Conventional Karyotype Analysis Research Articles

Expert consensus on the clinical application of efficient intelligent chromosomal karyotyping precise auxiliary diagnosis system

Chromosomal karyotyping analysis has been considered as the gold standard for cytogenetic diagnosis and an effective measure for preventing birth defects. However, conventional karyotyping analysis relies heavily on manual recognition, which is time-consuming and labor-intensive. The application of an efficient intelligent chromosomal karyotyping precise auxiliary diagnosis system in clinical practice can significantly reduce the analysis time and greatly improve the efficiency, increase the detection rate for low-percentage mosaicisms, and promote homogenization between laboratories. This can effectively enhance the capacity and level of cytogenetic diagnosis. With the continuous application of such system in the field of karyotyping analysis, a substantial amount of clinical application data and experience has been accumulated. This consensus has been formulated after multiple rounds of discussion by experts from the clinical application collaboration group of the efficient intelligent chromosomal karyotyping precise auxiliary diagnosis system. It aims to provide a reference for peers to better utilize intelligent auxiliary diagnosis systems during chromosomal karyotyping analysis and promote the standardized development of karyotyping analysis technology.

Chromosome 7 Isodisomy in a Child with Silver-Russell Síndrome

Silver-Rusell syndrome is a rare genetic disease. There is evidence that the genetic causes of the disorder are heterogeneous, with predominant alterations in the imprinted regions of chromosomes 11 and 7, in addition to other genomic alterations, such as chromosomal structural aberrations, single nucleotide polymorphisms, copy number variations, and small insertions and deletions. The most prevalent clinical manifestations include prenatal and postnatal growth retardation, dysmorphic features, and feeding difficulties. We present a case of a 4-year-old boy with phenotypic features consistent with Silver-Russell syndrome. The sample was subjected to conventional karyotyping analysis. The analysis was also conducted using the SALSA MLPA Probemix ME032-A1 UDP7-UDP14 and Applied Biosystems CytoScan 750K Suite. MS-MLPA analysis revealed the presence of hypermethylation in the <em>GRB-10</em> and <em>MEST</em> genes on chromosome 7. SNP-array analysis revealed a loss of heterozygosity (LOH) at 7q11.22q31.1 (38.7 Mb). The methylation of the genes involved in this epigenetic event, in conjunction with LOH and the clinical characterization of this child, indicates that the origin of the disease is due to an isodisomy of maternal chromosome 7. This report of a child who exhibits the clinical characteristics of SRS and presents a UPD of chromosome 7, most likely originating from the mother, once again demonstrates the involvement of these genes in SRS despite the incomplete understanding of the underlying mechanism. A multidisciplinary strategy has been proposed for the follow-up and treatment of this disease according to its etiology in the proband.

Genetic analysis of two families with abnormal findings upon prenatal diagnosis

To carry out genetic analysis on two families with carriers of small terminal translocations using karyotyping analysis and genomic copy number variation sequencing (CNV-seq). Two couples undergoing prenatal diagnosis at the Tianjin Central Hospital of Obstetrics and Gynecology respectively on April 12, 2020 and December 17, 2021 were selected as the study subjects. With informed consent, amniotic fluid and peripheral blood samples were collected and subjected to conventional karyotyping and CNV-seq analysis for the detection of chromosomal microdeletion/duplications. Both couples had given births to children with chromosomal aberrations previously, and both fetuses were found to have abnormal karyotypes. CNV-seq showed that they had harbored microdeletion/duplications, and their mothers had both carried balanced translocations involving terminal fragments of chromosomes. For fetuses with small chromosomal segmental abnormalities, their parental origin should be traced, and the diagnosis should be confirmed with combined genetic techniques.

Haematolymphoid Tumours Karyotypes: A Moroccan Population Retrospective Study from 1992 to 2021

Background: Haematolymphoid tumours are a heterogeneous group of pathologies involving cells of the haematopoietic lineage. Data reported here focus on the results of a descriptive, general, exclusive epidemiological study from a Moroccan population. Objective: Our research aimed to study the distribution of Haematolymphoid tumours in the regional context in Casablanca. Method: We focused on malignancies diagnosed between January 1992 and December 2021 at the Cytogenetics Department of the Pasteur institute of Morocco. Conventional karyotype analysis was performed on bone marrow samples from patients aged between 1 and 95 years and collected data were analyzed to perform statistical analyzes using SPSS20.0 software. Results: Of the karyotypes investigated 69.8% (1118/1601) were positive for haematolymphoid tumours. The mean age at diagnosis was 42.68 ± 18.20 years. The most frequent pathologies were myeloid neoplasms, with chronic myeloid leukaemia (CML) in first place at 69.8% (780/1118), followed by acute leukaemias (ALL, AML and ALAL) at 21.4% and myelodysplastic neoplasm (MDS) at 5.5%. The most recurrent clonal abnormalities were translocations t (9;22) with 74.2% in CML cases. Deletions 12.5% and additions 8% were found in all diseases with mainly trisomy’s 8 and 21 in AML and MDS and deletions in AML and ALAL. An association was also found in 12 cases of AML between t (8;21) translocation and sexual chromosome deletion (X and Y). The proportion of karyotype’s complexity (9.9%) seems to increase with ages. Conclusions: Haematolymphoid tumours occur at an earlier age in this cohort. Leukaemia represents the most frequent pathologies with a preponderance of chronic diseases, mainly affecting adults.

Fetal congenital talipes equinovarus: genomic abnormalities and obstetric follow-up results

Objective The etiology of congenital talipes equinovarus (CTEV) is unknown, and the relationship between chromosome microdeletion/microduplication and fetal CTEV is rarely reported. In this study, we retrospectively analyzed fetal CTEV to explore the relationship among the CTEV phenotype, chromosome microdeletion/microduplication, and obstetric outcomes. Methods Chromosome karyotype analysis and single nucleotide polymorphism (SNP) array were performed for the 68 fetuses with CTEV. Results An SNP array was performed for 68 fetuses with CTEV; pathogenic copy number variations (CNVs) were detected in eight cases (11.8%, 8/68). In addition to one case consistent with karyotype analysis, the SNP array revealed seven additional pathogenic CNVs, including three with 22q11.21 microdeletions, two with 17p12p11.2 microduplications, one with 15q11.2 microdeletions, and one with 7q11.23 microduplications. Of the seven cases carrying pathogenic CNVs, three were tested for family genetics; of these, one was de novo, and two were inherited from either the father or mother. In total, 68 fetuses with CTEV were initially identified, of which 66 cases successfully followed-up. Of these, 9 were terminated, 2 died in utero, and 55 were live births. In 9 cases, no clinical manifestations of CTEV were found at birth; the false-positive rate of prenatal ultrasound CTEVdiagnosis was thus 13.6% (9/66). Conclusion CTEV was associated with chromosome microdeletion/microduplication, the most common of which was 22q11.21 microdeletion, followed by 17p12p11.2 microduplication. Thus, further genomic detection is recommended for fetuses with CTEV showing no abnormalities on conventional karyotype analysis.

LAMP5 Is an AML-Restricted Immunotherapeutic Target Enriched in High-Risk Acute Myeloid Leukemia

LAMP5, Lysosomal Associated Membrane Protein Family Member 5, a neuronal protein that plays a role in synaptic plasticity is normally found primarily in the brain, however limited studies have identified it to be expressed in leukemia, particularly KMT2A-rearranged leukemias. Importantly, there is low expression of LAMP5 in normal hematopoiesis making it an ideal potential immunotherapeutic target for both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). As a newly identified aberrant protein in leukemias, there is little data available on the prevalence or clinical implications of LAMP5 expression. We interrogated the transcriptome and associated clinical and laboratory data from a large cohort of pediatric AML patients to define the prevalence, co-occurring cytomolecular aberrations and clinical outcomes associated with LAMP5 expression. Ribo-depleted RNA seq data from 2,241 patients with AML (Pediatric ages 0-29 years N=1,412, from recent Phase 3 COG clinical trials; Adult ages 18-88 years from BEAT AML N=451, SWOG N=199, TCGA-LAML N=179) with available cytogenetic, molecular, and clinical data were utilized. Gene fusions, internal tandem duplications (ITD) and partial tandem duplications (PTD) were detected by Cicero. Conventional karyotyping and mutational analysis including NGS were used to determine additional cytogenetic and molecular abnormalities. Response was measured by 5-year overall survival (OS), event-free survival (EFS) and relapse risk (RR). Of the 1,412 pediatric patients, LAMP5 was expressed (positive defined as mRNA transcripts per million (TPM) >2) in 396 patients (28%) with mRNA TPM ranging from 0-884 with similar prevalence found in adult AML (28%; BEAT AML N=126/452, SWOG N= 59/199 TCGA N=50/179), but with significantly higher TPM ranging from 0-3,200. Of the 396 pediatric patients with LAMP5 expression, there was a notable enrichment in KMT2A-r AML (N=203, 51%), which accounts for 59% of the total KMT2A-r cohort. Within the KMT2A-r cohort, there is enrichment in the MLLT1 (N=17/18, 94%), MLLT3 (N=61/93, 66%) and MLLT10 (N=58/69, 84%) fusion groups. Additionally, LAMP5 expression was seen in other high risk fusion subtypes including 100% of patients with KAT6A-CREBBP (N=12/12) and 29% of NUP98 fusions (N=45/154). There is absent LAMP5 expression in normal lymphoid and myeloid cells, but there is moderate expression seen in plasmacytoid dendritic cells, which could provide a therapeutic target for Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN). Given the association with high-risk AML fusion subtypes, the impact of LAMP5 on clinical outcome in AML pediatric patients was evaluated. High rates of relapse were seen in patients with LAMP5 expression, with a relapse rate of 45% compared to 36% in LAMP5-negative patients. 5 year event-free survival (EFS) was 36% for LAMP5-positive patients compared 45% for LAMP5-negative patients (p=0.0005). Similar poor outcomes were seen with overall survival 54% for LAMP5-positive patients vs. 62% for LAMP5-negative patients (p=0.001). In an analysis of a large cohort of AML patients, we found that LAMP5 expression is highly enriched in many high-risk fusion subtypes including KMT2A-rearranged AML, KAT6A-CREBBP and NUP98 fusions. Patients with LAMP5 expression have high rates of relapse with significantly worse outcomes compared to patients without LAMP5. Given low levels of LAMP5 in normal hematopoiesis, LAMP5 is an ideal AML-restricted immunotherapy target with broad applicability for many high-risk fusion subtypes of AML. Current efforts to generate a LAMP5 chimeric antigen receptor (CAR) T cell are underway which will be used to evaluate preclinical efficacy in AML via cell- and patient-derived xenograft models with the goal to translate this novel therapy into clinical use to improve the current dismal outcomes for these high-risk patients.

CD276 (B7-H3) Is an Immunotherapeutic Target in Acute Myeloid Leukemia with Preclinical Efficacy of Vobramitamab Duocarmazine, an Investigational CD276 Antibody-Drug Conjugate

CD276 (B7-H3) is an immune checkpoint inhibitor expressed in a majority of solid tumors and hematologic malignancies, however it has not been well-described in acute myeloid leukemia (AML). A variety of immunotherapies targeting CD276 including CAR T cells and antibody-drug conjugates (ADCs) are being explored in solid tumors, however their role in leukemia has not been well elucidated. We interrogated the transcriptome and associated clinical and laboratory data from a large cohort of pediatric and adult AML patients to define the prevalence, frequency of co-occurring genetic alterations and outcomes associated with CD276 expression. Lastly, we evaluated preclinical in vitro efficacy in AML of vobramitamab duocarmazine (vobra duo), an investigational ADC with a humanized B7-H3 mAb conjugated via a cleavable linker to the prodrug seco-DUocarmycin hydroxyBenzamide Azaindole. Ribo-depleted RNA sequencing data from 2,241 patients with AML (Pediatric ages 0-29 years N=1,412, from recent Phase 3 COG clinical trials; Adult ages 18-88 years BEAT AML N=451, SWOG N=199, TCGA-LAML N=179) with available cytogenetic, molecular, and clinical data were utilized. Conventional karyotyping and mutational analysis including NGS were used to determine additional cytomolecular aberrations. Response was measured by 5-year overall survival (OS), event-free survival (EFS) and relapse risk (RR). Of the 1,412 pediatric patients evaluated, CD276 mRNA expression was positive (defined as mRNA transcripts per million (TPM) >2) in 289 patients (20%). Higher prevalence (58%) was seen in adult AML patients (BEAT AML N=209/451, SWOG N=191/199, TCGA-LAML N=85/179), Fig 1A. Flow cytometry validated cell surface expression in pediatric AML, with 22% of patients expressing CD276. Cell surface expression was also observed in AML cell lines. CD276 mRNA expression is absent in normal hematopoietic cells (TPM <1) with absent cell surface expression in normal bone marrow as assessed by flow cytometry, allowing for targeted CD276 therapies to be utilized with no/minimal on-target off-tumor hematopoietic toxicity. Evaluation of co-occurring cytomolecular aberrations demonstrated a significant enrichment of CD276+ pediatric patients with high-risk genetic alterations, most notably KMT2A fusions (N=150/343, 43.7%), KAT6A-CREBBP (N=11/12, 91.6%) and CBFA2T3-GLIS2 (N=22/39, 56.4%). We evaluated the significance of CD276 mRNA expression on clinical outcomes in childhood AML. 5 year event free survival (EFS) for patients with CD276 expression was 35% compared to 44% for patients without CD276 expression (p=0.0022). Overall survival (OS) was 52% for patients with CD276 expression compared to 61% for patients without CD276 expression (p=0.0015). Relapse was seen in 40.5% of patients with CD276 expression compared to 35.8% of patients without CD276 expression and relapse occurred on average earlier in CD276 positive patients (0.89 years vs. 1.06 years, p=0.02). Given the poor clinical outcomes for patients with high CD276 expression, novel targeted therapies are needed for this high risk cohort. With multiple CD276 immunotherapies under clinical evaluation in solid tumors, we tested vobra duo in OCI-AML2, a CD276 positive KMT2A-rearranged AML cell line, which demonstrated robust cytolytic activity with an IC50 less than 1nM (IC50=0.9306 nM) supporting the potential efficacy of CD276 targeted therapies in AML (Fig 1B). In a large cohort of AML patients, we identified CD276 to be overexpressed in 20% of pediatric AML patients and 58% of adult AML patients with a significant enrichment in high risk fusion subtypes including KMT2A-rearranged AML, KAT6A-CREBBP and CBFA2T3-GLIS2 fusions. Patients with CD276 expression have poor outcomes with significantly worse EFS and OS compared to patients who do not have CD276 expression. With multiple CD276 immunotherapies under clinical evaluation for solid tumors, we hypothesized that targeting CD276 in AML would result in specific cytolytic activity. Vobra duo showed robust in vitro cytolytic activity against CD276 positive AML cells highlighting the need for ongoing preclinical evaluations of CD276 targeted therapies in AML. Given the established safety profile for vobra duo this provides a clear path for rapid translation to clinical use for high risk AML patients.

CLEC2A Is a Novel AML-Restricted Immunotherapeutic Target Enriched in KMT2A-Rearranged Acute Myeloid Leukemia

KMT2A rearrangements (KMT2A-r) are a frequent event in childhood acute myeloid leukemia (AML) and in treatment related AML in adults leading to a highly refractory and deadly subtype of AML regardless of age. Targeted therapies for KMT2A-r AML have had limited success with novel therapies urgently needed. Despite successes in other leukemias, immunotherapy in AML is in its infancy in part due to significant overlap of antigens expressed in AML and normal hematopoietic cells, which would limit efficacy and increase hematopoietic toxicity. We interrogated the AML transcriptome compared to normal hematopoiesis to identify optimal targets that are silent in normal hematopoieisis and are highly expressed in AML. Here we describe a novel immunotherapeutic target, CLEC2A, a member of the C-type lectin family, that is highly enriched in high-risk AML, particularly KMT2A-r AML and absent in normal hematopoeisis. We evaluated the association between KMT2A fusions and CLEC2A expression identifying CLEC2A as a putative oncoprotein and the potential for CLEC2A to serve as an immunotherapeutic target in high-risk AML via antibody-mediated cytotoxicity. Diagnostic specimens from 1,864 patients with AML (Pediatric 0-29 years from COG Phase III trials N=1486; Adult 18-88 years SWOG N=199 and TCGA-LAML N=179) were interrogated using contemporary NGS to identify genetic aberrations. Conventional karyotyping and mutational analysis were used to determine additional cytogenetic and molecular abnormalities. As CLEC2A was entirely absent in normal bone marrow (TPM 0-0.15), positive CLEC2A expression was defined as mRNA transcripts per million (TPM) >1. Although, CLEC2A was detected in only 18% of AML patients (252/1486; 17% pediatric and 90/378; 24% adult patients), it was highly enriched in those with KMT2A-r AML with 77% (N=193) of CLEC2A positive patients having KMT2A-r, accounting for 48% of the total KMT2A-r cohort, Fig 1A. CLEC2A expression was enriched in specific fusion subgroups: MLLT10 (80%), MLLT4 (71%), and MLLT3 (48%), Fig 1B. CLEC2A expression was low to absent in a majority of other patients, though was seen in other high-risk groups including non-KMT2A MLLT10 fusions, NUP98 fusions, CBFA2T3-GLIS2 fusions, and monosomy 7. CLEC2A is also expressed in a subtype of T-ALL (HOXA activated), which is a fusion driven T-ALL. We further evaluated a potential causal link between KMT2A fusions and CLEC2A expression and inquired whether KMT2A fusion proteins might directly bind to the CLEC2A promoter and induce CLEC2A expression. We used AutoCUT&RUN profiling of genome-wide occupancy of KMT2A oncoproteins to evaluate direct binding of the KMT2A fusion protein to the CLEC2A promoter in primary AML patient samples with three different KMT2A fusions (MLLT10, MLLT4 and MLLT3). We found direct binding of KMT2A fusion proteins to the CLEC2A promoter region, indicating a causal relationship between KMT2A fusions and CLEC2A expression. We then evaluated the potential of therapeutic targeting of CLEC2A by an antibody-drug conjugate using a HumZap cytotoxicy assay. CLEC2A antibody conjugated to saporin (a toxin with cytolytic activity when internalized) was incubated with CLEC2A+ and CLEC2A- cells with target-specific cytotoxicity evaluated by colorimetric assay. CLEC2A+ OCI-AML2 cells had significantly higher rate of cytotoxicity compared to CLEC2A- control cells at 10nM Ab-toxin conjugate. Clinical outcomes for patients with and without CLEC2A expression was evaluated. Patients with CLEC2A had significantly worse outcomes (EFS 23% vs. 45.5%, p<0.0001 and OS 38% vs. 63%, p<0.0001 for patients with and without CLEC2A expression, respectively). Given high rates of co-occurring KMT2A-r AML, particularly high-risk fusions (MLLT10 and MLLT4), we evaluated clinical outcomes within the KMT2A-r cohort as these patients have poor EFS and OS. KMT2A-r patients with CLEC2A had significantly worse EFS (24% vs. 39%, p=0.0063) and OS (38% vs. 60%, p=0.0002). Within the KMT2A-r cohort, high rates of relapse were seen for patients with CLEC2A expression compared to those without CLEC2A (72% vs. 55%, p=0.0039). Here we describe CLEC2A, a novel AML-restricted cell surface target that is an ideal immunotherapeutic target. CLEC2A is highly expressed in KMT2A-r AML, entirely absent in normal hematopoietic cells, directly and causally linked to the KMT2A fusion, and can be used for target-directed cytotoxicity.

Prenatal diagnosis and genetic etiology analysis of talipes equinovarus by chromosomal microarray analysis

BackgroundWith the advancement of molecular technology, fetal talipes equinovarus (TE) is believed to be not only associated with chromosome aneuploidy, but also related to chromosomal microdeletion and microduplication. The study aimed to explore the molecular etiology of fetal TE and provide more information for the clinical screening and genetic counseling of TE by Chromosomal Microarray Analysis (CMA).MethodsThis retrospectively study included 131 fetuses with TE identified by ultrasonography. Conventional karyotyping and SNP array analysis were performed for all the subjects. They were divided into isolated TE group (n = 55) and complex group (n = 76) according to structural anomalies.ResultsAmong the total of 131 fetuses, karyotype analysis found 12(9.2%) abnormal results, while SNP array found 27 (20.6%) cases. Trisomy 18 was detected most frequently among abnormal karyotypes. The detection rate of SNP array was significantly higher than that of traditional chromosome karyotype analysis (P < 0.05). SNP array detected 15 (11.5%) cases of submicroscopic abnormalities that karyotype analysis did not find. The most common CNV was the 22q11.2 microdeletion. For both analyses, the overall detection rates were significantly higher in the complex TE group than in the isolated TE group (karyotype: P < 0.05; SNP array: P < 0.05). The incremental yield of chromosomal abnormalities in fetuses with unilateral TE (22.0%) was higher than in fetuses with bilateral TE (19.8%), but this difference was not statistically significant (P > 0.05). Abnormal chromosomes were most frequently detected in fetuses with TE plus cardiovascular system abnormalities.ConclusionFetal TE is related to chromosomal microdeletion or microduplication. Prenatal diagnosis is recommended for fetuses with TE, and CMA testing is preferred. CMA can improve the detection rate of chromosomal abnormalities associated with fetal TE, especially in pregnancies with complex TE.

Prenatal diagnosis of a case with Schuurs-Hoeijmakers syndrome

To explore the genetic basis for a fetus with multiple malformations. Clinical data of the fetus was collected, Amniotic fluid sample of the fetus was subjected to conventional G-banded karyotyping, low-depth whole genome copy number variants detection and whole exome sequencing (WES). Candidate variant was verified by Sanger sequencing of the fetus and its parents. Prenatal ultrasound scan at 21+5 gestational weeks had revealed increased nuchal thickness (9.0 mm), enhanced echos of bilateral renal parenchyma, seroperitoneum, left pleural effusion and right displacement of the heart. The mother had a previous history of terminated pregnancy for multiple fetal anomalies. No abnormality was found by conventional karyotyping and CNV analysis, though WES revealed that the fetus has harbored a de novo heterozygous c.607C>T (p.Arg203Trp) variant of the ACS1 gene (NM_018026.3), and the result was validated by Sanger sequencing. Through WES and prenatal ultrasonography, the fetus was diagnosed with Schuurs-Hoeijmakers syndrome due to the heterozygous c.607C>T (p.Arg203Trp) variant of the PACS1 gene (NM_018026.3). For fetuses with multiple malformations, WES can help to reveal the genetic etiology when CNV result is negative.

An Integral R-Banded Karyotype Analysis System of Bone Marrow Metaphases Based on Deep Learning.

Conventional karyotype analysis, which provides comprehensive cytogenetic information, plays a significant role in the diagnosis and risk stratification of hematologic neoplasms. The main limitations of this approach include long turnaround time and laboriousness. Therefore, we developed an integral R-banded karyotype analysis system for bone marrow metaphases, based on deep learning. To evaluate the performance of the internal models and the entire karyotype analysis system for R-banded bone marrow metaphase. A total of 4442 sets of R-banded normal bone marrow metaphases and karyograms were collected. Accordingly, 4 deep learning-based models for different analytic stages of karyotyping, including denoising, segmentation, classification, and polarity recognition, were developed and integrated as an R-banded bone marrow karyotype analysis system. Five-fold cross validation was performed on each model. The whole system was implemented by 2 strategies of automatic and semiautomatic workflows. A test set of 885 metaphases was used to assess the entire system. The denoising model achieved an intersection-over-union (IoU) of 99.20% and a Dice similarity coefficient (DSC) of 99.58% for metaphase acquisition. The segmentation model achieved an IoU of 91.95% and a DSC of 95.79% for chromosome segmentation. The accuracies of the segmentation, classification, and polarity recognition models were 96.77%, 98.77%, and 99.93%, respectively. The whole system achieved an accuracy of 93.33% with the automatic strategy and an accuracy of 99.06% with the semiautomatic strategy. The performance of both the internal models and the entire system is desirable. This deep learning-based karyotype analysis system has potential in a clinical application.

Risk factor analysis for adverse prognosis of the fetal ventricular septal defect (VSD)

BackgroundVentricular septal defect (VSD) is the most common subtype of congenital heart disease. In the present study, we aimed to determine whether chromosome aberration was associated with the occurrence of VSD and evaluate the association of VSD size, location and chromosome aberration with adverse outcomes in the Chinese fetuses.MethodsFetuses with VSD and comprehensive follow-up data were included and evaluated retrospectively. Medical records were used to collect epidemiological data and foetal outcomes. For VSD fetuses, conventional karyotype and microarray analysis were conducted. After adjusting confounding factors by using multivariable logistic regression analyses, the association between chromosome variations and VSD occurrence was explored. The association between defect size, location and chromosome aberrations and adverse foetal outcomes was also investigated.ResultsChromosome aberration was the risk factor for VSD occurrence, raising 6.5-fold chance of developing VSD. Chromosome aberration, peri-membranous site and large defect size of VSD were significant risk factors of adverse fetal outcome. Chromosome aberrations, including pathogenic copy number variations (CNVs) and variations of uncertain significance (VUS), were both risk factors, increasing the risk of the adverse fetal outcome by 55.9 times and 6.7 times, respectively. The peri-membranous site would increase 5.3-fold risk and defects larger than 5 mm would increase the 7.1-fold risk for poor fetal outcome.ConclusionsThe current investigation revealed that chromosomal abnormalities, large defects, and the peri-membranous site were all risk factors for poor fetal outcomes. Our study also indicated that chromosome aberration was one of risk factors for the VSD occurrence.

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.

Polyploidy Phenomenon as a Cause of Early Miscarriages in Abortion Materials.

Chromosomal abnormalities are an important cause of especially early miscarriages. The aim of this study was to analyze the chromosomal aberrations and determine the frequencies of numerical and structural chromosome abnormalities in spontaneous abortion materials. This was a prospective research and ninety two abortion samples obtained from women who had one or more miscarriages were included in the study. Conventional karyotype analysis was performed on each sample to identify possible chromosomal abnormalities. By karyotype analysis, 11 polyploidy cases, (9 triploids and 2 tetraploids), 8 trisomies (one of which was mosaic), 2 monosomies (monosomy X), 1 isochromosome, 1 Xq deletion, and 4 translocations were detected in abortion materials. Isochromosome and Xq deletion cases were also mosaic. In addition, five polymorphic variants were revealed. We found higher paternal age in polyploidy cases. The most common anomaly we found in abortion materials was polyploidy. This was followed by aneuploidy (trisomy and monosomy). Polyploidy (triploidy or tetraploidy) emerged as an important cause in cases of spontaneous abortion. Paternal age may be associated with polyploidy especially triploidy.

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.

Karyo-Morphological analyses of &lt;em&gt;Drimia gigantea&lt;/em&gt; and&lt;em&gt; Drimia Viridula&lt;/em&gt;; Two of the three species in &lt;em&gt;Altissima&lt;/em&gt; complex of the family hyacinthaceae

Drimia is a heterogeneous, poorly understood genus that requires major rearrangement. Chromosome behavior of Drimia gigantea and Drimia viridula; two species in D. altissima complex was studied for karyotypes transmission of extra chromosomes pair and pollen viability. Meristematic cells at the root tips of D. gigantea and D. viridula were studied by conventional cytogenetic and karyotype analysis. Chromosome numbers were 2n = 22 and 2n = 20 for D. gigantea and D. viridula, respectively. Extra chromosome pair in D. gigantea resulted in 2n = 22. Chromosome length was 55.45 mm for D. viridula and 55.50 mm for D. gigantea. Bridge formation, laggards, chromosome exclusion and formation of 9 (3.26%), 10 (90.93%) and 11(5.81%) chromosome bodies were observed in D. gigantea, D. viridula consistently formed 10 bivalents. Pollen viability was 90.63% in D. gigantea and 94.95% in D. viridula. Karyotypic evidence suggests that an extra chromosome pair in D. gigantea evolved from fragmentation of the second-largest chromosome, the extra chromosome is not a B-chromosome because it participated in cell division. High pollen viability and frequent occurrence of ten bivalents, indicated that D. viridula is closer to the progenitors of a common genetic system within D. altissima complex. Furthermore, the karyo-morphological differences support the separation of the two taxa as distinct species.

Clinical, Molecular, and Histopathological Correlates in a Series of 24 Consecutive Cases of Suspected Myeloproliferative Neoplasms with a JAK2V617F Variant Allele Frequency ≤1%

Background: JAK2V617F is detected in >95% of polycythemia vera (PV) and 50-60% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). JAK2V617F variant allele frequency (VAF) is associated with different clinical characteristics and outcome. Highly sensitive molecular assays led to detect JAK2V617F mutation also in healthy subjects, associated with a higher risk of coronary heart disease (N Engl J Med. 2017;377(2):111-121) in the context of CHIP. To date, comprehensive data on suspected MPN patients with a JAK2V617F VAF≤1% are lacking. Aim and methods: The aim was to analyze clinical, molecular, and histopathological correlates in a series of suspected MPN patients (pts) with a JAK2V617F VAF≤1% (from 2017 to 2022) at our Center. JAK2V617F VAF was measured in granulocytes by highly sensitive RTQ-PCR. Conventional karyotype and target next-generation sequencing (NGS) analysis were performed (complete data will be presented at the meeting). Bone marrow (BM) biopsies were reviewed by two independent expert pathologists, R.S. and U.G., the latter included as an author in the International Consensus Classification (Blood 2022; 2022015850). Results: 21 males and 3 females (M/F=7:1), with a median age of 58 y (range, 22-81) were included. Median JAK2V617F VAF was 0.2% (0.1-1). In the suspicion of MPN, complete diagnostic work-up was carried out in pts presenting with erythrocytosis (n=15), thrombocytosis (n=4), history of atypical thrombosis (n=3), splenomegaly (n=1) and leukocytosis (n=1), (Fig.1). In pts with erythrocytosis, median (range) Hb and Hct values were 17 g/dL (15.8-18.5) and 51.5% (50.4-54.7), whereas leukocytes (6.6 x 109/L, 5-11) and platelet (258 x 109/L, 172-440) were within normal range. EPO level (10 mUI/mL; 1.8-12.5) was subnormal in 3 pts. Abnormal karyotype was documented in one case (45,X,-Y[7]/46,XY[13]). Additional mutations were documented in 3 pts: HBB T124N, SH2B3 S186I and cKIT D816V. In pts with thrombocytosis, platelet range was 460-1450 x 109/L; Hb, Hct and leucocytes values were normal. MPL W515K and CALR L367Tfs*46 additional mutations were in one and two pts, respectively. Considering pts with atypical thrombosis, one had a history of arterial events while the others experienced cerebral venous sinus and portal vein thrombosis. Thrombophilia screenings were negative. Karyotype was normal in two pts, whereas one had 45,X,-Y[3]/46,XY[20]. Additional CBL p.D460dup was detected in one case. The subjects investigated for splenomegaly/leukocytosis had normal karyotype, no additional mutations. Morphological features consistent with PV were documented in 4 pts (Fig. 2, A-B). In one of these cases, multifocal dense infiltrates of CD117+/CD25+ mast cells were detected, fulfilling the diagnostic criteria for systemic mastocytosis with associated hematological neoplasm. In the other cases, microscopic examination showed age-adjusted normal or focally increased cellularity, regular or mildly expanded erythroid lineage with no/only minor increase in granulocytic lineage and a higher number of megakaryocytes with PV-like features (i.e. polymorphic forms without atypia and no dense cluster formation) (Fig. 1, C-D). Neither reticulin fibrosis nor excess blasts were detected and these pts had a final diagnosis of MPN unclassifiable (MPN-U), with a PV-like morphology. In pts with thrombocytosis histology was consistent with ET in three cases and overt-PMF in one. Among ET, one pts harbored MPLW515K with grade 1 reticulin fibrosis (Fig. 2, E-F), whereas the overt PMF had CALR type 1 additional mutation. BM biopsies of pts presenting with atypical thrombosis, leukocytosis and mild splenomegaly showed non-specific MPN-like features (including focally increased cellularity, mild expansion of erythroid or granulocytic lineages, higher number of megakaryocytes with a TE-like or a PV-like morphology) and were diagnosed as MPN-U. Conclusions: This study highlights the importance of using highly sensitive assays to detect JAK2V617F, as well as the need to search for CALR and MPL mutations, particularly in pts with thrombocytosis. Moreover, in pts presenting with erythrocytosis and JAK2V617F positivity with VAF≤1%, histopathological diagnostic clues to MPN may be subtle as in the early phase of the disease, emphasizing the need to integrate histopathology with clinical and genetic data in order to address patients to proper clinical management. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical and Functional Implications of MYC Variants As a New Class of Pathogenic Variants in AML

MYC, a well-known oncogene, is implicated in a majority of cancers, but there is little data on its role in myeloid malignancies. We recently described a novel internal tandem duplication of MYC gene (MYC-ITD) in a subset of patients with AML. As the prevalence of MYC aberrations have not been well documented in AML, we interrogated the genome, transcriptome and associated clinical and laboratory data from the largest cohort of pediatric AML patients to define the prevalence, frequency of co-occurring cytomolecular alterations and outcomes associated with MYC variants. Mutation Profiling: Diagnostic specimens from 1,806 patients with AML (age 0-29 years) enrolled on COG Phase III trials were retrospectively interrogated using contemporary NGS techniques or a PCR based "Tandem Repeat" assay for MYC-ITD. Conventional karyotyping and mutational analysis, including NGS, were used to determine additional cytogenetic and molecular abnormalities. Of the 1,806 patients studied, MYC aberrations were identified in 58 patients (3.2%) with 50% (N=29) harboring MYC-ITD, 46.5% (N=27) with a SNV or indel (MYCSNV/indel) and 2 patients (3.5%) with dual MYC-ITD and SNV. For MYC-ITD, tandem duplications occurred in exon 3 (Max binding domain) with ITD lengths ranging from 252-588 bp. The majority of MYCSNV/indel (77%) were identified at the P72-P78 hotspot with 33.3% (N=9) harboring the P74>L mutation and 18.5% (N=5) having the T73>N mutation. Functional Studies: Given the novelty of the ITD variant, we initially characterized the functional properties of MYC-ITD in vitro. Similar to wild-type (MYCWT), MYC-ITD is localized to the nucleus and requires Max to bind to specific DNA sequences (E-boxes). However, in contrast to MYCWT, MYC-ITD fails to recognize sequences containing a single E-box in vitro, but exhibits higher affinity toward sequences with two or more E-boxes. MYC-ITD is consistently more efficient in activating promoters with multiple E-boxes than MYCWT. Thus, MYC-ITD may possess different DNA-binding and protein-interacting properties, potentially leading to altered transcriptional regulation and biological output. Given the alterations in DNA binding properties, we evaluated the functional consequences of MYC-ITD in our cord blood stem cell/endothelial cell (EC) co-culture system. Normal cord blood stem cells were transduced with MYC-ITD vs. MYCWT and grown in an EC co-culture system. MYC-ITD transduced cord blood stem cells showed significantly higher proliferation compared to MYCWT or control GFP-transduced cells with proliferation advantage of 37-and 45-fold after 14 and 21 days in co-culture, respectively (p<0.001). Clinical Impact: Analysis of cooperating cytomolecular lesions revealed a striking difference between MYC-ITD and MYCSNV/indel. MYC-ITD was highly enriched in core-binding factor (CBF) AML found in 86% (N=25)[(N=18, 62%) with inv(16) and (N=7, 24%) with t(8;21)] (Figure 1A), vs. 26% in the MYCWT cohort and had a paucity of somatic mutations. Conversely, MYCSNV/indel was enriched in NUP98-NSD1 fusions (N=7, 27%), FLT3-ITD (N=10, 38%) and NPM1 mutations (N=9, 33%) (Figure 1A), which was significantly higher than the WT cohort. Patients with MYC-ITD had a morphologic CR rate after course 1 of 92.3% vs. 80% (p=0.249) in patients with MYCSNV/indel with a corresponding relapse rate of 24% and 52% (p=0.046). Lower CR and higher relapse reflects the higher proportion of patients with high-risk lesions (FLT3-ITD, NUP98-NSD1 and WT1) in MYCSNV/indel and favorable CBF in MYCITD. Those with MYC-ITD and MYCSNV/indel had a 5-year event free survival (EFS) of 70% vs. 38.5%, respectively (p=0.018) with a corresponding overall survival (OS) of 76% and 47% (p=0.023) similar to patients with favorable or high risk disease (Figure 1B). Conclusion: Here we define MYC aberrations as a new class of pathogenic variants in childhood AML. We demonstrate functional and clinical characterization of a novel MYC-ITD and contrast this novel variant to that of established pathogenic MYCSNV/indel. We show the high prevalence of co-occurring core-binding factor fusions with MYC-ITD, and high frequency of co-occurring NUP98-NSD1, FLT3-ITD and NPM1 mutations in MYCSNV/indel. Patients with MYC-ITD have superior clinical outcomes while patients with MYCSNV/indel have clinical outcomes similar to high-risk patients, likely due to the associations with the disease associated fusions. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.