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Background and aims: Cerebrotendinous Xanthomatosis (CTX) is a treatable disorder of bile acid synthesis caused by deficiency of 27-sterol hydroxylase -encoded by CYP27A1- leading to gastrointestinal and progressive neuropsychiatric symptoms. Biochemically, CTX is characterized by accumulation of the bile alcohol cholestanetetrol glucuronide (GlcA-tetrol) and the deficiency of tauro-chenodeoxycholic acid (t-CDCA) and tauro-trihydroxycholestanoic acid (t-THCA). Materials and Methods: To ascertain the feasibility of CTX newborn screening (NBS) we performed a study with deidentified Dutch dried blood spots using reagents and equipment that is frequently used in NBS laboratories. 20,076 deidentified newborn blood spots were subjected to flow-injection (FIA)-MS/MS and UPLC-MS/MS analysis to determine the concentration of GlcA-tetrol and calculate the GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios. Results: Using UPLC-MS/MS analysis both GlcA-tetrol concentration and/or metabolite ratios GlcA-tetrol/t-CDCA proved to be informative biomarkers; newborn DBS results did not overlap with those of the CTX patients. For FIA-MS/MS, GlcA-tetrol also was an excellent marker but when using the combination of the GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios also did not yield any screen positives. Conclusion: Newborn screening for CTX using only metabolite ratios following the measurement of three CTX biomarkers is possible using either FIA-MS/MS or UPLC-MS/MS, which paves the way for introduction of CTX NBS.

Contains fulltext : 290609.pdf (Publisher’s version ) (Open Access) Genome sequencing (GS) can identify novel diagnoses for patients who remain undiagnosed after routine diagnostic procedures. We tested whether GS is a better first-tier genetic diagnostic test than current standard of care (SOC) by assessing the technical and clinical validity of GS for patients with neurodevelopmental disorders (NDD). We performed both GS and exome sequencing in 150 consecutive NDD patient-parent trios. The primary outcome was diagnostic yield, calculated from disease-causing variants affecting exonic sequence of known NDD genes. GS (30%, n = 45) and SOC (28.7%, n = 43) had similar diagnostic yield. All 43 conclusive diagnoses obtained with SOC testing were also identified by GS. SOC, however, required integration of multiple test results to obtain these diagnoses. GS yielded two more conclusive diagnoses, and four more possible diagnoses than ES-based SOC (35 vs. 31). Interestingly, these six variants detected only by GS were copy number variants (CNVs). Our data demonstrate the technical and clinical validity of GS to serve as routine first-tier genetic test for patients with NDD. Although the additional diagnostic yield from GS is limited, GS comprehensively identified all variants in a single experiment, suggesting that GS constitutes a more efficient genetic diagnostic workflow. 01 januari 2023

Dihydrofolate reductase (DHFR) is a critical enzyme in folate metabolism that reduces folic acid to dihydrofolic and tetrahydrofolic acid and provides an important target for antineoplastic, antimicrobial, and anti-inflammatory drugs. Defective DHFR activity leads to megaloblastic anemia syndrome combined with severe cerebral folate deficiency, and cerebral tetrahydrobiopterin deficiency due to a germ line missense mutation in DHFR has been reported.1,2 Folate represents a large family of water-soluble vitamins that play an important role in DNA synthesis, repair, and transmethylation pathways.3 Folate is also a substrate for purine and thymidine synthesis and a methyl donor for homocysteine to methionine conversion, with low folate status being reflected by elevated plasma homocysteine concentrations.4 Cerebral tetrahydrobiopterin is required for the formation of dopamine, serotonin, and norepinephrine and the hydroxylation of aromatic amino acids as a link to neurodevelopmental symptoms.

Contains fulltext : 235027.pdf (Publisher’s version ) (Open Access) Long-read sequencing (LRS) has the potential to comprehensively identify all medically relevant genome variation, including variation commonly missed by short-read sequencing (SRS) approaches. To determine this potential, we performed LRS around 15×-40× genome coverage using the Pacific Biosciences Sequel I System for five trios. The respective probands were diagnosed with intellectual disability (ID) whose etiology remained unresolved after SRS exomes and genomes. Systematic assessment of LRS coverage showed that ~35 Mb of the human reference genome was only accessible by LRS and not SRS. Genome-wide structural variant (SV) calling yielded on average 28,292 SV calls per individual, totaling 12.9 Mb of sequence. Trio-based analyses which allowed to study segregation, showed concordance for up to 95% of these SV calls across the genome, and 80% of the LRS SV calls were not identified by SRS. De novo mutation analysis did not identify any de novo SVs, confirming that these are rare events. Because of high sequence coverage, we were also able to call single nucleotide substitutions. On average, we identified 3 million substitutions per genome, with a Mendelian inheritance concordance of up to 97%. Of these, ~100,000 were located in the ~35 Mb of the genome that was only captured by LRS. Moreover, these variants affected the coding sequence of 64 genes, including 32 known Mendelian disease genes. Our data show the potential added value of LRS compared to SRS for identifying medically relevant genome variation.

Purpose: Structural variants (SVs) play an important role in inherited retinal diseases (IRD). Although the identification of SVs significantly improved upon the availability of genome sequencing, it is expected that involvement of SVs in IRDs is higher than anticipated. We revisited short-read genome sequencing data to enhance the identification of gene-disruptive SVs. Methods: Optical genome mapping was performed to improve SV detection in short-read genome sequencing−negative cases. In addition, reanalysis of short-read genome sequencing data was performed to improve the interpretation of SVs and to re-establish SV prioritization criteria. Results: In a monoallelic USH2A case, optical genome mapping identified a pericentric inversion (173 megabase), with 1 breakpoint disrupting USH2A. Retrospectively, the variant could be observed in genome sequencing data but was previously deemed false positive. Reanalysis of short-read genome sequencing data (427 IRD cases) was performed which yielded 30 pathogenic SVs affecting, among other genes, USH2A (n = 15), PRPF31 (n = 3), and EYS (n = 2). Eight of these (>25%) were overlooked during previous analyses. Conclusion: Critical evaluation of our findings allowed us to re-establish and improve our SV prioritization and interpretation guidelines, which will prevent missing pathogenic events in future analyses. Our data suggest that more attention should be paid to SV interpretation and the current contribution of SVs in IRDs is still underestimated., Genetics in Medicine, 25 (3)

Contains fulltext : 284807.pdf (Publisher’s version ) (Open Access) Massive parallel sequencing technology has become the predominant technique for genetic diagnostics and research. Many genetic laboratories have wrestled with the challenges of setting up genetic testing workflows based on a completely new technology. The learning curve we went through as a laboratory was accompanied by growing pains while we gained new knowledge and expertise. Here we discuss some important mistakes that have been made in our laboratory through 10 years of clinical exome sequencing but that have given us important new insights on how to adapt our working methods. We provide these examples and the lessons that we learned to help other laboratories avoid to make the same mistakes.

Massive parallel sequencing technology has become the predominant technique for genetic diagnostics and research. Many genetic laboratories have wrestled with the challenges of setting up genetic testing workflows based on a completely new technology. The learning curve we went through as a laboratory was accompanied by growing pains while we gained new knowledge and expertise. Here we discuss some important mistakes that have been made in our laboratory through ten years of clinical exome sequencing but that have given us important new insights on how to adapt our working methods. By providing these examples and the lessons that we learned from them, we hope that other laboratories do not need to make the same mistakes.

Long-read sequencing (LRS) has the potential to comprehensively identify all medically relevant genome variation, including variation commonly missed by short-read sequencing (SRS) approaches. To determine this potential, we performed LRS around 15×-40× genome coverage using the Pacific Biosciences Sequel I System for five trios. The respective probands were diagnosed with intellectual disability (ID) whose etiology remained unresolved after SRS exomes and genomes. Systematic assessment of LRS coverage showed that ~35 Mb of the human reference genome was only accessible by LRS and not SRS. Genome-wide structural variant (SV) calling yielded on average 28,292 SV calls per individual, totaling 12.9 Mb of sequence. Trio-based analyses which allowed to study segregation, showed concordance for up to 95% of these SV calls across the genome, and 80% of the LRS SV calls were not identified by SRS. De novo mutation analysis did not identify any de novo SVs, confirming that these are rare events. Because of high sequence coverage, we were also able to call single nucleotide substitutions. On average, we identified 3 million substitutions per genome, with a Mendelian inheritance concordance of up to 97%. Of these, ~100,000 were located in the ~35 Mb of the genome that was only captured by LRS. Moreover, these variants affected the coding sequence of 64 genes, including 32 known Mendelian disease genes. Our data show the potential added value of LRS compared to SRS for identifying medically relevant genome variation.

Summary The lack of molecular diagnoses in rare genetic diseases can be explained by limitations of current standard genomic technologies. Upcoming long-read techniques have complementary strengths to overcome these limitations, with a particular strength in identifying structural variants. By using optical genome mapping and long-read sequencing, we aimed to identify the pathogenic variant in a large family with X-linked choroideremia. In this family, aberrant splicing of exon 12 of the choroideremia gene CHM was detected in 2003, but the underlying genomic defect remained elusive. Optical genome mapping and long-read sequencing approaches now revealed an intragenic 1,752 bp inverted duplication including exon 12 and surrounding regions, located downstream of the wild-type copy of exon 12. Both breakpoint junctions were confirmed with Sanger sequencing and segregate with the X-linked inheritance in the family. The breakpoint junctions displayed sequence microhomology suggestive for an erroneous replication mechanism as the origin of the structural variant. The inverted duplication is predicted to result in a hairpin formation of the pre-mRNA with the wild-type exon 12, leading to exon skipping in the mature mRNA. The identified inverted duplication is deemed the hidden pathogenic cause of disease in this family. Our study shows that optical genome mapping and long-read sequencing have significant potential for the identification of (hidden) structural variants in rare genetic diseases., In a family with X-linked choroideremia, aberrant splicing of the gene CHM was detected previously, but the underlying genomic defect remained elusive. Long-read technologies revealed an inverted duplication as the hidden pathogenic cause of disease and thereby show significant potential for the identification of (hidden) SVs in rare genetic diseases.

Contains fulltext : 237894.pdf (Publisher’s version ) (Open Access) In a subset of pediatric cancers, a germline cancer predisposition is highly suspected based on clinical and pathological findings, but genetic evidence is lacking, which hampers genetic counseling and predictive testing in the families involved. We describe a family with two siblings born from healthy parents who were both neonatally diagnosed with atypical teratoid rhabdoid tumor (ATRT). This rare and aggressive pediatric tumor is associated with biallelic inactivation of SMARCB1, and in 30% of the cases, a predisposing germline mutation is involved. Whereas the tumors of both siblings showed loss of expression of SMARCB1 and acquired homozygosity of the locus, whole exome and whole genome sequencing failed to identify germline or somatic SMARCB1 pathogenic mutations. We therefore hypothesized that the insertion of a pathogenic repeat-rich structure might hamper its detection, and we performed optical genome mapping (OGM) as an alternative strategy to identify structural variation in this locus. Using this approach, an insertion of ~2.8 kb within intron 2 of SMARCB1 was detected. Long-range PCR covering this region remained unsuccessful, but PacBio HiFi genome sequencing identified this insertion to be a SINE-VNTR-Alu, subfamily E (SVA-E) retrotransposon element, which was present in a mosaic state in the mother. This SVA-E insertion disrupts correct splicing of the gene, resulting in loss of a functional allele. This case demonstrates the power of OGM and long-read sequencing to identify genomic variations in high-risk cancer-predisposing genes that are refractory to detection with standard techniques, thereby completing the clinical and molecular diagnosis of such complex cases and greatly improving counseling and surveillance of the families involved. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Accurate detection of somatic structural variation (SV) in cancer genomes remains a challenging problem. This is in part due to the lack of high-quality gold standard datasets that enable the benchmarking of experimental approaches and bioinformatic analysis pipelines for comprehensive somatic SV detection. Here, we approached this challenge by genome-wide somatic SV analysis of the paired melanoma and normal lymphoblastoid COLO829 cell lines using four different technologies: Illumina HiSeq, Oxford Nanopore, Pacific Biosciences and 10x Genomics. Based on the evidence from multiple technologies combined with extensive experimental validation, including Bionano optical mapping data and targeted detection of candidate breakpoint junctions, we compiled a comprehensive set of true somatic SVs, comprising all SV types. We demonstrate the utility of this resource by determining the SV detection performance of each technology as a function of tumor purity and sequence depth, highlighting the importance of assessing these parameters in cancer genomics projects and data analysis tool evaluation. The reference truth somatic SV dataset as well as the underlying raw multi-platform sequencing data are freely available and are an important resource for community somatic benchmarking efforts.

Contains fulltext : 225140.pdf (Publisher’s version ) (Open Access) OBJECTIVE: The purpose of this study was to explore the diagnostic yield and clinical utility of trio-based rapid whole exome sequencing (rWES) in pregnancies of fetuses with a wide range of congenital anomalies detected by ultrasound imaging. METHODS: In this observational study, we analyzed the first 54 cases referred to our laboratory for prenatal rWES to support clinical decision making, after the sonographic detection of fetal congenital anomalies. The most common identified congenital anomalies were skeletal dysplasia (n = 20), multiple major fetal congenital anomalies (n = 17) and intracerebral structural anomalies (n = 7). RESULTS: A conclusive diagnosis was identified in 18 of the 54 cases (33%). Pathogenic variants were detected most often in fetuses with skeletal dysplasia (n = 11) followed by fetuses with multiple major fetal congenital anomalies (n = 4) and intracerebral structural anomalies (n = 3). A survey, completed by the physicians for 37 of 54 cases, indicated that the rWES results impacted clinical decision making in 68% of cases. CONCLUSIONS: These results suggest that rWES improves prenatal diagnosis of fetuses with congenital anomalies, and has an important impact on prenatal and peripartum parental and clinical decision making.

// Junxiao Zhang 1 , Xiaoyan Wang 2 , Richarda M. de Voer 1 , Jayne Y. Hehir-Kwa 1 , Eveline J. Kamping 1 , Robbert D.A. Weren 1 , Marcel Nelen 1 , Alexander Hoischen 1 , Marjolijn J.L. Ligtenberg 1, 3 , Nicoline Hoogerbrugge 1 , Xiangling Yang 2 , Zihuan Yang 2 , Xinjuan Fan 4 , Lei Wang 2 , Huanliang Liu 2, 5 , Jianping Wang 2, * , Roland P. Kuiper 1, 6, * , Ad Geurts van Kessel 1, * 1 Department of Human Genetics, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands 2 Guangdong Provincial Key Laboratory of Colorectal and Pelvic Floor Diseases, Guangdong Institute of Gastroenterology and the Sixth Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong, China 3 Department of Pathology, Radboud University Medical Center, Nijmegen, The Netherlands 4 Department of Pathology, the Sixth Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong, China 5 Department of Clinical Laboratory, the Sixth Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong, China 6 Princess Maxima Center for Pediatric Oncology, Utrecht, The Netherlands * These authors contributed equally to this work Correspondence to: Roland P. Kuiper, email: r.kuiper@prinsesmaximacentrum.nl Jianping Wang, email: wangjpgz@126.com Keywords: molecular inversion probes, early-onset colorectal cancer, Mendelian colorectal cancer predisposition syndromes, next-generation sequencing Received: June 17, 2016 Accepted: February 12, 2017 Published: February 21, 2017 ABSTRACT The currently known Mendelian colorectal cancer (CRC) predisposition syndromes account for ~5–10% of all CRC cases, and are caused by inherited germline mutations in single CRC predisposing genes. Using molecular inversion probes (MIPs), we designed a targeted next-generation sequencing panel to identify mutations in seven CRC predisposing genes: APC, MLH1, MSH2, MSH6, PMS2, MUTYH and NTHL1 . From a consecutive series of 2,371 Chinese CRC patients, 140 familial and non-familial cases were selected that were diagnosed with CRC at or below the age of 35 years. Through MIP-based sequencing we identified pathogenic variants in six genes in 16 out of the 140 (11.4%) patients selected. In 10 patients, known pathogenic mutations in APC (five patients), MLH1 (three patients), or MSH2 (two patients) were identified. Three additional patients were found to carry novel, likely pathogenic truncating ( n = 2) and missense ( n = 1) mutations in the MSH2 gene and a concomitant loss of expression of both the MSH2 and MSH6 proteins in their respective tumor tissues. From our data, we conclude that targeted MIP-based sequencing is a reliable and cost-efficient approach to identify patients with a Mendelian CRC syndrome.

Contains fulltext : 169850.pdf (Publisher’s version ) (Closed access) Hearing impairment (HI) is genetically heterogeneous which hampers genetic counseling and molecular diagnosis. Testing of several single HI-related genes is laborious and expensive. In this study, we evaluate the diagnostic utility of whole-exome sequencing (WES) targeting a panel of HI-related genes. Two hundred index patients, mostly of Dutch origin, with presumed hereditary HI underwent WES followed by targeted analysis of an HI gene panel of 120 genes. We found causative variants underlying the HI in 67 of 200 patients (33.5%). Eight of these patients have a large homozygous deletion involving STRC, OTOA or USH2A, which could only be identified by copy number variation detection. Variants of uncertain significance were found in 10 patients (5.0%). In the remaining 123 cases, no potentially causative variants were detected (61.5%). In our patient cohort, causative variants in GJB2, USH2A, MYO15A and STRC, and in MYO6 were the leading causes for autosomal recessive and dominant HI, respectively. Segregation analysis and functional analyses of variants of uncertain significance will probably further increase the diagnostic yield of WES.

Background Diagnosis of primary immunodeficiencies (PIDs) is complex and cumbersome yet important for the clinical management of the disease. Exome sequencing may provide a genetic diagnosis in a significant number of patients in a single genetic test. Methods In May 2013, we implemented exome sequencing in routine diagnostics for patients suffering from PIDs. This study reports the clinical utility and diagnostic yield for a heterogeneous group of 254 consecutively referred PID patients from 249 families. For the majority of patients, the clinical diagnosis was based on clinical criteria including rare and/or unusual severe bacterial, viral, or fungal infections, sometimes accompanied by autoimmune manifestations. Functional immune defects were interpreted in the context of aberrant immune cell populations, aberrant antibody levels, or combinations of these factors. Results For 62 patients (24%), exome sequencing identified pathogenic variants in well-established PID genes. An exome-wide analysis diagnosed 10 additional patients (4%), providing diagnoses for 72 patients (28%) from 68 families altogether. The genetic diagnosis directly indicated novel treatment options for 25 patients that received a diagnosis (34%). Conclusion Exome sequencing as a first-tier test for PIDs granted a diagnosis for 28% of patients. Importantly, molecularly defined diagnoses indicated altered therapeutic options in 34% of cases. In addition, exome sequencing harbors advantages over gene panels as a truly generic test for all genetic diseases, including in silico extension of existing gene lists and re-analysis of existing data. Electronic supplementary material The online version of this article (10.1186/s13073-019-0649-3) contains supplementary material, which is available to authorized users.

Contains fulltext : 174063.pdf (Publisher’s version ) (Open Access) PURPOSE: Copy-number variation is a common source of genomic variation and an important genetic cause of disease. Microarray-based analysis of copy-number variants (CNVs) has become a first-tier diagnostic test for patients with neurodevelopmental disorders, with a diagnostic yield of 10-20%. However, for most other genetic disorders, the role of CNVs is less clear and most diagnostic genetic studies are generally limited to the study of single-nucleotide variants (SNVs) and other small variants. With the introduction of exome and genome sequencing, it is now possible to detect both SNVs and CNVs using an exome- or genome-wide approach with a single test. METHODS: We performed exome-based read-depth CNV screening on data from 2,603 patients affected by a range of genetic disorders for which exome sequencing was performed in a diagnostic setting. RESULTS: In total, 123 clinically relevant CNVs ranging in size from 727 bp to 15.3 Mb were detected, which resulted in 51 conclusive diagnoses and an overall increase in diagnostic yield of ~2% (ranging from 0 to -5.8% per disorder). CONCLUSIONS: This study shows that CNVs play an important role in a broad range of genetic disorders and that detection via exome-based CNV profiling results in an increase in the diagnostic yield without additional testing, bringing us closer to single-test genomics.Genet Med advance online publication 27 October 2016.

With the recent introduction of Poly(ADP‐ribose) polymerase inhibitors, a promising novel therapy has become available for ovarian carcinoma (OC) patients with inactivating BRCA1 or BRCA2 mutations in their tumor. To select patients who may benefit from these treatments, assessment of the mutation status of BRCA1 and BRCA2 in the tumor is required. For reliable evaluation of germline and somatic mutations in these genes in DNA derived from formalin‐fixed, paraffin‐embedded (FFPE) tissue, we have developed a single‐molecule molecular inversion probe (smMIP)‐based targeted next‐generation sequencing (NGS) approach. Our smMIP‐based NGS approach provides analysis of both strands of the open reading frame of BRCA1 and BRCA2, enabling the discrimination between real variants and formalin‐induced artefacts. The single molecule tag enables compilation of unique reads leading to a high analytical sensitivity and enabling assessment of the reliability of mutation‐negative results. Multiplex ligation‐dependent probe amplification (MLPA) and Methylation‐specific multiplex ligation‐dependent probe amplification (MS‐MLPA) were used to detect exon deletions of BRCA1 and methylation of the BRCA1 promoter, respectively. Here, we show that this combined approach allows the rapid and reliable detection of both germline and somatic aberrations affecting BRCA1 and BRCA2 in DNA derived from FFPE OCs, enabling improved hereditary cancer risk assessment and clinical treatment of ovarian cancer patients.

BACKGROUND Despite advances in next generation DNA sequencing (NGS), NGS-based single gene tests for diagnostic purposes require improvements in terms of completeness, quality, speed, and cost. Single-molecule molecular inversion probes (smMIPs) are a technology with unrealized potential in the area of clinical genetic testing. In this proof-of-concept study, we selected 2 frequently requested gene tests, those for the breast cancer genes BRCA1 and BRCA2, and developed an automated work flow based on smMIPs. METHODS The BRCA1 and BRCA2 smMIPs were validated using 166 human genomic DNA samples with known variant status. A generic automated work flow was built to perform smMIP-based enrichment and sequencing for BRCA1, BRCA2, and the checkpoint kinase 2 (CHEK2) c.1100del variant. RESULTS Pathogenic and benign variants were analyzed in a subset of 152 previously BRCA-genotyped samples, yielding an analytical sensitivity and specificity of 100%. Following automation, blind analysis of 65 in-house samples and 267 Norwegian samples correctly identified all true-positive variants (>3000), with no false positives. Consequent to process optimization, turnaround times were reduced by 60% to currently 10–15 days. Copy number variants were detected with an analytical sensitivity of 100% and an analytical specificity of 88%. CONCLUSIONS smMIP-based genetic testing enables automated and reliable analysis of the coding sequences of BRCA1 and BRCA2. The use of single-molecule tags, double-tiled targeted enrichment, and capturing and sequencing in duplo, in combination with automated library preparation and data analysis, results in a robust process and reduces routine turnaround times. Furthermore, smMIP-based copy number variation analysis could make independent copy number variation tools like multiplex ligation-dependent probes amplification dispensable.

Past large scale cancer genome sequencing efforts, including The Cancer Genome Atlas and the International Cancer Genome Consortium, have utilized short-read sequencing, which is well-suited for detecting single nucleotide variants (SNVs) but far less reliable for detecting variants larger than 20 base pairs, including insertions, deletions, duplications, inversions and translocations. Recent same-sample comparisons of short- and long-read human reference genome data have revealed that short-read resequencing typically uncovers only ~4,000 structural variants (SVs, ≥50 bp) per genome and is biased towards deletions, whereas sequencing with PacBio long-reads consistently finds ~20,000 SVs, evenly balanced between insertions and deletions. This discovery has important implications for cancer research, as it is clear that SVs are both common and biologically important in many cancer subtypes, including colorectal, breast and ovarian cancer. Without confident and comprehensive detection of structural variants, it is unlikely we have a sufficiently complete picture of all the genomic changes that impact cancer development, disease progression, treatment response, drug resistance, and relapse. To begin to address this unmet need, we have sequenced the COLO829 tumor and matched normal lymphoblastoid cell lines to 49- and 51-fold coverage, respectively, with PacBio SMRT Sequencing, with the goal of developing a high-confidence structural variant call set that can be used to empirically evaluate cost-effective experimental designs for larger scale studies and develop structural variation calling software suitable for cancer genomics. Structural variant calling revealed over 21,000 deletions and 19,500 insertions larger than 20 bp, nearly four times the number of events detected with short-read sequencing. The vast majority of events are shared between the tumor and normal, with about 100 putative somatic deletions and 400 insertions, primarily in microsatellites. A further 40 rearrangements were detected, nearly exclusively in the tumor. One rearrangement is shared between the tumor and normal, t(5;X) which disrupts the mismatch repeat gene MSH3, and is likely a driver mutation. Generating high-confidence call sets that cover the entire size-spectrum of somatic variants from a range of cancer model systems is the first step in determining what will be the best approach for addressing an ongoing blind spot in our current understanding of cancer genomes. Here the application of PacBio sequencing to a colorectal cancer cell line revealed thousands of previously overlooked variants, including a mutation likely involved in tumorogenesis. Citation Format: Aaron Wenger, Marcel Nelen, Meredith Ashby, Wigard P. Kloosterman. Structural variant detection with long read sequencing reveals driver and passenger mutationsin a melanoma cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1696.

Mutations in the αB-crystallin gene (CRYAB) have been reported in desmin-related myopathies, with or without cardiac involvement. Mutations in this gene have also been documented in large multi-generation families with autosomal dominant congenital posterior pole cataract (CPPC). In these congenital cataract families no cardiac or muscular phenotype was reported. This report describes a family with an unusual read-through mutation in CRYAB, leading to the elongation of the normal αB-crystallin protein with 19 amino acid residues. Affected family members combine a CPPC with an adult onset dilated cardiomyopathy (DCM), thereby expanding the αB-crystallinopathy phenotype. Repolarisation abnormalities preceded the onset of cardiomyopathy and were already present in childhood. No skeletal myopathy was observed. This report illustrates that congenital cataract can be a prelude to more severe disease even outside the context of inborn errors of metabolism. The identification of a CRYAB mutation in this family supports the notion that mutations in this gene are a rare cause of genetically determined DCM. The combined congenital cataract/cardiomyopathy phenotype adds to our understanding of the complex phenotypic spectrum of αB-crystallinopathies.

Next-generation sequencing (NGS) methods are being adopted by genome diagnostics laboratories worldwide. However, implementing NGS-based tests according to diagnostic standards is a challenge for individual laboratories. To facilitate the implementation of NGS in Dutch laboratories, the Dutch Society for Clinical Genetic Laboratory Diagnostics (VKGL) set up a working group in 2012. The results of their discussions are presented here. We provide best practice guidelines and criteria for implementing and validating NGS applications in a clinical setting. We introduce the concept of diagnostic yield as the main performance characteristic for evaluating diagnostic tests. We recommend that the laboratory procedures, including the tested genes, should be recorded in a publicly available document describing the complete diagnostic routing. We also propose that laboratories should use a list of core disease genes for specific genetic diseases. This core list contains the essential genes for each disease, and they should all be included in a diagnostic test to establish a reliable and accurate molecular diagnosis. The guidelines will ensure a clear and standardized quality of care provided by genetic diagnostic laboratories. The best practice guidelines and criteria that are presented here were adopted by the VKGL in January 2013. (C) 2013 Wiley Periodicals, Inc.

To identify novel candidate intellectual disability genes, we performed a meta-analysis on 2,637 de novo mutations, identified from the exomes of 2,104 ID trios. Statistical analyses identified 10 novel candidate ID genes, including DLG4, PPM1D, RAC1, SMAD6, SON, SOX5, SYNCRIP, TCF20, TLK2 and TRIP12. In addition, we show that these genes are intolerant to non-synonymous variation, and that mutations in these genes are associated with specific clinical ID phenotypes.

BACKGROUND The substantial technological advancements in next-generation sequencing (NGS), combined with dropping costs, have allowed for a swift diffusion of NGS applications in clinical settings. Although several commercial parties report to have broken the $1000 barrier for sequencing an entire human genome, a valid cost overview for NGS is currently lacking. This study provides a complete, transparent and up-to-date overview of the total costs of different NGS applications. METHODS Cost calculations for targeted gene panels (TGP), whole exome sequencing (WES) and whole genome sequencing (WGS) were based on the Illumina NextSeq500, HiSeq4000, and HiSeqX5 platforms, respectively. To anticipate future developments, sensitivity analyses are performed. RESULTS Per-sample costs were €1669 for WGS, € 792 for WES and €333 for TGP. To reach the coveted $1000 genome, not only is the long-term and efficient use of the sequencing equipment needed, but also large reductions in capital costs and especially consumable costs are also required. CONCLUSIONS WES and TGP are considerably lower-cost alternatives to WGS. However, this does not imply that these NGS approaches should be preferred in clinical practice, since this should be based on the tradeoff between costs and the expected clinical utility of the approach chosen. The results of the present study contribute to the evaluation of such tradeoffs.

Contains fulltext : 168105.pdf (Publisher’s version ) (Open Access) Sequencing of tumor DNA to detect genetic aberrations is becoming increasingly important, not only to refine cancer diagnoses but also to predict response to targeted treatments. Next-generation sequencing is widely adopted in diagnostics for the analyses of DNA extracted from routinely processed formalin-fixed, paraffin-embedded tissue, fine-needle aspirates, or cytologic smears. PCR-based enrichment strategies are usually required to obtain sufficient read depth for reliable detection of genetic aberrations. However, although the read depth relates to sensitivity and specificity, PCR duplicates generated during target enrichment may result in overestimation of library complexity, which may result in false-negative results. Here, we report the validation of a 23-gene panel covering 41 hotspot regions using single-molecule tagging of DNA molecules by single-molecule molecular inversion probes (smMIPs), allowing assessment of library complexity. The smMIP approach outperforms Sanger and Ampliseq-Personal Genome Machine-based sequencing in our clinical diagnostic setting. Furthermore, single-molecule tags allow consensus sequence read formation, allowing detection to 1% allele frequency and reliable exclusion of variants to 3%. The number of false-positive calls is also markedly reduced (>10-fold), and our panel design allows for distinction between true mutations and deamination artifacts. Not only is this technique superior, smMIP-based library preparation is also scalable, easy to automate, and flexible. We have thus implemented this approach for sequence analysis of clinical samples in our routine diagnostic workflow.

Contains fulltext : 167941.pdf (Publisher’s version ) (Open Access) To identify candidate genes for intellectual disability, we performed a meta-analysis on 2,637 de novo mutations, identified from the exomes of 2,104 patient-parent trios. Statistical analyses identified 10 new candidate ID genes: DLG4, PPM1D, RAC1, SMAD6, SON, SOX5, SYNCRIP, TCF20, TLK2 and TRIP12. In addition, we show that these genes are intolerant to nonsynonymous variation and that mutations in these genes are associated with specific clinical ID phenotypes.

Aims The occurrence of connexin40 (Cx40) minor polymorphism (−44 G → A) was increased in patients with idiopathic atrial fibrillation (AF), although its effect on atrial Cx40 protein expression is unknown. We aimed to evaluate whether alterations in Cx40 are directly linked to the development of AF, we studied the effect of this polymorphism on Cx40 expression and distribution in patients without any history of AF and in patients who developed post-operative AF. Methods and Results Hundred and eight patients (mean age 67 ± 9 years), without a history of AF or conditions that predispose to AF, were included. During heart surgery, 10 cc blood was collected for DNA genotyping and the right atrial appendage was partly excised. Ten patients (9%) were homozygous for the minor allele (AA, Group 1), 30 (28%) were heterozygous (AG, Group 2), and 68 (63%) were non-carriers (GG, Group 3). Ten age- and sex-matched tissue samples per group were analysed for Cx40 expression by: (i) real-time quantitative polymerase chain reaction (Q-PCR), (ii) western blotting, and (iii) immunohistochemistry on cryosections. Real-time quantitative polymerase chain reaction showed no significant differences of Cx40 mRNA among the groups. Western blot analysis, however, revealed a reduction in Cx40 protein in Groups 1 (−36.4%) and 2 (−39.5%) as compared with Group 3. Immunohistochemistry confirmed this reduction but indicated an unaltered subcellular distribution of the remaining Cx40. Incidence of post-operative AF (28%) was age-dependent but unrelated to the presence of the polymorphism or fibrosis. Conclusion Presence of the Cx40 minor allele (−44 G → A) results in a uniform down-regulation of right atrial appendage Cx40 protein which was not significantly related to development of post-operative AF.

Activation Delay and VT Parameters. Introduction: Desmosomal changes, electrical uncoupling, and surviving myocardial bundles embedded in fibrofatty tissue are hallmarks of activation delay in arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C). Currently, generally accepted task force criteria (TFC) are used for clinical diagnosis. We propose additional criteria based on activation delay and ventricular tachycardia (VT) to improve identification of affected individuals.Methods and Results: Activation delay and VT-related 12-lead electrocardiographic (ECG) criteria were studied, while off drugs, in 42 patients with proven ARVD/C according to TFC, and 27 controls with idiopathic VT from the RV outflow tract. Two of three measured TFC could only be identified in a small minority of ARVD/C patients. Additional ECG criteria proposed in this study included (a) prolonged terminal activation duration, an indicator of activation delay; (b) VT with LBBB morphology and superior axis; and (c) multiple different VT morphologies. These criteria were met in 30 (71%), 28 (67%), and 37 (88%) ARVD/C patients, respectively, and in one control patient (P Conclusions: The proposed additional criteria are specific for ARVD/C and more sensitive than the current TFC. Therefore, adding the newly proposed criteria to current TFC could improve ARVD/C diagnosis, independent of DNA analysis.

BACKGROUND Dideoxy-based chain termination sequencing developed by Sanger is the gold standard sequencing approach and allows clinical diagnostics of disorders with relatively low genetic heterogeneity. Recently, new next generation sequencing (NGS) technologies have found their way into diagnostic laboratories, enabling the sequencing of large targeted gene panels or exomes. The development of benchtop NGS instruments now allows the analysis of single genes or small gene panels, making these platforms increasingly competitive with Sanger sequencing. METHODS We developed a generic automated ion semiconductor sequencing work flow that can be used in a clinical setting and can serve as a substitute for Sanger sequencing. Standard amplicon-based enrichment remained identical to PCR for Sanger sequencing. A novel postenrichment pooling strategy was developed, limiting the number of library preparations and reducing sequencing costs up to 70% compared to Sanger sequencing. RESULTS A total of 1224 known pathogenic variants were analyzed, yielding an analytical sensitivity of 99.92% and specificity of 99.99%. In a second experiment, a total of 100 patient-derived DNA samples were analyzed using a blind analysis. The results showed an analytical sensitivity of 99.60% and specificity of 99.98%, comparable to Sanger sequencing. CONCLUSIONS Ion semiconductor sequencing can be a first choice mutation scanning technique, independent of the genes analyzed.

Implementation of next-generation DNA sequencing (NGS) technology into routine diagnostic genome care requires strategic choices. Instead of theoretical discussions on the consequences of such choices, we compared NGS-based diagnostic practices in eight clinical genetic centers in the Netherlands, based on genetic testing of nine pre-selected patients with cardiomyopathy. We highlight critical implementation choices, including the specific contributions of laboratory and medical specialists, bioinformaticians and researchers to diagnostic genome care, and how these affect interpretation and reporting of variants. Reported pathogenic mutations were consistent for all but one patient. Of the two centers that were inconsistent in their diagnosis, one reported to have found 'no causal variant', thereby underdiagnosing this patient. The other provided an alternative diagnosis, identifying another variant as causal than the other centers. Ethical and legal analysis showed that informed consent procedures in all centers were generally adequate for diagnostic NGS applications that target a limited set of genes, but not for exome- and genome-based diagnosis. We propose changes to further improve and align these procedures, taking into account the blurring boundary between diagnostics and research, and specific counseling options for exome- and genome-based diagnostics. We conclude that alternative diagnoses may infer a certain level of 'greediness' to come to a positive diagnosis in interpreting sequencing results. Moreover, there is an increasing interdependence of clinic, diagnostics and research departments for comprehensive diagnostic genome care. Therefore, we invite clinical geneticists, physicians, researchers, bioinformatics experts and patients to reconsider their role and position in future diagnostic genome care.European Journal of Human Genetics advance online publication, 28 January 2015; doi:10.1038/ejhg.2014.279.

Contains fulltext : 49344.pdf (Publisher’s version ) (Closed access) The mechanism of trinucleotide repeat expansion, an important cause of neuromuscular and neurodegenerative diseases, is poorly understood. We report here on the study of the role of flap endonuclease 1 (Fen1), a structure-specific nuclease with both 5' flap endonuclease and 5'-3' exonuclease activity, in the somatic hypermutability of the (CTG)(n)*(CAG)(n) repeat of the DMPK gene in a mouse model for myotonic dystrophy type 1 (DM1). By intercrossing mice with Fen1 deficiency with transgenics with a DM1 (CTG)(n)*(CAG)(n) repeat (where 104n110), we demonstrate that Fen1 is not essential for faithful maintenance of this repeat in early embryonic cleavage divisions until the blastocyst stage. Additionally, we found that the frequency of somatic DM1 (CTG)(n)*(CAG)(n) repeat instability was essentially unaltered in mice with Fen1 haploinsufficiency up to 1.5 years of age. Based on these findings, we propose that Fen1, despite its role in DNA repair and replication, is not primarily involved in maintaining stability at the DM1 locus.

Genetic testing is now considered the standard of care in the management of familial adenomatous polyposis (FAP). Non-carriers of mutations are excluded from endoscopic surveillance. During the systematic screening of the relatives of an affected member with FAP, one non-carrier of APC mutations was unexpectedly found with the typical Cowden syndrome phenotype. One large Algerian family with FAP was screened for an APC germline mutation. Moreover, we also performed a mutation search in the Cowden syndrome member for PTEN, BMPR1A or MADH4 (SMAD4) germline mutations. We identified a mutation in the APC gene that results in a truncated protein (Y935X) in the FAP proband, and subsequently in 12 FAP-affected members. Among the 12 APC mutation-negative members of this family, we found one member with the Cowden disease phenotype. However, the mutation analysis in the PTEN gene and the two other genes involved in juvenile polyposis, namely BMPR1A and MADH4 (SMAD4), in the Cowden syndrome patient failed to show any pathogenic mutation. Genetic testing is a powerful tool that can provide a definitive diagnosis for FAP. However, not all polyposes in the FAP family are adenomatous polyposes.

Contains fulltext : 174726.pdf (Publisher’s version ) (Open Access) Inherited eye disorders have a large clinical and genetic heterogeneity, which makes genetic diagnosis cumbersome. An exome-sequencing approach was developed in which data analysis was divided into two steps: the vision gene panel and exome analysis. In the vision gene panel analysis, variants in genes known to cause inherited eye disorders were assessed for pathogenicity. If no causative variants were detected and when the patient consented, the entire exome data was analyzed. A total of 266 Dutch patients with different types of inherited eye disorders, including inherited retinal dystrophies, cataract, developmental eye disorders and optic atrophy, were investigated. In the vision gene panel analysis (likely), causative variants were detected in 49% and in the exome analysis in an additional 2% of the patients. The highest detection rate of (likely) causative variants was in patients with inherited retinal dystrophies, for instance a yield of 63% in patients with retinitis pigmentosa. In patients with developmental eye defects, cataract and optic atrophy, the detection rate was 50, 33 and 17%, respectively. An exome-sequencing approach enables a genetic diagnosis in patients with different types of inherited eye disorders using one test. The exome approach has the same detection rate as targeted panel sequencing tests, but offers a number of advantages. For instance, the vision gene panel can be frequently and easily updated with additional (novel) eye disorder genes. Determination of the genetic diagnosis improved the clinical diagnosis, regarding the assessment of the inheritance pattern as well as future disease perspective.

We recently identified two genetic subtypes of high-grade oligodendroglial tumors (HG-OT): 1p − /19q − HG-OT are characterized by a loss of chromosome 1p32–36 (del(1)(p32–p36) and/or a del(19)(q13.3); whereas + 7/ − 10 HG-OT harbor a gain of chromosome 7 ( + 7) and/or a − 10 without a loss of 1p32–36 and 19q13.3. Because a − 10 and a + 7 are most frequently detected in glioblastomas (GBM), the genotype of + 7/ − 10 HG-OT suggests that these tumors are GBM with a prominent oligodendroglial phenotype rather than anaplastic oligodendrogliomas. PTEN is a tumor suppressor gene, located at 10q23.3, which is involved in tumor progression of GBM and other neoplasms. In this study, we screened for PTEN mutations in six low-grade oligodendroglial tumors (LG-OT), five 1p − /19q − HG-OT, seven + 7/ − 10 HG-OT, and nine xenografted GBM. PTEN mutations were detected in none of the LG-OT and 1p − /19q − HG-OT, once in + 7/ − 10 HG-OT, and frequently in GBM. As one of the + 7/ − 10 HG-OT harbored a PTEN mutation, this demonstrates that PTEN can be involved in the oncogenesis of this genetic subtype of HG-OT. The lower frequency of PTEN mutations in + 7/ − 10 HG-OT compared to GBM suggests that these tumors are of a distinct tumor type rather than GBM. Published by Elsevier Science Inc.

Cowden disease (CD) is characterised by multiple hamartomas in a variety of tissues. The pathological hallmark is the presence of a number of trichilemmomas. Several neurological symptoms are also part of CD with megalencephaly and Lhermitte-Duclos disease (LDD) as the most important features. Early recognition of CD patients is important because of the increased risk of developing malignancies. Breast cancer is the most frequent malignancy, but also urogenital, digestive tract, and thyroid cancers are found,vith higher frequencies. CD was localised to chromosome 10q23 and the PTEN gene (also known as MMAC1 or TEP1) was shown to be involved. Germline mutations were identified in both familial and sporadic CD patients. We identified eight PTEN mutations, of which seven were novel, in 13 CD patients. Combined with previous data we have identified 17 independent CD mutations. Gross DNA alterations in CD patients were not detected. Genotype-phenotype relations are discussed. The only correlation suggested to exist is that missense mutations are not detected in LDD patients. However, larger numbers are needed to confirm this. Association of PTEN mutations and the occurrence of malignant breast disease found in an earlier study cannot be confirmed. Clinical features of five CD patients without a PTEN mutation in the coding sequence do not differ from CD patients with a PTEN mutation. Furthermore, if is likely that we have identified the majority of CD patients in the Netherlands. From this we estimate that CD has a prevalence of about 1 in 250 000 in the Dutch population with a low mutation frequency.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al., [Background]: There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance. [Results]: A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization. [Conclusions]: The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups., This work was supported by funds provided through the Gene Partnership and the Manton Center for Orphan Disease Research at Boston Children’s Hospital and the Center for Biomedical Informatics at Harvard Medical School and by generous donations in-kind of genomic sequencing services by Life Technologies (Carlsbad, CA, USA) and Complete Genomics (Mountain View, CA, USA).

Contains fulltext : 124810.pdf (Publisher’s version ) (Open Access) The advent of massive parallel sequencing is rapidly changing the strategies employed for the genetic diagnosis and research of rare diseases that involve a large number of genes. So far it is not clear whether these approaches perform significantly better than conventional single gene testing as requested by clinicians. The current yield of this traditional diagnostic approach depends on a complex of factors that include gene-specific phenotype traits, and the relative frequency of the involvement of specific genes. To gauge the impact of the paradigm shift that is occurring in molecular diagnostics, we assessed traditional Sanger-based sequencing (in 2011) and exome sequencing followed by targeted bioinformatics analysis (in 2012) for five different conditions that are highly heterogeneous, and for which our center provides molecular diagnosis. We find that exome sequencing has a much higher diagnostic yield than Sanger sequencing for deafness, blindness, mitochondrial disease, and movement disorders. For microsatellite-stable colorectal cancer, this was low under both strategies. Even if all genes that could have been ordered by physicians had been tested, the larger number of genes captured by the exome would still have led to a clearly superior diagnostic yield at a fraction of the cost.

Item does not contain fulltext For the reasons discussed here, we think whole-genome- or exome-based approaches are currently most suited for diagnostic implementation in genetically heterogeneous diseases, initially to complement and later to replace Sanger sequencing, qPCR and genomic microarrays. Patients do need to be counseled for the possibility of receiving medically relevant information not related to the disease under investigation, but this chance can be minimized by a focused data-analysis process. Establishing the pathogenicity of individual genetic variants remains a daunting task, requiring novel bioinformatic tools and high-throughput functional approaches, but at least we can now be more sure that we have not missed relevant genetic variation.

Contains fulltext : 95713.pdf (Publisher’s version ) (Closed access) The core phenotype of Kleefstra syndrome (KS) is characterized by intellectual disability, childhood hypotonia, and a characteristic facial appearance. This can be caused by either submicroscopic 9q34 deletions or loss of function mutations of the EHMT1 gene. Remarkably, in three patients with a clinical suspicion of KS, molecular cytogenetic analysis revealed an interstitial 9q34 microdeletion proximal to the coding region of the EHMT1 gene based on the NM_ 024757.3 transcript. Because we found a mono-allelic EHMT1 transcript suggestive for haploinsufficiency of EHMT1 in two of these patients tested, we hypothesized that a deletion of regulatory elements or so far unknown coding sequences in the 5' region of the EHMT1 gene, might result in a phenotype compatible with KS. We further characterized the molecular content of deletions proximal to the transcript NM_ 024757.3 and confirmed presence of a novel predicted open reading frame comprising 27 coding exons (NM_ 024757.4). Further analysis showed that all three deletions included the presumed novel first exon of the EHMT1 gene. Subsequent testing of 75 individuals without previously detectable EHMT1 aberrations showed one additional case with a deletion comprising only this 5' part of the gene. These results have important implications for the genetic screening of KS and for studies of the functional significance of EHMT1.

Myotonic dystrophy is a multisystem disorder that is transmitted in an autosomal dominant fashion and is characterized by muscular weakness and atrophy, clinical and electromyographic evidence of myotonia, ocular cataract, and various other abnormalities, such as cardiac conduction disturbances, testicular atrophy in males, premature balding, increased risk from anesthesia, and mental retardation in cases with early onset1. It is the most common inherited muscular dystrophy of adulthood, with an incidence of approximately 1 per 7500 people. The clinical expression of myotonic dystrophy is variable, ranging from neonatal mortality to a complete absence of symptoms. Recently, the disorder has been . . .

PRADER—WILLI syndrome represents the most common form of genetic obesity and is associated with mental retardation, short stature, sexual infantilism, and hypotonia1 2 3 4 5 6 (Table 1). In about 60 percent of affected persons a microscopically visible interstitial deletion in chromosome 15 (band ql2) is observed,2 , 3 , 7 8 9 10 and in up to 75 percent deletions can be found at the molecular level.11 122 13 14 DNA studies with polymorphic markers have indicated that in Prader-Willi syndrome the aberrant chromosome 15 is always of paternal origin,11 , 12 suggesting that the two parental chromosomes are differently imprinte1515 and that the presence of a gene or genes on the paternally . . .

High resolution genomic copy-number analysis has shown that inherited and de novo copy-number variations contribute significantly to autism pathology, and that identification of small chromosomal aberrations related to autism will expedite the discovery of risk genes involved. Here, we report a microduplication of chromosome 15q11.2, spanning only four genes, co-segregating with autism in a Dutch pedigree, identified by SNP microarray analysis, and independently confirmed by FISH and MLPA analysis. Quantitative RT-PCR analysis revealed over 70% increase in peripheral blood mRNA levels for the four genes present in the duplicated region in patients, and RNA in situ hybridization on mouse embryonic and adult brain sections revealed that two of the four genes, CYFIP1 and NIPA1, were highly expressed in the developing mouse brain. These findings point towards a contribution of microduplications at chromosome 15q11.2 to autism, and highlight CYFIP1 and NIPA1 as autism risk genes functioning in axonogenesis and synaptogenesis. Thereby, these findings further implicate defects in dosage-sensitive molecular control of neuronal connectivity in autism. However, the prevalence of this microduplication in patient samples was statistically not significantly different from control samples (0.94% in patients vs. 0.42% controls, P = 0.247), which suggests that our findings should be interpreted with caution and indicates the need for studies that include large numbers of control subjects to ascertain the impact of these changes on a population scale.

Background— Desmosomal changes, electric uncoupling, and surviving myocardial bundles in fibrofatty tissue characterize arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C). Resultant activation delay is pivotal for reentry and thereby ventricular tachycardia (VT). Current task force criteria (TFC) for diagnosis have limited sensitivity. The aim of this study was to assess the diagnostic value of additional criteria on activation delay and VT to improve identification of affected individuals. Methods and Results— ECG criteria were studied, while off drugs, in 50 index patients with proven ARVD/C according to TFC (TFC ≥4 points) and 33 patients with probable ARVD/C (TFC 3 points, or TFC3), being 21 index patients and 12 family members of proven ARVD/C patients. Newly proposed additional criteria are (1) prolonged terminal activation duration in V 1 -V 3 , an indicator of activation delay, (2) VT with left bundle-branch block morphology and superior axis, and (3) multiple VT morphologies. All index patients were screened for mutations in ARVD/C-related genes encoding desmosomal proteins. Altogether, 23 of 33 (70%) TFC3 patients fulfilled ARVD/C diagnosis when newly proposed criteria were applied additionally to current TFC. VT with left bundle-branch block morphology and superior axis or multiple VT morphologies were recorded in 12 and 9 of 33 TFC3 patients, respectively, all being index patients. When applying prolonged terminal activation duration additionally to TFC on depolarization/conduction abnormalities, 14 (42%) TFC3 patients fulfilled ARVD/C diagnosis. Results were not significantly different between mutation carriers and noncarriers. Conclusions— Adding the newly proposed criteria to current TFC for ARVD/C will improve identification of affected individuals importantly, independent of outcome of DNA analyses.

A Duplication Including GATA4 Does Not Co-Segregate With Congenital Heart Defects Irene C. Joziasse,* Jasper J. van der Smagt, Martin Poot, Ron Hochstenbach, Marcel R. Nelen, Marielle van Gijn, Dennis Dooijes, Barbara J.M. Mulder, and Pieter A. Doevendans Department of Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands Department of Clinical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, The Netherlands Department of Cardiology, Academic Medical Center, Amsterdam, The Netherlands

Background - Atrial fibrillation (AF) is a major cause of cerebral infarction. Idiopathic AF is strongly associated with the human minor Connexin 40 (Cx40) promotor polymorphism. We examined the prevalence of the minor Cx40 allele in patients with cerebral ischemia and no other cardiovascular disease (CVD), as an indication of underlying idiopathic AF. Methods - In patients with cerebral ischemia without prior CVD (n=225), DNA analysis of the Cx40 minor allele (-44 G¿A) was performed. Patients were divided into those with a normal electrocardiographic (ECG) findings (group A, n=164), with ECG abnormalities (group B, n=51) and those with normal ECG and documented episodes of AF (group C, n=10). On the basis of echocardiography (ECHO) data availability, further subgroups were defined: normal ECG and ECHO (group D, n=45); ECG or ECHO abnormalities (group E, n=22); and normal ECG and ECHO and documented AF episodes (group F, n=8). The prevalence of Cx40 promotor polymorphism was compared among all the subgroups. Results - The average age was 58.7 years (±11.5) and 64.4% were men. Patients with episodes of AF and those with abnormal ECG or ECHO results (B+C or E+F) did not show a higher prevalence of the minor allele genotype (AA vs. GG) compared with the normal ECG/ECHO groups (A or D) (group A, odds ratio=1.04, 95% confidence interval: 0.26–4.11 and group D, odds ratio=0.38, 95% confidence interval: 0.04–3.63). Conclusion - In patients with cerebral ischemic events, without prior CVD, a higher prevalence of the Cx40 gene polymorphism, as a marker of underlying idiopathic AF appeared to be absent

PUBLISHED, Background Duplications and deletions in the human genome can cause disease or predispose persons to disease. Advances in technologies to detect these changes allow for the routine identification of submicroscopic imbalances in large numbers of patients. Methods We tested for the presence of microdeletions and microduplications at a specific region of chromosome 1q21.1 in two groups of patients with unexplained mental retardation, autism, or congenital anomalies and in unaffected persons. Results We identified 25 persons with a recurrent 1.35-Mb deletion within 1q21.1 from screening 5218 patients. The microdeletions had arisen de novo in eight patients, were inherited from a mildly affected parent in three patients, were inherited from an apparently unaffected parent in six patients, and were of unknown inheritance in eight patients. The deletion was absent in a series of 4737 control persons (P=1.1x10?7). We found considerable variability in the level of phenotypic expression of the microdeletion; phenotypes included mild-to-moderate mental retardation, microcephaly, cardiac abnormalities, and cataracts. The reciprocal duplication was enriched in nine children with mental retardation or autism spectrum disorder and other variable features (P=0.02). We identified three deletions and three duplications of the 1q21.1 region in an independent sample of 788 patients with mental retardation and congenital anomalies. Conclusions We have identified recurrent molecular lesions that elude syndromic classification and whose disease manifestations must be considered in a broader context of development as opposed to being assigned to a specific disease. Clinical diagnosis in patients with these lesions may be most readily achieved on the basis of genotype rather than phenotype., Supported in part by grants from the National Institutes of Health (NIH) (HD043569, to Dr. Eichler), the South Carolina Department of Disabilities and Special Needs (to Drs. Skinner, Stevenson, and Schwartz), the Wellcome Trust (061183, to Dr. Tassabehji), the Andre & Cyprien Foundation and the University Hospitals of Geneva (to Drs. Antonarakis, Bena, and Gallati), and the European Union (EU) (project 219250, to Dr. Sharp; AnEUploidy project 037627, to Drs. Leeuw, Armengol, Antonarakis, Estivill, Veltman, and de Vries). The Irish Autism Study was funded by the Wellcome Trust and the Health Research Board (a grant to Drs. Gallagher and Gill). Dr. Poot was supported by a grant from the Dutch Foundation for Brain Research (Hersenstichting grant 2008(1) 34); Drs. Regan and Knight, by the Oxford Partnership Comprehensive Biomedical Research Centre; Dr. Willatt, by the Cambridge Biomedical Research Centre, with funding from the United Kingdom Department of Health's National Institute for Health Research Biomedical Research Centres funding scheme; Drs. Huang and Maloney, as part of the National Genetics Reference Laboratory (Wessex) by the United Kingdom Department of Health; Ms. Buysse, as a research assistant of the Research Foundation?Flanders (FWO?Vlaanderen); and Dr. Eichler, as an investigator of the Howard Hughes Medical Institute.

Introduction: Plakophilin-2 (PKP2) is the major genetic determinant of ARVD/C in the Netherlands. Mutations have been found in 43 out of 82 index cases (52%), diagnosed in accordance with the 1994 Task Force criteria (TFC). A total of 15 different mutations were detected, most of them nonsense (7), frameshift (2) and splice mutations (3), suggesting haploinsufficiency (loss of function) of PKP2 as the predominant mechanism in the etiology of ARVD/C. Since many of these mutations are probably functional null alleles, it was hypothesized that large deletions within - or encompassing - the PKP2 gene can also predispose to ARVD/C. Aim: to determine whether large deletions in PKP2 underlie apparently PKP2 negative ARVD/C cases. Methods: a multiple ligation-dependent probe amplification kit was developed to determine copy number of all 14 exons of the PKP2 gene. In total 120 patients were included that had been referred for PKP2 testing, and that had tested negative with direct sequencing or denaturing gradient gel electrophoresis. Twenty-six patients had definite ARVD/C (TFC: ≥ 4 pts), 11 patients scored 3 pts and 83 patients scored less than 3 pts or had insufficient clinical data. Results: a single deletion was detected encompassing the first 4 exons and promoter region of the PKP2 gene. The patient, a 35 year old male, had experienced near-syncope during exercise. Mild dilation of the right ventricle (1 pt), negative T-waves in V1-V5 (1 pt) and short VT’s with LBBB morphology during exercise testing were found (1 pt). There were frequent PVC’s. The father also carried the deletion. A third degree paternal relative died suddenly at age 28 during exercise. Conclusion: large genomic deletions of PKP2, that go unnoticed with sequencing, can cause ARVD/C-like disease. Comprehensive testing of PKP2 should include assessment of copy number. However, the frequency of large deletions seems to be low. Since the detected deletion includes the promoter and the transcription initiation site, no protein can be derived from the affected allele. This patient demonstrates that complete loss of one PKP2 allele does not necessarily lead to more severe ARVD/C. This is in keeping with the concept of haploinsufficiency and the assumption that many PKP2 mutations cause functional null alleles.

Joubert syndrome (JBS) is a clinically variable and genetically heterogeneous developmental brain disorder with autosomal recessive inheritance. Five genes, AHI1, NPHP1, CEP290, MKS3, and RPGRIPIL, and two additional loci on chromosome 9 and I I have been identified so far. The relative contributions of AHI1 mutations and NPHP1 deletions have not yet been determined in a population-based JBS patient cohort. We therefore undertook a nationwide survey of JBS in the Netherlands and performed DNA analysis of the AHI1 and NPHP1 genes, as well as a new candidate gene CYCLIN D1. We obtained clinical data and DNA samples of 25 Dutch JBS patients. DNA analysis of AHI1 revealed pathogenic homozygous or compound heterozygous AHI1 mutations in four patients (16%). Based on the birth prevalence of about I in 100,000 for JBS in the Netherlands, we estimated a carrier frequency of AHI1 mutations of approximately 1 in 400. In another two patients, the AHI1 mutation Arg830Trp was identified (homozygously and heterozygously), a possible low penetrance allele. No deletions of NPHP1 or CYCLIN D1 mutations were detected in these 25 patients. In the four patients with AHI1 mutations, retinal disease (Leber congenital amaurosis or retinal dystrophy) was present in two, whereas none had renal disease. Pooling our data and data from the literature, retinal disease seems to occur in 75% of AHI1-associated JBS patients. Renal disease is present in 10% at most. We conclude that AHI1 mutations are an important cause of JBS in Dutch patients, and should always be looked for in patients suspected of JBS, especially when retinal dystrophy is present. Patients with AHI1 mutations should be regularly checked for retinal and renal disease up until adolescence. (c) 2007 Published by Elsevier Masson SAS.