Application value of low depth and high-throughput gene sequencing to detect chromosome copy number variations in prenatal diagnosis / 中国医师杂志

Objective:To explore the application value of low depth and high-throughput gene sequencing in detecting chromosome copy number variations (CNVs) in different risk indicators of prenatal diagnosis.Methods:We retrospectively analyzed the genetic testing results of 1 597 pregnant women who underwent amniocentesis in Maternal and Child Health Care of Zaozhuang from January 2017 to December 2020 to obtain amniotic fluid cells and undergo high-throughput gene sequencing for chromosome copy number variation (CNV-seq). The CNV-seq results was compared with G-banding karyotype analysis.Results:The success rate of CNV-seq detection in 1 597 cases of amniotic fluid cells was 100%, and 301 cases of chromosomal CNVs were found, with an abnormal rate of 18.85%. Among them, 208 cases of chromosomal CNVs with definite pathogenicity accounted for 69.10%; There were 93 cases of CNVs with unknown pathogenicity, accounting for 30.90%. Among 208 cases of CNVs with definite pathogenicity, 166 cases had abnormal chromosome aneuploidy, accounting for 79.81%; 42 cases of chromosomal deletion / duplication structural abnormality, accounting for 20.19%. The detection of chromosomal copy number abnormalities in different prenatal diagnosis indicators was different. The incidence of chromosomal CNVs in the NIPT screening risk group was the highest (53.09%, 163/307), followed by the ultrasonic structural abnormality group (22.38%, 32/143), the chromosomal abnormality carrying group (12.50%, 5/40), the other abnormality group (11.34%, 22/194), the serological prenatal screening high-risk group (9.04%, 74/819), and the elderly group (5.32%, 5/94). Compared with G-banding karyotype analysis, CNV-seq has a detection rate of 100% for 166 cases of chromosomal aneuploidy and 13 cases of unbalanced chromosomal structural abnormalities confirmed by G-banding karyotype analysis. In addition, and more pathogenicity specific chromosomal microdeletions / microduplication abnormalities can be found by CNV-seq.Conclusions:CNV-seq has high success rate and short time-consuming in the detection of chromosome CNVs, which can effectively avoid the failure of karyotype analysis and the problem of time-consuming; Moreover, CNV-seq can also find additional CNVs with clear pathogenicity, improve the positive detection rate, and effectively prevent the birth of defective children. Therefore, pregnant women with different prenatal diagnosis indications should be tested with CNV-seq at the same time of amniotic fluid karyotype analysis. CNV-seq can be used as a first-line auxiliary diagnostic technology in prenatal diagnosis for clinical application.

Analysis of a family with recurrent fetal copy number variations in 1p31.1 / 中华医学遗传学杂志

Objective@#To analyze a family with recurrent fetal copy number variations (microdeletion and microduplication, respectively) of 1p31.1 using single nucleotide polymorphism-based array (SNP-array) and G banding chromosomal karyotyping.@*Methods@#Amniocentesis and chorionic villus sampling were performed for a woman during the two pregnancies. Whole genome SNP-array was used to detect genomic imbalance of the fetus. The couple was also subjected to G-banding chromosomal analysis and SNP-array analysis.@*Results@#SNP-array showed a 1p31.1 (70 164 686-83 474 843)×1 and a 1p31.1 (70 164 686-83 479 747) × 3 in the fetuses during the two pregnancies, respectively. SNP array results of the couple appeared to be normal. The mother of the fetuses had a 46, XX, inv(1)(p31.1p32.1) karyotype.@*Conclusion@#The paracentric inversion in chromosome 1 in the gravida probably underlies the recurrent 1p31.1 copy number variations in the fetuses. SNP-array combined with G banding chromosomal analysis are suitable for prenatal diagnosis for recurrent microdeletion and microduplication in the same chromosomal region, and can provide detailed information for genetic counseling.

Identifying Mazama gouazoubira (Artiodactyla; Cervidae) chromosomes involved in rearrangements induced by doxorubicin

Abstract The process of karyotype evolution in Cervidae from a common ancestor (2n = 70, FN = 70) has been marked by complex chromosomal rearrangements. This ancestral karyotype has been retained by the current species Mazama gouazoubira (Fischer 1814), for which a chromosomal polymorphism (Robertsonian translocations and the presence of B chromosomes) has been described, presumably caused by a chromosome fragility. Thus, this study has identified doxorubicin-induced chromosome aberrations and mapped the regions involved in breaks, which may be related to the chromosome evolution process. G-banding pattern showed that 21 pairs of chromosomes presented chromosomal aberrations, 60% of the total chromosome number of the species M. gouazoubira. Among chromosomes that carry aberrations, the region where they were most frequently concentrated was distal relative to the centromere. These data suggest that certain chromosomal regions may be more susceptible to chromosome fragility and consequently could be involved in karyotype differentiation in species of the family Cervidae.

Detection of chromosome abnormality by next-generation sequencing technology of miscarried embryo in the first-trimester / 天津医药

Objective To investigate the clinical values of next-generation sequencing (NGS) technology in diagnosis of miscarried chorionic villi genetic disorders. Methods Patients who underwent miscarriage (n=87) were enrolled in this study. Among all patients, 32 cases were of recurrent miscarrage and 55 cases were of sporadic miscarriage. In all collected patients, 35 women were 35 years or older while other 52 women were less than 35 years old. Positive detection rate and the abnormal detection rate were compared between these two methods. Chromosomes abnormal rates were also compared among different types of miscarrage and different ages. All aborted villi tissue were analyzed by NGS of whole genome and G-band?ing karyotype. Results The successful detection rate of chorionic villi by NGS (100.00%) was higher than that of G-band?ing karyotype (74.71%), and the detection rate of abnormal chorionic villi by NGS (58.62%) was also higher than that of G-banding karyotype (50.77%). Three cases of chromosome structure anomaly were found in those 51 chromosome anomalies (5.88%). Other 48 cases of chromosome anomalies were aneuploidy anomalies (94.12%) include 39 cases of trisomy, 2 cases of double trisomy and 1 case of triple trisomy and 6 cases of monomer. On the other hand, 32 cases of chromosome aneuploi?dy anomalies were found in 33 chromosome anomalies by G-banding karyotype, which include 24 cases of trisomy, 2 cases of double trisomy, 1 case of triple trisomy, 5 cases of monomer and 1 case of chromosome structure anomaly. Most NGS re?sults (n=64) were in agreement with G-banding karyotype but with 1 case of discrepancy. Chromosomal abnormality rate de?tected by NGS in sporadic miscarrage group and recurrent spontaneous miscarrage group were 60.00%and 56.25%respective?ly. There was no significant difference (P>0.05). Chromosomal abnormality rate picked by NGS in women aged≥35 years old (71.43%) was higher than that in women<35 years old (50.00%) with statistically significant difference (P<0.05). Conclu?sion NGS technology showed highly accuracy in detecting chromosomal abnomality from villi tissue. Therefore, it could help to detect genetic disorders of miscarrage. It is useful to determine the reasons of miscarrage and guide the next pregnancy.

Loss of Y Chromosome in the Malignant Peripheral Nerve Sheet Tumor of a Patient with Neurofibromatosis Type 1

Neurofibromatosis type 1 (NF1) is one of the most commonly inherited autosomal dominant disorders. In order to determine whether genomic alterations and/or chromosomal aberrations involved in the malignant progression of NF1 were present in a Korean patient with NF1, molecular and cytogenetic analyses were performed on the pathologically normal, benign, and malignant tissues and primary cells cultured from those tissues of the patient. The comparative genomic hybridization (CGH) array revealed a Y chromosome loss in the malignant peripheral nerve sheet tumor (MPNST) tissue. G-banding analysis of 50 metaphase cells showed normal chromosomal patterns in the histopathologically normal and benign cultured cells, but a mosaic Y chromosome loss in the malignant cells. The final karyotype for the malignant cells from MPNST tissue was 45,X,-Y[28]/46,XY[22]. The data suggest that the somatic Y chromosome loss may be involved in the transformation of benign tumors to MPNSTs.

Comparison of I-FISH and G-banding for the detection of chromosomal abnormalities during the evolution of myelodysplastic syndrome

Myelodysplastic syndrome (MDS) patients with a normal karyotype constitute a heterogeneous group from a biological standpoint and their outcome is often unpredictable. Interphase fluorescence in situ hybridization (I-FISH) studies could increase the rate of detection of abnormalities, but previous reports in the literature have been contradictory. We performed I-FISH and conventional karyotyping (G-banding) on 50 MDS patients at diagnosis, after 6 and 12 months or at any time if a transformation to acute myeloid leukemia (AML) was detected. Applying a probe-panel targeting the centromere of chromosomes 7 and 8, 5q31, 5p15.2 and 7q31, we observed one case with 5q deletion not identified by G-banding. I-FISH at 6 and 12 months confirmed the karyotype results. Eight cases transformed to AML during follow-up, but no hidden clone was detected by I-FISH in any of them. The inclusion of I-FISH during follow-up of MDS resulted in a small improvement in abnormality detection when compared with conventional G-banding.

Controle genético das células-tronco humanas cultivadas / Genetic control of cultivated human stem cells

As células-tronco apresentam uma alta capacidade de autorregeneração, assim como, um potencial de diferenciação em uma variedade de tipos celulares. Estas células podem ser classificadas como embrionárias e adultas. Apesar de apresentar propriedades de células-tronco, as mesenquimais apresentam um certo grau de dificuldade no estabelecimento das culturas, podendo induzir a perda da expressão da enzima responsável pela imortalização ou enzima telomerase. A enzima telomerase é considerada um relógio biológico, um indicador que a senescência celular irá se instalar inevitavelmente. A questão mais atual e intrigante dos pesquisadores é se o suposto potencial de divisão, por um determinado período de tempo, das células-tronco cultivadas poderia levar ao acúmulo de alterações genéticas e epigenéticas, resultando em um processo neoplásico. Daí a importância do papel da citogenética humana no controle e monitoramento das células-tronco cultivadas que serão utilizadas na terapia em seres humanos. Alterações cromossômicas estruturais, tais como deleções, translocações e inversões, representam um mecanismo importante pelo qual as células cancerígenas desenvolvem-se gradualmente, uma vez que estas alterações cromossômicas podem levar a uma expressão anormal de muitos genes, podendo desencadear assim o processo neoplásico.

Stem cells have a high capacity of self-regeneration, as well as a potential to differentiate into several cell types. These cells can be classified as embryonic or adult. In spite of having inherent properties of stem cells, mesenchymal cells show a certain degree of difficulty to establish cultures. This might induce a loss of the expression of the telomerase enzyme which is considered to be a biological clock or an indicator of the senescence of the cells. The most current and intriguing question for researchers is whether the presumed division potential of cultivated stem cells, over a period of time could result in an accumulation of genetic alterations and consequently, in a neoplastic process. For this reason, cytogenetic techniques are very important to guarantee the control and safety of cultivated stem cells to be used in human therapy. Structural chromosomal alterations, such as for example, deletions, translocations and inversions represent an important mechanism by which cells might gradually transform in a neoplastic process. Thus, these chromosomal alterations could result in an abnormal expression of the genes and lead to cancer.

Karyology of the Atlantic forest rodent Juliomys (Cricetidae): a new karyotype from southern Brazil

Juliomys is a small rodent from the family Cricetidae which inhabits the Atlantic forest and forests from Argentina to eastern Brazil. The three species recognized so far have different karyotypes. In this paper, we describe a new karyotype with 2n = 32, FN = 48 found in Juliomys specimens from a high-altitude area in the Atlantic forest of southern Brazil. The karyotype was analyzed after G- and C-banding and silver staining of the nucleolus organizer regions (Ag-NOR) and its G-banding patterns were compared with those of the newly described species Juliomys ossitenuis (2n = 20, FN = 36). The 2n = 32 karyomorph presented peculiar features and was very different from those of the other species of the genus: J. pictipes (2n = 36, FN = 34), J. rimofrons (2n = 20, FN = 34) and J. ossitenuis (2n = 20, FN = 36). Differences were mostly due to centric and tandem fusions, pericentric inversion and loss of heterochromatin. The karyotype represents a powerful tool to differentiate Juliomys species and our data suggest that the karyotype described herein belongs to a new species.

Karyotypic characterization of Iheringichthys labrosus (Pisces, Pimelodidae): C-, G- and restriction endonuclease banding

Various chromosomal banding techniques were utilized on the catfish, Iheringichthys labrosus, taken from the Capivara Reservoir. C-banding regions were evidenced in telomeric regions of most of the chromosomes. The B microchromosome appeared totally heterochromatic. The restriction endonuclease AluI produced a banding pattern similar to C-banding in some chromosomes; the B microchromosome, when present, was not digested by this enzyme and remained stained. G-banding was conspicuous in almost all the chromosomes, with the centromeres showing negative G-banding. When the restriction endonuclease BamHI was used, most of the telomeres remained intact, while some centromeres were weakly digested. The B chromosome was also not digested by this enzyme. The first pair of chromosomes showed a pattern of longitudinal bands, both with G-banding and BamHI; this was more evident with G-banding. This banding pattern can be considered a chromosomal marker for this population of I. labrosus.

Analysis of Discrepancies Between G-banding and FISH in Hematologic Abnormalities / 대한임상병리학회지

BACKGROUND: The effective treatment of hematologic malignancies depends upon application of different therapeutic strategies by selecting patients known as the high risk group and the detection of malignant cells that can not be distinguished during following-up. We compared the results of G-banding and fluorescence in situ hybridization (FISH), which are used most frequently in detecting genetic changes, with the respect to investigating the discrepancies between these methods. METHODS: G-banding and FISH were performed on 919 consecutive specimens from 304 patients with hematologic malignancies. As for FISH, we covered most of the more frequent gene-tic changes, using 18 types of FISH probe. RESULTS: The average discrepancy between G-banding and FISH was 8.6% with a discrepancy at initial diagnosis of 6.0% and at follow-up of 11.9%, indicating greater discrepancy at follow-up after treatment. The chromosomal changes with especially large discrepancies were TEL/AML1, BCR/ABL & del(5q) (22.4%, 18.1%, and 16.2%, respectively). According to each disease, the discrepancies in acute biphenotypic leukemia (33.3%), acute lymphoblastic leukemia (14.7%), and chronic myelogenous leukemia (9.6%) were larger than average discrepancy. CONCLUSTIONS: We concluded that application of FISH is effective for detecting genetic changes in hematologic malignancies. Once genetic changes are detected, follow-up with FISH would be especially effective for making an accurate assessment of the likelihood of complete remission and recurrence.

The Usefulness of Fluorescence in Situ Hybridization(FISH) in the Diagnosis of Prader-Willi Syndrome

PURPOSE: To detect microdeletion of 15q11-13 region, high resolution cytogenetic analysis or FISH with probe at Prader-Willi syndrome region can be used. We tried to evaluate whether FISH with SNRPN is a more effective method than G-banding microscope in the diagnosis of Prader-Willi syndrome. MEHTODS: Peripheral blood sampling was done on five patients who we suspected of Prader-Willi syndrome clinically and lymphocytes from peripheral blood sampling were cultured. G-banding microscope was used to detect the microdeletion in chromosome 15 and FISH with SNRPN probe was used to detect signal defect in band q11-q13 in chromosome 15. RESULTS: There was a fluorescent signal defect in band 15 q11-q13 in one of chromosome 15 in 4 children with FISH method and only one patient was diagnosed with Prader-Willi syndrome with G-banding microscope. CONCLUSION: FISH analysis is more accurate, objective, and time saving than G-banding microscope, therefore it can be considered as a more adequate screening test for the diagnosis of Prader-Willi syndrome.

PCR-Based Detection Of Occult Y Chromosome Sequences In Turner Subjects.

Two hundred and sixty three patients with clinically suspected gonadal dysgenesis were analyzed cytogenetically using banded metaphase chromosomes. There were 61 cases exhibiting karyotypic changes; of these 28 showed 45, X and the rest were largely mosaics. The employment of FISH proved helpful to detect some of the unrecognizable chromosomal changes in selected cases. PCR analysis conducted with eleven sets of primers from the Y chromosome of 36 patients and 35 normal healthy individuals revealed the usefulness of the molecular investigations in conjunction with cytogenetic analysis. In conclusion, the application of molecular techniques to detect low level mosaicism allowed better management of the patients with dysgenesis of the gonads.

CYTOGENETIC STUDIES ON HPV-18-IMMORTALIZED CELLSFROM ESOPHAGEAL EPITHELIA OF HUMAN EMBRYO / 解剖学报

Objective To study the cytogenetic changes in HPV-immortalized cells(SHEE)for further investigating the esophageal carcinogenesis. Methods Giemsa staining was used to show the modal number of chromosomes.G-banding was done to observe the chromosomal aberrations.Fluorescence in situ hybridization(FISH)of the interphase nuclei with the centromere probes of chromosome 1,7 and 8 were performed to determine the numerical abnormalities. Results The modal numbers at passage 10 and 20 were 58-63 and 57-64, respectively.Passage 61 had a bimodal distribution 58-61 and 63-65.The structural aberrations could almost be found in all of karyotypes.The following structural aberrations were identified:1.del(1)(q12);2.del(1)(p32);3.der(4),t(4;?)(q31;?);4.der(5),t(5:?)(q31;?).FISH results showed that trisomy 1 and tetrasomy 7 markedly increased with the increase of passages and disomy 8 kept in faint change in all passages.Conclusion The modal number moved right slowly in the graph of the modal distribution and the increases of chromosomal numbers were in imbalance with increases of passages of SHEE.The structural aberrations of chromosome were found in most of karyotypes.Our results suggest that the cell immortalization is a multistep process involved in the multiple chromosomes.;

THE REGION BANDED IDIOGRAM AND IDENTIFICATION OF METAPHASE CHROMOSOME IN MOUSE / 解剖学报

It is necessary to determine the position of break points in order to study the structure rearrangement of chromosomes and gene mapping. Therefore it is indispensable to set up region, banded idiogram and nomenclature system. Nesbitt and Franke reported an idiogram and a nomenclature system for band patterns of mouse chromosomes which thereafter was accepted by the Committee on Standardized Genetic Nomenclture. Nevertheless, there are few data of systematical contrast studies in this domain. There are still difficulties for distinguishing between some chromosomes because they are similar and variability. In this paper, we compared our G-banding karyotypes with the banding patterns of Nesbitt's idiogram. Our 400 karyotypes come from bone marrow cells and fibroblasts of several normal inbred and outbred mice. Each banding obtained from each well banded chromosomes of different cells were contrasted to that of Nesbitt's idiogram. The results are as follows: 1. we have not found so many bands in one metaphase chromosome banded with trypsin-Giemsa technique as reported by Nesbitt. 2. The banding numbers and positions on each well banding chromosome picked out from different metaphase plates were consistent with those of Nesbitt's idiogrom. But some bands such as 1E1 and 1E2; 2E5-2F5; 4A3-4A5; 5C1-5C3 can't usually be distinguished in our data. 3. We observed that the major banding structure of each karyotype remains stability, but the minor banding structure appears to be variablitity in inbred mice. In addition, 6 variant pictures and region idiograms in the G-banding structure of each chromosome were showed and some notices for correct analysis of mouse karyotype were discussed.

ANALYSIS OF THE G-BANDING PATTERN OF THE CHROMOSOMES OF THE DBA/2 MOUSE / 解剖学报

Bone marrow cells and cultures of embryo skin and lung cells from DBA/2 mice were obtained for chromosome analysis in our studies. The specimens were banded by Giemsa staining following trysinization, which produced well-scattered and sharply banded mitotic figures ranging from early metaphase to mid-metaphase. Over 435 bands Within the mouse karyotype can be distinguished. Idiogram of G-banding patterns were constructed on the basis of large amount of karyotype analysis. The features of banding patterns of the individual chromosomes are presented and those chromosomes with similar banding patterns are contrasted to avoid possible misidentification.

STUDY OF HIGH-RESOLUTION G-BANDING PATTERN OF RABBIT(ORYCTOLAGUS CUNICULUS) / 解剖学报

Since rabbit (Oryctolagus cuniculus) possess many fine biological characteristics, they have been used in the studies of tumour, gene mapping and evolution. All of these need us to know the high-resolution G-banding pattern of rabbit chromosomes. Primary lung fibroblasts of new born rabbits were cultivated in RPMI 1640 medium supplemented with 20% new born bovine serum. In order to get the synchronized cells, chemical(MTX, TdR) method and physical (Cold Shock) method were used. By using trypsin-Giemsa banding-technique, we obtained fine high-resolution G-banding karyotypes of rabbit. The bands of rabbit chromosomes were analyzed and described. An idiogram which contains 583 bands of high-resolution G-banding chromosomes of rabbit has been made. The feature of rabbit choromosome bands and the method uesd to get the high resolution banding chromosomes were briefly discussed.