#NephMadness 2020: Genetics Region

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Selection Committee Member: Michelle Rheault @rheault_m

Michelle Rheault is the Director of the Division of Pediatric Nephrology and Medical Director of Dialysis at the University of Minnesota Masonic Children’s Hospital. Her clinical research interests are in Alport syndrome and pediatric glomerular diseases

Writer: Bethany Roehm @bethany_roehm

Bethany Roehm is a clinical research fellow at Tufts Medical Center in Boston, MA. She is also a current AJKD Editorial Intern. Her research focuses on understanding kidney function in patients with advanced heart failure with a current focus on patients with left ventricular assist device

Competitors for the Genetics Region

Women with X-Linked Alport Syndrome vs Autosomal Dominant Alport Syndrome

Genetic Counseling for Stones vs Genetic Counseling for Cystic Diseases

Women with X-Linked Alport Syndrome vs Autosomal Dominant Alport Syndrome

Women With X-Linked Alport Syndrome

X-linked Alport syndrome (XLAS) was once thought to only occur in men, but now it is increasingly being recognized as a disease that affects women as well. XLAS is caused by mutations in the gene COL4A5 encoding the alpha 5 chain of type IV collagen, α5(IV). COL4A5 lies on the X chromosome, and mutations interfere with collagen synthesis. To form collagen IV, α5 combines with α3 and α4 in a 1:1:1 ratio. Collagen IV interacts with proteins such as laminin, agrin, and nidogen to form the glomerular basement membrane (GBM). Since COL4A5 is on the X chromosome woman have two copies of the gene, of which only one is usually affected, heterozygotes. In the past, heterozygotes were described as “carriers”, but since nearly all of these women have some form of clinically apparent disease, “affected” is a more accurate description. The clinical severity of disease varies from patient to patient primarily due to varying patterns of lyonization, the random inactivation of one of the two X chromosomes in a cell. Since the proportion of cells with the chromosome carrying the mutated gene being active varies.  

Nearly all women with XLAS have microscopic hematuria for virtually their entire lives. Albuminuria occurs in 75% of these women and may be predictive of progression of chronic kidney disease (CKD). Often women recount stories like, “I’ve had blood in my urine forever but I had a cystoscopy that was normal and they told me not to worry about it.” Unfortunately, women may also experience declines in estimated glomerular filtration rate (eGFR) over time and end-stage kidney disease (ESKD) occurs in 15-30% of women with XLAS. Women with XLAS should not be considered for kidney donation.  

By middle age, some women with XLAS have some degree of bilateral high-tone sensorineural hearing loss. Similar to XLAS in men, women can have retinal or corneal disease. This can often be a clue to the diagnosis of XLAS in the absence of biopsy or genetic testing. Lenticonus, commonly seen in men with XLAS and those with autosomal recessive AS, is not seen in women with XLAS.

On kidney biopsy, women with XLAS show a thin basement membrane with or without lamellation (see figure below for electron microscopy with lamellation).

Thickened and lamellated glomerular basement membrane is the classic appearance of Alport syndrome by electron microscopy. Image courtesy Dr. Clifford Kashtan, University of Minnesota Masonic Children’s Hospital.

Immunostaining for α5(IV) collagen demonstrates a mosaic pattern of type IV collagen due to lyonization (see figure below), though normal α5(IV) may be found in mild presentations. 

Immunofluorescence microscopy in women with X-linked Alport syndrome can show a mosaic pattern of alpha 5 (IV) staining consistent with random X-inactivation. Red: alpha 2 (IV), Green: alpha 5 (IV). Image courtesy of Dr. Michelle Rheault, University of Minnesota Masonic Children’s Hospital.

An alternate way of diagnosing XLAS is through a skin biopsy. Because the α5(IV) and α6(IV) collagen trimer is present in the epidermal basement membrane of the skin, one can see the absence of or skip pattern of α5(IV) collagen immunostaining in affected women. If there is a high suspicion of XLAS, next generation sequencing can be done. Though there is wide variability in clinical presentation in affected women, mutation type does not seem to be predictive of clinical phenotype, perhaps due to unbalanced X chromosome inactivation. This is in contrast to men, where nonsense mutations and large deletions lead to severe disease. 

There are currently no specific treatments for XLAS. Women with suspected XLAS should be monitored annually for hypertension and albuminuria. Angiotensin-converting enzyme inhibitors or angiotensin receptor blockers are indicated in cases with albuminuria to slow progression of CKD. Pregnancy is a special concern in XLAS. Even if a woman has only mild proteinuria, this can progress to nephrotic syndrome, and further complicate the pregnancy with anasarca, pulmonary edema, pre-eclampsia, and permanent loss of kidney function, though long-term effects of pregnancy on glomerular filtration rate is not well studied. These clinical sequelae highlight the importance of recognizing XLAS as a potential cause of CKD in women.

X-linked Alport Syndrome in Women – Not Just a Carrier State!


Autosomal Dominant Alport Syndrome

The past few years have seen an explosion of new information about type IV collagen mutations. Every time you open a kidney journal, a new study is showing a high prevalence of heterozygous COL4A3 and COL4A4 mutations associated with focal segmental glomerulosclerosis (FSGS) lesions, or general populations with ESKD.

Visual Abstract by @Stones__ on Gast et al

Autosomal dominant Alport Syndrome (ADAS), arising from mutations in the COL4A3 and COL4A4 genes, is less common than XLAS and affects men and women equally. It was previously thought to account for only 5% of patients with AS, but more recent, unbiased studies showed that the numbers may be closer to 19-31%. Much like XLAS in women, the clinical course is often milder than XLAS in men or autosomal recessive AS. Hearing loss and ocular lesions are less common compared to other forms of AS. To date, there are no particular genotype-phenotype correlations, perhaps because there are so few studies of ADAS.  

In an observational study of 25 Japanese patients with ADAS, those with double mutations (also termed digenic) did not have a higher incidence of ESKD, nor was there any apparent difference between COL4A3 and COL4A4 mutations in terms of disease severity. Eighteen subjects had proteinuria; in many of these individuals the proteinuria was detected during childhood or adolescence, perhaps due to routine screening for proteinuria in children in Japan. Three developed ESKD, one at 33 years of age and the other two after 50 years of age. Only one person developed hearing loss, and this was in older age. Three had findings of FSGS upon kidney biopsy, and all but one had thin basement membrane on electron microscopy. These results demonstrate the heterogeneity of disease phenotype.

As described in the study above, 3 (12%) patients had findings of FSGS on kidney biopsy, and this has been demonstrated in other studies too. An observational study of 80 patients with FSGS or steroid-resistant nephrotic syndrome (SRNS) performed genetic testing to look for genetic etiologies. Twelve patients had definite pathogenic genetic variants, and of those 12 patients, 8 (56%) had type IV collagen mutations. While most had familial FSGS, two had sporadic FSGS, possibly indicating de novo mutations. This testing led to the diagnosis of ADAS in 6 patients and 2 patients with thin basement membrane disease. GBM findings on kidney biopsy were variable, with some showing classic lamellation and splitting of the GBM and others showing a normal GBM. Notably, not all patients with type IV collagen mutations had hematuria, though this is often thought to be a hallmark of ADAS and thin basement membrane disease. Other genetic studies of FSGS and SRNS have yielded similar findings and underscore the importance of including AS on the differential for FSGS or minimal change disease (MCD), as FSGS and MCD are often treated with immunosuppressive therapy while ADAS is not. 

There is some controversy as to whether thin basement membrane disease should be placed under the umbrella of ADAS. Thin basement membrane disease is also caused by a heterozygous COL4A3 or COL4A4 mutation. Some classify those diagnosed with thin basement membranes on a kidney biopsy as having ADAS, while others consider the two to be separate entities, citing that at least 1% of the population may be heterozygous for a pathogenic COL4A3 or COL4A4 mutation and that patients with thin basement membrane disease rarely progress to kidney failure, and when they do it may be due to other causes. Patients with thin basement membrane disease also typically lack lamellation of the GBM upon kidney biopsy. Others counter that thin basement membrane is a histologic diagnosis that does not delineate further important clinical features, such as proteinuria. By reclassifying those with thin basement membrane disease as having ADAS, they may be more likely to receive proper monitoring and treatment that could decrease the likelihood of developing ESKD. 

The risk factors for CKD progression in ADAS include:

  • Proteinuria
  • Family history of kidney disease progression and ESKD
  • FSGS
  • GBM thickening and lamellation

Patients suspected of having ADAS should be screened for hematuria and albuminuria annually, and started on angiotensin-converting enzyme inhibitors or angiotensin receptor blockers if there is significant albuminuria.

An ADAS Patient Perspective


Genetic Counseling for Stones vs Genetic Counseling for Cystic Diseases

Genetic Counseling for Stones

Kidney stones are common with a lifetime incidence of up to 5%. The first symptomatic kidney stone episode typically occurs around age 30. The traditional risk factors for kidney stones include low fluid intake, low dietary calcium intake, high dietary intake of oxalate and sodium, obesity, and diabetes mellitus. In addition to these factors, genetics can play a role in nephrolithiasis, especially in children and younger adults. Genetic testing may provide definitive diagnosis in 15-30% of affected patients. When counseling patients on whether to undergo genetic testing for stones, perhaps the most important consideration is whether or not genetic testing is likely to change management or prognosis. Below are some situations where a specific genetic diagnosis could make a meaningful difference for patients.

Cystine crystals on urinalysis in a patient with multiple kidney stones. Figure S4 from Cavanaugh and Perazella, AJKD © National Kidney Foundation.

One of the most notable genetic causes of kidney stones is cystinuria, accounting for 6%-8% of kidney stones in children (See figure 3). Implicated genes include SLC3A1 and SLC7A9. These genes encode subunits for the amino acid cotransporter responsible for cystine reabsorption in the kidney. Clinical findings include high urine cystine (which can be screened for with a spot urine cystine/creatinine ratio), recurrent cystine stones that can result in obstructive uropathy, pyelonephritis, and CKD. Cystine stones can be quite stubborn, and lithotripsy may be unable to break them up. In addition to high water intake, treatment includes urine alkalinization, cystine binding medications, and a diet low in animal protein and sodium.

Primary hyperoxaluria is caused by mutations that result in over-production of oxalate leading to high serum oxalate levels and resulting in an increased propensity to form calcium oxalate stones. Nephrocalcinosis and CKD that progresses to ESKD can also be seen. It is can be caused by mutations in one of three different gene mutations:

  1. Primary hyperoxaluria type 1 is the most common and often most severe form of the disease. It is caused by a loss of function mutation in the mutation of AGXT. This decreases the activity of alanine-glyoxylate aminotransferase (AGT), resulting in decreased conversion of glyoxylate to pyruvate; glyoxylate is instead oxidized by lactate dehydrogenase (LDH) into oxalate. Treatments include liver transplant and pyridoxine.
  2. Primary hyperoxaluria type 2 is caused by mutations in the GRHPR gene, resulting in a deficiency in glyoxylate reductase/hydroxypyruvate reductase (GR/HPR) which, like AGXT mutations, leads to a buildup of glyoxylate in the liver. The glyoxylate is ultimately converted to oxalate by LDH. Glyoxylate reductase/hydroxypyruvate reductase (GR/HPR) is found throughout the body so liver transplant was traditionally not considered an effective therapy, though there has been at least one case with success with this therapy. Otherwise usual stone prevention is the primary therapy.
  3. Primary hyperoxaluria type 3 is caused by mutations of HOGA1. The metabolic pathway hhere has not been fully worked out. Patients with PH3 can be treated by decreasing intake of foods that are rich in hydroxyproline like animal proteins, dairy, and eggs.

The metabolic pathways in primary hyperoxaluria. Figure 1 from Rumsby, AJKD © National Kidney Foundation.

Distal renal tubular acidosis is another rare genetic cause of kidney stones and nephrocalcinosis. Stones are predominantly calcium phosphate. Stone formation is promoted by alkaline urine, hypercalciuria and hypocitraturia. Hypercalciuria results mainly from decreased calcium reabsorption in the kidney, and increased calcium release from bone due to bones buffering the excess metabolic acidosis. Hypocitraturia occurs because metabolic acidosis causes increased citrate reabsorption in the proximal tubule. Potassium citrate can prevent stone recurrence by raising citrate levels in the urine, and treating the metabolic acidosis. However, too much potassium citrate may further alkalize the urine, promoting formation of calcium phosphate stones. Thus, treatment can be challenging to balance. Thiazide diuretics can also be used to decrease urine calcium.

Dent disease is an X-linked recessive disease affecting the CLCN5 or ORCL genes, resulting in low molecular weight proteinuria, hypercalciuria, and hyperphosphaturia. The latter two predispose patients to kidney stone formation and nephrocalcinosis. ESKD can occur fairly early in adulthood. Hypercalciuria is felt to be a driving factor in kidney stone formation, so treatment strategies center around reducing calcium excretion in the urine through decreased sodium in the diet and the administration of thiazide diuretics. Because of its X-linked inheritance pattern, both affected individuals and women of childbearing age with relatives who have Dent disease should be offered genetic testing.

These diseases highlight some of the genetic causes of kidney stones and, importantly, causes of kidney stones that are preventable. Moreover, the treatment for each of these diseases is quite different and some patients may be at risk of progressive CKD or ESKD, emphasizing the importance of obtaining a genetic diagnosis. Genetic testing may be most useful in obtaining a diagnosis in younger children with kidney stones, but may be less useful in older adolescents or young adults, as studies have shown lower detection rates of genetic causes in these groups. 

The table below summarizes of some of the most common monogenic causes of kidney stones:

Disease Gene Mutations Treatment
Cystinuria SLC3A1, SLC7A9 Urine alkalinization, cystine-binding medications, reduce sodium intake and animal protein intake
Primary hyperoxaluria AGXT, GRHPR, HOGA1 Depending on mutation liver transplant, pyridoxine, reduced hydroxyproline intake
Distal renal tubular acidosis SLC4A1, ATP6V1B1, ATP6V0A4, FOXI1 Potassium citrate, thiazide diuretics
Dent disease CLCN5, ORCL Thiazide diuretics, reduce sodium intake

Genetic Counseling for Cystic Diseases

In children with kidney cysts, the diagnosis is not always clear. Some cystic kidney disease in infancy may lead to progressive CKD and ESKD in childhood, whereas others may be static. In addition, some cystic kidney disease may have extrarenal manifestations that need to be screened for on a regular basis. Which children with kidney cysts need genetic testing?

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the polycystin 1 (PKD1) or polycystin 2 (PKD2) genes, and screening with ultrasound is recommended for adults at risk (ie, those with a family history), even if they are asymptomatic. However, the indications for screening in children who are at risk for the disorder, and what exactly that screening should entail, are less clear. Genetic testing in children can cause undue anxiety for the parents and child, and some screening tests like ultrasonography are not sufficient to rule out disease in children. Parents may also worry that their children could be denied insurance coverage for a pre-existing condition once they become adults. Alternatively, waiting too long to test can result in delays in treatment which could significantly slow the progression of kidney disease.

Current recommendations for screening in at-risk children are as follows: Parents with ADPKD should be counseled on how ADPKD is inherited and the potential risks and benefits of genetic testing in their children. After this, diagnostic screening with ultrasound can be offered, or in asymptomatic at-risk children parents can opt to annually monitor for disease manifestations like hypertension and proteinuria. At-risk children should be made aware of their risk of ADPKD by their parents upon reaching adulthood if not told already.

However, when a child has either very early onset or aggressive cystic kidney disease, genetic testing should be offered. In the case of very early onset ADPKD, multiple gene mutations may be present, such as a combination of two different PKD1 mutations, PKD1 and PKD2 mutations, or a combination of PKD1 and TSC2 (the gene responsible for tuberous sclerosis) mutations.  

There are also genetic mutations like HNF1B, PKHD1, and DZIP1L that can manifest similarly to ADPKD in young children. Patients with HNF1B mutations have a variable phenotype and commonly exhibit kidney malformations either in an autosomal dominant or sporadic pattern. On prenatal ultrasound HNF1B can present as bilateral hyperechoic kidneys, indicative of microscopic cysts, dysplasia, or tubular dilatation. Patients can suffer from gout, magnesium wasting, and renal carcinoma. Progression to ESKD in childhood is uncommon, but occurs. Extrarenal manifestations include mature onset diabetes of the young, developmental delay, and abnormalities of the female genitourinary tract. PKHD1 and DZIP1L result in medullary cysts that can be detected on prenatal ultrasound. Extrarenal manifestations include congenital hepatic fibrosis and malformations of the small bile ducts.

Other etiologies for cystic kidney disease include mutations in NPHP family of genes resulting in nephronophthisis. NPHP genes encode proteins called nephrocystins, with mutations resulting in ciliary impairment. Mutations in at least 20 different NPHP genes have been identified. In 60% of patients the exact mutation is unable to be identified. Nephronophthisis has a variable phenotype but is characterized by corticomedullary cysts, urinary concentrating defects, sodium reabsorption defects, small to normal size kidneys, and progressive kidney disease. Extrarenal manifestations may or may not be present but can include hepatic fibrosis, cerebellar vermis hypoplasia, skeletal dysplasia, retinitis pigmentosa, and pancreatic involvement, as well as other more rare syndromes such as Bardet-Biedl syndrome. Bardet-Biedl syndrome is characterized by obesity, blindness, polydactyly, and progressive CKD. ESKD in patients with nephronophthisis is common; one cohort study reported ESKD in 63% of patients with a median onset of 11.4 years.

Recommended genetic testing for cystic kidney disease in children with very early onset or aggressive disease includes next generation sequencing for PKD1, PKD2, PKHD1, DZIP1L, and HNF1B. While testing will hopefully provide diagnostic answers, sequencing for some mutations can be technically challenging and causative mutations may not yet be known. In one study, 78% of the patients who underwent genetic testing for cystic kidney diseases were found to have specific disease-causing mutations.

Visual Abstract by @Stones__ on Bullich et al

Both positive and negative test results can cause significant psychological distress. In addition, testing may not be readily available and may require reaching out to tertiary centers who perform specific testing. Genetic testing may also be expensive and may not be covered by insurance. Thus, when conducting genetic testing in children, it is important to communicate all of these potential risks and benefits of testing to their parents/guardians.

The Impact of Genetic Testing on A Person: A Patient Perspective


– Executive Team Member for this region: Anna Burgner, AJKDBlog Contributor. Follow her @anna_burgner.


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3 Trackbacks / Pingbacks

  1. #NephMadness 2020: An ADAS Patient Perspective – AJKD Blog
  2. #NephMadness 2020: X-linked Alport Syndrome in Women – Not Just a Carrier State! – AJKD Blog
  3. #NephMadness 2020 The Impact of Genetic Testing on A Person: A Patient Perspective – AJKD Blog

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