Cryptic Translocation Research Articles

Verification of a cryptic t(Y;15) translocation in a male with an apparent 45,X karyotype

BackgroundA rare disease is that an individual with a non-chimeric karyotype of 45,X develops into a male. We explored the genetic aetiology of an infertile male with an apparent 45,X karyotype, which was subsequently verified as cryptic translocation between chromosomes Y and 15.MethodsDNA was extracted from the patient's peripheral blood. A range of genetic testing was performed, including conventional chromosomal karyotyping, short tandem repeat (STR) analysis for azoospermia factor (AZF) region, fluorescence in situ hybridization (FISH) with specific probes groups of DXZ1/DYZ3, DYZ3/D15Z1/PML and SRY/D15Z1/PML, and chromosomal microarray analysis (CMA) for genomic copy number variations (CNVs).ResultsThe patient was found to have an apparent 45,X karyotype. STR analysis showed that he possessed a short arm of the Y chromosome, including the SRY gene; however, he was missing the long arm of the Y chromosome, including AZFa + b + c and Yqter. A FISH assay of DXZ1 and DYZ3 probes showed a green signal of the X centromere and a red of the Y centromeric signal on a D-group-sized chromosome. By FISH assaying with D15Z1 and DYZ3 probes, chromosomes 15 and Y centromeric signals appeared closely on a single chromosome, as the PML control probe ascertained. A further FISH assay with D15Z1 and SRY probes revealed a signal of the SRY gene at the end of one arm of chromosome 15. The result of the CMA indicated a deletion with an approximate size of 45.31 Mb spanning from Yq11 to Yter.ConclusionOur study enriched the karyotype-phenotype correlation of Y and 15 chromosomes translocation. It strengthened the critical roles of molecular genetic techniques in identifying the chromosomal breakpoints and regions involved. Genetic aetiology can guide early intervention in childhood and assisted reproduction in adulthood.

Genetic analysis of a recurrent abnormal pregnancy case caused by a cryptic reciprocal autosomal translocation

To provide genetic counseling for a couple with recurrent detection of fetal structural abnormality during second trimester pregnancy. The fetal tissue and peripheral blood samples of the couple were subjected to G banded chromosomal analysis, copy number variation sequencing (CNV-seq) and fluorescence in situ hybridization (FISH) assays. CNV-seq has detected a 6.59 Mb duplication at 7p22.3-p22.1 and a 3.81 Mb deletion at 4p16.3 in the fetal tissue, though conventional karyotyping results of both parents were normal. FISH has confirmed that the father has harbored a cryptic translocation of t(4;7)(7p+,4q+,4p+,7q+). The ultrasonographic abnormality of the fetuses may be attributed to the 7p microduplication and 4p microdeletion derived from the cryptic translocation carried by the father. Reciprocal translocation of tiny chromosomal segments should be suspected for couples with recurrent adverse pregnancies but apparently normal karyotypes.

Rare cryptic translocation seen acute myeloid leukemia – A case report

Rare cryptic translocation seen acute myeloid leukemia – A case report

Endothelial Cell Niche Promotes Leukemic Transformation of Human Cord Blood Stem/Progenitor Cells Expressing CBFA2T3-GLIS2 Oncogenic Fusion

The CBFA2T3-GLIS2 (CBF/GLIS) fusion is a product of a cryptic translocation exclusively seen in refractory infant AML. Lack of relevant model systems that accurately recapitulate this infant AML has limited progress. To overcome this barrier, we developed an endothelial cell (EC) co-culture system to support malignant transformation, self-renewal, and propagation of leukemia-initiating cells (LIC) in CBF/GLIS-transduced human cord blood hematopoietic stem/progenitor cells (CB HSPCs) ex vivo.Lack of recurrent cooperating mutations suggests that CBF/GLIS fusion might be sufficient for malignant transformation. To test this, we expressed the CBF/GLIS fusion or GFP control in CB HSPCs (CBF/GLIS-CB or GFP-CB) by lentiviral transduction and placed transduced cells in either EC co-culture or myeloid-promoting culture (MC). CBF/GLIS-CB cells expanded faster with prolonged lifespan in EC co-culture compared to MC (Figure 1A). Proliferation of CBF/GLIS-CB cells declined after transfer to either an EC trans-well culture or in suspension culture (Figure 1B), suggesting that direct contact as well as secreted factors are required for optimal growth of transduced cells.The CBF/GLIS fusion has been shown to confer enhanced megakaryocytic differentiation. At 6 weeks, CBF/GLIS-CB cells in EC co-culture formed significantly more megakaryocytic colonies than CBF/GLIS-CB cells grown in MC or CBF/GLIS-GFP cells grown in either condition (Figure 1C). At 12 weeks, CBF/GLIS-CB cells cultured in EC co-culture continued to produce numerous megakaryocytic colonies, demonstrating long lived self-renewal and enhance megakaryocytic differentiation of CBF/GLIS-CB cells co-cultured with ECs.To determine whether the EC niche promotes generation and propagation of LICs, we evaluated the murine engraftment of CBF/GLIS-CB cells expanded on ECs or in MC following 3, 6, 9 and 12 weeks of culture. CBF/GLIS-CB cells cultured in EC co-culture at each time point exhibited robust engraftment that progressed to frank leukemia in vivo (Figure 1D), demonstrating that EC co-culture promotes long-term maintenance of functional LICs. CBF/GLIS-CB cells grown in MC also induced leukemia from 3- and 6-week cultures but then became senescent at 9 and 12 weeks, suggesting limited preservation of the LICs.Flow cytometric analysis of CBF/GLIS-CB cells identified a malignant population that is of the RAM immunophenotype (CD56 hi, CD45 dim, and CD38 dim/-) previously reported in infants with CBF/GLIS AML in both culturing conditions. However, CBF/GLIS-CB cells in EC co-culture constituted an almost homogeneous population that expressed the RAM immunophenotype, whereas only a subset was detected in MC at week 6 (Figure 1E).To determine the fidelity of transformation to primary leukemia, we performed RNA-sequencing of CBF/GLIS-CB cells cultured with ECs or in MC. Unsurpervised clustering analysis demonstrated that the CBF/GLIS-CB cells from weeks 6 and 12 in EC co-culture clustered with primary CBF/GLIS-positive patient samples, but not CBF/GLIS-CB cells cultured in MC nor GFP controls (Figure 1F). Further transcriptome analysis revealed CBF/GLIS and HSC signature genes, previously identified to be associated with CBF/GLIS AML, were both significantly enriched in CBF/GLIS-CB cells grown in EC culture relative to MC (Figure 1G). These results suggested that the signaling pathways that are aberrantly dysregulated in primary CBF/GLIS leukemia are faithfully recapitulated in CBF/GLIS-CB cells co-cultured with ECs.Despite concerted efforts, previous attempts to model CBF/GLIS AML in murine hematopoietic cells have failed to generate overt leukemia. In this study, we demonstrate that in an EC co-culture system, the CBF/GLIS oncogenic fusion is sufficient to transform human CB HSPCs that faithfully recapitulates the morphology, transcriptome and immunophenotype of CBF/GLIS AML as well as highly aggressive leukemia in xenograft models. Furthermore, the EC co-culture system provides a tractable model system to further interrogate the mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy in CBF/GLIS AML. [Display omitted] DisclosuresHylkema: Quest Diagnostics Inc: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company. Pardo: Hematologics, Inc.: Current Employment. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: equity ownership. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.

QUANTITATIVE REAL-TIME PCR (RT-QPCR) COMPARING THE RELATIVE EXPRESSION LEVELS OF GENE TRANSCRIPTS INVOLVED IN A CRYPTIC THREE-WAY TRANSLOCATION T(9;11;19): AN ORIGINAL CASE OF AN INFANT WITH DISMAL PROGNOSIS ACUTE LYMPHOBLASTIC LEUKEMIA

KMT2A gene aberrations are more frequent in infants less than one year (yr) of age, accounting for about 70% of acute lymphoblastic leukemia (ALL) patients, and about 30% are diagnosed with acute myeloid leukemia (AML). The most common abnormality found in these patients is the translocation t(11;19)(q23;p13.3), corresponding to 22% of KMT2A rearranged ( KMT2A -r) positive cases. Related literature data shows that infants <1 yr with t(11;19)(q23;p13.3) present with up to 50% additional chromosomal changes. In this context, Meyer and coworkers (2007) showed that 25% of the patients with t(11;19)(q23;p13.3) and the KMT2A - MLLT1 fusion transcript presented three-way or more complex fusions, associated with a worse prognosis. In such cases, KMT2A -r must be assessed by more sensible techniques combining high-resolution cytogenetics and molecular approaches, providing a precise characterization for the correct choices in risk-adapted therapy. In this work, we show a cryptic three-way translocation t(9;11;19) harboring a KMT2A - MLLT1 and the novel SEC16A - KMT2A fusion, associated with SEC16A and KMT2A aberrant expression levels, in an infant with B-ALL presenting a poor prognosis. At diagnosis, a bone marrow sample from a 2-month-old boy was referred to the Laboratory of Cytogenetics – INCA. Immunophenotyping showed 94% of blast cells presenting lymphoid morphology and the diagnosis of pro-B lymphoblastic, being treated under a high-risk BFM INTERFANT 06 protocol. The patient could not experience any treatment response and died due to an early relapse. G-banding and FISH were performed on bone marrow and peripheral blood samples under standard protocols. LDI-PCR assays were used to identify the KMT2A partner genes and their corresponding breakpoints. RT-qPCR analyses were performed to verify the levels of transcript expression of genes involved in the rearrangements. G-banding showed the karyotype: 47,XY,+X,t(9;11)(q34;q23). The FISH analysis revealed a KMT2A -r. Molecular cytogenetic analyses revealed a cryptic three-way translocation. The LDI-PCR sequencing revealed a KMT2A - MLLT1 and the novel out-of-frame SEC16A - KMT2A . RT-qPCR showed standard expression levels for MLLT1 , whereas revealed a SEC16A reduced expression (75%), and KMT2A overexpression (3-fold). To relate the abovementioned data to the patient's clinical presentation, we hypothesized relation between the expression profiles of the fusion transcripts and the patient's dismal prognosis. Thus, we compared the relative expression among three different KMT2A regions, donor X patient. The first region, spanning exons 3 and 4, is present in both samples and was attributed the value 1. Spanning exons 11 and 12 ( SEC16A - KMT2A fusion region), despite its lower expression in the patient, no significant difference was observed. On the other hand, spanning exons 14 and 15 (downstream transcript of the KMT2A fusion) was significantly lower expressed in the patient (0.4-fold). These results confirm that increased KMT2A expression in the patient depends on the wild-type allele and that the KMT2A transcripts with fusions are probably expressed. Our case illustrates the importance of molecular tests in selecting cases for further investigations of posttranscriptional regulation mechanisms, which could open perspectives regarding novel therapeutic approaches for poor prognosis childhood leukemias.

IDENTIFICATION OF A PATIENT WITH A CRYPTIC TRANSLOCATION VIA PREIMPLANTATION GENETIC TESTING FOR ANEUPLOIDY: A CASE REPORT

One cause of recurrent pregnancy loss (RPL) is the presence of a balanced chromosome rearrangement in one of the biological parents. A standard RPL workup includes chromosomal analysis of both gamete sources via a karyotype1. However, cryptic translocations can evade detection via karyotype due to being sub-microscopic or a high degree of visual homology between the translocated regions. We describe here a family with RPL and a previously undetected cryptic translocation detected via Preimplantation Genetic Testing for Aneuploidy (PGT-A) using next generation sequencing (NGS).

P–540 A feasible diagnostic approach for the cryptic subtelomeric traslocations in early recurrent miscarriage patients by preimplantation genetic testing (PGT)

Abstract Study question Could cryptic subtelomeric traslocations in early recurrent miscarriage patients be diagnosed by preimplantation genetic testing? Summary answer PGT is a powerful tool to detect subtelomeric cryptic traslocations identifying the cause of early recurrent miscarriage and allowing subsequent genetic counselling. What is known already: Chromosome translocations are frequently associated with birth defects, spontaneous early pregnancy losses and infertility. However, submicroscopic traslocations (so-called cryptic traslocations) are too small to be detected by conventional karyotyping.. Due to balanced status, high resolution molecular techniques as arrayCGH are not able to detect it. Thus, cryptic traslocations detection is challenging. PGT is able to detect CNVs at higher resolution than routine karyotyping. Therefore, the recurrent diagnosis of CNV at embryo level could suggest a subchromosomal parental traslocation. The aim of this study is to investigate the feasibility of using PGT as an indicator of parental balanced cryptic traslocations. Study design, size, duration We included three couples who underwent PGT for unexplained repeated pregnancy loss (RPL) in our clinic from February 2020 to November 2020. Common established causes of RPL (uterine anomalies, antiphospholipid syndrome, immunological, hormonal and metabolic disorders) were previously rouled-out. Even couple karyotypes were normal. Twenty-three embryos from those couples were biopsied at blastocyst and analysed for CNVs detection using low coverage whole genome NGS. Participants/materials, setting, methods PGT by NGS was performed by Veriseq-NGS (Illumina), with previous whole genome amplification. Fluorescence in situ hybridization (FISH) using parental blood samples were performed to validate the origin of subchromosomal number variation. Commercially available subtelomeric specific probes were selected according to the CNV identified and the procedures were performed according to the manufacturer’s protocols. Main results and the role of chance Overall, CNVs of terminal duplication and deletion that imply unbalanced traslocation derivatives were detected in the 43.5% of biopsied embryos. For couple 1, 4 out of 5 embryos (80%) carried deletion of telomeric region on chromosomes 5 and 21. Three out of 6 biopsed embyos (50%) were diagnosed with subchromosomal copy variants at telomeric region on chromosomes 6 and 16 for couple 2. In the case of couple 3, three out of 12 embryos (25%) were carriers of CNV at subtelomeric region on chromosomes 2 and 6. The size of CNVs detected ranges from 8Mb to 20Mb. Accurate diagnosis with the parental study was made by FISH. The combination of probes to detect the structural chromosome alteration were: Tel5qter-LSI21q, Tel6pter-CEP16 and Tel6pter-CEP6 for each couple respectively. The FISH studies reveal that CNVs were inherited from one parent carrying the balanced cryptic traslocation. Ultimately, the abnormal karyotype from the carrier parent were 46,XY,t(5;21)(q33.2;q21.2) for couple 1, 46,XY,t(6;16)(p22.3;q22.1) for couple 2 and 46,XY,t(2;6)(p25.1;p24.2) for couple 3. Finally, each couple performed a cryotransfer of a single normal balanced embryo. Two pregnancies are ongoing. Limitations, reasons for caution The main limitation of this approach is the NGS- PGT resolution. CNVs smaller than 5Mb could not be detected. Wider implications of the findings: This study shows the value of PGT for unexplained RPL, followed by parental FISH to better characterize CNVs and identify couples in whom one partner carries a cryptic translocation. Accurate diagnosis of parental chromosome translocation can achieve with FISH only, but FISH would not be performed unless PGT showed CNVs. Trial registration number Not applicable

Fusion Transcripts without Corresponding Cytogenetic Abnormalities in Acute Myeloid Leukemia: Implications for AML Pathogenesis

Chromosomal translocations and inversions that create fusion genes are important causal events in AML. 7 recurrent fusion genes define AML entities in the 2016 revision of the WHO classification of hematologic malignancies. We have developed a comprehensive diagnostic AML platform based on next-generation mRNA sequencing (RNAseq). The adaptable integrated bioinformatic pipeline HAMLET (Human AML Expedited Transcriptomics) analyses the raw data simultaneously for fusion transcripts and for sequence variants and expression levels of genes with relevance to AML pathogenesis, prognosis, or actionability.Poly(A)+ RNA from 100 cryopreserved AML samples with a blast count of 10-99% (median: 75%) from 96 patients (3 diagnosis-relapse pairs, 1 de novo AML and subsequent tAML) was sequenced at a depth of 59x106 paired-end reads/sample with median read length of 126 bp in an ISO17025-accredited Illumina HiSeq 2500 pipeline. Fusion transcripts were called by STAR-Fusion and FusionCatcher. HAMLET detected the recurrent fusion transcripts RUNX1-RUNX1T1, CBFB-MYH11, PML-RARA, ERG-FUS, DEK-NUP214, and KMT2A-MLLT3 plus the variants KMT2A-MLLT4, KMT2A-MLLT1, and KMT2A-MLLT6 with 100% sensitivity and 100% specificity compared to metaphase cytogenetics, FISH, and RT-PCR (Griffioen, ASH 2016).In addition, HAMLET identified fusion transcripts from 54 combinations of genes in 57 AML samples that lacked corresponding chromosomal aberrations by metaphase cytogenetics. 14 “unusual” fusions involved genes located on different chromosomes; 40 fusions represented intrachromosomal events. 8 fusions were detected recurrently, i.e. in at least 2 cases (Fig. A). 32 AML carried at least 2 unusual fusions, with a maximum of 9 different fusions in an AML with a complex karyotype (Fig. B). Unusual fusions were not correlated with classical recurrent AML fusion genes. The 5 most prevalent fusions and an ETV6-LYN fusion were successfully validated in a total of 18 AML by RT-PCR. The only fusion that could not be validated was a YWHAZ-KLF10 fusion (2 cases).The most prevalent RT-PCR-validated fusion (seen in 30 AML cases) has been described in ALL (Roberts, 2012; Safavi, 2015) and was an intrachromosomal TPM4-KLF2 fusion between the tropomyosin 4 and an alternative reading frame (ARF) of the transcription factor Kruppel-like factor 2 genes. Unexpectedly, we detected this fusion in all 3 tested normal bone marrows (BM) but not in EBV-transformed B cells. The hitherto unvalidated C15orf57-CBX3 fusion may be generated by retrotransposition (Schrider, 2013) and was present in 18 AML as well as in 2/3 normal BM and 2/3 EBV B-cells with CBX3 in ARF. The intrachromosomal NCOR2-UBC fusion (9 AML) is listed in the database ChimerDB without further characterization and involves the genes of Nuclear corepressor 2 and Ubiquitin C in the normal reading frame (NRF). The NUP98-NSD1 fusion (3 AML) with NSD1 in NRF is frequently found in hematological malignancies including AML and originates from a cryptic t(5;11) due to telomeric positions of both genes. The PIM3-SCO2 fusion between the serine/threonine kinase PIM3 and an ARF of Cytochrome C oxidase assembly protein can originate from a 0.6 Mb inv(22) in chronic neutrophilic leukemia (Menezes, 2013) and occurs frequently in pediatric AML (Kuhn, 2013). The well-characterized ETV6-LYN fusion (1 AML) fusing the TEL1 oncogene to the SRC family kinase LYN in NRF originates from a complex translocation with inversion involving chromosomes 8 and 12 (Telford, 2016) and has oncogenic activity by activation of STAT5 (Takeda, 2011).Fusion transcripts without recognizable chromosomal abnormalities on metaphase cytogenetics occur frequently in AML and involve actionable targets. The upstream sequence can be fused to the normal or alternative reading frame of the downstream coding sequences and involve potential or validated proto-oncogenes. Apart from cryptic translocations or inversions, trans-splicing is suggested as an alternative mechanism by the presence of some fusion genes in non-malignant hematopoietic cells. Trans-splicing without DNA rearrangement can occur physiologically and alter cellular development towards potential premalignant differentiation (Yuan, 2013). Increasing use of RNAseq technology in routine AML diagnostics, e.g. by HAMLET, will permit to unravel the prognostic significance of unexpected fusion transcripts and to guide novel targeted therapies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

One healthy live birth after preimplantation genetic testing of a cryptic balanced translocation (9;13) in a family with cerebral palsy and glaucoma: a case report

BackgroundCryptic balanced translocations often evade detection by conventional cytogenetics. The preimplantation genetic testing (PGT) technique can be used to help carriers of balanced translocations give birth to healthy offspring; however, for carriers of cryptic balanced translocations, there is only one report about trying assisted reproduction using the PGT technique but with no pregnancy.Case presentationA couple had 3 births out of 4 pregnancies, and all died very young, with two of them having both cerebral palsy and glaucoma. The husband with oligoasthenospermia was found to be a cryptic balanced translocation carrier for t (9,13) (p24.3, q31.3) with G-banding, FISH (fluorescence in-situ hybridization), and MicroSeq techniques; live birth of a healthy baby girl was achieved with PGT/NGS (next-generation sequencing) for the couple.ConclusionHere, we report for the first time a successful live birth of a healthy baby through the PGT technique for a family in which the husband is a carrier of the cryptic balanced translocation t (9,13) (p24.3, q31.3), presumably causative for cerebral palsy and glaucoma. Our study showed that the PGT/NGS technique can effectively help families with a cryptic balanced translocation have healthy offspring.

Rapid Multi-Hybridisation FISH Screening for Balanced Porcine Reciprocal Translocations Suggests a Much Higher Abnormality Rate Than Previously Appreciated.

With demand rising, pigs are the world’s leading source of meat protein; however significant economic loss and environmental damage can be incurred if boars used for artificial insemination (AI) are hypoprolific (sub-fertile). Growing evidence suggests that semen analysis is an unreliable tool for diagnosing hypoprolificacy, with litter size and farrowing rate being more applicable. Once such data are available, however, any affected boar will have been in service for some time, with significant financial and environmental losses incurred. Reciprocal translocations (RTs) are the leading cause of porcine hypoprolificacy, reportedly present in 0.47% of AI boars. Traditional standard karyotyping, however, relies on animal specific expertise and does not detect more subtle (cryptic) translocations. Previously, we reported development of a multiple hybridisation fluorescence in situ hybridisation (FISH) strategy; here, we report on its use in 1641 AI boars. A total of 15 different RTs were identified in 69 boars, with four further animals XX/XY chimeric. Therefore, 4.5% had a chromosome abnormality (4.2% with an RT), a 0.88% incidence. Revisiting cases with both karyotype and FISH information, we reanalysed captured images, asking whether the translocation was detectable by karyotyping alone. The results suggest that chromosome translocations in boars may be significantly under-reported, thereby highlighting the need for pre-emptive screening by this method before a boar enters a breeding programme.

A New Recurrent Translocation, t(5;11)(q35;p15.5), Associated With del(5q) in Childhood Acute Myeloid Leukemia

AbstractPartial deletion of the long arm of chromosome 5, del(5q), is the cytogenetic hallmark of the 5q-syndrome, a distinct subtype of myelodysplastic syndrome-refractory anemia (MDS-RA). Deletions of 5q also occur in the full spectrum of other de novo and therapy-related MDS and acute myeloid leukemia (AML) types, most often in association with other chromosome abnormalities. However, the loss of genetic material from 5q is believed to be of primary importance in the pathogenesis of all del(5q) disorders. In the present study, we performed fluorescence in situ hybridization (FISH) studies using a chromosome 5-specific whole chromosome painting probe and a 5q subtelomeric probe to determine the incidence of cryptic translocations. We studied archival fixed chromosome suspensions from 36 patients with myeloid disorders (predominantly MDS and AML) and del(5q) as the sole abnormality. In 3 AML patients studied, this identified a translocation of 5q subtelomeric sequences from the del(5q) to the short arm of an apparently normal chromosome 11. FISH with chromosome 11-specific subtelomeric probes confirmed the presence of 11p on the shortened 5q. Further FISH mapping confirmed that the 5q and 11p translocation breakpoints were the same in all 3 cases, between the nucleophosmin (NPM1) and fms-related tyrosine kinase 4 (FLT4) genes on 5q35 and the Harveyras-1–related gene complex (HRC) and the radixin pseudogene (RDPX1) on 11p15.5. Importantly, all 3 patients with the cryptic t(5;11) were children: a total of 3 of 4 AML children studied. Two were classified as AML-M2 and the third was classified as M4. All 3 responded poorly to treatment and had short survival times, ranging from 10 to 18 months. Although del(5q) is rare in childhood AML, this study indicates that, within this subgroup, the incidence of cryptic t(5;11) may be high. It is significant that none of the 24 MDS patients studied, including 11 confirmed as having 5q-syndrome, had the translocation. Therefore, this appears to be a new nonrandom chromosomal translocation, specifically associated with childhood AML with a differentiated blast cell phenotype and the presence of a del(5q).

Biogeographic problem-solving reveals the Late Pleistocene translocation of a short-faced bear to the California Channel Islands

An accurate understanding of biodiversity of the past is critical for contextualizing biodiversity patterns and trends in the present. Emerging techniques are refining our ability to decipher otherwise cryptic human-mediated species translocations across the Quaternary, yet these techniques are often used in isolation, rather than part of an interdisciplinary hypothesis-testing toolkit, limiting their scope and application. Here we illustrate the use of such an integrative approach and report the occurrence of North America’s largest terrestrial mammalian carnivore, the short-faced bear, Arctodus simus, from Daisy Cave (CA-SMI-261), an important early human occupation site on the California Channel Islands. We identified the specimen by corroborating morphological, protein, and mitogenomic lines of evidence, and evaluated the potential natural and anthropogenic mechanisms of its transport and deposition. While representing just a single specimen, our combination of techniques opened a window into the behavior of an enigmatic species, suggesting that A. simus was a wide-ranging scavenger utilizing terrestrial and marine carcasses. This discovery highlights the utility of bridging archaeological and paleontological datasets to disentangle complex biogeographic scenarios and reveal unexpected biodiversity for island systems worldwide.

Truncated RUNX1 Generated by the Fusion of RUNX1 to Antisense GRIK2 via a Cryptic Chromosome Translocation Enhances Sensitivity to Granulocyte Colony-Stimulating Factor

Fusions of the Runt-related transcription factor 1 (RUNX1) with different partner genes have been associated with various hematological disorders. Interestingly, the C-terminally truncated form of RUNX1 and RUNX1 fusion proteins are similarly considered important contributors to leukemogenesis. Here, we describe a 59-year-old male patient who was initially diagnosed with acute myeloid leukemia, inv(16)(p13;q22)/CBFB-MYH11 (FAB classification M4Eo). He achieved complete remission and negative CBFB-MYH11 status with daunorubicin/cytarabine combination chemotherapy but relapsed 3 years later. Cytogenetic analysis of relapsed leukemia cells revealed CBFB-MYH11 negativity and complex chromosomal abnormalities without inv(16)(p13;q22). RNA-seq identified the glutamate receptor, ionotropic, kinase 2 (GRIK2) gene on 6q16 as a novel fusion partner for RUNX1 in this case. Specifically, the fusion of RUNX1 to the GRIK2 antisense strand (RUNX1-GRIK2as) generated multiple missplicing transcripts. Because extremely low levels of wild-type GRIK2 were detected in leukemia cells, RUNX1-GRIK2as was thought to drive the pathogenesis associated with the RUNX1-GRIK2 fusion. The truncated RUNX1 generated from RUNX1-GRIK2as induced the expression of the granulocyte colony-stimulating factor (G-CSF) receptor on 32D myeloid leukemia cells and enhanced proliferation in response to G-CSF. In summary, the RUNX1-GRIK2as fusion emphasizes the importance of aberrantly truncated RUNX1 in leukemogenesis.

Genetic analysis of a child with mental retardation and hypospadia

To carry out genetic testing for a boy presenting with mental retardation and hypoplasia. Conventional karyotyping, fluorescence in situ hybridization (FISH) and single nucleotide polymorphism based array (SNP-array) were used to analyze the boy and his parents. SNP-array has detected a 25.7 Mb microduplication at 2q33.3q36.3 in the boy. Chromosomal karyotyping and FISH analysis indicated that his mother had a karyotype of 46,XX,ish ins(11;2) (p15;q33q36), and that the boy has carried an abnormal chromosome 11 derived from the maternal translocation. The karyotype of the boy was ascertained as 46,XY,ish der(11)ins(11;2) (p15;q33q36)mat. SNP-array combined with G-banding and FISH can delineate the cryptic translocation and is valuable for the assessment of recurrence risk for subsequent pregnancies.

Ramp-up Treatment Strategy in Philadelphia Positive Acute Lymphoblastic Leukemia Is Associated with Deep Molecular Remissions and Favorable Outcomes

Introduction: The advent of Tyrosine Kinase Inhibitors (TKI) changed the treatment paradigm of Philadelphia chromosome Positive (Ph+) Acute Lymphoblastic Leukemia (ALL). Different treatment strategies exist including combinations of TKI with steroids or with intensive multi-agent chemotherapy regimens. We present here our institutional experience of a "ramp-up" strategy using dasatinib with steroids as induction followed by the combination of dasatinib plus the intensive chemotherapy schema of HyperCVAD alternating with HD MTX/cytarabine. Methods: We performed a retrospective review of the electronic medical records of adult patients (pts) treated in our institution for Ph+ ALL. We collected and analyzed data for pts with Ph+ ALL treated with dasatinib and steroids followed by dasatinib plus HyperCVAD alternating with HD-MTX/cytarabine. Results: We identified 25 pts that were treated with the ramp-up treatment approach between 2014-2019. The median age at diagnosis was 45 years old (range 21-65 years old). Fifty-two percent were women and 60% white. Diagnosis of Ph+ ALL was based on FISH analysis with karyotype being positive for the Ph+ chromosomal abnormality in 64% pts; 8% of pts had cryptic translocation and rest not available ( N/A). Sixty percent of pts had p190 Bcr/Abl transcript, 16% had p210 and 8% N/A (including 2 pts that Bcr/Abl transcript was undetectable despite morphological disease). The CD20 by multicolor flow cytometry (MFC) was positive in 16%, heterogenous in 20%, and low/negative in the rest (64%). Eighty-four percent of pts had a WBC less than 50K/cmm at diagnosis; median WBC was 17K/cmm (range 0.8 -131.4 K/cmm). All pts except one received induction with initial dose of 140 mg of dasatinib daily; 84% received prednisone and rest received dexamethasone followed by prednisone. For those pts on dasatinib 140 mg daily, only 2 required adjustment/brief interruption during induction. Eighty-four percent of pts achieved morphological CR, 8% CRp, and 8% had residual ALL post induction with dasatinib and steroids. Of the 23 pts achieving CR/CRp, 34% had minimal residual disease (MRD) by MFC, 26% by FISH analysis and 86% by PCR for Bcr/Abl transcript (21 evaluable pts). Median time from initiation of induction to response bone marrow assessment was 26 days (range 14 - 40 days;75% percentile 28 days). All 25 pts proceeded with HyperCVAD alternating with HD MTX/cytarabine, and the dose of dasatinib used was 70 mg daily (aside from 1 patient who received 100 mg daily). Median time from induction to initiation of HyperCVAD was 34 days (range 17-68 days). The median number of courses (HyperCVAD or HD MTX/cytarabine) for the 25 pts was 3. The 13 pts that ultimately went to transplant in CR1 had also a median of 3 courses. Sixty percent of pts ultimately underwent allo-HSCT. Median time to allo-HSCT from initiation of HyperCVAD was 106 days (range 66-294; 75% percentile 163 days). Overall, all pts achieved CR/CRp with 68% of pts (from the 22 evaluable for molecular remission) attaining also undetectable Bcr/Abl PCR (complete molecular response) as a best response. All pts with post-induction residual disease achieved CR with the dasatinib/intensive chemotherapy combination. Twenty percent of pts had morphological relapse; two pts were treated for non-morphological relapse. Median time to morphological relapse was 148 days from induction (range 106-796 days). The median follow-up was 20.4 months (range 1.5 -58.4; 75% percentile 36.8 months). At last follow up 72% of pts were alive; median OS (not censored for allo-HSCT) was not reached (figure 1). There were no deaths in the first 60 days. Conclusion: Induction therapy with dasatinib and steroids was overall well tolerated and was associated with a very favorable CR/CRp rate, with only a minority of pts requiring treatment adjustment. Subsequent consolidation with dasatinib and HyperCVAD alternating with HD-MTX/cytarabine (+/- allo-HSCT) led to deep remissions in 68% of the pts. Sixty percent of pts underwent allo-HSCT and the median OS was not reached and with no deaths in the first 60 days. Hence, induction therapy with dasatinib and steroids followed by dasatinib plus HyperCVAD alternating with HD-MTX/cytarabine can be considered for Philadelphia chromosome-positive ALL pts to achieve deep remissions with low early mortality. Disclosures Vachhani: AbbVie: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Speakers Bureau; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Costa:Fujimoto Pharmaceutical Corporation Japan: Other: Advisor; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy; Karyopharm: Consultancy. Erba:Amgen, Celgene, Daiichi Sankyo, ImmunoGen, Incyte, Jazz Pharmaceuticals, Millennium, Novartis, Ono, Pfizer, Seattle Genetics, Sunesis: Consultancy; Celgene, Incyte, Novartis: Speakers Bureau; Agios, Amgen, Astellas Pharma, Daiichi Sankyo, ImmunoGen, Janssen, Jazz Pharmaceuticals, Juno, Millennium, Seattle Genetics: Research Funding. Papadantonakis:Agios: Consultancy, Honoraria.

Identification of cryptic structural chromosomal aberrations in parents through detection of copy number variations in miscarriage tissues

To explore the genetic cause for abnormal pregnancies through detecting chromosomal copy number variations (CNVs) in abortic tissues by next generation sequencing (NGS). NGS technique was used to detect CNVs in abortion tissues. Parental chromosomal karyotypes were predicted based on the results. The aberrant chromosomal segments of the parents were accurately mapped by G-banding karyotyping analysis and fluorescence in situ hybridization (FISH). In addition to numerical chromosomal aberrations, 12 microdeletion/microduplications were detected by NGS. For 8 families where both parents accepted chromosomal karyotyping, 4 carriers of chromosomal abnormalities were identified. One marker chromosome was missed by karyotyping analysis, and a mother was confirmed to carry a cryptic balanced translocation by FISH. NGS can facilitate detection of cryptic chromosomal translocations in couples with repeated pregnancy failure and is of great value for detecting abnormal CNVs for its high sensitivity.

From cytogenetics to cytogenomics: whole-genome sequencing as a first-line test comprehensively captures the diverse spectrum of disease-causing genetic variation underlying intellectual disability

BackgroundSince different types of genetic variants, from single nucleotide variants (SNVs) to large chromosomal rearrangements, underlie intellectual disability, we evaluated the use of whole-genome sequencing (WGS) rather than chromosomal microarray analysis (CMA) as a first-line genetic diagnostic test.MethodsWe analyzed three cohorts with short-read WGS: (i) a retrospective cohort with validated copy number variants (CNVs) (cohort 1, n = 68), (ii) individuals referred for monogenic multi-gene panels (cohort 2, n = 156), and (iii) 100 prospective, consecutive cases referred to our center for CMA (cohort 3). Bioinformatic tools developed include FindSV, SVDB, Rhocall, Rhoviz, and vcf2cytosure.ResultsFirst, we validated our structural variant (SV)-calling pipeline on cohort 1, consisting of three trisomies and 79 deletions and duplications with a median size of 850 kb (min 500 bp, max 155 Mb). All variants were detected. Second, we utilized the same pipeline in cohort 2 and analyzed with monogenic WGS panels, increasing the diagnostic yield to 8%. Next, cohort 3 was analyzed by both CMA and WGS. The WGS data was processed for large (> 10 kb) SVs genome-wide and for exonic SVs and SNVs in a panel of 887 genes linked to intellectual disability as well as genes matched to patient-specific Human Phenotype Ontology (HPO) phenotypes. This yielded a total of 25 pathogenic variants (SNVs or SVs), of which 12 were detected by CMA as well. We also applied short tandem repeat (STR) expansion detection and discovered one pathologic expansion in ATXN7. Finally, a case of Prader-Willi syndrome with uniparental disomy (UPD) was validated in the WGS data.Important positional information was obtained in all cohorts. Remarkably, 7% of the analyzed cases harbored complex structural variants, as exemplified by a ring chromosome and two duplications found to be an insertional translocation and part of a cryptic unbalanced translocation, respectively.ConclusionThe overall diagnostic rate of 27% was more than doubled compared to clinical microarray (12%). Using WGS, we detected a wide range of SVs with high accuracy. Since the WGS data also allowed for analysis of SNVs, UPD, and STRs, it represents a powerful comprehensive genetic test in a clinical diagnostic laboratory setting.

Rapid detection of chromosomal translocation and precise breakpoint characterization in acute myeloid leukemia by nanopore long-read sequencing

Detection of chromosomal translocation is a key component in diagnosis and management of acute myeloid leukemia (AML). Targeted RNA next-generation sequencing (NGS) is emerging as a powerful and clinically practical tool, but it depends on expression of RNA transcript from the underlying DNA translocation. Here, we show the clinical utility of nanopore long-read sequencing in rapidly detecting DNA translocation with exact breakpoints. In a newly diagnosed patient with AML, conventional karyotyping showed translocation t(10;12)(q22;p13) but RNA NGS detected NUP98-NSD1 fusion transcripts from a known cryptic translocation t(5;11)(q35;p15). Rapid PCR-free nanopore whole-genome sequencing yielded a 26,194 bp sequencing read and revealed the t(10;12) breakpoint to be DUSP13 and GRIN2B in head-to-head configuration. This translocation was then classified as a passenger structural variant. The sequencing also yielded a 20,709 bp sequencing read and revealed the t(5;11) breakpoint of the driver NUP98-NSD1 fusion. The identified DNA breakpoints also served as markers for molecular monitoring, in addition to fusion transcript expression by digital PCR and sequence mutations by NGS. We illustrate that third-generation nanopore sequencing is a simple and low-cost workflow for DNA translocation detection.

The clinical benefit of array-based comparative genomic hybridization for detection of copy number variants in Czech children with intellectual disability and developmental delay

BackgroundChromosomal microarray analysis has been shown to be a valuable and cost effective assay for elucidating copy number variants (CNVs) in children with intellectual disability and developmental delay (ID/DD).MethodsIn our study, we performed array-based comparative genomic hybridization (array-CGH) analysis using oligonucleotide-based platforms in 542 Czech patients with ID/DD, autism spectrum disorders and multiple congenital abnormalities. Prior to the array-CGH analysis, all the patients were first examined karyotypically using G-banding. The presence of CNVs and their putative derivation was confirmed using fluorescence in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA) and predominantly relative quantitative polymerase chain reaction (qPCR).ResultsIn total, 5.9% (32/542) patients were positive for karyotypic abnormalities. Pathogenic/likely pathogenic CNVs were identified in 17.7% of them (96/542), variants of uncertain significance (VOUS) were detected in 4.8% (26/542) and likely benign CNVs in 9.2% of cases (50/542). We identified 6.6% (36/542) patients with known recurrent microdeletion (24 cases) and microduplication (12 cases) syndromes, as well as 4.8% (26/542) patients with non-recurrent rare microdeletions (21 cases) and microduplications (5 cases). In the group of patients with submicroscopic pathogenic/likely pathogenic CNVs (13.3%; 68/510) we identified 91.2% (62/68) patients with one CNV, 5.9% (4/68) patients with two likely independent CNVs and 2.9% (2/68) patients with two CNVs resulting from cryptic unbalanced translocations. Of all detected CNVs, 21% (31/147) had a de novo origin, 51% (75/147) were inherited and 28% (41/147) of unknown origin.In our cohort pathogenic/likely pathogenic microdeletions were more frequent than microduplications (69%; 51/74 vs. 31%; 23/74) ranging in size from 0.395 Mb to 10.676 Mb (microdeletions) and 0.544 Mb to 8.156 Mb (microduplications), but their sizes were not significantly different (P = 0.83). The pathogenic/likely pathogenic CNVs (median 2.663 Mb) were significantly larger than benign CNVs (median 0.394 Mb) (P < 0.00001) and likewise the pathogenic/likely pathogenic CNVs (median 2.663 Mb) were significantly larger in size than VOUS (median 0.469 Mb) (P < 0.00001).ConclusionsOur results confirm the benefit of array-CGH in the current clinical genetic diagnostics leading to identification of the genetic cause of ID/DD in affected children.

PS922 BENEFITS OF RNA SEQUENCING IN DETECTING RECURRENT AND NOVEL FUSION TRANSCRIPTS IN PATIENTS WITH ACUTE LYMPHOBLASTIC LEUKEMIA

Background:Chimeric transcripts are frequent genetic abnormalities in hematological malignancies often contributing to leukemogenesis. So far, analysis of chromosomal translocations giving rise to gene fusions is based on cytogenetic techniques such as chromosome banding analysis (CBA) and fluorescence in situ hybridization (FISH). However, these techniques only detect known gene fusions, while cryptic or novel translocations remain elusive. The impact of gene fusions is increasing as they are not only entity defining and important for risk stratification, but also potentially useful biomarkers for MRD monitoring or targeted therapy. Recently, RNA sequencing (RNAseq) has become a powerful tool to identify disease associated transcriptome profiles and determine recurrent and novel genomic gene fusions underlying the disease.Aims:Comprehensive analysis of the fusion gene landscape in ALL (acute lymphoblastic leukemia) patients and evaluation of the benefits of RNAseq in detecting recurrent and novel fusion transcripts.Methods:We screened the whole transcriptome of 288 patients with ALL (B‐ and T‐lineage) for the detection of recurrent and novel fusion transcripts and applied a combination of whole genome sequencing (WGS) and cytogenetics (CBA, FISH, and genomic arrays) for validation. Gene expression profiling was performed to determine expression signatures of emerging ALL subtypes.Results:The analysis included 288 patients that were screened for chimeric transcripts by RNAseq. After stringent filtering and validation by WGS data and/or cytogenetic analysis a total number of 122 fusion transcripts, corresponding to 61 unique gene fusions in 102 ALL patients, was identified with high confidence. These included 93/122 (76%) gene fusions comprising breakpoints located on different chromosomes while the remaining 29/122 (24%) fusion genes were located on the same chromosome. Among detected fusion events, 90/122 (74%) gene fusions were confirmed by WGS and cytogenetic data, 25/122 (20%) cases were confirmed by WGS only and 7/122 (6%) cases by cytogenetics only. Besides recurrent entity‐defining fusion gene families such as BCR‐ABL1 (n = 30), KMT2A/MLL‐r (n = 22), ETV6‐RUNX1 (n = 4), and TCF3‐PBX1 (n = 4), RNAseq detected so far unreported gene fusions or novel fusion partners of known genes in 38 cases including novel partners for RUNX1, ABL1, and MLLT10. Among patients lacking canonical chromosomal translocations, rearrangements involving 3’ ZNF384 were identified in 8/92 patients (9%) with either EP300 (n = 7) or EWSR1 (n = 1) as 5’ fusion partner, constituting the largest subtype in this subgroup. All ZNF384‐rearranged cases were validated by RT‐PCR. Notably, in none of the cases involving EP300 as fusion partner, standard cytogenetic techniques were able to detect the respective rearrangement as all cases displayed a normal karyotype. ZNF384‐rearrangements were recently defined as novel ALL subtype, based on gene expression signature and clinical features. As they are furthermore potentially targetable by small molecule inhibitors, accurate identification of patients harboring these and other cryptic aberrations by RNAseq is pivotal.Summary/Conclusion:Our data determine the capability of RNAseq to detect known as well as cytogenetically cryptic and novel fusion transcripts in ALL leading to a better characterization of patients with unresolved rearrangements and highlight its immediate importance as a valuable tool in routine diagnostics.