NephMadness 2015: Genetic Nephrology Region


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Genetics is a disparate collection of franchise players and rising stars. Just like in college basketball one dominant player can lift an entire team. Look at forward Frank Kaminsky from Wisconsin. While most people think he defines the prototypic “soft-shooting big man with quickness around the hoop”. Here at NephMadness headquarters we think that is a pretty apt description of epigenetics in nephrology, which squares off against the genetics of vesicoureteral reflux.
AD Tubulointerstitial Nephritis, medullary cystic disease, UMOD Nephropathy wow it has as many names as Ron Artest/Metta World Peace/Panda Friend. It faces off against arch enemy AR Tubulointerstitial Nephritis. The next matchup is the congenital form of a disease too familiar to basketball fans in its sporadic form, focal segmental glomerulosclerosis. Sean Elliott and Alonzo Mourning both had FSGS while in the NBA. APOL1 makes its second appearance in NephMadness after a strong showing in 2013 where it advanced to the elite 8 before falling to eventual tournament winner Kidney Transplant. APOL1 is going up against familial FSGS, a topic gaining strength with an explosion of basic science data. This one will be contested from tip-off to the final buzzer. Jahlil Okafor is a dominant force for Duke but like Magic Johnson and King James, he is known as much for his ability to score as his ability to find the open man and make his teammates better. This is similar to how genome wide sequencing and next-generation sequencing are merely the tools that will unlock todays secrets to discover tomorrow’s cures. #NephForward indeed.

Selection Committee member for the Genetic Nephrology Region:

O'Seaghdha, ConallConall O’Seaghdha, MB MRCPI

Dr. O’Seaghdha earned his medical degree from University College Dublin, Ireland and his nephrology fellowship training in Ireland and subsequently in Sydney, Australia. He also completed the Harvard fellowship in nephrology. During his fellowship, he worked as a clinical researcher for three years in the Framingham Heart Study where his interests were in the epidemiology of CKD in the general population, novel biomarkers of CKD, and the genetic epidemiology of kidney disease. He was also editor of the nephrology blog Renal Fellow Network during this time. After his fellowship he was an attending physician and Transplant Nephrologist in Massachusetts General Hospital and Instructor in Medicine in Harvard Medical School. He returned to Ireland in 2013 to take up his current position as Consultant Nephrologist and Transplant Physician in Beaumont Hospital, Dublin and Honorary Senior Lecturer in Medicine in the Royal College of Surgeons and in Trinity College Dublin. He is also the National Specialty Director for Nephrology higher training in Ireland.

Meet the Competitors for the Genetic Nephrology Region

AD Tubulointerstitial Nephritis
AR Tubulointerstitial Nephritis

Epigenetics in Nephrology
Vesicoureteral Reflux

Familial FSGS

GWAS in Nephrology
Next-Generation Sequencing

AD Tubulointerstitial Nephritis vs AR Tubulointerstitial Nephritis

This match-up of local rivals should be a humdinger! We have learned a lot more about the line-ups of both teams through recent genetic advances, although AD Tubulointerstitial Nephritis may be the pre-match favorite due to its star performer UMOD Nephropathy. Overall, however this appears to be an evenly matched contest and a highlight of the NephMadness first round.

AD Tubulointerstitial Nephritis

There have been a variety of names for these conditions, including medullary cystic kidney disease (MCKD), despite medullary cysts being far from universal, and familial juvenile hyperuricemic nephropathy. Modern genetic techniques have helped us hugely in characterizing these disorders and providing a molecular diagnosis in the face of nonspecific clinical data. Therefore, in the current era they are termed autosomal dominant (AD) tubulointerstitial nephritis.

AD tubulointerstitial nephritis comprises a group of familial disorders characterized by

  • Bland urinary sediment
  • Minimal hematuria
  • Minimal proteinuria
  • Progressive CKD

Histology is

  • Generally nonspecific
  • Tubulointerstitial pattern of injury
  • Variable amount of tubular atrophy and interstitial fibrosis depending on the point in the natural history of the condition that the biopsy is performed.
  1. UMOD Nephropathy

UMOD codes for uromodulin (also known as Tamm–Horsfall protein), which is expressed exclusively in the thick ascending limb of the loop of Henle and is the most common protein in normal urine.

Missense mutations in UMOD cause tubulointerstitial nephropathy with hyperuricemia, previously named MCKD type 2 or juvenile hyperuricemic nephropathy type 1. Common variants in UMOD have also been demonstrated in large genome-wide association studies (GWAS) to confer independent risk for both hypertension and kidney disease illustrating the shared risk for both phenotypes within this locus (see the GWAS in Nephrology team description for more).

The UMOD story got a lot more interesting when Trudu et al published an intriguing set of experiments establishing a link between uromodulin, hypertension, and kidney disease via activation of the renal sodium cotransporter NKCC2. UMOD risk variants identified in the above-mentioned GWAS are located in the promoter region of the gene leading to a theory that they altered UMOD expression. This was confirmed using human nephrectomy specimens and a large population cohort with urinary uromodulin levels. To model the effect in vivo, the authors used a transgenic mouse which over-expressed UMOD leading to salt-sensitive hypertension and interstitial nephritis. Moreover, they demonstrated that phosphorylated NKCC2 levels rose in tandem with UMOD gene dosage. In contrast to wild-type mice, the transgenic UMOD mice had marked improvement in blood pressure with furosemide (an inhibitor of NKCC2). Hypertensive humans with the variant showed a similar response to furosemide. We have known about the existence of uromodulin for some time but we are only beginning to understand it.

  1. MUC1 Nephropathy

This disease, previously referred to as MCKD type 1, is due to a mutation in the variable-number tandem repeat region of the MUC1 (Mucin 1) gene. The locus at chromosome 1q21 was identified by linkage mapping in 1998 but the gene has only recently been discovered due to difficulty with sequencing this highly repetitive region and was previously missed using next-generation sequencing. Mucin 1 lies on the tubular cell apical surface and has a role in signal transduction pathways. The frameshift mutation results in the formation of a truncated protein which cannot fold properly, promoting aggregation, and subsequent deposition in tubular cells. These mutations could also occur sporadically in which case the lack of a family history would make the diagnosis even more difficult. It is certain that there are individuals and families with MUC1 nephropathy who are labelled as having hypertensive (or other) nephropathy with bland urinalysis and tubulo-interstitial fibrosis on biopsy.

  1. Other Mutations

Mutations in the gene coding for renin (REN) also cause AD tubulo-interstitial kidney disease. Low renin expression has been demonstrated in renal biopsies of affected family members. It is thought that the toxic effects of the mutant protein reduce the viability of renin-expressing cells in and the juxtaglomerular apparatus, leading to nephron dropout and progressive tubulo-interstitial injury.

HNF1B encodes a transcription factor, hepatocyte nuclear factor 1β, involved in the early development of the kidney, liver, pancreas, and genital tract. Mutations in HNF1B may be sporadic or dominantly inherited and cause diabetes mellitus, pancreatic atrophy, abnormal liver function, early-onset gout, and mental retardation. Renal involvement may be evident early as cystic dysplastic kidneys, solitary kidney, or later as a tubulo-interstitial pattern of injury. The prevalence of spontaneous whole-gene HNF1B deletions may be as high as 50% in affected cases, explaining a lack of family history in many kindred. Some mutations may be incompatible with life and overall, HNF1B mutations appear to be the most frequent monogenic cause of developmental kidney disease. An excellent review of the spectrum of HNF1B nephropathy has recently been published.

With a big-name player like UMOD Nephropathy and rising stars such a MUC1 & HNF1B Nephropathy, AD Interstitial Nephritis may possess the right blend to go deep in this year’s tourney.

AR Tubulointerstitial Nephritis

Familial tubulo-interstitial nephritis may also be inherited as an autosomal recessive (AR) trait. It is usually termed nephronophthisis, a rare disorder but one of the most common causes of ESRD in pediatric populations. The incidence is estimated at 1–20 cases per 1,000,000 live births. It presents earlier than AD interstitial nephritis, occurring in the first 3 decades of life. It may also have extra-renal manifestations, the commonest being retinitis pigmentosa. There are many syndromal forms of nephronophthisis/AR tubulo-interstitial nephritis with Bardert-Biedl syndrome being perhaps the most well known and others being Jeune syndrome, Joubert syndrome, and Senior–Løken syndrome. Bardert-Biedl syndrome is characterized by retinal degeneration, obesity, learning difficulty, and a variety of other features such as polydactyly, hypogonadism, and hypercholesterolemia which show variable penetrance.

Histologically, nephronophthisis appears similar to AD interstitial nephritis with tubular atrophy, interstitial fibrosis and even corticomedullary cysts present. Genetic testing is the only way to distinguish nephronophthisis from AD interstitial nephritis, apart from mode of inheritance. Similar to AD tubulo-interstitial nephritis, there has been much progress recently in the molecular characterization of this phenotype.

The use of positional cloning and next-generation sequencing has facilitated the discovery of many nephronophthisis genes (NPHP). Their protein products, termed nephrocystins, localize to primary cilia placing nephronophthisis in the realm of other renal ciliopathies such as AD & AR polycystic kidney disease. Primary cilia are microtubule-like sensory organelles present on many cell types, including the apical surface of renal tubular cells. Currently 17 NPHP genes have been discovered, which together explain <50% of total cases (NPHP1 itself causes approximately 30% of cases). NPHP1-9 genes were discovered using a combination of genome-wide linkage and direct sequencing approaches in large pedigrees. More recent discoveries have been aided by next-generation sequencing. However, simultaneous analysis of all known mutations using massively parallel sequencing only led to a molecular diagnosis in 25% of cases, highlighted what remains to be discovered. Employing a candidate gene approach has proved useful (for NPHP16/ANKS6) by first identifying cilia gene products using proteomics and working back to the genes of interest. With >1000 known cilia proteins, this may enable the identification of many more nephronophthisis genes.

Nephronophthisis may also display polygenic inheritance, where mutations may be found in 2 or more susceptibility genes. Several families have been described that harbor mutations in several NPHP genes, which are known to interact. Furthermore, families with a nephronophthisis phenotype have been described having a single mutation in an isolated NPHP gene, suggesting they may have mutations in other, yet undiscovered NPNP genes as the condition is AR. These polygenic phenomena may also explain some of the incompletely penetrant extra-renal manifestations in certain individuals and families. For example, modifier effects of co-existing ANH1 and NPHP6 mutations have been suggested to cause extra-renal manifestations in Joubert syndrome due to NPHP1 mutations.

While not as celebrated as its bitter rival, AR Tubulointerstitial Nephritis has made big progress of late thanks to modern genetic advances. It will fancy its chances against its conference rival in this big first round matchup.

Table 1: NPHP Genes so far identified

NPHP Gene Name Location Gene product
NPHP1 NPHP1 2q13 Nephrocystin 1
NPHP2 INVS 9q31.1 Inversin
NPHP3 NPHP3 3q22.1 Nephrocystin 3
NPHP4 NPHP4 1p36.31 Nephrocystin 4
NPHP5 IQCB1 3q13.33 IQ motif-containing protein B1
NPHP6 CEP290 12q21.32 Centrosomal protein of 290 kDa
NPHP7 GLIS2 16p13.3 Zinc finger protein GLIS2
NPHP8 RPGRIP1L 16q12.2 Retinitis pigmentosa GTPase regulator-interacting protein 1‑like
NPHP9 NEK8 17q11.2 Serine/threonine-protein kinase Nek9
NPHP10 SDCCAG8 1q43 Serologically defined colon cancer antigen 8
NPHP11 TMEM67 8q22.1 Meckelin
NPHP12 TTC21B 2q24.3 Tetratricopeptide repeat protein 21B
NPHP13 WDR19 4p14 WD repeat-containing protein 19
NPHP14 ZNF423 16q12.1 Zinc finger protein 423
NPHP15 CEP164 11q23.2 Centrosomal protein of 164 kDa
NPHP16 ANKS6 9q22.33 Ankyrin repeat and SAM domain-containing protein 6
NPHP17 IFT172 2p23.3 Intraflagellar transport protein 172

Epigenetics in Nephrology vs Vesicoureteral Reflux 

This unlikely match-up sees 2 teams that have never met in the big dance face off in the first round. Both have had quiet pre-seasons but have certainly earned their right to this year’s tournament with a number of standout performances. We continue to learn more on the genetics of Vesicoureteral Reflux and Epigenetics in Nephrology is an exciting team for the future that has commentators buzzing.

Epigenetics in Nephrology

Epigenetics refers to alterations of gene expression at the level of gene transcription and translation without changes to gene sequence. These processes are modifiable by the cellular environment, potentially inheritable, and include DNA methylation, histone (major proteins in the chromatin) modifications, and regulatory changes induced by microRNAs (miRNAs). DNA methylation involves the addition of a methyl group to a cytosine base within CpG sites within promoter sequencing which influences gene expression, generally causing gene silencing. These mechanisms may, at least partly, explain how environmental factors interact with the genome to influence complex traits like kidney disease. The epigenome may also be thought of as a genetic-environmental footprint, explaining why in utero and early-life environmental conditions may lead to persistent lifetime and subsequent generation phenotypes (see Dutch Famine of 1944-45). Technologies to perform large-scale epigenetic analysis are evolving and have lagged behind traditional genomic techniques. However, the International Human Epigenome Consortium is creating a reference map of the human epigenome which will facilitate in-depth epigenome wide studies. The influences of miRNAs may be particularly exciting given their ability to be manipulated, either antagonized or over-expressed, it is methylation where much of the evidence currently exists.

With GWAS failing to explain much of the variability in blood pressure, epigenetics may uncover some of the missing heritability. A genome-wide animal study of salt-sensitive hypertension in rats has implicated hypermethylation of the renin promoter. A human epigenome-wide methylation study in young males with hypertension reported hypermethylation of the SULF1 gene which was confirmed in the validation sample for individuals ≤30 years old.

Transplantation is also an area where epigenetics play a large role. T regulatory cells (Tregs), so important in immune recognition and restricting self-reactive T cells, are regulated by their transcriptional factor FOXP3. The expression of FOXP3 is governed by methylation/demethylation of Tregs. This system may be crucial in achieving the holy grail of transplant medicine, operational tolerance.

The realm of epigenetic gene silencing in renal fibrosis is a standout topic in nephrology genetics research with huge translational potential. Fibrosis, a pathological wound repair process that persists even when the initial injury has been removed, is a final common pathway of many disease processes. There is accumulating evidence that the underlying molecular mechanism of fibrosis includes epigenetic processes, particularly gene hypermethylation. Bechtel et al demonstrated that hypermethylation of RASAL1 (an inhibitor of the Ras oncoprotein pathway) results in less inhibition of the Ras pathway and led to sustained fibroblast activation and subsequent renal fibrosis. The potential of using de-methylating agents to allow RASAL1 to inhibit the Ras pathway and thus lead to less fibrosis appears very attractive. As mentioned above, gene silencing of RASAL1 via methylation results in increased intrinsic Ras-GTPase activity in affected fibroblasts leading to fibrosis. Tampe et al showed successful inhibition of experimental renal fibrosis via reversal of aberrant RASAL1 hypermethylation. They achieved this using bone morphogenic protein 7, known to have anti-fibrotic activity.

This toss-up is difficult to call. With 2 relatively unknown teams facing off for the first time it’s anyone’s guess who will progress to the Round of 32. Is 2015 a year too early for the rookies of Team Epigenetics? We’ll have to wait and see.

Vesicoureteral Reflux

Vesicoureteral reflux (VUR) is another condition where modern genetic advances have revolutionized our understanding of pathogenesis and heritability. It is the most common type of congenital anomaly of the kidney and the urinary tract (CAKUT) with an estimated prevalence of 1–2%, but may well be even higher. It is characterized by retrograde flow of urine from the bladder back to the ureter and the kidney. VUR will often resolve with few significant sequelae but may be complicated by recurrent UTIs, scarring, and progressive renal disease. It remains unclear if the scars are a consequence of urine reflux/infections or if they represent co-existent developmental or dysplastic abnormalities. VUR may co-exist with other genitourinary abnormalities (ie, CAKUT) or as a part of syndromes with extra-renal developmental defects. Family studies have long supported the heritability of VUR. There is a 30–50% incidence in first-degree relatives, full concordance among monozygotic and 50% among dizygotic twins. The mode of inheritance is often AD but AR and X-linked pedigrees have been described. However, specific genetic causes of VUR remained elusive until recent technological advances.

Results of genome-wide linkage analysis in several families across various populations suggested linkage at multiple different loci. This is likely due to genetic heterogeneity of VUR in the families studied. GWAS data also demonstrate this heterogeneity with multiple SNPs across the genome giving significant or borderline significant association with VUR.

Whole-exome sequencing has brought the most productivity in discovering single-gene causes of VUR. Most of the genes reported have not been in families with syndromic VUR/CAKUT and required large kindreds with many affected individuals. An example of this is a 97-member pedigree with 16 affected individuals over 5 generations. Sequential genome–wide linkage and whole-exome sequencing was performed on the family. The causative mutation was discovered in TNXB, a gene associated with the joint hypermobility variant of Ehlers-Danlos syndrome. Other genes implicated using next-generation sequencing have included ROBO2, which may have multiple associated congenital abnormalities and HNF1B which may have liver, pancreas, and genital phenotypes (see Team AD Tubulointerstitial Nephritis above). RET may cause Hirshprung disease and multiple endocrine neoplasia type 2 as well as VUR and CAKUT. BMP4 mutations may cause defects in the eye, brain, and digits as well as CAKUT. PAX2 mutations cause renal coloboma syndrome and variants in this gene have also been described causing an FSGS phenotype (see Team Familial FSGS). These genes do not appear to play a major role in isolated, non-syndromic VUR. This underlines the complexity of genotype-phenotype interaction. It is likely that modifier genes with second “hits” or epigenetic alterations determine some of the varying phenotypes associated with certain gene variants and mutations.

The big clinical story regarding VUR in the past year was the RIVUR study published in NEJM (and covered on #NephJC). It demonstrated that prophylactic co-trimoxazole reduced the incidence of UTIs in children with VUR and a symptomatic UTI. However, this did not translate into less renal scarring at 2 years, which again questions the etiology of the “scars.”

Therefore, modern genetic techniques have helped us understand that VUR is a complex phenotype. It may be an isolated, non-syndromic finding or inherited as part of a myriad of non-renal developmental abnormalities. Some VUR can be considered a complex trait, influenced by multiple genes each having small effect sizes, as demonstrated using genome-wide linkage and association. It can also be inherited as a single-gene disorder in multiple different risk genes, as demonstrated using next-generation sequencing. This genetic complexity should not be surprising given multi-component nature of the lower urinary tract and its intricate development. A major challenge of clinical relevance that remains is to distinguish children who will have a benign course from those who will develop severe, complicated reflux nephropathy.

Familial FSGS vs APOL1

These 2 powerhouses know each other well. There is no love lost between the two with APOL1 being a franchise breakaway in recent years and has gone on to make a name for itself in the Genetics conference. APOL1 continues to captivate audiences although there is still a lot we don’t know about this exciting team. Familial FSGS is a conference stalwart dating back to the old “Podocyte Conference” with its breakthrough player Nephrin but has continued to attract new talent as discussed below.

Familial FSGS

FSGS is the third-leading cause of ESRD in the US with an increasing incidence in recent years. It describes a pattern of injury with many etiologies and proteinuria as the predominant clinical feature. It is caused by podocyte injury manifested by foot process effacement histologically. Several single-gene mutations have been identified that cause FSGS which has helped us understand the pathogenesis of glomerular disease. The genes have mostly been in podocyte-protein genes, a notable exception being LAMB2 which localizes to the glomerular basement membrane and causes Pierson syndrome (diffuse mesangial sclerosis, microcoria, and neurological anomalies).

Inheritance may be AD or AR, with AD conditions having a less severe and later onset phenotype and often exhibiting incomplete penetrance. FSGS due to single-gene mutations does not recur post-kidney transplantation. The first described gene was NPHS1 which codes for nephrin, an integral slit diaphragm protein, a mutation in which causes congenital nephrotic syndrome (so called “Finnish type”). This landmark study demonstrated the importance of the podocyte in congenital nephrotic syndrome/FSGS with multiple subsequent genes being described causing congenital nephrotic syndrome/FSGS (see Table below).  The proteins of interest are often integral slit diaphragm proteins (nephrin, podocin, CD2AP), foot process cytoskeleton components (ACTN4, INF2), or involved in regulation/expression of these proteins (WT1, perhaps PLCE1). Transient receptor potential cation channel type 6 (TRPC6) is a calcium channel located in the body of the foot process as well as the slit diaphragm. Mutations in TRPC6 are gain-of-function causing increased intracellular calcium influx. TRPC6 knockout mice are protected from albuminuria following angiotensin II infusion but how the gene causes podocyte injury remains unknown.

The advent of next-generation sequencing (see below section) has enabled the recent identification of additional single-gene causes of FSGS including ANLN, which codes for the F-actin binding protein Anillin. Potentially of more interest, next-generation sequencing has also expanded the phenotypic spectrum of known genes to include familial FSGS. These include PAX2, mutations in which were previously described to cause congenital abnormalities of the kidney and urinary tract and mutations in COL4A3 & COL4A4 which have recently been reported to be disease segregating in 10% of a large cohort of familial FSGS families, without an Alport phenotype. The Wilms Tumor 1 gene (WT1) encodes a zinc finger binding protein critical for kidney and genitourinary development. It is also involved in expression of essential slit diaphragm proteins such as nephrin, podocin, and podocalyxin. Renal phenotypes associated with WT1 mutations include Wilms tumor and several syndromic forms of FSGS associated with genitourinary anomalies and mental retardation. These include WAGR syndrome (with aniridia, genitourinary malformations, and mental retardation), Denys–Drash syndrome (with diffuse mesangial sclerosis, male pseudohermaphroditism), and Frasier syndrome (male pseudohermaphroditism, FSGS and gonadoblastoma). A recent study employed next-generation sequencing to identify WT1 mutations causing non-syndromic FSGS. Functional studies implicated WT1 in the transcriptional regulation of nephrin as well as synaptopodin expression, another crucial podocyte protein.

Genetic testing for familial FSGS has moved a step closer with the advent of next-generation sequencing although precisely when and how it may be useful remains a challenge. In transplantation, it may be helpful to assess risk of recurrence or to screen potential living related donors. In adolescents or young adults presenting with FSGS, having a molecular diagnosis may help tailor treatment as the presumption is that immunosuppression will not work in familial FSGS. However, it is not as simple as this, and certain agents, particularly cyclosporin (blocking calcineurin-mediated dephosphorylation of synaptopodin) and rituximab may have beneficial podocyte-specific effects, possibly regardless of etiology of the podocytopathy.

This team has strong comparisons and connections to Duke in the NCAA. FSGS is a perennial competitor with a rich tradition and will expect to go far in the tourney. APOL1 represents a huge early potential banana skin.

Table 2: List of major genes implicated in familial FSGS

Gene Phenotype
Autosomal Recessive
NPHS1 (Nephrin) FSGS; Congenital Nephrotic Syndrome
NPHS2 (Podocin) FSGS; Congenital Nephrotic Syndrome
LAMB2 FSGS; Pierson Syndrome
MYH9 FSGS; Sensorineural Deafness; Macrothrombocytopenia; Epstein, Fechtner & Sebastian Syndromes
PAX2 FSGS; Papillorenal Syndrome
Autosomal Dominant
INF2 FSGS; Charcot–Marie–tooth Disease
LMX1B FSGS; Nail-Patella Syndrome
WT-1 FSGS; Denys–Drash, WAGR & Frasier Syndrome

Apolipoprotein 1 (APOL1) related nephropathy is surely one of the biggest nephrology genetics stories in recent times. The APOL1 risk alleles, G1 and G2, are mutually exclusive (never occur on the same chromosome copy) and 2 copies are necessary to confer kidney disease risk (genotype may be G1/G1, G2/G2, or the compound heterozygous state of G1/G2). The alleles are common in individuals of West African ancestry and almost unheard of in those of European ancestry. Variation in these alleles is now known to be responsible for the vast majority excess risk of non-diabetic kidney disease including FSGS, HIV-associated nephropathy, severe lupus nephritis, and unspecified CKD (often previously labelled as hypertensive nephropathy in African Americans). The alleles are common, with about half of African Americans having either one or two risk alleles, and 10%–15% possessing both. The effect size is large, with a 7-10 fold increased risk of FSGS or unspecified ESRD, and an even higher risk for HIVAN. Despite this, they should be considered risk alleles rather than a single-gene disorder. The presence of the alleles is not enough to have the phenotype and additional “hits” are necessary, which may be genetic, environmental, or both. The origin of the APOL1 variants is a fascinating story, with initial genome-wide approaches suggesting MYH9 as the gene of interest in African American patients with FSGS. This was a reasonable theory given the fact that MYH9 is expressed in the podocyte and mutations in the gene cause syndromic FSGS (see Team Familial FSGS). However the excess risk was found to be due to variants in the nearby APOL1 gene. These alleles have risen to high frequency in individuals of African descent via a beneficial effect in resistance to Trypanosoma brucei rhodesiense. A succinct review of APOL1 (and other genetic nephropathies) is worth exploring.

A recent study reported in NEJM examined APOL1 variants in 2 large CKD cohorts, namely AASK and CRIC. AASK enrolled all African American patients with CKD attributed to hypertension that did not have diabetes. The CRIC study included black and white patients with CKD, approximately half of whom had diabetes. Interestingly this finding was also evident in the patients with diabetic kidney disease. Diabetic nephropathy has not been previously identified as phenotype influenced by APOL1 variation.

Little is known about the kidney-specific biology of APOL1. Only the genomes of humans and a few primate species carry the APOL1 gene making study in animal models difficult. Recent work has explored the role of innate viral immunity in over-expression of APOL1, particularly of the variant APOL1 which more injurious to cells than wild type. In the study, several patients (10/11 African American) were noted to develop a collapsing FSGS pattern of injury after treatment with interferon. Interferons and Toll-like receptor agonists hugely increased APOL1 expression. Note that HIV is a potent inducer of interferons, with HIV nephropathy being a particularly risk with possession APOL1 risk alleles. Lupus nephritis, another high interferon state, has been recently recognized as lying within the sphere of APOL1 nephropathies. Keeping with the viral pathway theme, another study demonstrated that in African Americans with both APOL1 risk variants, JC viruria was associated with a lower prevalence of kidney disease. How would JC virus protect from development of APOL1-associated nephropathy? Is it a clue to an environmental “second hit” whereby the JC virus may inhibit infection with other more nephrotoxic viruses? These questions and more will need to be answered in the coming years.

The effect of transplanting kidneys from APOL1 nephropathy risk donors demonstrated that deceased donor allografts possessing both APOL1 risk variants failed more rapidly than those with one or no risk alleles. This concept was well, but tragically, illustrated in a recent case report of a young Afro-Caribbean monozygotic twin transplant pair. The recipient had unspecified FSGS, the donor was normal at screening. There was clinical and histological evidence of FSGS at 30 months post-transplant and allograft failure occurred early. The donor subsequently developed proteinuria and renal dysfunction, undoubtedly aggravated by his reduced nephron mass. The twins were later genotyped confirming the presence of both APOL1 risk variants.

This leads on to the utility of testing for APOL1 variants. Certainly a case could be made in transplantation, illustrated by the case report described above. Also if the risk of allograft failure with possession of the APOL1 risk alleles in the donor could be validated, it would suggest genotyping donors of African origin could be beneficial. In the general CKD population, it is less clear. Certainly the alleles confer significant risk but that risk is not absolute so not all G1 & G2 carriers will develop kidney disease. Also, as there is no specific treatment for APOL1-related nephropathies, the utility for general testing in the African American CKD population is not evident.

Team APOL1 will be a tourney regular for years to come and may grow in coming seasons to be a big dance favorite. It’s a team full of potential but remains somewhat of an unknown quantity.

GWAS in Nephrology vs Next-Generation Sequencing

These conference rivals each have a loyal following who will relish this first-round contest. GWAS promised much when it exploded onto the scene some years back and many predicted several national championships which have failed to materialize. There is similar enthusiasm for Next-Generation Sequencing at present, a team who has huge aspirations and expects silverware this season.

GWAS in Nephrology

The human genome consists of approximately 3 billion nucleotides of DNA sequence. Areas of variance at an individual nucleotide, termed SNPs, occur across the genome at intervals of about one per 300 base pairs of DNA. SNPs in close physical proximity are more likely to be inherited together as part of a group (haplotype). This phenomenon, referred to as linkage disequilibrium, allows for one SNP to serve as a proxy for the presence of other SNPs in that haplotype. This is the concept of a “tag SNP” and obviates the need for individual genotyping of every variant. This is the principle behind GWAS. We have witnessed an explosion of GWAS for complex traits including renal function (eGFR), CKD, and hypertension. GWAS are usually designed to detect relatively common SNPs (minor allele frequency > a pre-determined level, for example 5%).

GWAS in Nephrology have not proven to be as clinically useful as initially hoped. Like GWAS in other complex diseases, many variants with tiny effect sizes have been uncovered but these variants only explain a small proportion of total heritability of the disorders. Large consortiums have been created to try to provide the necessary power to detect more variants but overall, the effect sizes remain small. Examples of these GWAS meta-analyses include the CHARGE consortium (n=29,163), the Global BPgen Consortium (n=34,433) and ICBP-GWAS (n=69,395 with validation in a further 133,661 individuals) for hypertension. ICBP-GWAS reported 29 SNPs independently associated with blood pressure but together they explained only 0.9% of the BP variation in the cohort. This reflects the genetic complexity nature of the trait. Large renal function GWAS collaborations have also been formed and have demonstrated similar findings of numerous SNPs with tiny effect sizes. These finding may one day lead to useful risk scores for CKD or hypertension but demonstrate the limited clinical relevance of individual or a small group of SNPs.

Despite these limitations, genome-wide approaches have proven useful in our field. The discovery of the APOL1 variants came from initial identification of variants in the nearby gene MYH9 gene on chromosome 22 (see Team APOL1). We have also learned much about UMOD nephropathy and the function of Tamm-Horsfall protein (Uromodulin) from a renal function GWAS in which UMOD variants popped up as being genome-wide significant for eGFR (see Team AD Tubulointerstitial Nephritis). It was mentioned above that this locus appears to confer shared risk for both hypertension and kidney disease (and also cardiovascular events). Another example of shared risk loci comes from the ICBP-GWAS study where a variant in the phospholipase C epsilon gene (PLCE1) was associated with blood pressure variance. A coding missense mutation in PLCE1 has been described causing steroid-resistant FSGS.

Variants in SHROOM3 have been consistently associated with CKD/eGFR in a large GWAS but any potential mechanism remained unclear. Due to this finding, a group from Mount Sinai performed a set of experiments in a transplant cohort which was hugely insightful. They genotyped transplant donors for the SHROOM3 variant which correlated with increased SHROOM3 protein expression and allograft fibrosis on protocol biopsies. It also associated with eGFR in the recipient. They identified the risk allele to be located in a sequence for the transcription factor TCF7–L2 which enhanced SHROOM3 expression that in turn regulated TGF-B induced renal fibrosis. In a mouse model, SHROOM3 knockdown strongly abrogated interstitial fibrosis. This exciting research with real translational potential was made possible by SNP associations in GWAS cohorts of the general population and firmly demonstrates the power of this approach.

Another demonstration of the power of GWAS comes from a study in >20,000 individuals of European and Asian ancestry which shed light on the complex genetic architecture of IgA nephropathy. Several genome-wide significant variants were identified which were predominantly located in pathways of immunity and inflammation including variants with overlapping susceptibility to autoimmune disorders such as inflammatory bowel disease. The study demonstrated that disease onset was related to the number of risk loci present, although they still only explained a small proportion of the variance in disease onset. A striking observation was the association of the genetic risk score with pathogen diversity, particularly helminth diversity. Helminths are common in the soil in Asia and may explain the increased incidence of IgA nephropathy in some geographical areas and the known association of mucosal infections as a trigger for IgA nephropathy.

Many SNPs from GWAS will have very small p values but will not reach genome-wide significance when corrected for multiple testing. This may be due to overly stringent criteria for genome-wide significance or underpowered studies. One method of using these variants in a clinical useful way is to perform pathway analysis not limited to only genome-wide significant or replicated variants. A recent paper in JASN employed pathway analysis on GWAS data examining the development of new-onset diabetes mellitus after transplant (NODAT) post renal transplantation. This study implicated β-cell dysfunction in the pathophysiology of NODAT, contrary to traditional thinking that the etiology was merely insulin resistance. Another example is from the Wellcome Trust Case Control Consortium where no genome-wide significant associations were observed for hypertension in the original study. However, pathway analysis revealed interconnected networks in dopamine signalling including genes coding for the AMPA, NMDA, and GABA-A receptors. This suggests that the regulation of vascular smooth muscle tone is important. There is inherent bias in pathway analysis, however, as it is reliant on accuracy and depth of input from the pathway databases but it does provide an additional use of GWAS data including non-genome-wide significant SNPs.

GWAS have revolutionized the search for genetic influences on complex diseases but it is far from a panacea. As a technique, GWAS are not designed to fully uncover the interplay of multiple genetic and environmental factors which cause CKD and hypertension. Genetic variant discovered by GWAS mostly have tiny independent effect sizes and none are likely to be obligatory for the phenotype to occur.

Team GWAS is a hot and cold side who can beat anyone on their day but also may succumb to unheralded opposition. This unpredictability makes them a fascinating team to follow. Their recent success will give them confidence. Will this be enough against the next-generation sequencing new kids?

Next-Generation Sequencing

Next-Generation Sequencing, including whole-exome sequencing (WES), and whole-genome sequencing (WGS), promises in-depth coverage of the exome/genome with improved coverage of rare variants and copy number variants (CNVs; large insertions and deletions). WES involves sequencing all exons, the coding proportion of the genes, which make up about 1% of the genome and where presumably most disease-causing variants lie. Deep sequencing projects, such as 1000 Genomes, demonstrate that rare variants, which are usually not covered in GWAS, constitute the majority of polymorphic sites in human populations.

An early example of the use of WES is the identification of a potassium channel mutation in the development of primary hyperaldosteronism, one of the more frequent causes of secondary hypertension. WES has facilitated gene discovery for several kidney diseases including FSGS and VUR (see Team VUR & Familial FSGS). It has also helped identify novel phenotypes for known genes in the case of COL4A mutations, which cause hereditary nephritis, presenting as familial FSGS. In nephronophthisis, an AR ciliopathy which causes tubulointerstitial nephritis, Next-Generation Sequencing has expanded the breadth of known causative genes. There are now 17 NPHP genes described, but despite this, many remain undiscovered.

The idea of WES is to remove much of the redundancy of the genome and maximise efficacy and cost-effectiveness. However, the sphere of epigenetics has taught us that non-coding portions of the genome can be vitally important, potentially heritable and influence expression of the genes and therefore phenotypes. Moreover, WES is not a perfect method for new gene discovery in familial disease and exonic regions may still prove difficult to sequence with the potential to miss causative variants. This is highlighted by the problems in sequencing MUC1 as a cause of AD interstitial nephritis due to multiple repetitive regions in and around the gene. Successful sequencing relies on multiple reads of the regions of interest, with depths of <10X showing inconsistent call rates.

The major challenge with Next-Generation Sequencing lies in identifying the specific disease-causing variants from all the benign variants we carry. WES in one individual will typically reveal approximately 20,000 variants and even when sequencing >1 individual in a family, multiple potentially pathogenic variants will be shared between family members. Filtering methods and in silico techniques may predict if a variant is likely to be damaging but have the potential also to inadvertently disregard pathogenic mutations. The issue is compounded in African American families which have known increased genetic diversity. African American families may have many more variants uncovered by WES, compared to non-African ancestry families. Therefore, even bigger pedigrees are needed to identify disease causing mutations.

Aside from research settings, the utility of WES in clinical practice for precise molecular diagnosis is unknown. The spectrum of childhood nephrotic syndrome is an area where it may be useful as several genes forming a large proportion of cases have been identified. Data on likely responsiveness to certain treatments is a potential indication for testing. Other indications include transplantation, both for assessment of potential recurrence and for disease in living related donors. The use of exon sequencing of 27 genes known to cause steroid resistant nephrotic syndrome (SRNS) that manifested before 25 years of age has been reported. A single-gene cause was detected in 29.5% (526 of 1783) of families, with younger presentations more likely to have a monogenic cause identified. A UK cohort of 36 patients (all <16 years at onset) with SRNS detected a pathogenic variant in 70% of familial causes and 15% of sporadic cases.

WGS is becoming more affordable and it is likely that very soon it may replace WES in the investigation of genetic diseases. So will WGS solve these issue that we shave with WES? As sequencing is not confined to the exome, gene regulatory regions, enhancers, and promoters will be covered. It will certainly add more complexity by sequencing the entire genome and will uncover millions of variants in each individual sequenced. Sophisticated filtering and bioinformatic methods will need to be employed to identify likely pathogenic variants. Current issues with WGS include a lower detection rates for CNVs versus single-nucleotide variants and incomplete concordance between different sequencing technologies. WGS will potentially provide information about countless medical conditions, many of undetermined significance. The huge volumes of data generated by such technologies will potentially greatly aid genetic interrogation of kidney diseases but will provide logistical and ethical challenges which must be overcome.

With a strong preseason behind it, Next-Generation Sequencing expects to win this contest with some to spare. GWAS has gone under the radar so far this season but has earned some notable recent victories and has a lot to prove to its doubters. This one will go down to the wire.

 – Post written and edited by Drs. Paul Phelan and Conall O’Seaghdha.


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