Anna Materna-Kiryluk

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.

Paper 6: EUROCAT member registries: organization and activities

BACKGROUND EUROCAT is a network of population-based congenital anomaly registries providing standardized epidemiologic information on congenital anomalies in Europe. There are three types of EUROCAT membership: full, associate, or affiliate. Full member registries send individual records of all congenital anomalies covered by their region. Associate members transmit aggregate case counts for each EUROCAT anomaly subgroup by year and by type of birth. This article describes the organization and activities of each of the current 29 full member and 6 associate member registries of EUROCAT. METHODS Each registry description provides information on the history and funding of the registry, population coverage including any changes in coverage over time, sources for ascertaining cases of congenital anomalies, and upper age limit for registering cases of congenital anomalies. It also details the legal requirements relating to termination of pregnancy for fetal anomalies, the definition of stillbirths and fetal deaths, and the prenatal screening policy within the registry. Information on availability of exposure information and denominators is provided. The registry description describes how each registry conforms to the laws and guidelines regarding ethics, consent, and confidentiality issues within their own jurisdiction. Finally, information on electronic and web-based data capture, recent registry activities, and publications relating to congenital anomalies, along with the contact details of the registry leader, are provided. CONCLUSIONS The registry description gives a detailed account of the organizational and operational aspects of each registry and is an invaluable resource that aids interpretation and evaluation of registry prevalence data.

Mutations in DSTYK and Dominant Urinary Tract Malformations

BACKGROUND Congenital abnormalities of the kidney and the urinary tract are the most common cause of pediatric kidney failure. These disorders are highly heterogeneous, and the etiologic factors are poorly understood. METHODS We performed genomewide linkage analysis and whole-exome sequencing in a family with an autosomal dominant form of congenital abnormalities of the kidney or urinary tract (seven affected family members). We also performed a sequence analysis in 311 unrelated patients, as well as histologic and functional studies. RESULTS Linkage analysis identified five regions of the genome that were shared among all affected family members. Exome sequencing identified a single, rare, deleterious variant within these linkage intervals, a heterozygous splice-site mutation in the dual serine-threonine and tyrosine protein kinase gene (DSTYK). This variant, which resulted in aberrant splicing of messenger RNA, was present in all affected family members. Additional, independent DSTYK mutations, including nonsense and splice-site mutations, were detected in 7 of 311 unrelated patients. DSTYK is highly expressed in the maturing epithelia of all major organs, localizing to cell membranes. Knockdown in zebrafish resulted in developmental defects in multiple organs, which suggested loss of fibroblast growth factor (FGF) signaling. Consistent with this finding is the observation that DSTYK colocalizes with FGF receptors in the ureteric bud and metanephric mesenchyme. DSTYK knockdown in human embryonic kidney cells inhibited FGF-stimulated phosphorylation of extracellular-signal-regulated kinase (ERK), the principal signal downstream of receptor tyrosine kinases. CONCLUSIONS We detected independent DSTYK mutations in 2.3% of patients with congenital abnormalities of the kidney or urinary tract, a finding that suggests that DSTYK is a major determinant of human urinary tract development, downstream of FGF signaling. (Funded by the National Institutes of Health and others.).

A novel nonsense mutation in CUL4B gene in three brothers with X-linked mental retardation syndrome

Badura‐Stronka M, Jamsheer A, Materna‐Kiryluk A, Sowińska A, Kiryluk K, Budny B, Latos‐Bieleńska A. A novel nonsense mutation in CUL4B gene in three brothers with X‐linked mental retardation syndrome.

Hirschsprung's disease prevalence in Europe : A register based study

BACKGROUND Hirschsprungs disease is a congenital gut motility disorder, characterised by the absence of the enteric ganglion cells along the distal gut. The aim of this study was to describe the epidemiology of Hirschsprungs disease, including additional congenital anomalies, total prevalence, trends, and association with maternal age. METHODS Cases of Hirschsprungs disease delivered during 1980 to 2009 notified to 31 European Surveillance of Congenital Anomaly registers formed the population-based case-series. Prevalence rates and 95% confidence intervals were calculated as the number of cases per 10,000 births. Multilevel Poisson regression was performed to investigate trends in prevalence, geographical variation and the association with maternal age. RESULTS There were 1,322 cases of Hirschsprungs disease among 12,146,210 births. The total prevalence was 1.09 (95% confidence interval, 1.03-1.15) per 10,000 births and there was a small but significant increase in prevalence over time (relative risk = 1.01; 95% credible interval, 1.00-1.02; p = 0.004). There was evidence of geographical heterogeneity in prevalence (p < 0.001). Excluding 146 (11.0%) cases with chromosomal anomalies or genetic syndromes, there were 1,176 cases (prevalence = 0.97; 95% confidence interval, 0.91-1.03 per 10,000 births), of which 137 (11.6%) had major structural anomalies. There was no evidence of a significant increased risk of Hirschsprungs disease in cases born to women aged ≥35 years compared with those aged 25 to 29 (relative risk = 1.09; 95% credible interval, 0.91-1.31; p = 0.355). CONCLUSION This large population-based study found evidence of a small increasing trend in Hirschsprungs disease and differences in prevalence by geographic location. There was also no evidence of an association with maternal age.

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

Frequency of 22q11.2 microdeletion in children with congenital heart defects in western poland

BackgroundThe 22q11.2 microdeletion syndrome (22q11.2 deletion syndrome -22q11.2DS) refers to congenital abnormalities, including primarily heart defects and facial dysmorphy, thymic hypoplasia, cleft palate and hypocalcaemia. Microdeletion within chromosomal region 22q11.2 constitutes the molecular basis of this syndrome. The 22q11.2 microdeletion syndrome occurs in 1/4000 births. The aim of this study was to determine the frequency of 22q11.2 microdeletion in 87 children suffering from a congenital heart defect (conotruncal or non-conotruncal) coexisting with at least one additional 22q11.2DS feature and to carry out 22q11.2 microdeletion testing of the deleted childrens parents. We also attempted to identify the most frequent heart defects in both groups and phenotypic traits of patients with microdeletion to determine selection criteria for at risk patients.MethodsThe analysis of microdeletions was conducted using fluorescence in situ hybridization (FISH) on metaphase chromosomes and interphase nuclei isolated from venous peripheral blood cultures. A molecular probe (Tuple) specific to the HIRA (TUPLE1, DGCR1) region at 22q11 was used for the hybridisation.ResultsMicrodeletions of 22q11.2 region were detected in 13 children with a congenital heart defect (14.94% of the examined group). Microdeletion of 22q11.2 occurred in 20% and 11.54% of the conotruncal and non-conotruncal groups respectively. Tetralogy of Fallot was the most frequent heart defect in the first group of children with 22q11.2 microdeletion, while ventricular septal defect and atrial septal defect/ventricular septal defect were most frequent in the second group. The microdeletion was also detected in one of the parents of the deleted child (6.25%) without congenital heart defect, but with slight dysmorphism. In the remaining children, 22q11.2 microdeletion originated de novo.ConclusionsPatients with 22q11.2DS exhibit wide spectrum of phenotypic characteristics, ranging from discreet to quite strong. The deletion was inherited by one child. Our study suggests that screening for 22q11.2 microdeletion should be performed in children with conotruncal and non-conotruncal heart defects and with at least one typical feature of 22q11.2DS as well as in the deleted childrens parents.

Trends in diagnosis and prevalence of critical congenital heart defects in the Podkarpacie province in 2002-2004, based on data from the Polish Registry of Congenital Malformations.

This paper presents results of data collection regarding congenital heart defects (CHD) for the Polish Registry of Congenital Malformations (PRCM) from the Podkarpacie province in 2002–2004. Routine methods (fetal echocardiography, clinical examination) and recently also unique methods (screening echocardiography and pulse oximetry) were used for early detection of critical cardiac malformations. Critical CHD were detected there in 107 newborns in 2002–2004, so the mean prevalence reached 1.71/1000. Within this group, death rate decreased from 8 deaths in 2002 to 2 deaths in 2004, mainly thanks to improved detection and treatment of CHD.

Epidemiology of isolated preaxial polydactyly type I: Data from the Polish Registry of Congenital Malformations (PRCM)

BackgroundPolydactyly represents a heterogeneous group of congenital hand and foot anomalies with variable clinical features and diverse etiology. Preaxial polydactyly type I (PPD1) is the most frequent form of preaxial polydactyly. The etiology of sporadic PPD1 remains largely unknown and the relative contribution of genetic and environmental factors is not clearly defined. The primary goals of this study are twofold: (1) to examine the epidemiology and clinical features of sporadic PPD1 in comparison to a healthy control group, and (2) to contrast the characteristics of sporadic PPD1 with familial forms of isolated polydactyly.MethodsAmong 2,530,349 live births registered in the Polish Registry of Congenital Malformations (PRCM), we identified 459 children with isolated sporadic PPD1 and 353 children with familial polydactyly, including 57 children with familial PPD1.ResultsIn comparison with the matched group of 303 controls, sporadic PPD1 cases had significantly lower birth order (P = 0.01) and birthweight (P < 0.0001). Similarly, when compared to familial cases of polydactyly, lower birth order (P = 0.047) and lower birthweight (P < 0.0001) were characteristic of sporadic PPD1 cases. Moreover, our analyses suggested several additional risk factors for sporadic PPD1, including lower paternal education levels (P = 0.01), upper respiratory tract infections during the first trimester of pregnancy (P = 0.049), and maternal history of epilepsy (P = 0.01).ConclusionsIn summary, our study provides support to the hypothesis that non-genetic factors play an important role in the etiology of non-familiar PPD1.