Integration of Genetic and Histopathology Data in Interpretation of Kidney Disease

Nephrol Dial Transplant (2020) 35: 1113–1132 doi: 10.1093/ndt/gfaa176

Integration of genetic and histopathology data in interpretation of kidney disease

Susan L. Murray 1,2, Neil K Fennelly3, Brendan Doyle3, Sally Ann Lynch4 and Peter J. Conlon1,2 REVIEW 1Department of Nephrology and Transplantation, Beaumont Hospital, Dublin, Ireland, 2Department of Medicine, Royal College of Surgeons, Dublin, Ireland, 3Department of Pathology, Beaumont Hospital, Dublin, Ireland and 4National Rare Disease Ofﬁce Mater Hospital Dublin, Downloaded from https://academic.oup.com/ndt/article/35/7/1113/5890560 by guest on 11 August 2020 Dublin, Ireland

Correspondence to: Susan L. Murray; E-mail: [email protected] This review was written in collaboration with NDT Educational.

ABSTRACT be described to one where the pathology and underlying mech- For many years renal biopsy has been the gold standard for di- anisms of disease could be understood [4, 5]. Over the last 65 years, the science of interpreting kidney bi- agnosis in many forms of kidney disease. It provides rapid, ac- opsies has developed gradually, aided by the development of curate and clinically useful information in most individuals three major diagnostic modalities: light microscopy (LM), im- with kidney disease. However, in recent years, other diagnostic munofluorescence (IF) and electron microscopy (EM) [6, 7]. It modalities have become available that may provide more de- has become a valuable tool in the diagnosis of native kidney dis- tailed and specific diagnostic information in addition to, or in- ease and the management of renal transplant. It is useful in stead of, renal biopsy. Genomics is one of these modalities. showing disease activity, such as crescent formation and inflam- Previously prohibitively expensive and time consuming, it is matory cell proliferation, and in establishing chronicity and de- now increasingly available and practical in a clinical setting for gree of fibrosis [8]. It has a high diagnostic yield and is thought the diagnosis of inherited kidney disease. Inherited kidney dis- to give useful information in up to 80% of cases [9, 10]. A pro- ease is a significant cause of kidney disease, in both the adult spective study by Turner et al.[11] showed that renal biopsy al- and paediatric populations. While individual inherited kidney tered the diagnosis in 44% and the therapeutic approach in 31% diseases are rare, together they represent a significant burden of of cases. Other studies have shown that treatment is modified in disease. Because of the heterogenicity of inherited kidney dis- up to 54% of patients [12]. It is an invasive procedure, but is ease, diagnosis and management can be a challenge and often generally considered very safe, though not entirely without risk, multiple diagnostic modalities are needed to arrive at a diagno- particularly the risk of bleeding [13, 14]. A recent study by sis. We present updates in genomic medicine for renal disease, Tøndel et al. [9] showed the risk of major bleeding to be 2% how genetic testing integrates with our knowledge of renal his- and the need for transfusion to be 1%. The study reported no topathology and how the two modalities may interact to en- deaths in >9000 patients. hance patient care. While a useful test, renal biopsy has drawbacks. Despite international guidelines and attempts at standardization, Keywords: genetics, inherited kidney disease, next-generation interobserver agreement between experienced renal patholo- sequencing, pathology, renal biopsy gists may be as low as 45% in some cases [15, 16]. Certain an- atomic abnormalities, such as a horseshoe kidney, single kidney or atrophic kidneys with thin renal cortices, may also INTRODUCTION preclude renal biopsy in everything but exceptional circum- Percutaneous renal biopsy was first described by Iversen and stances [2]. The accumulation of extracellular matrix in the Brun in 1951 [1]. It remains the gold standard for diagnosis in interstitium or fibrosis is common to all CKD, and while it is chronic kidney disease (CKD) [2, 3]. It helped to transform ne- a useful indicator of prognosis, it may obliterate useful path- phrology from a specialty where clinical syndromes could only ological architecture and make diagnosis difficult [17, 18].

VC The Author(s) 2020. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. 1113 Therefore renal biopsy may be a less useful diagnostic tool in Polycystic kidney disease (PKD) remains the most common late-presenting CKD. Approximately 15% of all incident inherited cause of CKD. It is thought to account for 7% of the patients in the UK who reach end-stage renal disease UK incident ESRD population and has a lifetime prevalence of (ESRD) lack a primary diagnosis [19]. at least 9.3 cases per 10 000 genes sequenced [26]. However, the Often in clinical practice, specific patterns of injury seen on improving cost-effectiveness and speed of genomic testing has biopsy are discussed as if they are diseases rather than descrip- led to recognition that other forms of inherited kidney disease tions of the histology at a given time. Focal segmental glomeru- are more common than previously assumed. Groopman et al. losclerosis (FSGS), for instance, is often treated as a single [27] reported that whole-exome sequencing (WES) of 3000 disease entity because of a relatively uniform histological pat- adult patients with CKD revealed a monogenic cause of disease tern. However, the onset, severity and response to treatment in in 10%, equivalent to rates of genetic diagnosis achieved in can- cases of FSGS can vary widely in patients with a very similar cer, and detected 66 separate monogenic disorders. Genomic histological pattern. Some cases of FSGS respond to corticoste- testing was particularly effective in diagnosing those with kid- roids, while some do not. It is much more likely that what we ney disease of unknown origin, in which they were able to make see are a number of disease entities unified by similar pathologi- a diagnosis in 18% of patients. cal findings rather than a single disease. Even in those where a clear monogenic form of kidney dis- Downloaded from https://academic.oup.com/ndt/article/35/7/1113/5890560 by guest on 11 August 2020 Overall, though it has been and remains a very useful diag- ease has not been identified, there is strong evidence of herita- nostic tool, renal biopsy can lack precision and may sometimes bility within families. In a survey of 1840 patients attending be inadequate for making a precise renal diagnosis. nephrology outpatient clinics or dialysis in Ireland in 2014, 34% Compared with renal biopsy, genetic testing for renal disease self-reported a family history of kidney disease [28]. Those with is a relatively new innovation. Previously prohibitively expen- an unknown underlying aetiology were 3 times more likely to sive, the introduction of next-generation sequencing (NGS) in report a family history of inherited kidney disease, while those the mid-2000s led to a 50 000-fold decrease in the cost of hu- with an unspecified tubulointerstitial kidney disease were man genome sequencing [20]. Since then, further technological 8 times more likely to report a family history. Other studies in advances have meant that the sequencing of specifically targeted different populations have shown similar results [29, 30]. There genes or an individual’s entire exome or genome can be per- is evidence that physiological parameters of the kidney are at formed efficiently and affordably. As we move towards a new least partially heritable. Glomerular filtration rate (GFR) is era of genomic medicine, we are faced with many new opportu- thought to be 30–60% heritable and handling of urinary cal- nities for diagnosis and management, and equally many new cium is thought to be 45% heritable [31–33]. challenges on how to integrate these new data safely and effec- As of 2019, >600 genes have been implicated in monogenic tively into our existing clinical practice. causes of kidney disease [34]. Testing and identification of This review examines new opportunities and challenges in many of these conditions may work in concert with, or in some the treatment in diseases of the kidney and, in particular, how cases replace, renal biopsy as the first-line diagnostic modality advances in genomic medicine can affect the utility of the per- in some renal diseases. cutaneous renal biopsy, how these two modalities interact with one another and how they can work in synthesis with one another to provide patient care. POLYGENIC KIDNEY DISEASE While monogenic causes contribute to a small fraction of all CKD, many more kidney diseases are influenced by a mixture of genetic, epigenetic and environmental risk factors. These kid- INHERITED KIDNEY DISEASE ney diseases are not inherited in a Mendelian fashion. Inherited kidney disease is an umbrella term that encompasses Correlation between phenotype and genotype is weak, but ge- many different disease states with a variety of different presen- netic factors may nevertheless play an important role in devel- tations. Given the broad variation of phenotypes, inherited kid- oping these diseases by conveying an increased relative risk. ney disease may cause disease that ranges in severity from Genome-wide association studies (GWASs) are a tool used extremely mild to incompatible with life and can present at any to look for associations between traits, including human disease time from antenatally to the last decades of life. There are many and single-nucleotide polymorphisms (SNPs). These are obser- kidney diseases that are inherited in a monogenic fashion due vational studies and pool the results of many individuals to look to a variant in a single gene, but there are equally as many kid- for common links between SNPs and traits. However, as most ney diseases that are influenced in a polygenic fashion. Even the GWAS loci tend to explain only a relatively small proportion of term ‘inherited’ is imprecise, as many disease-causing variants overall risk, translation of these findings into concrete clinical arise de novo in the proband, although once they arise they are benefit has proven a challenge. This has been compounded by hereditary thereafter. other limits, including technological challenges, small sample Most Mendelian inherited kidney diseases are rare, affecting size and allelic heterogeneity. Genome-wide polygenic risk only a tiny portion of the population. However, as a group they scores (GPSs) are designed to address these challenges by aggre- represent a significant burden of disease. They are the main gating the effects of millions of genetic loci across the genome, cause of CKD and ESRD in the paediatric population [21, 22]. including those that do not reach individual statistical signifi- In an adult population, inherited kidney disease is thought to cance. GPSs have now been shown to be able to predict risk in account for at least 10% of the CKD population [23–25]. well-studied polygenic conditions such as coronary artery

1114 S.L. Murray et al. disease, type 2 diabetes mellitus and atrial fibrillation [35]. nucleotide variations (SNVs) and insertions and deletions However, given the heterogenicity of CKD, the relative com- (indels) <10 base pairs in length and is therefore the gold stan- plexity of CKD diagnosis and the small numbers of patients in dard for confirmatory testing in single-gene disorders detected GWASs focused on specific diseases like IgA nephropathy by NGS [41]. In practice, it is used in cascade testing of affected (IgAN) and membranous nephropathy, GPSs may not be useful or at-risk family members once a pathogenic or likely patho- for the diagnosis of CKD [36]. genic variant has been identified in a proband. It is also used in Digenic inheritance is the simplest form of oligogenic inheri- segregation analysis where a variant of uncertain significance tance. It may be mistaken for polygenic disease, but in this case (VUS) is identified, in an attempt to clarify pathogenicity. only two separate loci are present that influence the expression NGS and severity of a phenotype. Digenic inheritance has been seen NGS refers to high-throughput, in-parallel sequencing and has in conditions including PKD and nephronophthisis [37, 38]. the ability to generate a very large volume of data very cheaply [42]. It can be used to code the entire exome or genome or to DIAGNOSTIC GENETIC TESTING code only areas of specific interest. There are numerous different NGS platforms that use a vari- The aim of diagnostic genetic testing is to identify disease- ety of sequencing technologies. However, all function using the Downloaded from https://academic.oup.com/ndt/article/35/7/1113/5890560 by guest on 11 August 2020 causing variants in an individual or family. The vastness of the core principle of the simultaneous parallel sequencing of mil- human genome makes this challenging. The human genome lions of small fragments of DNA, 1000–10 000 base pairs in contains 3.2 billion base pairs and the average genome differs length. Each fragment is sequenced multiple times (30 times is from the reference genome at 4–5 million sites [39]. There are the industry standard) to ensure a great depth of coverage and 22 000 known genes in the human genome, of which 4000 are accurate data when detecting unexpected DNA variations [43]. known to cause phenotypes that cause disease, susceptibility to The fragments are then pieced together using bioinformatics disease or benign changes in laboratory values [40]. to map the individual reads to a reference human genome [44]. The aim of diagnostic testing is not only to identify variants Algorithms are used to detect the best match between two or but also to determine which among the many variants detected more fragments of genetic sequencing [45]. Another set of bio- are predicted to be damaging. Several methods are used to try informatic algorithms detects mismatches between the refer- and determine this, including Sanger sequencing and NGS, ence genome and the mapped fragments to detect SNVs and each of which has specific advantages and disadvantages indels, in a process called variant calling [46]. Variant calling (Table 1). algorithms use statistical models of potential errors to detect which mismatches represent a true change and which are se- SANGER SEQUENCING quencing errors. The variants are then annotated with the addi- Developed in 1977 by Frederick Sanger, Sanger sequencing was tion of functional information such as the frequency of the the primary method of gene sequencing for >40 years. While in variant in human population databases and the predicted func- many scenarios Sanger sequencing has been succeeded by NGS tion of the variant upon the protein product of the gene [47]. due to enhanced speed and efficiency, it remains a useful and Databases such as ClinVar collate and publish information cost-effective tool in diagnostic testing. It provides targeted se- about previously interpreted variants. Large-scale genomic sequencing with high analytical validity in detecting causal single- quencing databases, such as the Genome Aggregation

Table 1. Advantages and disadvantages of forms of genetic testing

Technique Sanger sequencing Gene panel testing WES WGS Tests Single gene Multiple known genes Whole exome Whole genome Cost Cost-effective Cost-effective when testing Moderately expensive Most expensive known genes Advantages Quick and cost effective Can test a large range of known Can test all known genes Most complete analysis of Useful for very specific pheno- genes with high degree of Can be subsequently re-analysed genome types Unlikely to have a prob- accuracy as new knowledge accumulates Can be re-analysed at any point lem with a VUS Useful in disorders caused by in future as new knowledge many genes accumulates Disadvantages Only tests one gene at a time Can only test known genes Large data storage requirements Most expensive technique but May have to revisit genetic testing Challenges of discovering inci- getting cheaper if new genes are discovered. dental variants and VUS in Very large data storage VUS interpretation can be a inherited diseases requirements challenge Analytic techniques less developed than whole exome Challenges of discovering incidental variants. Challenge of VUSþþ Uses Cascade testing of family mem- Testing disorders with a specific Examine all CDS of the genome Examines all regions of the bers. Testing of single-gene dis- phenotype and multiple known genome orders, e.g. cystic fibrosis genes

Integrating histopathology and genetics in renal medicine 1115 Database, publish data on normal population variation and can have been detected in the exon coding regions, although patho- be used to help distinguish common from rare variants and genic variants have been detected in intronic regions [21, 57]. thus aid in determination of pathogenicity [48]. The American As massive parallel sequencing becomes less expensive, it College of Medical Genetics (ACMG) standards and guidelines has been argued that WES and WGS should replace targeted are widely used criteria that assess variants into one of five cate- NGS as the first line in genetic sequencing [34]. WES and WGS gories: pathogenic, likely pathogenic, VUS, likely benign and massively expand the search window for potential causative benign [49]. These data then need to be interpreted in a clinical variants. They may aid in detecting new variants and new geno- context with the input and expertise of clinicians [50]. type–phenotype correlations and may be more sensitive in There are many different methods of NGS. These may aim detecting patients with more than one monogenic disorder to sequence the entire exome or the entire genome or may focus causing their disease [58]. on specific gene sequences, as is the case in gene panels. TARGETED GENE PANELS Targeted gene panels are a form of NGS that targets only a spe- WES AND WHOLE GENOME SEQUENCING cific set of curated genes. While WES and WGS look at the en- Downloaded from https://academic.oup.com/ndt/article/35/7/1113/5890560 by guest on 11 August 2020 (WGS) tirety of the exome and genome, respectively, targeted panels The exome, that part of the genome that consists of exons, must be designed. Curated lists of genes are chosen by the test- accounts for only 1–2% of the entire genome. WES targets ing geneticist or through the use of a resource such as 22 000 known genes, or roughly 95% of human exons. It PanelApp, a curated crowdsourcing tool that allows gene panels examines only the protein-coding regions [CoDing Sequence to be shared and evaluated by the scientific community (https:// (CDS)]. WGS sequences 50–100 times more of the genome panelapp.genomicsengland.co.uk/). Because genes have to be than WES. It is untargeted and sequences exons, but also regu- selected, it means that panels are restricted by our current latory, intronic and intergenic regions. knowledge of the genes that cause disease, and must be updated The advantage of WES over WGS is that it is less expensive frequently to ensure that they remain current. Unlike WES and and the bioinformatic tools associated with it are more ad- WGS, it is not possible to retrospectively review the data panels vanced. It significantly reduces the number of variants one has provides, as our knowledge of the genes that cause disease con- to interpret (22 000 versus 2–3 million), and as 75% of patho- stantly expands. As WES and WGS become more cost effective, genic variants lie within the exome, it is a valuable approach. targeted NGS has less of a role. However, there are emerging whole-genome approaches that Nevertheless, panels have a role in testing homogenous con- detect structural variants and expansions of nucleotide repeats ditions caused by a small number of known genes, such as test- associated with disease that cannot be detected by WES. In ing for COL4A3, COL4A4 and COL4A5 in suspected Alport time, once the analysis of WGS is more robust, it is likely that syndrome. The advantages to gene panels over WES or WGS this will become the test of choice. are the reduced likelihood of finding an incidental finding or a WES and WGS may be used de novo or as a second-line test VUS. While VUSs still occur in panel testing, they are less com- when gene panel testing does not detect a disease-causing vari- mon. In addition, when a VUS occurs in a targeted gene test it ant [51]. Success rates are variable and depend on the disorder is more likely to be pathogenic than benign, as the more genes being sequenced, but have been reported to be >25% in consec- one tests the more likely the variant is benign. utive patients referred for genetic testing [52]. WES was used by Panel-based sequencing has been used with success in renal Lata et al. [53] to sequence 3067 patients with CKD, giving a di- disease. The Australian Renal Gene Panels service at the agnostic yield of 9.3%, encompassing 66 separate monogenic Children’s Hospital, Brisbane, QLD, Australia uses 10 targeted disorders, of which 39 were found in only a single patient. gene panels covering a curated range of 207 genes and was able Diagnostic rates are enhanced if the proband’s biological to achieve a 43% diagnosis rate in patients referred over a 3- parents are also tested in so-called trio testing [54]. This is par- year period [59]. This was effective in homogeneous conditions ticularly true if it is a de novo disorder; in contrast, where a par- such as Alport syndrome, but much less effective in heteroge- ent is also affected, triotesting will result in numerous inherited neous conditions such as congenital abnormalities of the kidney variants that can be difficult to filter. and urinary tract (CAKUT). Some laboratories offer homozygous exome testing on affected children of consanguineous relationships or if there are two affected siblings in a non-consanguineous relationship. BENEFITS AND RISKS OF GENETIC TESTING Filtering is performed looking for homozygous or compound Individuals undergoing genetic testing require pre- and post- heterozygous variants common to both affected siblings and testing counselling to ensure they understand the risks as well then segregation is completed on the parents using Sanger se- as the benefits of genetic testing [60]. Clinicians should docu- quencing. Indeed, diagnoses have been made in some lethal foe- ment that informed consent has been given and that the patient tal disorders by testing parents from consanguineous unions for is prepared for the possibility of uncertain and/or unexpected common homozygous variants whose phenotype fits with the findings [60]. A multidisciplinary approach with the availability findings in the foetus [55]. of nephrologists, clinical geneticists and genetic counsellors will There are fewer data on the use of WGS in the diagnosis of aid in safeguarding this process. Our team has developed short kidney disease [56]. The majority of disease-causing variants patient animation videos on variants of uncertain significance

1116 S.L. Murray et al. to aid in the consent process (http://bit.ly/VariantUS). Genetic There have been reports of people experiencing genetic dis- testing can be slow, often taking months or even years, thus it crimination as a result of their genetic diagnosis [66]. In may not be possible to make real-time clinical decisions based Europe, the European Union Charter of Fundamental Rights, on a genetic diagnosis. Article 21.1 prohibits discrimination based on ‘genetic features’, In general, genetic diagnosis has the potential to help answer but in practice the EU lacks a concrete legislative position and the questions that are most important to the patient: What is legislation is left to member states. In the USA, the Genetic the diagnosis? Why did it occur? What will be the future Information Nondiscrimination Act protects the right to health course? Will it reoccur post-transplant? Are other organs af- insurance and employment [67]. However, more subtle dis- fected? Is there effective treatment? Are family members at risk crimination may still occur. and can they consider becoming renal donors? [61]. The scope of some genetic counselling may be outside a The benefits of a genetic diagnosis include the ability to en- nephrologist’s usual practice and there is a need for specialized hance the specificity of diagnosis, allowing guidance of the genetic counsellors to work alongside physicians. These coun- prognosis and management. It allows for genetic counselling sellors can support physicians in helping to interpret data, an- and family planning and can provide reassurance for unaffected swer scientific queries and provide emotional support [68]. family members. On a population level, it allows us to under- Downloaded from https://academic.oup.com/ndt/article/35/7/1113/5890560 by guest on 11 August 2020 stand the disease process and its prevalence and provides opportunities to develop future therapies. It offers opportunities DISEASES OF THE KIDNEY for diagnosis in those who, by traditional methods, would not Advances in genomic medicine have affected diagnosis and have a diagnosis. It can potentially help risk stratification in treatment in several kidney diseases that previously relied solely clinical trials and identify patients unlikely to respond to treat- on renal biopsy as the primary diagnostic modality. These in- ment, such as avoiding steroid therapy in nephrotic syndrome clude ciliopathies, autosomal dominant tubulointerstitial kid- [62, 63]. ney disease (ADTKD), Alport syndrome and thin basement However, there are concerns regarding clinicians’ under- membrane nephropathy, FSGS and IgA nephropathy. standing of genetic test reports, particularly the limitations of our knowledge when a VUS is identified. Some laboratories have questioned whether non-clinical geneticists should be in- CILIOPATHIES formed of VUSs. The concern being that without a background Ciliopathies are a group of multisystem, inherited disorders understanding of normal human variation, someone will as- that cause functional or structural deficits in the cilia or the cilia sume any variant is significant no matter how the report is anchoring structures. Cilia are cell organelles present in almost worded. every cell with signalling or sensory functions [69, 70]. They There have been a number of high-profile legal cases where may be motile or non-motile. Those that are affected in renal clinicians have misinterpreted DNA variants with serious con- disease tend to be primary or non-motile cilia [71, 72]. Primary sequences. If one overinterprets a gene variant and determines cilia are present on the apical surface of the tubule and collect- it is pathogenic (when in fact it is not), then it can have conse- ing ducts. They act as a sensor that modulates urine osmolality quences not only to the proband, but also their at-risk relatives and composition via activation of intracellular signalling path- who might act on a result if they test positive. There have been ways [73]. cases where BRCA1 and BRCA2 were overinterpreted, leading Primary cilia are present in cell types across the body, so cil- to prophylactic surgery such as mastectomy, oophorectomy iopathies may affect any organ system in the body and and colorectal surgery. Potentially misinterpretation could lead extrarenal features relating to cilia dysfunction are commonly to a termination of a healthy pregnancy. Alternatively, if one associated with the renal phenotype. However, in practice, they underinterprets a variant, it can mean a patient misses vital op- primarily cause renal disease, retinal deterioration and cerebral portunities for treatment, an opportunity for pre-natal testing abnormalities [74]. Overlapping clinical features can make the or pre-implantation genetic diagnosis or avoidance of drugs diagnosis of ciliopathies challenging. contraindicated in that specific disorder. There are legal actions Most ciliopathies are single-gene disorders that alter the pending for all these scenarios. function of the cilium–centrosome complex [71]. Autosomal Non-targeted NGS, which examines the whole exome or ge- dominant PKD (ADPKD) and Von Hippel–Lindau disease are nome, may reveal unsolicited genetic findings, including some the main autosomal dominant ciliopathies that affect the kid- that may have a significant clinical impact on the patient. This ney. Others are primarily autosomal recessive. may include actionable variants that put the individual at risk Nephronophthisis, meaning wasting of the nephron, is an of developing cancer or cardiovascular disease [64]. It is impor- autosomal recessive ciliopathy. It is thought to be the most tant to appreciate that guidance from the European Society of common inherited kidney disease to cause ESRD in the paediat- Human Genetics states that the utility of the test and the diag- ric and adolescent population [75, 76]. Initial symptoms include nostic yield are considered before offering testing and whether polyuria, polydipsia and failure of urinary concentrating ability, testing will rule out or rule in a diagnosis. In contrast, the progressing insidiously to CKD and eventually ESRD, which ACMG lists 59 clinically actionable variants that should be may be recognized late due to the insidious nature of its onset. reported, regardless of the initial indication for sequencing, and It is distinguished in three forms: infantile, juvenile and adoles- this policy is observed throughout the USA [64, 65]. cent, based on the age of onset of ESRD. These three forms

Integrating histopathology and genetics in renal medicine 1117 manifest ESRD at the median ages of 1, 13 and 19 years, respec- but the rate of decline is very variable and ESRD can occur at tively [77–79]. any time between the ages of 17 and 75 years [97]. Urinalysis is It is characterized by renal cysts that occur at the bland, with mild or absent proteinuria, and there may be noctu- corticomedullary junction. Kidneys are usually normal or re- ria or enuresis in children as concentrating ability is lost. duced in size, though in the infantile form they may show a Histological features are equally non-specific. A kidney biopsy moderate increase in size [80–82]. Renal histology shows a may show interstitial scarring, but has no distinctive character- characteristic triad of tubulointerstitial nephropathy, tubular istic features [98]. Usual histological findings include interstitial basement membrane disruption and corticomedullary cysts fibrosis, tubular atrophy, microcyst formation and thickening [83]. Nephronophthisis may be isolated to the kidneys, how- and lamellation of the tubular basement membranes. There will ever, it is often a component part of syndromes with other be no evidence of glomerular disease and negative IF for immu- extrarenal features. Nearly 100 genes have been associated with noglobins and complement [95, 97]. nephronophthisis-related ciliopathies to date and as our knowl- Five genes are known to cause ADTKD: UMOD (ADTKD- edge of the genetic basis of nephronophthisis-related ciliopa- UMOD), MUC1 (ADTKD-MUC1), REN (ADTKD-REN), thies expands, it becomes clear that there is significant HNF1B (ADTKD-HNF1B) and, more rarely, SEC61A1 phenotypic overlap [84, 85]. Many multisystem ciliopathies (ADTKD-SEC61A1)[99–102]. ADTKD-UMOD, HNF1B, REN Downloaded from https://academic.oup.com/ndt/article/35/7/1113/5890560 by guest on 11 August 2020 may be caused by defects in multiple proteins with roles in cili- and SEC61A1 may have extrarenal features, while ADTKD- ary structure, function and biogenesis [86]. Some heterozygous MUC1 does not (Table 3). Another category, ADT not other- disorders such as Joubert syndrome have >30 known causative wise specified (ADT-NOS) is used to refer to those who have genes [87]. Our understanding of these disorders and the over- not undergone genetic testing or in whom testing is lap between them continues to shift rapidly. Some of the most inconclusive. common ciliopathies associated with renal disease are summa- Since renal biopsy will not provide a precise diagnosis in rized in Table 2. However, it is advised that all patients who pre- ADTKD, genetic testing is a viable alternative, particularly as sent with nephronophthisis should undergo screening for the autosomal dominant pattern means that multiple family ophthalmological and hepatic abnormalities at a minimum. members may be affected, and the variable age of onset and Autosomal recessive PKD (ARPKD) is caused by variants in bland radiological and urinary sediment findings mean it may PKHD1, which encodes the transmembrane protein fibrocystin be difficult to distinguish the affected from the unaffected [88]. Less common and more severe than ADPKD, it tends to through clinical screening alone. Histology alone will not distin- present in the first decade of life and is often detected prenatally guish the various pathologies. Commercial genetic testing is [89, 90]. Up to 30% of patients may present with oligohydram- available to look for pathogenic variants in UMOD, REN and nios, Potter’s sequence and pulmonary hypoplasia, leading to a HNF1B. However, MUC1 is an extraordinarily difficult gene to significant risk of neonatal death [91, 92]. sequence and cannot currently be sequenced through conven- Although not a true ciliopathy, disease-causing variants in tional means, therefore testing is conducted in a very small se- FAN1 may cause a rare autosomal recessive form of tubuloin- lection of centres worldwide. terstitial kidney disease called karyomegalic interstitial nephritis ADTKD-MUC1 is most commonly caused by a frameshift (KIN) [18, 93]. This is characterized by slowly progressive renal insertion in MUC1 that leads to the formation of a mutant pro- failure, progressing to ESRD in the fifth decade of life, bronchi- tein, MUC1-fs [100]. The cause of this frameshift is the inser- ectasis and deranged liver function tests. Karyomegaly describes tion of a single cytosine (c) into a sequence of seven c base pairs the presence of an enlarged cell nucleus, and in KIN the charac- in a variable nucleotide tandem repeat (VNTR) in the MUC1 teristic renal pathology is tubulointerstitial fibrosis and atrophy gene. A VNTR is a short sequence of nucleotides organized into and the presence of enlarged, hyperchromatic nuclei in the tu- a tandem repeat. In MUC1, the VNTR codes for a serine-, pro- bular epithelial cells. Karyomegaly can also be seen in other line- and threonine-rich sequence of 20 amino acids that make organs, including the brain and prostate [94]. up the extracellular part of the Mucin-1 protein. The VNTR is highly polymorphic and because of the high number of cytosine and guanine residues combined with the architecture of the ADTKD VNTR, direct sequencing is largely ineffective [103]. For these ADTKD can prove a diagnostic challenge for physicians. It is reasons, MUC1 is refractory to many NGS methods and a novel notable for an autosomal dominant inheritance pattern, but is mass spectrometer–based method has instead been developed otherwise characterized by a lack of distinctive clinical or histo- to detect the Cþ insertion in the VNTR [104]. More recently, logical features, particularly in the case of ADTKD-MUC1. single-model real-time sequencing has been used to Previously known by a number of names, but particularly by assemble the VNTR in affected and non-affected families and medullary cystic kidney disease, a 2015 Kidney Disease: identify the exact positions of causative changes, providing the Improving Global Outcomes consensus report recommended structural and positional information that cannot be captured the change to ADTKD, as cysts are not pathognomonic of the by this mass spectrometry method [103]. No truncating or mis- disease nor are they confined to the medulla when they do ap- sense variants have been found that cause ADTKD-MUC1. pear [95, 96]. The pathogenesis of ADTKD-MUC1 is thought to be due to ADTKD is characterized by nonspecific renal features the accumulation of MUC1-fs protein within the cell. However, (Figure 1A–C). It features progressive loss of kidney function, recent evidence suggests that the accumulation of MUC1-fs

1118 S.L. Murray et al. nertn itptooyadgntc nrnlmedicine renal in genetics and histopathology Integrating Table 2. Summary of ciliopathies associated with renal disease

Syndrome Inheritance Gene Renal features Extrarenal features Renal histology ADPKD Autosomal PKD1, PKD2, GANAB and DNAJB11 Polycystic kidneys, progression to Polycystic liver and pancreas, in- Saccular expansions or diver- dominant ESKD in third to fifth decade of creased risk of intracerebral ticula of all portions of renal life aneurysm tubule, secondary glomerulosclerosis ARPKD Autosomal PKHD1 and DZIP1L Polycystic liver, progressive CKD Polycystic liver, liver fibrosis, Elongated, radially arranged recessive in the first decade of life cysts formed from the collecting tubules Von Hippel–Lindau syndrome Autosomal VHL Clear cell carcinoma of the Haemangioblastomas in the spine, Benign and malignant cystic dominant kidney brain, pancreas and retina, pheo- lesions, clear cell carcinoma chromocytoma, pancreatic neuro- endocrine tumours Nephronophthisis Autosomal NPHP1-NPHP6, TMEM67, ANKS6, Polydipsia, polyuria, failure of None Cysts at the corticomedullary recessive TTC21B, GLIS2, CEP164, INVS, MAPKB1, concentrating ability, tubuloin- junction, severe tubular at- NEK8, CEP83, WDR19, ZNF423 and terstitial fibrosis and ESRD by rophy, interstitial fibrosis others second to third decade of life and chronic inflammation Senior–Løken syndrome Autosomal NPHP1, NPHP4, CEP290, IQCB1, SDCCAG8 Polydipsia, polyuria, failure of Retinal degeneration, retinitis pig- Cysts at the corticomedullary recessive and WDR19 concentrating ability, tubuloin- mentosa and leber congenital junction, severe tubular at- terstitial fibrosis and ESRD by amaurosis rophy, interstitial fibrosis second to third decade of life and chronic inflammation Joubert syndrome Autosomal INPP5E, TMEM216, CC2D2A, AHI1, CEP41, Polyuria, failure of concentrating Nephronophthisis, ataxia, hypotonia, Cystic dysplasia or cysts at the recessive RPGRIP1L and others ability, tubulointerstitial fibrosis learning difficulties and retinal corticomedullary junction, and ESRD by second to third dystrophy severe tubular atrophy, in- decade of life or multicystic terstitial fibrosis and chronic kidneys in first year of life inflammation Bardet–Biedl syndrome Autosomal BBS1-BBS12, NPHP6, CEP290, MKS1, Structural and urinary tract ab- Obesity, learning difficulties, retinitis Cystic lobulation of the kidney recessive SDCCAG8 and SEPT7 normalities, tubulointerstitial pigmentosa, post-axial polydactyly, disease behavioural difficulties, hypogo- nadism and nephronophthisis Meckel syndrome Autosomal B9D1, B9D2, CC2D2A, CEP290, MKS1, Enlarged cyst filled kidneys Occipital encephalocele, polydactyly Little corticomedullary differ- recessive RPGRIP1L, TMEM67 and TMEM216 and perinatal death entiation and deficient nephrons Alstro¨m syndrome Autosomal ALMS1 Progressive CKD in third decade Sensorineural hearing loss, nystag- Mixed tubulointerstitial and recessive of life, nephrocalcinosis mus, dilated cardiomyopathy, glomerular disease asthma, insulin-dependent diabetes hypercholesterolaemia and obesity short stature RHYNS syndrome Autosomal TMEM67 Polyuria, failure of concentrating Gaze palsy, sensorineural hearing Cysts at the corticomedullary recessive ability, tubulointerstitial fibro- loss, retinitis pigmentosa and skele- junction, severe tubular at- sis, ESKD in adolescence tal dysplasia rophy and interstitial fibrosis Oro-facial-digital syndrome X-linked, OFD1 and CPLANE1 Polycystic kidneys, ESRD in third Facial asymmetry, alae nasi hypopla- Cysts of the glomeruli and dis- Autosomal to fifth decade of life sia, tongue abnormalities and cleft tal tubules recessive lip

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FIGURE 1: Renal tissue under LM. (A) Interstitial inflammatory infiltrate and tubulitis on haematoxylin and eosin (H&E) stain in a patient with ADTKD-HNF1B.(B) Interstitial inflammatory infiltrate and tubulitis on H&E stain in a patient with ADTKD-UMOD. (C) Interstitial inflammatory infiltrate and tubulitis on H&E stain in a patient with ADTKD-unknown. (D) Global and segmental glomerulosclerosis on perisodic acid–schiff stain in a patient with FSGS due to autosomal dominant INF2 mutation. (E) Glomerulosclerosis on silver stain in a patient with a COL4A4 mutation. (F) Segmental glomerulosclerosis on H&E stain in a patient with primary FSGS, genetics unknown. (G) Capillary loop thickening and double contour formation on silver stain in a patient with hereditary C3 nephropathy. (H) Capillary loop thickening and double contour formation on silver stain in a patient with hereditary C3 nephropathy. protein alone is not immediately toxic and it is only when individuals, regardless of the stage of kidney disease. Staining the unfolded protein response pathway—a homoeostatic was shown to be as effective in those with normal renal mechanism that guards against the accumulation of unfolded function, advanced CKD and those on dialysis. In 173 patients proteins—becomes inactivated that it becomes harmful to tested for MUC1-fs, sensitivity was 94% and specificity was 89%. the cell [105]. This may help explain the variable age of onset. Seventeen families were identified who had MUC1-fs protein Because of the unique structure of MUC1-fs, it is possible to present in urinary cells. Variants other than the Cþ insertion into develop antibodies that detect MUC1-fs but not wild-type the MUC1 VNTR were identified. All encoded the same MUC1-fs MUC1. This has allowed for successful immunohistochemi- protein. While still only available on a research basis, urinary cal staining of the MUC1-fs protein on epithelial tissues and staining may become a useful non-invasive test. This may be of in urinary smears [106]. Zivna ´ et al. [101]wereabletoshow increasing importance as treatments become available for the presence of MUC1-fs protein in 11 of 12 renal biopsies of ADTKD-MUC1. Currently there is no effective treatment for the patients with ADTKD-MUC1 (Figure 2). Immunostaining of condition, but Dvela-Levitt et al. [105] recently reported promising the kidney was 92% sensitive and 81% specific. Skin biopsy results in animal studies and organoids with the small molecule was offered as a less invasive alternative, but MUC1-fs was transmebrane P24 trafficking protein 9, which has been shown to only present in the sebaceous glands, which are not consis- promote lysosomal degradation of the toxic MUC1-fs from tently obtained on skin biopsy. However, staining for MUC1- cells and reverse proteinopathy. The same therapy may have fs on urinary smears was shown to be very effective. There potential treatment implications for ADTKD-UMOD and other was strong intracellular staining in urothelial cells in affected proteinopathies.

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FIGURE 2: Immunohistochemical staining of renal biopsy tissue samples showing positive MUC1fs staining in affected individuals with ADTKD-MUC1 and negative MUC1fs staining in controls. (A) Kidney section from a negative control at 10 magnification. (B) Negative control at 40 magnification. (C) Kidney section from an individual affected by ADTKD-MUC1 at 10 magnification, showing positive staining of the distal tubules and collecting ducts at 20 magnification. (D) Kidney section from an individual affected by ADTKD-MUC1 at 20 magnification, showing positive staining of the distal tubules and collecting ducts at 20 magnification.

Alport syndrome be rare, is now believed to be more common than previously rec- Alport syndrome is a disorder of type IV collagen production. ognized [111]. Alport syndrome may also be inherited in a digenic Type IV collagen exists primarily in the basal lamina and is the fashion and patients with disease-causing variants in both COL4A5 and COL4A4 have been identified [112]. major collagenous component of the basement membranes of The earliest ultrastructural lesion seen in Alport syndrome is the cochlea of the ear, the glomerulus of the kidney and the cor- thinning of the GBM seen on EM [113]. In established Alport nea, lens and retina of the eye [107]. Alport syndrome is classi- syndrome, changes on LM tend to be non-specific, with glo- cally characterized by a triad of renal dysfunction, variable merulosclerosis, interstitial fibrosis and interstitial foam cells sensorineural deafness and ocular abnormalities, including an- [114]. Differences in collagen immunostaining can be detected terior dislocation of the lens, corneal opacities and temporal ret- between X-linked and recessive Alport syndrome. In X-linked inal thinning [108–110]. While an individual must have Alport syndrome there is often an absence of a5 type IV colla- persistent haematuria to meet the diagnostic criteria for Alport gen in males and patchy or reduced a5typeIVcollagenin syndrome, deafness and ocular abnormalities are not consis- females. Reduced levels of a5 type IV collagen may still be visi- tently found in patients with Alport syndrome and are no lon- ble in males with features of Alport syndrome and missense ger considered necessary for diagnosis in the presence of variants in COL4A5 [115]. In autosomal recessive Alport syn- evidence of disease-causing variants in the type IV collagen drome, a5 type IV collagen is visible in Bowman’s capsule and genes or characteristic abnormalities of the glomerular base- the distal tubules, but not the GBM. ment membrane (GBM) [109]. X-linked Alport syndrome tends to affect males more se- Alport syndrome occurs due to variants in COL4A3, COL4A4 verely than females. Males tend to have progressive CKD and and COL4A5, all of which affect the expression of the a chains of generally progress to ESRD between the second and third deca- type IV collagen, a3, a4anda5[110]. Approximately 85% of all des of life. Until recently, X-linked Alport syndrome in women Alport syndrome cases affect the gene COL4A5 and are inherited was considered a benign status and women were considered in an X-linked fashion, while 15% affect COL4A3 and COL4A4 only carriers. However, if a carrier is an individual or organism and are inherited in an autosomal recessive fashion. Autosomal who carriers a single copy or a recessive allele but does not dis- dominant inheritance of Alport syndrome, previously thought to play the trait, then only 5% of women who are heterozygous for

Integrating histopathology and genetics in renal medicine 1121 X-linked Alport syndrome can be said to be true carriers. The other 95% are affected by Alport syndrome. A study of 195 families with Alport syndrome and a proven COL4A5 disease- causing variant showed that 75% of women developed protein-

SEC61A1 uria, 28% developed hearing loss and 15% developed ocular

lesions defects. While the phenotype is usually milder than in males, out cysts 12% of women developed ESRD and 105 women developed None identiﬁed ADTKD- mental retardation deafness by the age of 40 years [116]. For this reason, in 2017, Progressive renal disease, tardation, polydactyly, mild ine and postnatal growth re- the Alport Syndrome Classification Working Group dispensed Small foci of tubulointerstitial small dysplastic kidneys with- Congenital anaemia, intrauter- with the term ‘carrier’. X-linked Alport syndrome should now be considered a dominant disease, one in which women have a lower, but not trivial, risk of progressing to serious kidney disease [109]. Even within families, the severity of presentation may vary among women. This is likely due to inactivation of HNF1B the X-chromosome in affected females [117, 118]. This may Downloaded from https://academic.oup.com/ndt/article/35/7/1113/5890560 by guest on 11 August 2020 members atrophy CAKUT lead to a mosaic pattern seen on the GBM [119]. hypokalaemia ADTKD- Pancreatic atrophy

Hypomagnesaemia, Autosomal recessive Alport syndrome affects both sexes bland urinary sediment

tests, genital malformation, equally and causes deafness and ESRD at a young age. Progressive renal disease with Non-speciﬁc tubulointerstitial Diabetes, deranged liver function Variable penetrance mong family Heterozygous individuals tend to have milder renal involve- ment. However, they have a higher lifetime risk of ESRD than the general population and a retrospective study suggests they benefit from renin–angiotensin–aldosterone system blockade [120]. It may be that the clinical significance of being a ‘carrier’ REN of a COL4A3, COL4A4 or COL4A5 disease-causing variant has been grossly underestimated. atrophy

ADTKD- Increasingly, it is recognized that Alport syndrome repre- Hyporeninaemia Mild hypotension

bland urinary sediment, sents a broader spectrum of disease than previously thought. cretion of uromodulin

Prone to acute kidney injury There is increasing recognition that variants in COL4A3, Progressive renal disease with Non-speciﬁc tubulointerstitial Hyperkalaemia,low urinary ex- COL4A4 and COL4A5 are a cause of FSGS in adults and children and may be among the most common causes of hereditary FSGS in children [121–123]. Currently treatment for Alport syndrome is limited to renin–angiotensin blockade, although bardoxalone is now in UMOD phase 3 trials as a potential treatment for Alport syndrome,

atrophy and some promise has also been seen in treatments with uromodulin microRNA-21 [124, 125]. ADTKD- bland urinary sediment low urinary excretion of Progressive renal disease with Non-speciﬁc tubulointerstitial THIN BASEMENT MEMBRANE NEPHROPATHY (TBMN) TBMN is characterized by the uniform thinning of the basement membrane seen on EM. A normal basement membrane is 350 nm, while a thin basement membrane is <250 nm MUC1 [126, 127].

atrophy TBMN presents with normal renal function, dysmorphic haematuria and <500 mg/day of proteinuria [128, 129]. The

bland urinary sediment most common presentation is that of microscopic haematuria, and proteinuria is not usually present. Because of this, the incidence of TBMN has not been described accurately, but some studies have suggested incidences as high as 1% of the population based on the percentage of children and adults who present with haematuria and no urological cause [130]. It tends to occur more commonly in women and this is thought to be because the GBM in men is naturally thicker [131]. At least two- thirds of those affected will have another affected relative [129]. Histopathology Non-specific tublointerstitial GoutOther electrolyte abnormalitiesOther clinical features None identified Low fractional excretion of urate, In advanced CKD None Early onset, frequent None Early onset, frequent Early onset, frequent Anaemia, In second decade of life InheritanceAge of onset of CKD (years)Onset in childhoodRenal features 18–50 Autosomal dominant Progressive renal disease with No Autosomal dominant 18–50 Autosomal dominant Gout rarely Autosomal dominant First year of life Autosomal dominant Frequent Variable, may be antenatal May have prenatal findings 10–50 Variable Condition ADTKD- At least 40% of patients with TBMN have a heterozygous

Table 3. Characteristics of the four major causes of ADTKD disease-causing variant in the COL4A4 or COL4A3 gene,

1122 S.L. Murray et al. which in homozygous patients or compound heterozygotes the genes associated with FSGS makes WES the preferred causes autosomal recessive Alport syndrome [119]. Recent choice for those with FSGS. studies suggest that variants in other collagen genes, such as Inheritance is variable and can be autosomal recessive, auto- COL4A1, may also be implicated in TBMN [132]. somal dominant or X-linked. The genes effected often produce There is ongoing debate as to whether TBMN is a term that proteins that localize to the podocyte and its slit diaphragm, should be discarded completely in favour of autospmal domi- such as nephrin (NPHS1), podocin (NPHS2), inverted formin 2 nant Alport syndrome in patients with an identified COL4A3, (INF2), a-actinin-4 (ACTN4), CD2-associated protein COL4A4 or COL4A5 variant and ‘recurrent and persistent hae- (CD2AP) and laminin-beta2 (LAMB2)[147–151]. maturia with other morphologic changes’ in those with evi- The syndromic forms of inherited FSGS tend to encode pro- dence of a thin GBM on biopsy and negative or no genetic teins that are expressed not only in the podocyte, but in other studies [109]. Proponents advise that this will help to avoid the tissue types. They include WT1, LAMB2, ITGB4 and LMX1B erroneous assumption that TBMN is an entirely benign condi- [152]. Major syndromes associated with hereditary FSGS in- tion, while opponents believe it will create unwarranted fear, clude Denys–Drash syndrome (WT1), nail–patella syndrome when only a small proportion progress to ESRD [133]. It is (LXM1B) and Pierson syndrome (LAMB2). Certain variants in probably advisable that all those with TBMN have lifelong genes thought to be syndromic may also cause only isolated re- Downloaded from https://academic.oup.com/ndt/article/35/7/1113/5890560 by guest on 11 August 2020 yearly assessment. nal disease—or perhaps cause extrarenal features so subtle that the clinical syndrome is not detectable—such as has been seen in patients with LXM1B variants that cause autosomal domi- FSGS nant FSGS without extrarenal manifestations [153]. Genetic FSGS has been thought of until recently as a single disease pro- variants may also cause an increased susceptibility to FSGS but cess. However, more accurately it should be considered as a his- may not present unless a second, environmental ‘hit’ occurs, as tological description that describes the underlying disease is the case with APOL1 risk variants. Determining the signifi- pathology of multiple genetically and clinically distinct condi- cance of detecting rare variants can also be challenging. A re- tions. There have been recent efforts to stratify FSGS and ne- cent study reported a 5.5% rate of rare, disease-causing variants phrotic syndrome into hereditary, immune-based and associated with FSGS in an asymptomatic control population circulating factor–based disease [134, 135]. FSGS is character- [154]. ized by hyalinosis, mesangial sclerosis, capillary destruction, Identification of the cause of FSGS may have a significant foam cells and adhesions between the glomerular tuft and impact on diagnosis and management. The majority of mono- Bowman’s capsule (Figure 1E–F)[136]. The underlying cause genic forms of FSGS do not respond to corticosteroids and have of this histological appearance is often not clinically apparent a very low risk of recurrence. Identifying an underlying genetic from histology alone and no non-genetic biomarker currently cause may avoid unnecessary exposure to toxic steroid- exists that can differentiate the forms of FSGS. Therefore mo- containing treatment regimens. There is also increased interest lecular diagnosis is likely key to understanding the underlying in therapies including ciclosporin, coenzyme Q10 and vitamin pathogenesis and treatment. B12 to treat certain forms of hereditary FSGS [155, 156]. While The prevalence of FSGS varies widely with the population previously genetic testing was only recommended for the paedi- and is thought to be responsible for 5–20% of ESRD [137, 138]. atric population, it has now been suggested that genetic testing It was previously thought that genetic testing was only of use in be considered in any case where the cause of FSGS cannot be a paediatric population, as the likelihood of identifying a patho- classified by clinicopathological assessment [156]. genic variant correlated inversely with age. Disease-causing variants are identified in 60–100% of those diagnosed during the first year of life, 40–60% of young children, 25–40% of older children and 10–25% of adolescents [139–142]. Hereditary APOL1 forms were long thought to account for only 1% of FSGS in APOL1 is a gene that codes for apolipoprotein L1. Variants adults [143]. However, recent literature has challenged this, in these genes have been identified as initiating factors in the in- with Yao et al. [144] achieving a genetic diagnosis rate of 11% creased risk for hypertension and kidney disease found in those in a recent study of 193 individuals with biopsy-proven FSGS. of sub-Saharan African ancestry. The discovery of APOL1 has Some of these variants will present with steroid-resistant ne- been instrumental in understanding the 4-fold risk of CKD in phrotic syndrome (SRNS), while others will have significantly the population with African ancestry [157]. less proteinuria. Of the pathogenic variants detected, 55% were There are two known risk alleles in APOL1, G1 and G2, both in COL4A3, COL4A4 and COL4A5, 40% were in podocyte of which are only known to be present in those of African an- genes and 5% were in CAKUT genes. More than 40 genes have cestry [158]. Much as the sickle cell trait is protective of malaria, now been identified that present with both syndromic and non- asinglecopyofanAPOL1 risk allele is thought to be protective syndromic forms of hereditary FSGS (Table 4)andnewFSGS- of African sleeping sickness, a parasitic infection transmitted by causing genes continue to be discovered [145, 146]. Disease- Trypanosoma brucei rhodesiense that is endemic in sub- causing variants in COL4A3, COL4A4 and COL4A5 are more Saharan Africa [159]. However, the presence of two risk alleles classically associated with Alport syndrome but are now (G1/G1, G1/G2 or G2/G2) leads to an increased risk of FSGS thought to be one of the most common causes of hereditary and of human immunodeficiency virus–associated nephropa- FSGS in adults [119, 145]. Our rapidly expanding knowledge of thy (HIVAN).

Integrating histopathology and genetics in renal medicine 1123 1124 Table 4. Genes causing syndromic and non-syndrome FSGS

Gene Protein Inheritance Syndrome Renal phenotype Non-syndromic forms of FSGS Cell signalling PLCE1 1-phosphatidylinositol-4,5-bisphosphate phos- AR No Steroid-resistant nephrotic syndrome phodiesterase epsilon-1 KANK2 KN motif and ankyrin repeat domain-containing AR No Early onset, steroid-resistant nephrotic syn- protein 2 drome, haematuria KANK4 KN motif and ankyrin repeat domains 4 AD No VUS, may contribute to FSGS TRPC6 Transient receptor potential cation channel, sub- AD No FSGS family C, member 6 Slit diaphragm-associated proteins NPHS1 Nephrin AR No Congenital nephrotic syndrome or FSGS NPHS2 Podocin AR No Early-onset FSGS CD2AP CD2-associated protein AD/AR No Early-onset FSGS MYO1E Myosin 1E AR No Early-onset FSGS MAGI2 Membrane-associated guanylate kinase, WW AR No Steroid-resistant congenital nephrotic syndrome and PDZ domain-containing 2 Nuclear pore complex proteins XPO5 Exportin 5 AR No Childhood-onset steroid nephrotic syndrome NUP85 Nucleoporin 85 kDa AR No Childhood-onset FSGS NUP93 Nucleoporin 93 kDa AR No Childhood-onset FSGS NUP205 Nucleoporin 205 kDa AR Childhood-onset FSGS NUP160 Nucleoporin 160 kDa AR No Steroid-resistant nephrotic syndrome in the second decade Cell membrane-associated proteins PTPRO Protein tyrosine phosphatase receptor-type, O AR No Steroid resistant nephrotic syndrome in childhood MP2 Epithelial membrane protein 2 AR No Childhood-onset steroid-resistant nephrotic syndrome Mitochondrial function COQ8B Coenzyme Q8B AR No Steroid-resistant nephrotic syndrome Cilia TTC21B Tetratricopeptide repeat domain-containing pro- AR No Nephronophthisis, adolescent-onset FSGS with tein 21B tubulointerstitial lesions AVIL Advillin AR No Childhood-onset FSGS Cytoskeleton, cell polarity adhesion INF2 Inverted foramin 2 AD No FSGS, other, severe histological appearances ACTN4 Actinin-a-4 AD No Congenital steroid-resistant nephrotic syndrome ARHGAP24 Rho GTPase activating protein 24 AD No Adolescent-onset FSGS

..Murray S.L. ANLN Actin binding protein anillin AD No FSGS with variable age of onset CRB2 Crumbs cell polarity complex component 2 AR No Childhood-onset FSGS, congenital steroid-resistant nephrotic syndrome ARHGDIA Rho GDP dissociation inhibitor a AR No Congenital or early-onset steroid-resistant nephrotic syndrome

tal. et FAT1 Fat atypical cadherin AR No VUS, may contribute to steroid-resistant ne-

phrotic syndrome Downloaded from https://academic.oup.com/ndt/article/35/7/1113/5890560 by guest on 11 August 2020 August 11 on guest by https://academic.oup.com/ndt/article/35/7/1113/5890560 from Downloaded nertn itptooyadgntc nrnlmedicine renal in genetics and histopathology Integrating

DNA repair, transcription NXF5 Nuclear RNA export factor 5 X-linked No Adult-onset nephrotic syndrome Cell vesicles TBC1D8B TBC1 domain family protein 8 X-linked No Early-onset steroid-resistant nephrotic syndrome Syndromic forms of FSGS Lysosome SCARB2 Lysosomal integral membrane protein 2 AR Action myoclonus-renal failure syndrome FSGS (ataxia, myoclonus, collapsing FSGS) DNA repair, transcription WT1 Wilm’s tumour 1 AD Denys–Drash syndrome (Wilms’ tumour, male Childhood-onset FSGS pseudohermaphroditism, FSGS), Frasier syndrome (FSGS, gonadoblastoma, male pseudohermaphroditism isolated congenital or childhood-onset diffuse mesangial sclerosis) PAX2 Paired box 2 AD Renal coloboma syndrome (renal hypoplasia, Adult-onset FSGS childhood-onset FSGS, optic nerve colobomas) EYA1 Eyes absent homolog 1 AD Branchio-oto-renal syndrome (abnormalities in Adult-onset FSGS branchial, ear and renal development) LMX1B LIM homoeobox transcription factor 1b AD Nail–Patella syndrome (hypoplastic or absent FSGS patella, dysplasia of elbows, short stature dys- trophic nails, frequently glaucoma) SMARCAL1 SMARCA-like protein 1 AR Schimke immuno-osseous dysplasia (immuno- Childhood-onset FSGS deﬁciency, skeletal dysplasia, childhood-onset FSGS) LMNA Lamin A AD Familial partial lipodystrophy with adult-onset Adult-onset FSGS FSGS, other syndromes WDR73 WD repeat-containing protein 73 AR Galloway–Mowat syndrome (microcephaly joint Childhood-onset steroid-resistant nephrotic contractures and developmental delay) syndrome Cell matrix COL4A3 a3 Type 4 collagen AR, AD ATS (deafness, renal failure, ocular FSGS, ATS abnormalities) COL4A4 a4 Type 4 collagen AR, AD ATS (deafness, renal failure, ocular FSGS, ATS abnormalities) COL4A5 a5 Type 4 collagen X-linked ATS (deafness, renal failure, ocular FSGS, ATS abnormalities) ITGB4 Integrin-b4 AR Epidermolysis bullosa Steroid-resistant nephrotic syndrome LAMB2 Laminin-b2 AR Pierson syndrome (microcoria, neuromuscular Childhood-onset steroid-resistant nephrotic junction defects, early-onset FSGS or diffuse syndrome mesangial sclerosis) Cytoskeleton, cell polarity adhesion MYH9 Myosin heavy chain 9 AD Epstein–Fechtner syndrome (FSGS, cataracts, Childhood-onset FSGS sensorineural deafness macrothrombocytopae- nia leucocyte inclusions) Mitochondrial function MT-TL1 mitochondrial tRNA for leucine 1 Mitochondrial mitochondrial encephalomyopathy, lactic acido- Secondary FSGS sis, stroke-like episodes (MELAS) MT-TL2 Mitochondrially encoded tRNA leucine 2 Mitochondrial MELAS Secondary FSGS

1125 MT-TY Mitochondrially encoded tRNA tyrosine Mitochondrial Ophthalmoplaegia, dilated cardiomyopathy, Secondary FSGS FSGS

Continued Downloaded from https://academic.oup.com/ndt/article/35/7/1113/5890560 by guest on 11 August 2020 August 11 on guest by https://academic.oup.com/ndt/article/35/7/1113/5890560 from Downloaded APOL1 nephropathy may present with a number of histopathological presentations, including tubulointerstitial injury and arteriosclerosis, FSGS, HIVAN, lupus nephritis, solidified glomerulosclerosis and sickle cell nephritis [158, 160, 161]. Individuals are often labelled as having hypertensive or non- specific forms of CKD, leading to disease misclassification [162]. In a study of 196 non-diabetic, non-nephrotic, African American patients with progressive CKD, characterized by chronic tubulointerstitial injury and arteriosclerosis, those with Childhood-onset FSGS Childhood-onset FSGS Childhood-onset FSGS Childhood-onset FSGS two APOL1 risk variants tended to be younger and display a

Childhood and adult-onset FSGS greater degree of interstitial fibrosis and microcystic tubular di- Steroid resistant nephrotic syndrome Steroid-resistant nephrotic syndrome latation. They displayed solidified fibrosis and thyroidization- type tubular atrophy [160]. In a study of 138 children and young adults with FSGS, those with two APOL1 risk variants Downloaded from https://academic.oup.com/ndt/article/35/7/1113/5890560 by guest on 11 August 2020 had more segmental glomerulosclerosis, more total glomerulosclerosis and more tubular atrophy and interstitial fibrosis. They were more likely to develop the collapsing variant of FSGS [163]. The lifetime risk of FSGS in patients of African ancestry is thought to be 0.2, 0.3 and 4.25% in those with none, one or two APOL1 risk alleles, respectively [157]. In those who developed FSGS, progression to ESRD was also faster, with a hazard ratio phrotic syndrome ¨sbeck syndrome (childhood-on- zyme Q10 deﬁciency zyme Q10 deﬁciency of 2.3. Those who develop APOL1-related FSGS seem to have similar sensitivity to corticosteroids as the general population, ciency and neurological defects anaemia, peripheral neuropathy) with 30% responding to steroids [157]. lay, microcephaly and early onset FSGS) lay, microcephaly and early onset FSGS)

set nephrotic syndrome, and megaloblastic There is little prospective data on the outcomes of recipients or donors with two APOL1 risk alleles. Survival of deceased do- nor renal allografts with two APOL1 risk alleles was significantly shorter than those without [164]. Transplantation of a kidney from a healthy living donor with two APOL1 risk alleles has been associated with an increased rate of decline in GFR, with ESRD occurring 10 years post-donation, however, further research into this area is required [165].

FABRY DISEASE Fabry disease is an X-linked, multisystem disorder. A disorder of glycosphingolipid metabolism, it is caused by disease- causing variants in the GLA gene on chromosome X [166]. This leads to a deficiency in lysosomal a-galactosidase A, which causes an accumulation of globotriaosylceramide (GL-3) within cell lysosomes [167]. Accumulation of GL-3 affects the heart, causing arrhythmia Cubilin AR Imerslund–Gra and cardiomyopathy; the peripheral nervous system, causing pain and burning; the skin, causing angiokeratomas; and the Nucleoporin 107 kDa AR Galloway–Mowat syndrome (developmental de- Nucleoporin 133 kDa AR Galloway–Mowat syndrome (developmental de- Podocalyxin-like protein ARkidney, Microcoria, omphalocele, steroid-resistant ne- causing proteinuria and nephropathy [168]. It affects Coenzyme Q6 monooxygenase AR Sensorineural deafness, childhood FSGS, coen- Sphingosine-1-phosphate lyase-1 AR Adrenal insufﬁciency, ichthyosis, immunodeﬁ- males more severely, but may also cause significant symptoms Coenzyme Q2 polyprenyltransferase AR Sensorineural deafness, childhood FSGS, coen- in heterozygous females. It is often not detected before adulthood. Nephropathy in Fabry disease is because of the accumulation of GL-3 in the podocytes, which leads to their destruction [169]. Podocytes have a limited ability to regenerate, leading to segmental or global glomerulosclerosis and to CKD [170]. The characteristic pathological features are myelin-like inclusions in the podocyte cytoplasm that are seen on EM [171]. These inclu- protein NUP107 PODXL NUP133 COQ2 SGPL1 CUBN COQ6 Nuclear pore complex Gene Protein Inheritance Syndrome Renal phenotype Cell membrane sions may also be seen less prominently in glomerular endothe-

Table 4. Continued lial cells, tubular epithelial cells and mesangial cells [172, 173].

1126 S.L. Murray et al. LM shows vacuolization of the podocyte cytoplasm and glo- genetic testing that is peculiar to renal disease, and this lies in merular sclerosis [171]. IF is unremarkable, although Fabry dis- the field of renal transplantation. ease has been reported in conjunction with IgAN and pauci- Allogenic renal transplantation remains the gold standard immune glomerulonephritis [174, 175]. for management of ESRD, conveying increased survival and an An enzymatic blood test of a-galactosidase A activity can be improved quality of life compared with dialysis as well as im- used to confirm the diagnosis and genomic testing can be used proved cost-effectiveness [188–191]. Long-term mortality in to identify the culprit variant [176]. Enzyme replacement ther- patients who have undergone transplant is 50% lower than in apy with recombinant galactosidase can stabilize disease, with those still on dialysis. enzyme therapy ameliorating the decrease in GFR but not en- Organs are a vital and scarce resource and living donor tirely halting it [177]. Even with enzyme therapy, nephropathy transplants are an essential part of a successful renal transplant often progresses over time to ESRD. programme. They offer a significant number of benefits to the IgAN transplant recipient. Individuals who receive a living transplant IgAN is the most common form of glomerulonephritis in the enjoy a lower risk of rejection, better allograft function and lon- world [178]. It is characterized by haematuria, proteinuria and ger life than those who receive a deceased donor transplant considerable heterogenicity in the progression of CKD. Its his- [192]. They enjoy less fatigue and better quality of life [193]. Downloaded from https://academic.oup.com/ndt/article/35/7/1113/5890560 by guest on 11 August 2020 tological appearance is characterized by a variable appearance They spend less time on dialysis than those awaiting deceased on LM and IgA-dominant deposits on IF [179]. donor transplants and avoid the attendant risks [194]. The causes of IgAN are poorly defined and it is generally not Donor nephrectomy is not without risk and potential donors considered to be hereditary. However, there have been multiple should undergo rigorous screening prior to donation to ensure reports of families presenting with biopsy-proven IgAN and a that no harm comes to the donor. The worst-case scenario is for Mendelian inheritance, although penetrance is often incom- a donor to go on to develop significant CKD or even ESRD plete [180]. There is also evidence of geographic and ethnic themselves following donation. For many years it was thought clustering, with the prevalence of IgAN being high within Asian that donation was a benign procedure, as the rate of decline in and Native American ethnic groups and low in those with GFR in donors was comparable to that of the general popula- African ancestry [181]. An Italian study examined 54 unaf- tion [195]. However, because of the intense screening they un- fected family members of patients with IgAN and found micro- dergo, donors are a rarefied patient group, and when compared scopic haematuria present in 24%. The same group found a 16- with a group of well-selected, non-donor controls, the risk of fold increased risk of IgAN in first-degree relatives of those with ESRD is 8–11 times higher [196, 197]. IgAN in a cohort of northern Italians [182]. However, even Many living donors are first or second-degree relatives of the among families who display familial clustering, there is signifi- kidney recipient. It is known that those with a first- or second- cant heterogenicity, with variable penetrance and no specific degree relative are at higher risk of developing ESRD even in pattern of inheritance [183]. the absence of known Mendelian disorders [198, 199]. The genetic locus of IgAN remains elusive, made more com- Approximately 15% of those who reach ESRD currently do plex by the heterogenicity of the disease. More than 130 candi- so without a primary renal diagnosis. If a genetic cause of dis- date gene association studies for IgAN have been conducted ease can be identified in any of these cases, it will not only give without success [180]. GWASs in European, Chinese and the patient a diagnosis, it will allow for potential testing of any Japanese cohorts have shown associations at the 6p21 human living donors and appropriate counselling as to the risks of liv- leucocyte antigen and 22q12, but a definitive risk allele or a ing donation. monogenic form of IgAN has not yet been discovered [184]. This is also true in those who may have a higher lifetime risk Most susceptibility loci that have been identified via GWAS en- of developing kidney disease due to genetic factors, for instance, code genes involved in the mucosal pathogen response, suggest- those who have two APOL1 risk alleles. Renal donors with two ing the intestinal immune network may be involved in the risk alleles are at much higher risk for progressing to ESRD pathogenesis of IgAN [185]. This supports the notion that than those without [165]. A study of African American kidney IgAN occurs due to a complex interplay of genetic and environ- donors says that 96% of donors would want transplant centres mental factors. to offer routine APOL1 testing to donors, but even in the event Other hereditary forms of kidney disease, such as Alport they had two risk alleles, 61% would still wish to donate [200]. syndrome may masquerade as hereditary IgAN [186, 187]. A survey of 383 American nephrologists and transplant surgeons report that only 4% routinely send APOL1 testing, but 87% thought it would help donors make an informed decision and 67% would begin sending the test in the next year if avail- GENETIC TESTING IN RENAL able [201]. Identification of at-risk individuals would help in TRANSPLANTATION choosing appropriate candidates and stratifying risk. Genetic testing offers many benefits to individuals opting to undergo it. It may provide reassurance, have diagnostic and therapeutic benefits and allow for genetic counselling and family planning. These benefits are valuable but not unique to patients INDICATIONS FOR GENOMIC ASSESSMENT with renal disease, and they apply across the spectrum of organ Genetic testing is now increasingly available in the clinical set- systems and diseases. However, there is one potential benefit of ting and availability is only likely to increase. Therefore it is

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