SNP Microarray Analysis Research Articles

Prenatal detection of mosaicism for a genome wide uniparental disomy cell line in a cohort of patients: Implications and outcomes.

To investigate the prenatal detection rate of mosaicism by SNP microarray analysis, in which an individual has not one, but two, complete genomes (sets of DNA) in their body, a normal biparental line with a Genome Wide Uniparental Disomy (GWUPD) cell line was used. This study retrospectively examines the prenatal detection of GWUPD in a cohort of ∼90,000 prenatal specimens and ∼20,000 products of conceptions (POCs) that were studied by SNP microarray. In total, 25 cases of GWUPD were detected; 16 cases were detected prenatally with GWUPD (∼0.018%) and 9 POCs revealed GWUPD (0.045%). The nine POC specimens presented with placental abnormalities. The 12 amniotic fluid specimens were ascertained because of abnormal ultrasound findings. Nine of 12 pregnancies had findings consistent with Beckwith-Wiedemann syndrome or because of abnormal placentas. However, three pregnancies were detected with GWUPD of maternal origin, with less common findings and demonstrated maternal origin. Four other pregnancies showed GWUPD in a chorionic villus sample, but normal findings in amniotic fluid and apparently normal fetal development. This cohort with GWUPD mosaicism expands our understanding of GWUPD and has implications for prenatal care and counseling. Additional studies are necessary to understand the rarer maternal GWUPD.

The yield of SNP microarray analysis for fetal ultrasound cardiac abnormalities.

Chromosomal microarray analysis (CMA) has emerged as a critical instrument in prenatal diagnostic procedures, notably in assessing congenital heart diseases (CHD). Nonetheless, current research focuses solely on CHD, overlooking the necessity for thorough comparative investigations encompassing fetuses with varied structural abnormalities or those without apparent structural anomalies. This study sought to assess the relation of single nucleotide polymorphism-based chromosomal microarray analysis (SNP-based CMA) in identifying the underlying causes of fetal cardiac ultrasound abnormalities. A total of 2092 pregnant women who underwent prenatal diagnosis from 2017 to 2022 were included in the study and divided into four groups based on the presence of ultrasound structural abnormalities and the specific type of abnormality. The results of the SNP-Array test conducted on amniotic fluid samples from these groups were analyzed. Findings from the study revealed that the non-isolated CHD group exhibited the highest incidence of aneuploidy, overall chromosomal abnormalities, and trisomy 18, demonstrating statistically significant differences from the other groups (p < 0.001). Regarding the distribution frequency of copy number variation (CNV) segment size, no statistically significant distinctions were observed between the isolated CHD group and the non-isolated CHD group (p > 0.05). The occurrence rates of 22q11.2 and 15q11.2 were also not statistically different between the isolated CHD group and the non-isolated congenital heart defect group (p > 0.05). SNP-based CMA enhances the capacity to detect abnormal CNVs in CHD fetuses, offering valuable insights for diagnosing chromosomal etiology and facilitating genetic counseling. This research contributes to the broader understanding of the utility of SNP-based CMA in the context of fetal cardiac ultrasound abnormalities.

An Integrated Approach Including CRISPR/Cas9-Mediated Nanopore Sequencing, Mate Pair Sequencing, and Cytogenomic Methods to Characterize Complex Structural Rearrangements in Acute Myeloid Leukemia.

Complex structural chromosome abnormalities such as chromoanagenesis have been reported in acute myeloid leukemia (AML). They are usually not well characterized by conventional genetic methods, and the characterization of chromoanagenesis structural abnormalities from short-read sequencing still presents challenges. Here, we characterized complex structural abnormalities involving chromosomes 2, 3, and 7 in an AML patient using an integrated approach including CRISPR/Cas9-mediated nanopore sequencing, mate pair sequencing (MPseq), and SNP microarray analysis along with cytogenetic methods. SNP microarray analysis revealed chromoanagenesis involving chromosomes 3 and 7, and a pseudotricentric chromosome 7 was revealed by cytogenetic methods. MPseq revealed 138 structural variants (SVs) as putative junctions of complex rearrangements involving chromosomes 2, 3, and 7, which led to 16 novel gene fusions and 33 truncated genes. Thirty CRISPR RNA (crRNA) sequences were designed to map 29 SVs, of which 27 (93.1%) were on-target based on CRISPR/Cas9 crRNA nanopore sequencing. In addition to simple SVs, complex SVs involving over two breakpoints were also revealed. Twenty-one SVs (77.8% of the on-target SVs) were also revealed by MPseq with shared SV breakpoints. Approximately three-quarters of breakpoints were located within genes, especially intronic regions, and one-quarter of breakpoints were intergenic. Alu and LINE repeat elements were frequent among breakpoints. Amplification of the chromosome 7 centromere was also detected by nanopore sequencing. Given the high amplification of the chromosome 7 centromere, extra chromosome 7 centromere sequences (tricentric), and more gains than losses of genomic material, chromoanasynthesis and chromothripsis may be responsible for forming this highly complex structural abnormality. We showed this combination approach's value in characterizing complex structural abnormalities for clinical and research applications. Characterization of these complex structural chromosome abnormalities not only will help understand the molecular mechanisms responsible for the process of chromoanagenesis, but also may identify specific molecular targets and their impact on therapy and overall survival.

The association of gene polymorphisms with milk production and mastitis resistance phenotypic traits in dairy cattle

Abstract The aim of this study was to evaluate the association between gene polymorphisms (SNPs) and mastitis indicators and their relationship with milk production profitability in dairy herd. A functional analysis was also performed of five genes containing the studied SNPs and those located close by. DNA was isolated from the hair bulb of 320 dairy cows kept in three herds and SNP-microarray analysis was performed. The data on 299 cows was subjected to final statistical analysis using AI-REML method with one-trait repeatability test-day animal model and pedigree information using the DMU4 package. Five from 35 SNPs significantly associated with mastitis indicators or production traits and located within a gene or no more than 500,000 nucleotides from the gene were selected for the functional and economic analysis. A questionnaire was also developed to collect associated economic data of 219 cows from three herds, such as the value of milk production and direct costs incurred over three years; this allowed the gross margin, direct profitability index and direct costs incurred to produce one liter of milk to be determined, among others. None of the five studied SNPs were related to protein content. The rs110785912(T/A), found near CXCR4, and rs136813430(T/C), located in the TLR4 gene exon, were associated with lnSCC, while rs110455063(C/G), located near IGF-I, was associated with milk yield, fat and total solid contents. rs109421300(T/C), associated with fat/protein content ratio, as well as fat and total solid content, is located in the DGAT1 gene intron. rs41587003(A/C), located in the DLG2 gene intron, was associated with lactose content. The economic analysis revealed differences between the variants of the three tested SNPs. The T/C variant of the rs136813430(T/C) SNP was characterized by the highest gross margin, the highest direct profitability index and the lowest costs incurred to produce 1 liter of milk. The T/A variant of rs110785912(T/A) was related to low lnSC and was characterized by the highest direct profitability index. In turn, the C/C variant of the rs41587003(A/C) was related to the lowest level of lactose and the highest costs of milk production. It appears that rs136813430(T/C) may be the most promising of the tested SNPs for increasing the profitability of milk production. To our knowledge, it is the first effort to assess directly a correlation between the DNA polymorphism and economic output of a dairy enterprise.

Abstract 15501: Rare Genomic Copy Number Variants Implicate New Candidate Genes for Bicuspid Aortic Valve

Introduction: Bicuspid Aortic Valve (BAV), the most common adult congenital heart defect, is a major cause of aortic insufficiency or stenosis requiring valve replacement and thoracic aortic aneurysms predisposing to acute aortic dissections. The spectrum of BAV ranges from early onset valve and aortic complications to sporadic late onset disease. We determined the frequency and gene content of rare genomic copy number variants (CNVs) in isolated BAV cases. Methods: We performed genome-wide SNP microarray analysis of familial BAV cases with early onset complications in the UTHealth BAV Research Registry (EBAV, n=394) and elderly probands from the International BAV Consortium (BAVCON, n=4216). CNVs were detected in Illumina genotypes using the PennCNV, QuantiSNP and cnvPartition algorithms. BAV cases were compared to 16,576 controls without known cardiovascular disease from the Database of Genotypes and Phenotypes. Only CNV calls with &gt;6 consecutive variants, &gt;20 kb in length, identified by at least two algorithms and intersected with known genes were included. For comparison, we assessed a cohort with left ventricular outflow tract obstructive lesions including BAV (LVOTO, n=1561) using identical methods. CNV burden and associations were tested using PLINK. Results: We identified 84 large recurrent CNVs in BAV cases that are absent or rare (&lt;0.1%) in controls. 34 rare CNVs overlap between BAV and LVOTO cases and involve candidate genes that interact with each other during heart development. The largest and most prevalent of the recurrent CNVs are 8p23 duplications involving GATA4 (OR 340, CI: 42-2700). CNVs of genes that cause BAV when mutated were significantly enriched in cases (n=135, P =1x10 -27 ). The overall burden of rare genic CNVs, specifically large deletions, was also increased in BAV cases (empiric P &lt; 1x10 -5 ) and was higher in younger EBAV cases than in older BAVCON cases. Conclusion: We identified likely pathogenic CNVs in more than 10% of BAV cases, implicating alterations of candidate genes at these loci in the pathogenesis of BAV.

Establishment and identification of six wheat-Thinopyrum ponticum disomic addition lines derived from partial amphiploid Xiaoyan 7430.

Six wheat-Thinopyrum ponticum disomic addition lines derived from partial amphiploid Xiaoyan 7430 were identified using in situ hybridization and SNP microarray, the homoeologous group and stripe rust resistance of each alien chromosome were determined, and Th. ponticum chromosome-specific markers were developed. Xiaoyan 7430 is a significant partial amphiploid, which is used to set up a bridge for transferring valuable genes from Thinopyrum ponticum (Podp.) Barkworth & D.R. Dewey into common wheat. To accelerate the application of these useful genes in enriching the genetic variability of cultivated wheat by chromosome engineering, a complete set of derived addition lines has been created from Xiaoyan 7430. The chromosome composition of each line was characterized by the combination of genomic in situ hybridization and multicolor fluorescence in situ hybridization (mc-FISH), and the homoeology of each alien chromosome was determined by wheat SNP microarray analysis. Addition line WTA55 with alien group-6 chromosome was evaluated resistant to stripe rust isolates at both the seedling and grain-filling stages (Zadoks scale at z.11 and z.73). Diagnostic marker analysis proved that it could carry a novel stripe rust resistance gene derived from Th. ponticum. Furthermore, a FISH probe and 45 molecular markers specific for alien chromosomes were developed based on specific-locus amplified fragment sequencing (SLAF-seq). Of which 27 markers were separately located on single alien chromosome, and some of them could be used to identify the derived translocation lines. This set of addition lines as well as the molecular markers and the FISH probe will promote the introgression of abundant variation from Th. ponticum into wheat in wheat improvement programs.

14. Four-way translocation in a patient with mild clitoromegaly

Balanced reciprocal translocations are frequent chromosomal rearrangements between two nonhomologous chromosomes with an approximate incidence of 1/500 and can be inherited or de novo. Individuals carrying these rearrangements are usually phenotypically normal but with increased risk for infertility, miscarriages, stillbirths, and abnormal liveborn offspring. More complex rearrangements such as double reciprocal, triple reciprocal, and three-way balanced translocations are rare events. Complex constitutional rearrangements involving more than three chromosomes are extremely rare and a recent review of the literature described less than 15 patients. We report here a one-day-old full-term female with prominent labial folds and enlarged clitoris that was referred for cytogenetic analysis. Chromosome studies demonstrated the appearance of either two reciprocal translocations or a single four-way translocation. Metaphase FISH and SNP microarray analyses were performed on the patient to determine the complexity of the rearrangement. FISH analysis confirmed the presence of a four-way translocation without deletions or duplications at the breakpoints as determined by SNP array. Further, the SNP array detected a 4.5 Mb deletion of 1p31.1 and a triplication of 1q43 characterized as a variant of unknown significance (VUS). Parental peripheral blood has been requested to determine the origin of the rearrangement, the 1p31.1 deletion, and the 1q34 VUS and these results will be presented accordingly.

10. SNP microarray analysis of over 7,500 myeloid patients: Implications, importance and suggestions for standard of care

We report on one of the largest and definitive studies of Myeloid disorders using microarray with analysis of over 7,500 patients with suspected myeloid disorders. Overall, microarray analysis revealed that 71% of the AML patients had a detectable abnormality; 65% of the diagnosed MDS/MPN patients, and ∼49% of the patients with a suggested MDS/MPN disorder. When chromosomes were normal, 57% of AML patients had an abnormality detected by the array. When chromosomes were abnormal the array provided better information in 81% of patients.

A Turner syndrome case associated with dic(Y;22)

BackgroundConstitutional telomeric associations are very rare events and the mechanism underlying their development is not well understood.Case presentationWe here describe a female case of Turner syndrome with a 45,X,add(22)(p11.2)[25]/45,X[5]. We reconfirmed this karyotype by FISH analysis as 45,X,dic(Y;22)(p11.3;p11.2)[28]/45,X[2].ish dic(Y;22)(SRY+,DYZ1+). A possible mechanism underlying this mosaicism was a loss of dic(Y;22) followed by a monosomy rescue of chromosome 22. However, SNP microarray analysis revealed no loss of heterozygosity (LOH) in chromosome 22, although a mosaic pattern of LOH was clearly detectable at the pseudoautosomal regions of the sex chromosomes.ConclusionsOur results suggest that the separation of the dicentric chromosome at the junction resulted in a loss of chromosome Y without a loss of chromosome 22, leading to this patient’s unique mosaicism. Although telomere signals were not detected by FISH at the junction, it is likely that the original dic(Y;22) chromosome was generated by unstable telomeric associations. We propose a novel “pulled apart” mechanism as the process underlying this mosaicism.

19. De novo homozygous variant due to uniparental isodisomy results in a recessive disorder

A 10-month old male presented with developmental delay, hypotonia, microcephaly, and sparse, coarse hair. He was born at 38 weeks’ gestation to a 33-year-old G1P0 mother. Brain MRI revealed delayed myelination but otherwise normal appearance of the brain. In addition, he has had significant delay in reaching developmental milestones but has been making improvements with physical and speech therapy. In order to look for an underlying genetic cause for his clinical features, we performed whole-genome SNP microarray analysis.

A case report of familial 4q13.3 microdeletion in three individuals with syndromic intellectual disability

BackgroundInterstitial 4q deletions are rare chromosomal alterations. Most of the previously reported deletions involving the 4q13.3 region are large chromosomal alterations with a common loss of band 4q21 resulting in marked growth restriction, severe intellectual disability, and absent or severely delayed speech. A microdeletion of 4q13.3 hasn’t been previously reported. We discuss the involvement of genes and the observed phenotype, comparing it with that of previously reported patients.Case presentationWe report on a 4q13.3 microdeletion detected in three affected individuals of a Lithuanian family. The clinical features of two affected children and their affected mother are very similar and include short stature, congenital heart defect, skeletal anomalies, minor facial anomalies, delayed puberty, and intellectual disability. Whole genome SNP microarray analysis of one child revealed an interstitial 4q13.3 microdeletion, 1.56 Mb in size. FISH analysis confirmed the deletion in the proband and identified the same deletion in her affected sib and mother, while it was not detected in a healthy sib. Deletion includes ADAMTS3, ANKRD17, COX18, GC, and NPFFR2 protein-coding genes.ConclusionsOur findings suggest that 4q13.3 microdeletion is a cause of a recognizable phenotype of three affected individuals. The detected microdeletion is the smallest interstitial deletion in 4q13. We highlight ADAMTS3, ANKRD17 and RNU4ATAC9P as candidate genes for intellectual disability, growth retardation and congenital heart defect.

Enrichment of atypical hyperdiploidy and IKZF1 deletions detected by SNP-microarray in high-risk Australian AIEOP-BFM B-cell acute lymphoblastic leukaemia cohort

Acute lymphoblastic leukaemia (ALL) is the most common childhood malignancy with the majority of patients being classified as B-cell lineage (B-ALL). The sub-classification of B-ALL is based on genomic architecture. Recent studies have demonstrated the capability of SNP-microarrays to detect genomic changes in B-ALL which cannot be observed by conventional cytogenetic methods. In current clinical trials, B-ALL patients at high risk of relapse are mainly identified by adverse cancer genomics and/or poor response to early therapy. To test the hypothesis that inclusion of SNP-microarrays in frontline diagnostics could more efficiently and accurately identify adverse genomic factors than conventional techniques, we evaluated the Australian high-risk B-ALL cohort enrolled on AIEOP-BFM ALL 2009 study (n = 33). SNP-microarray analysis identified additional aberrations in 97% of patients (32/33) compared to conventional techniques. This changed the genomic risk category of 24% (8/33) of patients. Additionally, 27% (9/33) of patients exhibited a ‘hyperdiploid’ genome, which is generally associated with a good genomic risk and favourable outcomes. An enrichment of IKZF1 deletions was observed with one third of the cohort affected. Our findings suggest the current classification system could be improved and highlights the need to use more sensitive techniques such as SNP-microarray for cytogenomic risk stratification in B-ALL.

Spinal dysraphism, club feet, and dextrocardia with situs inversus totalis in a neonate: a rare association and review

AbstractObjectivesThe spinal dysraphism and situs inversus are a rare association. Since 1909, reports on the coincidence of malformations of the spine and gastrointestinal tract have been published. So far there is no plausible explanation for the association.Case presentationWe report a term female infant with spinal dysraphism with club feet associated with dextrocardia and situs inversus totalis. Whole genome SNP microarray analysis was normal. However, there are extended contiguous regions of allele homozygosity [&gt;8 Mb[megabase]) observed in chromosome 6 and 14.ConclusionsWe report a rare association of spinal dysraphism and situs inversus totalis in a neonate. We review the literature. There have recently been theorized by some to in fact represent nothing more than the presence of two or more polytopic field defects, with all the anomalies present sharing a common molecular basis.

Prenatal diagnosis of Kleefstra syndrome

Abstract Objectives To describe the prenatal diagnosis of Kleefstra syndrome (KS), a rare panethnic disorder characterized by mental and developmental delays, distinct facial features, congenital heart and urogenital defects, among others. KS is caused by haploinsusufficiency and loss of function of euchromatin histone methyltransferase 1 (EHMT1) due to deletions or mutations in the chromosomal region 9q34.3. Case presentation The prenatal diagnosis of KS in this case report includes the description of subtle fetal phenotypic abnormalities detected by the fetal ultrasound examination as well as the results of the amniotic fluid microarray analysis that confirmed a fetal denovo deletion in the chromosomal region 9q34.3. These results confirmed the prenatal diagnosis of KS. Conclusions This case is noteworthy because of the late development of very subtle ultrasound abnormalities that triggered prenatal diagnostic studies in amniotic fluid cells, including SNP microarray analysis, that defined the diagnosis of KS. It allowed us to obtain the necessary antepartum consultations with neonatology, other pediatric subspecialties and arrange for the patient’s appropriate place of delivery to optimize the fetal and neonatal outcome.

Title: SNP Microarray Reveals Predicted Outcomes of a Novel High Risk AML Subgroup with ERG Amplification

Increased expression of ERG (ETS-related gene), a proto-oncogene within the ETS (erythroblast transformation specific) family, is associated with an unfavorable clinical outcome in AML patients. A mechanism for ERG over-expression is ERG amplification. ERG amplification is present in 4-6% of adult AML. This study investigates the genetic profile of ERG-amplified AML by assessment of genomic copy number changes and mutational status of AML-related genes and how this correlates to treatment response and overall outcome. Currently copy number analysis is not recommended by ELN or WHO as a diagnostic standard in AML. An institutional retrospective review of 205 adult AML cases from 2015 to 2018 provided two cohorts: a discovery cohort and control cohort. The discovery cohort includes 11 cases (4%) with ERG amplification. All cases in the discovery cohort include either TP53 mutation and/or loss/LOH of 17p. Thus, we developed a control cohort of 39 cases with TP53 abnormalities without ERG amplifications to determine the role TP53 plays in the observed outcomes. The 179 adult AML cases from the TCGA public database comprises the validation cohort, which incorporates both copy number and molecular sequencing data. Approximately 4% of AML cases in the TCGA dataset show ERG amplification. For our discovery and control cohorts, genomic DNA was extracted (Maxwell, Promega, Madison, WI) from fresh bone marrow aspirates. Copy number/LOH analysis was performed using the CytoSNP microarray (Illumina, San Diego, CA). Sequence variants were evaluated in hotspot regions of 49 genes using the ThunderBolts Myeloid Panel library preparation (RainDance Technologies, Billerica, MA) and paired-end sequencing (MiSeq, Illumina, San Diego, CA). Alignment and variant calling was performed with MiSeq Reporter (Illumina) and NextGene (Soft Genetics, State College, PA) and sequences were annotated using VarSeq (Golden Helix, Bozman, MT). SNP microarray analysis of the discovery cohort revealed a distinct pattern of complex cytogenetics including chromothripsis, TP53 loss/LOH and loss of 5q. Molecular sequencing was available for 10 of 11 patients. All specimens analyzed for mutational status demonstrated TP53 loss of function mutations. The majority of cases (9/11) demonstrated complete loss of function of TP53 by mutation in conjunction with a "second hit" in the form of loss due to deletion or LOH of 17p. These nine patients failed to achieve any response with chemotherapy with a median survival time of 3.8 months. The two surviving patients had a single TP53 mutation with an intact second allele. Both of these patients are post-allogeneic stem cell transplant with no evidence of relapse. There is an association of ERG amplification with MDS as 8 of the 11 patients transformed from very-high risk MDS. The interaction between ERG and TP53 allows one to predict treatment response and outcomes based on the presence or absence of an intact TP53 allele. Within the control group, there were no significant differences in survival or response to treatment based on number of intact TP53 alleles. The validation cohort of TCGA patients confirmed the strong positive correlation between ERG amplification and TP53 mutations. There were 8 cases with ERG amplification. Of these, 6 cases had a similar genetic pattern found in the discovery cohort including TP53 mutations, loss of 5q and complex cytogenetics. The median overall survival (OS) of this cohort of ERG amplified + TP53 mutation was 2.5 months. ERG amplification classifies a high-risk population of AML detected by copy number/LOH analysis with a distinct genetic signature including loss of 5q, complex cytogenetics including chromothripsis and loss of function TP53 mutation. The successive loss of the second TP53 allele in the setting of ERG amplification identifies a subset of patients with resistance to cytarabine and a very poor prognosis. These patients are unlikely to benefit from cytotoxic chemotherapy and may have improved outcomes with alternative regimens and/or clinical trial. ERG amplification is associated with AML that transforms from very-high risk MDS. Recognizing these patients before transforming to AML could allow for early transplant. This high-risk subgroup is currently un-recognized by standard cytogenetics and genetic mutation profiling; therefore, we recommend that copy number/LOH analysis become part of the standard diagnostic testing for MDS and AML. Disclosures No relevant conflicts of interest to declare.

The Prolonged Inhibition of Chk1/Chk2 Kinases Enhances Genetic Instability and Compromises the Efficacy of Chemotherapy Against Acute Lymphoblastic Leukemia Cells

Acute lymphoblastic leukemia (ALL) cells respond to chemotherapy, or more generally to DNA damages, by activating different DNA Damage Response (DDR) pathways. DDR-pathways regulate cell cycle progression and DNA damages repair. Molecular and functional alterations in key DDR-related genes drastically affect the effectiveness of DNA-damaging treatments in cancer cells. For this reasons selective DDR-inhibitors have been developed in order to sensitize cancer cells against conventional chemotherapy. Despite the proven efficacy of DDR-inhibitors in cancer treatment, only few studies have highlighted the biological consequences the prolonged inhibition of DDR-pathways in cancer cells. We hypothesized that the protracted inhibition of the DDR pathways may generate resistant clones characterized by an increased genetic instability. The aim of the study was to evaluate biological consequences of the prolonged inhibition of two crucial DDR-related kinase such as cell cycle checkpoint kinase 1 (Chk1) and 2 (Chk2) in B cells ALL. In particular, we investigated the consequences of Chk1/Chk2 inhibition in term of increase of genetic instability and in term of responsiveness to chemotherapy agents. Starting from B-ALL cell line NALM-6, we generated a resistant model (hereafter referred as N6R-PF8) by treating the parental cells with increasing concentration of PF-00477736 (Chk1/Chk2 inhibitor) for more than a year and increasing the IC50 value of 10-folds. From a molecular point of view, the N6R-PF8 accumulated significant molecular alterations. SNP microarray analysis highlighted different alterations in DDR-related genes and, in particular, in the ATM/CHK2 pathway. Three regions in copy number LOSS (CN=1) containing several genes involved in cell cycle checkpoint regulation (ATM and NPAT) and in the apoptosis (BIRC2, CASP1 and CASP5) were detectable only in NALM-6 parental cell lines and were copy number neutral in the resistant model. Immunoblotting analysis confirmed that in N6R-PF8 cells the ATM/CHK2 and ATR/CHK1 down-stream pathways were significant over-expressed and activated in comparison to the parental cell. Whole exome sequencing analysis showed that the two cell lines were characterized by different mutational profiles and that N6R-PF8 cells harboured significantly more genetic alterations in comparison with NALM-6 cells. Interestingly, crucial genes involved in DNA repair pathway (MLH3, NBN, POLD1 and PMS2) have been found altered only in N6R-PF8 cells. From a functional point of view, the molecular alterations characterizing the N6R-PF8 significantly compromised the cytotoxicity of PF-00477736 and of different DNA damaging agents in comparison to parental cells. Furthermore, the treatment with ATR/ATM inhibitor restored the sensitivity of N6R-PF8 to PF-00477736 and to different chemotherapy agents. In this scenario the level of expression of these two kinases seems to correlate with the sensitivity to DNA damaging agents and to PF-00477736. Finally, we confirmed that the protracted inhibition of crucial DDR-related kinase may increase the overall genetic instability in ALL cells and compromise the efficacy of DNA damaging based therapies. Disclosures Martinelli: Roche: Consultancy; ARIAD: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; BMS: Consultancy.

Stable transmission of complex chromosomal rearrangements involving chromosome 1q derived from constitutional chromoanagenesis

BackgroundChromoanagenesis events encompassing chromoanasynthesis, chromoplexy, and chromothripsis are described in cancers and can result in highly complex chromosomal rearrangements derived from ‘all-at-once’ catastrophic cellular events. The complexity of these rearrangements and the original descriptions in cancer cells initially led to the assumption that it was an acquired anomaly. While rare, these phenomena involving chromosome 1 have been reported a few individuals in a constitutional setting.Case presentationHere, we describe a newborn baby who was initially referred for cytogenetic testing for multiple congenital anomalies including cystic encephalomalacia, patent ductus arteriosus, inguinal hernia, and bilateral undescended testicles. Chromosome analysis was performed and revealed a derivative chromosome 1 with an 1q24-q31 segment inserted into 1q42.13 resulting in gain of 1q24-q31. Whole genome SNP microarray analysis showed a complex pattern of copy number variants with four gains and one loss involving 1q24-q31. Mate pair next-generation sequencing analysis revealed 18 chromosome breakpoints, six gains along an 1q24-q31 segment, one deletion of 1q31.3 segment and one deletion of 1q42.13 segment, which is strongly evocative of a chromoanasynthesis event for developing this complex rearrangement. Parental chromosome analyses were performed and showed the same derivative chromosome 1 in the mother.ConclusionsTo our knowledge, our case is the first case with familial constitutional chromoanagenesis involving chromosome 1q24-q42. This report emphasizes the value of performing microarray and mate pair next-generation sequencing analysis for individuals with germline abnormal or complex chromosome rearrangements.

Haploseek: A new 24-hour all-in-one method for combined PGT of monogenic diseases (PGT-M) chromosomal rearrangements (PGT-SR) and aneuploidy (PGT-A)

Introduction Recent technological advances have enabled combined gene mutation detection and comprehensive chromosome screening (CCS) from a single embryo biopsy by means of microarray or high coverage next generation sequencing (NGS) technologies. However, these methods are still challenging, very expensive, time consuming, and therefore not widely applied. Therefore, the aim of this study was to develop an economical, user-friendly, all-in-one NGS-based workflow for gene mutation detection combined with CCS screening while maintaining the highest standard for accuracy in a 24 hour workflow protocol. Materials & Methods Three families, of 3-4 children each, were recruited at the Shaare Zedek Medical Center, Jerusalem, Israel. For each family, whole genome haplotype phasing of the parents was accomplished by SNP microarray genotyping analysis of the couple and one child. From the other children in each family, lymphoblast cultures were prepared and single cells were isolated so that each child would be represented by one cell only. Subsequently, each child's single cell isolate was subjected to low genomic coverage (1x) single read (1 × 50) whole genome sequencing on a NextSeq 500 instrument followed by combined chromosome copy number assessment (PGT-A/PGT-SR) and whole genome haplotype phasing for PGT-M. Sequencing reads were aligned to the reference human genome followed by crude variant calling at SNP positions matching those on the trio-phased SNP microarrays. Custom R scripts were devised to integrate trio-phased microarray data and low coverage sequencing data so as to predict paternal and maternal haplotypes throughout the genomes of each sequenced sample. In addition, a custom user-friendly software, termed, ‘haploseek’, was created to visualize data output on a web browser in two plots depicting haplotype prediction and marginal haplotype probability, respectively. For copy number variant (CNV) prediction, a custom CNV detection R script was devised and plotted by web browser. All single cell haplotype and CNV predictions were validated by SNP microarray analysis of bulk genomic peripheral blood DNA from the corresponding tissue culture donor. The entire single cell sequencing assay was completed within 24 hours, beginning from single cell isolation until test result conclusion. Results Whole genome copy number assessment and haplotype phasing of the single cell sequencing data was completely (100%) concordant with known, microarray determined, whole genome chromosome copy numbers and haplotypes in each child from all 3 families. Interestingly, a relatively long segmental deletion of chr21q was identified in the single cell sequencing data of one of the children together with haplotype information suggesting that the deleted allele was paternal in origin. This result and all other copy number assessments in the low coverage data were confirmed by array validation as well. Conclusions Our results demonstrate the establishment of a reliable method for all-in-one molecular and chromosomal diagnosis of single cells. Importantly, the whole ‘Haploseek’ process was predicated upon rapid sample processing, rapid sequencing, stream-lined analysis, and user-friendly result reporting so as to expedite clinical implementation. Thus, our novel NGS –based method may become the ultimate, economically feasible, 24 hour end-to-end solution for combined PGT-M, PGT-SR and PGT-A application.

Pediatric acute myeloid leukemia with t(7;21)(p22;q22)

The t(7;21)(p22;q22) resulting in RUNX1-USP42 fusion, is a rare but recurrent cytogenetic abnormality associated with acute myeloid leukemia (AML) and myelodysplastic syndromes. The prognostic significance of this translocation has not been well established due to the limited number of patients. Herein, we report three pediatric AML patients with t(7;21)(p22;q22). All three patients presented with pancytopenia or leukopenia at diagnosis, accompanied by abnormal immunophenotypic expression of CD7 and CD56 on leukemic blasts. One patient had t(7;21)(p22;q22) as the sole abnormality, whereas the other two patients had additional numerical and structural aberrations including loss of 5q material. Fluorescence in situ hybridization analysis on interphase cells or sequential examination of metaphases showed the RUNX1 rearrangement and confirmed translocation 7;21. Genomic SNP microarray analysis, performed on DNA extracted from the bone marrow from the patient with isolated t(7;21)(p22;q22), showed a 32.2 Mb copy neutral loss of heterozygosity (cnLOH) within the short arm of chromosome 11. After 2-4 cycles of chemotherapy, all three patients underwent allogeneic hematopoietic stem cell transplantation (HSCT). One patient died due to complications related to viral reactivation and graft-versus-host disease. The other two patients achieved complete remission after HSCT. Our data displayed the accompanying cytogenetic abnormalities including del(5q) and cnLOH of 11p, the frequent pathological features shared with other reported cases, and clinical outcome in pediatric AML patients with t(7;21)(p22;q22). The heterogeneity in AML harboring similar cytogenetic alterations may be attributed to additional uncovered genetic lesions.

Unbalanced X;9 translocation in an infertile male with de novo duplication Xp22.31p22.33.

Male carriers of an X-autosome translocation are generally infertile, regardless of the position of the breakpoint on the X chromosome while the pathogenicity of Xp22.3 subtelomeric duplications is under debate. To shed light into this controversy, we present a rare case, of an azoospermic male with no other significant clinical findings, in whom classical cytogenetics revealed additional unbalanced chromosomal material, at the telomere of the long arm of one homolog of chromosome 9. In peripheral blood specimens of the index case and his parents, we performed GBanding, Inverted-DAPI Banding, AgNOR staining, Telomere specific Fluorescence in Situ Hybridization (FISH), Molecular karyotyping by Multi-color FISH, whole genome SNP microarrays, sub-telomeric MLPA, and transcription analysis of the expression of KAL1 gene by RT-PCR. Multi-color FISH revealed an unbalanced translocation involving the short arm of chromosome X. SNP microarray analysis combined to classical cytogenetics and MLPA demonstrated a de novo 8.796Mb duplication of Xp22.31-p22.33. Compared to three control specimens, the patient presented significantly elevated expression levels of KAL1 mRNA in peripheral blood, suggesting transcriptional functionality of the duplicated segment. The duplicated segment contains the pseudo-autosomal region PAR1 and more than 30 genes including SHOX, ARSE, STS, KAL1, and FAM9A and is not listed as polymorphic. Our data advocate that duplications of the Xp22.3 region may not be associated with a clinical consequence.