c/em\u003e genes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe are investigating more than 100 Turkish Cypriot families with evidence of CKD in at least two generations. The majority of families exhibit autosomal dominant (AD) inheritance. Our work has been aided by the number of large families with 6\u0026ndash;10 siblings in up to four generations.\u003c/p\u003e \u003cp\u003eNonetheless, investigations of these families via massive parallel sequencing, either involving whole-exome sequencing or targeting of a specific panel of glomerular genes, were disappointing and yielded diagnostic findings in only 12 of the 57 families studied (genes were \u003cem\u003eUMOD\u003c/em\u003e (4 cases), \u003cem\u003eCOL4A1 (1), COL4A3 (3), COL4A4 (2), and COL4A5\u003c/em\u003e (2)), leaving 45 unsolved families. Among the latter, we observed that a common missense variant (COL4A4:p. G545A), which was hitherto considered to be benign or hypomorphic, was present in four of the 45 unsolved and one of the solved families. Further investigation via PCR-RFLP analysis of DNA from the families revealed that this variant cosegregated with clinical evidence of kidney disease in the affected family members.\u003c/p\u003e \u003cp\u003eThe renal disease in these families is characterized by mild hypertension, variable hematuria and minimal proteinuria, more suggestive of tubulointerstitial kidney disease and consistent with patients described as having hypertensive nephropathy.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eStudy population\u003c/h2\u003e\n \u003cp\u003eCyprus is the third largest island in the Mediterranean inhabited by the majority of the Greek Cypriot and the minority of the Turkish Cypriot. Genetic studies indicate that Greek and Turkish Cypriots have similar origins with close affinities to southeastern Anatolia and the Near East and genetic continuity with the earliest inhabitants that first colonized the island approximately 10,000 years ago[\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eTurkish Cypriots are currently centred on northern Cyprus in the Turkish-administered Northern Cyprus (TRNC). A 2006 census of the TRNC population revealed that of 178,031 \u003cem\u003ede jure\u003c/em\u003e citizens, 120,007 were born to parents who were both born in Cyprus[\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eThe study was conducted at Nicosia State Hospital in northern Cyprus, a multispecialty tertiary care hospital that provides renal services to all of northern Cyprus.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eFamilies\u003c/h2\u003e\n \u003cp\u003eAll patients were referred to the Nephrology Department of the Nicosia State Hospital for investigation of kidney disease. Family trees were created for families with 2 or more members with evidence of CKD [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e] [see Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(a) \u0026minus;\u0026thinsp;1(e)].\u003c/p\u003e\n \u003cp\u003eFamilies were contacted and were either visited in their villages or at the hospital. After providing written informed consent, all volunteer family members underwent urinalysis and renal function evaluation, and DNA was donated.\u003c/p\u003e\n \u003cp\u003eThe strict requirement for the use of family names (surnames) in northern Cyprus was introduced only in 1974, which made tracking families before that date more difficult. On the other hand, families of 10 children were common until the 1970s.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eMethods\u003c/h2\u003e\n \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\n \u003ch2\u003eDNA Isolation and PCR-RFLP\u003c/h2\u003e\n \u003cp\u003eDNA extractions were carried out using the PureLinkTM Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer\u0026rsquo;s procedure. All DNA samples were quantified using a Qubit 2.0 fluorometer (Invitrogen, Thermo Fisher Scientific) and a Qubit dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA, USA).\u003c/p\u003e\n \u003cp\u003eThe index patients from a total of 53 families underwent NGS, which included parallel analysis of a 5-gene panel containing \u003cem\u003eCOL4A3, COL4A4, COL4A5, CFHR5, and FN1\u003c/em\u003e as previously described [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e],. Twenty-four patients had previously undergone whole-exome sequencing (WES), 20 of whom also underwent next-generation sequencing (NGS) via a 5-gene panel. WES analysis was performed as previously described, and patients with a minor allele frequency (MAF)\u0026thinsp;\u0026gt;\u0026thinsp;0.005 in the 1000 Genomes Project database were excluded [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eThe nucleotide sequence around the variant COL4A4:p. G545A (rs1800516) was obtained from the NCBI database. The novel primers for PCR amplification were designed using the Primer-Blast software tool (NCBI). The sequences of primers used were as follows:\u003c/p\u003e\n \u003cp\u003eforward primer: 5\u0026rsquo;-GAGACCAAGAATGCCAAGCT-3\u0026rsquo;, and\u003c/p\u003e\n \u003cp\u003ereverse primer: 5\u0026rsquo;-AATAAACACCAGCCAAGCCA-3\u0026rsquo;.\u003c/p\u003e\n \u003cp\u003ePCR was carried out in a volume of 25 \u0026micro;l containing 1x standard buffer (Amplicon, Denmark; containing 15 mM MgCl\u003csub\u003e2\u003c/sub\u003e), 12.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 16 mM dNTPs, 7.5 pmol from each primer, 0.75 U of \u003cem\u003eTaq\u003c/em\u003e DNA polymerase (Amplicon, Denmark), approximately 25 ng of genomic DNA and distilled water. The primers used to amplify 491 bp fragments of the \u003cem\u003eCOL4A4\u003c/em\u003e gene were generated by using the following PCR cycling conditions: initial denaturation at 95\u0026deg;C for 2 min; 30 cycles of denaturation at 95\u0026deg;C for 30 sec, annealing for 30 sec at 52\u0026deg;C and elongation at 72\u0026deg;C for 3 sec; and a final extension for 5 min at 72\u0026deg;C. The amplification products (491 bp) were visualized and photographed with a BioDoc-It Imaging System (Cole-Parmer\u0026trade;, Hanwell, London), an ultraviolet transilluminator, on a 1% agarose gel that was stained with ethidium bromide.\u003c/p\u003e\n \u003cp\u003eRestriction fragment length polymorphism (RFLP) analysis was performed in a 20 \u0026micro;l total reaction volume containing 1x restriction enzyme tango buffer (33 mM Tris-acetate [pH 7.9 at 37\u0026deg;C], 10 mM Mg-acetate, 66 mM K-acetate and 0.1 mg/mL BSA), 10 U of \u003cem\u003eXceI (NspI)\u003c/em\u003e (Thermo Fisher Scientific, USA), 10 \u0026micro;l of PCR product and nuclease-free water at 37\u0026deg;C for six hours. The digested products were electrophoresed for 45 min at 100 V on a 2.5% agarose gel and visualized under ultraviolet light after staining with ethidium bromide. The G allele was identified as an undigested 491 bp product, as opposed to the 349 and 142 bp products of XceI (\u003cem\u003eNsp I)\u003c/em\u003e digestion yielded by the C allele. PCR products of heterozygous and mutant genotypes were verified by direct sequencing using the same set of primers (Applied Biosystems 3500xl DNA Analyser, USA) to assess the reliability of the presented assay.\u003c/p\u003e\n \u003cp\u003eA schematic illustration of the \u003cem\u003eCOL4A4\u003c/em\u003e p.G545A PCR-RFLP assay is given in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. A 491 bp PCR product was digested with \u003cem\u003eXceI (NspI)\u003c/em\u003e to determine the presence of the G-to-C transition (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe genotyping results of individuals with CC and heterozygous genotypes were confirmed by Sanger sequencing, and the data obtained were consistent with the RFLP findings and precisely verified the reliability of the PCR restriction digestion assay.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eDatasets and mutation predictions\u003c/h2\u003e\n \u003cp\u003eMutation predictions for \u003cem\u003eCOL4A4\u003c/em\u003e:p. G545A was obtained from the gnomAD browser for exome data (GRCh37).\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u0026nbsp;\u003cspan class=\"RefSource\"\u003ehttps://gnomad.broadinstitute.org/variant/2-227946893-C-G?dataset=gnomad_r2_1\u003c/span\u003e \u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eand for genomic data (GRCh38)\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u0026nbsp;\u003cspan class=\"RefSource\"\u003ehttps://gnomad.broadinstitute.org/variant/2-227082177-C-G?dataset=gnomad_r3\u003c/span\u003e \u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eInformation regarding previous reports of clinical significance was obtained from ClinVar\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u0026nbsp;\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/clinvar/variation/255015/?new_evidence=false\u003c/span\u003e \u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eand from the Leiden Open Variation Database (LOVD3)\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u0026nbsp;\u003cspan class=\"RefSource\"\u003ehttps://databases.lovd.nl/shared/genes/COL4A4\u003c/span\u003e \u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eThe mutation was also reviewed in Mutation Taster\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u0026nbsp;\u003cspan class=\"RefSource\"\u003ehttps://www.genecascade.org/MutationTaster2021/#example1\u003c/span\u003e \u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eClinical investigations\u003c/h2\u003e\n \u003cp\u003eCreatinine was the upper limit of normal for men (97 \u0026micro;mol/l), for women (80 \u0026micro;mol/l), and for blood urea nitrogen (BUN) (7.1 mmol/l). Kidney function (eGFR) was estimated by the MDR method.\u003c/p\u003e\n \u003cp\u003eProteinuria was initially measured via 24-hr urine collection, and a normal status was defined as a protein concentration\u0026thinsp;\u0026lt;\u0026thinsp;140 mg/day and an albumin concentration\u0026thinsp;\u0026lt;\u0026thinsp;30 mg/day. More recently, proteinuria has also been measured as the protein and albumin/creatinine ratio (Uacr\u0026thinsp;\u0026lt;\u0026thinsp;30 mg/mg and Upcr\u0026thinsp;\u0026lt;\u0026thinsp;200 mg/mg).\u003c/p\u003e\n \u003cp\u003eHematuria was investigated historically by urine microscopy and more recently by urine dipstick tests. Haematuria was deemed positive and equivalent to \u0026lsquo;a trace\u0026rsquo; on a dipstick if there were 3 or more red blood cells per high-power field.\u003c/p\u003e\n \u003cp\u003eKidney biopsies were performed in two families. Biopsy specimens were routinely examined by light microscopy and immunofluorescence, and in one case electron microscopy was available.\u003c/p\u003e\n \u003cp\u003eCKD staging as shown in the family tree figures: stage 0, no evidence of kidney disease; stage 1, microscopic haematuria only; stage 2, microscopic haematuria and/or proteinuria; stage 3, eGFR in the range of 59\u0026thinsp;\u0026minus;\u0026thinsp;30 ml/min; stage 4, eGFR 29\u0026thinsp;\u0026minus;\u0026thinsp;15; and stage 5, kidney failure. The diagnostic criteria were based on KDIGO guidelines[\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eStudy groups investigated\u003c/h2\u003e\n \u003cp\u003eAfter we observed the p.G545A variant in our sequencing results, a member from all the families with some evidence of a glomerular phenotype (at least one person with hematuria or proteinuria) whose DNA sample was donated was tested for p.G545A (n\u0026thinsp;=\u0026thinsp;85 families). Twelve families (14%) had a member with this variant.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eGenotype and allele frequencies for both p.G545A and the wild type were calculated via the genotype counting method. The observed and expected genotype frequencies for both alleles were compared according to the Hardy‒Weinberg equilibrium by using controls and the chi-square test (2-tailed test).\u003c/p\u003e\n \u003cp\u003eThe analysis of cosegregation of the disease with the variant in families was tested with cosegregation analysis to evaluate whether there is evidence that a current variant underlies the distribution of a given phenotypic trait.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003eFamilies with COL4A4:p. G545A variant\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eCOL4A4:\u003c/em\u003ep. G545A was initially found in 5 families investigated by DNA sequencing. Subsequently, we examined 85 families with evidence of familial kidney disease most likely with a glomerular phenotype (at least one person with hematuria or proteinuria) and found 12 families (14%) with the G545A variant. We investigated the 5 largest families and showed that the variant G545A cosegregated with kidney disease [Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e]. The clinical data from these 5 families are summarized in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and by family trees [See Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e (a) \u0026ndash; (e)].\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eAnalysis of observed cosegregation under the null hypothesis of no association of phenotype with the variant (i.e., assuming 50% chance of transmission of the allele in each informative meiosis) in families with p.G545A variation.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFamily (Fig)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eInformative meioses\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eConcordant\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eDiscordant\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eProbability of N or more concordant under null\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e1 (a)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0002594\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e1 (b)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.30361938\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e1 (c)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.01757813\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e1 (d)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.01544189\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e1 (e)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.3125\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\"\u003e\u003cstrong\u003eLegend\u003c/strong\u003e:\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\"\u003eBinomial probability of observed cosegregation under the null hypothesis of random transmission of \u003cem\u003eCOL4A4\u003c/em\u003e:p. G545A at each informative meiosis was calculated using the binomial cumulative distribution function, and a single affected founder was assumed for each pedigree. Combined p\u0026thinsp;=\u0026thinsp;3.5 x10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e.\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eClinical information from family members shown on five family trees (FTs)\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFamily\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eNumber (N) of family\u003c/p\u003e\n\u003cp\u003emembers on FT\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eAll clinically affected (CKD stages 1\u0026ndash;5)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eClinically affected (CKD stages 3\u0026ndash;5)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eClinically unaffected (CKD stage 0)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003csup\u003ec\u003c/sup\u003eNo clinical Info\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003csup\u003ed\u003c/sup\u003eAge at first CKD\u003c/p\u003e\n\u003cp\u003eStage 3/KRT\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003csup\u003ee\u003c/sup\u003eAffected and age\u0026thinsp;\u0026gt;\u0026thinsp;50\u0026nbsp;year KRT %\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003eN (variant present)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eN (variant present)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eN (variant present)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1 (a)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e32\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20 (13/13)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9 (5/5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7 (1/5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e60/65\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1/11 (9%)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1 (b)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11 (8/9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8 (7/7)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8 (6/6)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e63/0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0/7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1 (c)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e18 (8/9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10 (6/6)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6 (2/3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e67/67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4/14 (29%)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1 (d)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e44\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20 (6/9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10 (4/5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8 (1/6)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e61/60\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3/15 (20%)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1 (e)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5 (2/3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3 (2/2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9 (0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e57/61\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1/4 (25%)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"8\"\u003e\u003csup\u003e\u003cstrong\u003eLEGEND\u003c/strong\u003e\u003c/sup\u003e:\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"8\"\u003eFamily trees (FTs) \u0026ndash; see Supplementary Figs.\u0026nbsp;1a-1e).\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"8\"\u003eThe first generation is omitted from Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e because there are no clinical data.\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"8\"\u003e\u003csup\u003ea\u003c/sup\u003eClinically affected: evidence of renal disease (stages 1\u0026ndash;5), including all reported cases of \u0026lsquo;kidney disease\u0026rsquo; but no survival data. \u003csup\u003eb\u003c/sup\u003e Variant present (x/y): y is the number of family members tested, and x is the number showing the heterozygous mutation. \u003csup\u003ec\u003c/sup\u003e No clinical information: no information available because of death or diaspora. \u003csup\u003ed\u003c/sup\u003e Median age at CKD stage 3/KRT. \u003csup\u003ee\u003c/sup\u003e Affected and aged\u0026thinsp;\u0026gt;\u0026thinsp;50 years. KRT%: percentage who received KRT after the age of 50 years.\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003eHypertension\u003c/h2\u003e\n\u003cp\u003eMildly raised blood pressure was universal and was easily controlled with 1\u0026ndash;2 drugs. There was no case of accelerated hypertension considered relevant to deteriorating kidney function.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003eMicroscopic hematuria\u003c/h2\u003e\n\u003cp\u003eHematuria can be intermittent or absent. Among the 53 people whose urine was tested on one or more occasions, 40 had a positive result at least once (75%).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003eProteinuria\u003c/h2\u003e\n\u003cp\u003eWe found that the proteinuria test was negative according to a dipstick test until the eGFR was less than 30 ml/min. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e shows proteinuria versus eGFR, documenting the observation that proteinuria was not greater than 1 g/d until the eGFR was less than 30 ml/min. We noted that for proteinuria (more than 0.2 g/d) in patients with an eGFR\u0026thinsp;\u0026gt;\u0026thinsp;50 ml/min, approximately 50% of the urine protein was albumin, and this percentage increased to 80\u0026ndash;90% when the proteinuria exceeded 2.0 g/d with eGFR values less than 30 ml/min.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eOnset of CKD Stage 3 and end-stage kidney disease (ESKD)\u003c/h2\u003e\n\u003cp\u003eTable\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e shows the age of onset of CKD and ESKD in the 5 families. The median age of the 38 patients with a GFR\u0026thinsp;\u0026lt;\u0026thinsp;60 ml/min at first detection was 61 years (range 34\u0026ndash;84). Among family members with clinical evidence of renal disease and aged older than 50 years, 19.6% had progressed to ESKD at a median age of 66 years (range 48\u0026ndash;80 years).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003eRenal biopsy\u003c/h2\u003e\n\u003cp\u003eHistology was performed for 2 families and revealed tubulointerstitial disease with interstitial fibrosis, tubular atrophy and extensive global glomerular sclerosis, with some glomeruli exhibiting segmental sclerosis (FSGS). On one of the biopsies, electron microscopy was performed and showed patchy thinning of the glomerular basement membrane (GBM) measured at 159 nm (a value less than 260 nm is diagnostic for thin glomerular basement membranes). In one family (see Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. C), a 52-year-old female developed nephrotic syndrome, and a renal biopsy showed membranous nephropathy. She is now in remission following a course of immunosuppression. Her data are not included in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003eOther clinical features\u003c/h2\u003e\n\u003cp\u003eIn Cyprus, diabetes mellitus is very common, and we have not specifically evaluated this topic in these families. The data recorded show that of the 65 family members over the age of 50 years with clinical information available, 40% with no renal disease (stage 0) had diabetes, and 40% with stage 1\u0026ndash;5 disease were diabetic.\u003c/p\u003e\n\u003cp\u003eNo specific routine investigation of hearing loss or eye problems was performed, but we are not aware of any issue with these features of Alport\u0026rsquo;s syndrome.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003eDigenic family\u003c/h2\u003e\n\u003cp\u003eIn one of the five families [see Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e (a)], DNA sequencing identified a second novel variant, \u003cem\u003eCOL4A3\u003c/em\u003e:p. G877R (c.2629G\u0026thinsp;\u0026gt;\u0026thinsp;A) that is classified as likely pathogenic by Franklin and Varsome. CKD stages 3\u0026ndash;4 were present in 4 patients with both variants, and CKD stage 3 was present once in a patient with only the p.G545A variant. In the latter case, there was no proteinuria despite an eGFR of 50 ml/min. In the former, all had proteinuria, but the maximum was 1.2 g/day, with an eGFR of 20 ml/min.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003eComparison of three different groups\u003c/h2\u003e\n\u003cp\u003eThree different groups were studied:\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGroup 1: Family members with the p.G545A variant (see\u003c/em\u003e Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea\u003cem\u003e) \u0026ndash; e) for family trees).\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn total, 63 patients were tested for the p.G545A variant, and the minor allele frequency was 38% (\u003cem\u003eX\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;10, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001). Affected individuals were assigned to CKD Stages 1\u0026ndash;5. All clinical information about the families is given in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003eGroup 2: Other Patients with Kidney Disease\u003c/h2\u003e\n\u003cp\u003eThis group consisted of three different populations (n\u0026thinsp;=\u0026thinsp;309):\u003c/p\u003e\n\u003col style=\"list-style-type: lower-roman;\"\u003e\n\u003cli\u003e\n\u003cp\u003eThe primary renal diagnosis of 223 patients who started kidney replacement therapy (KRT) was either unknown or had diabetes or hypertension. Eighteen individuals (8%) had the p.G545A variant, so the variant allele frequency was 4% (\u003cem\u003eX\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.39, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.53).\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eSixty-nine patients with clinical evidence of a haematuric glomerular disease or a renal biopsy showed secondary FSGS but no family history; 2 (3%) of these patients had p.G545A. The variant allele frequency was 1% (\u003cem\u003eX\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.01, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.90).\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eSeventeen individuals from 17 different families with kidney disease were considered to have a tubulointerstitial phenotype, but none had the p.G545A variant (0%).\u003c/p\u003e\n\u003c/li\u003e\n\u003c/ol\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n\u003ch2\u003eGroup 3: Healthy Controls\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eA total of\u003c/em\u003e 172 unrelated healthy volunteers, with a mean age of 56 years (range 22\u0026ndash;103), were studied. The stages of kidney disease (CKD) in the 172 individuals in the control group ranged from 99 individuals with CKD stage 0 to 63 individuals with CKD1, 8 with CKD2 and 2 with CKD3. The minor allele frequency was 2% for the control group (\u003cem\u003eX\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.07, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.78). There were 7 individuals with the variant allele; the median age was 51 years (range 33\u0026ndash;80); 5 had CKD stage 0, one had CKD2, and one had CKD3.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe large number of people affected in our five families has allowed us to perform a detailed analysis of the natural history of this kidney disease, which is indolent and asymptomatic until the disease is advanced and it constitutes an important cause of ESKD, usually in the seventh decade of life.\u003c/p\u003e \u003cp\u003eFrom our 5 families, with 73 members with clinical evidence of renal disease, we observed the following:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eMild hypertension is easily treated with 1\u0026ndash;2 drugs.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eProteinuria does not exceed 1 g/d until the eGFR is less than 30 ml/min (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eMicroscopic haematuria is variable, not always present and was found at least once in 75% of those tested.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eESKD occurs in 19.6% of those affected who are aged\u0026thinsp;\u0026gt;\u0026thinsp;50 years and has a median age of 66 years.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eUntil CKD stage 4 is reached, patients can present with a tubulointerstitial phenotype (i.e., renal impairment with minimal or no proteinuria).\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eThe majority of families thus far have been reviewed retrospectively, and late presentation of an asymptomatic condition might be expected.\u003c/p\u003e \u003cp\u003eHistology revealed tubulointerstitial disease with interstitial fibrosis and tubular atrophy and extensive global glomerular sclerosis, with some glomeruli exhibiting segmental sclerosis. Most likely, more important than focal and segmental glomerular sclerosis (FSGS) is the large percentage of global glomerular sclerosis evident at an early stage when the eGFR is still in excess of 50 ml/min [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA review of renal diseases leading to ESKD in the Middle East and North Africa revealed that in all countries, the sum of those with \u0026lsquo;\u003cem\u003eunknown aetiology\u0026rsquo;\u003c/em\u003e and \u0026lsquo;\u003cem\u003ehypertensive nephropathy\u0026rsquo;\u003c/em\u003e was approximately 50%[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This finding implies that the cause of renal failure is unknown in at least half of all patients. This finding contrasts with the European view that approximately 17% of patients with ESKD do not have a known renal diagnosis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the past 20 years, there have been an increasing number of reports of AD kidney failure associated with heterozygous pathogenic variants of \u003cem\u003eCOL4A3\u003c/em\u003e or \u003cem\u003eCOL4A4\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Although initially considered to be a benign condition (benign familial haematuria), end-stage renal failure was first reported in 1985 in elderly patients [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe observed significant enrichment in the frequency of \u003cem\u003eCOL4A4\u003c/em\u003e:p. G545A among the 85 families studied here compared with controls, and renal disease in the 5 families reported here cosegregated substantially more than would be expected by chance with this variant. It is common, and in gnomAD (v4.0), which is a register of whole-genome sequencing data, the variant is most commonly found in people from the \u0026lsquo;Middle East\u0026rsquo;, with an allele frequency of 0.082.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn silico\u003c/em\u003e variant predictions suggest that \u003cem\u003eCOL4A4\u003c/em\u003e:p. G545A is pathogenic based on the use of Polyphen as a \u0026lsquo;probably damaging\u0026rsquo;, SIFT as a \u0026lsquo;deleterious\u0026rsquo; variant and a Grantham score of 60. The evolutionary model of variant effects yielded a high EVE score of 0.966[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The other predictor scores were as follows: REVEL: 0.776; CADD: 23.5; and PrimateAI: 0.575. There are also reports that it is a variant of unknown significance (VUS) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Franklin and Varsome tools classify the variant as \u0026ldquo;benign\u0026rdquo; with the use of the ACMG criteria [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBecause the variant p.G545A is so common, it cannot be regarded as a monogenic cause of renal disease, but it is better considered a predisposing or risk factor rather than a pathogenic variant. The absence of other genetic causes of kidney disease identified in all but one of the families studied here argues against the p.G545A variant acting only as a genetic modifier of disease.\u003c/p\u003e \u003cp\u003eOur investigation of Greek Cypriots with familial kidney disease has shown that the majority of patients have haematuric nephropathy, often accompanied by proteinuria (termed here the \u0026lsquo;glomerular phenotype\u0026rsquo;) caused by heterozygous mutations of the \u003cem\u003eCOL4A3\u003c/em\u003e, \u003cem\u003eCOL4A4\u003c/em\u003e or \u003cem\u003eCFHR5\u003c/em\u003e genes[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In the Greek Cypriot population, the p.G545A variant was significantly more common in patients referred for testing because of haematuria (n\u0026thinsp;=\u0026thinsp;468) than in controls in the general population (n\u0026thinsp;=\u0026thinsp;368), p\u0026thinsp;=\u0026thinsp;0.037. On the other hand, \u003cem\u003eCOL4A4\u003c/em\u003e:p.G545A, either demonstrates incomplete penetrance or it acts as a genetic factor conferring higher predisposition to hematuria and CKD rather than as a causative mutation following a Mendelian inheritance. Our previous work (Deltas C and colleagues, unpublished results), revealed this variant in cohorts as follows: in people of the general population, where there was no known evidence for microscopic haematuria; in patients referred to our lab for genetic testing of microscopic haematuria but there was no clearcut family segregation when it was possible to test; and in patients who had the \u003cem\u003eCOL4A4\u003c/em\u003e:p.G545A variant co-inherited with another clearly pathogenic variant\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMembers of our five families fulfilled the clinical KDIGO criteria for ADTKD, which include CKD with bland urinary sediment, no to moderate proteinuria, and normal or decreased kidneys, with their pedigree consisting of at least 2 affected family members in 2 successive generations[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBoth AD and X-linked Alport syndrome have recently been reported as causes of autosomal dominant tubulointerstitial disease (ADTKD)[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and several mild pathogenic variants in the \u003cem\u003eCOL4A5\u003c/em\u003e gene lead to a much attenuated form of Alport syndrome with late-onset kidney failure[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur findings do not provide a genetic explanation for kidney disease but suggest that p.G545A may, like APOL1[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], play a permissive polygenic role in this disease.\u003c/p\u003e \u003cp\u003eAlthough many individuals in our 5 families fulfil the KDIGO criteria for ADTKD, we believe that further in-depth genetic investigation will reveal evidence of pathogenic variants in \u003cem\u003eCOL4A3\u003c/em\u003e and \u003cem\u003eCOL4A4\u003c/em\u003e, as well as a polygenic role for common variants such as p.G545A. In summary, we highlight a novel renal condition that phenocopies \u0026lsquo;hypertensive nephropathy\u0026rsquo; and is likely to be a common cause of renal failure in elderly people in the eastern Mediterranean region.\u003c/p\u003e"},{"header":"List of abbreviations","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"548\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\" valign=\"bottom\"\u003e\n \u003cp\u003eAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"81.02189781021897%\" valign=\"bottom\"\u003e\n \u003cp\u003eAutosomal dominant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\" valign=\"bottom\"\u003e\n \u003cp\u003eADTKD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"81.02189781021897%\" valign=\"bottom\"\u003e\n \u003cp\u003eAD tubulointerstitial disease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\" valign=\"bottom\"\u003e\n \u003cp\u003eBUN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"81.02189781021897%\" valign=\"bottom\"\u003e\n \u003cp\u003eBlood urea nitrogen\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\" valign=\"bottom\"\u003e\n \u003cp\u003eCKD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"81.02189781021897%\" valign=\"bottom\"\u003e\n \u003cp\u003eChronic kidney disease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\" valign=\"bottom\"\u003e\n \u003cp\u003eeGFR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"81.02189781021897%\" valign=\"bottom\"\u003e\n \u003cp\u003eEstimated glomerular filtration rate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\" valign=\"bottom\"\u003e\n \u003cp\u003eERA-EDTA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"81.02189781021897%\" valign=\"bottom\"\u003e\n \u003cp\u003eEuropean Renal Association\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\" valign=\"bottom\"\u003e\n \u003cp\u003eESKD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"81.02189781021897%\" valign=\"bottom\"\u003e\n \u003cp\u003eEnd stage kidney disease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\" valign=\"bottom\"\u003e\n \u003cp\u003eFSGS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"81.02189781021897%\" valign=\"bottom\"\u003e\n \u003cp\u003eFocal and segmental sclerosis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\" valign=\"bottom\"\u003e\n \u003cp\u003eGBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"81.02189781021897%\" valign=\"bottom\"\u003e\n \u003cp\u003eGlomerular basement membrane\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\" valign=\"bottom\"\u003e\n \u003cp\u003eKDIGO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"81.02189781021897%\" valign=\"bottom\"\u003e\n \u003cp\u003eKidney Disease: \u003cstrong\u003eImproving Global Outcomes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\" valign=\"bottom\"\u003e\n \u003cp\u003eKRT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"81.02189781021897%\" valign=\"bottom\"\u003e\n \u003cp\u003eKidney replacement therapy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\" valign=\"bottom\"\u003e\n \u003cp\u003eMDRD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"81.02189781021897%\" valign=\"bottom\"\u003e\n \u003cp\u003eModification of Diet in Renal Disease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\" valign=\"bottom\"\u003e\n \u003cp\u003eNGS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"81.02189781021897%\" valign=\"bottom\"\u003e\n \u003cp\u003eNext generation seuqencing\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\" valign=\"bottom\"\u003e\n \u003cp\u003ePCR-RFLP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"81.02189781021897%\" valign=\"bottom\"\u003e\n \u003cp\u003ePolymerase chain reaction-restriction fragment length polymorphism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\" valign=\"bottom\"\u003e\n \u003cp\u003eTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"81.02189781021897%\" valign=\"bottom\"\u003e\n \u003cp\u003eTurkish Cypriot\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\" valign=\"bottom\"\u003e\n \u003cp\u003eTRNC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"81.02189781021897%\" valign=\"bottom\"\u003e\n \u003cp\u003eTurkish administered Northern Cyprus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.978102189781023%\" valign=\"bottom\"\u003e\n \u003cp\u003eWES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"81.02189781021897%\" valign=\"bottom\"\u003e\n \u003cp\u003eWhole exome sequencing\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003ch3\u003eEthics Approval\u003c/h3\u003e\n\u003cp\u003eAll samples were collected in accordance with the ethical principles for medical research involving human subjects \u003cem\u003eand according to\u003c/em\u003e the Declaration of Helsinki of the World Medical Association. This study was approved by the TRNC Ministry of Health Ethics Committee [Decision Number YTK.1.01 (Ek 002/19)].\u003c/p\u003e\n\u003cp\u003eAll the research involving human participants was performed with written informed consent. and was approved by the ethics committee of Dr. Burhan Nalbantoğlu State Hospital, Nicosia.\u003c/p\u003e\n\u003ch3\u003eData availability\u003c/h3\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003ch3\u003eCompeting Interests\u003c/h3\u003e\n\u003cp\u003eThe following authors declare no competing interests: FO, DDO, AB, SA, SB, MY, GP, AM, CD, DPG, CG, or GHN.\u003c/p\u003e\n\u003ch3\u003eFunding\u003c/h3\u003e\n\u003cp\u003eFunding for FO was provided by Eastern Mediterranean University. This work was partly funded by EMU. DPG is supported by St. Peter\u0026rsquo;s Trust for Kidney, Bladder, \u0026amp; Prostate Research. This work was partly funded by the Cyprus Research and Innovation Foundation, program RESTART 2016-2020/INTEGRATED/0918/0043, to CD. Additionally, CD was funded by the \u003cem\u003eCY\u003c/em\u003e-Biobank, an EU Horizon 2020 Research and Innovation Programme, under Grant Agreement No. 857122, the Republic of Cyprus, and the University of Cyprus.\u003c/p\u003e\n\u003ch3\u003eAuthor contributions\u003c/h3\u003e\n\u003cp\u003eResearch idea and study design: DDO, GHN; clinical care, data and sample collection: DDO, AB, SA, SB, MY, FO; laboratory studies: FO, CG; data analysis/interpretation: FO, GHN, DPG, CG; statistical analysis: AM, DPG, CD. ; manuscript writing: GHN, CD, DPG, CG, FO.\u003c/p\u003e\n\u003cp\u003eAll the authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003ch3\u003eAcknowledgements\u003c/h3\u003e\n\u003cp\u003eWe would like to thank the families included in the study. We also thank Dr. Huseyin Sevay for his help with the pedigree drawings.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eERA-EDTA Registry Annual Report 2019. Amsterdam UMC, Department of Medical Informatics, Amsterdam, the Netherlands, 2021.\u003c/li\u003e\n\u003cli\u003eConnor TMF, Oygar DD, Gale DP, Steenkamp R, Nitsch D, Neild GH, et al. Incidence of End-Stage Renal Disease in the Turkish-Cypriot Population of Northern Cyprus: A Population Based Study. PLoS One. 2013;8:e54394.\u003c/li\u003e\n\u003cli\u003eAthanasiou Y, Voskarides K, Gale DP, Damianou L, Patsias C, Zavros M, et al. Familial C3 Glomerulopathy Associated with CFHR5 Mutations: Clinical Characteristics of 91 Patients in 16 Pedigrees. Clin J Am Soc Nephrol. 2011;6:1436\u0026ndash;46.\u003c/li\u003e\n\u003cli\u003eVoskarides K, Damianou L, Neocleous V, Zouvani I, Christodoulidou S, Hadjiconstantinou V, et al. COL4A3/COL4A4 mutations producing focal segmental glomerulosclerosis and renal failure in thin basement membrane nephropathy. J Am Soc Nephrol. 2007;18:3004\u0026ndash;16.\u003c/li\u003e\n\u003cli\u003ePierides A, Voskarides K, Athanasiou Y, Ioannou K, Damianou L, Arsali M, et al. Clinico-pathological correlations in 127 patients in 11 large pedigrees, segregating one of three heterozygous mutations in the COL4A3 COL4A4 genes associated with familial haematuria and significant late progression to proteinuria and chronic kidney dise. Nephrol Dial Transplant. 2009;24:2721\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003ePapazachariou L, Demosthenous P, Pieri M, Papagregoriou G, Savva I, Stavrou C, et al. Frequency of COL4A3/COL4A4 Mutations amongst families segregating glomerular microscopic hematuria and evidence for activation of the unfolded protein response. Focal and segmental glomerulosclerosis is a frequent development during ageing. PLoS One. 2014;9:1\u0026ndash;25.\u003c/li\u003e\n\u003cli\u003eStavrou C, Koptides M, Tombazos C, Psara E, Patsias C, Zouvani I, et al. Autosomal-dominant medullary cystic kidney disease type 1: Clinical and molecular findings in six large Cypriot families. Kidney Int. 2002;62:1385\u0026ndash;94.\u003c/li\u003e\n\u003cli\u003eVoskarides K, Mazi\u0026egrave;res S, Hadjipanagi D, Di Cristofaro J, Ignatiou A, Stefanou C, et al. Y-chromosome phylogeographic analysis of the Greek-Cypriot population reveals elements consistent with Neolithic and Bronze Age settlements. Investig Genet. 2016;7:1.\u003c/li\u003e\n\u003cli\u003eGurkan C, Sevay H, Demirdov DK, Hossoz S, Ceker D, Teralı K, et al. Turkish Cypriot paternal lineages bear an autochthonous character and closest resemblance to those from neighbouring Near Eastern populations. Ann Hum Biol. 2017;44:164\u0026ndash;74.\u003c/li\u003e\n\u003cli\u003eKidd KK, Bulbul O, Gurkan C, Dogan M, Dogan S, Neophytou PI, et al. Genetic relationships of Southwest Asian and Mediterranean populations. Forensic Sci Int Genet. 2021;53.\u003c/li\u003e\n\u003cli\u003eCensus. The Final results of TRNC General Population and 2006, Census. http://nufussayimi.devplan.org/Kesin-sonuc-index_en.html.\u003c/li\u003e\n\u003cli\u003eLevey AS, Coresh J, Balk E, Kausz AT, Levin A, Steffes MW, et al. National Kidney Foundation Practice Guidelines for Chronic Kidney Disease: Evaluation, Classification, and Stratification. 2003.\u003c/li\u003e\n\u003cli\u003eLin F, Bian F, Zou J, Wu X, Shan J, Lu W, et al. Whole exome sequencing reveals novel COL4A3 and COL4A4 mutations and resolves diagnosis in Chinese families with kidney disease. BMC Nephrol. 2014;15:1\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eNieuwhof CMG, De Heer F, De Leeuw P, Van Breda Viriesman PJC. Thin GBM nephropathy: Premature glomerular obsolescence is associated with hypertension and late onset renal failure. Kidney Int. 1997;51:1596\u0026ndash;601.\u003c/li\u003e\n\u003cli\u003eNeild GH, Oygar DD, Hmida MB. Can we improve diagnosis of renal failure? A revised coding system for Middle East and North Africa. Saudi J Kidney Dis Transpl. 2011;22:651\u0026ndash;61.\u003c/li\u003e\n\u003cli\u003eJefferson JA, Lemmink HH, Hughes AE, Hill CM, Smeets HJM, Doherty CC, et al. Nephrology Dialysis Transplantation Autosomal dominant Alport syndrome linked to the type IV collage a3 and a4 genes ( COL4A3 and COL4A4 ). Nephrol Dial Transplant. 1997;12:1595\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003ePescucci C, Mari F, Longo I, Vogiatzi P, Caselli R, Scala E, et al. Autosomal-dominant Alport syndrome: Natural history of a disease due to COL4A3 or COL4A4 gene. Kidney Int. 2004;65:1598\u0026ndash;603.\u003c/li\u003e\n\u003cli\u003eFurlano M, Mart\u0026iacute;nez V, Pybus M, Arce Y, Cresp\u0026iacute; J, Venegas MDP, et al. Clinical and Genetic Features of Autosomal Dominant Alport Syndrome: A Cohort Study. Am J Kidney Dis. 2021;78:560-570.e1.\u003c/li\u003e\n\u003cli\u003eSavige J. Heterozygous Pathogenic COL4A3 or COL4A4 Variants (Autosomal Dominant Alport Syndrome) Is Common, and Not Typically Associated With End-Stage Kidney Failure, Hearing Loss, or Ocular Abnormalities. Kidney Int Reports. 2022;7:1933\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eMatthaiou A, Poulli T, Deltas C. Prevalence of clinical, pathological and molecular features of glomerular basement membrane nephropathy caused by COL4A3 or COL4A4 mutations: a systematic review. Clin Kidney J. 2020;13:1025\u0026ndash;36.\u003c/li\u003e\n\u003cli\u003eGarc\u0026iacute;a-Aznar JM, De la Higuera L, Besada Cerecedo L, Gandiaga NP, Vega AI, Fern\u0026aacute;ndez-Fresnedo G, et al. New Insights into Renal Failure in a Cohort of 317 Patients with Autosomal Dominant Forms of Alport Syndrome: Report of Two Novel Heterozygous Mutations in COL4A3. J Clin Med. 2022;11:4883.\u003c/li\u003e\n\u003cli\u003eDische FE, Weston MJ, Parsons V. Abnormally thin glomerular basement membranes associated with hematuria, proteinuria or renal failure in adults. Am J Nephrol. 1985;5:103\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eFrazer J, Notin P, Dias M, Gomez A, Min JK, Brock K, et al. Disease variant prediction with deep generative models of evolutionary data. Nature. 2021;599:91\u0026ndash;5.\u003c/li\u003e\n\u003cli\u003eGibson J, Fieldhouse R, Chan MMY, Sadeghi-Alavijeh O, Burnett L, Izzi V, et al. Prevalence estimates of predicted pathogenic COL4A3-COL4A5 variants in a population sequencing database and their implications for alport syndrome. J Am Soc Nephrol. 2021;32:2273\u0026ndash;90.\u003c/li\u003e\n\u003cli\u003eRichards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405\u0026ndash;24.\u003c/li\u003e\n\u003cli\u003eEckardt KU, Alper SL, Antignac C, Bleyer AJ, Chauveau D, Dahan K, et al. Autosomal dominant tubulointerstitial kidney disease: Diagnosis, classification, and management - A KDIGO consensus report. Kidney Int. 2015;88:676\u0026ndash;83.\u003c/li\u003e\n\u003cli\u003eWopperer FJ, Knaup KX, Stanzick KJ, Schneider K, Jobst-Schwan T, Ekici AB, et al. Diverse molecular causes of unsolved autosomal dominant tubulointerstitial kidney diseases. Kidney Int. 2022;102:405\u0026ndash;20.\u003c/li\u003e\n\u003cli\u003ePopp B, Ekici AB, Knaup KX, Schneider K, Uebe S, Park J, et al. Prevalence of hereditary tubulointerstitial kidney diseases in the German Chronic Kidney Disease study. Eur J Hum Genet. 2022;30:1413\u0026ndash;22.\u003c/li\u003e\n\u003cli\u003ePierides A, Voskarides K, Kkolou M, Hadjigavriel M, Deltas C. X-linked, COL4A5 hypomorphic Alport mutations such as G624D and P628L may only exhibit thin basement membrane nephropathy with microhematuria and late onset kidney failure. Hippokratia. 2013;17:207\u0026ndash;13.\u003c/li\u003e\n\u003cli\u003eDaneshpajouhnejad P, Kopp JB, Winkler CA, Rosenberg AZ. The evolving story of apolipoprotein L1 nephropathy: the end of the beginning. Nat Rev Nephrol. 2022;18:307\u0026ndash;20.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alport syndrome, autosomal dominant, COL4A4, tubulointerstitial disease, hypertensive nephropathy","lastPublishedDoi":"10.21203/rs.3.rs-2844330/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2844330/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIn Cyprus, chronic kidney disease (CKD) is very common and often presents as a haematuric nephropathy caused by autosomal dominant pathogenic variants in the \u003cem\u003eCOL4A3\u003c/em\u003e or \u003cem\u003eCOL4A4\u003c/em\u003e genes. We investigated 57 Turkish Cypriots (TCs) with familial CKD for pathogenic variants in the \u003cem\u003eCOL4A3\u003c/em\u003e and \u003cem\u003eCOL4A4\u003c/em\u003e genes.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eProbands from 53 families underwent massive parallel DNA sequencing using a glomerular gene panel for familial haematuria (\u003cem\u003eCOL4A3, COL4A4, COL4A5, CFHR5\u003c/em\u003e, and \u003cem\u003eFN1\u003c/em\u003e), and whole exome sequencing (WES) was performed for 24 families. Twenty families were subjected to both procedures. Variants of interest were validated and tested in other family members by Sanger DNA sequencing or polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) and agarose gel electrophoresis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe diagnostic yield from these families was disappointing, and likely pathogenic variants were identified in only 12 of the 57 patients (genes, including \u003cem\u003eCOL4A3 (3), COL4A4 (2)\u003c/em\u003e, and \u003cem\u003eCOL4A5\u003c/em\u003e (2)), leaving 45 unsolved families. Among the latter, a common missense variant (\u003cem\u003eCOL4A4\u003c/em\u003e:p. G545A), was present in four of the 45 unsolved and one of the solved families. Subsequently, we examined at least one member from a total of 85 families with evidence of familial kidney disease and a probable glomerular phenotype (at least one person with hematuria or proteinuria) and found 12 families (14%) with the p.G545A variant, which seemed to cosegregate with renal disease more often than would be expected by chance. All these families demonstrate an autosomal dominant (AD) inherited susceptibility to kidney disease associated with hypertension, variable and intermittent microscopic hematuria, and minimal proteinuria that remains at \u0026lt;\u0026thinsp;1 g/day until the estimated glomerular filtration rate (eGFR) falls below 30 ml/min, after which it may increase.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eWe suggest that \u003cem\u003eCOL4A4\u003c/em\u003e:p. G545A may play a permissive polygenic role in a novel renal condition that phenocopies \u0026lsquo;hypertensive nephropathy\u0026rsquo;. This variant may be a common contributor to renal failure in the eastern Mediterranean region, thus justifying further investigation in appropriate families.\u003c/p\u003e","manuscriptTitle":"Autosomal dominant kidney disease phenocopying hypertensive nephropathy in Turkish Cypriot Families","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2024-01-16 20:44:48","doi":"10.21203/rs.3.rs-2844330/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2023-04-28 03:43:12","doi":"10.21203/rs.3.rs-2844330/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ef8654e5-a8f6-4f10-b6ac-e916510ddfe2","owner":[],"postedDate":"January 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-23T12:52:14+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-16 20:44:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-2844330","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2844330","identity":"rs-2844330","version":["v2"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}