Fluorescence in situ hybridization for the diagnosis of NPHP1 deletion–related nephronophthisis on renal biopsy

Accepted Manuscript Fluorescence in situ hybridization for the diagnosis of NPHP1 deletion-related nephronophthisis on renal biopsy

Christopher P Larsen, Stephen M Bonsib, Marjorie L Beggs, Jon D Wilson PII: DOI: Reference:

S0046-8177(18)30234-X doi:10.1016/j.humpath.2018.06.021 YHUPA 4620

To appear in:

Human Pathology

Received date: Revised date: Accepted date:

16 April 2018 8 June 2018 9 June 2018

Please cite this article as: Christopher P Larsen, Stephen M Bonsib, Marjorie L Beggs, Jon D Wilson , Fluorescence in situ hybridization for the diagnosis of NPHP1 deletion-related nephronophthisis on renal biopsy. Yhupa (2018), doi:10.1016/j.humpath.2018.06.021

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ACCEPTED MANUSCRIPT Fluorescence In Situ Hybridization for the diagnosis of NPHP1 deletion-related nephronophthisis on renal biopsy

Christopher P Larsen MD1, Stephen M Bonsib MD1, Marjorie L Beggs PhD1, Jon D Wilson MD1 1

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Arkana Laboratories, Little Rock, AR 72211

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Corresponding author: Christopher P Larsen Arkana Laboratories 10180 Executive Center Drive, Suite 100 Little Rock, AR 72211 [email protected] Phone: 501-604-2695 Fax: 501-604-2699

Running title: NPHP1 in situ hybridization

ACCEPTED MANUSCRIPT Abstract

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Nephronophthisis is an autosomal recessive tubulointerstitial nephropathy that is a leading genetic etiology of end stage renal disease in children and young adults. Approximately 60% of patients with a known genetic etiology of nephronophthisis are due to homozygous deletion of the NPHP1 gene. We identified a total of 45 renal biopsies from young patients with chronic kidney disease of undetermined etiology and analyzed them for the possibility of nephronophthisis due to NPHP1 deletion using interphase fluorescence in situ hybridization and/or polymerase chain reaction. Homozygous NPHP1 deletion was identified in nine patients (20%). In cases with adequate tissue, both assays were performed and showed 100% agreement. Blinded histopathologic analysis was then performed and identified six lesions that were significantly more common in biopsies from patients with NPHP1 deletion-proven nephronophthisis than chronic kidney injury of other known etiologies. Many of the classically described nephronophthisis biopsy lesions such as tubular basement membrane duplication, presence of cysts, and mononuclear interstitial inflammation were not significantly associated with this disease when compared with biopsies from patients with chronic kidney injury due to other etiologies. There were, however, morphologic lesions that were strongly associated with NPHP1 deletion including tubular abnormalities such as diverticulum, florets, and macula densa-like change as well as interstitial Tamm-Horsfall aggregates, periglomerular fibrosis, and the absence of arteriosclerosis. Awareness of the histopathologic pattern of injury in nephronophthisis combined with testing for NPHP1 deletion enables renal pathologists to provide a definitive pathologic and genetic diagnosis in a subset of patients with this disease.

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Key words: nephronophthisis, NPHP, chronic kidney disease, renal biopsy, genetics, cystic kidney disease

ACCEPTED MANUSCRIPT Introduction

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Nephronophthisis is an autosomal recessive tubulointerstitial nephropathy that is a leading genetic etiology of end stage kidney disease in children and young adults, with a prevalence of approximately 5% of all children with renal failure[1,2]. At least 20 genes are known to be associated with nephronophthisis in humans [3]. Almost all of these genes encode proteins that localize to primary cilia, leading to the classification of nephronophthisis as a ciliopathy. Despite the large number genes described in association with this disease, the underlying genetic etiology is unexplained in the majority of cases [4]. When the genetic etiology is identified, NPHP1 is by far the most commonly recognized gene involved. Among a large case series detailing 365 families with nephronophthisis in whom both disease-causing alleles were identified, 64% of families had disease causing alleles in the NPHP1 gene. Among these the most common genetic change was homozygous deletion of the entire NPHP1 gene, affecting 93% of families [5].

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Material and Methods

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The histopathology of nephronophthisis is characterized by alterations in the tubulointerstitial compartment. Histopathologic lesions commonly described on biopsy in this disease include interstitial fibrosis, inflammation, and tubular basement membrane abnormalities with emphasis on thickening, thinning, and duplication of the tubular basement membrane [6]. However, these biopsy findings are nonspecific and commonly seen in the setting chronic kidney injury regardless of the underlying etiology. As a result, until now, it has not been possible to make a conclusive diagnosis of nephronophthisis on renal biopsy. In this report, we empirically determine the morphologic phenotype of nephronophthisis due to homozygous deletion of NPHP1 gene. Additionally, we demonstrate that a fluorescent in situ hybridization (FISH) and/or polymerase chain reaction (PCR) assay can be used to reach a definitive genetic diagnosis of this most common type of nephronophthisis with a high degree of sensitivity and specificity.

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All data were collected according to protocols approved by Solutions Institutional Review Board. All cases were processed by light, immunofluorescence, and electron microscopy using routine techniques described below[7]. Light microscopy

Kidney biopsies were fixed in buffered formalin, dehydrated in graded alcohols, and embedded in paraffin using standard techniques. Serial 3 μm-thick sections were cut and treated with hematoxylin and eosin, Jones methenamine silver, Masson trichrome, and periodic acid-Schiff reagent. Immunofluorescence Samples were transported in Michel’s media, washed in buffer, and frozen in a cryostat. Sections, cut at 5 μm, were rinsed in buffer and reacted with fluorescein-tagged polyclonal rabbit anti-human antibodies to IgG, IgA, IgM, C3, C4, C1q, fibrinogen, and κ-, and λ-light chains (Dako, Carpenteria, CA, USA) for 1 h,

ACCEPTED MANUSCRIPT rinsed, and a coverslip applied using aqueous mounting media. The stains were evaluated by standard immunofluorescence microscopy using a Leica L5 filter cube. Electron microscopy

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The ends of the renal biopsy specimen were removed as 1 mm cubes, dehydrated using graded alcohols and embedded in epon/araldite resin. Sections 1-μm thick were cut using an ultramicrotome, stained with toluidine blue and examined with a light microscope. Thin sections were examined in a Jeol JEM1011 electron microscope (Jeol, Tokyo, Japan). Photomicrographs were routinely taken at ×5000, ×12,000, and ×20,000 magnifications. Hump-like deposits were defined as large deposits with a rounded configuration which set on the surface of the GBM with minimal associated basement membrane response. In general, these deposits stand as high above the GBM as they are wide along its surface. Hinge deposits were defined as hump-like deposits overlying mesangium between folds of glomerular basement membranes.

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Fluorescence In Situ Hybridization

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Sections of 4um thickness from formalin-fixed-paraffin embedded tissue blocks were placed onto charges slides. The sections were dewaxed by placing in a 60oC oven for 30 minutes, and then placed in 3 changes of xylene for 3 minutes each, followed by rehydration in a series of graded alcohols into distilled water. The dewaxed sections were then placed in 1x Wash Buffer (Dako/Agilent) for 2 minutes. Sections were then probed according to Dako/Agilent SureFish protocol as follows. Slides were placed in 1x Pre-Treatment Solution (Dako/Agilent: K5799) and microwaved for 10 minutes. The slides were allowed to cool for 15 minutes, and then two 3-minute changes of Wash Buffer at room temperature were performed. The slides were immersed into pre-warmed 37oC 1x Pepsin Solution (Dako/Agilent: K5799), and then rinsed in two 3-minute changes of Wash Buffer at room temperature. The slides were dehydrated in graded alcohol: 70% alcohol for 2 minutes, 95% alcohol for 2 minutes, and 100% alcohol for 2 minutes, and then air dried for 10 – 15 minutes. 10ul of probe cocktail (1ul of each probe plus 8 ul of Hyb buffer per slide) containing CEP2 and NPHP1 probes (Dako/Agilent: G101089G and G101207R) in 1x IQFISH Fast Hyb Buffer (Dako/Agilent: G9416A) were applied onto the section(s). The section(s) were then coverslipped, sealed with sealant, and placed onto the hybridizer (Dako/Agilent Dakocytomation) that had been loaded with humidity strips. The hybridizer cycled as follows: 80oC 10:00 minutes, followed by 45oC 1:30 minutes, cooled to 37oC. The coverslips were removed and the slides were placed into glass coplin jar containing Stringent Wash ((Dako/Agilent) pre-warmed to 63oC for 10 minutes. The slides were then rinsed with two 3-minute changes of Wash Buffer. The sections were then dehydrated in graded alcohol: 70% alcohol for 2 minutes, 95% alcohol for 2 minutes, and 100% alcohol for 2 minutes, air dried in the dark (protected from light) for 10 – 15 minutes, and coverslipped using 15ul of Fluorescence Mounting Media with DAPI (Dako/Agilent). The probe signals were visualized under fluorescence microscopy with FITC (for CEP2) and CY3 (for NPHP1) filters (Figure 1). CEP2 and NPHP1 probe signals were counted for 100 individual cells, and the NPHP1/CEP2 ratio was calculated (normal/homozygous intact ratio >0.90 [range 0.87 – 1.32]; homozygous deletion ratio <0.02 [range 0.0 – 0.02]).

ACCEPTED MANUSCRIPT PCR detection of NPHP1

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Identification of control samples for morphologic comparison

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To determine the presence or absence of the NPHP1 gene PCR was performed with the following primers to exon 20 of the NPHP1 and exon 24 of COL4A6 genes: 5’-TCTGTGTAACTCTGCTCACTTCC-3’ NPHP1_E20F and 5’-CGTGGAGGATCCATCTGATTC-3’ NPHP1_E20R (expected amplicon size of 517 bp), 5’TGGTTGGTGGGCATATCTGG-3’ COL4A6_E24F and 5’-GGAGCCTCTGAAGCAGTGTT-3’ COL4A6_E24R (expected amplicon size 329 bp). PCR was performed using PrimeSTAR GXL reagents from Takara with the following cycle parameters: 35 cycles of melt 98oC for 1 second; anneal 60oC for 15 seconds; and extension 72oC for 30 seconds; followed by a final period of time at 72oC for 5min). The PCR products were cleaned-up with Agencourt beads as per manufacturer’s instructions. The products of each gene for each sample were run side by side on a D1000 TapeStation (Agilent) and images analyzed for bands of the correct size. A sample negative for an NPHP1 band but positive for a COL4A6 band was considered to have homozygous loss of the NPHP1 gene.

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Histopathologic scoring criteria

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The database at Arkana Laboratories was searched to identify patients with at least moderate chronic kidney disease of known etiology for comparison. Three general disease processes were chosen for comparison as they represent common etiologies of chronic kidney disease in young patients in the absence of immune complex deposition. These included cases with evidence of chronic injury due to APOL1-associated nephropathy, diabetic nephropathy, and reflux nephropathy. The APOL1-associated nephropathy biopsies were identified from a previous study examining the morphologic findings of African American patients with chronic kidney disease who had subnephrotic proteinuria[8]. A total of 13 biopsies with two APOL1 risk alleles were randomly selected for inclusion in the current histopathologic comparison.

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All cases (known NPHP1 deletion and controls) were scored in a blinded fashion by two renal pathologists (CPL and SMB). Periglomerular fibrosis was defined as PAS-positive thickening surrounding Bowman’s capsule within intact glomeruli. Tubular basement membrane duplication was assessed in both atrophic and intact tubules. A tubular diverticulum was defined as perpendicular outpouching with overlying TBM change (thickening or thinning) within intact tubules. Tubular florets profiles were defined as complicated tubular profiles with branching in at least 4 directions. A cyst was defined as being great than >200 microns (approximately the diameter of Bowman’s capsule). Macula densa-like lesions were defined as tubular profiles in which there is a transition to a grouping of cells with crowded, hyperchromatic nuclei and a high nuclear:cytoplasmic ratio. Cases were assessed for Tamm-Horsfall aggregates within the interstitium. Arteriosclerosis, interstitial fibrosis, tubular atrophy, and interstitial inflammation within intact and atrophic areas were defined according to those defined in the Banff working classification.[9] Representative photomicrographs of many of these lesions are shown in Figure 2. Renal biopsies with NPHP1 deletion versus controls with chronic kidney injury had phenotypes assessed by Fisher’s exact text. We considered p values <0.05 as significant.

ACCEPTED MANUSCRIPT Results

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A total of 45 renal biopsies from young patients with chronic kidney disease of undetermined etiology were identified in our database for inclusion in this study. Nine of these forty-five patients (20%) were identified to have homozygous NPHP1 deletion by either FISH and/or PCR. There were a total of eighteen patients with adequate formalin-fixed paraffin embedded (FFPE) tissue for FISH and DNA extracted from fresh frozen renal biopsy tissue for PCR. Six of these eighteen had evidence of homozygous NPHP1 deletion by both FISH (Figure 1) and PCR analysis. There was 100% agreement between the two assays. Fourteen additional patients had sufficient DNA available for NPHP1 deletion testing by PCR but inadequate residual FFPE renal biopsy tissue for FISH. One of these fourteen patients showed absence of PCR amplicon, indicating deletion of NPHP1 gene. Control PCR primers for exon 24 of the COL4A6 gene yielded appropriately sized amplicon. There were twelve patients with sufficient residual FFPE renal biopsy tissue for FISH but insufficient DNA available for PCR testing. Two of these patients showed absence of the NPHP1 gene by FISH. Control CEP2 probe signals were intact in these samples.

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The patients identified with NPHP1 deletion-associated nephronophthisis had a mean age of 16.6 years at the time of biopsy (range 3-26 years). There were 7 males and 2 females. The patients had a mean serum creatinine (Cr) of 6.7 mg/dl (range 2.0-22 mg/dl). Most had normal levels of protein in their urine although there were two with increased proteinuria at 0.6 and 1.1 grams per day.

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The histopathologic findings in the 9 patients with nephronophthisis due to homozygous NPHP1 deletion were compared with 38 biopsies with chronic kidney diseases due to other etiologies without immune complex deposition. These included 13 from patients with chronic kidney disease due to APOL1-associated nephropathy, 14 from patients with diabetic nephropathy, and 11 from patients with reflux nephropathy. Results from this histopathologic comparison are presented in Table 1. A total of 6 lesions were significantly associated with nephronophthisis due to NPHP1 deletion (Figure 2). The lesions that were significantly more common in nephronophthisis include interstitial Tamm Horsfall protein aggregates, tubular diverticulae, tubular florets, macula densa-like change, periglomerular fibrosis, and no more than mild arterial intimal fibrosis. Among these 6 lesions identified to be significantly associated with homozygous deletion of NPHP1, the presence of macula densa-like lesions was the alteration most commonly present. The remaining lesions, including tubular basement membrane duplication and interstitial inflammation, were not significantly associated with nephronophthisis.

Discussion The clinical features of nephronophthisis include renal failure, polyuria, polydipsia, secondary enuresis and growth retardation[3]. However, this disease is often not detected clinically. A recent genetic analysis of adults with end stage renal disease highlights this fact[10]. They found that NPHP1 deletionassociated nephronophthisis was found to be the etiology of kidney failure in 0.5% of adult patients. Among the twenty-six patients who were ultimately diagnosed with NPHP1-deletion associated

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nephronophthisis, only three (12%) were clinically diagnosed as having nephronophthisis. The other 88% were recorded as chronic kidney disease of other etiologies including CKD with unknown etiology (n=11), cystic disease (n=2), hypertensive nephrosclerosis (n=2), tubular and interstitial disease (n=2), glomerular disease (n=1), GN (histologically examined; n=1), sporadic primary reflux nephropathy (n=1), vascular nephropathy (n=1), urate nephropathy (n=1), and autosomal dominant polycystic kidney disease (n=1; no mutation in PKD1 or PKD2). [10]. Since it is not always clinically diagnosed, patients with nephronophthisis will occasionally undergo renal biopsy in an attempt to determine the etiology of kidney failure. Unfortunately, the morphologic lesions most commonly described on renal biopsy from patients with nephronophthisis are nonspecific in nature, making a definitive diagnosis based on histopathology alone impossible. We present the largest biopsy case series published to date of nephronophthisis due to NPHP1 gene deletion. The histopathologic lesions that are significantly associated with this disease are described as well as the utility of FISH and/or PCR to cost effectively establish a definitive diagnosis on renal biopsy tissue.

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We found in situ hybridization to be both sensitive and specific for the detection of NPHP1 deletion in renal biopsies suspected to represent nephronophthisis. We routinely use the in situ hybridization technique in our laboratory, rather than the PCR assay, when nephronophthisis is suspected on renal biopsy. In situ hybridization is preferred since it fits into the workflow and test utilization routinely used by the anatomic pathology laboratory and does not require involvement of the molecular laboratory. Additionally, the in situ hybridization assay cost is comparable to the PCR assay. The absence of NPHP1 deletion will not rule out the possibility of nephronophthisis due to other genetic etiologies. However, it does provide an accurate and relatively inexpensive technique to diagnose the most common known genetic etiology of this disease. Among 365 families with nephronophthisis, homozygous deletion for NPHP1 was responsible for disease in 60% of families[5]. Homozygous deletion of NPHP1 caused a relatively mild disease phenotype compared with the null mutations in other known nephronophthisis causing genes, and resulted in isolated kidney disease in 77% of the affected families[5]. Since it is the most common etiology of this disease, testing for NPHP1 gene deletion is recommended as an initial step in patients who are >4 years of age and suspected to have nephronophthisis prior to more comprehensive genetic testing by additional methodologies for other nephronophthisis genetic variants[5,11] [12]. In this study, we performed FISH and/or PCR on renal biopsy tissue. However, these same assays could be performed on peripheral blood specimens as well. If genetic testing for this variant turns out to be negative in the face of strong clinical suspicion for nephronophthisis, broad gene panels are available for more comprehensive testing [4,13,14]. We sought to determine which morphologic lesions are significantly more common in patients with NPHP1 deletion nephronophthisis in order that we might identify those most likely to benefit from targeted genetic testing such as FISH or PCR for the NPHP1 deletion. The renal histopathology of nephronophthisis as described in reviews of this disease as well as case reports and small case series typically includes interstitial fibrosis, tubular atrophy, mononuclear interstitial inflammation, periglomerular fibrosis, and tubular basement membrane irregularities such as thickening with duplication [1,6,15-20]. However, most experienced renal pathologists will agree that these histopathologic findings are ubiquitous in renal parenchyma with chronic injury and therefore not useful

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diagnostically as a tool to identify a specific disease entity. We sought to empirically determine if any lesions were significantly more common in nephronophthisis than in other biopsies with chronic injury of known etiology. Through blinded scoring of 9 patients with nephronophthisis and 38 patients with chronic kidney injury due to other etiologies, we identified 6 morphologic lesions that are significantly more common in nephronophthisis (Figure 2). Many of the classically described nephronophthisis biopsy lesions such as tubular basement membrane duplication, presence of cysts, and mononuclear interstitial inflammation were not significantly associated with this disease when compared with biopsies from patients with chronic kidney injury due to other etiologies. There were, however, morphologic lesions that were strongly associated with NPHP1 deletion including tubular abnormalities such as diverticulum, florets, and macula densa-like change as well as interstitial Tamm-Horsfall aggregates, periglomerular fibrosis, and the absence of arteriosclerosis. Awareness of these histopathologic patterns enables the pathologist to maintain a high index of suspicion for the diagnosis of nephronophthisis.

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Nephronophthisis is a common genetic etiology of end stage kidney disease in children and young adults. While often detected based on the clinical presentation alone, these patients occasionally undergo biopsy in an effort to discover the etiology of the unexplained chronic kidney injury. We present here, to our knowledge, the largest biopsy cohort to date of proven NPHP1-deletion related nephronophthisis. The results of this analysis identify biopsy lesions significantly associated with nephronophthisis that could be used to prompt consideration for targeted genetic testing. Awareness of the histopathologic pattern of injury in nephronophthisis as well as the availability of relatively cost effective FISH and/or PCR assays for diagnosis of the most common genetic form of this disease will enable definitive diagnosis in many nephronophthisis patients who undergo kidney biopsy.

Disclosures none

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Acknowledgments

ACCEPTED MANUSCRIPT Figure legends

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Figure 1. Fluorescence In-Situ Hybridization for the detection of NPHP1 gene deletion in renal biopsy tissue. (A) Cases without NPHP1 deletion show nuclear profiles (DAPI blue) with two probe signals for both the control centromeric probe CEP2 (FITC green) and NPHP1 (CY3 orange). An arrow indicates a nuclear profile with two signals of both probes. Not all cells have two green and orange signals due to nuclear cut-through inherent in three-micron tissue sections (B) Cases with NPHP1 deletion reveal nuclear profiles with two probe signals for only CEP2- FITC conjugate (green) probe, and no signal for the NPHP1 CY3- conjugated probe (orange). The arrow indicates a nuclear profile with two CEP2 probe signals and no signals for the NPHP1 probe. Not all cells have two green signals due to nuclear cutthrough inherent in three-micron tissue sections. (Separate DAPI, CY3, and FITC images merged using Image-J software; original magnification 100x oil immersion).

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Figure 2. Histopathologic lesions associated with NPHP1 deletion. (A) A tubular floret profile with complicated tubular branching in at least 4 directions (PAS; original magnification ×400). (B) Macula densa-like lesions (arrows) are tubular profiles in which there is a transition to a grouping of cells with crowded, hyperchromatic nuclei and a high nuclear:cytoplasmic ratio (H&E; original magnification ×400). (C) A tubular diverticulum (arrow) with perpendicular outpouching of the tubular cytoplasm and associated overlying TBM thinning (PAS; original magnification ×600). (D) Tamm-Horsfall aggregates within the interstitium (PAS; original magnification ×400). (E) Periglomerular fibrosis with PAS-positive thickening surrounding Bowman’s capsule of an intact glomerulus (PAS; original magnification ×400). (F) A normal artery from a biopsy of a patient with NPHP1-deletion associated nephronophthisis (Masson trichrome; original magnification ×600).

ACCEPTED MANUSCRIPT References

[11] [12] [13]

[14]

[15] [16] [17] [18]

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Hildebrandt F, Attanasio M, Otto E. Nephronophthisis: disease mechanisms of a ciliopathy. J Am Soc Nephrol 2009; 20: 23-35. Wolf MT, Hildebrandt F. Nephronophthisis. Pediatr Nephrol 2011; 26: 181-194. Srivastava S, Sayer JA. Nephronophthisis. J Pediatric genetics 2014; 3: 103-114. Halbritter J, Porath JD, Diaz KA, et al. Identification of 99 novel mutations in a worldwide cohort of 1,056 patients with a nephronophthisis-related ciliopathy. Hum Genet 2013; 132: 865-884. Chaki M, Hoefele J, Allen SJ, et al. Genotype-phenotype correlation in 440 patients with NPHPrelated ciliopathies. Kidney Int 2011; 80: 1239-1245. Wolf MT. Nephronophthisis and related syndromes. Curr Opin Pediatr 2015; 27: 201-211. Walker PD, Cavallo T, Bonsib SM. Practice guidelines for the renal biopsy. Mod Pathol 2004; 17: 1555-1563. Larsen CP, Beggs ML, Saeed B, et al. Histopathologic findings associated with APOL1 risk variants in chronic kidney disease. Mod Pathol 2014; 28: 95-102. Racusen LC, Solez K, Colvin RB, et al. The Banff 97 working classification of renal allograft pathology. Kidney Int 1999; 55: 713-723. Snoek R, van Setten J, Keating BJ, et al. NPHP1 (Nephrocystin-1) Gene Deletions Cause AdultOnset ESRD. J Am Soc Nephrol. 2018 Apr 13. doi: 10.1681/ASN.2017111200. [Epub ahead of print] Simms RJ. Nephronophthisis. Eur J Hum Gen 2009; 17: 406-416. Stokman M, Lilien M, Knoers N. Nephronophthisis. GeneReviews(R) 2016; https://www.ncbi.nlm.nih.gov/books/NBK368475/. Halbritter J, Diaz K, Chaki M, et al. High-throughput mutation analysis in patients with a nephronophthisis-associated ciliopathy applying multiplexed barcoded array-based PCR amplification and next-generation sequencing. J Med Genet 2012; 49: 756-767. Otto EA, Ramaswami G, Janssen S, et al. Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy. J Med Genet 2011; 48: 105-116. Benzing T, Schermer B. Clinical spectrum and pathogenesis of nephronophthisis. Curr Opin Nephrol Hypertens 2012; 21: 272-278. Blowey DL, Querfeld U, Geary D, Warady BA, Alon U. Ultrasound findings in juvenile nephronophthisis. Pediatr Nephrol 1996; 10: 22-24. Chamberlin BC, Hagge WW, Stickler GB. Juvenile nephronophthisis and medullary cystic disease. Mayo Clin Proc 1977; 52: 485-491. Fillastre JP, Guenel J, Riberi P, Marx P, Whitworth JA, Kunh JM. Senior-Loken syndrome (nephronophthisis and tapeto-retinal degeneration): a study of 8 cases from 5 families. Clin Nephrol 1976; 5: 14-19. Mongeau JG, Worthen HG. Nephronophthisis and medullary cystic disease. Am J Med 1967; 43: 345-355. Waldherr R, Lennert T, Weber HP, et al. The nephronophthisis complex. A clinicopathologic study in children. Virchows Arch (Pathol Anat) 1982; 394: 235-254.

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ACCEPTED MANUSCRIPT Highlights A fluorescence in situ hybridization assay for the detection of nephronophthisis due to homozygous deletion of NPHP1 was validated. Homozygous NPHP1 deletion was detected in nine (20%) of 45 renal biopsies from young patients with chronic kidney disease of undetermined etiology.

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Six histopathologic lesions were found to be more common in biopsies from patients with NPHP1 deletion-proven nephronophthisis than chronic kidney injury of other known etiologies.

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Morphologic lesion

APOL1associated CKD n=13

Reflux nephropathy n=11

Diabetic nephropathy n=14

P (NPHP1 deletion vs total of other diseases)

Interstitial Tamm Horsfall aggregates # positive (%)

0 (0%)

3 (27%)

6 (67%)

0.008

TBM duplication, intact tubular profiles # positive (%)

1 (8%)

4 (36%)

3 (21%)

8 (21%)

4 (44%)

0.2

TBM duplication, atrophic tubular profiles # positive (%)

11 (85%)

10 (91%)

14 (100%)

35 (92%)

Tubular diverticulum # positive (%)

1 (8%)

3 (27%)

1 (7%)

Tubular floret # positive (%)

0 (0%)

1 (9%)

0 (0%)

Cysts # positive (%)

3 (23%)

2 (18%)

0 (0%)

Macula dense-like change # positive (%)

0 (0%)

1 (9%)

1 (7%)

Cases with > mild interstitial inflammation within intact tissue # positive (%)

2 (15%)

1 (9%)

3 (21%)

Cases with > mild interstitial inflammation within fibrotic tissue # positive (%)

7 (54%)

5 (45%)

Cases with >mild arteriosclerosis # positive (%)

7(54%)

4 (36%)

Periglomerular fibrosis # positive (%)

1 (8%)

0.5

5 (13%)

6 (67%)

0.002

1 (3%)

5 (56%)

0.0004

5 (13%)

2 (22%)

0.6

2 (5%)

7 (78%)

0.00002

6 (16%)

1 (9%)

1

9 (64%)

21(55%)

6 (67%)

0.7

10 (71%)

21 (55%)

0 (0%)

0.002

2 (14%)

3 (8%)

4 (44%)

0.02

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Biopsies with NPHP1 deletion n=9

4 (29%)

Total for biopsies without NPHP1 deletion n=38 7 (18%)

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Table 1. Histopathologic comparison of nephronophthisis with other etiologies of chronic kidney injury

Figure 1

Figure 2