Alport syndrome and Alport kidney diseases - elucidating the disease spectrum.

PURPOSE OF REVIEW: With the latest classification, variants in three collagen IV genes, COL4A3 , COL4A4 , and COL4A5 , represent the most prevalent genetic kidney disease in humans, exhibiting diverse, complex, and inconsistent clinical manifestations. This review breaks down the disease spectrum and genotype-phenotype correlations of kidney diseases linked to genetic variants in these genes and distinguishes "classic" Alport syndrome (AS) from the less severe nonsyndromic genetically related nephropathies that we suggest be called "Alport kidney diseases". RECENT FINDINGS: Several research studies have focused on the genotype-phenotype correlation under the latest classification scheme of AS. The historic diagnoses of "benign familial hematuria" and "thin basement membrane nephropathy" linked to heterozygous variants in COL4A3 or COL4A4 are suggested to be obsolete, but instead classified as autosomal AS by recent expert consensus due to a significant risk of disease progression. SUMMARY: The concept of Alport kidney disease extends beyond classic AS. Patients carrying pathogenic variants in any one of the COL4A3/A4/A5 genes can have variable phenotypes ranging from completely normal/clinically unrecognizable, hematuria without or with proteinuria, or progression to chronic kidney disease and kidney failure, depending on sex, genotype, and interplays of other genetic as well as environmental factors.

Is there a dominant-negative effect in individuals with heterozygous disease-causing variants in COL4A3/COL4A4?

Alport syndrome (AS) shows a broad phenotypic spectrum ranging from isolated microscopic hematuria (MH) to end-stage kidney disease (ESKD). Monoallelic disease-causing variants in COL4A3/COL4A4 have been associated with autosomal dominant AS (ADAS) and biallelic variants with autosomal recessive AS (ARAS). The aim of this study was to analyze clinical and genetic data regarding a possible genotype-phenotype correlation in individuals with disease-causing variants in COL4A3/COL4A4. Eighty-nine individuals carrying at least one COL4A3/COL4A4 variant classified as (likely) pathogenic according to the American College of Medical Genetics guidelines and current amendments were recruited. Clinical data concerning the prevalence and age of first reported manifestation of MH, proteinuria, ESKD, and extrarenal manifestations were collected. Individuals with monoallelic non-truncating variants reported a significantly higher prevalence and earlier diagnosis of MH and proteinuria than individuals with monoallelic truncating variants. Individuals with biallelic variants were more severely affected than those with monoallelic variants. Those with biallelic truncating variants were more severely affected than those with compound heterozygous non-truncating/truncating variants or individuals with biallelic non-truncating variants. In this study an association of heterozygous non-truncating COL4A3/COL4A4 variants with a more severe phenotype in comparison to truncating variants could be shown indicating a potential dominant-negative effect as an explanation for this observation. The results for individuals with ARAS support the, still scarce, data in the literature.

Whole exome sequencing approach for identification of the molecular etiology in pediatric patients with hematuria.

BACKGROUND: Hematuria is a common condition in clinical practice of pediatric patients. It is related to a wide spectrum of disorders and has high heterogeneity both clinically and genetically, which contributes to challenges of diagnosis and lead many pediatric patients with hematuria not to receive accurate diagnosis and early management. METHODS: In this single center study, 42 children with hematuria were included in Tianjin Children's Hospital between 2019 and 2020. We analyzed the clinical information and performed WES (Whole exome sequencing) for all cases. Then the classification of identified variants was performed according to the American College of Medical Genetics and Genomics (ACMG) guidelines for interpreting sequence variants. For the fragment deletion, qPCR was performed to validate and confirm the inherited pattern. RESULTS: For the 42 patients, 16 cases had gross hematuria and 26 had microscopic hematuria. Molecular genetic causes were uncovered in 9 (21.4%) children, including 7 with Alport syndrome (AS), one with polycystic nephropathy and one with lipoprotein glomerulopathy. The genetic causes for other patients were not related with hematuria. CONCLUSIONS: WES is a rapid and effective way to evaluate patients with hematuria. The analysis of genotype-phenotype correlations of patients with AS indicated that severe variants were associated with early kidney failure. Secondary findings were not rare in Chinese children, thus the clinician should pay more attention to the clinical interpretation of sequencing results and properly interaction with patients and their family.

GWAS for the composite traits of hematuria and albuminuria.

Our GWAS of hematuria in the UK Biobank identified 6 loci, some of which overlap with loci for albuminuria suggesting pleiotropy. Since clinical syndromes are often defined by combinations of traits, generating a combined phenotype can improve power to detect loci influencing multiple characteristics. Thus the composite trait of hematuria and albuminuria was chosen to enrich for glomerular pathologies. Cases had both hematuria defined by ICD codes and albuminuria defined as uACR > 3 mg/mmol. Controls had neither an ICD code for hematuria nor an uACR > 3 mg/mmol. 2429 cases and 343,509 controls from the UK Biobank were included. eGFR was lower in cases compared to controls, with the exception of the comparison in females using CKD-EPI after age adjustment. Variants at 4 loci met genome-wide significance with the following nearest genes: COL4A4, TRIM27, ETV1 and CUBN. TRIM27 is part of the extended MHC locus. All loci with the exception of ETV1 were replicated in the Geisinger MyCode cohort. The previous GWAS of hematuria reported COL4A3-COL4A4 variants and HLA-B*0801 within MHC, which is in linkage disequilibrium with the TRIM27 variant (D' = 0.59). TRIM27 is highly expressed in the tubules. Additional loci included a coding sequence variant in CUBN (p.Ala2914Val, MAF = 0.014 (A), p = 3.29E-8, OR = 2.09, 95% CI = 1.61-2.72). Overall, GWAS for the composite trait of hematuria and albuminuria identified 4 loci, 2 of which were not previously identified in a GWAS of hematuria.

Genetic Modifiers of Mendelian Monogenic Collagen IV Nephropathies in Humans and Mice.

Familial hematuria is a clinical sign of a genetically heterogeneous group of conditions, accompanied by broad inter- and intrafamilial variable expressivity. The most frequent condition is caused by pathogenic (or likely pathogenic) variants in the collagen-IV genes, COL4A3/A4/A5. Pathogenic variants in COL4A5 are responsible for the severe X-linked glomerulopathy, Alport syndrome (AS), while homozygous or compound heterozygous variants in the COL4A3 or the COL4A4 gene cause autosomal recessive AS. AS usually leads to progressive kidney failure before the age of 40-years when left untreated. People who inherit heterozygous COL4A3/A4 variants are at-risk of a slowly progressive form of the disease, starting with microscopic hematuria in early childhood, developing Alport spectrum nephropathy. Sometimes, they are diagnosed with benign familial hematuria, and sometimes with autosomal dominant AS. At diagnosis, they often show thin basement membrane nephropathy, reflecting the uniform thin glomerular basement membrane lesion, inherited as an autosomal dominant condition. On a long follow-up, most patients will retain normal or mildly affected kidney function, while a substantial proportion will develop chronic kidney disease (CKD), even kidney failure at an average age of 55-years. A question that remains unanswered is how to distinguish those patients with AS or with heterozygous COL4A3/A4 variants who will manifest a more aggressive kidney function decline, requiring prompt medical intervention. The hypothesis that a subgroup of patients coinherit additional genetic modifiers that exacerbate their clinical course has been investigated by several researchers. Here, we review all publications that describe the potential role of candidate genetic modifiers in patients and include a summary of studies in AS mouse models.

[COL4A5 genotypes and clinical characteristics of children with Alport syndrome]. / 儿童Alport综合征COL4A5åŸºå› åž‹åŠä¸´åºŠç‰¹ç‚¹åˆ†æž.

OBJECTIVES: To investigate the genotypes of the pathogenic gene COL4A5 and the characteristics of clinical phenotypes in children with Alport syndrome (AS). METHODS: A retrospective analysis was performed for the genetic testing results and clinical data of 19 AS children with COL4A5 gene mutations. RESULTS: Among the 19 children with AS caused by COL4A5 gene mutations, 1 (5%) carried a new mutation of the COL4A5 gene, i.e., c.3372A>G(p.P1124=) and presented with AS coexisting with IgA vasculitis nephritis; 3 children (16%) had large fragment deletion of the COL4A5 gene, among whom 2 children (case 7 had a new mutation site of loss51-53) had gross hematuria and albuminuria at the onset, and 1 child (case 13 had a new mutation site of loss3-53) only had microscopic hematuria, while the other 15 children (79%) had common clinical phenotypes of AS, among whom 7 carried new mutations of the COL4A5 gene. Among all 19 children, 3 children (16%) who carried COL4A5 gene mutations also had COL4A4 gene mutations, and 1 child (5%) had COL4A3 gene mutations. Among these children with double gene mutations, 2 had gross hematuria and proteinuria at the onset. CONCLUSIONS: This study expands the genotype and phenotype spectrums of the pathogenic gene COL4A5 for AS. Children with large fragment deletion of the COL4A5 gene or double gene mutations of COL4A5 with COL4A3 or COL4A4 tend to have more serious clinical manifestations.

Development and Multicenter Case-Control Validation of Urinary Comprehensive Genomic Profiling for Urothelial Carcinoma Diagnosis, Surveillance, and Risk-Prediction.

PURPOSE: Urinary comprehensive genomic profiling (uCGP) uses next-generation sequencing to identify mutations associated with urothelial carcinoma and has the potential to improve patient outcomes by noninvasively diagnosing disease, predicting grade and stage, and estimating recurrence risk. EXPERIMENTAL DESIGN: This is a multicenter case-control study using banked urine specimens collected from patients undergoing initial diagnosis/hematuria workup or urothelial carcinoma surveillance. A total of 581 samples were analyzed by uCGP: 333 for disease classification and grading algorithm development, and 248 for blinded validation. uCGP testing was done using the UroAmp platform, which identifies five classes of mutation: single-nucleotide variants, copy-number variants, small insertion-deletions, copy-neutral loss of heterozygosity, and aneuploidy. UroAmp algorithms predicting urothelial carcinoma tumor presence, grade, and recurrence risk were compared with cytology, cystoscopy, and pathology. RESULTS: uCGP algorithms had a validation sensitivity/specificity of 95%/90% for initial cancer diagnosis in patients with hematuria and demonstrated a negative predictive value (NPV) of 99%. A positive diagnostic likelihood ratio (DLR) of 9.2 and a negative DLR of 0.05 demonstrate the ability to risk-stratify patients presenting with hematuria. In surveillance patients, binary urothelial carcinoma classification demonstrated an NPV of 91%. uCGP recurrence-risk prediction significantly prognosticated future recurrence (hazard ratio, 6.2), whereas clinical risk factors did not. uCGP demonstrated positive predictive value (PPV) comparable with cytology (45% vs. 42%) with much higher sensitivity (79% vs. 25%). Finally, molecular grade predictions had a PPV of 88% and a specificity of 95%. CONCLUSIONS: uCGP enables noninvasive, accurate urothelial carcinoma diagnosis and risk stratification in both hematuria and urothelial carcinoma surveillance patients.

Evaluation of Sensitive Urine DNA-Based PENK Methylation Test for Detecting Bladder Cancer in Patients with Hematuria.

Hematuria is a prevalent symptom associated with bladder cancer (BC). However, the invasiveness and cost of cystoscopy, the current gold standard for BC diagnosis in patients with hematuria, necessitate the development of a sensitive and accurate noninvasive test. This study introduces and validates a highly sensitive urine-based DNA methylation test. The test improves sensitivity in detecting PENK methylation in urine DNA using linear target enrichment followed by quantitative methylation-specific PCR. In a case-control study comprising 175 patients with BC and 143 patients without BC with hematuria, the test's optimal cutoff value was determined by distinguishing between two groups, achieved an overall sensitivity of 86.9% and a specificity of 91.6%, with an area under the curve of 0.892. A prospective validation clinical study involving 366 patients with hematuria scheduled for cystoscopy assessed the test's performance. The test demonstrated an overall sensitivity of 84.2% in detecting 38 cases of BC, a specificity of 95.7%, and an area under the curve of 0.900. Notably, the sensitivity for detecting Ta high grade and higher stages of BC reached 92.3%. The test's negative predictive value was 98.2%, and the positive predictive value was 68.7%. These findings highlight the potential of the PENK methylation in urine DNA using linear target enrichment followed by quantitative methylation-specific PCR test in urine as a promising molecular diagnostic tool for detecting primary BC in patients with hematuria, which may reduce the need for cystoscopy.

Predicting the germline dependence of hematuria risk in prostate cancer radiotherapy patients.

BACKGROUND AND PURPOSE: Late radiation-induced hematuria can develop in prostate cancer patients undergoing radiotherapy and can negatively impact the quality-of-life of survivors. If a genetic component of risk could be modeled, this could potentially be the basis for modifying treatment for high-risk patients. We therefore investigated whether a previously developed machine learning-based modeling method using genome-wide common single nucleotide polymorphisms (SNPs) can stratify patients in terms of the risk of radiation-induced hematuria. MATERIALS AND METHODS: We applied a two-step machine learning algorithm that we previously developed for genome-wide association studies called pre-conditioned random forest regression (PRFR). PRFR includes a pre-conditioning step, producing adjusted outcomes, followed by random forest regression modeling. Data was from germline genome-wide SNPs for 668 prostate cancer patients treated with radiotherapy. The cohort was stratified only once, at the outset of the modeling process, into two groups: a training set (2/3 of samples) for modeling and a validation set (1/3 of samples). Post-modeling bioinformatics analysis was conducted to identify biological correlates plausibly associated with the risk of hematuria. RESULTS: The PRFR method achieved significantly better predictive performance compared to other alternative methods (all p < 0.05). The odds ratio between the high and low risk groups, each of which consisted of 1/3 of samples in the validation set, was 2.87 (p = 0.029), implying a clinically useful level of discrimination. Bioinformatics analysis identified six key proteins encoded by CTNND2, GSK3B, KCNQ2, NEDD4L, PRKAA1, and TXNL1 genes as well as four statistically significant biological process networks previously shown to be associated with the bladder and urinary tract. CONCLUSION: The risk of hematuria is significantly dependent on common genetic variants. The PRFR algorithm resulted in a stratification of prostate cancer patients at differential risk levels of post-radiotherapy hematuria. Bioinformatics analysis identified important biological processes involved in radiation-induced hematuria.

COL4A gene variants are common in children with hematuria and a family history of kidney disease.

BACKGROUND: Inherited kidney diseases are a common cause of chronic kidney disease (CKD) in children. Identification of a monogenic cause of CKD is more common in children than in adults. This study evaluated the diagnostic yield and phenotypic spectrum of children who received genetic testing through the KIDNEYCODE sponsored genetic testing program. METHODS: Unrelated children < 18 years of age who received panel testing through the KIDNEYCODE sponsored genetic testing program from September 2019 through August 2021 were included (N = 832). Eligible children met at least one of the following clinician-reported criteria: estimated GFR ≤ 90 ml/min/1.73 m2, hematuria, a family history of kidney disease, or suspected or biopsy confirmed Alport syndrome or focal segmental glomerulosclerosis (FSGS) in the tested individual or family member. RESULTS: A positive genetic diagnosis was observed in 234 children (28.1%, 95% CI [25.2-31.4%]) in genes associated with Alport syndrome (N = 213), FSGS (N = 9), or other disorders (N = 12). Among children with a family history of kidney disease, 30.8% had a positive genetic diagnosis. Among those with hematuria and a family history of CKD, the genetic diagnostic rate increased to 40.4%. CONCLUSIONS: Children with hematuria and a family history of CKD have a high likelihood of being diagnosed with a monogenic cause of kidney disease, identified through KIDNEYCODE panel testing, particularly COL4A variants. Early genetic diagnosis can be valuable in targeting appropriate therapy and identification of other at-risk family members. A higher resolution version of the Graphical abstract is available as Supplementary information.

Genetic features and kidney morphological changes in women with X-linked Alport syndrome.

BACKGROUND: X-linked Alport syndrome (XLAS) caused by COL4A5 pathogenic variants usually has heterogeneous phenotypes in female patients. The genetic characteristics and glomerular basement membrane (GBM) morphological changes in women with XLAS need to been further investigated. METHODS: A total of 83 women and 187 men with causative COL4A5 variants were enrolled for comparative analysis. RESULTS: Women were more frequently carrying de novo COL4A5 variants compared with men (47% vs 8%, p=0.001). The clinical manifestations in women were variable, and no genotype-phenotype correlation was observed. Coinherited podocyte-related genes, including TRPC6, TBC1D8B, INF2 and MYH9, were identified in two women and five men, and the modifying effects of coinherited genes contributed to the heterogeneous phenotypes in these patients. X-chromosome inactivation (XCI) analysis of 16 women showed that 25% were skewed XCI. One patient preferentially expressing the mutant COL4A5 gene developed moderate proteinuria, and two patients preferentially expressing the wild-type COL4A5 gene presented with haematuria only. GBM ultrastructural evaluation demonstrated that the degree of GBM lesions was associated with the decline in kidney function for both genders, but more severe GBM changes were found in men compared with women. CONCLUSIONS: The high frequency of de novo variants carried by women indicates that the lack of family history tends to make them susceptible to be underdiagnosed. Coinherited podocyte-related genes are potential contributors to the heterogeneous phenotype of some women. Furthermore, the association between the degree of GBM lesions and decline in kidney function is valuable in evaluating the prognosis for patients with XLAS.

Combined Alport syndrome, Klinefelter syndrome and Fanconi syndrome in a Chinese boy.

Alport syndrome (AS) is a progressive renal disease characterized by hematuria and progressive renal failure. X-linked dominant (XLAS) is the major inheritance form, accounting for almost 80% of the cases, caused by mutations in COL4A5 genes. Klinefelter syndrome (KS) is the most common genetic cause of human male gonadal dysgenesis. AS and KS are both rare disease, there are only three cases of combined AS and KS in the literatures. Fanconi syndrome (FS) caused by AS is also very rare. We report here the first case combined AS, KS and FS in a Chinese boy. We suggest that the severe renal phenotype and FS might be due to the two homozygous COL4A5 variants in our boy, and cases of AS combined KS will be good research objects for X chromosome inactivation.

Clinical and diagnostic utility of genomic sequencing for children referred to a Kidney Genomics Clinic with microscopic haematuria.

BACKGROUND: Microscopic haematuria in children is associated with the risk of progression to chronic kidney disease. Genetic disease is an important potential aetiology. Genomic sequencing presents the most effective diagnostic route for these conditions, but access remains inequitable internationally. METHODS: We conducted a retrospective review of the electronic medical records of a Kidney Genomics Clinic (KGC) from January 2016 to December 2021. RESULTS: Sixty patients were referred to the KGC with haematuria over this period. Forty-three percent of patients had analysis of a limited haematuria panel (COL4A1, COL4A3, COL4A4, COL4A5, MYH9) with 58% receiving a genetic diagnosis. Forty-two percent of referred patients had further analysis of genes implicated in the development of kidney disease, and 36% received a diagnosis. Eight percent of patients underwent cascade testing for a known familial variant, and all received a diagnosis. Children with the highest levels of haematuria (> 500 × 106/L red blood cells) had the highest diagnostic yield (67%). Proteinuria, defined as a urinary protein to creatinine ratio > 20, increased the diagnostic yield from 31 to 65%. Importantly, negative genetic analysis can still have significant clinical utility for patients by altering surveillance and further management; the genetic result had clinical utility in 60% of patients. CONCLUSIONS: Our KGC review highlights the substantial clinical utility and diagnostic yield of genomic analysis for microscopic haematuria in paediatric patients. Whilst the management of variants of uncertain significance can be challenging, a multidisciplinary team including genetic counselling can help ensure these patients are followed up meaningfully. A higher resolution version of the Graphical abstract is available as Supplementary information.

Urinary Analysis of FGFR3 and TERT Gene Mutations Enhances Performance of Cxbladder Tests and Improves Patient Risk Stratification.

PURPOSE: Cxbladder tests are urinary biomarker tests for detection of urothelial carcinoma. We developed enhanced Cxbladder tests that incorporate DNA analysis of 6 single nucleotide polymorphisms for the FGFR3 and TERT genes, in addition to the current 5 mRNA biomarkers and clinical risk factors. MATERIALS AND METHODS: Two multicenter, prospective studies were undertaken in: (1) U.S. patients with gross hematuria aged ≥18 years and (2) Singaporean patients with gross hematuria or microhematuria aged >21 years. All patients provided a midstream urine sample and underwent cystoscopy. Samples were retrospectively analyzed using enhanced Cxbladder-Triage (risk stratifies patients), enhanced Cxbladder-Detect (risk stratifies patients and detects positive patients), and the combination enhanced Cxbladder-Triage × Cxbladder-Detect. RESULTS: In the pooled cohort (N=804; gross hematuria: n=484, microhematuria: n=320), enhanced Cxbladder-Detect had a sensitivity of 97% (95% CI 89%-100%), specificity of 90% (95% CI 88%-92%), and negative predictive value of 99.7% (95% CI 99%-100%) for detection of urothelial carcinoma. Overall, 83% of patients were enhanced Cxbladder-Detect-negative (ie, needed no further work-up). Of 133 enhanced Cxbladder-Detect-positive patients, 59 had a confirmed tumor, of which 19 were low-grade noninvasive papillary carcinoma or papillary urothelial neoplasm of low malignant potential. In total, 40 tumors were high-grade Ta, T1-T4, Tis, including concomitant carcinoma in situ. Of the 74 patients with normal cystoscopy, 41 were positive by single nucleotide polymorphism analysis. Enhanced Cxbladder-Triage and enhanced Cxbladder-Detect had significantly better specificity than the first-generation Cxbladder tests (P < .001). CONCLUSIONS: This study in ethnically diverse patients with hematuria showed the analytical validity of the enhanced Cxbladder tests.

Renal Involvement in H Syndrome, A Rare Cause of Diabetes Mellitus: Case Report.

BACKGROUND: H syndrome is a rare genodermatosis deriving from a mutation in the SLC29A3 gene and affecting numerous systems, particularly the skin. The syndrome exhibits different clinical characteristics involving several systems, most beginning with the letter "H." The most common clinical findings are cutaneous hyperpigmentation, flexion contracture in the fingers, hearing loss, short stature, insulin-dependent diabetes mellitus, heart anomalies, hepatosplenomegaly, and hypogonadism. Fewer than 150 cases have been reported so far and vast majority of them consisted with patients with Arab ethnicity. CASE PRESENTATION: We describe a patient presenting with short stature, developing diabetes mellitus at follow-ups, with homozygous deletion determined in exon 3 of the SLC29A3 gene, and diagnosed with H syndrome, reported due to the presence and rarity of renal involvement (hematuria and proteinuria). CONCLUSION: In conclusion, despite its rarity, endocrinologists, rheumatologists/nephrologists, and dermatologists need to be aware of H syndrome as a pleiotropic syndrome. H syndrome should be considered in the differential diagnosis of patients with cutaneous hyperpigmentation (particularly in the bilateral thigh and calf region) together with proteinuria/hematuria. In addition, periodic urine analysis should be performed in patients with H syndrome.

Variants in genes coding for collagen type IV α-chains are frequent causes of persistent, isolated hematuria during childhood.

BACKGROUND: Children with persistent, isolated microscopic hematuria typically undergo a limited diagnostic workup and are monitored for signs of kidney disease in long-term longitudinal follow-up, which can delay diagnosis and allow disease progression in some cases. METHODS: To determine the clinical utility of genetic screening in this population, we performed targeted genetic testing using a custom, 32-gene next-generation sequencing panel for progressive kidney disease on children referred to the Texas Children's Hospital Pediatric Nephrology clinic for persistent, microscopic hematuria (n = 30; cohort 1). Patients with microscopic hematuria identified by urinalysis on at least two separate occasions were eligible for enrollment, but those with other evidence of kidney disease were excluded. Results were analyzed for sequence variants using the American College of Medical Genetics and Genomics (ACMG) guideline for data interpretation and were validated using a secondary analysis of a dataset of children with hematuria and normal kidney function who had undergone genetic testing as part of an industry-sponsored program (cohort 2; n = 67). RESULTS: In cohort 1 33% of subjects (10/30) had pathogenic or likely pathogenic (P/LP) variants in the type IV collagen genes (COL4A3/A4/A5), and 10% (3/30) had variants of uncertain significance in these genes. The high diagnostic rate in type IV collagen genes was confirmed in cohort 2, where 27% (18/67) of subjects had P/LP variants in COL4A3/A4/A5 genes. CONCLUSIONS: Children with persistent, isolated microscopic hematuria have a high likelihood of having pathogenic variants in type IV collagen genes and genetic screening should be considered. A higher resolution version of the Graphical abstract is available as Supplementary information.

Highly Sensitive and Specific Detection of Bladder Cancer via Targeted Ultra-deep Sequencing of Urinary DNA.

BACKGROUND: There is an unmet need for an accurate, validated, noninvasive test for diagnosing and monitoring bladder cancer (BC). Detection of BC-associated mutations in urinary DNA via targeted deep sequencing could meet this need. OBJECTIVE: To test the ability of mutational analysis of urinary DNA to noninvasively detect BC within the context of haematuria investigations and non-muscle-invasive BC (NMIBC) surveillance. DESIGN, SETTING, AND PARTICIPANTS: Capture-based ultra-deep sequencing was performed for 443 somatic mutations in 23 genes in 591 urine cell-pellet DNAs from haematuria clinic patients and 293 from NMIBC surveillance patients. Variant calling was optimised to minimise false positives using urine samples from 162 haematuria clinic patients without BC. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The sensitivity and specificity for BC diagnosis were determined. RESULTS AND LIMITATIONS: Mutational analysis of urinary DNA detected 144 of the 165 haematuria patients diagnosed with incident BC from two independent cohorts, yielding overall sensitivity of 87.3% (95% confidence interval [CI] 81.2-92.0%) at specificity of 84.8% (95% CI 79.9-89.0%). The sensitivity was 97.4% for grade 3, 86.5% for grade 2, and 70.8% for grade 1 BC. Among NMIBC surveillance patients, 25 out of 29 recurrent BCs were detected, yielding sensitivity of 86.2% (95% CI 70.8-97.7%) at specificity of 62.5% (95% CI 56.1-68.0%); a positive urine mutation test in the absence of clinically detectable disease was associated with a 2.6-fold increase in the risk of future recurrence. The low number of recurrences in the NMIBC surveillance cohort and the lower sensitivity for detecting grade 1 pTa BC are limitations. CONCLUSIONS: Detection of mutations in a small panel of BC-associated genes could facilitate noninvasive BC testing and expedite haematuria investigations. Following further validation, the test could also play a role in NMIBC surveillance. PATIENT SUMMARY: Identification of alterations in genes that are frequently mutated in bladder cancer appears to be a promising strategy for detecting disease from urine samples and reducing reliance on examination of the bladder via a telescopic camera inserted through the urethra.

A Urine-based Genomic Assay Improves Risk Stratification for Patients with High-risk Hematuria Stratified According to the American Urological Association Guidelines.

BACKGROUND: According to the recent American Urological Association (AUA) guideline on hematuria, patients are stratified into groups with low, intermediate, and high risk of urothelial carcinoma (UC). These risk groups are based on clinical factors and do not incorporate urine-based tumor markers. OBJECTIVE: To evaluate whether a urine-based genomic assay improves the redefined AUA risk stratification for hematuria. DESIGN, SETTING, AND PARTICIPANTS: We selected patients with complete biomarker status, as assessed on urinary DNA, from a previously collected prospective Dutch hematuria cohort (n = 838). Patients were stratified into the AUA risk categories on the basis of sex, age, and type of hematuria. Biomarker status included mutation status for the FGFR3, TERT, and HRAS genes, and methylation status for the OTX1, ONECUT2, and TWIST1 genes. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary endpoint was the diagnostic model performance for different hematuria risk groups. Further analyses assessed the pretest and post-test UC probability in the hematuria subgroups using a Fagan nomogram. RESULTS AND LIMITATIONS: Overall, 65 patients (7.8%) were classified as low risk, 106 (12.6%) as intermediate risk, and 667 (79.6%) as high risk. The UC incidence differed significantly between the gross hematuria (21%, 98/457) and microscopic hematuria (4%, 14/381) groups (p < 0.001). All cancer cases were in the high-risk group, which had UC incidence of 16.8% (112/667). Application of the diagnostic model revealed robust performance among all risk groups (area under the receiver operating characteristic curve 0.929-0.971). Depending on the risk group evaluated, a negative urine assay was associated with post-test UC probability of 0.3-2%, whereas a positive urine assay was associated with post-test UC probability of 31-42%. CONCLUSIONS: This study shows the value that a urine-based genomic assay adds to the AUA guideline stratification for patients with hematuria. It seems justified to safely withhold cystoscopy for patients with AUA low risk who have a negative urine assay. In addition, evaluation should be expedited for patients with AUA intermediate or high risk and a positive urine assay. PATIENT SUMMARY: Patients who have blood in their urine (hematuria) can be classified as having low, intermediate, or high risk of having cancer in their urinary tract. We found that use of a urine-based genetic test improves the accuracy of predicting which patients are most likely to have cancer. Patients with a negative test may be able to avoid invasive tests, while further tests could be prioritized for patients with a positive test.

A COL4A4-G394S Variant and Impaired Collagen IV Trimerization in a Patient with Mild Alport Syndrome.

Background: Missense variants in COL4A genes are often found in patients with an Alport syndrome-like presentation, but their pathogenicity is not always clear. We encountered a woman with microscopic hematuria and proteinuria at 33 years of age with a diagnosis of thin basement membrane disease who was approaching end stage kidney disease at 59 years of age. We hypothesized that this patient's kidney disease was within the spectrum of Alport syndrome. Methods: We used histologic, genetic, and biochemical approaches to investigate the mechanisms of kidney disease. By immunofluorescence, we investigated collagen IV chain composition of the glomerular basement membrane (GBM). We employed targeted sequencing to search for pathogenic variants in COL4A and other relevant genes. We utilized N- and C-terminal split NanoLuciferase assays to determine the effect of a novel COL4A4 variant of uncertain significance (VUS) on collagen IV heterotrimer formation in vitro. We transfected COL4A4 expression constructs with split NanoLuciferase fragment-fused COL4A3 and COL4A5 constructs into human embryonic kidney 293T cells. To assay for α3α4α5(IV) heterotrimer formation and secretion, we measured luminescence in cell lysates and culture supernatants from transfected cells. Results: Immunostaining suggested that the collagen α3α4α5(IV) network was present throughout the patient's GBMs. DNA sequencing revealed a novel homozygous VUS: COL4A4 c.1180G>A (p. Gly394Ser). In the C-terminal split luciferase-based α3α4α5(IV) heterotrimer formation assays, luminescence levels for G394S were comparable to WT, but in the N-terminal tag assays, the extracellular luminescence levels for G394S were decreased by approximately 50% compared with WT. Conclusions: Our cell-based assay provides a platform to test COL4 VUS and shows that G394S impairs assembly of the α3α4α5(IV) N-terminus and subsequent trimer secretion. These data suggest that the COL4A4-G394S variant is pathogenic and causes an atypical mild form of autosomal recessive Alport syndrome.

BladMetrix: a novel urine DNA methylation test with high accuracy for detection of bladder cancer in hematuria patients.

BACKGROUND: Cystoscopy is the gold standard for bladder cancer detection, but is costly, invasive and has imperfect diagnostic accuracy. We aimed to identify novel and accurate DNA methylation biomarkers for non-invasive detection of bladder cancer in urine, with the potential to reduce the number of cystoscopies among hematuria patients. RESULTS: Biomarker candidates (n = 32) were identified from methylome sequencing of urological cancer cell lines (n = 16) and subjected to targeted methylation analysis in tissue samples (n = 60). The most promising biomarkers (n = 8) were combined into a panel named BladMetrix. The performance of BladMetrix in urine was assessed in a discovery series (n = 112), consisting of bladder cancer patients, patients with other urological cancers and healthy individuals, resulting in 95.7% sensitivity and 94.7% specificity. BladMetrix was furthermore evaluated in an independent prospective and blinded series of urine from patients with gross hematuria (n = 273), achieving 92.1% sensitivity, 93.3% specificity and a negative predictive value of 98.1%, with the potential to reduce the number of cystoscopies by 56.4%. CONCLUSIONS: We here present BladMetrix, a novel DNA methylation urine test for non-invasive detection of bladder cancer, with high accuracy across tumor grades and stages, and the ability to spare a significant number of cystoscopies among patients with gross hematuria.