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Pre- and postsurgical differentiation between follicular thyroid adenoma (FTA) and follicular thyroid cancer (FTC) represents a significant diagnostic challenge. Furthermore, it remains unclear whether they share a common or distinct background and what the mechanisms underlying follicular thyroid lesions malignancy are. The study aimed to compare FTA and FTC by the comprehensive microarray and to identify recurrent regions of loss of heterozygosity (LOH). We analyzed formalin-fixed paraffin-embedded (FFPE) samples acquired from 32 Caucasian patients diagnosed with FTA (16) and FTC (16). We used the OncoScan™ microarray assay (Affymetrix, USA), using highly multiplexed molecular inversion probes for single nucleotide polymorphism (SNP). The total number of LOH was higher in FTC compared with FTA (18 vs. 15). The most common LOH present in 21 cases, in both FTA (10 cases) and FTC (11 cases), was 16p12.1, which encompasses many cancer-related genes, such as TP53, and was followed by 3p21.31. The only LOH present exclusively in FTA patients (56% vs. 0%) was 11p11.2-p11.12. The alteration which tended to be detected more often in FTC (6 vs. 1 in FTA) was 12q24.11-q24.13 overlapping FOXN4, MYL2, PTPN11 genes. FTA and FTC may share a common genetic background, even though differentiating rearrangements may also be detected.

Contains fulltext : 237830.pdf (Publisher’s version ) (Open Access) Genome-wide association studies (GWAS) have generated unprecedented insights into the genetic etiology of orofacial clefting (OFC). The moderate effect sizes of associated noncoding risk variants and limited access to disease-relevant tissue represent considerable challenges for biological interpretation of genetic findings. As rare variants with stronger effect sizes are likely to also contribute to OFC, an alternative approach to delineate pathogenic mechanisms is to identify private mutations and/or an increased burden of rare variants in associated regions. This report describes a framework for targeted resequencing at selected noncoding risk loci contributing to nonsyndromic cleft lip with/without cleft palate (nsCL/P), the most frequent OFC subtype. Based on GWAS data, we selected three risk loci and identified candidate regulatory regions (CRRs) through the integration of credible SNP information, epigenetic data from relevant cells/tissues, and conservation scores. The CRRs (total 57 kb) were resequenced in a multiethnic study population (1061 patients; 1591 controls), using single-molecule molecular inversion probe technology. Combining evidence from in silico variant annotation, pedigree- and burden analyses, we identified 16 likely deleterious rare variants that represent new candidates for functional studies in nsCL/P. Our framework is scalable and represents a promising approach to the investigation of additional congenital malformations with multifactorial etiology.

Single nucleotide polymorphism (SNP) is the most common type of genetic sequence variations and known to associate with the development of various diseases including cancer. Accurate detection of SNP is often necessary to determine the susceptibility of patients to diseases as well as their response to the medical treatments. Molecular inversion probe (MIP) has been widely utilized as an alternative method to detect the SNP as compared to the conventional PCR/genome sequencing method. However, base-pairing based detection of SNP fundamentally lacks target specificity toward the single mismatch of bases and often fails to meet the accuracy requirement for SNP detection. In this study, we report the development of new SNP detection method using a dumbbell-probe with a gap-filling approach. Two separate DNA strands are designed to self-assemble to form a dumbbell DNA probe for the selective detection of EGFR 21 point mutation. Unlike the direct binding of MIP to the target sequence, our system has an additional process for the enhancement of sequence specificity through a base matched gap-filling. In the presence of target sequences, the dumbbell-probe undergoes base-pair hybridization to the target sequence followed by sequence specific gap-filling and ligation in single-step. The detection of EGFR 21 point mutation is achieved with a highly specific and selective manner. This simple and robust detection method offers promising future potential in the diagnosis of various genetic mutations.

Genomic chimerism represents co-existing cells with different genotypes and has diagnostic significance in transplant engraftment monitoring, residual cancer detection, and other contexts. We previously described an approach to chimerism detection by interrogating variably present or absent genomic loci using single-molecule molecular inversion probes (smMIPs) and next-generation sequencing, which provided ultrasensitive limits of detection (

Introduction Risk stratification of children with ependymomas of the posterior fossa in current therapeutic protocols is mainly based on clinical criteria. We aimed to identify independent outcome predictors for this disease entity by a systematic integrated analysis of clinical, histological and genetic information in a defined cohort of patients treated according to the German HIT protocols. Methods Tumor samples of 134 patients aged 0.2–15.9 years treated between 1999 and 2010 according to HIT protocols were analyzed for histological features including mitotic activity, necrosis and vascular proliferation and genomic alterations by SNP and molecular inversion probe analysis. Survival analysis was performed by Kaplan-Meier method with log rank test and multivariate Cox regression analysis. Results Residual tumor after surgery, chromosome 1q gain and structural genomic alterations were identified as predictors of significantly shorter event-free (EFS) and overall survival (OS). Furthermore, specific histological features including vascular proliferation, necrosis and high mitotic activity were predictive for shorter OS. Multivariate Cox regression revealed residual tumor, chromosome 1q gain and mitotic activity as independent predictors of both EFS and OS. Using these independent predictors of outcome, we were able to build a 3-tiered risk stratification model that separates patients with standard, intermediate and high risk, and which outperforms current stratification procedures. Conclusion The integration of defined clinical, histological and genetic parameters led to an improved risk-stratification model for posterior fossa ependymoma of childhood. After validation in independent cohorts this model may provide the basis for risk-adapted treatment of children with ependymomas of the posterior fossa.

Microsatellite instability (MSI) testing of colorectal cancers (CRCs) is used to screen for Lynch syndrome (LS), a hereditary cancer‐predisposition, and can be used to predict response to immunotherapy. Here, we present a single‐molecule molecular inversion probe and sequencing‐based MSI assay and demonstrate its clinical validity according to existing guidelines. We amplified 24 microsatellites in multiplex and trained a classifier using 98 CRCs, which accommodates marker specific sensitivities to MSI. Sample classification achieved 100% concordance with the MSI Analysis System v1.2 (Promega) in three independent cohorts, totaling 220 CRCs. Backward–forward stepwise selection was used to identify a 6‐marker subset of equal accuracy to the 24‐marker panel. Assessment of assay detection limits showed that the 24‐marker panel is marginally more robust to sample variables than the 6‐marker subset, detecting as little as 3% high levels of MSI DNA in sample mixtures, and requiring a minimum of 10 template molecules to be sequenced per marker for >95% accuracy. BRAF c.1799 mutation analysis was also included to streamline LS testing, with all c.1799T>A variants being correctly identified. The assay, therefore, provides a cheap, robust, automatable, and scalable MSI test with internal quality controls, suitable for clinical cancer diagnostics.

Background Mutations in the RAS/RAF pathway predict resistance to anti-epidermal growth factor receptor antibodies in colorectal cancer (CRC), and may be targets for future therapies. This study investigates concordance of BRAF, HRAS, KRAS, NRAS and PIK3CA mutation status in primary CRC with matched liver (n = 274), lung (n = 114) or combined liver and lung metastases (n = 14). Methods Next generation sequencing was performed on DNA from formalin-fixed paraffin embedded CRC and matched liver and/or lung metastases, for recurrent mutations in BRAF, HRAS, KRAS, NRAS and PIK3CA and using the single-molecule molecular inversion probe method. Results Paired sequencing results on all five genes were reached in 249 of the 402 cases (62%). The obtained number of unique reads was not always sufficient to confidently call the absence or presence of mutations for all regions of interest. The mutational status of matched pairs was highly concordant; 91.1% concordance for all five genes, 95.5% for KRAS, 99.1% for NRAS. Lung metastases more often harboured RAS mutations compared to liver metastases (71% vs. 48%, p Conclusions In this large series of CRC we show that both primary tumors and corresponding metastases can be used to determine the mutational status for targeted therapy, given the high concordance rates. Next generation sequencing including a single molecule tags is feasible, however in combination with archival formalin-fixed paraffin embedded material is limited by coverage depth.

Aim: This study assesses the technical performance and cost of a targeted next-generation sequencing (NGS) multigene pharmacogenetic (PGx) test. Materials & methods: A genetic test was developed for 21 PGx genes using molecular inversion probes to generate library fragments for NGS. Performance of this test was assessed using 53 unique reference control cell lines from the Genetic Testing Reference Materials Coordination Program (GeT-RM). Results: 93.7% of variants were successfully called and the repeatability rate was 99.9%. Reference calls were available for 78.4% of diplotype calls resulting from PGx testing, and concordance for the test was 85.7%. Cost per sample was $32–$56. Conclusion: A targeted NGS assay using molecular inversion probe technology is able to characterize the pharmacogenome efficiently.

Next generation sequencing (NGS) technologies have enabled the possibility of analyzing a large number of SNPs simultaneously from multiple samples in a single experiment, for complementing the shortcomings of STR based methods. To efficiently genotype the desired SNPs, it is critical to optimize the library construction procedures. In this study, we formulated a strategy combining the molecular inversion probe (MIP) based target region capture method and NGS for genotyping 1245 SNPs. All the SNPs we selected exhibited high heterozygosity (minor allele frequency (MAF) > 0.3) according to 1000 genomes data. We applied the method to genotype a population of 210 unrelated individuals from the Hubei province of China and assessed the allele frequencies, Hardy-Weinberg equilibrium and linkage disequilibrium. The MAFs of more than 95% of the SNPs were ≥0.2, and no significant deviation or strong linkage was observed for 98% of the SNPs. The data indicated that, even within a relatively confined region, our SNP panel is suitable for individual identifications. Furthermore, we performed paternity test for 7 trio families using low quality DNA samples. The conclusions are in total agreement with these of STR-based analyses, with higher confidence indexes. Finally, we evaluated the performance of the MIP-NGS method with mock degraded DNA samples. We were able to genotype most of the SNPs even when the genomic DNA was sonicated to ˜100 bp range. In summary, we established a highly accurate and cost-effective method of SNP genotyping, which is potentially capable of solving complex issues encountered in forensic practices.

A substantial proportion of subjects with autosomal recessive retinitis pigmentosa (arRP) or Usher syndrome type II (USH2) lacks a genetic diagnosis due to incomplete USH2A screening in the early days of genetic testing. These cases lack eligibility for optimal genetic counseling and future therapy. USH2A defects are the most frequent cause of USH2 and are also causative in individuals with arRP. Therefore, USH2A is an important target for genetic screening. The aim of this study was to assess unscreened or incompletely screened and unexplained USH2 and arRP cases for (likely) pathogenic USH2A variants. Molecular inversion probe (MIP)-based sequencing was performed for the USH2A exons and their flanking regions, as well as published deep-intronic variants. This was done to identify single nucleotide variants (SNVs) and copy number variants (CNVs) in 29 unscreened or partially pre-screened USH2 and 11 partially pre-screened arRP subjects. In 29 out of these 40 cases, two (likely) pathogenic variants were successfully identified. Four of the identified SNVs and one CNV were novel. One previously identified synonymous variant was demonstrated to affect pre-mRNA splicing. In conclusion, genetic diagnoses were obtained for a majority of cases, which confirms that MIP-based sequencing is an effective screening tool for USH2A. Seven unexplained cases were selected for future analysis with whole genome sequencing.

AimsHaematopoietic clones caused by somatic mutations with ≥2% variant allele frequency (VAF), known as clonal haematopoiesis of indeterminate potential (CHIP), increase with age and have been linked to risk of haematological malignancies and cardiovascular disease. Recent observations suggest that smaller clones are also associated with adverse clinical outcomes. Our aims were to determine the prevalence of clonal haematopoiesis driven by clones of variable sizes, and to examine the development of clones over time in relation to age and metabolic dysregulation over up to 20 years in individuals with obesity.Methods and ResultsWe used an ultrasensitive single-molecule molecular inversion probe sequencing assay to identify clonal haematopoiesis driver mutations (CHDMs) in blood samples from individuals with obesity from the Swedish Obese Subjects study. In a single-timepoint dataset with samples from 1050 individuals, we identified 273 candidate CHDMs in 216 individuals, with VAF ranging from 0.01% to 31.15% and CHDM prevalence and clone sizes increasing with age. Longitudinal analysis over 20 years in CHDM-positive samples from 40 individuals showed that small clones can grow over time and become CHIP. VAF increased on average by 7% (range -4% to 27%) per year. Rate of clone growth was positively associated with insulin resistance (R=0.40, P=0.025) and low circulating levels of high-density lipoprotein-cholesterol (HDL-C) (R=-0.68, P=1.74E-05).ConclusionOur results show that haematopoietic clones can be detected and monitored before they become CHIP and indicate that insulin resistance and low HDL-C, well-established cardiovascular risk factors, are associated with clonal expansion in individuals with obesity.Translational perspectivesClonal haematopoiesis-driver mutations are somatic mutations in haematopoietic stem cells that lead to clones detectable in peripheral blood. Haematopoietic clones with a variant allele frequency (VAF) ≥2%, known as clonal haematopoiesis of indeterminate potential (CHIP), are recognized as an independent cardiovascular risk factor. Here, we show that smaller clones are prevalent, and also correlate with age. Our longitudinal observations in individuals with obesity over 20 years showed that more than half of all clone-positive individuals show growing clones and clones with VAF Translational outlook 1: Haematopoietic clones can be detected and monitored before they become CHIP.Translational outlook 2: The association between insulin resistance and low HDL-C with growth of haematopoietic clones opens the possibility that treatments improving metabolism, such as weight loss, may reduce growth of clones and thereby cardiovascular risk.One Sentence SummaryIn obesity, the growth rate of mutation-driven haematopoietic clones increased with insulin resistance and low HDL-C, both known risk factors for cardiovascular disease.

Adult medulloblastomas (MB) are rare. We investigated the genetic landscape and prognostic impact of genetic aberrations in a cohort of 117 adult medulloblastomas. Histological features and pathway activation were evaluated at the protein level; 14.5% showed wingless-type activation, 63.3% SHH activation, and 22.2% were classified as non-WNT/non-SHH-MB. Genome-wide copy number analysis was performed by molecular inversion probe array technology. MB-related genes were sequenced in WNT- and SHH-activated MBs. 79.7% of SHH-MBs showed desmoplastic/nodular histology; all other MBs had classic histology. WNT-MBs carried oncogenic CTNNB1 mutations in 88.2% and had monosomy 6 in 52.9%. In SHH-MBs, TERT promoter mutations occurred in 97%, mutations in PTCH1 in 38.2%, SMO in 15.5%, SUFU in 7.4%, and TP53-mutations in 4.1%. In all, 84.6% of non-WNT/non-SHH-MBs had an isochromosome 17q. A whole chromosomal aberration (WCA) signature was present in 45.1% of SHH-TP53-wild type (wt)-MBs and 65.4% of non-WNT/non-SHH-MBs. In 98 cases with survival data, WNT-MBs had a 5-year overall survival (OS) of 68.6%. SHH-MBs TP53wt type and non-WNT/non-SHH-MBs showed 5-year OS of 80.4% and 70.8%, respectively. TP53-mutant SHH-MBs represented a prognostically unfavorable entity; all patients died within 5 years. Patients with a WCA signature showed significantly increased OS (p = 0.011 for SHH-TP53wt-MBs and p = 0.048 for non-WNT/non-SHH-MBs).

Simple Summary Cell lines are widely used for research, but even commonly used cell lines may not be well characterized, making validation of published data and generalizing the results to the clinic problematic. Here, we have established a head and neck squamous cell carcinomas (HNSCC) cell line database. We believe that these cell lines are suitable models to study HNSCC disease in vitro and in vivo and may provide a valuable tool for investigators wishing to study the relation between e.g., hypoxia and radiosensitivity in HNSCC. Abstract To study head and neck squamous cell carcinomas (HNSCC) in vitro, a large variety of HNSCC cell lines have been developed. Here, we characterize a panel of 22 HNSCC cell lines, thereby providing a tool for research into tumor-specific treatment options in HNSCC. Both human papillomavirus (HPV) positive and HPV negative tumor cell lines were collected from commercial and collaborative sources. Short tandem repeat profiling was used to confirm or characterize the identity of the cell lines. Targeted sequencing was performed using a standard pathology single molecule Molecular Inversion Probe panel to detect mutations for 23 tumor suppressors and oncogenes. HPV status, p16 status, radiosensitivity data, and hypoxia data are summarized from all cell lines. We detected HPV transcripts in five cell lines, all of which overexpressed p16. One HPV negative cell line was also p16 positive. We detected mutations in KIT (SCCNij185), PIK3CA (SCCNij185), and CDKN2A (UT-SCC-5 and UT-SCC-38). TP53 mutations were the most frequent, occurring in 16/22 cell lines. HPV infection and TP53 mutations were almost mutually exclusive, with the exception of 93-VU-147T. The cell lines exhibited a wide range of sensitivities towards hypoxia and irradiation. Here, we provide a description of a set of frequently used HNSCC cell lines with diverse characteristics as found in HNSCC patients.

Malaria diagnostic testing in Africa is threatened by Plasmodium falciparum parasites lacking histidine-rich protein 2 (pfhrp2) and 3 (pfhrp3) genes. Among 12,572 subjects enrolled along Ethiopia’s borders with Eritrea, Sudan, and South Sudan and using multiple assays, we estimate HRP2-based rapid diagnostic tests would miss 9.7% (95% CI 8.5-11.1) of falciparum malaria cases due to pfhrp2 deletion. Established and novel genomic tools reveal distinct subtelomeric deletion patterns, well-established pfhrp3 deletions, and recent expansion of pfhrp2 deletion. Current diagnostic strategies need to be urgently reconsidered in Ethiopia, and expanded surveillance is needed throughout the Horn of Africa.

BackgroundInterleukin(IL)-1 signaling is of major importance in human innate cytokine responses. Common variants in related genes have been linked to various inflammation-mediated diseases and stimulation-induced cytokine responses, but the role of rare variants remains to be elucidated.MethodsIn this study, we characterize the role of rare and common genetic variation, as identified by molecular inversion probe-based sequencing, in 48 genes related to the IL-1 pathway. Using a systems biology approach, we examined the inter-individual variability of in vitro stimulation-specific human cytokine responses from 463 healthy individuals of the Human Functional Genomics Project and assessed the role of rare and common genetic variants, separately and combined, by means of the Sequence Kernel Association Test.ResultsWe identified strong associations for rare genetic variants in NCF4 (adjP=7.2E−05) and CASP1 (adjP=3.0E−05) with IL-6 production in response to PHA and LPS stimulation, respectively. In addition, common variants in IL36A and IL38 were associated to both C. albicans-induced IL-1β (IL36AadjP=0.0442; IL38adjP=0.0092) and IL-6 production (IL36AadjP=0.0037; IL38adjP=0.0082), an effect that was stronger at the subpathway level both for IL-1β (adjP=0.0017) and IL-6 (adjP=1.8E−04). The common variant signature for the IL-1β and IL-6 response to C. albicans was confirmed by an association with all anti-inflammatory genes (adjP=1.87E−03 and adjP=5.75E−04), and we validated this finding for non-coding common variants. Lastly, we identified a burden of rare variants in pro-inflammatory genes and LPS-induced IL-6 production (adjP=2.42E−04), and a new role for anti-inflammatory rare variants on S. aureus-stimulated IL-6 production (adjP=6.71E−03).ConclusionsIn conclusion, we show that both common and rare genetic variation in genes of the IL-1 pathway, separately and combined, differentially influence in vitro cytokine responses to various stimuli in healthy individuals. This study therefore accentuates potential mechanisms that are translatable into new hypothesis-driven characterization of common and rare variant involvement in a wide variety of inflammatory and immunological mechanisms and diseases.

Resolving the genetic architecture of painful neuropathy will lead to better disease management strategies. We aimed to develop a reliable method to re-sequence multiple genes in a large cohort of painful neuropathy patients at low cost. In this study, we compared sensitivity, specificity, targeting efficiency, performance and cost effectiveness of Molecular Inversion Probes-Next generation sequencing (MIPs-NGS) and TruSeq® Custom Amplicon-Next generation sequencing (TSCA-NGS). Capture probes were designed to target nine sodium channel genes (SCN3A, SCN8A-SCN11A, and SCN1B-SCN4B). One hundred sixty-six patients with diabetic and idiopathic neuropathy were tested by both methods, 70 patients were validated by Sanger sequencing. Sensitivity, specificity and performance of both techniques were comparable, and in agreement with Sanger sequencing. The average targeted regions coverage for MIPs-NGS was 97.3% versus 93.9% for TSCA-NGS. MIPs-NGS has a more versatile assay design and is more flexible than TSCA-NGS. The cost of MIPs-NGS is >5 times cheaper than TSCA-NGS when 500 or more samples are tested. In conclusion, MIPs-NGS is a reliable, flexible, and relatively inexpensive method to detect genetic variations in a large cohort of patients. In our centers, MIPs-NGS is currently implemented as a routine diagnostic tool for screening of sodium channel genes in painful neuropathy patients.

Targeted sequencing of genomic regions is a cost- and time-efficient approach for screening patient cohorts. We present a fast and efficient workflow to analyze highly imbalanced, targeted next-generation sequencing data generated using molecular inversion probe (MIP) capture. Our Snakemake pipeline performs sample demultiplexing, overlap paired-end merging, alignment, MIP-arm trimming, variant calling, coverage analysis and report generation. Further, we support the analysis of probes specifically designed to capture certain structural variants and can assign sex using Y-chromosome-unique probes. In a user-friendly HTML report, we summarize all these results including covered, incomplete or missing regions, called variants and their predicted effects. We developed and tested our pipeline using the hemophilia A & B MIP design from the “My Life, Our Future” initiative. HemoMIPs is available as an open-source tool on GitHub at: https://github.com/kircherlab/hemoMIPs, Author summary Next generation sequencing techniques enable researchers to identify causal variants for patients in large cohorts. Targeted sequencing approaches capture genomic regions of interest to allow high throughput and cost efficient patient-specific data generation. HemoMIPs is an open-source software that analyses targeted sequencing datasets generated using molecular inversion probes (MIPs) and provides HTML reports of pathogenic and benign variants, patient sex, existence of known structural variants as well as performance statistics on the sequencing run.

Background Sensitive and reliable molecular diagnostics is needed to guide therapeutic decisions for cancer patients. Although less material becomes available for testing, genetic markers are rapidly expanding. Simultaneous detection of predictive markers, including mutations, gene amplifications and MSI, will save valuable material, time and costs. Methods Using a single-molecule molecular inversion probe (smMIP)-based targeted next-generation sequencing (NGS) approach, we developed an NGS panel allowing detection of predictive mutations in 33 genes, gene amplifications of 13 genes and microsatellite instability (MSI) by the evaluation of 55 microsatellite markers. The panel was designed to target all clinically relevant single and multiple nucleotide mutations in routinely available lung cancer, colorectal cancer, melanoma, and gastro-intestinal stromal tumor samples, but is useful for a broader set of tumor types. Results The smMIP-based NGS panel was successfully validated and cut-off values were established for reliable gene amplification analysis (i.e. relative coverage ≥3) and MSI detection (≥30% unstable loci). After validation, 728 routine diagnostic tumor samples including a broad range of tumor types were sequenced with sufficient sensitivity (2.4% drop-out), including samples with low DNA input (EGFR, BRAF, MET, ERBB2, KIT, PDGFRA). Amplifications were observed in 5.5% of the samples, comprising clinically relevant amplifications (e.g. MET, ERBB2, FGFR1). 1.5% of the tumor samples were classified as MSI-high, including both MSI-prone and non-MSI-prone tumors. Conclusions We developed a comprehensive workflow for predictive analysis of diagnostic tumor samples. The smMIP-based NGS analysis was shown suitable for limited amounts of histological and cytological material. As smMIP technology allows easy adaptation of panels, this approach can comply with the rapidly expanding molecular markers.

In this study, a simple fluorescent detection of survival motor neuron gene (SMN) in diagnosis of spinal muscular atrophy (SMA) based on nucleic acid amplification test and the poly-T luminescent copper nanoclusters (CuNCs) was established. SMA is a severely genetic diseases to cause infant death in clinical, and detection of SMN gene is a powerful tool for pre- and postnatal diagnosis of this disease. This study utilized the molecular inversion probe for recognition of nucleotide variant between SMN1 (c.840 C) and SMN2 (c.840 C > T) genes, and rolling circle amplification with a universal primer for production of poly-T single-strand DNA. Finally, the fluorescent CuNCs were formed on the poly-T single-strand DNA template with addition of CuSO4 and sodium ascorbate. The fluorescence of CuNCs was only detected in the samples with the presence of SMN1 gene controlling the disease of SMA. After optimization of experimental conditions, this highly efficient method was performed under 50 °C for DNA ligation temperature by using 2U Ampligase, 3 h for rolling circle amplification, and the formation of the CuNCs by mixing 500 μM Cu2+ and 4 mM sodium ascorbate. Additionally, this highly efficient method was successfully applied for 65 clinical DNA samples, including 4 SMA patients, 4 carriers and 57 wild individuals. This label-free detection strategy has the own potential to not only be a general method for detection of SMN1 gene in diagnosis of SMA disease, but also served as a tool for detection of other single nucleotide polymorphisms or nucleotide variants in genetic analysis through designing the different sensing probes.

Introduction Young age is an adverse prognostic factor in children with ependymomas. Treatment of these infants is challenging since beneficial therapeutic options are limited. As ependymomas are considered a biologically heterogeneous group, we aimed to characterize infant ependymomas with regard to their histological and genetic features. Materials and methods We analyzed 28 ependymomas occurring in children younger than 18 months at diagnosis enrolled into the HIT2000-E protocols with the aim to postpone irradiation until the age of 18 months if possible. All cases underwent neuropathological review, including immunohistochemical characterization. Genome-wide copy number alterations (CNA) were assessed by molecular inversion probe assays, and RELA and YAP1 fusions were detected by RT-PCR and sequencing. Results All infant ependymomas were anaplastic (WHO grade III). Twenty-one (75%) cases were located in the posterior fossa. Gross total resection was accomplished in 12 (57%) of these cases. All posterior fossa tumors showed loss of H3-K27me3 characteristic of PFA ependymomas. CNA analysis showed a stable genome in all cases with lack of chromosome 1q gain, an adverse prognostic marker in PFA ependymomas of older children. However, after a median follow-up of 5.4 years, 15 (71%) relapsed, and 9 (43%) died. Seven ependymomas (25%) occurred in the supratentorial region. Gross total resection could be achieved in only two of these cases. Four tumors carried C11orf95-RELA fusions, and two cases had typical YAP1-MAMLD1 fusions (one case was not analyzable). The RELA-fused cases did not display CDKN2A loss as an adverse indicator of prognosis in this disease entity. Although three infants (43%) with supratentorial ependymomas relapsed, all patients survived (median follow-up, 8.0 years). Conclusion Infant ependymomas seem to fall into three biological entities, with supratentorial tumors carrying RELA or YAP fusions and PFA posterior fossa ependymomas. The latter showed a poor outcome even though chromosome 1q gain was absent.

Ependymoma with YAP1-MAMLD1 fusion is a rare, recently described supratentorial neoplasm of childhood, with few cases published so far. We report on 15 pediatric patients with ependymomas carrying YAP1-MAMLD1 fusions, with their characteristic histopathology, immunophenotype and molecular/cytogenetic, radiological and clinical features. The YAP1-MAMLD1 fusion was documented by RT-PCR/Sanger sequencing, and tumor genomes were studied by molecular inversion probe (MIP) analysis. Significant copy number alterations were identified by GISTIC (Genomic Identification of Significant Targets in Cancer) analysis. All cases showed similar histopathological features including areas of high cellularity, presence of perivascular pseudo-rosettes, small to medium-sized nuclei with characteristic granular chromatin and strikingly abundant cells with dot-like cytoplasmic expression of epithelial membrane antigen. Eleven cases presented features of anaplasia, corresponding to WHO grade III. MRI showed large supratentorial multinodular tumors with cystic components, heterogeneous contrast enhancement, located in the ventricular or periventricular region. One of two variants of YAP1-MAMLD1 fusions was detected in all cases. The MIP genome profiles showed balanced profiles, with focal alterations of the YAP1 locus at 11q22.1-11q21.2 (7/14), MAMLD1 locus (Xp28) (10/14) and losses of chromosome arm 22q (5/14). Most patients were female (13/15) and younger than 3 years at diagnosis (12/15; median age, 8.2 months). Apart from one patient who died during surgery, all patients are alive without evidence of disease progression after receiving different treatment protocols, three without postoperative further treatment (median follow-up, 4.84 years). In this to date, largest series of ependymomas with YAP1-MAMLD1 fusions we show that they harbor characteristic histopathological, cytogenetic and imaging features, occur mostly in young girls under 3 years and are associated with good outcome. Therefore, this genetically defined neoplasm should be considered a distinct disease entity. The diagnosis should be confirmed by demonstration of the specific fusion. Further studies on large collaborative series are warranted to confirm our findings.

BACKGROUND Microsatellite instability (MSI) is an emerging actionable phenotype in oncology that informs tumor response to immune checkpoint pathway immunotherapy. However, there remains a need for MSI diagnostics that are low cost, highly accurate, and generalizable across cancer types. We developed a method for targeted high-throughput sequencing of numerous microsatellite loci with pan-cancer informativity for MSI using single-molecule molecular inversion probes (smMIPs). METHODS We designed a smMIP panel targeting 111 loci highly informative for MSI across cancers. We developed an analytical framework taking advantage of smMIP-mediated error correction to specifically and sensitively detect instability events without the need for typing matched normal material. RESULTS Using synthetic DNA mixtures, smMIPs were sensitive to at least 1% MSI-positive cells and were highly consistent across replicates. The fraction of identified unstable microsatellites discriminated tumors exhibiting MSI from those lacking MSI with high accuracy across colorectal (100% diagnostic sensitivity and specificity), prostate (100% diagnostic sensitivity and specificity), and endometrial cancers (95.8% diagnostic sensitivity and 100% specificity). MSI-PCR, the current standard-of-care molecular diagnostic for MSI, proved equally robust for colorectal tumors but evidenced multiple false-negative results in prostate (81.8% diagnostic sensitivity and 100% specificity) and endometrial (75.0% diagnostic sensitivity and 100% specificity) tumors. CONCLUSIONS smMIP capture provides an accurate, diagnostically sensitive, and economical means to diagnose MSI across cancer types without reliance on patient-matched normal material. The assay is readily scalable to large numbers of clinical samples, enables automated and quantitative analysis of microsatellite instability, and is readily standardized across clinical laboratories.

Background Adjuvant chemotherapy (ACT) provides modest benefit in resected non-small cell lung cancer (NSCLC) patients. Genome-wide studies have identified gene copy number aberrations (CNA), but their prognostic implication is unknown. Methods DNA from 1,013 FFPE tumor samples from three pivotal multicenter randomized trials (ACT vs. control) in the LACE-Bio consortium (median follow-up: 5.2 years) was successfully extracted, profiled using a molecular inversion probe SNP assay, normalized relative to a pool of normal tissues and segmented. Minimally recurrent regions were identified. P values were adjusted to control the false discovery rate (Q values). Results A total of 976 samples successfully profiled, 414 (42%) adenocarcinoma (ADC), 430 (44%) squamous cell carcinoma (SCC) and 132 (14%) other NSCLC; 710 (73%) males. We identified 431 recurrent regions, with on average 51 gains and 43 losses; 253 regions (59%) were ≤3 Mb. Most frequent gains (up to 48%) were on chr1, 3q, 5p, 6p, 8q, 22q; most frequent losses (up to 40%) on chr3p, 8p, 9p. CNA frequency of 195 regions was significantly different (Q≤0.05) between ADC and SCC. Fourteen regions (7p11-12, 9p21, 18q12, and 19p11-13) were associated with disease-free survival (DFS) (univariate P≤0.005, Q

Background: Atypical ductal hyperplasia (ADH) is common finding in the mammographic era, but has wide variation in diagnosis and treatment. ADH is a high risk factor for invasive breast ductal carcinoma (IDC), and is also considered to be a non-obligate precursor to IDC. Although a few studies have revealed some of the common genomic characteristics of ADH, a clear understanding of the molecular changes associated with breast cancer progression has been limited by inadequately powered studies and low resolution methodology (Lopez-Garcia et al., 2010, Histopathology 57: 171-192). Copy number alterations (CNAs), one of the major genetic alterations in cancer, are present in IDC and ductal carcinoma in situ (DCIS) and given a suitable detection method could be used in research and potentially in the clinical setting to predict patient prognosis. Method: We optimised a robust, cost effective low-coverage whole genome sequencing (LCWGS) method for CNA detection, using as little as 5 ng of formalin-fixed paraffin-embedded tissue derived DNA. We applied this novel method to 21 cases of pure ADH (not associated with cancer) as well as 20 ADH with synchronous DCIS and/or IDC. Cases were reviewed independently by two pathologists, followed by micro-dissection and DNA extraction. CNA were analysed using the LCWGS method, using 5-10 ng total DNA input per sample. Results: Highly accurate copy number profiles produced by low input LCWGS are comparable to those obtained from an alternative method, Molecular Inversion Probe arrays, while requiring 10 fold less input DNA (Kader et al., 2016, Genome Medicine 8: 121). Genetic analysis of pure ADH found that 95% had at least one copy number event and the median fraction of the genome altered was 8% (0-19%). Surprisingly, while 29% of pure ADH showed 16q loss and 1q gain (CNAs common in low-grade (LG) breast cancer), 29% of pure ADH showed 8q gain, an event more frequently observed in high grade (HG) cancer. Analysis of ADH with synchronous DCIS and/or IDC showed that 67% of ADH was clonally related with both LG (6/8) and HG carcinoma (4/7). The final analysis of 20 synchronous cases (9 LG, 11 HG) will be presented. Conclusion: Here we report the most detailed molecular taxonomy of this high risk pre-malignant breast lesion with the largest cohort undertaken worldwide. Our observation that 67% of ADH is clonal to breast carcinoma suggests that both LG and HG carcinoma can evolve from a similar ancestor lesion. As ADH has the potential to develop both LG and HG carcinoma, a biomarker signifying breast cancer progression from pre-malignant lesions is highly desirable for the more precise treatment of individual patients diagnosed with ADH. Citation Format: Kader T, Hill P, Opeskin K, Goode DL, Elder K, Pang J-M, Fox SB, Mann GB, Campbell IG, Gorringe KL. Identification of copy number alterations associated with the progression of high risk premalignant breast lesions to breast carcinoma [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD1-12.

Objective To establish a high throughput, low cost, and simple nanotechnology-based method for the detection of single nucleotide polymorphism (SNP) loci in type 2 diabetes mellitus (T2DM). Methods Multiplex ligase detection reaction (LDR) amplification was performed using fluorescently labeled magnetic nanosphere-bound upstream LDR probes and downstream probes labeled with a unique fluorescent group for each SNP locus. The amplified LDR products were separated by magnetic nanospheres and then scanned by fluorescence spectroscopy. Four SNP loci associated with T2DM were detected, including the rs13866634 locus in SLC30A8, rs10811661in CDKN2A/2B, rs1111875 in the HHEX gene, and rs7903146 in the TCF7L2 gene. The SNP genotype was also determined by DNA sequencing as a control. Results The SNP genotypes of the four gene loci determined by the nanosphere-based multiplex LDR method were consistent with the DNA sequencing results. The accuracy rate was 100%. Conclusion A method based on multiplex PCR and LDR was established for simultaneous detection of four SNP loci of T2DM susceptibility genes.

Uterine carcinosarcoma, also known as Malignant Mixed Mullerian Tumour, is a high-grade biphasic neoplasm composed of sarcomatous elements thought to originate via transdifferentiation from high-grade endometrial carcinoma. To identify molecular factors contributing to the histogenesis of this tumour, we analyzed DNA extracted from matched carcinoma and sarcoma components from 12 cases of carcinosarcoma by a molecular inversion probe microarray to assess genomic copy number alterations (CNAs) and allelic imbalances. Widespread CNAs were identified in tumours with serous histology in the carcinoma component (9/12), while the remaining three cases with endometrioid carcinoma were near-diploid. Quantification of the extent of genomic aberrations revealed a significant increase in sarcoma relative to carcinoma in tumours with well-delineated histologic components. Focal amplification of 13q31.3 was identified in 6/12 profiled tumours, of which four harboured the aberration exclusively in the sarcoma component. This result was verified by fluorescence in situ hybridization against GPC5, the only gene situated within the minimal region of amplification. In a validation cohort composed of 97 carcinosarcomas and other uterine sarcomas, amplification of GPC5 (GPC5/CEP13 ratio ≥ 2.2) was identified in 11/97 (11.3%) cases (9/64 carcinosarcoma, 1/3 rhabdomyosarcoma, 1/21 leiomyosarcoma, 0/8 adenosarcoma, 0/1 undifferentiated endometrial sarcoma) and an additional 4 (2.8%) cases had low level gains (GPC5/CEP13 ratio ≥1.5 but

Seventeen clusters of Shiga toxin-producing Escherichia coli O157:H7/- (O157) strains, determined by cluster analysis of pulsed-field gel electrophoresis patterns, were analyzed using whole genome sequence (WGS) data to investigate this pathogen's molecular epidemiology. The 17 clusters included 136 strains containing strains from nine outbreaks, with each outbreak caused by a single source contaminated with the organism, as shown by epidemiological contact surveys. WGS data of these strains were used to identify single nucleotide polymorphisms (SNPs) by two methods: short read data were directly mapped to a reference genome (mapping derived SNPs) and common SNPs between the mapping derived SNPs and SNPs in assembled data of short read data (common SNPs). Among both SNPs, those that were detected in genes with a gap were excluded to remove ambiguous SNPs from further analysis. The effectiveness of both SNPs was investigated among all the concatenated SNPs that were detected (whole SNP set); SNPs were divided into three categories based on the genes in which they were located (i.e., backbone SNP set, O-island SNP set, and mobile element SNP set); and SNPs in non-coding regions (intergenic region SNP set). When SNPs from strains isolated from the nine single source derived outbreaks were analyzed using an unweighted pair group method with arithmetic mean tree (UPGMA) and a minimum spanning tree (MST), the maximum pair-wise distances of the backbone SNP set of the mapping derived SNPs were significantly smaller than those of the whole and intergenic region SNP set on both UPGMAs and MSTs. This significant difference was also observed when the backbone SNP set of the common SNPs were examined (Steel-Dwass test, P≤0.01). When the maximum pair-wise distances were compared between the mapping derived and common SNPs, significant differences were observed in those of the whole, mobile element, and intergenic region SNP set (Wilcoxon signed rank test, P≤0.01). When all the strains included in one complex on an MST or one cluster on a UPGMA were designated as the same genotype, the values of the Hunter-Gaston Discriminatory Power Index for the backbone SNP set of the mapping derived and common SNPs were higher than those of other SNP sets. In contrast, the mobile element SNP set could not robustly subdivide lineage I strains of tested O157 strains using both the mapping derived and common SNPs. These results suggested that the backbone SNP set were the most effective for analysis of WGS data for O157 in enabling an appropriation of its molecular epidemiology.

Summary Wheat breeders and academics alike use single nucleotide polymorphisms (SNPs) as molecular markers to characterize regions of interest within the hexaploid wheat genome. A number of SNP‐based genotyping platforms are available, and their utility depends upon factors such as the available technologies, number of data points required, budgets and the technical expertise required. Unfortunately, markers can rarely be exchanged between existing and newly developed platforms, meaning that previously generated data cannot be compared, or combined, with more recently generated data sets. We predict that genotyping by sequencing will become the predominant genotyping technology within the next 5–10 years. With this in mind, to ensure that data generated from current genotyping platforms continues to be of use, we have designed and utilized SNP‐based capture probes from several thousand existing and publicly available probes from Axiom® and KASP™ genotyping platforms. We have validated our capture probes in a targeted genotyping by sequencing protocol using 31 previously genotyped UK elite hexaploid wheat accessions. Data comparisons between targeted genotyping by sequencing, Axiom® array genotyping and KASP™ genotyping assays, identified a set of 3256 probes which reliably bring together targeted genotyping by sequencing data with the previously available marker data set. As such, these probes are likely to be of considerable value to the wheat community. The probe details, full probe sequences and a custom built analysis pipeline may be freely downloaded from the CerealsDB website (http://www.cerealsdb.uk.net/cerealgenomics/CerealsDB/sequence_capture.php).

Biodiversity has suffered a dramatic global decline during the past decades, and monitoring tools are urgently needed providing data for the development and evaluation of conservation efforts both on a species and on a genetic level. However, in wild species, the assessment of genetic diversity is often hampered by the lack of suitable genetic markers. In this article, we present Random Amplicon Sequencing (RAMseq), a novel approach for fast and cost-effective detection of single nucleotide polymorphisms (SNPs) in nonmodel species by semideep sequencing of random amplicons. By applying RAMseq to the Eurasian otter (Lutra lutra), we identified 238 putative SNPs after quality filtering of all candidate loci and were able to validate 32 of 77 loci tested. In a second step, we evaluated the genotyping performance of these SNP loci in noninvasive samples, one of the most challenging genotyping applications, by comparing it with genotyping results of the same faecal samples at microsatellite markers. We compared (i) polymerase chain reaction (PCR) success rate, (ii) genotyping errors and (iii) Mendelian inheritance (population parameters). SNPs produced a significantly higher PCR success rate (75.5% vs. 65.1%) and lower mean allelic error rate (8.8% vs. 13.3%) than microsatellites, but showed a higher allelic dropout rate (29.7% vs. 19.8%). Genotyping results showed no deviations from Mendelian inheritance in any of the SNP loci. Hence, RAMseq appears to be a valuable tool for the detection of genetic markers in nonmodel species, which is a common challenge in conservation genetic studies.

Follicular dendritic cell sarcoma is a rare malignant neoplasm of dendritic cell origin that is currently poorly characterized by genetic studies. To investigate whether recurrent genomic alterations may underlie the biology of follicular dendritic cell sarcoma and to identify potential contributory regions and genes, molecular inversion probe array analysis was performed on 14 independent formalin-fixed, paraffin-embedded samples. Abnormal genomic profiles were observed in 11 out of 14 (79%) cases. The majority showed extensive genomic complexity that was predominantly represented by hemizygous losses affecting multiple chromosomes. Alterations of chromosomal regions 1p (55%), 2p (55%), 3p (82%), 3q (45%), 6q (55%), 7q (73%), 8p (45%), 9p (64%), 11q (64%), 13q (91%), 14q (82%), 15q (64%), 17p (55%), 18q (64%), and 22q (55%) were recurrent across the 11 samples showing abnormal genomic profiles. Many recurrent genomic alterations in follicular dendritic cell sarcoma overlap deletions that are frequently observed across human cancers, suggesting selection, or an active role for these alterations in follicular dendritic cell sarcoma pathogenesis. In support of a tumor suppressor-driven biology, homozygous deletions involving tumor suppressor genes CDKN2A, RB1, BIRC3, and CYLD were also observed. Neither recurrent gains nor amplifications were observed. This genomic characterization provides new information regarding follicular dendritic cell sarcoma biology that may improve understanding about the underlying pathophysiology, provide better prognostication, and identify potential therapeutic markers for this rare disease.

High-throughput sequencing methods for genotyping genome-wide markers are being rapidly adopted for phylogenetics of non-model organisms in conservation and biodiversity studies. However, the reproducibility of SNP genotyping and degree of marker overlap or compatibility between datasets from different methodologies have not been tested in non-model systems. Using double-digest restriction site associated DNA sequencing, we sequenced a common set of 22 specimens from the butterfly genus Speyeria on two different Illumina platforms, using two variations of library preparation. We then used a de novo approach to bioinformatic locus assembly and SNP discovery for subsequent phylogenetic analyses. We found a high rate of locus recovery despite differences in library preparation and sequencing platforms, as well as overall high levels of data compatibility after data processing and filtering. These results provide the first application of NGS methods for phylogenetic reconstruction in Speyeria, and support the use and long-term viability of SNP genotyping applications in non-model systems. This article is protected by copyright. All rights reserved.

We presented here the application of two in-plate SNP (single nucleotide polymorphism) genotyping platforms for soybean plants [Glycine max (L.) Merr.], KASP® (Kompetitive Allele Specific PCR genotyping, LGC Genomics) and TaqMan® (Life Technologies) respectively. These two systems offer us an ability to determine the genotypes of 384 individual samples accurately and efficiently by allele specific PCR in a single plate using typical PCR conditions. Both of the systems require small quantity of genomic DNA obtained from a simple DNA extraction. The genomic sequences containing target SNPs can easily be used as a basic blueprint to design the probes and primers of KASP® and TaqMan® assays whether the sequences are obtained from the genome sequence of soybean William 82 (Wm82.a2.v1), Illumina Soy50k SNPs, or parallel resequencing. Moreover, we listed the pros and cons of the two systems and explained the principles behind the platforms. The high call rate and clear clustering separation of the SNPs can be readily obtained from these platforms without conducting any assay optimization processes. These platforms can routinely be performed on 96/384-well plate format with or without an automation procedure. Therefore, these platforms are especially suitable for the SNP genotyping on a particular trait with a large sample size, gene fine mapping, and marker assisted selection. Further, they require little hands-on experience and achieve per-site and per-individual costs below that of current SSR, AFLP, RFLP, and SNP chip technologies. The platforms can be used for genotyping on a wide range of organisms due to their simplicity and flexibility of handling. Meanwhile, we also especially presented some of the advantages using KASP® SNP genotyping pipeline, which was cost effective in the selection of allele specific assay and therefore, efficiently facilitated the soybean genotyping across large numbers (thousands or more) of individual lines for a great range of markers (hundreds to thousands) in our laboratory.

The identification of minimal residual disease is the primary diagnostic finding which predicts relapse in patients treated for acute myeloid leukemia. Ultrasensitive detection of minimal residual disease would enable better patient risk stratification and could open opportunities for early therapeutic intervention. Herein we apply single molecule molecular inversion probe capture, a technology combining multiplexed targeted sequencing with error correction schemes based on molecular barcoding, in order to detect mutations identifying minimal residual disease with ultrasensitive and quantitative precision. We designed a single molecule molecular inversion probe capture panel spanning >50 kb and targeting 32 factors relevant to acute myeloid leukemia pathogenesis. We demonstrate linearity and quantitative precision over 100-fold relative abundance of mutant cells (1 in 100 to 1 in 1,500), with estimated error rates approaching 1 in 1,200 base pairs sequenced and maximum theoretical limits of detection exceeding 1 in 60,000 mutant alleles. In 3 of 4 longitudinally collected specimens from patients with acute myeloid leukemia, we find that single molecule molecular inversion probe capture detects somatic mutations identifying minimal residual disease at substantially earlier time points and with greater sensitivity than clinical diagnostic approaches used as current standard of care (flow cytometry and conventional molecular diagnosis), and identifies persisting neoplastic cells during clinical remission. In 2 patients, single molecule molecular inversion probe capture detected heterogeneous, subclonal acute myeloid leukemia populations carrying distinct mutational signatures. Single molecule molecular inversion probe technology uniquely couples scalable target enrichment with sequence read error correction, providing an integrated, ultrasensitive approach for detecting minimal residual disease identifying mutations.

Due to the recent growth of the alien population in Korea, tracking the ethnic origin of human specimens has gained importance in genetic forensics. To address this issue, we developed a method to analyze single nucleotide polymorphisms (SNPs) based on next-generation sequencing (NGS) technology, which is now being used for personal identification in forensics. We designed a panel of 153 Korean-specific high-performance multiplexed SNPs and performed NGS using 233 DNA samples collected from eight different ethnic groups around the world. Eight Korean-specific genetic markers (rs28777, rs1010872, rs6043841, rs6034433, rs885479, rs2503107, rs4530059, and rs214955) that were screened showed significant variability among the ethnic groups. Three markers were novel SNPs that were absent from our multiplexed SNP panel, and two markers were associated with specific phenotypes. Our high-performance multiplexed SNP panel allows efficient screening of Korean-specific SNP alleles in populations that are genetically similar to the Korean population (e.g., Japanese and northeast Asians including Chinese and Eurasians), which will be useful for personal identification, paternity testing, and forensic investigations.

// 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.