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 validate a fetus with high risk for trisomy 13 suggested by non-invasive prenatal testing (NIPT). METHODS: The fetus was selected as the study subject after the NIPT detection at Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences on February 18, 2019. Clinical data of the pregnant woman was collected. Fluorescence in situ hybridization (FISH), chromosomal karyotyping analysis and chromosomal microarray analysis (CMA) were carried out on amniotic fluid and umbilical cord blood and the couple's peripheral blood samples. Copy number variation sequencing (CNV-seq) was also performed on the placental and amniotic fluid samples following induced labor. RESULTS: The pregnant woman, a 38-year-old G4P1 gravida, was found to have abnormal fetal development by prenatal ultrasonography. NIPT test suggested that the fetus has a high risk for trisomy 13. Chromosomal karyotyping analysis of fetal amniotic fluid and umbilical cord blood were 46,XN,add(13)(p10). The result of CMA was arr[hg19]1q41q44(223937972_249224684)×3, with the size of the repeat fragment being approximately 25.29 Mb, the fetal karyotype was thereby revised as 46,XN,der(13)t(1;13)(q41;p10). Chromosomal karyotyping analysis and CMA of the parents' peripheral blood samples showed no obvious abnormality. The CNV-seq analysis of induced placenta revealed mosaicisms of normal karyotype and trisomy 13. The CNV-seq test of induced amniotic fluid confirmed a duplication of chr1:22446001_249220000 region spanning approximately 24.75 Mb, which was in keeping with the CMA results of amniotic fluid and umbilical cord blood samples. CONCLUSION NIPT may yield false positive result due to placenta mosaicism. Invasive prenatal diagnosis should be recommended to women with a high risk by NIPT test. And analysis of placenta can explain the inconsistency between the results of NIPT and invasive prenatal diagnosis.
Humans ; Female ; Pregnancy ; Trisomy 13 Syndrome/genetics* ; DNA Copy Number Variations ; Placenta ; Chromosomes, Human, Pair 1 ; In Situ Hybridization, Fluorescence ; Prenatal Diagnosis/methods* ; Fetus ; Amniotic Fluid ; Chromosome Aberrations ; Trisomy/genetics*

OBJECTIVE: To retrospectively analyze sex chromosomal abnormalities and clinical manifestations of children with disorders of sex development (DSD). METHODS: A total of 14 857 children with clinical features of DSD including short stature, cryptorchidism, hypospadia, buried penis and developmental delay were recruited from Zhengzhou Children's Hospital from January 2013 to March 2022. Fluorescence in situ hybridization (FISH) and chromosomal karyotyping were carried out for such children. RESULTS: In total 423 children were found to harbor sex chromosome abnormalities, which has yielded a detection rate of 2.85%. There were 327 cases (77.30%) with Turner syndrome and a 45,X karyotype or its mosaicism. Among these, 325 were females with short stature as the main clinical manifestation, 2 were males with short stature, cryptorchidism and hypospadia as the main manifestations. Sixty-two children (14.66%) had a 47,XXY karyotype or its mosaicism, and showed characteristics of Klinefelter syndrome (KS) including cryptorchidism, buried penis and hypospadia. Nineteen cases (4.49%) had sex chromosome mosaicisms (XO/XY), which included 11 females with short stature, 8 males with hypospadia, and 6 cases with cryptorchidism, buried penis, testicular torsion and hypospadia. The remainder 15 cases (3.55%) included 9 children with a XYY karyotype or mosaicisms, with main clinical manifestations including cryptorchidisms and hypospadia, 4 children with a 47,XXX karyotype and clinical manifestations including short stature and labial adhesion, 1 child with a 46,XX/46,XY karyotype and clinical manifestations including micropenis, hypospadia, syndactyly and polydactyly, and 1 case with XXXX syndrome and clinical manifestations including growth retardation. CONCLUSION Among children with DSD due to sex chromosomal abnormalities, sex chromosome characteristics consistent with Turner syndrome was most common, among which mosaicism (XO/XX) was the commonest. In terms of clinical manifestations, the females mainly featured short stature, while males mainly featured external genital abnormalities. Early diagnosis and treatment are particularly important for improving the quality of life in such children.
Humans ; Male ; Female ; Turner Syndrome/genetics* ; In Situ Hybridization, Fluorescence ; Cryptorchidism ; Hypospadias ; Retrospective Studies ; Quality of Life ; Sex Chromosome Aberrations ; Karyotyping ; Mosaicism ; Disorders of Sex Development/genetics*

OBJECTIVE: To present on a prenatally diagnosed case with complex structural rearrangements of chromosome 8. METHODS: Chromosome karyotyping, chromosomal microarray analysis (CMA) and fluorescence in situ hybridization (FISH) were carried out for a fetus with increased nuchal thickness. RESULTS: The karyotype of the amniotic fluid sample showed extra materials on 8p. FISH revealed a centromeric signal at the terminal of 8p with absence of telomeric signal. CMA revealed partial deletion of 8p23.3 [(208049_2256732)×1], partial duplication of 8p23.3p23.2 [(2259519_3016818)×3], and partial duplication of 8q [8q11.1q12.2(45951900_60989083)×3]. CONCLUSION The complex structural rearrangements of chromosome 8 in this case has differed from the commonly seen inv dup del(8p).
Female ; Pregnancy ; Humans ; Chromosomes, Human, Pair 8/genetics* ; In Situ Hybridization, Fluorescence ; Gene Rearrangement ; Prenatal Diagnosis ; Centromere