Marcin Zaniew

Copy-Number Disorders Are a Common Cause of Congenital Kidney Malformations

We examined the burden of large, rare, copy-number variants (CNVs) in 192 individuals with renal hypodysplasia (RHD) and replicated findings in 330 RHD cases from two independent cohorts. CNV distribution was significantly skewed toward larger gene-disrupting events in RHD cases compared to 4,733 ethnicity-matched controls (p = 4.8 × 10(-11)). This excess was attributable to known and novel (i.e., not present in any database or in the literature) genomic disorders. All together, 55/522 (10.5%) RHD cases harbored 34 distinct known genomic disorders, which were detected in only 0.2% of 13,839 population controls (p = 1.2 × 10(-58)). Another 32 (6.1%) RHD cases harbored large gene-disrupting CNVs that were absent from or extremely rare in the 13,839 population controls, identifying 38 potential novel or rare genomic disorders for this trait. Deletions at the HNF1B locus and the DiGeorge/velocardiofacial locus were most frequent. However, the majority of disorders were detected in a single individual. Genomic disorders were detected in 22.5% of individuals with multiple malformations and 14.5% of individuals with isolated urinary-tract defects; 14 individuals harbored two or more diagnostic or rare CNVs. Strikingly, the majority of the known CNV disorders detected in the RHD cohort have previous associations with developmental delay or neuropsychiatric diseases. Up to 16.6% of individuals with kidney malformations had a molecular diagnosis attributable to a copy-number disorder, suggesting kidney malformations as a sentinel manifestation of pathogenic genomic imbalances. A search for pathogenic CNVs should be considered in this population for the diagnosis of their specific genomic disorders and for the evaluation of the potential for developmental delay.

Th1/Th2 balance and CD45-positive T cell subsets in primary nephrotic syndrome

Abstract T cells are involved in the pathogenesis of nephrotic syndrome (NS). The aim of the study was to determine whether the activity of T-helper-1 (Th1) and T-helper-2 (Th2) cells and the distribution of the lymphocyte subsets, namely CD45RA+CD4+ (”naive” helper T cells, suppressor-inducer), CD45RA+CD8+ (”naive” suppressor T cells, suppressor-effector), CD45RO+CD4+ (”memory” helper T cells), are predictive for steroid sensitivity in children with primary NS. These parameters were assessed at the onset of disease, before initiation of steroid therapy. Two groups of NS children were retrospectively formed according to steroid sensitivity (SS) or resistance (SR). The activity of Th1 and Th2 cells was defined by the production of interleukin-2 (IL-2), interferon-γ, IL-4, and IL-10 in the supernatants of CD4+ T cell cultures activated with autologous monocytes presenting tetanus toxoid (TT). Peripheral lymphocyte subsets were determined using double- or triple-color flow cytometry. In SS children with NS we found a decreased proliferative response of CD4+ T cells to TT stimulation, cytokine synthesis indicating the predominance of Th2 activity, and an increased percentage of activated suppressor-inducer (CD45RA+ CD4+CD25+, 5.18±0.8, P<0.001) and suppressor-effector (CD45RA+CD8+CD25+, 2.05±0.6, P<0.01) cells, with the concomitant reduction of activated memory cells (CD45RO+CD4+CD25+, 0.2±0.1, P<0.001). In children with SRNS we found an increased proliferative response of CD4+ T cells to TT, a rise in activated memory (CD45RO+CD4+CD25+, 3.82±0.7, P<0.01) and suppressor-inducer peripheral T cells (CD45RA+ CD4+CD25+, 3.85±0.6, P<0.01), but a low percentage of activated suppressor-effector (CD45RA+CD8+ CD25+, 0.5±0.2, P<0.05) T cells. We conclude that prior to treatment the distribution of lymphocyte subpopulations in peripheral blood together with Th1 and Th2 cell activity provides a useful tool for evaluating the likelihood of steroid sensitivity in patients with primary NS.

Increased apoptosis of peripheral blood lymphocytes in children with nephrotic syndrome.

Abstract Nephrotic syndrome is accompanied by and probably related to abnormal T-lymphocyte function. Decreased stimulation of survival factors and increased levels of ”dead signals” may lead to the malfunction of many cells, including lymphocytes. In our study, we investigated the process of apoptosis within T cells in children with a first attack of nephrotic syndrome (NS). We found that the number of apoptotic T cells is greater in these patients than in both children in remission from NS and in controls. The percentage of annexin-V-fluorescein isothiocyanate (FITC)-positive CD3+ cells was 27.30± 12.13% in children with a first attack of NS, 19.22± 15.16% (P=0.006) in children in remission and 16.20± 6.13% (P=0.004) in controls. The percentage of annexin-V-FITC-positive CD3+CD4+ cells was 7.35±7.72% in children with a first attack of NS, 3.80±3.75% (P=0.0001) in children in remission and 3.82±2.01% (P=0.0002) in controls. We conclude that abnormal number and function of T lymphocytes found in NS patients may be related to an increased apoptotic rate of circulating lymphocytes.

Genetic Drivers of Kidney Defects in the DiGeorge Syndrome

Background The DiGeorge syndrome, the most common of the microdeletion syndromes, affects multiple organs, including the heart, the nervous system, and the kidney. It is caused by deletions on chromosome 22q11.2; the genetic driver of the kidney defects is unknown. Methods We conducted a genomewide search for structural variants in two cohorts: 2080 patients with congenital kidney and urinary tract anomalies and 22,094 controls. We performed exome and targeted resequencing in samples obtained from 586 additional patients with congenital kidney anomalies. We also carried out functional studies using zebrafish and mice. Results We identified heterozygous deletions of 22q11.2 in 1.1% of the patients with congenital kidney anomalies and in 0.01% of population controls (odds ratio, 81.5; P=4.5×10‐14). We localized the main drivers of renal disease in the DiGeorge syndrome to a 370‐kb region containing nine genes. In zebrafish embryos, an induced loss of function in snap29, aifm3, and crkl resulted in renal defects; the loss of crkl alone was sufficient to induce defects. Five of 586 patients with congenital urinary anomalies had newly identified, heterozygous protein‐altering variants, including a premature termination codon, in CRKL. The inactivation of Crkl in the mouse model induced developmental defects similar to those observed in patients with congenital urinary anomalies. Conclusions We identified a recurrent 370‐kb deletion at the 22q11.2 locus as a driver of kidney defects in the DiGeorge syndrome and in sporadic congenital kidney and urinary tract anomalies. Of the nine genes at this locus, SNAP29, AIFM3, and CRKL appear to be critical to the phenotype, with haploinsufficiency of CRKL emerging as the main genetic driver. (Funded by the National Institutes of Health and others.)

Whole-Exome Sequencing in Adults With Chronic Kidney Disease: A Pilot Study

Chronic kidney disease (CKD) affects an estimated 14% of Americans (1, 2). These persons have 10- to 15-fold higher morbidity and mortality rates than the general population (1, 3). In most patients with CKD, the diagnosis is based on standard office work-up and sometimes kidney biopsy findings. However, early-stage CKD is often clinically silent, and subtypes can be difficult to distinguish on the basis of clinical data alone. Thus, in many persons, the precise cause of kidney failure remains unknown. Approximately 10% to 25% of patients with CKD note a family history of nephropathy (46), suggesting that in many cases the disease has a hereditary component. Recent advances in genomic technologies, such as chromosomal microarray and massively parallel (next-generation) sequencing, enable genome-wide analysis at a modest cost and precise definition of the molecular cause of many complex diseases (713). Application of these methods has suggested opportunities for individualized diagnosis and risk stratification, including targeted work-up and surveillance for associated disease complications (1113) and sometimes precision therapy (1215). However, studies to date have focused mainly on a limited range of disorders in pediatric cohorts or on cancer in adults (717); thus, the clinical utility of these approaches for a broader spectrum of diseases, particularly among adults, remains unclear. Applying chromosomal microarray analysis, we recently showed that 7.4% of 419 children with various forms of CKD had a major known pathogenic genomic imbalance that was not suspected after clinical assessment (18). These disorders were evenly distributed among patients clinically diagnosed with congenital and noncongenital forms of CKD, indicating that genetic analysis has utility across broad clinical categories. In most of these cases, the genetic findings either reclassified the disease or provided information that could guide subsequent clinical care, such as evaluation for metabolic or neuropsychiatric disease. Similarly, next-generation sequencing has been shown to have great utility for diagnosing genetic forms of nephrotic syndrome or congenital kidney defects in pediatric populations, albeit mainly in the context of targeted panels (1921). Whole-exome sequencing (WES) is a genome-wide testing approach that allows selective sequencing of the protein-coding regions of the genome, which are enriched for disease-associated variants (1215). Because of its genome-wide coverage, WES enables screening of most genes associated with kidney disease and can therefore be applied across diverse categories of renal disorders. Moreover, it can potentially identify novel etiologic genes for nephropathy or detect actionable incidental mutations unrelated to the primary indications for testing. For these reasons, WES is emerging as a preferred diagnostic tool for hereditary disorders (1215, 22, 23). In pediatric cohorts, WES recently identified diagnostic mutations in up to 11.5% of patients with congenital kidney anomalies and 26% of patients with steroid-resistant nephrotic syndrome, supporting its diagnostic utility for early-onset CKD (24, 25). However, the value of this sequencing method for the diagnosis and management of CKD in adults has not been adequately studied. We did a pilot study to test the utility of WES in adults referred for evaluation of CKD or hypertension. Methods Study Design The results of WES in a convenience sample of patients referred for evaluation of CKD were reviewed for their potential to inform clinical practice. To facilitate diagnostic interpretation of WES data, we compiled a list of genes encompassing most common Mendelian forms of kidney and hypertensive disorders. We next annotated the exomes for diagnostic variants in nephropathy genes and then analyzed other genes, including those recommended by the American College of Medical Genetics (ACMG), for return of medically actionable incidental findings (26). Patient Population and Setting The study sample was selected from a group of 344 patients seen at outpatient nephrology clinics between October 2013 and May 2014 at Columbia University Medical Center, a tertiary care medical center with a nephrology division offering highly specialized care for glomerular disorders. These 344 patients were referred for evaluation and management of kidney disease and consented to a general genetic research and biobanking protocol. Supplement Table 1 presents the characteristics of these 344 patients. From this group, we selected 81 adult patients (aged >18 years) (Supplement Table 2) for WES who fulfilled 1 of the following inclusion criteria: a family history of kidney disease (defined as any family member with urinary abnormalities or impaired kidney function, as reported by the patient), undiagnosed kidney disease, or clinical suspicion of a genetic kidney disease (for example, in a proband with young age of onset and no family history of nephropathy). The PKD1 gene is not well-captured by WES because of gene duplication (27), so patients fulfilling clinical diagnostic criteria for autosomal dominant polycystic kidney disease were not included in the WES study. Supplement. Supplementary Tables In addition to these 81 patients from Columbia University Medical Center, we also included 11 patients referred for suspected inherited kidney disease or hypertension from outside institutions. Three patients with familial tubulointerstitial nephropathy and 1 with early-onset hypertension were referred from 3 local practices in the United States (New York University and nephrology practices in suburban New York and Delaware). Four were referred for evaluation of Mendelian hypertension from the Polish Kidney Genetics Network (POLYGENES, www.polygenes.org), centered in the Department of Genetics at Pozna University of Medical Sciences and The Center of Medical Genetics GENESIS (Pozna, Poland). Three other patients were referred from Gaslini Institute (Genova, Italy) for evaluation of glomerulonephritis with nondiagnostic kidney biopsies. All participants gave informed consent, and the study was approved by the Columbia University Institutional Review Board and local ethics committees. WES and Sequence Interpretation Staff extracted DNA from whole blood. Telomere length was measured using genomic DNA from whole blood, as previously described (28, 29). For WES, fragment libraries using 200 ng of genomic DNA were constructed from each sample, following the Agilent standard library preparation protocol for TruSeq (Illumina). Exome capture was done with the SureSelectXT Human All Exon V4 (51 Mb) kit (Agilent), and sequencing was done using the HiSeq 2000 or 2500 (Illumina) at the Columbia Genome Center. On average, 92.83 million independent paired-end reads (18.56-Gb bases) were generated per sample to provide an average coverage of 110-fold, with 99.17% of target regions being covered at least 10-fold. The paired-end reads (read size, 101 bp) were mapped to the human reference genome National Center for Biotechnology Information build 37 using BurrowsWheeler Aligner, version 0.5.9. The Genome Analysis Toolkit, version 1.6-13, was used to call germline single nucleotide variants and insertions or deletions (indels). Variants were annotated for predicted effect on protein function (using ANNOVAR and SnpEff); allele frequency in public databases (ExAC, dbSNP, and the 1000 Genomes Project); and predicted pathogenicity with in silico algorithms, including PolyPhen and Combined Annotation Dependent Depletion scores (3036). Evidence for disease causality was assessed using ClinVar and the Human Genome Mutation Database (Qiagen), followed by manual review of the cited primary literature (33, 36). In addition, we developed a curated priority list of 287 Online Mendelian Inheritance in Man (OMIM; http://omim.org) genes implicated in Mendelian forms of kidney disorders and hypertension to facilitate clinical annotation (hereon, we refer to this gene list as nephropathy genes; see Supplement Table 3. A known limitation of exome sequencing is that some segments of the genome are not amenable to capture (23). Among the 287 nephropathy genes, 29 were identified with at least 1 exon that is not captured by the Agilent kit, representing potential blind spots in the analysis (Supplement Table 3). Variant interpretation was done by a panel of nephrologists or molecular geneticists with domain expertise in inherited kidney diseases (K.K., S.S.C., C.A., L.R., E.G., and A.G.G.), bioinformaticians (S.L. and D.A.F.), and a clinical molecular geneticist (V.J.), using the ACMG guidelines for clinical sequence interpretation (37). Detailed classification criteria for pathogenic and likely pathogenic variants are in Supplement Table 4. We also reviewed potentially pathogenic mutations in OMIM genes associated with other heritable disorders and in the ACMGs 59 actionable genes (26). All diagnostic variants were confirmed by Sanger sequencing. Finally, we verified the distribution of potentially functional variants in nephropathy genes in each exome. These potentially functional variants were defined as missense, nonsense, splice site, or indel variants with a minor allele frequency less than 1% in ExAC (a database of genetic variation in >60000 persons) and a Combined Annotation Dependent Depletion score greater than 10 (indicating a variant score in the top 10% of deleteriousness in a large reference data set of variants). We also verified allele frequencies using an anonymized in-house control data set derived from 9012 persons who had undergone WES for indications other than nephropathy; these control data included healthy parents of children with a developmental disorder and participants from genetic studies of amyotrophic lateral sclerosis or seizure disorders. Role of the Funding Source The study was funded by the New York State Empire Clinical Research Investigator Program, the Renal Research Institut

Retrospective cohort study of familial hypomagnesaemia with hypercalciuria and nephrocalcinosis due to CLDN16 mutations

BACKGROUND Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare autosomal recessive tubular disorder exhibiting a high risk for progressive chronic kidney disease (CKD). METHODS This is a retrospective multicentre study of 25 paediatric cases with FHHNC in Poland. Median age at diagnosis was 4 years and median follow-up time was 4.8 years. RESULTS All cases of FHHNC carried recessive mutations in CLDN16. The founder mutation in CLDN16, Leu151Phe, was the most frequent cause of FHHNC in Polish patients, with 13 (52%) cases being homozygous and 5 (20%) carrying Leu151Phe allele in compound heterozygosity. All cases showed nephrocalcinosis, increased urinary fractional excretion of magnesium and hypercalciuria. Other disease features included hypomagnesaemia (76%), hyperparathyroidism (76%), hyperuricaemia (56%) and hypocitraturia (60%). Treatment with thiazides effectively reduced hypercalciuria in most cases. During follow-up, renal function declined in 60% of patients; 12% of patients reached CKD stage 3 or 4 and one patient developed end-stage renal failure. CONCLUSIONS We report one of the largest cohorts of FHHNC cases caused by CLDN16 mutations. A missense variant of CLDN16, Leu151Phe, is the most common mutation responsible for FHHNC in Poland. Additionally, we found that normomagnesaemia does not exclude FHHNC and the calculation of fractional excretion of Mg can be diagnostic in the setting of normomagnesaemia. We also demonstrate the efficacy of a treatment with thiazides in terms of hypercalciuria in the majority of patients.

Dent disease in children: diagnostic and therapeutic considerations.

BACKGROUND Dent disease (DD) is a rare X-linked tubulopathy characterized by a proximal tubular dysfunction leading to nephrocalcinosis/nephrolithiasis and progressive renal failure. The disease is associated with a mutation either in CLCN5 or OCRL genes. We aim to define clinical and genetic disease characteristics and summarize treatments of Polish patients with DD. METHODS The study cohort consists of 10 boys (aged 5 - 16.5 years) whose data were collected through POLtube Registry. RESULTS All of the patients had tubular proteinuria, hypercalciuria, and nephrocalcinosis/nephrolithiasis. Renal impairment and growth deficiency were found in 3 patients and rickets in 2 patients. In total, 9 of 10 patients carried a mutation in the CLCN5 gene. Five of 9 detected mutations were novel. In 1 patient with a clinical phenotype of DD, no mutations in either CLCN5 or OCRL were discovered. Therapy consisted of thiazides in 7 patients, and phosphate supplements and enalapril in 3 cases. Growth hormone therapy was initiated in 3 patients and resulted in improved growth rate. CONCLUSIONS We report clinical and molecular characterization of Polish children with DD. Our study suggests that this tubulopathy may be generally under-diagnosed in Poland. The study revealed variable treatments, demonstrating a need for therapeutic guidelines.

Long-term renal outcome in children with OCRL mutations: retrospective analysis of a large international cohort

Background Lowe syndrome (LS) and Dent-2 disease (DD2) are disorders associated with mutations in the OCRL gene and characterized by progressive chronic kidney disease (CKD). Here, we aimed to investigate the long-term renal outcome and identify potential determinants of CKD and its progression in children with these tubulopathies. Methods Retrospective analyses were conducted of clinical and genetic data in a cohort of 106 boys (LS: 88 and DD2: 18). For genotype-phenotype analysis, we grouped mutations according to their type and localization. To investigate progression of CKD we used survival analysis by Kaplan-Meier method using stage 3 CKD as the end-point. Results Median estimated glomerular filtration rate (eGFR) was lower in the LS group compared with DD2 (58.8 versus 87.4 mL/min/1.73 m2, P < 0.01). CKD stage II-V was found in 82% of patients, of these 58% and 28% had moderate-to-severe CKD in LS and DD2, respectively. Three patients (3%), all with LS, developed stage 5 of CKD. Survival analysis showed that LS was also associated with a faster CKD progression than DD2 (P < 0.01). On multivariate analysis, eGFR was dependent only on age (b = -0.46, P < 0.001). Localization, but not type of mutations, tended to correlate with eGFR. There was also no significant association between presence of nephrocalcinosis, hypercalciuria, proteinuria and number of adverse clinical events and CKD. Conclusions CKD is commonly found in children with OCRL mutations. CKD progression was strongly related to the underlying diagnosis but did not associate with clinical parameters, such as nephrocalcinosis or proteinuria.

Familial juvenile hyperuricemic nephropathy as rare cause of dialysis-dependent chronic kidney disease—a series of cases in two families

Abstract Hyperuricemia is a common symptom in adult population. It usually accompanies the chronic kidney disease. Less frequently, it is a primary phenomenon causing later serious clinical consequences. Familial juvenile hyperuricemic nephropathy (FJHN) is one of the hereditary conditions associated with high levels of serum uric acid and leading to dialysis in young adult age. It results from mutation in the UMOD gene, encoding the uromodulin protein, that is, Tamm–Horsfall protein. The aim of this paper was to present two families (7 affected members) with FJHN, in whom standard nephrological diagnostics did not provide clear cause of dialysis-dependent chronic kidney disease, until genetic testing was performed.

A classic twin study of lower urinary tract obstruction: Report of 3 cases and literature review

The aim of the present study was to investigate genetic effects in the formation of congenital lower urinary tract obstruction (LUTO) comprising posterior urethral valves (PUV), urethral atresia, and urethras with variable degrees of stenosis.