Clinical Application of Chromosomal Microarray Analysis in Fetuses with Congenital Heart Disease

Background: Congenital heart disease (CHD) is an important birth defect, but its mechanism is still unclear. In recent years, genetic causes including chromosomal abnormalities are associated with the occurrence of congenital heart disease. In this study, CMA technology is applied to explore the genetic causes of congenital heart disease, so as to further clarify the correlation between genotype and phenotype and prepare for late pregnancy intervention and postnatal diagnosis and treatment.Objective: To explore the chromosomal abnormalities and copy number variation (CNVs) of fetuses with CHD by CMA technology, and to clarify the clinical application value of CMA technology as a detection method of first-tier antenatal CHD.Methods: Amniotic fluid sample from 155 pregnant women diagnosed with fetus CHD by prenatal ultrasound from 2018 to 2021 are collected for SNP-array detection and karyotype analysis. According to the detected CNVs results, FISH, CMA or karyotype analysis are further selected for parental verification.Results: Among the 155 fetuses with CHD, a total of 32 (20.6%) cases of chromosomal abnormalities are detected, of which 31.3% are chromosome number abnormalities. CNVs of likely pathogenicity and unknown significance are 2.5% and 5.2% respectively. The detection rate of chromosomal abnormalities in CHD of different subtypes is different, among which the high detection rate is complex CHD (31.2%), right ventricular outflow tract obstruction (30.7%) and conotruncal defects (25%). The detection rate of chromosomal abnormalities in CHD with extracardiac structural abnormalities is significantly higher than that in isolated CHD (52.4% vs 11.3%, p<0.05). In addition, the detection rate of CHD with abnormal extracardiac structure is significantly higher than that of CHD with soft markers (52.4% vs 17.8%, p<0.05), which is statistically significant. There is no significant difference in detection rate between CHD with soft markers and isolated CHD (17.8% vs 11.3%). Of the 155 pregnant women with fetus CHD, 59 chose to terminate their pregnancies, some of which were terminated according to the results of SNP-array, and some of which were terminated according to the severity of CHD.Conclusion: SNP-array technology can be used to detect chromosomal abnormalities of first-tier antenatal CHD fetuses, with high resolution, short reporting period and high efficiency. Meanwhile, pregnancy intervention can be taken according to the results.

MLL-SEPT5 Fusion Transcript in Myelodysplastic Syndrome Patient With t(11;22)(q23;q11)

Objectives: This study aimed to identify unknown mixed lineage leukemia (MLL) translocation partner genes in a de novo patient with myelodysplastic syndrome (MDS) with t(11;22)(q23;q11) and investigate the clinical and molecular features of this patient.Methods: Bone marrow cells were assessed by karyotype analysis to reveal chromosomal abnormalities. Fluorescence in situ hybridization (FISH) was performed to detect MLL gene rearrangement using an MLL-specific break-apart probe. LDI-PCR and RT-PCR were performed, and the PCR products were sequenced using an Illumina MiSeq sequencer (Illumina, San Diego, CA, USA). The sequence data of the PCR products were analyzed using bioinformatics tools. Meanwhile, clinical data were collected to evaluate the prognosis of the patient.Results: Chromosomal karyotype analysis showed that the karyotype of the patient was 46, XX, t(11;22)(q23;q11)[10]/46, XX[1]. Subsequently, FISH data confirmed MLL gene rearrangement in the patient. LDI-PCR precisely showed that SEPT5 was the MLL translocation partner gene. RT-PCR and sequencing analysis disclosed the presence of MLL-SEPT5 fusion transcript and confirmed the fusion between MLL exon 8 and SEPT5 exon 3. Moreover, the patient had a recurrence shortly after allogeneic hematopoietic stem cell transplantation.Conclusion: Although the MLL-SEPT5 fusion transcript was occasionally described in acute myeloid leukemia, it was first identified in MDS. Patients with MLL-SEPT5 fusion gene exhibited a poor prognosis even with an aggressive treatment.

Fetal Aberrant Right Subclavian Artery: Associated Anomalies, Genetic Etiology, and Postnatal Outcomes in a Retrospective Cohort Study

Aberrant right subclavian artery (ARSA) is becoming more common in fetuses. However, there are relatively few studies on the genetic etiology of ARSA. We performed genetic analysis on fetuses with ARSA and followed up the pregnancy outcome to evaluate the prognosis of the fetuses, providing information for prenatal and eugenics consultations. A retrospective study was conducted on 112 pregnant women with fetuses diagnosed with ARSA from December 2016 to February 2021. Karyotype analysis and single-nucleotide polymorphism array (SNP-array) were performed in 112 fetuses. The 112 fetuses were divided into two groups: ARSA group, 48 (42.9%) and ARSA with other ultrasound abnormalities group, 64 (57.1%) cases. The total rate of pathogenic copy number variation (CNV) was 7.1% (8/112) using karyotype analysis (3/8) and SNP-array (5/8). The rate of pathogenic CNV in isolated ARSA and ARSA combined with other ultrasound abnormalities were 4.2% (2/48) and 9.4% (6/64), respectively. There was no significant difference between the two groups (P=0.463). The results of genetic analysis influence parents' decision to terminate the pregnancy. During follow-up, fetuses with ARSA without pathogenic CNV were found to have normal growth and development after birth. Therefore, prenatal genetic counseling and SNP-array should be recommended to better assess fetal prognosis.

Novel Homozygous Nonsense Mutation Associated with Bardet-Biedl Syndrome in Fetus with Congenital Renal Malformation

Background: Bardet-Biedl syndrome (BBS) is a rare autosomal recessive genetic disorder with clinical and genetic heterogeneity. BBS is more commonly reported in adults and children than in fetuses. Method: Here, a retrospective study of 210 fetuses with congenital renal malformation was performed. These fetuses were performed invasive prenatal diagnosis. Chromosome karyotype analysis, whole exome sequencing (WES), and a single nucleotide polymorphism array (SNP-array) were used.Results: We found the intrauterine phenotype of a fetus with enlarged kidneys, enhanced echo, and oligohydramnios, and the molecular characterizations of the fetus with BBS. The results of chromosome karyotype analysis and SNP-array on the fetus were normal. WES, however, revealed homozygous mutation of c.1177C>T (p.Arg393*) on exon 12 of the BBS1 gene, and heterozygous variation of c.2704G>A (p.Asp902Asn) on exon 22 of the CC2D2A gene. According to ACMG guidelines, c.1177C> T was identified as a pathogenic mutation and c.2704G>A was identified as an uncertain significance. Sanger sequencing showed that there was heterozygous mutation of c.1177C>T and heterozygous variation of c.2704G>A in the parents of the fetus. Conclusion: WES identified a novel homozygous nonsense mutation c.1177C>T in the BBS1 gene of a Chinese fetus with congenital renal malformation. The finding provides more insight into BBS1 mutations in Asian populations in general, and provides a basis for genetic counseling.

TP53 Allelic Status in Myeloid Malignancies

Introduction TP53 is a tumor suppressor gene involved in regulating cell division and apoptosis in response to DNA damage. In hematologic malignancies, TP53 alterations are present in both myeloid and lymphoid malignancies. TP53 alterations including both sequence level mutations and deletions occur in 8-10% of de novo acute myeloid leukemia (AML), and are significantly enriched in patients with therapy-related myeloid neoplasms with a frequency of 25-40%. TP53 alterations are associated with complex karyotype, resistance to traditional cytotoxic chemotherapy and dismal outcome, and are well established poor prognostic markers for both AML and myelodysplastic syndrome (MDS). While both biallelic and monoallelic TP53 alterations are seen, biallelic TP53 alteration is more frequent in both AML and MDS. In the instances of monoallelic TP53 alterations, the remaining wild-type allele can be inhibited by the dominant negative effect of the mutant p53. The clinical implication of TP53 allelic status remains controversial. Biallelic TP53 alterations and/or high TP53 mutation variant allele frequency (VAF) are associated with older patients, complex karyotype, few co-occurring mutations and poor outcome in both AML and MDS. Other studies demonstrate that biallelic vs. monoallelic TP53 alterations or high vs. low TP53 VAF have similar prognosis in myeloid malignancies. Current European LeukemiaNet (ELN) recommends testing TP53 deletion using karyotype analysis and TP53 mutations by molecular testing. Methods: We performed a retrospective review of patients with myeloid malignancies, myeloid next generation sequencing (NGS) panel and cytogenetic tests performed at ARUP Laboratories. We identified 18 patients with myeloid malignancies and TP53 mutations. We used a combination of karyotype analysis, FISH, chromosomal microarray (CMA) and NGS to determine the TP53 allelic status. Results: 18 patients diagnosed with myeloid malignancies and TP53 mutations were identified. Among them, 6 were diagnosed with AML, 10 had MDS, one with CMML and one had post essential thrombocythemia myelofibrosis. Age range is from 31 to 78 with a median age 65.5 years. 23 TP53 mutations were identified among 18 patients. The majority (78%) of TP53 mutations are located in the DNA binding domain. Co-occurring mutations are uncommon in patients with TP53 mutations. 11 out of 18 patients did not have co-occurring mutations in other myeloid malignancy related genes. Three cases had 1, three cases had 2, and one case had 3 co-occurring mutations at the time of diagnosis. Karyotype analysis and FISH were performed on all 18 patients. CMA was performed on 9 patients. 17p abnormalities, defined by the deletion or copy-neutral loss of heterozygosity (CN-LOH) of 17p, were seen in 9 out of 18 cases. The 17p abnormalities in 8 out of these 9 cases were visible by karyotype. Case 11 had a CN-LOH identified by CMA which was cryptic by karyotype. Typical complex karyotype was seen in 16 out of 18 cases. Biallelic TP53 alterations are defined by either the presence of a TP53 mutation with a 17p abnormality (deletion or CN-LOH), or two TP53 mutations with similar VAF, or one TP53 mutation with VAF &gt;50%. Biallelic TP53 alterations were seen more frequently compared with monoallelic TP53 alterations (14 vs. 4 patients) and enriched in older patients (Figure 1). All 6 AML patients had biallelic TP53 alterations. Outcome data is available in 10 patients including 8 patients with biallelic and 2 patients with monoallelic TP53 alterations. Patients with biallelic TP53 alterations have dismal outcome (Figure 2). Patient 7 represents an atypical patient with TP53 alteration in our study cohort. She was diagnosed with MDS at the age of 31. She had monoallelic TP53 mutation with a relatively low VAF and normal karyotype through her disease course. Her MDS never progressed and she remained in remission after transplant for more than four years and still doing well. Conclusion: In this study, we evaluated the TP53 allelic status in 18 patients with myeloid malignancies. Biallelic TP53 alterations are more frequent than monoallelic TP53 alterations, and are associated with older patients, and fewer co-occurring mutations. Biallelic TP53 alterations are associated with typical complex karyotype and dismal outcome. Our study supports the importance to differentiate between biallelic and monoallelic TP53 alterations. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Identification of a Novel Homozygous Nonsense Mutation in a Fetus with Bardet-Biedl Syndrome

Background: Bardet-Biedl syndrome (BBS) is a rare autosomal recessive genetic disorder with clinical and genetic heterogeneity. BBS is more commonly reported in adults and children than in fetuses. Here, we reported the intrauterine phenotype and molecular characterizations of a fetus with BBS. Methods: Chromosome karyotype analysis, whole exome sequencing (WES), and a single nucleotide polymorphism array (SNP-array) were used to analyze the genetic etiology of a fetus with enlarged kidneys, enhanced echo, and oligohydramnios. Results: The results of chromosome karyotype analysis and SNP-array on the fetus were normal. WES, however, revealed homozygous mutation of c.1177C>T (p.Arg393*) on exon 12 of the BBS1 gene, and heterozygous variation of c.2704G>A (p.Asp902Asn) on exon 22 of the CC2D2A gene. According to ACMG guidelines, c.1177C> T was identified as a pathogenic mutation and c.2704G>A was identified as an uncertain significance. Sanger sequencing showed that there was heterozygous mutation of c.1177C>T and heterozygous variation of c.2704G>A in the parents of the fetus. Conclusions: WES identified a novel homozygous nonsense mutation c.1177C>T in the BBS1 gene of a Chinese fetus. The finding provides more insight into BBS1 mutations in Asian populations in general, and provides a basis for genetic counseling.

The Genetic Etiology Diagnosis of Fetal Growth Restriction Using Single-Nucleotide Polymorphism-Based Chromosomal Microarray Analysis

Background: An increase in pathogenic copy number variants (pCNVs) has been recognized to associate with fetal growth restriction (FGR). Here, we aim to explore the application value of chromosomal microarray analysis (CMA) in prenatal diagnosis of FGR.Methods: Prenatal ultrasound was applied to identify FGR. A total of 149 pregnant women with FGR were enrolled in our study. All subjects underwent karyotype analysis and CMA to reveal the chromosomal abnormalities.Results: In this study, all subjects were successfully detected by karyotype and CMA analyses. Of these subjects, the chromosomal abnormalities detection rate was 5.37% (8/149) for karyotyping and 13.42% (20/149) for CMA, respectively. Among them, an 8.05% (12/149) incremental yield of CMA over karyotype analysis was observed (p = 0.004). In addition, a significant difference of pCNV detection rate was observed between the groups with different high-risk factors (p = 0.005). The FGR with structural anomalies group showed the highest pCNV detection rate (33.33%), followed by the FGR with non-structural anomalies group (8.77%) and the isolated FGR group (8.06%).Conclusion: In conclusion, CMA technology showed an effective application value in etiology diagnosis of FGR. We believe that CMA should be recommended as first-line detection technology for prenatal diagnosis in FGR.

Cellular and Molecular Genetic Analysis of 1980 Male Infertility Patients

Background The aims of this study were to investigate the distribution of chromosome karyotype abnormality and azoospermia factor (AZF) microdeletion on Y chromosome in male infertility patients and its effect on infertility. Further, the study aimed to guide fertility in patients. Methods A total of 1980 azoospermic and oligoospermic male infertility patients were selected from the male outpatient department of our hospital from January 2016 to December 2019. Peripheral blood was collected from the patients for karyotype analysis. Further, AZF microdeletion analysis on Y chromosome was performed by capillary electrophoresis. Results Among the patients of male infertility, 178 had chromosomal abnormality (8.99%, 178/1980). Among them, 98 had an abnormal chromosome number. Among the 98 patients, 47, XXY was the most common (80 cases), accounting for 44.99 % (80/178) of abnormal karyotypes. There were 211 cases of AZF microdeletion on Y chromosome, with a total deletion rate of 10.66% (211/1980). The most common type was AZFb/c deletion (sY1192 140 cases), accounting for 66.3 % (140/211). Conclusion Karyotype abnormality and AZF gene microdeletion are important causes of male infertility. Men with Yqh-, del(Y) (q11) have a higher risk of AZF microdeletion. Infertility patients should routinely undergo cell and molecular genetic tests to guide patient fertility.

PSX-B-16 Recovery of live cells from refrigerated sheep skin after different days of cold storage

Cryopreservation of tissues from domesticated and wild relatives has been suggested to conserve genetic diversity. Ensuring that the tissues have live cells prior to preservation, especially in postmortem tissues, is an essential stem for success. How long cells live after clinical death is not precisely known in animals. The objective of this study was to evaluate the limits of cell survival in sheep skin stored at 4°C postmortem. Ear skin was procured from six random but healthy slaughtered animals and stored at 4°C in the lab. Ten explants (2–3 mm2) were cultured from each animal in DMEM media with 10% FBS, 50 units/mL of penicillin, 50 µg/mL of streptomycin, and 2.5 µg/mL of fungizone on two 60 mm dishes after 0, 10, 20, 27, 30, 35, 38, 41, 45, 50, 55, 60, 65 and 70 days of storage. Outgrowth of fibroblast-like cells around the explants was scored after 10 days of culture in a CO2 incubator. Results show outgrowth of cells up to 65 days of postmortem storage. Out of 476 explants adhered to dish surface, 374 (78.58%) exhibited outgrowth. The number of outgrowing cells decreased with increasing postmortem storage time interval. To test the differences between cell cultures obtained from postmortem fresh and stored tissues, we established secondary cultures from primary cells of 0-dpm and 65-dpm time points from selected cell lines. Both cultures exhibited similar growth morphology and growth curve, could be cryopreserved with &gt;80% post freezing cell viability, lasted in cultures up to 35 passages, and expressed GFP gene upon transfection with a GFP gene containing plasmid vector. The karyotype analysis of 65-dpm tissue derived cells revealed a normal female karyotype without any genetic aberrations. These results suggest that normal proliferative cells can be recovered from sheep skin up to about 2 months postmortem, if kept refrigerated.

Tools for Drawing Informative Idiograms

Each species has a typical karyotype, which represents the phenotypic appearance of the somatic chromosomes including number, size, and morphology. Idiogram is a diagrammatic representation of the chromosomes showing their relative size, homologous groups and different cytogenetic landmarks. Chromosomal analysis of cytological preparations is an essential component of many investigations which involves the calculation of karyotypic parameters and generation of idiogram. Although various tools are available for karyotype analysis, here we demonstrate karyotype analysis using our recently developed tool named KaryoMeasure. KaryoMeasure is a semi-automated free and user-friendly karyotype analysis software that facilitates data collection from different digital images of the metaphase chromosome spreads and calculates a wide variety of chromosomal and karyotypic parameters along with the related standard errors. KaryoMeasure draws idiograms of both diploid and allopolyploid species into a vector-based SVG or PDF image file.