OBJECTIVE: To explore the molecular cytogenetic characteristics of three fetuses with psu idic(Y)(q11.22) using a combination of multiple methods. METHODS: A total of 11 000 pregnant women who underwent prenatal diagnosis at the Prenatal Diagnosis Center of Taizhou City from January 2019 to October 2024 were selected as the study subjects. Chromosome karyotype analysis (G-banding) and copy number variation analysis based on next-generation sequencing (NGS) were performed on the amniotic fluid/cord blood samples of the 11 000 fetuses. For cases suspected of Y chromosome abnormalities, C-banding and/or fluorescence in situ hybridization (FISH) and AZF microdeletion testing were additionally conducted. This study has been reviewed and approved by the Medical Ethics Committee of Taizhou Hospital, Zhejiang Province (Ethics No. KL20240860). RESULTS: Among the 11,000 prenatal samples undergoing concurrent karyotype and copy number variation analysis, two fetuses with 45,X/46,X,psu idic(Y)(q11.22) mosaicism and one fetus with 46,X,psu idic(Y)(q11.22) were detected. FISH detection indicated that approximately 66.7% of the cells in fetus 2 exhibited a dicentric Y chromosome, and the metaphase karyotype supported the presence of a pseudodicentric chromosome. AZF testing revealed complete deletion of the AZFb+AZFc regions in fetus 2 and fetus 3. CONCLUSION Conventional G-banding karyotype analysis for psu idic(Y)(q11.22) is prone to misdiagnosis or missed diagnosis. The combined application of chromosome karyotype analysis (G+C banding), copy number variation analysis, and FISH detection in clinical practice can accurately diagnose fetuses with psu idic(Y).
Humans ; Female ; Pregnancy ; Prenatal Diagnosis/methods* ; DNA Copy Number Variations/genetics* ; Adult ; Chromosomes, Human, Y/genetics* ; Karyotyping ; In Situ Hybridization, Fluorescence ; Cytogenetic Analysis/methods* ; Fetus ; High-Throughput Nucleotide Sequencing ; Male

OBJECTIVE: To assess the value of chromosomal microarray analysis (CMA) for the detection of low-level mosaicisms in amniotic fluid samples, and to retrospectively analyze the rare cases of mosaicisms. METHODS: Chromosomal karyotype of the fetus was determined by G-banding analysis of cultured amniotic fluid cells. CMA was used to detect copy number variation of fetal chromosomes, and fluorescence in situ hybridization (FISH) was used to determine the proportion of fetal chromosomal mosaicisms in uncultured amniotic fluid cells. RESULTS: Among 825 prenatal samples, 4 cases of true fetal mosaicisms were detected, which yielded an incidence of 0.48%. Two cases were sex chromosomal mosaicisms, and two were autosomal mosaicisms, which involved chromosomes 8 and 9, respectively. All cases were verified by G-banding analysis of cultured amniotic fluid cells, CMA, and/or FISH. CONCLUSION CMA has a great value for detecting low-level mosaicisms in amniotic fluid samples, though the positive results need to be verified by other techniques and should be interpreted with caution. The review of rare cases can provide a basis for prenatal genetic counseling.
Humans ; Female ; Amniotic Fluid/metabolism* ; Pregnancy ; Mosaicism/embryology* ; Prenatal Diagnosis/methods* ; Adult ; In Situ Hybridization, Fluorescence ; Microarray Analysis/methods* ; Karyotyping ; Retrospective Studies ; Male

OBJECTIVE: To explore the clinical characteristics and genetic factors in four patients with Disorder of sex development (DSD). METHODS: Four patients who visited Tianjin Medical University General Hospital between January 2023 and January 2024, presenting with short stature, abnormal external genitalia, or infertility as their chief complaints, were selected as the study subjects. Clinical data were collected, and peripheral or umbilical cord blood samples were obtained for karyotyping analysis and low-depth whole-genome sequencing (CNV-seq). Quantitative fluorescence PCR (QF-PCR) was used to detect the sex-determining region Y (SRY) gene and azoospermia factor (AZF) on the Y chromosome, while fluorescence in situ hybridization (FISH) was employed to determine the location of the SRY gene. Whole exome sequencing (WES) was performed for genetic testing, and Sanger sequencing was used for familial validation of the candidate variants. The study procedure and protocol were approved by the Medical Ethics Committee of Tianjin Medical University General Hospital (Ethics No.: IRB2024-WZ-006). RESULTS: Case 1 had a karyotype of 45,X[22]/46,XY[8], with CNV-seq indicating a mosaic deletion of 7.44 Mb (copy number = 0.2) at Yp11.31-p11.2, a mosaic deletion of 5.32 Mb (copy number = 0.3) at Yq11.1-q11.221, and a deletion of 10.26 Mb (copy number = 0) at Yq11.221-q11.23. Y chromosome microdeletion analysis showed SRY and AZFa (+), AZFb+c (-). Case 2 had a karyotype of 45,X[12]/46,X,del(X)(q26.3)[18], with CNV-seq indicating a mosaic deletion of 132.62 Mb (copy number = 1.4) at Xp22.33-q26.3 and a deletion of 19.62 Mb (copy number = 1) at Xq26.3-q28. Case 3 had a karyotype of 46,XX, with CNV-seq showing two copies of the X chromosome and no Y chromosome. Y chromosome microdeletion analysis showed SRY (+) and AZFa+b+c (-), and FISH confirmed a translocation of the SRY gene to the terminal end of the short arm of the X chromosome. Case 4 had a karyotype of 46,XY, with CNV-seq showing one copy each of the X and Y chromosomes. Y chromosome microdeletion analysis showed SRY(+) and AZFa+b+c (+), and WES revealed a c.1103del variant in the AR gene (maternal origin), which was classified as a pathogenic variant based on the guidelines from the American College of Medical Genetics and Genomics (ACMG) (PVS1+PP1+PM2_Supporting). CONCLUSION The combined application of multiple detection techniques such as chromosomal karyotyping analysis, CNV-seq, QF-PCR, and WES can identify the genetic etiology of DSD patients, providing a basis for clinical consultation and treatment plan formulation.
Humans ; Male ; Female ; Chromosomes, Human, Y/genetics* ; Disorders of Sex Development/genetics* ; Sex-Determining Region Y Protein/genetics* ; Karyotyping ; In Situ Hybridization, Fluorescence ; Exome Sequencing ; Adult ; Child

OBJECTIVE: To explore the reasons for false negative results by Optical genome mapping (OGM) analysis of three cases and propose strategies for handling them. METHODS: Three patients presented at the Affiliated Hospital of Nantong University between July 2022 and July 2024 were selected as study subjects. The patients included a 37-year-old female with two miscarriages, a 1.5-year-old boy with delayed motor development, and a 35-year-old male whose son had intellectual disability. The patients had undergone comprehensive evaluation with chromosomal karyotyping analysis, single nucleotide polymorphism microarray (SNP array) assay, fluorescence in situ hybridization (FISH), and methylation-specific multiple ligation-dependent probe amplification (MS-MLPA). A retrospective analysis was also carried out on the results of OGM testing. This study was approved by the Medical Ethics Committee of the Hospital (Ethics No.: 2020-K004). RESULTS: The chromosomal karyotype of patient 1 was 46,XX,4qs, and no abnormality was found by SNP array, FISH, and OGM testing. Patient 2 had a normal chromosomal karyotype, and SNP array analysis did not reveal any copy number abnormalities of chromosomal fragments but the presence of a homozygous region of approximately 79.58 Mb at 15q11.2-q26.3 (chr15: 22817871_102397317). MS-MLPA detection indicated that the copy number of the 15q11.2-q13 region was 2, and the degree of methylation was relatively high (average ratio = 1.0). OGM detection confirmed the presence of approximately 67.97 Mb of homozygosity in the chr15:33814680_101787650 [hg38] region of 15q14-q26.3. Patient 3 had a chromosomal karyotype of 46,XY,t(9;14)(q13;q11.2). No abnormality was found by OGM testing for patients 1 and 3. CONCLUSION As a novel cytogenetic technique, OGM can achieve high-resolution and high-precision analysis for numerical and structural genomic abnormalities. Nevertheless, it also has certain limitations, as its false negative results are related to factors such as the type of genomic variation, the chromosomal regions involved in the variation, the type of disease, and the version of human reference genome. Currently, it cannot be used as an independent method for the diagnosis of genetic diseases.
Humans ; Male ; Female ; Adult ; Polymorphism, Single Nucleotide/genetics* ; Karyotyping ; Chromosome Mapping/methods* ; Infant ; False Negative Reactions ; In Situ Hybridization, Fluorescence

OBJECTIVE: To investigate the clinical characteristics and genetic etiology of a male with a normal phenotype and SRY gene-positive 46,XX/46,XY tetrazoospermia chimerism. METHODS: A male patient with an abnormal peripheral blood chromosomal karyotype detected at the Infertility Center of Taizhou Hospital of Zhejiang Province on December 2, 2013 was selected as the study subject. Peripheral venous blood samples were collected from the proband and his family members, together with a semen sample from the proband. Chromosomal karyotype analysis, red blood cell blood group identification, chromosomal microarray analysis (CMA), fluorescence in situ hybridization (FISH), sex-determining region Y (SRY) gene detection, and short tandem repeat (STR) microsatellite marker analysis were performed on the peripheral venous blood sample from the proband. Routine semen analysis, sperm FISH, and STR testing were also conducted. STR verification was performed on both parents. This study was approved by the Medical Ethics Committee of the hospital (Ethics No.: k20201009). RESULTS: The proband, a 37-year-old male, had normal secondary sexual characteristics and external genitalia development. The chromosomal karyotype of his peripheral blood sample was 46,XX[94]/46,XY[6]. ABO blood group typing was positive for Rh(D) type O and negative for Rh(D) type A, indicating the presence of two red blood cell populations. CMA result was arr[GRCh37](1-22)×2,(XX)×1. Autosomal and X chromosome SNP genotypes were BB-BB, AB-AB, and AA-AA, making it impossible to identify homozygous/heterozygous chimerism. FISH detection of interphase nuclei showed nuc ish XX[92]/XY[8]. Testing of the SRY gene was positive. STR analysis showed a single X peak (no Y peak) at the AMEL locus, 10/12 at the Penta D locus, and no third allele at other loci. Routine semen analysis were normal. Sperm FISH detection showed haploid nuclei nuc ish X[53]/Y[47]. Sperm STR analysis revealed an X/Y bimodal distribution at the AMEL locus and a 9/14 distribution at the Penta D locus, with no third allele observed at other loci. Above results suggested that the proband's blood and germ cell lines had originated from a heterozygous chimera formed by the fusion of two different zygotes. CONCLUSION Combined genetic techniques confirmed that the proband's peripheral blood AMEL genotype is X/X, while the sperm is X/Y. The Penta D locus showed a bi-allelic heterozygous pattern of 10/12 in the peripheral blood sample and 9/14 in the sperm sample, suggesting that the proband is a tetrazygotic chimera resulted from the fusion of 46,XX/46,XY zygotes.
Humans ; Male ; Adult ; Chimerism ; Microsatellite Repeats ; Sex-Determining Region Y Protein/genetics* ; Phenotype ; Genes, sry ; In Situ Hybridization, Fluorescence ; Karyotyping

Objective To summarize the clinicopathological features,immunohistochemical characteristics,HOX transcript antisense RNA(HOTAIR)in situ hybridization status,treatment,and prognosis of myxopapillary ependymoma(MPE). Methods A total of 17 patients diagnosed with MPE based on pathological evidence in the Department of Pathology of Peking Union Medical College Hospital from November 2006 to July 2023 were selected,and the clinicopathological data of these patients were collected.Immunohistochemical staining for trimethylation at lysine 27 of histone H3 (H3K27me3),glial fibrillary acidic protein(GFAP),and epithelial membrane antigen(EMA)and alcian blue-periodic acid Schiff(AB-PAS)staining were performed in all the patients.Sixteen patients with spinal ependymomas were selected as the control group.Tissue microarrays were prepared from 17 MPE patients and the control group.HOTAIR ISH was performed and semi-quantitatively scored,and the scores of the two groups were compared by the Wilcoxon rank-sum test. Results The 17 MPE patients aged 14-64 years,with the mean age of(37.48±16.10)years and the male-to-female ratio of 0.7∶1.Their clinical manifestations mainly included lumbosacral and lower limb pains.Microscopically,tumor cells were arranged in a papillary pattern around fibrovascular axis,with abundant myxoid materials,and tumor cells were arranged in a loose meshwork in some patients.The immunohistochemical staining results showed that 17(100%),10(58.82%),and 8(47.06%)patients expressed GFAP,EMA,and D2-40,respectively,and 2(11.76%)patients lacked expression of H3K27me3.AB-PAS staining showed blue myxoid materials in all the 17(100%)patients.HOTAIR was expressed in both MPE and control groups,with higher semi-quantitative score in the MPE group than in the control group(P=0.004).Twelve patients were followed up,with a median follow-up period of 65.50 months,during which three patients showed recurrence.Conclusions MPE exhibits typical pathological features,and the combination with immunohistochemical staining for GFAP and EMA as well as AB-PAS staining facilitates diagnosis of this disease.A small number of patients loss the expression of H3K27me3.HOTAIR is highly expressed in MPE but lacks specificity,which limits its auxiliary diagnostic value.The overall prognosis of MPE is favorable,with a few patients experiencing recurrence.
Humans ; Ependymoma/metabolism* ; Male ; Adult ; Female ; Adolescent ; Middle Aged ; In Situ Hybridization ; Young Adult ; RNA, Antisense/genetics* ; Immunohistochemistry ; Prognosis

Background and Objective: Retinoblastoma is one of the most common intraocular cancers among children usually caused by the loss of retinoblastoma protein function. Despite being a highly heritable disease, conventional diagnostic and prognostic methods depend on clinical examination, with limited consideration of cancer genetics in the standard of care. CD133, KRT19, and MUC1 are commonly explored genes for their utility in liquid biopsies of cancer including lung adenocarcinoma. To date, there are few extensive molecular studies on retinoblastoma in Filipino patients. To this end, the study aimed to describe the copy number of CD133, KRT19, and MUC1 in retinoblastoma samples from a Filipino patient and quantitate the respective expression level of these genes. Methods: Hematoxylin & Eosin (H&E) staining was utilized to characterize the retinoblastoma tissue while fluorescence in situ hybridization (FISH) using probes specific to CD133, KRT19, and MUC1 was performed to determine the copy number of genes in retinoblastoma samples from a Filipino patient (n = 1). The gene expression of CD133, MUC1, and KRT19 was quantitated using RT-qPCR. Results: The H&E staining in the retinoblastoma tissue shows poorly differentiated cells with prominent basophilic nuclei. CD133 was approximately 1.5-fold overexpressed in the retinoblastoma tissue with respect to the normal tissue, while MUC1 and KRT19 are only slightly expressed. Multiple intense signals of each probe were localized in the same nuclear areas throughout the retinoblastoma tissue, with high background noise. Conclusion These findings suggest that CD133 is a potential biomarker for the staging and diagnosis of retinoblastoma in Filipino cancer patients. However, further optimization of the hybridization procedures is recommended.
Retinoblastoma ; Biomarkers ; In Situ Hybridization

OBJECTIVE: To explore the coincidence rate of G-banding karyotype analysis and fluorescence in situ hybridization (FISH) for the diagnosis of children with sex chromosome mosaicisms. METHODS: A retrospective analysis was carried out for 157 children with suspected sex chromosome abnormalities who had presented at Shenzhen Children's Hospital from April 2021 to May 2022. Interphase sex chromosome FISH and G-banding karyotyping results were collected. The coincidence rate of the two methods in children with sex chromosome mosaicisms was compared. RESULTS: The detection rates of G-banding karyotype analysis and FISH were 26.1% (41/157) and 22.9% (36/157) , respectively (P > 0.05). The results of G-banding karyotype analysis showed that 141 cases (89.8%) were in the sex chromosome homogeneity group, of which only 5 cases (3.5%) were inconsistent with the results of FISH. There were 16 cases (10.2%) in the sex chromosome mosaicism group, of which 11 cases (68.8%) were inconsistent with the results of FISH. There was a statistical difference between the two groups in the coincidence rate of the results of the two methods (P < 0.05). CONCLUSION No significant difference was found between G-banding karyotype analysis and FISH in the detection rate of chromosome abnormalities. The coincidence rate in the mosaicism group was lower than that in the homogeneity group, and the difference was statistically significant. The two methods should be combined for clinical diagnosis.
Humans ; Mosaicism ; In Situ Hybridization, Fluorescence/methods* ; Retrospective Studies ; Karyotyping ; Chromosome Aberrations ; Sex Chromosome Aberrations ; Karyotype ; Chromosome Banding ; Sex Chromosomes

OBJECTIVE: To explore the genetic basis for a fetus with club foot detected upon mid-pregnancy ultrasonography. METHODS: Amniotic fluid of the fetus and peripheral blood samples of its parents were collected and subjected to G-banding karyotype analysis and copy number variation sequencing (CNV-seq). The result was verified by fluorescence in situ hybridization (FISH). RESULTS: The fetus and its parents all had a normal karyotype. CNV-seq analysis revealed that the fetus has harbored a 23.12 Mb on chromosome 5 and a 21.46 Mb duplication on chromosome 7. FISH assay has verified that its mother has carried a cryptic t(5;7)(p14.3;q33) translocation. CONCLUSION CNV-seq combined with FISH can effectively detect cryptic chromosome aberrations, and can help to reduce severe birth defects and provide a basis for prenatal genetic counseling.
Pregnancy ; Female ; Humans ; Cri-du-Chat Syndrome ; In Situ Hybridization, Fluorescence ; DNA Copy Number Variations ; Prenatal Diagnosis ; Fetus ; Amniotic Fluid ; Chromosome Deletion

OBJECTIVE: To explore the genetic basis, clinical phenotype and pathogenesis for a child with mosaicism ring chromosome 4. METHODS: Clinical data of the child was collected. Peripheral blood chromosomal karyotype G banding analysis, chromosomal microarray analysis (CMA), fluorescence in situ hybridization (FISH) were carried out for the child, in addition with a review of the literature. RESULTS: The child was born full-term with low birth weight, facial dysmorphism, patent ductus arteriosus and ventricular septal defect. His karyotype was determined as mos46,XY,r(4)(p16.3q35.2)[259]/45,XY,-4[25]/47,XY,r(4)(p16.3q35.2), +r(4)(p16.3q35.2)[8]/46,XY,der(4)del(4)(p16.3)inv(4)(p16.3q31.1)[6]/46,XY,dic?r(4;4)(p16.3q35.2;p16.3q35.2)[4]/48,XY,r(4)(p16.3q35.2),+r(4)(p16.3q35.2)×2[3]/46,XY,r(4)(p1?q2?)[2]; CMA result was arr[GRCH37]4p16.3(68 345-2 981 614)×1; FISH result was 45,XY,-4[12]/45,XY,-4×2,+mar1.ish r1(4)(WHS-,D4Z1+)[1]/ 46,XY,-4,+mar1.ishr1(4)(WHS-,D4Z1+)[73]/46,XY,-4,+mar2.ishr2(4)(WHS-,D4Z1++)[1]/47,XY,-4,+mar1×2.ishr1(4) (WHS-, D4Z1+)×2[4]/46,XY,del(4)(p16.3).ish del(4)(p16.3)(WHS-,D4Z1+)[9]. CONCLUSION In this case, the ring chromosome 4 as a de novo variant has produced a number of cell lines during embryonic development and given rise to mosaicism. The clinical phenotype of ring chromosome 4 is variable. The instability of the ring chromosome itself, presence of mosaicism, chromosome breakpoint and range of deletion and/or duplication may all affect the ultimate phenotype.
Humans ; Pregnancy ; Female ; Ring Chromosomes ; In Situ Hybridization, Fluorescence ; Karyotyping ; Karyotype ; Mosaicism