Conventional Chromosome Analysis Research Articles

Karyotype evolutionary diversification in marine fishes. First classical and molecular cytogenetic data on four Atlantic species

ABSTRACT Fishes present an exceptional level of diversification among vertebrates, whose adaptive instructions contained in their genome are physically organised in their chromosomes. Although cytogenetic data are useful to help understand the evolutionary processes, they are restricted or unavailable for many fish families. Cytogenetic characteristics of one Atlantic member of the Kyphosidae, Cirrhitidae, Pempheridae and Bythitidae families – about which little or no prior data is known – are described here. Conventional chromosomal analysis and the mapping of the 18S and 5S ribosomal DNA sequences revealed substantial variations in the diploid number (2n = 38–48) and structure of the chromosomes (FN = 48–84; i.e. number of chromosome arms), as well as in the rDNA distribution (simple, multiple and polymorphic sites). The findings extend the cytogenetic data for reef fish groups across a wider phylogenetic range and reveal the various ways in which karyotype evolution has taken place in the marine environment.

Novel Four-Way Variant Translocation, t(1;9;22;16)(q21;q34;q11.2;q24), in a Patient with Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is characterized by the Philadelphia (Ph) chromosome resulting from the translocation of t(9;22)(q34;q11), producing the BCR::ABL1 fusion gene. Variant Ph chromosome translocations, involving rearrangements in chromosomes other than 9 and 22, occur in 5–10% of CML cases. Herein, we report a unique case of a 36-year-old male with a four-way variant Ph chromosome. Conventional chromosomal analysis performed on bone marrow aspirate samples showed 46, XY, t(1;9;22;16)(q21;q34;q11.2;q24). Nested RT-PCR of the BCR::ABL1 gene revealed a major BCR::ABL rearrangement. The treatment with nilotinib achieved a complete hematologic, cytogenetic, and molecular response after 12 months.

Olfactory receptor genes and chromosome 11 structural aberrations: Players or spectators?

The largest multi-gene family in metazoans is the family of olfactory receptor (OR) genes. Human ORs are organized in clusters over most chromosomes and seem to include >0.1% of the human genome. Because 369 out of 856 OR genes are mapped on chromosome 11 (HSA11), we sought whether they mediate structural rearrangements involving this chromosome. To this aim, we analyzed 220 specimens collected during diagnostic procedures involving structural rearrangements of chromosome 11. A total of 222 chromosomal abnormalities were included, consisting of inversions, deletions, translocations, duplications, and one insertion, detected by conventional chromosome analysis and/or FISH and array-CGH. We verified by bioinformatics and statistical approaches the occurrence of breakpoints in cytobands with or without OR genes. We found that OR genes are not involved in chromosome 11 reciprocal translocations, suggesting that different DNA motifs and mechanisms based on homology or non-homology recombination can cause chromosome 11 structural alterations. We also considered the proximity between the chromosomal territories of chromosome 11 and its partner chromosomes involved in the translocations by using the deposited Hi-C data concerning the possible occurrence of chromosome interactions. Interestingly, most of the breakpoints are located in regions highly involved in chromosome interactions. Further studies should be carried out to confirm the potential role of chromosome territories’ proximity in promoting translocations, to help understand mechanisms underlying genome structural variation possibly associated to genetic diseases, but also fundamental to promote genome evolution.

Ring Chromosomes in Hematological Malignancies Are Associated with TP53 Gene Mutations and Characteristic Copy Number Variants.

Ring chromosomes (RC) are present in <10% of patients with hematological malignancies and are associated with poor prognosis. Until now, only small cohorts of patients with hematological neoplasms and concomitant RCs have been cytogenetically characterized. Here, we performed a conventional chromosome analysis on metaphase spreads from >13,000 patients diagnosed with hematological malignancies at the Johns Hopkins University Hospital and identified 98 patients with RCs-90 with myeloid malignancies and 8 with lymphoid malignancies. We also performed a targeted Next-Generation Sequencing (NGS) assay, using a panel of 642 cancer genes, to identify whether these patients harbor relevant pathogenic variants. Cytogenetic analyses revealed that RCs and marker chromosomes of unknown origin are concurrently present in most patients by karyotyping, and 93% of patients with NGS data have complex karyotypes. A total of 72% of these individuals have pathogenic mutations in TP53, most of whom also possess cytogenetic abnormalities resulting in the loss of 17p, including the loss of TP53. All patients with a detected RC and without complex karyotypes also lack TP53 mutations but have pathogenic mutations in TET2. Further, 70% of RCs that map to a known chromosome are detected in individuals without TP53 mutations. Our data suggest that RCs in hematological malignancies may arise through different mechanisms, but ultimately promote widespread chromosomal instability.

Clinical features and genetic analysis of two fetuses with ring chromosome 21 mosaicism

To investigate the perinatal clinical phenotype and genetic characteristics of two fetuses with ring chromosome 21 mosaicisms. Two fetuses who were diagnosed at the Xiamen Maternal and Child Health Care Hospital in November 2021 were selected as the study subjects. Clinical data of the two fetuses were collected. Conventional G-banded karyotyping and chromosomal microarray analysis (CMA) were carried out for the fetuses and their parents. Prenatal ultrasonography of fetus 1 has revealed absence of nasal bone, ventricular septal defect, persistent left superior vena cava, and mild tricuspid regurgitation. Chromosomal karyotyping was 46,X?,dic r(21;21)(p12q22;q22p12)[41]/45,X?,-21[9]. CMA has revealed a 30.00 Mb quadruplication at 21q11.2q22.3 and a 3.00 Mb deletion at 21q22.3. For fetus 2, ultrasonography has revealed pointed echo of the nasal bone. The fetus was found to have a karyotype of 46,X?,r(21)(p12q22)[83]/45,X?,-21[14]/46,X?,dic r(21;21)(p12q22;q22p12)[3]. CMA has revealed a 5.10 Mb quadruplication at 21q22.12q22.3 and a 2.30 Mb deletion at 21q22.3. The perinatal phenotype of the two fetuses with ring chromosome 21 mosaicisms is related to the duplication of chromosomal segments near the breakpoints of the chromosomal deletions. The combined chromosomal karyotyping and CMA has enabled prenatal diagnosis and genetic counseling for these families.

Prenatal diagnosis of a case with complete and uniform tetrasomy 12p by the utility of noninvasive prenatal testing.

To report a rare type of Pallister-Killian syndrome (PKS) diagnosed prenatally by the utility of non-invasive prenatal testing (NIPT). NIPT was performed in the first trimester. Conventional karyotyping and chromosomal microarray analysis (CMA) were performed on the amniotic samples in the second trimester. Copy number variation sequencing (CNV-seq) was used for the validation of fetal skin and the placental tissue after pregnancy termination. NIPT results showed increased signal from chromosome 12p. Subsequent prenatal diagnostic testing by karyotype revealed 47, XY, +i (12p), and CMA displayed four copies of 12p: 12p13.33-12p11.1(173786_34835641) × 4. The CNV-seq results of the fetal skin and the fetal side of placenta showed four copies of 12p13.33-p11 and an estimated chimeric duplication of 34.08 Mb (chimerism ratio: 10%) in 12 p13.33-p11, respectively. However, no abnormality was detected by CNV-seq at the maternal side of placenta. Our findings suggest that a positive signal from chromosome 12p on NIPT should raise suspicion for PKS. With the wide application of NIPT, the true positive of incidental finding is expected to increase.

Genetic Characterization of Rat Hepatic Stellate Cell Line PAV-1.

The rat hepatic stellate cell line PAV-1 was established two decades ago and proposed as a cellular model to study aspects of hepatic retinoic acid metabolism. This cell line exhibits a myofibroblast-like phenotype but also has the ability to store retinyl esters and synthesize retinoic acid from its precursor retinol. Importantly, when cultured with palmitic acid alone or in combination with retinol, the cells switch to a deactivated phenotype in which the proliferation and expression of profibrogenic marker genes are suppressed. Despite these interesting characteristics, the cell line has somehow fallen into oblivion. However, based on the fact that working with in vivo models is becoming increasingly complicated, genetically characterized established cell lines that mimic aspects of hepatic stellate cell biology are of fundamental value for biomedical research. To genetically characterize PAV-1 cells, we performed karyotype analysis using conventional chromosome analysis and multicolor spectral karyotyping (SKY), which allowed us to identify numerical and specific chromosomal alteration in PAV-1 cells. In addition, we used a panel of 31 species-specific allelic variant sites to define a unique short tandem repeat (STR) profile for this cell line and performed bulk mRNA-sequencing, showing that PAV-1 cells express an abundance of genes specific for the proposed myofibroblastic phenotype. Finally, we used Rhodamine-Phalloidin staining and electron microscopy analysis, which showed that PAV-1 cells contain a robust intracellular network of filamentous actin and process typical ultrastructural features of hepatic stellate cells.

Genetic analysis of a fetus with de novo 46,X,der(X)t(X;Y)(q26;q11)

To carry out prenatal genetic testing for a fetus with de novo 46,X,der(X)t(X;Y)(q26;q11). A pregnant woman who had visited the Birth Health Clinic of Lianyungang Maternal and Child Health Care Hospital on May 22, 2021 was selected as the study subject. Clinical data of the woman was collected. Peripheral blood samples of the woman and her husband and umbilical cord blood of the fetus were collected and subjected to conventional G-banded chromosomal karyotyping analysis. Fetal DNA was also extracted from amniotic fluid sample and subjected to chromosomal microarray analysis (CMA). For the pregnant women, ultrasonography at 25th gestational week had revealed permanent left superior vena cava and mild mitral and tricuspid regurgitation. G-banded karyotyping analysis showed that the pter-q11 segment of the fetal Y chromosome was connected to the Xq26 of the X chromosome, suggesting a Xq-Yq reciprocal translocation. No obvious chromosomal abnormality was found in the pregnant woman and her husband. The CMA results showed that there was approximately 21 Mb loss of heterozygosity at the end of the long arm of the fetal X chromosome [arr [hg19] Xq26.3q28(133912218_154941869)×1], and 42 Mb duplication at the end of the long arm of the Y chromosome [arr [hg19] Yq11.221qter(17405918_59032809)×1]. Combined with the search results of DGV, OMIM, DECIPHER, ClinGen and PubMed databases, and based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the deletion of arr[hg19] Xq26.3q28(133912218_154941869)×1 region was rated as pathogenic, and the duplication of arr[hg19] Yq11.221qter(17405918_59032809)×1 region was rated as variant of uncertain significance. The Xq-Yq reciprocal translocation probably underlay the ultrasonographic anomalies in this fetus, and may lead to premature ovarian insufficiency and developmental delay after birth. Combined G-banded karyotyping analysis and CMA can determine the type and origin of fetal chromosomal structural abnormalities as well as distinguish balanced and unbalanced translocations, which has important reference value for the ongoing pregnancy.

Clinical outcomes and characteristics of patients with TP53-mutated myelodysplastic syndromes

ABSTRACTObjectiveTo explore the clinical outcomes and characteristics of TP53-mutated primary myelodysplastic syndromes (MDS).MethodsA total of 74 de novo primary MDS patients who were diagnosed and treated in the Department of Hematology of our hospital from January 2018 and September 2021 were analyzed retrospectively. All patients had evaluable blood cell counts, mean corpuscular volume (MCV), lactate dehydrogenase (LDH), bone marrow (BM) morphology, biopsy, and MDS-related 20-gene mutations sequencing. In addition, 69 of 74 patients had complete cytogenetic analysis through conventional chromosome analysis and fluorescence in-situ hybridization.ResultsPatients were divided into two cohorts, the TP53-mutated type (TP53Mut) group (n = 19) and TP53 wild type (TP53WT) group (n = 55). Compared with the TP53WT group, patients in the TP53Mut group had higher ratios of cytogenetic abnormalities (82.4% vs. 30.8%, P < 0.001), with 5q- karyotype (64.70% vs. 38.5%, P < 0.001), complex karyotype(CK) (64.70% vs. 38.5%, P < 0.001), HR-MDS (94.7% vs. 61.8%, P = 0.008), and acute myelogenous leukemia (AML) transformation (26.3% vs. 12.7%, P < 0.001). Interestingly, patients in the TP53Mut group had lower median MCV than the TP53WT group (94.40 fl vs. 101.90 fl, P = 0.008). Furthermore, MCV = 100 fl as cutoff, and found that MCV ≤ 100 fl was more common in the TP53Mut group (73.7% vs. 38.2%, P < 0.001). After 1–4 courses of HMA ± chemotherapy, the overall response rate of the TP53Mut group was higher than the TP53WT group (83.3% vs. 71.4%, P = 0.012). With the median follow-up 12.0 months (1–46 months), the results show that the median OS and leukemia-free survival (LFS) of TP53Mut group was significantly shorter than the TP53WT group (P = 0.0018; P = 0.0310). Results of multivariate Cox proportional hazard analyses show TP53 mutation was an independent prognostic factor for the OS (HR 2.724, 95%CI 1.099–6.750, P = 0.030).ConclusionTP53-mutated primary MDS patients were associated with higher frequency of cytogenetic abnormalities, with 5q- karyotype, CK, AML transformation, higher risk IPSS-R, lower MCV and sensitive to HMA treatment, but worse survival.

Complementary Approaches in Fetal Genetic Diagnosis: Decision-Making Process and Alternative Choices for Clinicians in a Secondary Health Care Institution

Objectives The aim of this study was to determine indications of invasive, genetic results of conventional karyotyping and chromosomal microarray analysis and culture failure rates to discuss possible solution options and guide our clinical choices. Materials and methods Fetal samples were analyzed by conventional karyotyping, array comparative genomic hybridization, fluorescence in situ hybridization. Results Failure rates of chorionic villus sampling (CVS) and amniocentesis were as follows, respectively: 4.5% and 0.4%. The rates of abnormal genetic results in fetuses with only thickened nuchal translucency and thickened nuchal translucency + USG abnormality were %4.2 and %40, respectively. Conclusions Abnormal genetic results showed a significant increase in cases of thickened nuchal translucency accompanied by USG abnormalities. Although culture failure rates in the CVS were higher, none of the cases remained inconclusive. Centers with prenatal invasive genetic diagnosis should offer a wide spectrum of genetic tests by medical genetics specialists.

Identification of EWSR1 rearrangements in patients with immature hematopoietic neoplasms: A case series and review of literature

Rearrangement of the EWSR1 gene (22q12.2) is a well-recognized genetic lesion in bone and soft tissue tumors. However, few reports have suggested that EWSR1 rearrangements may also occur in the setting of hematopoietic tumors. We herein describe two cases of immature hematopoietic neoplasms presenting with EWSR1 rearrangements. The first occurred in a 41-year-old female diagnosed with mixed-phenotype acute leukemia, B/T/myeloid, in which conventional chromosome analysis revealed a t(2;22)(q35;q12). Further analysis with whole genome sequencing revealed that this rearrangement led to an EWSR1::FEV gene fusion. The second case was identified in an 18-year-old male with a high-grade B-cell lineage malignant neoplasm with immature features in which conventional chromosome analysis revealed a t(17;22)(q25;q12). Mate-pair sequencing, a next generation sequencing-based assay, was performed and revealed three in-frame chimeric gene fusions involving the EWSR1, TEF and STRADA gene regions. This report further expands the repertoire of hematopoietic neoplasms with EWSR1 fusions and partner genes involved in these rearrangements.

56. Optical genome mapping reveals genomic complexity and detects novel genetic abnormalities in T-Lymphoblastic Leukemia

Background: Few recurrent chromosomal abnormalities have been recognized in T-lymphoblastic leukemia (T-ALL) by conventional chromosomal analysis (CCA), largely due to the poor growth of T-ALL cells under in vitro culture and/or abnormalities are cryptic by CCA. Optical Genome Mapping (OGM) enable us to map the whole genome and identify numerical and structural chromosomal abnormalities with high resolution.

Chromosomal abnormalities and neurological outcomes in fetal cerebral ventriculomegaly: a retrospective cohort analysis.

This study investigated the incidences of chromosomal abnormalities and the neurological outcomes according to the degree of fetal cerebral ventriculomegaly. All women with antenatal ultrasound diagnosis of fetal cerebral ventriculomegaly were retrospectively identified from two maternal-fetal medicine units in Hong Kong from January 2014 to December 2018. Degrees of fetal ventriculomegaly were classified as mild (10-11.9 mm), moderate (12-14.9 mm), or severe (≥15 mm). Genetic investigation results were reviewed, including conventional karyotyping and chromosomal microarray analysis (CMA); correlations between chromosomal abnormalities and the degree of fetal ventriculomegaly were explored. The neurological outcomes of subsequent live births were analysed to identify factors associated with developmental delay. Of 84 cases (ie, pregnant women and their fetuses) included, 46 (54.8%) exhibited isolated fetal ventriculomegaly, 55 (65.5%) had mild cerebral ventriculomegaly, and 29 (34.5%) had moderate or severe cerebral ventriculomegaly. Overall, 20% (14/70) of cases had chromosomal abnormalities. Moreover, 12% (3/25) of mild isolated ventriculomegaly cases had abnormal karyotype or CMA results. The CMA provided an incremental diagnostic yield of 8.6% (6/70), compared with conventional karyotyping; 4.3% exhibited pathogenic variants and 4.3% exhibited variants of uncertain significance. Among the 53 live births in the cohort, fewer cases of mild isolated ventriculomegaly were associated with developmental delay than more severe isolated ventriculomegaly (9.7% vs 41.7%, P<0.03). Chromosomal microarray analysis testing should be offered to all women with fetal cerebral ventriculomegaly, including women with isolated mild ventriculomegaly. The incidence of developmental delay after birth increases with the degree of prenatal cerebral ventriculomegaly.

Somatic mosaicism detected by genome-wide sequencing in 500 parent-child trios with suspected genetic disease: clinical and genetic counseling implications.

Identifying genetic mosaicism is important in establishing a diagnosis, assessing recurrence risk, and providing accurate genetic counseling. Next-generation sequencing has allowed for the identification of mosaicism at levels below those detectable by conventional Sanger sequencing or chromosomal microarray analysis. The CAUSES Clinic was a pediatric translational trio-based genome-wide (exome or genome) sequencing study of 500 families (531 children) with suspected genetic disease at BC Children's and Women's Hospitals. Here we present 12 cases of apparent mosaicism identified in the CAUSES cohort: nine cases of parental mosaicism for a disease-causing variant found in a child and three cases of mosaicism in the proband for a de novo variant. In six of these cases, there was no evidence of mosaicism on Sanger sequencing—the variant was not detected on Sanger sequencing in three cases, and it appeared to be heterozygous in three others. These cases are examples of six clinical manifestations of mosaicism: a proband with classical clinical features of mosaicism (e.g., segmental abnormalities of skin pigmentation or asymmetrical growth of bilateral body parts), a proband with unusually mild manifestations of a disease, a mosaic proband who is clinically indistinguishable from the constitutive phenotype, a mosaic parent with no clinical features of the disease, a mosaic parent with mild manifestations of the disease, and a family in which both parents are unaffected and two siblings have the same disease-causing constitutional mutation. Our data demonstrate the importance of considering the possibility of mosaicism whenever exome or genome sequencing is performed and that its detection via genome-wide sequencing can permit more accurate genetic counseling.

Acquisition Patterns and Interaction of Molecular, Obvious and Cryptic Cytogenetic Abnormalities in Myelodysplastic Syndromes

Abstract Introduction: The acquisition of cytogenetic aberrations (CA) and/or of molecular mutations (clonal evolution, CE) is known to be associated with poor outcome in pts with myelodysplastic syndromes (MDS) (Jabbour et al, Am J Hematol, 2013; Cevik et al, abstract, Onkologie, 2013). The aim of our study was to identify additional cases with CE by taking cryptic CA, which cannot be detected by conventional chromosomal banding analysis (CCB), and their interaction with molecular mutations, into account. Methods: We retrospectively reviewed 103 pts with proven MDS (10x RCUD, 40x RCMD, 10x RAEB-1, 16x RAEB-2, 11x sAML after MDS, 3x MDS-U, 7x CMML, 6x MDS with unknown subtype). Karyotype from CCB and molecular karyotype from SNP-array analysis (SNP-A) were available for all pts. SNP-A and fluorescence in situ hybridization (FISH) analyses were used to detect cryptic CA. For 57 pts results from mutational analysis from a 17-genes Sanger and/or a 54-genes next generation sequencing panel were included. Genetic follow-up and survival data were available for 57 pts, 22 pts were genetically analyzed under treatment with disease modifying therapies. Genetic follow-up comprised analysis of bone marrow and/or frequent sequential genetic monitoring of CD34+ peripheral blood cells. Results: CA were detected at diagnosis in 65/103 pts (63%). By CCB (aberrant in 55/103 pts; 53%) the median number of CA was 1 (range: 0-4). One to 6 (median 1) cryptic CA were detected in 31/103 (30%) pts, 10/31 pts (32%) showed a normal karyotype in CCB. The cryptic CA included cryptic copy number variations (CNV) in 12 pts and CN neutral losses of heterozygosity (CN-LOH) in 19 pts as the sole cryptic CA (14x) or in addition to cryptic CNVs (5x). In 12 pts with CN-LOHs we could identify a homozygous molecular mutation or a cryptic deletion in the region of a CN-LOH. Genes mutated in the region of a CN-LOH were DNMT3A (2p23.3, 2 pts), EZH2 (7q36.1, 2 pts), CBL (11q23.3, 2 pts), RUNX1 (21q22.12, 2 pts), TET2 (4q24), IKZF1 (7p12.2), TP53 (17p13.1), and SRSF2 (17q25.1). The formation of this phenomenon was observed in consecutive samples of 3 pts. From the 57 pts with follow-up data available, 8 showed manifestations of antecedent CE at first diagnosis (2x sub-clones; 6x homozygous mutations in CN-LOHs), from which 7 showed further genetic progression during the course of the disease. CE after first diagnosis was also observed in 20 of the 49 other pts with available follow-up data. The new aberrations of the biggest clone at first CE after diagnosis were partial or complete LOH of chromosome (chr) 7 (6 pts), trisomy 8 (5 pts), chr 21 abnormalities (2 pts), chr 12 abnormalities (2 pts), CN-LOH in 4q, 11p-, 20q-, -X, an additional marker chromosome, tetraploid cells, and mutations in ASXL1 (2 pts), CDKN2A, ETV6, IDH1, and JAK2 . Further steps of CE in these pts are currently being evaluated. Median survival time from detection of the first CE after diagnosis (25 months, 12/20 deaths) did not differ significantly from median survival time from first diagnosis for pts with signs of antecedent CE at first diagnosis (25 months, 5/8 deaths, P=0.92, n.s.). Median survival time of pts without detection of CE was significantly longer (173 months, 8/29 deaths, P=0.005 and Conclusions: In our cohort of MDS pts the frequency of cryptic CA was 30%. A homozygous mutation in a region of CN-LOH, which indicates antecedent CE, was detected in 12% of pts. The presence of such evolutionary patterns at first diagnosis might be indicative of a genetically instable disease as these pts had a shorter survival time and a shorter time to further genetic progression. There is a clear tendency that cytogenetic LOHs preferentially affect regions with molecularly mutated genes, resulting in homozygous mutations. The development of these patterns seems to be an important step towards progression. Our data imply that the relevance of cryptic CA for CE and disease progression in MDS has been underestimated as yet. Cryptic CA should be taken into account for developing prediction models for clinical progress and for improving individualized patients care. Download : Download high-res image (124KB) Download : Download full-size image Disclosures Al-Ali: Gilead: Consultancy, Honoraria; Alexion: Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Metz: Celgene: Other: study projects, advanced training support; Novartis: Consultancy, Other: study projects, advanced training support. Germing: Janssen: Honoraria; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Platzbecker: Janssen: Consultancy, Honoraria, Research Funding; Acceleron: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Haase: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

The genetic abnormalities investigation of leukemia in past few decades

Cytogenetic and molecular analysis of each patient’s leukemia cells has become an essential component of diagnosis prior to treatment. It has furthered our understanding of leukemogenesis at a molecular level. Specific and well-characterized recurring chromosomal abnormalities facilitate diagnosis, confirm subtype classification, and have major prognostic value for treatment planning. Conventional chromosome analysis is a basic way for diagnosis and treatment. In addition in this way evaluation of disease progression is important and so it is the only method that can identify the presence of clonal evolution, particularly in accelerated and relapse phase in the disease. Also conventional cytogenetic can detect chromosomal abnormality associated with its advanced phase. Anyhow, the value of translocation rates in interphase and metaphase nuclei in monitoring leukemia is at the time of diagnosis and after treatment additionally. Genomic profiling transformed our understanding of the genetic basis of leukemia particularly in acute leukemia, which is a malignant clonal proliferation in lymphoid stem cells or myeloid progenitor cells.

Optimizing the Diagnostic Strategy to Identify Genetic Abnormalities in Miscarriage.

IntroductionThe single most common cause of miscarriage is genetic abnormality.ObjectiveWe conducted a prospective cohort study to compare the performance of conventional karyotyping and chromosomal microarray analysis (CMA) using array comparative genomic hybridization (array-CGH) and single nucleotide polymorphism array (SNP-array) to identify genetic abnormalities in miscarriage specimens.MethodsA total of 63 miscarriage specimens were included. Conventional karyotyping, array-CGH, and SNP-array were performed and the results compared.ResultsGenetic abnormalities were detected in 31 cases (49.2%) by at least one testing modality. Single autosomal trisomy was the most common defect (71.0%), followed by polyploidy (16.1%), multiple aneuploidy (9.7%), and monosomy X (3.2%). Mosaicisms were identified in four cases and confirmed by fluorescence in situ hybridization (FISH) using appropriate probes. SNP-array had a higher detection rate of genetic abnormalities than array-CGH (93.5 vs. 77.4%), and conventional karyotyping had the lowest detection rate (76.0%). SNP-array enabled the detection of all types of genetic abnormalities, including polyploidy.ConclusionsAlthough conventional karyotyping and FISH are still needed, SNP-array represents the first choice for miscarriage because the technique showed excellent performance in the detection of genetic abnormalities and minimized the probability of testing failure as well as time, costs, and labor.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40291-021-00523-9.

Application of Copy Number Variation Sequencing in Genetic Analysis of Miscarriages in Early and Middle Pregnancy

High-throughput sequencing based on copy number variation (CNV-seq) is commonly used to detect chromosomal abnormalities. This study identifies chromosomal abnormalities in aborted embryos/fetuses in early and middle pregnancy and explores the application value of CNV-seq in determining the causes of pregnancy termination. High-throughput sequencing was used to detect chromosome copy number variations (CNVs) in 116 aborted embryos in early and middle pregnancy. The detection data were compared with the Database of Genomic Variants (DGV), the Database of Chromosomal Imbalance and Phenotype in Humans using Ensemble Resources (DECIPHER), and the Online Mendelian Inheritance in Man (OMIM) database to determine the CNV type and the clinical significance. High-throughput sequencing results were successfully obtained in 109 out of 116 specimens, with a detection success rate of 93.97%. In brief, there were 64 cases with abnormal chromosome numbers and 23 cases with CNVs, in which 10 were pathogenic mutations and 13 were variants of uncertain significance. An abnormal chromosome number is the most important reason for embryo termination in early and middle pregnancy, followed by pathogenic chromosome CNVs. CNV-seq can quickly and accurately detect chromosome abnormalities and identify microdeletion and microduplication CNVs that cannot be detected by conventional chromosome analysis, which is convenient and efficient for genetic etiology diagnosis in miscarriage.

38. A rare constitutional 13q deletion associated with retinoblastoma and chronic lymphocytic leukemia

The 13q deletion syndrome is a rare congenital disorder characterized by a wide phenotypic spectrum depending on the size and location of the deletion, with clinical features spanning intellectual disability, craniofacial dysmorphism, congenital malformations, and retinoblastoma (Mitter et al., 2011). In the somatic setting, deletion of 13q14 is the most common cytogenetic abnormality in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), and is associated with a favorable outcome when present as the sole cytogenetic abnormality (Van Dyke et al., 2010). To date, published reports and review of the literature have not associated constitutional 13q deletions to an increased risk of CLL/SLL. Here, we describe the molecular and cytogenetic analysis of a 47-year-old male referred to our laboratory for CLL genetic analysis. By fluorescence in situ hybridization (FISH), peripheral blood nuclei exhibited a homozygous 13q14 deletion in ∼63% of nuclei, and a heterozygous deletion in the remaining ∼37% of nuclei. FISH also identified heterozygous loss of the RB1 gene region in ∼97% of nuclei, consistent with the patient's history of retinoblastoma. Conventional chromosome analysis identified a large del(13)(q14.1q21.2), and chromosome microarray (CMA) mapped the deletion to 13:43,773,101-69,897,491 (26.1Mb, hg19) in a perfect heterozygous state. In essential agreement with the FISH results, CMA also identified a homozygous 796Kb deletion including MIR16-1, MIR15A, and DLEU1/2 in ∼50% of cells; this homozygous deletion likely represents the CLL cell population. To our knowledge this is the first case of a constitutional 13q deletion associated with adult CLL. Given that monoallelic deletions of RB1 constitute ∼6% of RB1’s mutational spectrum (Albretch et al., 2005; Castéra et al., 2013), a better understanding of this patient may help explore the risk for adult-onset CLL in those individuals with an infantile history of retinoblastoma who carry an RB1 deletion rather than a point mutation.

Clinical Utility of Targeted Next-Generation Sequencing Assay to Detect Copy Number Variants Associated with Myelodysplastic Syndrome in Myeloid Malignancies

Copy number variants (CNVs) and gene mutations are important for diagnosis and treatment of myeloid malignancies. In a routine clinical setting, somatic gene mutations are detected by targeted next-generation sequencing (NGS) assay, but CNVs are commonly detected by conventional chromosome analysis and fluorescence in situ hybridization (FISH). The aim of this proof-of-principle study was to investigate the feasibility of using targeted NGS to simultaneously detect both somatic mutations and CNVs. Herein, we sequenced 406 consecutive patients with myeloid malignancies by targeted NGS and performed a head-to-head comparison with the results from a myelodysplastic syndrome (MDS) FISH and conventional chromosome analysis to detect CNVs. Among 91 patients with abnormal MDS FISH results, the targeted NGS revealed all 120 CNVs detected by MDS FISH (including -5/5q-, -7/7q-, +8, and 20q-) and 193 extra CNVs detected by conventional chromosome analysis. The targeted NGS achieved 100% concordance with the MDS FISH. The lower limit of detection of MDS CNVs by the targeted NGS was generally 5% variant allele fraction for DNA, based on the lowest percentages of abnormal cells detected by MDS FISH in this study. This proof-of-principle study demonstrated that the targeted NGS assay can simultaneously detect both MDS CNVs and somatic mutations, which can provide a more comprehensive genetic profiling for patients with myeloid malignancies using a single assay in a clinical setting.