Publications by authors named "Isaac J Nijman"

66 Publications

Investigation of Genetic Modifiers of Copper Toxicosis in Labrador Retrievers.

Life (Basel) 2020 Oct 31;10(11). Epub 2020 Oct 31.

Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 108, 3584 CM Utrecht, The Netherlands.

Copper toxicosis is a complex genetic disorder in Labrador retrievers characterized by hepatic copper accumulation eventually leading to liver cirrhosis. The variation of hepatic copper levels in Labrador retrievers has been partly explained by mutations in c.980C>T and c.4358G>A. To further elucidate the genetic background of this disease, we used targeted Next Generation Sequencing (NGS) in a cohort of 95 Labrador retrievers to analyze 72 potential modifier genes for variations associated with hepatic copper levels. Variants associated with copper levels were subsequently evaluated in a replication cohort of 144 Labrador retrievers. A total of 44 variants in 25 different genes were identified, of which four showed significant association with copper levels. Of the four variants found associated with hepatic copper levels in the NGS cohort, one was validated in the replication cohort. The non-reference allele of the variant NC_006602.3.g.52434480C>T in resulting in amino-acid change p.Leu7Phe was associated with decreased hepatic copper levels. In humans, resistin is associated with severity of non-alcoholic fatty liver disease, fibrosis, cirrhosis and mitochondrial dysfunction in hepatocytes. Further studies are needed to investigate the biological function of p.Leu7Phe in the development of copper toxicosis in Labrador retrievers.
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http://dx.doi.org/10.3390/life10110266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693796PMC
October 2020

The role of rare compound heterozygous events in autism spectrum disorder.

Transl Psychiatry 2020 06 22;10(1):204. Epub 2020 Jun 22.

Department of Psychiatry, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.

The identification of genetic variants underlying autism spectrum disorders (ASDs) may contribute to a better understanding of their underlying biology. To examine the possible role of a specific type of compound heterozygosity in ASD, namely, the occurrence of a deletion together with a functional nucleotide variant on the remaining allele, we sequenced 550 genes in 149 individuals with ASD and their deletion-transmitting parents. This approach allowed us to identify additional sequence variants occurring in the remaining allele of the deletion. Our main goal was to compare the rate of sequence variants in remaining alleles of deleted regions between probands and the deletion-transmitting parents. We also examined the predicted functional effect of the identified variants using Combined Annotation-Dependent Depletion (CADD) scores. The single nucleotide variant-deletion co-occurrence was observed in 13.4% of probands, compared with 8.1% of parents. The cumulative burden of sequence variants (n = 68) in pooled proband sequences was higher than the burden in pooled sequences from the deletion-transmitting parents (n = 41, X = 6.69, p = 0.0097). After filtering for those variants predicted to be most deleterious, we observed 21 of such variants in probands versus 8 in their deletion-transmitting parents (X = 5.82, p = 0.016). Finally, cumulative CADD scores conferred by these variants were significantly higher in probands than in deletion-transmitting parents (burden test, β = 0.13; p = 1.0 × 10). Our findings suggest that the compound heterozygosity described in the current study may be one of several mechanisms explaining variable penetrance of CNVs with known pathogenicity for ASD.
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http://dx.doi.org/10.1038/s41398-020-00866-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7308334PMC
June 2020

Modifier genes in SCN1A-related epilepsy syndromes.

Mol Genet Genomic Med 2020 04 7;8(4):e1103. Epub 2020 Feb 7.

Department of Genetics, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht, The Netherlands.

Background: SCN1A is one of the most important epilepsy-related genes, with pathogenic variants leading to a range of phenotypes with varying disease severity. Different modifying factors have been hypothesized to influence SCN1A-related phenotypes. We investigate the presence of rare and more common variants in epilepsy-related genes as potential modifiers of SCN1A-related disease severity.

Methods: 87 patients with SCN1A-related epilepsy were investigated. Whole-exome sequencing was performed by the Beijing Genomics Institute (BGI). Functional variants in 422 genes associated with epilepsy and/or neuronal excitability were investigated. Differences in proportions of variants between the epilepsy genes and four control gene sets were calculated, and compared to the proportions of variants in the same genes in the ExAC database.

Results: Statistically significant excesses of variants in epilepsy genes were observed in the complete cohort and in the combined group of mildly and severely affected patients, particularly for variants with minor allele frequencies of <0.05. Patients with extreme phenotypes showed much greater excesses of epilepsy gene variants than patients with intermediate phenotypes.

Conclusion: Our results indicate that relatively common variants in epilepsy genes, which would not necessarily be classified as pathogenic, may play a large role in modulating SCN1A phenotypes. They may modify the phenotypes of both severely and mildly affected patients. Our results may be a first step toward meaningful testing of modifier gene variants in regular diagnostics for individual patients, to provide a better estimation of disease severity for newly diagnosed patients.
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http://dx.doi.org/10.1002/mgg3.1103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196470PMC
April 2020

Dutch genome diagnostic laboratories accelerated and improved variant interpretation and increased accuracy by sharing data.

Hum Mutat 2019 12 3;40(12):2230-2238. Epub 2019 Sep 3.

Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

Each year diagnostic laboratories in the Netherlands profile thousands of individuals for heritable disease using next-generation sequencing (NGS). This requires pathogenicity classification of millions of DNA variants on the standard 5-tier scale. To reduce time spent on data interpretation and increase data quality and reliability, the nine Dutch labs decided to publicly share their classifications. Variant classifications of nearly 100,000 unique variants were catalogued and compared in a centralized MOLGENIS database. Variants classified by more than one center were labeled as "consensus" when classifications agreed, and shared internationally with LOVD and ClinVar. When classifications opposed (LB/B vs. LP/P), they were labeled "conflicting", while other nonconsensus observations were labeled "no consensus". We assessed our classifications using the InterVar software to compare to ACMG 2015 guidelines, showing 99.7% overall consistency with only 0.3% discrepancies. Differences in classifications between Dutch labs or between Dutch labs and ACMG were mainly present in genes with low penetrance or for late onset disorders and highlight limitations of the current 5-tier classification system. The data sharing boosted the quality of DNA diagnostics in Dutch labs, an initiative we hope will be followed internationally. Recently, a positive match with a case from outside our consortium resulted in a more definite disease diagnosis.
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http://dx.doi.org/10.1002/humu.23896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900155PMC
December 2019

The molecular genetic make-up of male breast cancer.

Endocr Relat Cancer 2019 10;26(10):779-794

Department of Pathology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.

Male breast cancer (MBC) is extremely rare and accounts for less than 1% of all breast malignancies. Therefore, clinical management of MBC is currently guided by research on the disease in females. In this study, DNA obtained from 45 formalin-fixed paraffin-embedded (FFPE) MBCs with and 90 MBCs (52 FFPE and 38 fresh-frozen) without matched normal tissues was subjected to massively parallel sequencing targeting all exons of 1943 cancer-related genes. The landscape of mutations and copy number alterations was compared to that of publicly available estrogen receptor (ER)-positive female breast cancers (smFBCs) and correlated to prognosis. From the 135 MBCs, 90% showed ductal histology, 96% were ER-positive, 66% were progesterone receptor (PR)-positive, and 2% HER2-positive, resulting in 50, 46 and 4% luminal A-like, luminal B-like and basal-like cases, respectively. Five patients had Klinefelter syndrome (4%) and 11% of patients harbored pathogenic BRCA2 germline mutations. The genomic landscape of MBC to some extent recapitulated that of smFBC, with recurrent PIK3CA (36%) and GATA3 (15%) somatic mutations, and with 40% of the most frequently amplified genes overlapping between both sexes. TP53 (3%) somatic mutations were significantly less frequent in MBC compared to smFBC, whereas somatic mutations in genes regulating chromatin function and homologous recombination deficiency-related signatures were more prevalent. MDM2 amplifications were frequent (13%), correlated with protein overexpression (P = 0.001) and predicted poor outcome (P = 0.007). In conclusion, despite similarities in the genomic landscape between MBC and smFBC, MBC is a molecularly unique and heterogeneous disease requiring its own clinical trials and treatment guidelines.
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http://dx.doi.org/10.1530/ERC-19-0278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6938562PMC
October 2019

Amplicon-Based Targeted Next-Generation Sequencing of Formalin-Fixed, Paraffin-Embedded Tissue.

Methods Mol Biol 2019 ;1908:1-17

Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.

Next-generation sequencing (NGS) is rapidly becoming the method of choice for mutation analysis in both research and diagnostics. The benefit of targeted NGS compared to whole-genome and whole-exome sequencing is that smaller amounts of input material can be used as well as qualitatively suboptimal tissue samples, like formalin-fixed, paraffin-embedded archival tissue.Here, we describe the protocol for targeted next-generation sequencing using the Ion Torrent PGM platform in combination with Ion Ampliseq NGS gene panels for formalin-fixed, paraffin-embedded tissues. Both the manual and the automated workflow are described as well as the bioinformatics for data analysis.
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http://dx.doi.org/10.1007/978-1-4939-9004-7_1DOI Listing
June 2019

Somatic alterations compromised molecular diagnosis of DOCK8 hyper-IgE syndrome caused by a novel intronic splice site mutation.

Sci Rep 2018 11 13;8(1):16719. Epub 2018 Nov 13.

Chair and Institute of Environmental Medicine, UNIKA-T, Technical University of Munich and Helmholtz Zentrum Munich, Munich/Augsburg, Munich, Germany.

In hyper-IgE syndromes (HIES), a group of primary immunodeficiencies clinically overlapping with atopic dermatitis, early diagnosis is crucial to initiate appropriate therapy and prevent irreversible complications. Identification of underlying gene defects such as in DOCK8 and STAT3 and corresponding molecular testing has improved diagnosis. Yet, in a child and her newborn sibling with HIES phenotype molecular diagnosis was misleading. Extensive analyses driven by the clinical phenotype identified an intronic homozygous DOCK8 variant c.4626 + 76 A > G creating a novel splice site as disease-causing. While the affected newborn carrying the homozygous variant had no expression of DOCK8 protein, in the index patient molecular diagnosis was compromised due to expression of altered and wildtype DOCK8 transcripts and DOCK8 protein as well as defective STAT3 signaling. Sanger sequencing of lymphocyte subsets revealed that somatic alterations and reversions revoked the predominance of the novel over the canonical splice site in the index patient explaining DOCK8 protein expression, whereas defective STAT3 responses in the index patient were explained by a T cell phenotype skewed towards central and effector memory T cells. Hence, somatic alterations and skewed immune cell phenotypes due to selective pressure may compromise molecular diagnosis and need to be considered with unexpected clinical and molecular findings.
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http://dx.doi.org/10.1038/s41598-018-34953-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233225PMC
November 2018

Assessment of parental mosaicism in -related epilepsy by single-molecule molecular inversion probes and next-generation sequencing.

J Med Genet 2019 02 27;56(2):75-80. Epub 2018 Oct 27.

Department of Genetics, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht, The Netherlands.

Background: Dravet syndrome is a severe genetic encephalopathy, caused by pathogenic variants in Low-grade parental mosaicism occurs in a substantial proportion of families (7%-13%) and has important implications for recurrence risks. However, parental mosaicism can remain undetected by methods regularly used in diagnostics. In this study, we use single-molecule molecular inversion probes (smMIP), a technique with high sensitivity for detecting low-grade mosaic variants and high cost-effectiveness, to investigate the incidence of parental mosaicism of variants in a cohort of 90 families and assess the feasibility of this technique.

Methods: Deep sequencing of was performed using smMIPs. False positive rates for each of the proband's pathogenic variants were determined in 145 unrelated samples. If parents showed corresponding variant alleles at a significantly higher rate than the established noise ratio, mosaicism was confirmed by droplet digital PCR (ddPCR).

Results: Sequence coverage of at least 100× at the location of the corresponding pathogenic variant was reached for 80 parent couples. The variant ratio was significantly higher than the established noise ratio in eight parent couples, of which four (5%) were regarded as true mosaics, based on ddPCR results. The false positive rate of smMIP analysis without ddPCR was therefore 50%. Three of these variants had previously been considered de novo in the proband by Sanger sequencing.

Conclusion: smMIP technology combined withnext generation sequencing (NGS) performs better than Sanger sequencing in the detection of parental mosaicism. Because parental mosaicism has important implications for genetic counselling and recurrence risks, we stress the importance of implementing high-sensitivity NGS-based assays in standard diagnostics.
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http://dx.doi.org/10.1136/jmedgenet-2018-105672DOI Listing
February 2019

Mosaicism of de novo pathogenic SCN1A variants in epilepsy is a frequent phenomenon that correlates with variable phenotypes.

Epilepsia 2018 03 20;59(3):690-703. Epub 2018 Feb 20.

Department of Genetics, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht, the Netherlands.

Objective: Phenotypes caused by de novo SCN1A pathogenic variants are very variable, ranging from severely affected patients with Dravet syndrome to much milder genetic epilepsy febrile seizures plus cases. The most important determinant of disease severity is the type of variant, with variants that cause a complete loss of function of the SCN1A protein (α-subunit of the neuronal sodium channel Nav1.1) being detected almost exclusively in Dravet syndrome patients. However, even within Dravet syndrome disease severity ranges greatly, and consequently other disease modifiers must exist. A better prediction of disease severity is very much needed in daily practice to improve counseling, stressing the importance of identifying modifying factors in this patient group. We evaluated 128 participants with de novo, pathogenic SCN1A variants to investigate whether mosaicism, caused by postzygotic mutation, is a major modifier in SCN1A-related epilepsy.

Methods: Mosaicism was investigated by reanalysis of the pathogenic SCN1A variants using single molecule molecular inversion probes and next generation sequencing with high coverage. Allelic ratios of pathogenic variants were used to determine whether mosaicism was likely. Selected mosaic variants were confirmed by droplet digital polymerase chain reaction and sequencing of different tissues. Developmental outcome was classified based on available data on intelligence quotient and school functioning/education.

Results: Mosaicism was present for 7.5% of de novo pathogenic SCN1A variants in symptomatic patients. Mosaic participants were less severely affected than nonmosaic participants if only participants with truncating variants are considered (distribution of developmental outcome scores, Mann-Whitney U, P = .023).

Significance: Postzygotic mutation is a common phenomenon in SCN1A-related epilepsies. Participants with mosaicism have on average milder phenotypes, suggesting that mosaicism can be a major modifier of SCN1A-related diseases. Detection of mosaicism has important implications for genetic counseling and can be achieved by deep sequencing of unique reads.
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http://dx.doi.org/10.1111/epi.14021DOI Listing
March 2018

Longitudinal analysis of colon crypt stem cell dynamics in sulindac treated Familial Adenomatous Polyposis patients.

Sci Rep 2017 09 20;7(1):11972. Epub 2017 Sep 20.

Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.

The non-steroidal anti-inflammatory drug sulindac decreases size and number of adenomas after 4-6 months of treatment for familial adenomatous polyposis (FAP) patients. However, the underlying mechanism remains unknown. As stem cells are thought to be the tumor precursor cells, visualizing their behavior is crucial for monitoring tumor progression. Increased tag diversity in inactive genes is indicative of a protracted clonal evolution and consequently, increased risk for tumor formation. Therefore, the effect of sulindac on stem cell dynamics was studied. Normal appearing single crypts were laser microdissected in placebo- and sulindac- treated FAP patient tissue after which the methylation patterns were visualized by Next Generation Sequencing. A significant difference in tag diversity over time was found in the sulindac group compared to the placebo group (*p = 0.018), indicative of a shortened clonal evolution treated sulindac. The rate of change in tag diversity over time was correlated with polyp number change over time. No significant difference over time was observed in the percent methylation when comparing placebo vs sulindac. In conclusion, daily sulindac administration in FAP patients significantly altered colorectal stem cell dynamics, which might explain the chemopreventive action of this drug indicating that tag diversity may be used as a predictive biomarker.
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http://dx.doi.org/10.1038/s41598-017-11865-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5607292PMC
September 2017

Ankyrin repeat and zinc-finger domain-containing 1 mutations are associated with infantile-onset inflammatory bowel disease.

J Biol Chem 2017 05 16;292(19):7904-7920. Epub 2017 Mar 16.

From the Division of Pediatrics, Wilhelmina Children's Hospital,

Infantile-onset inflammatory bowel disease (IO IBD) is an invalidating illness with an onset before 2 years of age and has a complex pathophysiology in which genetic factors are important. Homozygosity mapping and whole-exome sequencing in an IO IBD patient and subsequent sequencing of the candidate gene in 12 additional IO IBD patients revealed two patients with two mutated ankyrin repeat and zinc-finger domain-containing 1 () alleles (homozygous R585Q mutation and compound heterozygous E152K and V32_Q87del mutations, respectively) and two patients with one mutated allele. Although the function of ANKZF1 in mammals had not been previously evaluated, we show that ANKZF1 has an indispensable role in the mitochondrial response to cellular stress. ANKZF1 is located diffusely in the cytoplasm and translocates to the mitochondria upon cellular stress. ANKZF1 depletion reduces mitochondrial integrity and mitochondrial respiration under conditions of cellular stress. The mutations identified in IO IBD patients with two mutated alleles result in dysfunctional ANKZF1, as shown by an increased level of apoptosis in patients' lymphocytes, a decrease in mitochondrial respiration in patient fibroblasts with a homozygous R585Q mutation, and an inability of ANKZF1 R585Q and E152K to rescue the phenotype of yeast deficient in Vms1, the yeast homologue of ANKZF1. These data indicate that loss-of-function mutations in result in deregulation of mitochondrial integrity, and this may play a pathogenic role in the development of IO IBD.
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http://dx.doi.org/10.1074/jbc.M116.772038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5427269PMC
May 2017

Dwarfism with joint laxity in Friesian horses is associated with a splice site mutation in B4GALT7.

BMC Genomics 2016 10 28;17(1):839. Epub 2016 Oct 28.

Animal Breeding and Genomics Centre, Wageningen University, PO Box 338, NL-6700 AH, Wageningen, The Netherlands.

Background: Inbreeding and population bottlenecks in the ancestry of Friesian horses has led to health issues such as dwarfism. The limbs of dwarfs are short and the ribs are protruding inwards at the costochondral junction, while the head and back appear normal. A striking feature of the condition is the flexor tendon laxity that leads to hyperextension of the fetlock joints. The growth plates of dwarfs display disorganized and thickened chondrocyte columns. The aim of this study was to identify the gene defect that causes the recessively inherited trait in Friesian horses to understand the disease process at the molecular level.

Results: We have localized the genetic cause of the dwarfism phenotype by a genome wide approach to a 3 Mb region on the p-arm of equine chromosome 14. The DNA of two dwarfs and one control Friesian horse was sequenced completely and we identified the missense mutation ECA14:g.4535550C > T that cosegregated with the phenotype in all Friesians analyzed. The mutation leads to the amino acid substitution p.(Arg17Lys) of xylosylprotein beta 1,4-galactosyltransferase 7 encoded by B4GALT7. The protein is one of the enzymes that synthesize the tetrasaccharide linker between protein and glycosaminoglycan moieties of proteoglycans of the extracellular matrix. The mutation not only affects a conserved arginine codon but also the last nucleotide of the first exon of the gene and we show that it impedes splicing of the primary transcript in cultured fibroblasts from a heterozygous horse. As a result, the level of B4GALT7 mRNA in fibroblasts from a dwarf is only 2 % compared to normal levels. Mutations in B4GALT7 in humans are associated with Ehlers-Danlos syndrome progeroid type 1 and Larsen of Reunion Island syndrome. Growth retardation and ligamentous laxity are common manifestations of these syndromes.

Conclusions: We suggest that the identified mutation of equine B4GALT7 leads to the typical dwarfism phenotype in Friesian horses due to deficient splicing of transcripts of the gene. The mutated gene implicates the extracellular matrix in the regular organization of chrondrocyte columns of the growth plate. Conservation of individual amino acids may not be necessary at the protein level but instead may reflect underlying conservation of nucleotide sequence that are required for efficient splicing.
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http://dx.doi.org/10.1186/s12864-016-3186-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5084406PMC
October 2016

Heterozygous KIDINS220/ARMS nonsense variants cause spastic paraplegia, intellectual disability, nystagmus, and obesity.

Hum Mol Genet 2016 06 22;25(11):2158-2167. Epub 2016 Mar 22.

Department of Genetics

We identified de novo nonsense variants in KIDINS220/ARMS in three unrelated patients with spastic paraplegia, intellectual disability, nystagmus, and obesity (SINO). KIDINS220 is an essential scaffold protein coordinating neurotrophin signal pathways in neurites and is spatially and temporally regulated in the brain. Molecular analysis of patients' variants confirmed expression and translation of truncated transcripts similar to recently characterized alternative terminal exon splice isoforms of KIDINS220 KIDINS220 undergoes extensive alternative splicing in specific neuronal populations and developmental time points, reflecting its complex role in neuronal maturation. In mice and humans, KIDINS220 is alternative spliced in the middle region as well as in the last exon. These full-length and KIDINS220 splice variants occur at precise moments in cortical, hippocampal, and motor neuron development, with splice variants similar to the variants seen in our patients and lacking the last exon of KIDINS220 occurring in adult rather than in embryonic brain. We conducted tissue-specific expression studies in zebrafish that resulted in spasms, confirming a functional link with disruption of the KIDINS220 levels in developing neurites. This work reveals a crucial physiological role of KIDINS220 in development and provides insight into how perturbation of the complex interplay of KIDINS220 isoforms and their relative expression can affect neuron control and human metabolism. Altogether, we here show that de novo protein-truncating KIDINS220 variants cause a new syndrome, SINO. This is the first report of KIDINS220 variants causing a human disease.
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http://dx.doi.org/10.1093/hmg/ddw082DOI Listing
June 2016

Tissue-specific mutation accumulation in human adult stem cells during life.

Nature 2016 Oct 3;538(7624):260-264. Epub 2016 Oct 3.

Center for Molecular Medicine, Cancer Genomics Netherlands, Department of Genetics, University Medical Center Utrecht, Heidelberglaan 100, 3584CX Utrecht, The Netherlands.

The gradual accumulation of genetic mutations in human adult stem cells (ASCs) during life is associated with various age-related diseases, including cancer. Extreme variation in cancer risk across tissues was recently proposed to depend on the lifetime number of ASC divisions, owing to unavoidable random mutations that arise during DNA replication. However, the rates and patterns of mutations in normal ASCs remain unknown. Here we determine genome-wide mutation patterns in ASCs of the small intestine, colon and liver of human donors with ages ranging from 3 to 87 years by sequencing clonal organoid cultures derived from primary multipotent cells. Our results show that mutations accumulate steadily over time in all of the assessed tissue types, at a rate of approximately 40 novel mutations per year, despite the large variation in cancer incidence among these tissues. Liver ASCs, however, have different mutation spectra compared to those of the colon and small intestine. Mutational signature analysis reveals that this difference can be attributed to spontaneous deamination of methylated cytosine residues in the colon and small intestine, probably reflecting their high ASC division rate. In liver, a signature with an as-yet-unknown underlying mechanism is predominant. Mutation spectra of driver genes in cancer show high similarity to the tissue-specific ASC mutation spectra, suggesting that intrinsic mutational processes in ASCs can initiate tumorigenesis. Notably, the inter-individual variation in mutation rate and spectra are low, suggesting tissue-specific activity of common mutational processes throughout life.
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http://dx.doi.org/10.1038/nature19768DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5536223PMC
October 2016

Targeted sequencing of 351 candidate genes for epileptic encephalopathy in a large cohort of patients.

Mol Genet Genomic Med 2016 Sep 30;4(5):568-80. Epub 2016 Jul 30.

Department of Genetics UMC Utrecht Utrecht The Netherlands.

Background: Many genes are candidates for involvement in epileptic encephalopathy (EE) because one or a few possibly pathogenic variants have been found in patients, but insufficient genetic or functional evidence exists for a definite annotation.

Methods: To increase the number of validated EE genes, we sequenced 26 known and 351 candidate genes for EE in 360 patients. Variants in 25 genes known to be involved in EE or related phenotypes were followed up in 41 patients. We prioritized the candidate genes, and followed up 31 variants in this prioritized subset of candidate genes.

Results: Twenty-nine genotypes in known genes for EE (19) or related diseases (10), dominant as well as recessive or X-linked, were classified as likely pathogenic variants. Among those, likely pathogenic de novo variants were found in EE genes that act dominantly, including the recently identified genes EEF1A2, KCNB1 and the X-linked gene IQSEC2. A de novo frameshift variant in candidate gene HNRNPU was the only de novo variant found among the followed-up candidate genes, and the patient's phenotype was similar to a few recent publications.

Conclusion: Mutations in genes described in OMIM as, for example, intellectual disability gene can lead to phenotypes that get classified as EE in the clinic. We confirmed existing literature reports that de novo loss-of-function HNRNPUmutations lead to severe developmental delay and febrile seizures in the first year of life.
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http://dx.doi.org/10.1002/mgg3.235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5023942PMC
September 2016

Destabilized SMC5/6 complex leads to chromosome breakage syndrome with severe lung disease.

J Clin Invest 2016 08 18;126(8):2881-92. Epub 2016 Jul 18.

The structural maintenance of chromosomes (SMC) family of proteins supports mitotic proliferation, meiosis, and DNA repair to control genomic stability. Impairments in chromosome maintenance are linked to rare chromosome breakage disorders. Here, we have identified a chromosome breakage syndrome associated with severe lung disease in early childhood. Four children from two unrelated kindreds died of severe pulmonary disease during infancy following viral pneumonia with evidence of combined T and B cell immunodeficiency. Whole exome sequencing revealed biallelic missense mutations in the NSMCE3 (also known as NDNL2) gene, which encodes a subunit of the SMC5/6 complex that is essential for DNA damage response and chromosome segregation. The NSMCE3 mutations disrupted interactions within the SMC5/6 complex, leading to destabilization of the complex. Patient cells showed chromosome rearrangements, micronuclei, sensitivity to replication stress and DNA damage, and defective homologous recombination. This work associates missense mutations in NSMCE3 with an autosomal recessive chromosome breakage syndrome that leads to defective T and B cell function and acute respiratory distress syndrome in early childhood.
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http://dx.doi.org/10.1172/JCI82890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4966312PMC
August 2016

De novo 14q24.2q24.3 microdeletion including IFT43 is associated with intellectual disability, skeletal anomalies, cardiac anomalies, and myopia.

Am J Med Genet A 2016 06 19;170(6):1566-9. Epub 2016 Feb 19.

Department of Genetics, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht, The Netherlands.

We report an 11-year-old girl with mild intellectual disability, skeletal anomalies, congenital heart defect, myopia, and facial dysmorphisms including an extra incisor, cup-shaped ears, and a preauricular skin tag. Array comparative genomic hybridization analysis identified a de novo 4.5-Mb microdeletion on chromosome 14q24.2q24.3. The deleted region and phenotype partially overlap with previously reported patients. Here, we provide an overview of the literature on 14q24 microdeletions and further delineate the associated phenotype. We performed exome sequencing to examine other causes for the phenotype and queried genes present in the 14q24.2q24.3 microdeletion that are associated with recessive disease for variants in the non-deleted allele. The deleted region contains 65 protein-coding genes, including the ciliary gene IFT43. Although Sanger and exome sequencing did not identify variants in the second IFT43 allele or in other IFT complex A-protein-encoding genes, immunocytochemistry showed increased accumulation of IFT-B proteins at the ciliary tip in patient-derived fibroblasts compared to control cells, demonstrating defective retrograde ciliary transport. This could suggest a ciliary defect in the pathogenesis of this disorder. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/ajmg.a.37598DOI Listing
June 2016

Effectiveness of whole-exome sequencing and costs of the traditional diagnostic trajectory in children with intellectual disability.

Genet Med 2016 09 4;18(9):949-56. Epub 2016 Feb 4.

Department of Genetics, Utrecht University Medical Center, Utrecht, The Netherlands.

Purpose: This study investigated whole-exome sequencing (WES) yield in a subset of intellectually disabled patients referred to our clinical diagnostic center and calculated the total costs of these patients' diagnostic trajectory in order to evaluate early WES implementation.

Methods: We compared 17 patients' trio-WES yield with the retrospective costs of diagnostic procedures by comprehensively examining patient records and collecting resource use information for each patient, beginning with patient admittance and concluding with WES initiation. We calculated cost savings using scenario analyses to evaluate the costs replaced by WES when used as a first diagnostic tool.

Results: WES resulted in diagnostically useful outcomes in 29.4% of patients. The entire traditional diagnostic trajectory average cost was $16,409 per patient, substantially higher than the $3,972 trio-WES cost. WES resulted in average cost savings of $3,547 for genetic and metabolic investigations in diagnosed patients and $1,727 for genetic investigations in undiagnosed patients.

Conclusion: The increased causal variant detection yield by WES and the relatively high costs of the entire traditional diagnostic trajectory suggest that early implementation of WES is a relevant and cost-efficient option in patient diagnostics. This information is crucial for centers considering implementation of WES and serves as input for future value-based research into diagnostics.Genet Med 18 9, 949-956.
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http://dx.doi.org/10.1038/gim.2015.200DOI Listing
September 2016

Mice lacking functional CD95-ligand display reduced proliferation of the intestinal epithelium without gross homeostatic alterations.

Med Mol Morphol 2016 Jun 23;49(2):110-8. Epub 2015 Dec 23.

Department of Surgery, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.

Homeostasis of the continuously self-renewing intestinal tract involves cell proliferation, migration, differentiation along the crypt-villus-axis and shedding of cells into the gut lumen. CD95-ligand (FAS-ligand, CD95L) is a cytokine that is known for its capacity to induce apoptosis by binding its cognate receptor, CD95 (Fas). More recently, it was discovered that CD95L can also induce other cellular responses, such as proliferation, differentiation and cell migration. CD95L is highly expressed in Paneth cells of the small intestine which are in close contact with intestinal stem cells. This suggests a potential role for CD95L in controlling stem cell function and, possibly, intestinal homeostasis. We analyzed the intestines of mice deficient for functional CD95L (gld) for potential alterations in the diversity of stem-cell-lineages and parameters of intestinal homeostasis. Stem cell diversity was assessed by analyzing methylation patterns of the non-transcribed mMYOD gene. Proliferation was analyzed by BrdU labeling and differentiation was assessed by immunohistochemistry. Of all parameters analyzed, only epithelial cell proliferation was significantly reduced in the small intestines of gld-mice, but not in their colons which lack CD95L expression. We conclude that CD95L has a proliferation-stimulating role during normal turnover of the small intestine, but has a marginal effect on overall intestinal homeostasis.
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http://dx.doi.org/10.1007/s00795-015-0129-9DOI Listing
June 2016

Prioritization and burden analysis of rare variants in 208 candidate genes suggest they do not play a major role in CAKUT.

Kidney Int 2016 Feb;89(2):476-86

The leading cause of end-stage renal disease in children is attributed to congenital anomalies of the kidney and urinary tract (CAKUT). Familial clustering and mouse models support the presence of monogenic causes. Genetic testing is insufficient as it mainly focuses on HNF1B and PAX2 mutations that are thought to explain CAKUT in 5–15% of patients. To identify novel, potentially pathogenic variants in additional genes, we designed a panel of genes identified from studies on familial forms of isolated or syndromic CAKUT and genes suggested by in vitro and in vivo CAKUT models. The coding exons of 208 genes were analyzed in 453 patients with CAKUT using next-generation sequencing. Rare truncating, splice-site variants, and non-synonymous variants, predicted to be deleterious and conserved, were prioritized as the most promising variants to have an effect on CAKUT. Previously reported disease-causing mutations were detected, but only five were fully penetrant causal mutations that improved diagnosis. We prioritized 148 candidate variants in 151 patients, found in 82 genes, for follow-up studies. Using a burden test, no significant excess of rare variants in any of the genes in our cohort compared with controls was found. Thus, in a study representing the largest set of genes analyzed in CAKUT patients to date, the contribution of previously implicated genes to CAKUT risk was significantly smaller than expected, and the disease may be more complex than previously assumed.
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http://dx.doi.org/10.1038/ki.2015.319DOI Listing
February 2016

A nonsense mutation in B3GALNT2 is concordant with hydrocephalus in Friesian horses.

BMC Genomics 2015 Oct 9;16:761. Epub 2015 Oct 9.

Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, PO Box 80154, 3508 TD, Utrecht, The Netherlands.

Background: Hydrocephalus in Friesian horses is a developmental disorder that often results in stillbirth of affected foals and dystocia in dams. The occurrence is probably related to a founder effect and inbreeding in the population. The aim of our study was to find genomic associations, to investigate the mode of inheritance, to allow a DNA test for hydrocephalus in Friesian horses to be developed. In case of a monogenic inheritance we aimed to identify the causal mutation.

Results: A genome-wide association study of hydrocephalus in 13 cases and 69 controls using 29,720 SNPs indicated the involvement of a region on ECA1 (P <1.68 × 10(-6)). Next generation DNA sequence analysis of 4 cases and 6 controls of gene exons within the region revealed a mutation in β-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2) as the likely cause of hydrocephalus in Friesian horses. The nonsense mutation XM_001491545 c.1423C>T corresponding to XP_001491595 p.Gln475* was identical to a B3GALNT2 mutation identified in a human case of muscular dystrophy-dystroglycanopathy with hydrocephalus. All 16 available cases and none of the controls were homozygous for the mutation, and all 17 obligate carriers (= dams of cases) were heterozygous. A random sample of the Friesian horse population (n = 865) was tested for the mutation in a commercial laboratory. One-hundred and forty-seven horses were carrier and 718 horses were homozygous for the normal allele; the estimated allele frequency in the Friesian horse population is 0.085.

Conclusions: Hydrocephalus in Friesian horses has an autosomal recessive mode of inheritance. A nonsense mutation XM_001491545 c.1423C>T corresponding to XP_001491595 p.Gln475* in B3GALNT2 (1:75,859,296-75,909,376) is concordant with hydrocephalus in Friesian horses. Application of a DNA test in the breeding programme will reduce the losses caused by hydrocephalus in the Friesian horse population.
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http://dx.doi.org/10.1186/s12864-015-1936-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4600337PMC
October 2015

Loss-of-Function Mutations in the WNT Co-receptor LRP6 Cause Autosomal-Dominant Oligodontia.

Am J Hum Genet 2015 Oct 17;97(4):621-6. Epub 2015 Sep 17.

Department of Medical Genetics, Centre for Molecular Medicine, University Medical Centre Utrecht, 3508 AB Utrecht, the Netherlands. Electronic address:

Tooth agenesis is one of the most common developmental anomalies in man. Oligodontia, a severe form of tooth agenesis, occurs both as an isolated anomaly and as a syndromal feature. We performed exome sequencing on 20 unrelated individuals with apparent non-syndromic oligodontia and failed to detect mutations in genes previously associated with oligodontia. In three of the probands, we detected heterozygous variants in LRP6, and sequencing of additional oligodontia-affected individuals yielded one additional mutation in LRP6. Three mutations (c.1144_1145dupAG [p.Ala383Glyfs(∗)8], c.1779dupT [p.Glu594(∗)], and c.2224_2225dupTT [p.Leu742Phefs(∗)7]) are predicted to truncate the protein, whereas the fourth (c.56C>T [p.Ala19Val]) is a missense variant of a conserved residue located at the cleavage site of the protein's signal peptide. All four affected individuals harboring a LRP6 mutation had a family history of tooth agenesis. LRP6 encodes a transmembrane cell-surface protein that functions as a co-receptor with members from the Frizzled protein family in the canonical Wnt/β-catenin signaling cascade. In this same pathway, WNT10A was recently identified as a major contributor in the etiology of non-syndromic oligodontia. We show that the LRP6 missense variant (c.56C>T) results in altered glycosylation and improper subcellular localization of the protein, resulting in abrogated activation of the Wnt pathway. Our results identify LRP6 variants as contributing to the etiology of non-syndromic autosomal-dominant oligodontia and suggest that this gene is a candidate for screening in DNA diagnostics.
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http://dx.doi.org/10.1016/j.ajhg.2015.08.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4596913PMC
October 2015

Effective Therapeutic Intervention and Comprehensive Genetic Analysis of mTOR Signaling in PEComa: A Case Report.

Anticancer Res 2015 Jun;35(6):3399-403

Department of Molecular Oncology, Netherlands Cancer Institute, Amsterdam, the Netherlands.

Background/aim: Perivascular epithelioid cell tumors (PEComas) are rare mesenchymal neoplasms. The exact genetic alterations underlying the pathophysiology of PEComas are largely unknown, although it has been shown that activation of the Mammalian target of rapamycin (mTOR) signaling pathway plays a pivotal role. Herein we describe the successful treatment of a patient with metastatic PEComa with the mTOR inhibitor everolimus and a comprehensive analysis to identify mechanisms for response.

Materials And Methods: Immunohistochemistry, array comparative genomic hybridization (aCGH) and genetic analyses were performed.

Results: Immunohistochemistry confirmed constitutive activation of mTOR. aCGH revealed a hyperdiploid karyotype affecting large regions of the genome. Next-generation sequencing did not reveal any tumor-specific mutations in mTOR-related genes.

Conclusion: Our results show the complexity of determining causal genetic alterations that can predict responsiveness to mTOR inhibition, even for a tumor with a complete remission to this specific treatment.
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June 2015

Familial Ehlers-Danlos syndrome with lethal arterial events caused by a mutation in COL5A1.

Am J Med Genet A 2015 Jun 2;167(6):1196-203. Epub 2015 Apr 2.

Department of Medical Genetics, University Medical Center Utrecht (UMCU), Utrecht, The Netherlands.

Different forms of Ehlers-Danlos syndrome (EDS) exist, with specific phenotypes and associated genes. Vascular EDS, caused by heterozygous mutations in the COL3A1 gene, is characterized by fragile vasculature with a high risk of catastrophic vascular events at a young age. Classic EDS, caused by heterozygous mutations in the COL5A1 or COL5A2 genes, is characterized by fragile, hyperextensible skin and joint laxity. To date, vessel rupture in four unrelated classic EDS patients with a confirmed COL5A1 mutation has been reported. We describe familial occurrence of a phenotype resembling vascular EDS in a mother and her two sons, who all died at an early age from arterial ruptures. Diagnostic Sanger sequencing in the proband failed to detect aberrations in COL3A1, COL1A1, COL1A2, TGFBR1, TGFBR2, SMAD3, and ACTA2. Next, the proband's DNA was analyzed using a next-generation sequencing approach targeting 554 genes linked to vascular disease (VASCULOME project). A novel heterozygous mutation in COL5A1 was detected, resulting in an essential glycine substitution at the C-terminal end of the triple helix domain (NM_000093.4:c.4610G>T; p.Gly1537Val). This mutation was also present in DNA isolated from autopsy material of the index's brother. No material was available from the mother, but the mutation was excluded in her parents, siblings and in the father of her sons, suggesting that the COL5A1 mutation occurred in the mother's genome de novo. In conclusion, we report familial occurrence of lethal arterial events caused by a COL5A1 mutation.
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http://dx.doi.org/10.1002/ajmg.a.36997DOI Listing
June 2015

Sambamba: fast processing of NGS alignment formats.

Bioinformatics 2015 Jun 19;31(12):2032-4. Epub 2015 Feb 19.

Department of Statistical Simulation, St. Petersburg State University, St. Petersburg, Russia, Illumina Cambridge, Cambridge, UK, Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences, Utrecht, The Netherlands, Department of Medical Genetics, Institute for Molecular Medicine, University Medical Centre Utrecht, Utrecht, The Netherlands and Department of Nematology, Wageningen University, Wageningen, The Netherlands Department of Statistical Simulation, St. Petersburg State University, St. Petersburg, Russia, Illumina Cambridge, Cambridge, UK, Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences, Utrecht, The Netherlands, Department of Medical Genetics, Institute for Molecular Medicine, University Medical Centre Utrecht, Utrecht, The Netherlands and Department of Nematology, Wageningen University, Wageningen, The Netherlands.

Unlabelled: Sambamba is a high-performance robust tool and library for working with SAM, BAM and CRAM sequence alignment files; the most common file formats for aligned next generation sequencing data. Sambamba is a faster alternative to samtools that exploits multi-core processing and dramatically reduces processing time. Sambamba is being adopted at sequencing centers, not only because of its speed, but also because of additional functionality, including coverage analysis and powerful filtering capability.

Availability And Implementation: Sambamba is free and open source software, available under a GPLv2 license. Sambamba can be downloaded and installed from http://www.open-bio.org/wiki/Sambamba.Sambamba v0.5.0 was released with doi:10.5281/zenodo.13200.
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http://dx.doi.org/10.1093/bioinformatics/btv098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4765878PMC
June 2015

Genetic etiology of renal agenesis: fine mapping of Renag1 and identification of Kit as the candidate functional gene.

PLoS One 2015 18;10(2):e0118147. Epub 2015 Feb 18.

McArdle Laboratory for Cancer Research, Department of Oncology, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin, United States of America; Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, Nebraska, United States of America; University of Wisconsin Carbone Cancer Center, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin, United States of America.

Congenital anomalies of the kidney and urogenital tract (CAKUT) occur in approximately 0.5% of live births and represent the most frequent cause of end-stage renal disease in neonates and children. The genetic basis of CAKUT is not well defined. To understand more fully the genetic basis of one type of CAKUT, unilateral renal agenesis (URA), we are studying inbred ACI rats, which spontaneously exhibit URA and associated urogenital anomalies at an incidence of approximately 10%. URA is inherited as an incompletely dominant trait with incomplete penetrance in crosses between ACI and Brown Norway (BN) rats and a single responsible genetic locus, designated Renag1, was previously mapped to rat chromosome 14 (RNO14). The goals of this study were to fine map Renag1, identify the causal genetic variant responsible for URA, confirm that the Renag1 variant is the sole determinant of URA in the ACI rat, and define the embryologic basis of URA in this rat model. Data presented herein localize Renag1 to a 379 kilobase (kb) interval that contains a single protein coding gene, Kit (v-kit Hardy-Zukerman 4 feline sarcoma viral oncogene homolog); identify an endogenous retrovirus-derived long terminal repeat located within Kit intron 1 as the probable causal variant; demonstrate aberrant development of the nephric duct in the anticipated number of ACI rat embryos; and demonstrate expression of Kit and Kit ligand (Kitlg) in the nephric duct. Congenic rats that harbor ACI alleles at Renag1 on the BN genetic background exhibit the same spectrum of urogenital anomalies as ACI rats, indicating that Renag1 is necessary and sufficient to elicit URA and associated urogenital anomalies. These data reveal the first genetic link between Kit and URA and illustrate the value of the ACI rat as a model for defining the mechanisms and cell types in which Kit functions during urogenital development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0118147PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4333340PMC
December 2015

Next-generation sequencing-based genome diagnostics across clinical genetics centers: implementation choices and their effects.

Eur J Hum Genet 2015 Sep 28;23(9):1142-50. Epub 2015 Jan 28.

Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

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.
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http://dx.doi.org/10.1038/ejhg.2014.279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4538197PMC
September 2015

Organoid models of human and mouse ductal pancreatic cancer.

Cell 2015 Jan 31;160(1-2):324-38. Epub 2014 Dec 31.

Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA; Lustgarten Foundation Pancreatic Cancer Research Laboratory, Cold Spring Harbor, NY 11724, USA.

Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.
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http://dx.doi.org/10.1016/j.cell.2014.12.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4334572PMC
January 2015

Simultaneous detection of clinically relevant mutations and amplifications for routine cancer pathology.

J Mol Diagn 2015 Jan 24;17(1):10-8. Epub 2014 Oct 24.

Department of Medical Oncology, University Medical Center Utrecht, Heidelberglaan Utrecht, Utrecht, the Netherlands; Netherlands Center for Personalized Cancer Treatment, Universiteitsweg Utrecht, Utrecht, the Netherlands.

In routine cancer molecular pathology, various independent experiments are required to determine mutation and amplification status of clinically relevant genes. Most of these tests are designed to identify a limited number of genetic aberrations, most likely in a given tumor type. We present a modified version of a multiplexed PCR and IonTorrent-based sequencing approach that can replace a large number of existing assays. The test allows for the simultaneous detection of point mutations and gene amplifications in 40 genes, including known hotspot regions in oncogenes (KRAS, BRAF), inactivating mutations in tumor suppressors (TP53, PTEN), and oncogene amplifications (ERBB2, EGFR). All point mutations were confirmed using certified diagnostic assays, and a sensitivity and specificity of 100% (95% CI, 0.875-1.0) and 99% (95% CI, 0.960-0.999), respectively, were determined for amplifications in FFPE material. Implementation of a single assay to effectively detect mutations and amplifications in clinically relevant genes not only improves the efficiency of the workflow within diagnostic laboratories but also increases the chance of detecting (rare) actionable variants for a given tumor type that are typically missed in routine pathology. The ability to obtain comprehensive and rapid mutational overviews is key for improving the efficiency of cancer patient care through tailoring treatments based on the genetic characteristics of individual tumors.
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http://dx.doi.org/10.1016/j.jmoldx.2014.09.004DOI Listing
January 2015