G-banded Karyotype Research Articles

Genetic analysis of a Chinese pedigree with rare mosaic 11q partial duplication and a literature review

To explore the genetic characteristics of a Chinese pedigree with rare mosaic 11q partial duplication and its pathogenetic mechanisms. A pedigree which underwent prenatal diagnosis at Wenzhou Central Hospital between September 25, 2015 and November 30, 2023 was selected for the study. Clinical data were collected from the pedigree. Peripheral blood samples from the parents, amniotic fluid from the fetus, and peripheral blood sample from the neonate were obtained. Genetic testing was carried out by using G-banded chromosomal karyotyping and single nucleotide polymorphism array (SNP-array) technology. Relevant literature was searched in the CNKI, Wanfang Data Knowledge Service Platform, and PubMed databases to summarize the clinical phenotypes of patients with 11q partial duplication. This study was approved by the Medical Ethics Committee of Wenzhou Central Hospital (Ethics No. L2024-07-080). The pregnant woman (G3) had a history of adverse pregnancy outcomes. During her first pregnancy (G1), prenatal ultrasound indicated intrauterine growth restriction and a Dandy-Walker variant. Follow-up at 8 years of age showed developmental delays and mild intellectual disability. During her second pregnancy (G2), prenatal ultrasound revealed nasal bone hypoplasia, and the pregnancy was terminated at 23rd gestational week. During her third pregnancy (G3), all prenatal tests were normal, and the neonate showed normal growth and development at 4 months of age. The karyotype of amniotic fluid of her first pregnancy was 46,X?, and the SNP-array analysis of neonatal peripheral blood showed arr[GRCh37/hg19]11q13.4q25(70432450_134607121)×2~3, with a mosaicism rate being approximately 40%. The karyotype for her second pregnancy was 46,X?,rec(11)dup(11q)inv(11)(p15q13)dmat[6]/46,X?[27], and the SNP-array result was arr[GRCh38]11q13.4q25(71406636_135067522)×2~3, with a mosaicism rate being approximately 75%. The karyotype for her third pregnancy was 46,X?,inv(11)(p15q13)mat, and the SNP-array result was arr(XN)×1,(1~22)×2. The karyotype of the woman was 46,XX,inv(11)(p15q13), and that of her husband was 46,XY. A review of 12 similar cases (including G1) from the literature revealed that the common clinical phenotypes of 11q partial duplication included intellectual disability (12/12), developmental delay (12/12), ear abnormalities (12/12), microcephaly (10/12), seizures (8/12), hypotonia (8/12), and congenital heart malformations (7/12). Mosaic partial duplication of 11q may underlie the genetic etiology of this pedigree. The pregnant woman is a carrier of an inversion on chromosome 11, which might have formed the mosaic 11q partial duplication through meiotic errors and mitotic trisomy rescue mechanisms during reproduction.

Maternal X chromosome pericentric inversion resulting in the genetic analysis of offspring pedigrees with deletions at Xp22.33 and Xp22.33p11.3, and duplications at Xq27.3q28: Case report.

This study investigates the genetic cause of primary infertility and short stature in a woman, focusing on maternal X chromosome pericentric inversion and its impact on offspring genetic outcomes, including deletions at Xp22.33 and Xp22.33p11.3, and duplications spanning Xq27.3 to the distal end of the X chromosome's long arm. The proband presented with primary infertility, menstrual irregularities, and ultrasound findings indicating a small uterus. Peripheral blood G-banded karyotype analysis and single nucleotide polymorphism array analysis revealed a 46,X,rec(X)dup(Xq)inv(X)(p11.3q27)dmat karyotype in the proband, inherited from her mother. Genetic testing identified pathogenic deletions at Xp22.33 and Xp22.33p11.3, and a pathogenic duplication at Xq27.3q28. Genetic counseling and pedigree analysis were conducted to trace the maternal origin of the pericentric inversion and assess recurrence risks. The study confirmed the maternal X chromosome pericentric inversion caused the observed genetic abnormalities, with a 50% recurrence risk for X-linked inheritance. Maternal X chromosome pericentric inversion significantly affects offspring genetic outcomes. Assisted reproductive technologies, including in vitro fertilization with preimplantation genetic testing, are recommended to reduce recurrence risks in future pregnancies. Prenatal genetic testing is advised for natural conception to ensure fetal genetic health.

Genetic Analysis of 17q Terminal Partial Trisomy.

Chromosomal trisomy syndrome is associated with diverse clinical phenotypes, including intellectual disability. Partial trisomy of the distal 17q is a rare anomaly with similar clinical features, including psychomotor and growth deficits, facial dysmorphism, and microcephaly. Here, we describe three patients from two unrelated families with terminal trisomy 17q. We performed G-banding karyotype and chromosomal microarray analyses. The child in Family 1 had a 31.3 Mb mosaic duplication on chromosome 17. Family 2 comprised dizygotic twins with a 263 kb deletion on chromosome 15 and a 9.2 Mb duplication on chromosome 17; however, normal karyotyping results were obtained for both parents. We also analyzed the genetic mechanisms underlying the occurrence of these chromosomal aberrationsand summarized the literature describing known genotype-phenotype correlations. Given the rarity of partial trisomy of terminal 17q, these cases will provide new insights into the diagnosis of this condition and genotype-phenotype correlations, which can aid in the detection of such conditions and genetic counseling.

Prenatal diagnosis analysis of three cases of Turner syndrome fetuses with complex mosaic small supernumerary marker chromosomes

To explore the value of applying multiple genetic testing techniques for the prenatal diagnosis of Turner syndrome fetuses with complex mosaic small supernumerary marker chromosomes (sSMC). Chromosomal karyotypes of amniotic fluid samples from 5 030 pregnant women who had undergone amniocentesis at the Prenatal Diagnosis Center of the Third Affiliated Hospital of Zhengzhou University from January to December 2022 were retrospectively reviewed. Three fetuses with complex mosaicism fetuses (carrying 2 types of sSMC) were selected as the study subjects. Genetic tests including G-banded chromosomal karyotyping analysis, fluorescence in situ hybridization (FISH), chromosomal microarray analysis (CMA), and copy number variation sequencing (CNV-seq) were used to clarify the origin and mosaic status of the sSMC. This study has been approved by the Medical Ethics Committee of the Third Affiliated Hospital of Zhengzhou University (No. 2023-159-01). G-banded chromosomal analysis of fetus 1 showed a karyotype of 45,X[64]/46,X,+mar1[13]/46,X,+mar2[3]. FISH results showed that 52% of of its cells had contained one X chromosome signal, whilst 48% contained two X chromosome signals. CMA results revealed the fetus had harbored a 32.32 Mb and a 50.93 Mb deletion in Xp22.33p21.1 and Xq22.2q28 regions, respectively, in addition with mosaic deletions of approximately 1.43 copies, 1.78 copies and 1.43 copies in the Xp21.1p11.1, Xq11.1q21.1 and Xq21.2q22.2 regions, respectively. The fetus 2 had a karyotype of 45,X[27]/46,X,+mar1[14]/46,X,+mar2[12]. FISH results indicated that 88% of its cells contained one X chromosomes signal and two Y chromosome signals, and 12% contained signals for one X chromosomes signal and one Y chromosome signal. CNV-seq results revealed a deletion of 7.74 Mb in the Yq11.222q11.23 region and a mosaic duplication of approximately 1.738 copies in the Yp11.31q11.221 region. The fetus 3 had a karyotype of 45,X[60]/46,X,+mar1[11]/46,X,+mar2[6]. FISH results showed that 28% of its cells contained one X chromosome signal, and 72% contained tow X chromosome signals. CNV-seq results revealed deletions of 55.60 Mb and 53.50 Mb in the Xp22.33p11.1 and Xq22.1q28 regions, respectively, along with a mosaic deletion of approximately 1.85 copies in the Xp11.1q13.2 region and a mosaic repeats of approximately 2.66 copies in the Xq13.2q22.1 region. The sSMCs in the 3 fetuses had all originated from sex chromosomes and were of complex mosaic type. After genetic counseling, the three couples had all opted to terminate the pregnancy. The combined use of multiple genetic testing techniques has determined the origin and structure of complex mosaic sSMCs and provided a basis for prenantal diagnosis and genetic counseling.

Prenatal diagnosis of a fetus with mosaicism ring chromosome 2

To explore the genetic basis for a fetus with increased risk for Down syndrome and cardiac anomalies discovered by prenatal ultrasonography. A pregnant woman presented at the Women and Children's Hospital of Ningbo University on August 21, 2023 were selected as the study subject. Clinical data were retrospectively analyzed. Maternal peripheral blood sample was collected for non-invasive prenatal testing (NIPT) based on fetal free DNA. Amniotic fluid sample was collected for G-banded chromosomal karyotyping analysis. Trio-whole exome sequencing (WES) was also carried out on the amniotic fluid sample and peripheral blood samples from the couple. Copy number variation (CNV) identified by the WES was validated by real-time fluorescent quantitative PCR (qPCR). Chromosomal karyotyping was also carried out for the couple. This study has been approved by the Medical Ethics Committee of Women and Children's Hospital of Ningbo University (No. EC2020-048). Ultrasound examination at 22+6 gestational weeks had indicated intrauterine growth retardation (IUGR). The fetus was also found to have ventricular septal defect, overriding aorta and pulmonary stenosis. NIPT indicated a low risk for aneuploidy of chromosomes 13, 18 and 21. G-banding analysis revealed that the fetus had a karyotype of 45,XY,-2[5]/46,XY,r(2)(p25q37)[55]. WES has identified a deletion of approximately 1 614.28 kb in the 2p25.3 region, namely seq[hg38]del(2)(p25.3p25.3)chr2: g.10500_1624775del. The same deletion was found in neither parent, suggesting a de novo origin. qPCR results confirmed the expression of target genes in the fetal sample to be significantly reduced, whilst no similar anomaly was found in either parent. The mosaicism ring chromosome 2 probably underlay the IUGR and cardiovascular malformations in this fetus.

Clinical phenotype and genetic analysis of a child with partial duplication of 10q and a literature review

To explore the clinical phenotype and pathogenesis of a child with partial duplication in the long arm of chromosome 10 (10q), and conduct a review of relevant literature. A child presented at Lianyungang Maternal and Child Health Care Hospital in April 2018 for growth retardation, intellectual disability, and autism spectrum disorder (ASD) was selected as the study subject. Peripheral blood samples were collected from the child and his parents for G-banded chromosomal karyotyping analysis. Genomic DNA was also extracted for chromosomal microarray analysis (CMA). The clinical phenotype and relevant genes were searched in the Online Mendelian Inheritance in Man (OMIM) and the UK Database of Genomic Variation and Phenotype in Humans using Ensembl Resources (DECIPHER). The pathogenicity of chromosomal variation was analyzed based on guidelines from the American College of Medical Genetics and Genomics (ACMG). Relevant literature was searched from the CNKI, Wanfang Data, and PubMed databases by using keywords such as "10q" "duplication" and "trisomy", with the time set as from the establishment of database to December 1, 2023. This study has been approved by the Medical Ethics Committee of the Lianyungang Maternal and Child Health Care Hospital (No. XM2023030). The clinical phenotype of child had included growth retardation, intellectual disability, and ASD. G-banded chromosomal analysis suggested that the child has a karyotype of 46,XY,dup(10)(q23.31q24.33), whilst both of his parents were normal. CMA analysis of the child revealed that the child was arr[19]10q23.31q24.33(87603382_104948862)×3, with a 17.34 Mb duplication in the 10q23.31q24.33 region. Search of the OMIM database suggested that the duplicated segment has contained 171 genes associated with various diseases, and search of the DECIPHER database has identified cases with overlapping with the duplication. A search of the PubMed database has identified 2 publications involving 2 patients with chromosomal duplications overlapping the 10q23.31q24.33 region with a segment length of > 10 Mb. The 2 patients had mainly manifested growth retardation, intellectual disability, ASD, and facial and limb malformations. The main pathogenic genes had included PTEN, WNT8B, LZTS2, NFKB2, PAX2, KIF11, FRA10AC1, and CNNM2. No similar case was retrieved from the CNKI and Wanfang Data databases. The partial 10q duplication as a novel CNV involving genes such as PTEN and WNT8B probably underlay the growth retardation, intellectual disability and ASD in child 1 . This study has enriched the genotype-phenotype spectrum of patients with partial 10q23.31q24.33 duplications.

Genetic analysis of a child with 18q terminal deletion and aortic regurgitation and a literature review

To explore the genetic characteristics of a child with 18q terminal deletion syndrome. Clinical data of a child presented at the Lianyungang Maternal and Child Health Care Hospital on July 20, 2023 was collected. Peripheral blood sample from the child was subjected to G-banded chromosomal karyotyping and chromosomal microarray analysis (CMA). Relevant literature was searched from CNKI, WanFang and PubMed databases over the past decade (from November 1, 2013 to November 1, 2023) using keywords including "18q-syndrome", "18q deletion syndrome" and "18q terminal deletion". This study was approved by the Lianyungang Maternal and Child Health Care Hospital (Ethics No. LYG-MER2021017). The child, a 4-year-and-6-month-old female, had manifested short stature, intellectual disability, distinctive facial features, aortic regurgitation, auditory canal atresia, and white matter lesions. She was found to have a karyotype of 46,XX,del(18)(q21), whilst the result of CMA was arr[GRCh37]18q21.33q23(60065821_77317445)×1. Both of her parents were found to have a normal karyotype. Literature review has retrieved 7 reports which involved 11 cases with a terminal 18q23 deletion. The phenotypes of cardiac abnormalities have been diverse, with pulmonary stenosis, atrial septal defect and ventricular septal defect being most common. The 18q terminal deletion probably underlay the multiple congenital anomalies and mental retardation in this child.

Prenatal diagnosis of a fetus with 15q11q13 complex duplication syndrome and a literature review

To explore the clinical features and genetic etiology of a fetus with 15q11q13 complex duplication syndrome. A fetus diagnosed with 15q11q13 duplication syndrome at Ningbo Women and Children's Hospital on April 19, 2023 was selected as the study subject. Clinical data was collected, and the fetus was subjected to invasive prenatal diagnosis including G-banded karyotyping and chromosomal microarray analysis (CMA). Following the discovery of chromosomal duplication, trio-whole exome sequencing was carried out to exclude single base variants and confirm the parental original of the duplication. Optical genome mapping was also performed to delineate the structural arrangement of the duplication. Relevant literature was searched in the PubMed, Wanfang Medical Network and CNKI databases using "15q11q13", "duplication", "hexasomy" and "Six fold repetition" as the key words from January 1, 2000 to August 1, 2023 for a review of previously reported 15q11q13 hexasomy cases. This study was approved by the Ningbo Women & Children's Hospital (Ethics No. EC2020-048). The fetus was found to have a mosaicism karyotype of 48,X?,+mar,+idic(15)(q13)[33]/47,X?,+idic(15)(q13)[17]. CMA and trio-WES have all shown a six-fold duplication in the PWS/AS critical region (PWACR) at 15q11.2q13.2 and quadruple duplication of 15q13.2q13.3 region, which have derived from its mother and formed supernumerary marker chromosomes (SMCs). Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the 15q11.2q13.2 sixfold duplication was classified as pathogenic, whilst the 15q13.2q13.3 quadruple duplication was classified as variant of uncertain significance. Literature search has identified 11 cases of 15q11q13 duplication involving hexasomy of the PWACR, with all cases showing mental retardation, language delay and hypotonia, and most of them also had motor retardation, epilepsy and mild facial dysmorphism. Hexasomy for the PWACR combined with tetrasomy of 15q13.2q13.3 probably underlay the left hand polydactyly, polyhydramnios and intrauterine growth retardation in this fetus.

Clinical phenotype and genetic analysis of a rare case with 6p duplication and terminal deletion syndrome

To explore the genetic basis for a child with developmental delay and intellectual deficit (DD/ID). A child who was admitted to the Maternal and Child Health Care Hospital of Longhua District of Shenzhen City on June 3, 2023 due to DD/ID, craniofacial malformations, and recurrent infections of upper respiratory tract was selected as the study subject. G-banded chromosomal karyotyping was carried out for the child and her parents. Low-depth genome-wide copy number variation sequencing (CNV-seq) and chromosomal microarray analysis (CMA) were used to screen for genome-wide copy number variations (CNV), and fluorescence in situ hybridization (FISH) was used to verify the origin of candidate CNV. The child, an 8-year-old girl, had featured unexplained growth and intellectual development delay, multiple craniofacial malformations, and recurrent infections of the upper respiratory tract. She was found to have a karyotype of 46,XX,der(6)add(6)(q23), while both of her parents were normal. Both CNV-seq and CMA showed that the child has harbored a 21.38 Mb interstitial duplication at 6p25.3p22.3 and a 0.78 Mb terminal deletion at 6p25. FISH verified that both the duplication and deletion had occurred de novo. The abnormal phenotype of the child may be attributed to the 6p duplication and terminal deletion.

Comprehensive diagnosis and genetic analysis of two children with ring chromosome 18

To clarify the genetic diagnosis of two children with ring chromosome 18 and explore their mechanisms and clinical phenotypes. Two patients treated at the Children's Hospital of Henan Province respectively in June 2022 and March 2023 were selected as the study subjects. Genetic testing and diagnosis were carried out through copy number variation sequencing (CNV-seq), G-banded chromosomal karyotyping, and whole exome sequencing (WES). Child 1 had mainly manifested developmental delay, white matter hypoplasia, type 1 diabetes mellitus, and micropenis. He was found to have a chromosomal karyotype of 46,XY,r(18)(p11.21q22.1)[40]/46,XY[7], and CNV-seq results showed that he has a 14.86 Mb deletion at 18p11.21p11.32 and a 14.02 Mb deletion at 18q22.1q23. Child 2 had peculiar facial features, delayed white matter myelination, developmental delay, atrial septal defect, severe sensorineural deafness, and congenital laryngeal stridor. He was found to have a chromosomal karyotype of 46,XY,r(18)(p11.2q23). CNV-seq result proved that he had a 14.86 Mb deletion at 18p11-21p11.32 and a 20.74 Mb deletion at 18q21.32q23. WES has failed to detect single nucleotide variants (SNVs) in either child, but revealed a large segmental deletion at chromosome 18 in both of them. Both children were diagnosed with ring chromosome 18 syndrome. The different size of the deletional fragments in the 18q region and mosaicism of ring chromosome 18 in child 1 may underlay the variation in their clinical phenotypes. The type 1 diabetes mellitus and micropenis noted in both children are novel features for ring chromosome 18 syndrome.

Genetic analysis of a child with mos 46,X,psu idic(X)(q21.3)[40]/45,X[3]

To explore the correlation between structural chromosomal abnormality and clinical characteristics of a child featuring gonadal dysplasia. A 13-year-old child who was admitted to Lianyungang Maternal and Child Health Care Hospital on February 7, 2023 for primary amenorrhoea and occasional abdominal pain was selected as the study subject. Clinical data of the child was collected, and peripheral blood samples of the child and her parents were collected. G-banding chromosomal karyotyping and copy number variation sequencing (CNV-seq) were carried out. "Pseudodual centromere isochromosome X" and "psu idic(X)" were used as keywords to search the CNKI, Wanfang and PubMed databases, and the search period was set as from January 1, 2002 to June 1, 2023. Relevant literature on the structural abnormality of X chromosome was searched and analyzed retrospectively. The child has a height of 153 cm and weighed 45 kg. She has no obvious facial dysmorphism. Laboratory tests showed that she had higher FSH and luteinizing hormone, and lower E2. Ultrasonography showed that she had small ovaries and rudimentary uterus. She was found to have a karyotype of 46,X,psu idic(X)(q21.3)[40]/mos 45,X[3], whilst both of her parents had a normal karyotype. CNV-seq showed that she had a 63.27 Mb deletion in Xq21.32q28 and a 91.59 Mb duplication in Xp22.33q21.32 (mosaicism rate = 74%). A total of 11 relevant literature were retrieved. Clinical phenotypes of patients with similar structural chromosomal abnormalities were diverse, which was closely related to the mosaicism rate of the 45,X karyotype and the location of the breaking point. 46,X,psu idic(X)(q21.3)/45,X probably underlay the dysplasia of uterus and ovary and sex hormone abnormalities in this child, while her height was spared. Deletion of Xq21.32q28 is a key factor leading to Turner syndrome-like phenotype such as rudimentary uterus and ovarian dysplasia.

The establishment of pulp polyp-derived mesenchymal stem cells with normal karyotype

Background/PurposePulp polyp is often eliminated as dental waste. Pulp polyp cells were reported to have high proliferation activity which might be comprised of stem cells. However, little has been known on the presence of stem cells in the pulp polyp. Moreover, pulp polyp cells might contain chromosomal abnormality. The present study was conducted to investigate the presence of pulp polyp stem cells, which could later be propagated and confirmed as normal/non-pathogenic cells using karyotype analysis. Materials and methodsCollected pulp polyps were minced, enzymatically digested, and cultured. Expression of mesenchymal stem cell (MSC) markers on pulp polyp cells were analyzed using flow cytometry. Multilineage differentiation capacity was assessed by culturing the cells in osteogenic, chondrogenic, and adipogenic differentiation media. Genomic stability of the cells was evaluated with G-banded and molecular karyotype analyses. ResultsPulp polyp cells appeared as fibroblasts-like cells. The cells were positive for cluster of differentiation (CD)105, CD90, and CD73, and negative for CD45, CD34, CD11b, CD19, and human leukocyte antigen (HLA)-DR. The cells were capable of osteogenic, chondrogenic, and adipogenic differentiation. G-banded karyotype analysis showed that there was no abnormality in the number or structure of chromosomes in pulp polyp-derived MSCs (PP-MSCs). Molecular karyotype analysis revealed that all copy number variations identified in PP-MSCs were not pathogenic. ConclusionPP-MSCs, which fulfill the minimal criteria for MSCs and are proven to have normal karyotype, have been successfully established. PP-MSCs might be a promising and safe candidate that can be considered for pulp-dentin complex regeneration.

Optimal prenatal genetic diagnostic approach for posterior fossa malformation: karyotyping, copy number variant testing, or whole-exome sequencing?

BackgroundPosterior fossa malformation (PFM) is a relatively uncommon prenatal brain malformation. Genetic diagnostic approaches, including chromosome karyotyping, copy number variant (CNV) testing, and whole-exome sequencing (WES), have been applied in several cases of fetal structural malformations. However, the clinical value of appropriate genetic diagnostic approaches for different types of PFMs has not been confirmed. Therefore, in this study, we aimed to analyze the value of different combined genetic diagnostic approaches for various types of fetal PFMs.MethodsThis retrospective study was conducted at Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing Maternal and Child Health Care Hospital. Fifty-one pregnant women diagnosed with fetal PFMs who underwent genetic testing in our hospital from January 1, 2017 to December 31, 2022 were enrolled; women with an isolated enlarged cisterna magna were excluded. All participants were categorized into two groups according to the presence of other abnormalities: isolated and non-isolated PFMs groups. Different combined approaches, including karyotype analysis, CNV testing, and trio-based WES, were used for genetic analysis. The detection rates of karyotype analysis, CNV testing, and WES were measured in the isolated and non-isolated groups.ResultsIn isolated PFMs, pathogenic/likely pathogenic (P/LP) CNVs were detected in four cases (36.36%, 4/11), whereas G-banding karyotyping and WES showed negative results. In non-isolated PFMs, a sequential genetic approach showed a detection rate of 47.5% (19/40); karyotyping revealed aneuploidies in five cases (16.67%, 5/30), CNV testing showed P/LP CNVs in five cases (16.13%, 5/31), and WES identified P/LP variants (in genes CEP20, TMEM67, OFD1, PTPN11, ARID1A, and SMARCA4) in nine cases (40.91%, 9/22). WES showed a detection rate of 83.33% (5/6) in fetuses with Joubert syndrome. Only six patients (five with Blake’s pouch cyst and one with unilateral cerebellar hemisphere dysplasia) survived.ConclusionsWe recommend CNV testing for fetuses with isolated PFMs. A sequential genetic approach (karyotyping, CNV testing, and WES) may be beneficial in fetuses with non-isolated PFMs. Particularly, we recommend WES as the first-line genetic diagnostic tool for Joubert syndrome.

P-529 Optical Genomic Mapping (OGM) enable cryptic chromosomal rearrangements in a couple with recurrent miscarriages undergoing preimplantation genetic diagnosis

Abstract Study question Could Optical Genomic Mapping (OGM) detect chromosomal structural variations (SVs) below the detection limit of conventional cytogenetic diagnostics in couples with recurrent miscarriages? Summary answer The Next-generation cytogenetics of Optical Genomic Mapping confirmed a cryptic chromosomal translocation in a couple with recurrent miscarriages undergoing preimplantation genetic testing for aneuploidies (PGT-A). What is known already Chromosomal structural variants (SVs) significantly contribute to infertility, recurrent pregnancy losses and congenital defects... Approximately 2-4% of couples facing recurrent miscarriages carry chromosomal abnormalities. Currently, their detection is based on convencional cytogenetic methods (karyotyping, FISH or array-CGH), however cryptic chromosomal rearrangements are difficult to identify. Next-generation cytogenetics such as OGM is able to detect SVs with a resolution of < 500 kb, allowing a better understanding of the cause of patient’s infertility. In our study, we utilized OGM to confirm a cryptic translocation in a couple experiencing recurrent miscarriages, aiming to enhance our understanding of the underlying causes of their infertility. Study design, size, duration Our study focuses on a non-consanguineous couple diagnosed with recurrent pregnancy loss that attended to our private fertility clinic to undergo assisted reproductive treatment (ART) with their autologous semen, oocytes and PGT-A. Conventional cytogenetic technique was performed on both partners by G-banding karyotyping. PGT-A was performed by NGS (Veriseq, Illumina). Optical Genomic Mapping (OGM) served as a diagnostic tool, complemented by lymphocyte FISH analysis to confirm chromosomal structural variations in these infertile patients. Participants/materials, setting, methods Ultra High Molecular Weight genomic DNA (UHMW gDNA) was extracted from frozen human peripheral blood of both partners. UHMW gDNA was quantified using Qubit dsDNA assay BR kit and Qubit 4 Fluorometer (Thermo Fisher Scientific). We used Saphyr system (Bionano Genomics) for cytogenetic diagnosis. The consensus genomic map was constructed by de novo assembly in the Bionano Solve data analysis software. SVs were identified by aligning Optical Genomic Map with the reference genome GRCh38/hg38. Main results and the role of chance The couple had suffered four prior miscarriages and undergoing three PGT-A cycles. Both partners provided a normal karyotype. Among 13 biopsied embryos, four showed CNVs on chromosomes 2(p24.3è25.3, 12Mb) and 6(p24.1è25.3, 11Mb). Following this result we suspected that the couple might has a cryptic chromosomal structural abnormalities. To test our hypothesis, we performed OGM. We analysed UHMW gDNA samples from both partners. OGM generated an average of 823.67Gb. The average N50 molecule length (≥150kb) was 318kb. The average mapping rate was 90.75% and the average labeling rate was 15.30 labels per 100kb. The average effective coverage depth was 242X. OGM successfully identified a cryptic translocation in the male partner ogm[GRch38]t(2;6)(p24.3;24.1), while the female partner showed no abnormalities. Translocation breakpoints identified by OGM were Chr2:12.560.508 and Chr6:11.622.593. The translocated segments were near to the detection limit of conventional cytogenetic analysis and with similar pattern that could be the reason for missed detection by karyotyping. Importantly, we identified one gene disrupted by OGM rearrangement breakpoint: MIR3681HG. A previous study considered that CNV loci of 2p24.3, including MIR3681HG gene, could be associated with spermatogenesis. The lymphocyte FISH analysis finding corroborated the OGM result. Limitations, reasons for caution OGM is a very powerful method for detecting chromosomal structural variants in human genomes and is able to detect cryptic structural variants. However, this is a very new diagnostic application, so there is limited experience with OGM for correlating structural variations results to infertility diagnosis in couples with recurrent miscarriages. Wider implications of the findings A balanced cryptic translocation has been identified in a couple with recurrent miscarriages. Our case report demonstrates the high diagnostic capability of Optical Genomic Mapping for cryptic SVs. OGM technology allows a more accurate cytogenetic diagnosis by detecting cryptic structural variants with high resolution in infertile patients with recurrent miscarriages. Trial registration number not applicable

The impact of comparative genomic hybridization/single-nucleotide polymorphism microarray in risk stratification of pediatric acute lymphoblastic leukemia.

The objective of this study is to assess the concordance and added value of combined comparative genomic hybridization plus single-nucleotide polymorphism microarray (CGH/SNP) analyses in pediatric acute lymphoblastic leukemia (ALL) risk stratification compared to conventional cytogenetic methods. This is a retrospective study that included patients aged 1-18years diagnosed with de novo ALL at Sainte-Justine Hospital between 2016 and 2021. Results from conventional cytogenetic and molecular analyses were collected and compared to those of CGH/SNP. A total of 135 ALL patients were included. Sample failures or non-diagnostic analyses occurred in 17.8% cases with G-banding karyotypes versus 1.5% cases with CGH/SNP. The mean turnaround time for results was significantly faster for CGH/SNP than karyotype with 5.8 versus 10.7days, respectively. The comparison of ploidy assessment by CGH/SNP and G-banding karyotype showed strong concordance (r=.82, p<.001, r2=.68). Furthermore, G-banding karyotype did not detect additional clinically relevant aberrations that were missed by the combined analysis of CGH/SNP and fluorescence in situ hybridization. The most common gene alterations detected by CGH/SNP were deletions involving CDKN2A (35.8%), ETV6 (31.3%), CDKN2B (28.4%), PAX5 (20.1%), IKZF1 (12.7%), and copy-neutral loss of heterozygosity (CN-LOH) of 9p (9.0%). Among these, only ETV6 deletion was found to have a significant prognostic impact with superior event-free survival in both univariate and multivariate analyses (adjusted hazard ratio 0.08, 95% confidence interval: 0.01-0.50, p=.02). CGH/SNP provided faster, reliable, and highly concordant results than those obtained by conventional cytogenetics. CGH/SNP identified recurrent gene deletions in pediatric ALL, of which ETV6 deletion conferred a favorable prognosis.

Comparative analysis of the application with the combination of CMA and karyotype in routine and late amniocentesis.

This is a retrospective comparative study. We aimed to analyze the results of karyotype and chromosomal microarray analysis (CMA) of amniotic fluid across different gestational weeks and evaluate the clinical value in prenatal diagnosis, particularly in the late pregnancies. Samples from 580 pregnant women of 18-23weeks of gestation (mid-gestation group) and 196 pregnant women of 24-32weeks of gestation (late group) were performed both standard G-band karyotype analysis and CMA. Among the 580 pregnant women in the routine group, the most common indications were positive Down's screening (213/580, 36.7%), followed by advanced maternal age (196/580, 33.8%); while fetal structural anomalies on ultrasonography were the top reason for amniocentesis in the late group (56/196, 28.6%). In the routine group, the total detection rate was 12.1% (70/580), of which 4.1% (24/580) were identified by karyotype analysis and 11.2% (65/580) by CMA. The total detection rate was 15.3% (30/196) in the late group, of which 5.1% (10/196) were detected by karyotype analysis, and 14.3% (28/196) by CMA. Karyotype analysis and CMA are complementary in detecting chromosomal abnormalities. Amniotic cavity puncture in the karyotype analysis in 18-23weeks of gestation and 24-32weeks of gestation is safe and effective, more obvious effect on the latter.

Clinical validation of an abbreviated karyotype analysis protocol for fertility evaluation

Conventional G-banded karyotype is an essential tool for detecting chromosomal variants in patients undergoing fertility evaluation. In Australia, 15 cells are traditionally analysed or counted, to enhance detection of mosaic chromosomal variants. However, this protocol is not backed by clinical evidence. This study aims to assess the test performance of an abbreviated 5-cell karyotype analysis protocol in adult patients undergoing fertility evaluation. A retrospective review of 53,293 blood karyotype tests, performed between 2019 and 2023, was conducted on a patient cohort primarily referred by reproductive endocrinology specialists. There were 513 variants reported in this cohort. Low level mosaic variants, where the variant was observed in less than 40% of cells, were reported in 13 cases, or one in 4,100 patients. Due to reduced sensitivity for low level mosaic variants, a 5-cell protocol is estimated to have a test sensitivity of 97.3% and a negative predictive value of 99.97%. A decision-making flowchart is proposed and we show that additional chromosome analysis and/or counts would be triggered in fewer than one in 10 cases using a 5-cell protocol, whilst remaining appropriate for detecting clinically significant mosaicism. A 5-cell karyotype analysis protocol therefore maintains analytical and clinical validity in adult patients undergoing fertility-related blood karyotyping. Future research is recommended to validate these findings across laboratories and to explore their application to other clinical contexts.

Establishment of Canine Oral Mucosal Melanoma Cell Lines and Their Xenogeneic Animal Models.

Canine oral melanoma is the most prevalent malignant tumor in dogs and has a poor prognosis due to its high aggressiveness and high metastasis and recurrence rates. More research is needed into its treatment and to understand its pathogenic factors. In this study, we isolated a canine oral mucosal melanoma (COMM) cell line designated as COMM6605, which has now been stably passaged for more than 100 generations, with a successful monoclonal assay and a cell multiplication time of 22.2 h. G-banded karyotype analysis of the COMM6605 cell line revealed an abnormal chromosome count ranging from 45 to 74, with the identification of a double-armed chromosome as the characteristic marker chromosome of this cell line. The oral intralingual and dorsal subcutaneous implantation models of BALB/c-nu mice were successfully established; Melan-A (MLANA), S100 beta protein (S100β), PNL2, tyrosinase-related protein 1 (TRP1), and tyrosinase-related protein 2 (TRP2) were stably expressed positively in the canine oral tumor sections, tumor cell lines, and tumor sections of tumor-bearing mice. Sublines COMM6605-Luc-EGFP and COMM6605-Cherry were established through lentiviral transfection, with COMM6605-Luc-EGFP co-expressing firefly luciferase (Luc) and enhanced green fluorescent protein (EGFP) and COMM6605-Cherry expressing the Cherry fluorescent protein gene. The COMM6605-Luc-EGFP fluorescent cell subline was injected via the tail vein and caused lung and lymph node metastasis, as detected by mouse live imaging, which can be used as an animal model to simulate the latter steps of hematogenous spread during tumor metastasis. The canine oral melanoma cell line COMM6605 and two sublines isolated and characterized in this study can offer a valuable model for studying mucosal melanoma.

Evaluation the Application of Karyotype Analysis and Chromosome Microarray in Prenatal Diagnosis.

We aimed to compare the difference of the chromosomal abnormalities using karyotype analysis and chromosomal microarray (CMA) as well as to evaluate their application in different prenatal diagnosis indications. Overall, 3007 pregnant women with prenatal diagnosis indications from Medical Genetics Department of Linyi Women and Children's Health Care Hospital, who underwent standard G-banded karyotype analysis and CMA, were enrolled from 2018-2022. G-banded karyotype analysis and CMA were undergone simultaneously. All fetuses with genetic variants were enrolled for further analyzing. The frequency and differences of chromosomal abnormalities of the two methods were compared in different prenatal diagnosis indications groups. CMA improved 4.09% (123/3007) of genetic changes compared karyotype analysis. CMA is on par with karyotyping for detection of aneuploidies and gross unbalanced rearrangements. Serological screening and ultrasound abnormalities were the main indications of prenatal diagnosis. The detection rate of chromosomal abnormalities was highest in non-invasive prenatal testing (NIPT) abnormal group. In the ultrasound abnormality group, the detection rate of genetic variants in nuchal translucency (NT) increased group was higher than other subgroups and there was statistically significant difference in the detection rate of pCNVs. CMA can detect 5.57% (40/718) more genetic abnormalities in ultrasound abnormality group on the normal karyotype. CMA improved 0.67% (20/3007) of genetic changes with clinically significant compared karyotype, brought 3.42% (103/3007) of variants with uncertain significance (VOUS). CMA identified additional, clinically significant genetic variants on the basis of normal karyotype analysis, brought a proportion of unclear significant variants. All the pregnant women accepted amniocentesis should be informed about their characteristics of karyotype analysis and CMA by genetic counselors.

Genetic analysis of a fetus with Pitt-Hopkins syndrome due to a 18q21.2q21.31 microdeletion

To carry out invasive prenatal diagnosis for a fetus with ultrasound-indicated agenesis of corpus callosum and explore its genetic etiology. A pregnant woman presented at the Affiliated Hospital of Putian College on December 16, 2022 was selected as the study subject. Amniotic fluid and peripheral blood samples from the fetus and the couple were collected. Conventional G-banded chromosomal karyotyping was carried out, and whole-genome copy number variation analysis was performed using single nucleotide polymorphism microarray (SNP-array). The karyotypes of the fetus and the couple were normal by the G-banding analysis. SNP-array analysis of the amniotic fluid sample revealed a 4.5 Mb microdeletion in the 18q21.2q21.31 region of the fetus. SNP-array analysis of peripheral blood samples from the couple did not find any abnormality. Through G-banded chromosomal karyotyping and SNP-array analysis, a fetus with 18q21.2q21.31 microdeletion was identified, which has conformed to the diagnosis of Pitt-Hopkins syndrome. Above finding has provided a basis for genetic counseling for the couple.