Next Generation Sequencing in Pediatric Epilepsy Using Customized Panels: Size Matters.

INTRODUCTION: Next generation sequencing (NGS) with customized gene panels is a helpful tool to identify monogenic epilepsy syndromes. The number of genes tested within a customized panel may vary greatly. The aim of the present study was to compare the diagnostic yield of small (<25 kb) and large (>25 kb) customized epilepsy panels. METHODS: This retrospective cohort study investigated data of 190 patients of 18 years or younger, with the diagnosis of an epilepsy of unknown etiology who underwent NGS using customized gene panels. Small (<25 kb) and large (>25 kb) panels were compared regarding the distribution of benign/likely benign and pathogenic/likely pathogenic variants and variants of unclear significance. In addition, differences of the diagnostic yield with respect to epilepsy severity, i.e., developmental and epileptic encephalopathy [DEE] vs. non-DEE, were analyzed. RESULTS: The diagnostic yield defined as pathogenic or likely pathogenic variants in large panels was significantly increased (29% [n = 14/48] vs. 13% [n = 18/142], p = 0.0198) compared with smaller panels. In non-DEE patients the increase of the diagnostic yield in large panels was significant(35% n = 6/17 vs. 13% n = 12/94, p = 0.0378), which was not true for DEE patients. DISCUSSION: This study indicates that large panels are superior for pediatric patients with epilepsy forms without encephalopathy (non-DEE). For patients suffering from DEE small panels of a maximum of 10 genes seem to be sufficient. The proportion of unclear findings increases with rising panel sizes. CONCLUSION: Customized epilepsy panels of >25 kb compared with smaller panels show a significant higher diagnostic yield in patients with epilepsy especially in non-DEE patients.

Mutations in INF2 may be associated with renal histology other than focal segmental glomerulosclerosis.

BACKGROUND: In 2010, INF2 mutations were associated with autosomal-dominant focal segmental glomerulosclerosis (FSGS), clinically presenting with moderate proteinuria in adolescence. However, in the meantime, cases with more severe clinical courses have been described, including progression to end-stage renal disease (ESRD) during childhood. INF2 mutations in patients with isolated FSGS are clustered in exons 2 to 4, encoding the diaphanous inhibitory domain, involved in the regulation of the podocyte actin cytoskeleton. METHODS: We report a family with 14 affected individuals (autosomal-dominant mode of inheritance), most of whom presented with nephrotic-range proteinuria, hypertension, and progressive renal failure. Four members received a kidney transplant without disease recurrence. Two patients underwent renal biopsy with the result of minimal-change glomerulopathy and IgA nephropathy respectively. We performed mutational analysis of ACTN4, CD2AP, COQ6, INF2, LAMB2, NPHS1, NPHS2, PLCE1, TRPC6, and WT1 in the index patient by next-generation sequencing. Additionally, in 6 affected and 2 unaffected family members target diagnostics were performed. RESULTS: We identified a novel heterozygous mutation c.490G>C (p.(Ala164Pro) in exon 3 of the INF2 gene in the index patient and 6 additionally examined affected family members. In silico analysis predicted it as "probably damaging". Additionally, three patients and 2 unaffected relatives harbored a novel heterozygous variant in ACTN4 (c.1149C>G, p.(Ile383Met)) with uncertain pathogenicity. CONCLUSION: Mutations in INF2 are associated with familial proteinuric diseases - irrespective of the presence of FSGS and in the case of rapid disease progression. Therefore, mutational analysis should be considered in patients with renal histology other than FSGS and severe renal phenotype.

Severe bleeding complications and multiple kidney transplants in a patient with tuberous sclerosis complex caused by a novel TSC2 missense variant.

We presented an extremely severe case of tuberous sclerosis complex (TSC) in a female patient with recurring, life-threatening bleeding complications related to renal angiomyolipomas. Massive intratumoral hemorrhage required surgical removal of both angiomyolipomatous kidneys and kidney transplantation. During the follow-up period, the patient developed severe metrorrhagia that eventually led to hysterectomy and salpingo-oophorectomy. Bleeding from the operative sites caused the loss of the first kidney transplant received from the mother, and immediate hemorrhagic shock led to the loss of the second, cadaveric kidney allograft. The third kidney transplant had a successful outcome. Pathological analysis of all tissue specimens showed TSC-associated lesions and deformed blood vessels in the surgically removed organs. Molecular genetic analysis of TSC1 and TSC2 in the DNA of peripheral leukocytes identified a novel TSC2 c.3599G>C (p.R1200P) variant. Functional assessment confirmed the likely pathogenicity of the TSC2 c.3599G>C (p.R1200P) variant. To the best of our knowledge, this is the first report of the c.3599G>C (p.R1200P) variant in exon 29 of the TSC2 gene related to a severe clinical course and multiple kidney transplants in a patient with TSC.

Dealing with the incidental finding of secondary variants by the example of SRNS patients undergoing targeted next-generation sequencing.

BACKGROUND: Steroid-resistant nephrotic syndrome (SRNS) is a severe cause of progressive renal disease. Genetic forms of SRNS can present with autosomal recessive or autosomal dominant inheritance. Recent studies have identified mutations in multiple podocyte genes responsible for SRNS. Improved sequencing methods (next-generation sequencing, NGS) now promise rapid mutational testing of SRNS genes. METHODS: In the present study, a simultaneous screening of ten SRNS genes in 37 SRNS patients was performed by NGS. RESULTS: In 38 % of the patients, causative mutations in one SRNS gene were found. In 22 % of the patients, in addition to these mutations, a secondary variant in a different gene was identified. CONCLUSIONS: This high incidence of accumulating sequence variants was unexpected but, although they might have modifier effects, the pathogenic potential of these additional sequence variants seems unclear so far. The example of molecular diagnostics by NGS in SRNS patients shows that these new sequencing technologies might provide further insight into molecular pathogenicity in genetic disorders but will also generate results, which will be difficult to interpret and complicate genetic counseling. Although NGS promises more frequent identification of disease-causing mutations, the identification of causative mutations, the interpretation of incidental findings and possible pitfalls might pose problems, which hopefully will decrease by further experience and elucidation of molecular interactions.

Identification of 47 novel mutations in patients with Alport syndrome and thin basement membrane nephropathy.

BACKGROUND: Alport syndrome (ATS) is a progressive hereditary nephropathy characterized by hematuria and proteinuria. It can be associated with extrarenal manifestations. In contrast, thin basement membrane nephropathy (TBMN) is characterized by microscopic hematuria, is largely asymptomatic, and is rarely associated with proteinuria and end-stage renal disease. Mutations have been identified in the COL4A5 gene in ATS and in the COL4A3 and COL4A4 genes in ATS and TBMN. To date, more than 1000 different mutations in COL4A5, COL4A3, and COL4A4 are known. METHODS: In this study mutational analysis by exon sequencing and multiplex ligation-dependent probe amplification was performed in a large European cohort of families with ATS and TBMN. RESULTS: Molecular diagnostic testing of 216 individuals led to the detection of 47 novel mutations, thereby expanding the spectrum of known mutations causing ATS and TBMN by up to 10 and 6%, respectively, depending on the database. Remarkably, a high number of ATS patients with only single mutations in COL4A3 and COL4A4 were identified. Additionally, three ATS patients presented with synonymous sequence variants that possible affect correct mRNA splicing, as suggested by in silico analysis. CONCLUSIONS: The results of this study clearly broaden the genotypic spectrum of known mutations for ATS and TBMN, which will in turn now facilitate future studies into genotype-phenotype correlations. Further studies should also examine the significance of single heterozygous mutations in COL4A3 and COL4A4 and of synonymous sequence variants associated with ATS.

Development and clinical validation of a novel algorithmic score (GAAD) for detecting HCC in prospective cohort studies.

BACKGROUND: Alpha-fetoprotein (AFP) and des-gamma carboxyprothrombin (DCP), also known as protein induced by vitamin K absence-II (PIVKA-II [DCP]) are biomarkers for HCC with limited diagnostic value when used in isolation. The novel GAAD algorithm is an in vitro diagnostic combining PIVKA-II (DCP) and AFP measurements, age, and gender (biological sex) to generate a semi-quantitative result. We conducted prospective studies to develop, implement, and clinically validate the GAAD algorithm for differentiating HCC (early and all-stage) and benign chronic liver disease (CLD), across disease stages and etiologies. METHODS: Patients aged ≥18 years with HCC or CLD were prospectively enrolled internationally into algorithm development [n = 1084; 309 HCC cases (40.7% early-stage) and 736 controls] and clinical validation studies [n = 877; 366 HCC cases (47.6% early-stage) and 303 controls]. Serum samples were analyzed on a cobas® e 601 analyzer. Performance was assessed using receiver operating characteristic curve analyses to calculate AUC. RESULTS: For algorithm development, AUC for differentiation between early-stage HCC and CLD was 90.7%, 84.4%, and 77.2% for GAAD, AFP, and PIVKA-II, respectively. The sensitivity of GAAD for the detection of early-stage HCC was 71.8% with 90.0% specificity. Similar results were shown in the clinical validation study; AUC for differentiation between early-stage HCC and CLD was 91.4% with 70.1% sensitivity and 93.7% specificity. GAAD also showed strong specificity, with a lower rate of false positives regardless of disease stage, etiology, or region. CONCLUSIONS: The GAAD algorithm significantly improves early-stage HCC detection for patients with CLD undergoing HCC surveillance. Further phase III and IV studies are warranted to assess the utility of incorporating the algorithm into clinical practice.

Performance evaluation of the Elecsys PIVKA-II and Elecsys AFP assays for hepatocellular carcinoma diagnosis.

Background and Aims: Prothrombin induced by vitamin K absence-II (PIVKA-II) is a serum biomarker linked to hepatocellular carcinoma (HCC), showing superiority to alpha-fetoprotein (AFP) for early disease detection. We aimed to assess the clinical and analytical performance of the Elecsys® PIVKA-II immunoassay in diagnosing HCC and evaluate PIVKA-II's technical performance. Methods: Serum samples from adult cases (i.e. patients with a first-time HCC diagnosis; n = 168) and disease controls (i.e. patients without HCC with an at-risk condition; n = 208) were assessed. An AFP cut-off of 20 ng/mL was used to differentiate between HCC cases and disease controls. Clinical performance of the Elecsys PIVKA-II assay was compared with that of comparator assays (Lumipulse G PIVKA-II, µTASWako DCP, ARCHITECT PIVKA-II) using receiver operating characteristic curve analysis to determine the area under the curve (AUC) values. Results: The Elecsys PIVKA-II assay compared favorably with comparator assays. Using a 28.4 ng/mL cut-off, the Elecsys PIVKA-II assay detected HCC with 86.9% sensitivity and 83.7% specificity. Clinical performance of the Elecsys PIVKA-II assay (AUC: 90.8%) was equivalent to that of comparator assays (AUC: 88.3-89.6%). Relatively high PIVKA-II concentrations were observed for cholangiocarcinoma and pancreatic cancer with the Elecsys assay in specificity panel analyses, indicating that high PIVKA-II concentrations should not be used alone in the absence of other clinical data. Conclusions: The Elecsys PIVKA-II assay showed good analytical performance under routine laboratory conditions, comparing favorably with comparator assays. These findings support the suitability of the Elecsys PIVKA-II assay as an aid in HCC diagnosis.

Novel PKD1 and PKD2 mutations in autosomal dominant polycystic kidney disease (ADPKD).

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic renal disorder with an incidence of 1:1000. Mutations in two genes (PKD1 and PKD2) have been identified as causative. Eighty-five percent of patients with ADPKD carry their mutation in the PKD1 gene. So far, > 500 mutations for PKD1 and > 120 mutations for PKD2, respectively, are known. METHODS: In this study, we performed mutation analysis of PKD1 and PKD2 by exon sequencing in patients during routine molecular diagnostics for ADPKD. RESULTS: In total, 60 mutations were identified in 93 patients representing a mutation detection efficiency of 64.5%. Fifty-two mutations were identified in PKD1 (86.7%) and 8 in PKD2 (13.3%). These include 41 novel mutations detected in PKD1 and 5 novel mutations in PKD2. Accordingly, our data expand the spectrum of known PKD mutations by 8% for PKD1 (41/513) and 4.2% for PKD2 (5/120). These results are in agreement with the detection ranges of 42%, 63% and 64% for definitive disease-causing mutations, and 78%, 86% and 89% for all identified variants reported in several comprehensive mutation screening reports. CONCLUSIONS: The increased number of known mutations will facilitate future studies into genotype-phenotype correlations.

Novel KCNH1 Mutations Associated with Epilepsy: Broadening the Phenotypic Spectrum of KCNH1-Associated Diseases.

Here, we describe four patients suffering from a rather broad spectrum of epilepsy-related disorders, ranging from developmental and epileptic encephalopathy with intellectual disability (DEE) to genetic generalized epilepsy (GGE), which all harbor novel KCNH1 mutations. In one family, we found a weak association of a novel nonsense mutation with epilepsy, suggesting reduced penetrance, and which shows, in agreement with previous findings, that gain-of-function effects rather than haploinsufficiency are important for the pathogenicity of mutations. De novo missense variants in the pore region of the channel result in severe phenotypes presenting usually with DEE with various malformations. The potential pathogenicity of a novel KCNH1 germline mutation located outside of the critical pore domain observed in a GGE patient with a milder phenotype is supported by the fact that the very same amino acid exchange was detected as a somatic mutation in the resected brain tissue of a patient suffering from a focal cortical dysplasia type IIb. Thus, our case series broadens the phenotypic spectrum of KCNH1-associated diseases.

Understanding attitudes and behaviors towards cell-free DNA-based noninvasive prenatal testing (NIPT): A survey of European health-care providers.

Cell-free DNA-based noninvasive prenatal testing (cfDNA) is a relatively new screening tool that analyzes cfDNA circulating in maternal plasma to screen for aneuploidies. Since its introduction, cfDNA has been rapidly adopted by health care providers (HCPs). This rapid adoption, as well as progressive developments in the technology, requires professional societies to continuously update their guidelines to indicate the broadening scope both in terms of test indications and patient populations for whom it has become the appropriate primary test. CfDNA testing, initially applied to high-risk patients, is now largely considered an option for all patients. For HCPs, the rapid introduction of cfDNA into clinical practice has come with the requirement to stay up-to-date and accurately informed. We performed a survey to understand the current practices and views of European HCPs on the use of cfDNA. European HCPs were surveyed on several topics such as familiarity with cfDNA-based noninvasive prenatal testing (NIPT), current usage, patient counseling, test menu expansion, and future perspectives. The results of this survey demonstrate increasing usage and awareness of cfDNA-based NIPT in five European countries (UK, France, Germany, Spain and Italy). Major barriers to implementation include cost and a lack of physician education on NIPT.

The FBN2 gene: new mutations, locus-specific database (Universal Mutation Database FBN2), and genotype-phenotype correlations.

Congenital contractural arachnodactyly (CCA) is an extremely rare disease, due to mutations in the FBN2 gene encoding fibrillin-2. Another member of the fibrillin family, the FBN1 gene, is involved in a broad phenotypic continuum of connective-tissue disorders including Marfan syndrome. Identifying not only what is in common but also what differentiates these two proteins should enable us to better comprehend their respective functions and better understand the multitude of diseases in which these two genes are involved. In 1995 we created a locus-specific database (LSDB) for FBN1 mutations with the Universal Mutation Database (UMD) tool. To facilitate comparison of identified mutations in these two genes and search for specific functional areas, we created an LSDB for the FBN2 gene: the UMD-FBN2 database. This database lists 26 published and six newly identified mutations that mainly comprise missense and splice-site mutations. Although the number of described FBN2 mutations was low, the frequency of joint dislocation was significantly higher with missense mutations when compared to splice site mutations.

Variant panorama in 1,385 index patients and sensitivity of expanded next-generation sequencing panels in arrhythmogenic disorders.

BACKGROUND: Arrhythmogenic disorders occur in a broad spectrum of cardiac pathologies in the general population with a prevalence of 1:10,000 to 1:500. Genetic studies conducted during the past 20 years have markedly illuminated the genetic basis of inherited cardiac disorders. However, uncertainty exists regarding which genes should be included and routinely assessed on genetic testing panels. Here, we review the genetic basis of the most important arrhythmogenic disorders found in our laboratory since 2016 by next-generation sequencing (NGS) analysis. METHODS: We analyzed sequence data from 1,385 clinical index cases with a suspected diagnosis of long QT syndrome (LQTS), Brugada syndrome (BrS), catecholaminergic polymorphic ventricular tachycardia (CPVT), hypertrophic cardiomyopathy (HCM), dilatative cardiomyopathy (DCM) or arrhythmogenic right ventricular cardiomyopathy (ARVC). Genetic testing was performed by NGS using a custom design based on an Agilent SureSelectQXT. RESULTS: The detection rate of pathogenic or likely pathogenic variants was in the range of 16% for BrS to 40% for HCM. Only the few well known core genes and some additional side genes substantially contribute to the diagnostic sensitivity. CONCLUSIONS: Clinical testing provides a definitive diagnosis for many patients. The genetic result may be important for risk stratification, genetic counseling and, in some cases, treatment planning. Diagnostic panels should not be further expanded as inclusion of many genes rather produces variants of unclear significance and confusing reports.

Heterozygous COL4A3 Variants in Histologically Diagnosed Focal Segmental Glomerulosclerosis.

Introduction: Steroid-resistant nephrotic syndrome (SRNS) is one of the most frequent causes for chronic kidney disease in childhood. In ~30% of these cases a genetic cause can be identified. The histological finding in SRNS is often focal segmental glomerulosclerosis (FSGS). In rare cases, however, pathogenic variants in genes associated with Alport syndrome can be identified in patients with the histological finding of FSGS. Materials and Methods: Clinical information was collected out of clinical reports and medical history. Focused molecular genetic analysis included sequencing of COL4A5 and COL4A3 in the index patient. Segregation analysis of identified variants was performed in the parents and children of the index patient. Results: The female index patient developed mild proteinuria and microscopic hematuria in childhood (12 years of age). The histological examination of the kidney biopsies performed at the age of 21, 28, and 32 years showed findings partly compatible with FSGS. However, immunosuppressive treatment of the index patient did not lead to a sufficient reduction of in part nephrotic-range proteinuria. After the patient developed hearing impairment at the age of 34 years and her daughter was diagnosed with microscopic hematuria at the age of 6 years, re-examination of the index's kidney biopsies by electron microscopy revealed textural changes of glomerular basement membrane compatible with Alport syndrome. Molecular genetic analysis identified two missense variants in COL4A3 in a compound heterozygous state with maternal and paternal inheritance. One of them is a novel variant that was also found in the 6 year old daughter of the index patient who presented with microscopic hematuria. Discussion: We were able to show that a novel variant combined with a previously described variant in compound heterozygous state resulted in a phenotype that was histologically associated with FSGS. Molecular genetic analysis therefore can be essential to solve difficult cases that show an unusual appearance and therefore improve diagnostic accuracy. Additionally, unnecessary and inefficient treatment with multiple side effects can be avoided.

Genetic heterogeneity of cytogenetically normal AML with mutations of CEBPA.

Biallelic mutations of the CCAAT/enhancer binding protein α (CEBPA) gene define a distinct genetic entity of acute myeloid leukemia (AML) with favorable prognosis. The presence of GATA2 and CSF3R mutations that are specifically associated with this subgroup but not mutated in all samples suggests a genetic heterogeneity of biCEBPA-mutated AML. We characterized the mutational landscape of CEBPA-mutated cytogenetically normal AML by targeted amplicon resequencing. We analyzed 48 biallelically mutated CEBPA (biCEBPA), 32 monoallelically mutated CEBPA (moCEBPA), and 287 wild-type CEBPA (wtCEBPA) patient samples from German AML Cooperative Group studies or registry. Targeted sequencing of 42 genes revealed that moCEBPA patients had significantly more additional mutations and additional mutated genes than biCEBPA patients. Within the group of biCEBPA patients, we identified 2 genetic subgroups defined by the presence or absence of mutations in chromatin/DNA modifiers (C), cohesin complex (C), and splicing (S) genes: biCEBPA CCSpos (25/48 [52%]) and biCEBPA CCSneg (23/48 [48%]). Equivalent subgroups were identified in 51 biCEBPA patients from the Cancer Genome Project. Patients in the biCEBPA CCSpos group were significantly older and had poorer overall survival and lower complete remission rates following intensive chemotherapy regimens compared with patients in the biCEBPA CCSneg group. Patients with available remission samples from the biCEBPA CCSpos group cleared the biCEBPA mutations, but most had persisting CCS mutations in complete remission, suggesting the presence of a preleukemic clone. In conclusion, CCS mutations define a distinct biological subgroup of biCEBPA AML that might refine prognostic classification of AML. This trial was registered at www.clinicaltrials.gov as #NCT00266136 and NCT01382147.

Truncating Wilms Tumor Suppressor Gene 1 Mutation in an XX Female with Adult-Onset Focal Segmental Glomerulosclerosis and Streak Ovaries: A Case Report.

About 30% of children with nephrotic syndrome (NS) have inherited forms. Among them, mutations in Wilms tumor suppressor gene 1 (WT1) are a well characterized cause associated with steroid-resistant NS, Wilms tumor, and urogenital malformation in males. However, the role of WT1 mutations in adult-onset focal segmental glomerulosclerosis (FSGS) is unclear. We report the case of a 38-year-old female with FSGS. She had been diagnosed with streak ovaries during diagnostic workup for infertility. Mutational analysis identified the heterozygous mutation c.1372C>T (p.Arg458*) in WT1 and the heterozygous non-neutral polymorphism c.868G>A (p.Arg229Gln) in NPHS2. Chromosomal analysis revealed a normal 46,XX female karyotype. Our case highlights that WT1 mutations should be considered in XX females with adult-onset FSGS, especially if urogenital abnormalities are present.

Lessons From the EThIGII Trial: Proper Putative Benefit Assessment of Low-Molecular-Weight Heparin Treatment in M2/ANXA5 Haplotype Carriers.

This study presents sample size considerations derived from the Efficacy of Thromboprophylaxis as an Intervention during Gravidity (EThIGII) trial (ClinicalTrials.gov: NCT00400387) to address the question of low-molecular-weight heparin (LMWH) treatment in women with recurrent pregnancy loss (RPL) depending on the M2/ANXA5 haplotype. To evaluate the possible influence of such treatment on miscarriage rates of trial participants, a post hoc analysis of ANXA5 promoter genotypes in the light of M2/ANXA5 (RPRGL3) distribution was performed using logistic models. DNA for genotyping was available from 129 LMWH and 95 control patients, 44 (19.6%) of whom were M2/ANXA5 carriers. Miscarriages occurred in 1 (4.0%) of 25 M2/ANXA5 carriers from the LMWH group compared to 4 (21.1%) of 19 in the control group, resulting in an odds ratio (95% confidence interval) for miscarriage of 0.16 (0.016-1.5) for women treated with LMWH. In noncarriers, miscarriage rates were 6 (5.8%) of 104 versus 7 (9.2%) of 76 for the LMWH and the control groups, respectively, corresponding to an odds ratio for miscarriage of 0.60 (0.19-1.9). The apparent beneficial effects of miscarriage rate reduction in M2/ANXA5 carriers with RPL concur with biological considerations about improvement in reduced ANXA5 function through LMWH treatment in an adequate murine model. The data obtained were instrumental to design proper assessment of the existence and magnitude of this effect.

Rare co-occurrence of osteogenesis imperfecta type I and autosomal dominant polycystic kidney disease.

BACKGROUND: There are several clinical reports about the co-occurrence of autosomal dominant polycystic kidney disease (ADPKD) and connective tissue disorders. A simultaneous occurrence of osteogenesis imperfecta (OI) type I and ADPKD has not been observed so far. METHODS: This report presents the first patient with OI type I and ADPKD. RESULTS: Mutational analysis of PKD1 and COL1A1 in the index patient revealed a heterozygous mutation in each of the two genes. Mutational analysis of the parents indicated the mother as a carrier of the PKD1 mutation and the father as a carrier of the COL1A1 mutation. The simultaneous occurrence of both disorders has an estimated frequency of 3.5:100 000 000. CONCLUSION: In singular cases, ADPKD can occur in combination with other rare disorders, e.g. connective tissue disorders.

Expanding the mutation spectrum for Fraser syndrome: identification of a novel heterozygous deletion in FRAS1.

Fraser syndrome (FS) is a rare autosomal recessive inherited disorder characterized by cryptophthalmos, laryngeal defects and oral clefting, mental retardation, syndactyly, and urogenital defects. To date, 250 patients have been described in the literature. Mutations in the FRAS1 gene on chromosome 4 have been identified in patients with Fraser syndrome. So far, 26 mutations have been identified, most of them are truncating mutations. The mutational spectrum includes nucleotide substitutions, splicing defects, a large insertion, and small deletions/insertions. Moreover, single heterozygous missense mutations in FRAS1 seem to be responsible for non-syndromic unilateral renal agenesis. Here we report the first case of a family with two patients affected by Fraser syndrome due to a deletion of 64 kb (deletion 4q21.21) and an additional novel frameshift mutation in exon 66 of the FRAS1 gene. To date, large deletions of the FRAS1 gene have not yet been described. Large deletions seem to be a rare cause for Fraser syndrome, but should be considered in patients with a single heterozygous mutation.

Next generation sequencing as a useful tool in the diagnostics of mosaicism in Alport syndrome.

Alport syndrome (ATS) is a progressive hereditary nephropathy characterized by hematuria and/or proteinuria with structural defects of the glomerular basement membrane. It can be associated with extrarenal manifestations (high-tone sensorineural hearing loss and ocular abnormalities). Somatic mutations in COL4A5 (X-linked), COL4A3 and COL4A4 genes (both autosomal recessive and autosomal dominant) cause Alport syndrome. Somatic mosaicism in Alport patients is very rare. The reason for this may be due to the difficulty of detection. We report the case of a boy and his mother who presented with Alport syndrome. Mutational analysis showed the novel hemizygote pathogenic mutation c.2396-1G>A (IVS29-1G>A) at the splice acceptor site of the intron 29 exon 30 boundary of the COL4A5 gene in the boy. The mutation in the mother would not have been detected by Sanger sequencing without the knowledge of the mutational analysis result of her son. Further investigation of the mother using next generation sequencing showed somatic mosaicism and implied potential germ cell mosaicism. The mutation in the mother has most likely occurred during early embryogenesis. Analysis of tissue of different embryonic origin in the mother confirmed mosaicism in both mesoderm and ectoderm. Low grade mosaicism is very difficult to detect by Sanger sequencing. Next generation sequencing is increasingly used in the diagnostics and might improve the detection of mosaicism. In the case of definite clinical symptoms of ATS and missing detection of a mutation by Sanger sequencing, mutational analysis should be performed by next generation sequencing.

A novel interstitial deletion of 10q24.2q24.32 in a patient with renal coloboma syndrome.

Renal coloboma syndrome (RCS) is considered to be a rare autosomal dominant inherited disorder characterized by renal malformations and optic disc coloboma. Ocular anomalies range from asymptomatic abnormalities in retinal blood vessel patterning to large excavations of the optic nerve associated with reduced visual acuity. Commonly observed manifestations of the kidney are renal hypoplasia and vesicoureteric reflux leading to end-stage renal disease. Mutations in the PAX2 gene on chromosome 10 have been identified in patients with RCS. Up to date, nucleotide substitutions, insertions, small deletions, one de novo translocation, and one 240 kb deletion of the coding region of the PAX2 gene have been described to be responsible for RCS. We report here a new case of a patient with RCS due to a deletion of 3.8 Mb on chromosome 10q. Deletions on the long arm of chromosome 10 harboring the PAX2 gene seem to be a rare cause for RCS. Nevertheless, array-CGH testing should represent an important and valuable addition to PAX2 gene sequencing in diagnostic of RCS.