Detection Of Duplications Research Articles

Prenatal Diagnosis of Chromosome Abnormalities: Past, Present, and Future

For decades, conventional chromosome analysis using G-banded karyotyping has been the gold standard for detecting cytogenetic abnormalities in fetuses for prenatal diagnosis and pregnancy loss. Although chromosome studies easily identify such chromosome abnormalities as aneuploidy (gain or loss of an entire chromosome), balanced rearrangements, and large unbalanced structural rearrangements, they have limited resolution. Pathogenic copy number changes smaller than approximately 5–10 Mb are not detectable; however, recent advances in chromosomal microarray (CMA)2 analysis now allow the detection of chromosomal deletions and duplications at much higher resolutions (down to only a few kilobases, depending on the microarray), producing a higher diagnostic yield. In fact, a recently published study that compared standard karyotyping with postnatal CMA testing for patients with unexplained developmental delay, autism spectrum disorders, or multiple congenital anomalies demonstrated an increased diagnostic yield of about 12%–15% with CMA analysis, vs. 4% with karyotyping. These findings led to the recommendation by the American College of Medical Genetics that CMA testing replace chromosome analysis as the first-tier test for such patients (1, 2). Given these findings in the postnatal setting, the incremental diagnostic yields of CMA analysis and chromosome analysis are now being evaluated for prenatal genetic testing. Recent publications in the New England Journal of Medicine have described approaches to answering this question, not only with prenatal samples (3) but also with stillbirths (defined as pregnancy loss at or after 20 weeks' gestation) (4). The multicenter study by Wapner et al. successfully analyzed 4182 samples of amniotic fluid and chorionic villi with both chromosome and CMA analysis (3). The women were tested for indications that included advanced maternal age, a positive result in maternal-serum screening, and fetal anomalies detected on ultrasonography. CMA was equally efficacious in identifying all aneuploidies and unbalanced rearrangements that had been identified by karyotyping. …

Pinda: A Web service for detection and analysis of intraspecies gene duplication events

We present Pinda, a Web service for the detection and analysis of possible duplications of a given protein or DNA sequence within a source species. Pinda fully automates the whole gene duplication detection procedure, from performing the initial similarity searches, to generating the multiple sequence alignments and the corresponding phylogenetic trees, to bootstrapping the trees and producing a Z-score-based list of duplication candidates for the input sequence. Pinda has been cross-validated using an extensive set of known and bibliographically characterized duplication events. The service facilitates the automatic and dependable identification of gene duplication events, using some of the most successful bioinformatics software to perform an extensive analysis protocol. Pinda will prove of use for the analysis of newly discovered genes and proteins, thus also assisting the study of recently sequenced genomes. The service's location is http://orion.mbg.duth.gr/Pinda. The source code is freely available via https://github.com/dgkontopoulos/Pinda/.

Adult-onset autosomal dominant leukodystrophy without early autonomic dysfunctions linked to lamin B1 duplication: a phenotypic variant

The early presentation of autonomic dysfunctions at the disease onset has been considered the mandatory clinical feature in adult-onset autosomal dominant leukodystrophy, which is a rarely recognised leukodystrophy caused by duplication of the lamin B1 gene. We report the first family with adult-onset autosomal dominant leukodystrophy and lamin B1 duplication, without the distinguishing early-appearing autonomic dysfunctions. Subjects from three consecutive generations of a multi-generational Serbian family affected by adult-onset autosomal dominant leukodystrophy underwent clinical, biochemical, neurophysiological, neuroradiological, and genetic studies. The patients atypically exhibited late autonomic dysfunctions commencing at the disease end-stages in some. Genetic findings of lamin B1 duplication verified adult-onset autosomal dominant leukodystrophy, which was supported also by neuroimaging studies. Exclusively, proton magnetic spectroscopy of the brain revealed a possibility of neuro-axonal damage in the white matter lesions, while magnetic resonance imaging of the spinal cord excluded spinal myelin affection as a required finding in this leukodystrophy. The detection of lamin B1 duplication, even when autonomic dysfunctions do not precede the other symptoms of the disease, proves for the first time that lamin B1-duplicated adult-onset autosomal dominant leukodystrophy may have a phenotypic variant with delayed autonomic dysfunctions. Prior to this report, such a phenotype had been speculated to represent an entity different from lamin B1-duplicated leukodystrophy. Hereby we confirm the underlying role of lamin B1 duplication, regardless of the autonomic malfunction onset in this disorder. It is the only report on adult-onset autosomal dominant leukodystrophy from Southeastern Europe.

Development of a comprehensive real-time PCR assay for dystrophin gene analysis and prenatal diagnosis of Chinese families

BackgroundTo develop a comprehensive method to analyze deletions or duplications of the dystrophin gene in both patients and carriers of Duchenne muscular dystrophy (DMD), likewise applied to prenatal diagnosis. MethodsA total of thirty Chinese families were recruited, composed of 29 DMD affected males and 38 female relatives containing four pregnant women. Deletions were previously screened by multiplex PCR. A comprehensive real-time PCR assay using SYBR Green I dye was performed for the initial detection of duplications in patients with a seven-exon primer set, carrier detection for female relatives and prenatal diagnosis for the 4 of them. The results were later confirmed by multiple ligation-dependent probe amplification (MLPA) and linkage analysis. ResultsThree out of 4 duplications were first discovered by real-time PCR. Carrier status was ascertained in 22 and rejected in the remaining sixteen female relatives. Furthermore, 4 fetuses were diagnosed as two normal females, one normal male and one female carrier, respectively. ConclusionsOur real-time PCR assay is useful in duplication screen with a detection rate of >70%, as well as rapid and reliable in both carrier detection and prenatal diagnosis of DMD families with known deletions and duplications.

Region duplication detection based on Harris corner points and step sector statistics

Region duplication is a simple and effective operation for digital image forgeries. The detection of region duplication is very important in digital image forensics. Most existing detection methods for region duplication are based on exhaustive block-matching of image pixels or transform coefficients. They may not be effective when the duplicate regions have gone through some geometrical transformations. In this paper, a novel region duplication detection method that is robust to general geometrical transformations is proposed. Firstly, the Harris corner interest points in an image are detected. Then, an image region description method based on step sector statistics is developed to represent the small circle image region around each Harris point with a feature vector. Finally, the small circle image regions are matched using the best-bin-first algorithm to reveal duplicate regions. Experimental results show that the proposed method can work effectively on the forged images from two image databases, and it is also robust to several geometrical transformations and image degradations.

Detection of Image Region Duplication Using Spin Image

Detection of Image Region Duplication Using Spin Image

Improved Detection of Deletions and Duplications in the DMD Gene Using the Multiplex Ligation-Dependent Probe Amplification (MLPA) Method

The multiplex ligation-dependent probe amplification (MLPA) assay is the most powerful tool in screening for deletions and duplications in the dystrophin gene in patients with Duchenne and Becker muscular dystrophy (DMD/BMD). The efficacy of the assay was validated by testing 20 unrelated male patients with DMD/BMD who had already been screened by multiplex PCR (mPCR). We detected two duplications that had been missed by mPCR. In one DMD patient showing an ambiguous MLPA result, a novel mutation (c.3808_3809insG) was identified. MLPA improved the mutation detection rate of mPCR by 15 %. The results of our study (1) confirmed MLPA to be the method of choice for detecting DMD gene rearrangements in DMD/BMD patients, (2) showed that ambiguous MLPA amplification products should be verified by other methods, and (3) indicated that the MLPA method could be used in screening even for small mutations located in the probe-binding regions.

Denaturing high-performance liquid chromatography coupled with multiplex PCR for rapid detection of large duplications or deletions in patients with Duchenne muscular dystrophy and spinal muscular atrophy

To assess the value of multiplex PCR-denaturing high-performance liquid chromatography (PCR-DHPLC) method for screening large duplications or deletions in patients with Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA). DNA was extracted from peripheral venous blood samples from 35 DMD and 6 SMA patients. Large duplications or deletions were screened with multiplex PCR coupled with DHPLC method. The results were validated with testing of positive and negative controls. Known duplications or deletions in all controls were reliably detected with multiple PCR coupled with DHPLC. Large duplications or deletions were found in 71.4% of 35 DMD patients, which included 5 large duplications and 20 large deletions. For SMA patients, deletions of SMN1 exon 7 were detected in 16 samples. Multiplex PCR coupled with DHPLC method is an effective and reliable method for detecting large genomic duplications or deletions in patients with DMD or SMA.

Detection of FLT3 Internal Tandem Duplication in Targeted, Short-Read-Length, Next-Generation Sequencing Data

A recurrent somatic mutation frequently found in cytogenetically normal acute myeloid leukemia (AML) is internal tandem duplication (ITD) in the fms-related tyrosine kinase 3 gene (FLT3). This mutation is generally detected in the clinical laboratory by PCR and electrophoresis-based product sizing. As the number of clinically relevant somatic mutations in AML increases, it becomes increasingly attractive to incorporate FLT3 ITD testing into multiplex assays for many somatic mutations simultaneously, using next-generation sequencing (NGS). However, the performance of most NGS analysis tools for identifying medium-size insertions such as FLT3 ITD mutations is largely unknown. We used a multigene, targeted NGS assay to obtain deep sequence coverage (>1000-fold) of FLT3 and 26 other genes from 22 FLT3 ITD-positive and 29 ITD-negative specimens to examine the performance of several commonly used NGS analysis tools for identifying FLT3 ITD mutations. ITD mutations were present in hybridization-capture sequencing data, and Pindel was the only tool out of the seven tested that reliably detected these insertions. Pindel had 100% sensitivity (95% CI = 83% to 100%) and 100% specificity (95% CI = 88% to 100%) in our samples; Pindel provided accurate ITD insertion sizes and was able to detect ITD alleles present at estimated frequencies as low as 1%. These data demonstrate that FLT3 ITDs can be reliably detected in panel-based, next-generation sequencing assays.

The frequency of previously undetectable deletions involving 3′ Exons of the PMS2 gene

Lynch syndrome is characterized by mutations in one of four mismatch repair genes, MLH1, MSH2, MSH6, or PMS2. Clinical mutation analysis of these genes includes sequencing of exonic regions and deletion/duplication analysis. However, detection of deletions and duplications in PMS2 has previously been confined to Exons 1-11 due to gene conversion between PMS2 and the pseudogene PMS2CL in the remaining 3' exons (Exons 12-15). We have recently described an MLPA-based method that permits detection of deletions of PMS2 Exons 12-15; however, the frequency of such deletions has not yet been determined. To address this question, we tested for 3' deletions in 58 samples that were reported to be negative for PMS2 mutations using previously available methods. All samples were from individuals whose tumors exhibited loss of PMS2 immunohistochemical staining without concomitant loss of MLH1 immunostaining. We identified seven samples in this cohort with deletions in the 3' region of PMS2, including three previously reported samples with deletions of Exons 13-15 (two samples) and Exons 14-15. Also detected were deletions of Exons 12-15, Exon 13, and Exon 14 (two samples). Breakpoint analysis of the intragenic deletions suggests they occurred through Alu-mediated recombination. Our results indicate that ∼12% of samples suspected of harboring a PMS2 mutation based on immunohistochemical staining, for which mutations have not yet been identified, would benefit from testing using the new methodology.

1066PD - Overexpression of Enhancer of Zeste Homolog 2 Is Associated with Clinical Outcome in Acute Myeloid Leukemia Patients

ABSTRACT Background Enhancer of zeste homolog 2 (EZH2), a subunit of polycomb repressive complex 2 functioned as methyltransferase causing trimethylation at lysine-27 of histone 3, is involved in stem cell regulation and various cancers. It was reported two faces that both increased expression and mutation/deletion were implicated in cancer development and progression of different malignancies. For acute myeloid leukemia (AML), the clinical significance of EZH2 was not explored. In this study, we examined EZH2 expression and mutation and evaluated the association between EZH2 and patient survival. Methods A total of 105 patients, who were newly diagnosed as de novo AML and received intensive chemotherapy between 2004 January and 2010 December, were retrospectively analyzed. All patients received French-American-British (FAB) classification, karyotype analyses, immunophenotyping, detection of NPM mutation and FLT3 internal tandem duplication (FLT3/ITD). The mutation of EZH2 was identified by direct sequencing. An immunohistochemical analysis of EZH2 was performed on bone marrow biopsy samples, and overexpression was defined as more than 50% positive staining among the leukemic marrow. Results EZH2 overexpression was identified in 57 patients (54.3%), and no associated with age, gender, FAB classification, karyotype, FLT3/ITD and NPM mutation. Only two mutations were detected in this cohort. Patients with EZH2 overexpression had a significant shorter median overall survival (OS; 18.5 vs 40.7 months, p = 0.047) as well as a higher trend of relapsed/refractory disease (47.9% vs 66.7%, p = 0.052). Karyotype and FLT3/ITD were also the significant prognostic factors for the entire cohort and NPM mutation was a trend of favorable prognostic factor in intermediate-karyotype patients by univariate analysis. We defined a prognostic classification based on these four factors that was able to classify patients into four risk groups showing significant different survivals (median OS: not reached, not reached, 22.0 and 10.6 months, respectively; p Conclusion EZH2 overexpression is a novel poor prognostic biomarker in AML patients. The current result indicated that EZH2 expression status may be incorporated into prognostic classification. Disclosure All authors have declared no conflicts of interest.

Application of next-generation sequencing technology for genetic diagnosis of Duchenne muscular dystrophy

To detect genetic causes of Duchenne muscular dystrophy (DMD). Next-generation sequencing was used to detect 6 DMD patients in whom no exonic deletions were detected by multiplex PCR. Sanger sequencing and multiplex ligation-dependent probe amplification were used to confirm the results. One case was found to have deletions of exons 10 and 11, 1 had exons 16 and 17 duplication, 4 cases have 8 point mutations including c.2776C>T, c.5475delA, c.6391_6392delCA, IVS64+1G>A, c.2645A>G, c.5244G>A, c.7728T>C, c.8729A>T, c.8734A>G and c.8810G>A. The former 4 mutations are suspicious pathogenicity, the other 6 mutations are polymorphisms in population. Three novel mutations (IVS64+1G>A, c.6391_6392delCA (p.Q2131NfsX3) and p.Q926X (CAG>TAG) were not reported before. Next-generation sequencing technology is a useful tool for the detection of deletion, duplication and point mutation, which is valuable for clinical application.

Comprehensive FLT3 analysis in Indian acute myeloid leukaemia

Abstract Aim: Activating mutations of FLT3 are commonly found in AML patients and reported to be associated with poor clinical outcome. We aimed to evaluate the incidence of FLT3 mutations along with FLT3 mRNA and CD135 protein expression in AML patients of western India and their role in prognosis of disease. Method: Analysis for the detection of FLT3 internal tandem duplication (ITD), Tyrosine kinase domain (TKD) point mutations and quantification of mRNA level was carried out in total 174 de novo patients diagnosed with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and aplastic anemia using PCR and RT-PCR methods. FLT3 protein was quantified by flow cytometry on leukemic blasts. Results: The incidence of FLT3 ITD, FLT3 TKD mutations and CD135 protein expression was found to be 19%, 7% and 62% respectively in AML patients. In MDS, only FLT3 ITD mutation and CD135 protein over expression could be analyzed, incidence of which was 22% and 60%. In aplastic anemia, FLT3 mutations, FLT3 mRNA and protein over expression were not detected. FLT3 mutations as well as FLT3 mRNA and protein over expression were prominently noted in AML subtypes associated with myelo-monocytic lineage. CD135 protein over expression was significantly associated with reduced Disease Free Survival (DFS) whereas WBC and blasts emerged as poor prognostic factors with respect to Disease Free Survival (DFS) and Overall Survival (OS) respectively in multivariate analysis. Conclusion: Our data suggest that CD135 receptor protein over expression is a potential prognostic marker as well as molecular target for FLT3 inhibitors in AML patients.

Mutation analysis of PMP22 in Slovak patients with Charcot-Marie-Tooth disease and hereditary neuropathy with liability to pressure palsies

Charcot-Marie-Tooth disease (CMT) and related peripheral neuropathies are the most commonly inherited neurological disorders in humans, characterized by clinical and genetic heterogeneity. The most prevalent clinical entities belonging to this group of disorders are CMT type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP). CMT1A and HNPP are predominantly caused by a 1.5 Mb duplication and deletion in the chromosomal region 17p11.2, respectively, and less frequently by other mutations in the peripheral myelin protein 22 (PMP22) gene. Despite being relatively common diseases, they haven't been previously studied in the Slovak population. Therefore, the aim of this study was to identify the spectrum and frequency of PMP22 mutations in the Slovak population by screening 119 families with CMT and 2 families with HNPP for causative mutations in this gene. The copy number determination of PMP22 resulted in the detection of CMT1A duplication in 40 families and the detection of HNPP deletion in 7 families, 6 of which were originally diagnosed as CMT. Consequent mutation screening of families without duplication or deletion using dHPLC and sequencing identified 6 single base changes (3 unpublished to date), from which only c.327C>A (Cys109X) present in one family was provably causative. These results confirm the leading role of PMP22 mutation analysis in the differential diagnosis of CMT and show that the spectrum and frequency of PMP22 mutations in the Slovak population is comparable to that seen in the global population.

Detection of α-globin gene deletion and duplication using quantitative multiplex PCR of short fluorescent fragments

The aim of this study was to establish a sensitive method that can detect the presence of not only the common but also the unusual or unknown α-globin gene deletions for screening of α-thalassemia. We used quantitative multiplex PCR of short fluorescent fragments (QMPSF) for the α-globin genes (HBA) to screen α-thalassemia deletions. We set up and validated HBA-QMPSF using 50 negative and 100 positive controls of deletional α-thalassemia. To evaluate its ability to detect the presence of the common and unusual or unknown α-globin gene deletions, 579 unrelated samples were simultaneously analyzed using this assay and multiplex Gap polymerase chain reaction (Gap-PCR). The inconsistent results were further confirmed by multiplex ligation-dependent probe amplification (MLPA). HBA-QMPSF was capable of detecting α-globin gene deletions with an acceptable variability as shown by mean values (SD) of allele dosage for the heterozygous deleted control obtained from intra- and inter-experimental replicates [0.63 (0.01) and 0.61 (0.03)]. In 572 out of the 579 unrelated subjects, HBA-QMPSF and multiplex Gap-PCR gave consistent results. In seven cases which were finally proved to be composed of one rare deletion--Thai/-α3.7, one novel deletion--SEA/-α2.8, four αααanti3.7/αα and one αααanti4.2/αα triplications, HBA-QMPSF showed deletion or duplication in the α-globin gene while multiplex Gap-PCR failed to give the correct diagnosis. HBA-QMPSF is able to detect the presence of the common and unusual or unknown α-thalassemia deletions and duplications. It can be used as an initial screening test for α-thalassemia caused by HBA gene copy number alteration.

P1-09-03: Prevalence of Germline TP53 Mutations in Young Women with HER2−Positive Breast Cancer.

Abstract Background: Li Fraumeni syndrome is a rare inherited cancer susceptibility condition associated with germline mutations in the TP53 gene, in which breast cancer (BC) is the most frequent tumor. The prevalence of TP53 mutations in population-based series of very young onset BC (<30 years at diagnosis) ranges from <1% to approximately 7%1-4. Recent data show that BC in patients carrying a germline TP53 mutation are commonly HER2 amplified (63-83%)5-7. In this study, we assessed the prevalence of germline TP53 mutations in women with HER2 positive BC diagnosed age ≤ 50 years. Material & Methods: We identified 347 women with invasive HER2 positive BC diagnosed at age ≤ 50 years using the Clinical Operations and Research Information System (CORIS) at the Dana Farber Cancer Institute. Information on age at diagnosis, histology, hormone receptor and HER2 status as well as personal and family cancer history was confirmed from medical records. 129 patients were excluded for various reasons, including a cancer diagnosis prior to the BC and a documented BRCA1/2 mutation. A combination of Exon Grouping Analysis (EGAN) and Sanger sequencing for detection of TP53 mutations in exons 2–11 including surrounding intronic sequence was performed on 218 germline DNA samples. Multiplex Ligation-dependent Probe Amplification (MLPA) analysis for the detection of TP53 deletions or duplications is ongoing. Results: A germline TP53 mutation was identified in 4 women diagnosed at age ≤ 50 years (1.8%, 95%CI 0.5−4.6). At BC diagnosis, they were 23, 32, 44 and 50 years. Two BC were ER+/PR+, HER2+ and 2 were ER-/PR-, HER2+. Estimate of prevalence of germline TP53 mutations by age at BC diagnosis are: age ≤ 35, 2/41 (4.9%, 95%CI 0.6−16.6), and age ≤ 45 3/168 (1.8%, 95%CI 0.4−5.1). Among the women with germline TP53 mutations, 2 met the Chompret criteria8 and none the classic LFS criteria. Discussion: TP53 mutations were identified in a cohort of women with HER2+ BC at young age. As expected, the frequency is higher in younger women, but mutations were seen in all age groups that were evaluated. None of these women met classic LFS criteria by family history. Consideration of TP53 testing should be given to women diagnosed below age 35 who are negative for BRCA1/2 mutations regardless of family history. Analysis of other series will be helpful in reaching more stable estimates of the prevalence of mutation carriers among patients with HER2+ BC at young age. 1. Sidransky D et al. Cancer Res. 1992; 52:2984–2986. 2. Borresen AL et al. Cancer Res.1992; 52:3234–3236. 3. Lalloo F et al. Lancet 2003; 361:1101–02 4. Gonzalez KD et al. J Clin Oncol 2009;27(8):1250–6 5. Wilson JR et al. J Med Genet 2010;47(11):771–774. 6. Melhem-Bertrandt A et al: San Antonio Breast Cancer Symposium 2010: P3-12-01. 7. Masciari S et al: J Clin Oncol 29: 2011 (suppl; abstr 1519) 8. Tinat J et al. J Clin Oncol. 2009;27(26):e108–9 Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-09-03.

Detection of FLT3 Internal Tandem Duplications in Acute Myeloid Leukemia by Targeted Multi-Gene Next Generation Sequencing,

Abstract 3547 Introduction:Recurrent somatic mutations are valuable prognostic markers in cytogenetically normal Acute Myeloid Leukemia (AML). The most common of these mutations is a 9 to ∼150 bp internal tandem duplication (ITD) in the fms-related tyrosine kinase 3 (FLT3) gene, which is typically identified via PCR amplification and capillary electrophoresis. Since testing for individual mutations in this manner will become laborious and expensive as the number of clinically relevant mutations increases, we and others have proposed using targeted next-generation sequencing (NGS) for comprehensive detection of somatic mutations in multiple genes simultaneously. Successful application of this approach will require automated analysis methods capable of sensitive detection of a variety of mutation types, including single-base substitutions and insertions/deletions, with a low false-positive rate. However, the accuracy of current methods for identifying medium-sized insertions such as the FLT3 ITDs has not been established. Therefore, we sought to determine the ability of several common analysis tools to identify FLT3 ITDs from Illumina NGS sequence data. Methods:We performed targeted sequencing of 10 samples with known FLT3 ITDs ranging between 17 and 93 base-pairs (bp) as part of a larger test panel of 28 genes commonly mutated in AML and other malignancies. Nine of the FLT3 ITD-positive samples were from patients with newly diagnosed AML and were confirmed by PCR and capillary electrophoresis. A cancer cell line known to be heterozygous for a 30 bp FLT3 ITD, MV4-11, was also included. Indexed Illumina sequencing libraries were generated using automated library preparation and enriched for target regions using solution-phase hybridization-capture with biotinylated cRNA probes targeting exons +/− 200 bp plus 1 kb flanking the FLT3 gene and the 27 other genes in the panel. Enriched libraries were sequenced in multiplex on an Illumina HiSeq instrument using 2 × 101 bp reads. Demultiplexed reads were mapped to the hg19 reference sequence with novoalign, and indels were called in a 1 kilobase-pair region surrounding the FLT3 ITD with samtools, GATK, maq, CLC Genomics Workbench, PINDEL, and DINDEL using default parameters, in addition to de novo assembly of reads with partial similarity to the region using phrap. Insertion calls were then compared to results from PCR and capillary electrophoresis. Results:Multiplex sequencing resulted in 585 to 1,000-fold raw coverage of the FLT3 gene for the 10 study samples (Table 1). No FLT3 ITD insertions were detected in any sample using the common NGS analysis tools samtools, GATK, maq, DINDEL, and CLC Genomics Workbench. However, PINDEL identified insertions between 17 and 72 bp in 9 of 10 FLT3 ITD-positive samples. PINDEL failed to detect a 93 bp ITD insertion (the largest insertion in this set) in one patient sample, as well as an 84 bp insertion in a patient with two insertions (81 and 54 bp) detected by standard methods. De novo assembly of the FLT3 ITD region also resulted in detection of insertions in 9 of the 10 cases. No insertions were called in an additional set of 15 samples without known FLT3 ITDs. Conclusions:We evaluated the ability of several NGS analysis tools to detect previously known FLT3 ITDs in multi-gene targeted NGS data. Most of the general-purpose analysis tools we tested were unable to detect FLT3 ITD insertions. However, two approaches detected known FLT3 ITD insertions in 90% of the samples tested in this study, including the program PINDEL and de-novo assembly of the FLT3 ITD region using phrap. These results demonstrate that medium-sized FLT3 ITD insertions can be detected in clinical samples by high coverage NGS sequencing with the appropriate analysis pipeline. However, further methods for reliable detection of larger (>70bp) insertions must be developed before clinical NGS-based methods can be applied to the detection of the full spectrum of somatic mutations present in leukemias and other malignancies.CaseFLT3 coverageFLT3 ITD size PCR/Capillary electrophoresis (bp)FLT3 ITD size PINDELFLT3 ITD size de novo assembly18971821212917424545377793N.D.96492281/51b5484/545874363939693369/21b72/2172/2771,09787/66b69/21N.D.88315754549585171818MV4-11624303130N.D. = Not detectedbCapillary electrophoresis revealed two ITD mutant alleles in these specimensaFLT3 ITD locus defined as chr13:28,607,800–28,608,800 Disclosures:No relevant conflicts of interest to declare.

Detection of internal tandem duplications in the FLT3 gene by different electrophoretic methods

In acute myeloid leukemia (AML), the internal tandem duplication (ITD) in the juxtamembrane domain of the FLT3 (Fms-like tyrosine kinase 3) gene is one of the most frequent genetic alterations associated with poor prognosis. A complex evaluation of the analytical properties of the three most frequently used detection methods--PCR followed by agarose (AGE), polyacrylamide (PAGE) or capillary electrophoresis (CE)--was performed on 95 DNA samples obtained from 73 AML patients. All the three methods verified the presence of a mutant allele in 20 samples from 18 patients. AGE and PAGE could detect the presence of 1%-2% mutant allele, while the detection limit of CE was 0.28%. However, acceptable reproducibility (inter-assay CV <25%) of the mutant allele rate determination was only achievable above 1.5% mutant/total allele rate. The reproducibility of the ITD size determination by CE was much better, but the ITD size calculated by PeakScanner or GeneScan analysis was 7% lower as compared to values obtained by DNA sequencing. The presence of multiple ITD was over-estimated by PAGE and AGE due to the formation of heteroduplexes. This study suggests the use of PCR+CE in the diagnostics and the follow-up of AML patients. The data further supports the importance of proper analytical evaluation of home-made molecular biological diagnostic tests.

Molecular characterization of MPS IIIA, MPS IIIB and MPS IIIC in Tunisian patients

Sanfilippo syndrome (mucopolysaccharidosis type III, MPS III) is a progressive disorder in which patients are characterized by severe central nervous system degeneration together with mild somatic disease. MPS III results from a deficiency in one of the four enzymes involved in the heparan sulfate degradation, with sulfamidase (SGSH), α-N-acetylglucosaminidase (NAGLU), acetyl-coenzyme A: α-glucosaminide N-acetyltransferase (HGSNAT), and N-acetylglucosamine-6-sulfatase (GNS) being deficient respectively in MPS IIIA, MPS IIIB, MPS IIIC and MPS IIID. Mutation screening using PCR reaction/sequencing analysis on genomic DNA fragments was performed in seven Tunisian index cases with MPS IIIA, three with MPS IIIB and two with MPS IIIC. QMPSF (Quantitative Multiplex PCR of Short fluorescent Fragments) analysis was developed for the detection of genomic deletions and duplications in the SGSH gene. These approaches allowed the identification of 11 mutations, 8 of them were novel including a mutation involving the start codon (p.Met1?), one small duplication (p.Leu11AlafsX22), one small deletion (p.Val361SerfsX52) and a large deletion of exon 1 to exon 5 in the SGSH gene, one missense mutation (p.Pro604Leu) and one nonsense mutation (p.Tyr558X) in the NAGLU gene and, finally, one missense mutation (p.Trp627Cys) and one nonsense mutation (p.Trp403X) in the HGSNAT gene.

Genome-wide Profiling in AML Patients Relapsing after Allogeneic Hematopoietic Cell Transplantation

Molecular pathogenesis of relapse after allogeneic hematopoietic cell transplantation is poorly understood. Data regarding relapse mechanisms after transplantation is scarcely available. We investigated genomic aberrations (GAs) in 21 patients undergoing related and unrelated HLA-matched transplantation in leukemic blasts before transplant and at relapse after transplantation. We found a higher number of GAs after transplantation, suggesting increased genomic instability during relapse. Two of 21 patients showed a large homozygous region spanning the whole HLA-locus on chromosome 6p in the relapse sample. In both patients sequence-based HLA typing of the blasts revealed a loss of the patient-specific allele at the mismatched locus leading to homozygosity for the HLA haplotype shared by the patient and the donor. In addition, GAs were found in critical regions such as 12p13, 13q12.2, and 17p13. Our results suggest that escape from immunologic surveillance may be a relevant mechanism of relapse after transplantation in patients with GAs on chromosome 6p. A combination of continuous immunologic pressure mediated by donor T cells and clonal evolution of myeloid leukemia may result in acquired GAs after transplantation.