Pathogenicity of missense variants affecting the collagen IV α5 carboxy non-collagenous domain in X-linked Alport syndrome.

X-linked Alport syndrome is a genetic kidney disease caused by pathogenic COL4A5 variants, but little is known of the consequences of missense variants affecting the NC1 domain of the corresponding collagen IV α5 chain. This study examined these variants in a normal (gnomAD) and other databases (LOVD, Clin Var and 100,000 Genomes Project) to determine their pathogenicity and clinical significance. Males with Cys substitutions in the collagen IV α5 NC1 domain reported in LOVD (n = 25) were examined for typical Alport features, including age at kidney failure. All NC1 variants in LOVD (n = 86) were then assessed for structural damage using an online computational tool, Missense3D. Variants in the ClinVar, gnomAD and 100,000 Genomes Project databases were also examined for structural effects. Predicted damage associated with NC1 substitutions was then correlated with the level of conservation of the affected residues. Cys substitutions in males were associated with the typical features of X-linked Alport syndrome, with a median age at kidney failure of 31 years. NC1 substitutions predicted to cause structural damage were overrepresented in LOVD (p < 0.001), and those affecting Cys residues or 'buried' Gly residues were more common than expected (both p < 0.001). Most NC1 substitutions in gnomAD (88%) were predicted to be structurally-neutral. Substitutions affecting conserved residues resulted in more structural damage than those affecting non-conserved residues (p < 0.001). Many pathogenic missense variants affecting the collagen IV α5 NC1 domain have their effect through molecular structural damage and 3D modelling is a useful tool in their assessment.

Digenic Alport Syndrome.

Digenic Alport syndrome refers to the inheritance of pathogenic variants in COL4A5 plus COL4A3 or COL4A4 or in COL4A3 plus COL4A4 Where digenic Alport syndrome includes a pathogenic COL4A5 variant, the consequences depend on the sex of the affected individual, COL4A5 variant "severity," and the nature of the COL4A3 or COL4A4 change. A man with a pathogenic COL4A5 variant has all his collagen IV α3α4α5-heterotrimers affected, and an additional COL4A3 or COL4A4 variant may not worsen disease. A woman with a pathogenic COL4A5 variant has on average 50% of her heterotrimers affected, which is increased to 75% with a further COL4A3 or COL4A4 variant and associated with a higher risk of proteinuria. In digenic Alport syndrome with pathogenic COL4A3 and COL4A4 variants, 75% of the heterotrimers are affected. The COL4A3 and COL4A4 genes occur head-to-head on chromosome 2, and inheritance is autosomal dominant when both variants affect the same chromosome (in cis) or recessive when they affect different chromosomes (in trans). This form of digenic disease results in increased proteinuria and a median age of kidney failure intermediate between autosomal dominant and autosomal recessive Alport syndrome. Previous guidelines have suggested that all pathogenic or likely pathogenic digenic variants should be identified and reported. Affected family members should be identified, treated, and discouraged from kidney donation. Inheritance within a family is easier to predict if the two variants are considered independently and if COL4A3 and COL4A4 variants are known to be inherited on the same or different chromosomes.

Genotype-phenotype correlations for COL4A3-COL4A5 variants resulting in Gly substitutions in Alport syndrome.

Alport syndrome is the commonest inherited kidney disease and nearly half the pathogenic variants in the COL4A3-COL4A5 genes that cause Alport syndrome result in Gly substitutions. This study examined the molecular characteristics of Gly substitutions that determine the severity of clinical features. Pathogenic COL4A5 variants affecting Gly in the Leiden Open Variation Database in males with X-linked Alport syndrome were correlated with age at kidney failure (n = 157) and hearing loss diagnosis (n = 80). Heterozygous pathogenic COL4A3 and COL4A4 variants affecting Gly (n = 304) in autosomal dominant Alport syndrome were correlated with the risk of haematuria in the UK 100,000 Genomes Project. Gly substitutions were stratified by exon location (1 to 20 or 21 to carboxyl terminus), being adjacent to a non-collagenous region (interruption or terminus), and the degree of instability caused by the replacement residue. Pathogenic COL4A5 variants that resulted in a Gly substitution with a highly destabilising residue reduced the median age at kidney failure by 7 years (p = 0.002), and age at hearing loss diagnosis by 21 years (p = 0.004). Substitutions adjacent to a non-collagenous region delayed kidney failure by 19 years (p = 0.014). Heterozygous pathogenic COL4A3 and COL4A4 variants that resulted in a Gly substitution with a highly destabilising residue (Arg, Val, Glu, Asp, Trp) were associated with an increased risk of haematuria (p = 0.018), and those adjacent to a non-collagenous region were associated with a reduced risk (p = 0.046). Exon location had no effect. In addition, COL4A5 variants adjacent to non-collagenous regions were over-represented in the normal population in gnomAD (p < 0.001). The nature of the substitution and of nearby residues determine the risk of haematuria, early onset kidney failure and hearing loss for Gly substitutions in X-linked and autosomal dominant Alport syndrome.

Guidelines for Genetic Testing and Management of Alport Syndrome.

Genetic testing for pathogenic COL4A3-5 variants is usually undertaken to investigate the cause of persistent hematuria, especially with a family history of hematuria or kidney function impairment. Alport syndrome experts now advocate genetic testing for persistent hematuria, even when a heterozygous pathogenic COL4A3 or COL4A4 is suspected, and cascade testing of their first-degree family members because of their risk of impaired kidney function. The experts recommend too that COL4A3 or COL4A4 heterozygotes do not act as kidney donors. Testing for variants in the COL4A3-COL4A5 genes should also be performed for persistent proteinuria and steroid-resistant nephrotic syndrome due to suspected inherited FSGS and for familial IgA glomerulonephritis and kidney failure of unknown cause.

Humoral and cellular immunity to SARS-CoV-2 vaccination in renal transplant versus dialysis patients: A prospective, multicenter observational study using mRNA-1273 or BNT162b2 mRNA vaccine.

BACKGROUND: Dialysis and kidney transplant patients are vulnerable populations for COVID-19 related disease and mortality. METHODS: We conducted a prospective study exploring the eight week time course of specific cellular (interferon-γ release assay and flow cytometry) or/and humoral immune responses (ELISA) to SARS-CoV-2 boost vaccination in more than 3100 participants including medical personnel, dialysis patients and kidney transplant recipients using mRNA vaccines BNT162b2 or mRNA-1273. RESULTS: SARS-CoV-2-vaccination induced seroconversion efficacy in dialysis patients was similar to medical personnel (> 95%), but markedly impaired in kidney transplant recipients (42%). T-cellular immunity largely mimicked humoral results. Major risk factors of seroconversion failure were immunosuppressive drug number and type (belatacept, MMF-MPA, calcineurin-inhibitors) as well as vaccine type (BNT162b2 mRNA). Seroconversion rates induced by mRNA-1273 compared to BNT162b2 vaccine were 97% to 88% (p < 0.001) in dialysis and 49% to 26% in transplant patients, respectively. Specific IgG directed against the new binding domain of the spike protein (RDB) were significantly higher in dialysis patients vaccinated by mRNA-1273 (95%) compared to BNT162b2 (85%, p < 0.001). Vaccination appeared safe and highly effective demonstrating an almost complete lack of symptomatic COVID-19 disease after boost vaccination as well as ceased disease incidences during third pandemic wave in dialysis patients. CONCLUSION: Dialysis patients exhibit a remarkably high seroconversion rate of 95% after boost vaccination, while humoral response is impaired in the majority of transplant recipients. Immunosuppressive drug number and type as well as vaccine type (BNT162b2) are major determinants of seroconversion failure in both dialysis and transplant patients suggesting immune monitoring and adaption of vaccination protocols.

Consensus statement on standards and guidelines for the molecular diagnostics of Alport syndrome: refining the ACMG criteria.

The recent Chandos House meeting of the Alport Variant Collaborative extended the indications for screening for pathogenic variants in the COL4A5, COL4A3 and COL4A4 genes beyond the classical Alport phenotype (haematuria, renal failure; family history of haematuria or renal failure) to include persistent proteinuria, steroid-resistant nephrotic syndrome, focal and segmental glomerulosclerosis (FSGS), familial IgA glomerulonephritis and end-stage kidney failure without an obvious cause. The meeting refined the ACMG criteria for variant assessment for the Alport genes (COL4A3-5). It identified 'mutational hotspots' (PM1) in the collagen IV α5, α3 and α4 chains including position 1 Glycine residues in the Gly-X-Y repeats in the intermediate collagenous domains; and Cysteine residues in the carboxy non-collagenous domain (PP3). It considered that 'well-established' functional assays (PS3, BS3) were still mainly research tools but sequencing and minigene assays were commonly used to confirm splicing variants. It was not possible to define the Minor Allele Frequency (MAF) threshold above which variants were considered Benign (BA1, BS1), because of the different modes of inheritances of Alport syndrome, and the occurrence of hypomorphic variants (often Glycine adjacent to a non-collagenous interruption) and local founder effects. Heterozygous COL4A3 and COL4A4 variants were common 'incidental' findings also present in normal reference databases. The recognition and interpretation of hypomorphic variants in the COL4A3-COL4A5 genes remains a challenge.

Ecto-5' -Nucleotidase CD73 (NT5E), vitamin D receptor and FGF23 gene polymorphisms may play a role in the development of calcific uremic arteriolopathy in dialysis patients - Data from the German Calciphylaxis Registry.

INTRODUCTION: Calciphylaxis/calcific uremic arteriolopathy affects mainly end-stage kidney disease patients but is also associated with malignant disorders such as myeloma, melanoma and breast cancer. Genetic risk factors of calciphylaxis have never been studied before. METHODS: We investigated 10 target genes using a tagging SNP approach: the genes encoding CD73/ ecto-5'-nucleotidase (purinergic pathway), Matrix Gla protein, Fetuin A, Bone Gla protein, VKORC1 (all related to intrinsic calcification inhibition), calcium-sensing receptor, FGF23, Klotho, vitamin D receptor, stanniocalcin 1 (all related to CKD-MBD). 144 dialysis patients from the German calciphylaxis registry were compared with 370 dialysis patients without history of CUA. Genotyping was performed using iPLEX Gold MassARRAY(Sequenom, San Diego, USA), KASP genotyping chemistry (LGC, Teddington, Middlesex, UK) or sequencing. Statistical analysis comprised logistic regression analysis with adjustment for age and sex. RESULTS: 165 SNPs were finally analyzed and 6 SNPs were associated with higher probability for calciphylaxis (OR>1) in our cohort. Nine SNPs of three genes (CD73, FGF23 and Vitamin D receptor) reached nominal significance (p< 0.05), but did not reach statistical significance after correction for multiple testing. Of the CD73 gene, rs4431401 (OR = 1.71, 95%CI 1.08-2.17, p = 0.023) and rs9444348 (OR = 1.48, 95% CI 1.11-1.97, p = 0.008) were associated with a higher probability for CUA. Of the FGF23 and VDR genes, rs7310492, rs11063118, rs13312747 and rs17882106 were associated with a higher probability for CUA. CONCLUSION: Polymorphisms in the genes encoding CD73, vitamin D receptor and FGF23 may play a role in calciphylaxis development. Although our study is the largest genetic study on calciphylaxis, it is limited by the low sample sizes. It therefore requires replication in other cohorts if available.

Calcific uraemic arteriolopathy (calciphylaxis): data from a large nationwide registry.

Background: Calcific uraemic arteriolopathy (CUA, calciphylaxis) is a rare disease predominantly in dialysis patients and associated with high mortality. Painful skin ulcerations and calcification of cutaneous arterioles characterize calciphylaxis. Methods: We established an observational, Internet-based registry allowing online notification for all German CUA cases. The registry recorded data about patient characteristics, biochemistry and therapies. Blood samples were stored in a central biobank. Results: Between 2006 and 2015, 253 CUA patients were recorded: median age 70 [interquartile range (IQR) 61-76] years, 60% females and 86% ( n = 207) dialysis patients, translating into an estimated annual incidence rate of 0.04% in German dialysis patients. Fifty-two per cent received vitamin K antagonists (VKAs) prior to CUA. Skin lesions were localized in 71% on the legs or gluteal region. In dialysis CUA patients median total serum calcium was 2.20 (IQR 2.06-2.37) mmol/L, phosphorus 1.67 (IQR 1.35-2.03) mmol/L, intact parathyroid hormone 147 (IQR 72-276) pg/mL and fetuin-A 0.21 (IQR 0.16-0.26) g/L (normal range 0.35-0.95). Median sclerostin, osteoprotegerin, TRAP5b, bone-specific alkaline phosphatase and c-terminal FGF23 levels were all elevated. The most frequently recorded therapeutic procedures in dialysis CUA patients were as follows: wound debridement (29% of cases), stopping VKA (25%), lowering calcium supply (24%), sodium thiosulphate (22%), application of vitamin K (18%), increase of dialysis duration/frequency (17%) and stoping active vitamin D (16%). Conclusions: Approximately 50% of CUA patients used VKA. Our data suggest that uncontrolled hyperparathyroidism is not the key determinant of calciphylaxis. Therapeutic strategies were heterogeneous. The experience of the German registry will help substantially to initiate a large-scale multinational CUA registry.

Shedding new light on vasculitis: how the LAMP story unfolded.

The last couple of years have brought some major advances both in our understanding of antineutrophil cytoplasmic antibody (ANCA)-positive vasculitis pathogenesis mechanisms and in its treatment options. Recent discoveries of completely new antigens such as lysosome-associated membrane protein-2 (LAMP-2) have meant a huge step forward, and the fact that this antigen is homologous to proteins of bacterial fimbria caused a shift in the focus regarding underlying pathomechanisms of ANCA vasculitis toward bacterial infections, mainly with Klebsiella or Escherichia species, possibly playing a role in triggering the disease. So nephrology has seen real progress in understanding of glomerulonephritis disease mechanisms - not only regarding primary membranous glomerulonephritis (with the recent identification of the phospholipase A2 receptor being the underlying antigen) but also regarding secondary pauci-immune glomerulonephritis due to ANCA-positive vasculitis.

Antilipolytic effect of calcimimetics depends on the allelic variant of calcium-sensing receptor gene polymorphism rs1042636 (Arg990Gly).

Calcium-sensing receptor polymorphism rs1042636 (Arg990Gly) affects the response to the calcimimetic cinacalcet, used to treat hypercalcemia in secondary hyperparathyroidism (sHPT) or parathyroid carcinoma. Carriers of the Arg allelle, show less parathyroid hormone secretion suppression in response to the drug. This effect was reproducible in transfected cultured human embryonic kidney cells, supporting a causal relationship on the protein level. We previously established that cinacalcet has an antilipolytic effect in isolated human adipocytes; however, there were a number of samples that did not respond to the treatment. The present work aimed to investigate whether the variable antilipolytic response to cinacalcet in adipocytes was consistent with the effect reported for the rs1042636 polymorphism. Lipolysis was assessed by measuring glycerol release after exposure to cinacalcet (10 µM) or vehicle in adipocytes isolated from 38 donors. Responsiveness was defined as lipolysis suppression (cinacalcet vs vehicle control) greater than 20%. Genotype analysis showed that 23 adipocyte donors were homozygous for Arg at position 990, 14 heterozygous and 1 homozygous Gly-Gly. Among the Arg homozygotes, one was responsive to cinacalcet, whereas five Gly carriers responded to the calcimimetic. In all, 83% of adipocytes showing response to cinacalcet carried the glycine allele, whereas in 96% of Arg-Arg individuals adipocytes did not respond to the calcimimetic (P=0.027, Fisher's exact test). Confirming sHPT observations, adipocytes from rs1042636 Gly-allele carriers show higher sensitivity to the antilipolytic action of cinacalcet. The potential benefit of cinacalcet as a suppressor of basal lipolysis and free fatty acid release in uremic patients needs to consider the rs1042636 single-nucleotide polymorphism.

Mechanisms and treatment of extraosseous calcification in chronic kidney disease.

Strong and unidirectional associations exist between the severity of cardiovascular calcifications and mortality in patients with advanced chronic kidney disease. In the past 10 years, a wealth of experimental and clinical information has been published on the key pathophysiological events that contribute to the development and progression of vascular and soft-tissue calcifications. These processes involve a sensitive balance of calcification inhibition, induction and removal. The traditional view of regarding secondary hyperparathyroidism and elevated calcium × phosphate product as the pivotal risk factors for calcification has been challenged by data demonstrating a role for other, more subtle and complex pathomechanisms. These mechanisms include the loss of endogenous calcification inhibitors, deficient clearance of calcified debris, effects of vitamin K and vitamin D, and the action of calcification inducers as in osteogenic transdifferentiation. In this Review, we describe our current knowledge of the factors involved in the passive and active regulation of extraosseous calcification processes, with an assessment of their importance as targets for future diagnostic and therapeutic interventions.

CaSR polymorphism Arg990Gly and response to calcimimetic agents in end-stage kidney disease patients with secondary hyperparathyroidism and in cell culture.

AIMS: Calcimimetics are effective in reducing parathyroid hormone (PTH) levels in patients with secondary hyperparathyroidism, but variability in dose response has been noted. We examined SNP Arg990Gly of the calcium-sensing receptor as a possible cause. MATERIALS & METHODS: We performed a dose-response study with cinacalcet on 23 hemodialysis patients (with PTH >300 pg/ml). Intact (i)PTH levels were measured at baseline and over time post-dose; 17 patients had iPTH measured at 24 h post-dose (60 mg). Arg990Gly status was established by sequencing a section from exon 7 of the CaSR gene. RESULTS: Only 33% of patients homozygous for the arginine allele showed an iPTH suppression of at least 5% of baseline at 24 h, while 88% of patients with one or two glycine alleles achieved this target (p < 0.05). CONCLUSION: We conclude that Arg990Gly influences the response to calcimimetics in patients with secondary hyperparathyroidism with an odds ratio of 2.6. This corresponds with in vitro data testing the effect of calcimimetic agent R-568 in HEK-293 cells transfected with the two alleles of Arg990Gly: HEK-293 cells expressing glycine-type CaSR were more sensitive to R-568 than arginine-type CaSR (p = 0.0001).

Calcium-sensing receptor gene polymorphism Arg990Gly and its possible effect on response to cinacalcet HCl.

Cinacalcet, a novel calcimimetic compound, is effective in reducing parathyroid hormone (PTH) levels in approximately 70% of patients with secondary hyperparathyroidism. However, interindividual variations in the dose required to achieve the treatment goal have been noted in clinical studies. Our investigation examined the genetic polymorphisms of the calcium-sensing receptor (CaSR) gene as one possible cause of the different responses to cinacalcet. We report data on seven end-stage renal failure patients who were treated with regular haemodialysis and who participated in clinical trials of cinacalcet. All patients had secondary hyperparathyroidism with baseline intact PTH (iPTH) levels greater than 600 pg/ml. Three patients were male and four female with a mean+/-SD age of 60+/-12 years. DNA was extracted from peripheral lymphocytes. An area in exon 7 of the CaSR gene was amplified by the polymerase chain reaction and sequenced. Mean+/-SD baseline iPTH was 1086+/-189 pg/ml. The five patients without Arg990Gly demonstrated a 29.7+/-4.0% (+/-SEM) reduction in iPTH from individual baseline. One patient was found to be homozygous for the Arg990Gly polymorphism and another was heterozygous for both arginine and glycine alleles. The homozygous patient showed a significantly higher sensitivity to cinacalcet compared to the other patients (P=0.003) with a 76.3+/-7.7% reduction in iPTH from baseline. No polymorphisms were noted in codons 986 or 1011. This preliminary study points to the possibility that patients homozygous for glycine at the 990 position in exon 7 of the CaSR may be more sensitive to the calcimimetic drug cinacalcet compared to those who are homozygous for arginine at that location.