Ligation-dependent Probe Amplification Assay Research Articles

Contribution of Influenza Viruses, Other Respiratory Viruses and Viral Co-Infections to Influenza-like Illness in Older Adults

Influenza-like illness (ILI) can be caused by a range of respiratory viruses. The present study investigates the contribution of influenza and other respiratory viruses, the occurrence of viral co-infections, and the persistence of the viruses after ILI onset in older adults. During the influenza season 2014–2015, 2366 generally healthy community-dwelling older adults (≥60 years) were enrolled in the study. Viruses were identified by multiplex ligation–dependent probe-amplification assay in naso- and oropharyngeal swabs taken during acute ILI phase, and 2 and 8 weeks later. The ILI incidence was 10.7%, which did not differ between vaccinated and unvaccinated older adults; influenza virus was the most frequently detected virus (39.4%). Other viruses with significant contribution were: rhinovirus (17.3%), seasonal coronavirus (9.8%), respiratory syncytial virus (6.7%), and human metapneumovirus (6.3%). Co-infections of influenza virus with other viruses were rare. The frequency of ILI cases in older adults in this 2014–2015 season with low vaccine effectiveness was comparable to that of the 2012–2013 season with moderate vaccine efficacy. The low rate of viral co-infections observed, especially for influenza virus, suggests that influenza virus infection reduces the risk of simultaneous infection with other viruses. Viral persistence or viral co-infections did not affect the clinical outcome of ILI.

Gene copy number alterations in Indian children with B-acute Lymphoblastic Leukemia: Correlation with survival outcome

BackgroundAcute Lymphoblastic Leukemia (ALL) is the most common lymphoid malignancy occurring in children. Copy number alterations like CDKN2A/B, IKZF1, ETV6, RB1 and BTG1 gene deletions have been shown to be associated with differential survival outcome. The literature on survival outcome of children with each gene deletion is very limited. The targeted therapy for some of these gene deletions are under trial in adult ALL. Our study will provide insight in the role of such genes in disease biology. MethodsA total of 91 children with newly diagnosed ALL were enrolled and monitored for response to treatment and were followed up for 4.0 years and analyzed for copy number alterations by Multiplex ligation dependent probe amplification assay (MLPA) using SALSA MLPA probemix P335–B2 ALL-IKZF1 kit. Survival analysis was performed using event free survival (EFS) as the end point.Survival curves between two groups were calculated using Kaplan-Meier analysis and compared using log-rank tests. Other comparisons were performed using the Chi-squared or Fisher's exact test. All analyses were performed using Stata 14.0. ResultsWe observed gene copy number alterations in 51.6% of children with B-ALL subgroup. CDKN2A/B was the most commonly observed gene deletion followed by PAX5, ETV6, IKZF1, RB1, BTG1 and EBF1. Children with CDKN2A/B, IKZF1, RB1 and BTG1 gene deletion had significantly lesser event free survival as compared to those without gene deletions in short term follow up while ETV6 and EBF1 gene deletions and PAR1 gene alterations did not have any impact on survival outcome. DiscussionEvent free survival in children with CDKN2A/B, IKZF1, RB1 and BTG1 gene deletions was significantly lower in short term possibly due to aggressive disease phenotype with faster progression. We found PAR1 gene alterations to be associated with better event free survival in long term follow up though it did not show significant association. ConclusionsCDKN2A/B, IKZF1 and RB1 gene deletions were independently associated with poor event free survival. We opine that individual gene deletions, and not integrated copy number alterations profile, may be used for risk stratification and the CDKN2A/B, IKZF1 and RB1 may serve as poor prognostic markers.

Gene expression pattern analysis using dual-color RT-MLPA and integrative genome-wide association studies of eQTL for tuberculosis suscepitibility

BackgroundWhen infected with Mycobacterium tuberculosis, only a small proportion of the population will develop active TB, and the role of host genetic factors in different TB infection status was not fully understood.MethodsForty-three patients with active tuberculosis and 49 with latent tuberculosis were enrolled in the prospective cohort. Expressing levels of 27 candidate mRNAs, which were previously demonstrated to differentially expressed in latent and active TB, were measured by dual color reverse transcription multiplex ligation dependent probe amplification assay (dcRT-MLPA). Using expression levels of these mRNAs as quantitative traits, associations between expression abundance and genome-wild single nucleotide polymorphisms (SNPs) were calculated. Finally, identified candidate SNPs were further assessed for their associations with TB infection status in a validation cohort with 313 Chinese Han cases.ResultsWe identified 9 differentially expressed mRNAs including il7r, il4, il8, tnfrsf1b, pgm5, ccl19, il2ra, marco and fpr1 in the prospective cohort. Through expression quantitative trait loci mapping, we screened out 8 SNPs associated with these mRNAs. Then, CG genotype of the SNP rs62292160 was finally verified to be significantly associated with higher transcription levels of IL4 in LTBI than in TB patients.ConclusionWe reported that the SNP rs62292160 in Chinese Han population may link to higher expression of il4 in latent tuberculosis. Our findings provided a new genetic variation locus for further exploration of the mechanisms of TB and a possible target for TB genetic susceptibility studies, which might aid the clinical decision to precision treatment of TB.

BRCAness digitalMLPA profiling predicts benefit of intensified platinum-based chemotherapy in triple-negative and luminal-type breast cancer

BackgroundWe previously showed that BRCA-like profiles can be used to preselect individuals with the highest risk of carrying BRCA mutations but could also indicate which patients would benefit from double-strand break inducing chemotherapy. A simple, robust, and reliable assay for clinical use that utilizes limited amounts of formalin-fixed, paraffin-embedded tumor tissue to assess BRCAness status in both ER-positive and ER-negative breast cancer (BC) is currently lacking.MethodsA digital multiplex ligation-dependent probe amplification (digitalMLPA) assay was designed to detect copy number alterations required for the classification of BRCA1-like and BRCA2-like BC. The BRCA1-like classifier was trained on 71 tumors, enriched for triple-negative BC; the BRCA2-like classifier was trained on 55 tumors, enriched for luminal-type BC. A shrunken centroid-based classifier was developed and applied on an independent validation cohort. A total of 114 cases of a randomized controlled trial were analyzed, and the association of the classifier result with intensified platinum-based chemotherapy response was assessed.ResultsThe digitalMLPA BRCA1-like classifier correctly classified 91% of the BRCA1-like samples and 82% of the BRCA2-like samples. Patients with a BRCA-like tumor derived significant benefit of high-dose chemotherapy (adjusted hazard ratio (HR) 0.12, 95% CI 0.04–0.44) which was not observed in non-BRCA-like patients (HR 0.9, 95% CI 0.37–2.18) (p = 0.01). Analysis stratified for ER status showed borderline significance.ConclusionsThe digitalMLPA is a reliable method to detect a BRCA1- and BRCA2-like pattern on clinical samples and predicts platinum-based chemotherapy benefit in both triple-negative and luminal-type BC.

Hypomagnesemia with Hypercalciuria Leading to Nephrocalcinosis, Amelogenesis Imperfecta, and Short Stature in a Child Carrying a Homozygous Deletion in the CLDN16 Gene

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare autosomal recessive disease caused by mutations in the CLDN16 or CLDN19 gene; however, few cases develop classical amelogenesis imperfecta. Herein, we report the case of a boy with early clinical renal manifestations that started at 1year of age and presenting with dental hypoplasia and growth delay. The patient presented with vomiting, polyuria, and polydipsia. Apart from recurrent sterile leukocyturia, erroneously treated as infectious, he was normal, except for short stature and amelogenesis imperfecta with gradually discolored teeth. Laboratory tests revealed hyperparathyroidism, hypomagnesemia, severe hypercalciuria, and hypermagnesuria on 24-h urine testing. Helical computed tomography confirmed nephrocalcinosis. We performed whole-exome sequencing (WES) to test the hypothesis of FHHNC and oligogenic inheritance of amelogenesis. Analysis of the WES binary sequence alignment/map file revealed the presence of exon 1 of the CLDN16 and absence of the other exons [c.325_c918*? (E2_E5del)]. We confirmed a CLDN16 E2_E5 homozygous deletion by multiplex ligation-dependent probe amplification and polymerase chain reaction assays. Although most mutations causing FHHNC are missense and nonsense mutations in the CLDN16 or CLDN19 gene, large deletions occur and may be misled by WES, which is generally used for genetic screening of oligogenic disorders. The patient received cholecalciferol, magnesium oxide and potassium citrate. Later, the combination with hydrochlorothiazide plus amiloride was prescribed, with a good response during follow-up. Our report broadens the phenotype of FHHNC, including severe early-onset amelogenesis and short stature, and reinforces the phenotype-genotype correlation of the large deletion found in CLDN16.

Haploinsufficiency of A20 with a novel mutation of deletion of exons 2–3 of TNFAIP3

Objectives Haploinsufficiency of A20 (HA20) due to loss-of-function mutations of TNFAIP3 leads to an autoinflammatory disease. These mutations produce a premature termination codon in most cases of HA20. However, exon deletion has not been reported. Methods Genomic DNA was extracted from the peripheral blood of the patient clinically suspected of HA20. We examined autoinflammatory disease-causing genes and performed a multiplex ligation-dependent probe amplification (MLPA) assay for copy number analysis. Next, to determine the disconnection point, genomic DNA was amplified with long-range PCR and sequenced. Finally, western blotting was carried out to measure A20 protein expression in mitogen phytohaemagglutinin (PHA)-induced T-cell blasts from the patient and a healthy volunteer. Results Targeted next-generation sequencing found no pathogenic mutation, but copy number variation (CNV) analysis suggested a heterozygous deletion of exons 2–3. The MLPA assay and long-range PCR confirmed the mutation. Western blotting analysis indicated a marked decrease in expression of A20 protein from the patient compared to a normal control. The results showed that this deletion was a pathogenic mutation. Conclusion This study demonstrates a novel mutation resulting in a deletion of exons 2–3 of TNFAIP3. MLPA analysis is a useful initial screening method for HA20 patients.

Phenotype and genotype of FXIII deficiency in two unrelated probands: identification of a novel F13A1 large deletion mediated by complex rearrangement

BackgroundInherited Factor XIII deficiency (FXIIID) is one of the most severe and under-diagnosed rare bleeding disorders. Only 5 large deletions involving one or more exons in F13A1 have been reported, and lacking of multiplex ligation-dependent probe amplification (MLPA) assay might underestimate the copy number variations (CNVs) in F13A1 and F13B. We had characterized the clinical presentation of two unrelated severe FXIIID probands and explored the pathogenic mechanisms.ResultsBoth probands experienced several episodes of fatal bleeding and delayed wound healings prior to diagnosis. FXIII activity was measured by the ammonia release assay, and FXIII-A and FXIII-B antigens were determined by ELISA. All the exons including exon-intron boundaries and promoter regions of F13A1 and F13B were amplified and directly sequenced. Copy number variations (CNVs) of F13A1 and F13B were detected by the CNVplex® method. Breakpoints of the F13A1 large deletion were identified by quantitative primer walking combined long-range PCR (LR-PCR) strategies. Proband 1 was found to have compound heterozygous mutations of a novel small deletion (c.1147del) and a missense mutation p.Arg383Ser. Proband 2 was compound heterozygous for a novel large deletion (g.[77815_112815del;112837_116628del]) and a missense mutation p.Arg716Gly in F13A1. Bioinformatics analysis of the large deletion breakpoints predicted that two fork stalling and template switching and/or microhomology-mediated break-induced replication (FoSTeS/MMBIR) events with two homologies of TCT and C might be responsible for the complex rearrangement. Prophylactic replacement therapy was immediately administered for the two probands upon establishment of the diagnosis.ConclusionsWe detected two type I FXIIID pedigrees and adopted CNVplex® method to detect CNVs of F13A1 and F13B for the first time. A large heterozygous deletion of g.[77815_112815del;112837_116628del] in F13A1, mediated by two FoSTeS/MMBIR events, was identified.

Abstract B35: Australian Ovarian Cancer Assortment Trial–Allocating ovarian cancer patients into clinical trials based on molecular profiling

Abstract Background: The Australian Ovarian Cancer Assortment Trial (ALLOCATE) was designed as a pilot study to demonstrate feasibility of molecularly profiling patients with recurrent ovarian cancer with the aim of allocating patients to targeted therapies based on the genomic profile of their tumors. Materials and Methods: Two next-generation sequencing (NGS) panels, as well as a BRCA1 methylation assay, were used for molecular profiling of most common subtypes of ovarian cancer. A custom Illumina TruSeq Amplicon Low Input (v2) panel with dual-strand coverage was designed to target 38 genes commonly mutated and clinically important in ovarian cancer. The second assay was a NGS modification of the Multiplex Ligation-dependent Probe Amplification (MLPA) assay that was designed to target 11 genes with common copy number alterations (CNA) in ovarian cancer, including extensive BRCA1/2 coverage for large exonic deletions (Kondrashova et al., 2015). A thorough analytic validation was performed to ensure that both tests were fit for diagnostic use. Patients with recurrent epithelial ovarian cancer were eligible for the study. Where feasible, patients underwent biopsies of recurrent tumor that were snap frozen. Otherwise, archival FFPE tumor blocks were retrieved. Sequencing was performed using Illumina Miseq and HiSeq 2500 with target median coverage of 2000x (amplicon panel) and 800x (MLPA-Seq). Data were analyzed using an internally built pipeline, an upgraded version of AmpliVar (Hsu et al 2015), with Variant Effect Predictor (Mclaren et al., 2016) used for variant annotation. Results: Between December 2013 and October 2016, 113 patients with recurrent ovarian cancer were recruited from two tertiary hospitals, with 15 cases (13%) excluded due to insufficient tumor material or poor-quality DNA. Ninety-eight cases (87%) were analyzed and reports issued back to the referring clinician. Fifty-six patients (61%) in the study had recurrent high-grade serous ovarian cancer (HGSC). Of these, TP53 mutations were identified in 91%. Events in genes other than TP53 were detected in 44 cases, most commonly MYC and CCNE1 amplifications and BRCA1/2 mutations. BRCA1/2 reversions were identified in two cases, explaining their lack of response to platinum/PARPi. Fifteen patients (16%) had recurrent low-grade serous ovarian cancer (LGSC), with KRAS or BRAF mutations identified in four cases. Two HGSC tumors were reclassified as LGSC on the basis of a lack of TP53 mutation, presence of KRAS mutation, and subsequent pathology review. Other cases in the study included mucinous, clear cell, and mixed-histology carcinomas and a metastatic carcinosarcoma. In terms of clinical utility, 6 patients (7%) received a matched therapy. Three HGSC patients with somatic BRCA1/2 mutations were treated with PARP inhibitors. Another HGSC patient with ERBB2 amplification was treated with trastuzumab. One LGSC patient with a BRAF mutation was enrolled on a BRAF inhibitor clinical trial. A second LGSC patient was enrolled in a trial of anastrazole. Furthermore, 7 patients (14%) with HGSC who were previously untested were found to have a germline BRCA1/2 mutation and were subsequently referred to a familial cancer clinic for further management and cascade testing. The limitations in the study included the turnaround time and advanced stage of disease at enrolment, which significantly affected the clinical utility of the test. Conclusion: We demonstrated that molecular profiling of recurrent ovarian cancer using the ALLOCATE panel was feasible and reflected the known genomic characteristics of the different subtypes. However, challenges remain, including appropriate patient selection and efficient turnaround time for reporting. Furthermore, improved access to targeted therapies or clinical trials will also enhance the clinical utility of the ALLOCATE panel. Citation Format: Olga Kondrashova, George Au-Yeung, Leakhena Leas, Gwo-Yaw Ho, Sebastian Lunke, Kathryn Alsop, Clare Scott, Anne Hamilton, Sumitra Ananda, Alison Freimund, Michael Quinn, Orla McNally, Nadia Traficante, Tiffany Cowie, Matthew Wakefield, Arthur Hsu, Alex Dobrovic, Michael Christie, Graham Taylor, David Bowtell, Linda Mileshkin, Paul Waring. Australian Ovarian Cancer Assortment Trial–Allocating ovarian cancer patients into clinical trials based on molecular profiling. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B35.

Cyclin dependent kinase inhibitor 2A/B gene deletions are markers of poor prognosis in Indian children with acute lymphoblastic leukemia.

Cyclin dependent kinase inhibitor 2A/B (CDKN2A/B) genes are implicated in many malignancies including acute lymphoblastic leukemia (ALL). These tumor suppressor genes, with a key regulatory role in cell cycle are located on chromosome 9p21.3. Previous studies involving CDKN2A/B gene deletions have shown mixed associations with survival outcome in childhood ALL. Hundred and four newly diagnosed children with ALL (1-14 years) were enrolled in this study. Genomic DNA from pretreatment bone marrow/peripheral blood samples of these children was investigated for copy number alterations in CDKN2A/B genes using multiplex ligation dependent probe amplification assay. Immunophenotype subtyping and cytogenetic and molecular analysis of ALL was performed at start of induction chemotherapy in all children. Children were monitored for response to prednisolone (Day 8), complete morphological remission, and minimal residual disease at the end of induction. The minimum postinduction follow-up period was 6 months. CDKN2A/B deletions were seen in 19.8% (18/91) of B lineage acute lymphoblastic leukemia (B-ALL) and 38.5% (5/13) of T lineage acute lymphoblastic leukemia (T-ALL). Monoallelic CDKN2A/B deletions were found in 61.1% of total deletions in B-ALL while all the children with T-ALL harbored biallelic deletions. The prevalence of CDKN2A/B gene deletions was found to be significantly higher in older children (P=0.002), in those with higher leukocyte count (P=0.037), and in National Cancer Institute high risk group patients (P=0.001) in the B-ALL subgroup. Hazard ratio was significantly high for children with CDKN2A/B deletions in total cohort (P=0.004). Children with CDKN2A/B deletion had significantly lesser event free survival (P=0.03). CDKN2A/B deletions were significantly more prevalent in T-ALL subgroup and were found to have higher hazard ratio and lesser event free survival in total cohort in our study.

High frequency of intermediate and poor risk copy number abnormalities in pediatric cohort of B-ALL correlate with high MRD post induction

Copy number abnormalities (CNAs) and recurrent fusion transcripts are important genetic events which define and prognosticate B-Cell Acute Lymphoblastic Leukemia (B-ALL). We evaluated CNAs and fusion transcripts in 67 pediatric B-ALL cases and correlated the data with standard risk factors and early treatment outcome parameters. Common fusion transcripts ETV6-RUNX1, E2A-PBX, BCR-ABL1 and MLL-AF4 were examined by RT-PCR and noted in 15%, 15%, 13% and 1.4% of all cases respectively. CNAs in IKZF1, PAX5, EBF1, BTG1, RB1, CDKN2A/B and genes from PAR1 region viz., CSF2RA, IL3RA,P2RY8, SHOX region and CRLF2 were analyzed by multiplex ligation dependent probe amplification assay and were detected in 70% (47/67) of cases, with predominantly deletions in CDKN2A/B (36%), PAX5 (18%) and IKZF1 (16%). A statistically significant association of intermediate/poor risk CNAs was noted with high WBC count (p = 0.001), NCI group (p = 0.02) and minimal residual disease at Day35 (p < 0.0001). IKZF1 and CDKN2A/B deletion revealed poor EFS of 56% at 24 months as compared to EFS of 80% in rest of the cases (p = 0.05) suggesting their potential role in early risk stratification.

ID: 1048 A novel nonsense mutation in the CYP21A2 gene of a Vietnamese patient with congenital adrenal hyperplasia

Inactivating mutations in the CYP21A2 gene which encodes the protein involved in steroid synthesis have been reported in the patients with congenital adrenal hyperplasia (CAH). An infant who diagnosed with the severe phenotype of CAH such as increasing testicular volume, elevating of 17-hydroxyprogesteron, testosterone and progesterone and his family were subjected for genetic studies. Initially, we used PCR and direct sequencing to screen mutations in the CYP21 gene in the proband and his family. We identified a novel nonsense mutation c.374C&gt;G predicts a substitution of serine for a stop codon at codon 125 (p.S125*) within exon 3 in the proband. However, the inheritance pattern of the mutation was not consistent with disease causation because of a heterozygous mutation carrier in father and sibling, wild-type alleles in mother but mutant alleles in proband. This inspired us to find deletions of exon using multiplex ligation-dependent probe amplification (MLPA) assay. In the profiles of MLPA electropherogram, the proband had a large deletion in exon 3, but his mother did not have. It means that the proband inherited a normal allele from his mother and a mutant allele from his father, but the deletion of a normal allele occurred in the proband. Therefore, mutation c.374C&gt;G (p.S125*) in exon 3 in the proband is considered as a heterozygous deletion mutation. In addition, a large deletion in exon 1 in the maternal allele in the proband is observed. Taking together, the proband carried a nonsense mutation accompanied with two deletions in exon 1 and exon 3 in the CYP21A2 gene affect the CAH phenotype severity. These mutations also expand the CYP21A2 mutation spectrum in CAH disorder. This case also highlights the need of caution when interpreting results of molecular genetics and biochemical testing during genetic counseling.

Mutational analysis of a Chinese family with oculocutaneous albinism type 2.

Oculocutaneous albinism (OCA) is an autosomal recessive disorder characterized by hypopigmentation of the skin, hair, and eyes accompanied with ophthalmologic abnormalities. Molecular genetic test can confirm the diagnosis of the four subtypes of OCA (OCA1-4). Herein, we report a Chinese family with two patients affected by OCA. Mutations of TYR, OCA2, TYRP1, and SLC45A2 were examined by using PCR-sequencing. Large deletions or duplications of TYR and OCA2 were examined by Multiplex Ligation-dependent Probe Amplification (MLPA) assay. Compound heterozygous mutations of OCA2, (c.808-3C>G and c.2080-2A>G), were identified in both patients characterized with yellow hair and milky skin, heterochromia iridis, and nystagmus. Several computer-assisted approaches predicted that c.808-3C>G and c.2080-2A>G in OCA2 might potentially be pathogenic splicing mutations. No exon rearrangement (deletion/duplication) of TYR and OCA2 was observed in the patients by MLPA analysis. This study suggests that compound heterozygous mutations, (c.808-3C>G and c.2080-2A>G), in OCA2 may be responsible for partial clinical manifestations of OCA.

Viral prevalence increases with regional colony abundance in honey bee drones (Apis mellifera L)

Transmission among colonies is a central feature for the epidemiology of honey bee pathogens. High colony abundance may promote transmission among colonies independently of apiary layout, making colony abundance a potentially important parameter determining pathogen prevalence in populations of honey bees. To test this idea, we sampled male honey bees (drones) from seven distinct drone congregation areas (DCA), and used their genotypes to estimate colony abundance at each site. A multiplex ligation dependent probe amplification assay (MLPA) was used to assess the prevalence of ten viruses, using five common viral targets, in individual drones. There was a significant positive association between colony abundance and number of viral infections. This result highlights the potential importance of high colony abundance for pathogen prevalence, possibly because high population density facilitates pathogen transmission. Pathogen prevalence in drones collected from DCAs may be a useful means of estimating the disease status of a population of honey bees during the mating season, especially for localities with a large number of wild or feral colonies.

Abstract 3474: Prevalence of BRCA1 and BRCA2 small mutation and large genomic rearrangements in breast cancer patients visiting a genetic counseling clinic

Abstract BACKGROUND: This study was conducted to investigate mutation prevalence of the BRCA1 and BRCA2 genes in breast cancer patients, including both small mutations and large genomic rearrangements (LGRs) following the patients in the process of genetic counseling. METHODS: A total of 358 breast cancer patients who visited genetic counseling clinic and screened for BRCA1/2 mutation at the National Cancer Center were included. Among the non-carriers by direct sequencing, a subset of patients who were agreed on participation of the research were screened for the presence of LGRs using a multiple ligation-dependent probe amplification (MLPA) assay. Clinico-pathologic characteristics of cancer were evaluated thorough medical record review and characteristics for those who found BRCA mutation through MLPA were described. RESULTS: 53 (14.8%) mutation carriers were identified. The frequency of BRCA small mutations in specific subgroups was as follows: 18.0% of patients with a family history, 12.8% of early-onset breast cancer, and 20.6% of bilateral breast cancer patients. MLPA identified BRCA1 LGRs in 3 (1.5%) out of 204 patients with BRCA1 and BRCA2 small mutation-negative results. Those with LGRs showed higher probabilities of BRCA mutation carriers estimated by risk assessment models and receptor negatives of tumor immunohistochemistry. CONCLUSIONS: The prevalence of BRCA1/2 mutation prevalence in breast cancer patients with family history or personal risk factors were comparable to previous Korean studies. MLPA to screen for mutations in the BRCA1 gene could be recommended for breast cancer patients negative for small mutations. To specify proper targets of MLPA, probabilities of carrying BRCA1/2 mutation and tumor receptor status could be considered. Citation Format: Ji Yeon Sohn, Boyoung Park, Kyong-Ah Yoon, Soo Jin Park, Moo Hyun Lee, Eun Hae Cho, Keun Seok Lee, Myong Cheol Lim, Sun-Young Kong, Eun Sook Lee. Prevalence of BRCA1 and BRCA2 small mutation and large genomic rearrangements in breast cancer patients visiting a genetic counseling clinic. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3474.

Abstract A1-67: Prognostic significance of copy number aberrations in Wilms tumor

Abstract Background and Purpose: Treatment of Wilms tumor (WT) patients under International Society of Paediatric Oncology (SIOP) protocols is currently stratified by staging and histopathology at nephrectomy after neoadjuvant chemotherapy. However, most relapses occur in cases without specific histopathological risk factors, and there is a clinical need for better prognostic biomarkers. Combined loss of heterozygosity (LOH) of 1p and 16q has recently been introduced in the US as an adverse prognostic indicator, while previous work in our and other laboratories suggests that 1q gain may have a similar association with poor outcome. To examine the clinical significance of 1q gain and assess its potential as a WT biomarker, we developed a simple, effective assay that measures its genomic copy number together with that of several other loci of interest, and applied it to a large tumor series. Methods: 686 frozen tumor samples from the SIOP WT 2001 trial (from a total of 7 countries) were assayed using a rapid multiplex ligation-dependent probe amplification (MLPA) assay that was developed and optimized in association with MRC-Holland b.v. to assess the copy number status of 1p, 1q, 16q, WT1, WTX, TP53, MYCN and FBXW7. Analyses were conducted in 3 laboratories, with exchange of a blinded quality assurance sample set. Results: 1q gain was present in 28% (190/686) of the cases. The 5- year Event Free Survival (EFS) rate was 72.6% (95% Confidence Interval (CI), 66.3%-85%) for those with 1q gain and 86.4% (95% CI, 83.4%-89.6%) for those who lacked 1q gain (p=&amp;lt;0.0001). The Overall Survival (OS) rate for 1q gain cases was 86.9% (95% CI, 82.1%-92.1%) compared to 93.8% (95% CI, 91.7%-92.1%) for cases without the aberration (p=0.01). Both the EFS and OS analysis showed a statistically significant poorer outcome for cases with 1q gain, but there was no association with disease stage. 1q gain was associated with a significantly increased risk of disease recurrence (HR = 2.18, p&amp;lt;0.0001). In this analysis the proportions of samples with 1p and 16q loss were respectively 8% and 16%. Both were marginally significant for EFS but in the OS analysis 1p lost its significance. MYCN gain, 4q (FBXW7) loss and 17p (TP53) loss were also associated with adverse outcome. Conclusion: Gain of 1q is a potential adverse biomarker for WT. Its association with high risk histological features after pre-operative chemotherapy and independent impact on survival require assessment in a larger number of patients before consideration for clinical use. Citation Format: Tasnim Chagtai, Christina Zill, Linda Dainese, Jenny Wegert, Mariana Maschietto, Gordan Vujanic, Neil Sebire, Ivo Leuschner, Peter Ambros, Maureen O'Sullivan, Christophe Bergeron, David Gisselsson, Marcel Kool, Marry van den Heuvel-Eibrink, Norbert Graf, Harm van Tinteren, Aurore Coulomb, Manfred Gessler, Richard Williams, Kathy Pritchard-Jones. Prognostic significance of copy number aberrations in Wilms tumor. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-67.

Abstract 2964: Histology-specific patterns of DNA methylation in Lynch-associated and sporadic ovarian cancer

Abstract Epithelial ovarian cancer is a heterogeneous group of cancers, and molecular tools are urgently needed for a better understanding and targeted management of this often lethal disease. Since epigenetic methods can offer new tools for the management of ovarian cancer, our aim was to investigate epigenetic mechanisms in ovarian tumorigenesis representing different histological types. Expression profiling of ovarian and endometrial cancer cell lines treated with demethylating agents as well as literature were used to select gene candidates for epigenetic regulation. A methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay was constructed for thirteen genes to study methylation in 104 (85 sporadic and 19 Lynch syndrome-associated) ovarian carcinomas. Increased methylation (hypermethylation) was characteristic of ovarian cancer tissues relative to the corresponding normal tissues and hypermethylation was consistently more prominent in non-serous than serous tumors. The highest frequencies of hypermethylation were detected in Lynch-associated clear cell ovarian carcinomas. An interesting finding among endometrioid ovarian carcinomas was a significant association of lower levels of methylation in HOXA9, RSK4 and SPARC with higher tumor grade; thus, the methylation patterns of high-grade endometrioid ovarian carcinomas resembled epigenetic characteristics of high-grade serous tumors. Overall, non-serous ovarian tumors of Lynch and sporadic origin (as opposed to serous tumors) showed frequent epigenetic inactivation in RSK4, SPARC, PROM1, HOXA10, HOXA9, WT1-AS, SFRP2, SFRP5, OPCML, and MIR34B. Our findings provide important new information regarding some critical genes affected by epigenetic dysregulation in ovarian tumorigenesis. The results will be utilized in future experiments aiming to define the genetic and epigenetic origins of ovarian carcinomas of different types. Citation Format: Anni Niskakoski, Sippy Kaur, Synnove Staff, Laura Renkonen-Sinisalo, Heini Lassus, Heikki J. Järvinen, Jukka-Pekka Mecklin, Ralf Bützow, Päivi Peltomäki. Histology-specific patterns of DNA methylation in Lynch-associated and sporadic ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2964. doi:10.1158/1538-7445.AM2015-2964

Abstract 5264: A novel strategy for accuurate and unambiguous mutation detection of the highly homologous PMS2 gene

Abstract Background: Heterozygous mutations in the DNA mismatch repair gene, PMS2 contribute to Lynch syndrome. Carrier individuals are at increased risk of developing cancers, including colorectal and endometrial cancers. Biallelic mutations in PMS2 can cause constitutional mismatch-repair-deficiency syndrome, which is characterized by childhood onset malignancy, including brain cancers, hematologic malignancies, colorectal cancers, and multiple intestinal polyposis. Molecular analysis of the PMS2 gene is complicated by the presence of a large family of highly homologous sequences in other genomic regions and frequent gene conversion between the active gene and pseudogenes. Although the combination of multiplex ligation-dependent probe amplification (MLPA) assay and Sanger sequencing can largely overcome this problem, copy number variations (CNVs) at the 3′ end cannot be unambiguously differentiated when gene conversion is involved. Here we described a novel approach that combines long range PCR (LR-PCR), next generation sequencing (NGS), and MLPA to identify sequence and copy number changes of the PMS2 gene. Method: A comprehensive workflow incorporating MLPA, LR-PCR, and NGS was carefully designed for sequence and CNV analyses of the PMS2 gene. MLPA was used to detect CNVs in PMS2 and PMS2CL. In order to distinguish mutations in the active gene from changes in the pseudogene, the active gene was specifically enriched by LR-PCR, followed by whole gene NGS analysis to detect point mutations and CNVs. CNVs can be further confirmed by MLPA. Allele dropout is overcome by capture-based NGS. Results: Using the comprehensive workflow described above, we have identified CNVs in all coding exons of the PMS2 gene: exonic deletions (E2-E9, E5-E9, E8, E10, E14), exonic duplications (E11-E12), whole gene deletion (E1-E15), and a nonsense mutation, S22X. We identified an exon 14 deletion (c.2276-113_c.2445+1596del) in the active gene that could not be differentiated originally by the combination of MLPA and Sanger sequencing due to a gene conversion event in the 3′ region between PMS2 and PMS2CL, however, with LR-PCR followed by NGS, the deletion was unequivocally identified. Breakpoint analysis of multiple samples with exon 14 deletions revealed that frequent exon 14 deletions were mediated by homologous Alu sequences. We also identified a potential LINE1 sequence mediated intragenic duplication flanking exons 11 and 12. Conclusion: The combined strategies of long range PCR, MLPA, and deep coverage NGS ensure accurate and unambiguous identification of a wide spectrum of mutations, including point mutations, small indels, and copy number changes, in addition to the determination of breakpoints. The novel strategy of comprehensive workflow provides a reliable tool for NGS based accurate molecular analysis of genes containing multiple copies of highly homologous sequences such as PMS2. Citation Format: Jianli Li, Hongzheng Dai, Yangming Feng, Jia Tang, Stella Chen, Xia Tian, Elizabeth Gorman, Eric S. Schmitt, Jing Wang, Victor Wei Zhang, Lee-Jun Wong. A novel strategy for accuurate and unambiguous mutation detection of the highly homologous PMS2 gene. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5264. doi:10.1158/1538-7445.AM2015-5264

Abstract O.16: Comprehensive Genotyping Of The FCGR2/3 Locus Reveals A Novel Association Of FCGR2C-ORF With Susceptibility To Kawasaki Disease

The human FCGR2/3 locus contains highly homologous genes encoding the five major receptors for IgG (Fc-gamma receptors, FcγRs). In two prior GWAS on Kawasaki disease (KD), a SNP in FCGR2A (131H&gt;R; rs1801274) was identified to be associated with disease susceptibility. However, the FCGR2/3 locus contains multiple single nucleotide polymorphisms (SNPs) and copy number variations (CNVs), which were not covered by the detection platforms used in the GWAS. In this study we therefore focused on further fine-mapping of this locus to investigate the association of the different genetic variations with KD susceptibility. A highly accurate and validated multiplex ligation-dependent probe amplification (MLPA) assay was used to analyze all functionally relevant SNPs and CNVs within this locus. In a genetic association study involving case-control and family-based testing with 1028 patients with KD, the previous finding of FCGR2A-131H as a susceptibility marker for KD was confirmed (OR 1.16; 95%CI 1.08-1.32, meta-P = 0.01). In addition, we found a novel significant association of the FCGR2C-ORF haplotype with susceptibility to KD (OR 1.34; 95% confidence interval 1.11-1.62, meta-P = 0.003). FCGR2C-ORF leads to the expression of an extra, functionally activating FcγR (i.e. FcγRIIc) on myeloid cell types and NK cells. Being absent in Asian individuals, the FCGR2C-ORF haplotype only contributed to KD susceptibility in European subjects, independent of the established association with FCGR2A-H131R. We did not find any significant association of CNV of the locus with susceptibility to KD. Our data point to an important role of the activating FcγRs in KD pathology. We hypothesize that the identified functional SNPs might alter the balance between the activating and inhibitory FcγRs leading to unbalanced inflammation and KD.

Accurate classification of GCB/ABC Diffuse Large B cell Lymphoma using a simple and rapid RT-MLPA assay: a CALYM study

Diffuse large B-cell lymphoma, the most common non-Hodgkin lymphoma, is subdivided into germinal center B-cell–like and activated B-cell–like subtypes. Unfortunately, these lymphomas are difficult to differentiate in routine diagnosis, impeding the development of treatments. Patients with these lymphomas can benefit from specific therapies. We therefore developed a simple and rapid classifier based on a reverse transcriptase multiplex ligation dependent probe amplification assay and 14 gene signature. When compared to the Affymetrix U133+2 gold standard, all 46 samples 95% CI [92%, 100%]) of a validation cohort classified by both techniques were attributed to the expected subtype. Similarly, 93% of the 55 samples (95% CI [82%, 98%]) of a second independent series characterized using a mid-throughput gene expression profiling method were classified correctly. Unclassifiable sample proportions reached 13.2% and 13.8% in these cohorts, comparable to the frequency originally reported. The developed assay was also sensitive enough to obtain reliable results from preserved samples and flexible enough to include prognostic factors such as MYC/BCL2 co-expression. Finally, in a series of 135 patients, both overall (P =0.01) and progression-free (P =0.004) survival differences between the two subtypes were confirmed. As the multiplex ligation dependent probe amplification methodology is already in use and requires only common instruments and reagents, it could easily be applied to clinical trial patient stratification to help in treatment decisions.

Fc-dependent mechanisms of action: roles of FcγR and FcRn.

Fc-dependent mechanisms of action: roles of FcγR and FcRn.