[Clinical features and genetic analysis of two fetuses with ring chromosome 21 mosaicism].

OBJECTIVE: To investigate the perinatal clinical phenotype and genetic characteristics of two fetuses with ring chromosome 21 mosaicisms. METHODS: Two fetuses who were diagnosed at the Xiamen Maternal and Child Health Care Hospital in November 2021 were selected as the study subjects. Clinical data of the two fetuses were collected. Conventional G-banded karyotyping and chromosomal microarray analysis (CMA) were carried out for the fetuses and their parents. RESULTS: Prenatal ultrasonography of fetus 1 has revealed absence of nasal bone, ventricular septal defect, persistent left superior vena cava, and mild tricuspid regurgitation. Chromosomal karyotyping was 46,X?,dic r(21;21)(p12q22;q22p12)[41]/45,X?,-21[9]. CMA has revealed a 30.00 Mb quadruplication at 21q11.2q22.3 and a 3.00 Mb deletion at 21q22.3. For fetus 2, ultrasonography has revealed pointed echo of the nasal bone. The fetus was found to have a karyotype of 46,X?,r(21)(p12q22)[83]/45,X?,-21[14]/46,X?,dic r(21;21)(p12q22;q22p12)[3]. CMA has revealed a 5.10 Mb quadruplication at 21q22.12q22.3 and a 2.30 Mb deletion at 21q22.3. CONCLUSION: The perinatal phenotype of the two fetuses with ring chromosome 21 mosaicisms is related to the duplication of chromosomal segments near the breakpoints of the chromosomal deletions. The combined chromosomal karyotyping and CMA has enabled prenatal diagnosis and genetic counseling for these families.

Prenatal Genetic Diagnosis of Fetal Cystic Hygroma: A Retrospective Single-Center Study from China.

Fetal cystic hygroma (CH) is associated with poor prognosis and chromosomal anomalies. Recent studies have suggested that the genetic background of affected fetuses is essential for predicting pregnancy outcomes. However, the detection performance of different genetic approaches for the etiological diagnosis of fetal CH remains unclear. In this study, we aimed to compare the diagnostic efficiency of karyotyping and chromosomal microarray analysis (CMA) in a local fetal CH cohort, and tried to propose an optimized testing strategy that may help improve the cost-effectiveness of disease management. We reviewed all pregnancies that underwent invasive prenatal diagnosis between January 2017 and September 2021 at one of the largest prenatal diagnostic centers in Southeast China. We collected cases identified by the presence of fetal CH. Prenatal phenotypes and laboratory records of these patients were audited, collated, and analyzed. The detection rates of karyotyping and CMA were compared, and the concordance rate of these two methods was calculated. A total of 157 fetal CH cases were screened from 6,059 patients who underwent prenatal diagnosis. Diagnostic genetic variants were identified in 44.6% (70/157) of the cases. Karyotyping, CMA, and whole-exome sequencing (WES) identified pathogenic genetic variants in 63, 68, and 1 case, respectively. The Cohen's κ coefficient between karyotyping and CMA was 0.96, with a concordance of 98.0%. Of the 18 cases in which cryptic copy number variants <5 Mb were detected by CMA, 17 were interpreted as variants of uncertain significance, and the remaining cases were interpreted as pathogenic. Trio exome sequencing revealed a pathogenic homozygous splice site mutation in the PIGN gene in a case undiagnosed by CMA and karyotyping. Our study demonstrated that chromosomal aneuploidy abnormalities are the main genetic cause of fetal CH. Based on this, we recommend karyotyping combined with rapid aneuploidy detection as a first-tier approach for the genetic diagnosis of fetal CH. WES and CMA could improve the diagnostic yield when routine genetic tests fail to determine the cause of fetal CH.

Highly Sensitive Detection of Low-Abundance BRAF V600E Mutation in Fine-Needle Aspiration Samples by Zip Recombinase Polymerase Amplification.

Papillary thyroid carcinoma (PTC) is the most common thyroid cancer with high incidence in endocrine tumors, which emphasizes the significance of accurate diagnostics. Still, the commonly used cytological method (fine-needle aspiration (FNA) cytology) and molecular diagnostic methods (such as PCR and sequencing) are limited in terms of diagnostic time, sensitivity, and user-friendliness. In this study, we introduce a novel Zip recombinase polymerase amplification (Z-RPA) strategy to efficiently detect rare mutant alleles in PTC fine-needle aspiration samples, which is sensitive, fast, and simple to manipulate. Using Zip nucleic acid (ZNA) probes to clamp the mutation region, the phi 29 polymerase could selectively displace mismatched ZNA probes and start amplification, while leaving complementary ZNA probes untouched and blocking amplification according to genotype. We demonstrated the good sensitivity and specificity of this strategy with optimized conditions and design, which enabled detection of BRAF V600E mutation in a total 4 ng of genomic DNA within 40 min (≈1 copy). Robust behavior in clinical specimen analysis was also demonstrated. The Z-RPA strategy provides a pragmatic approach to rapidly, sensitively, and easily detect BRAF V600E mutation in clinical fine-needle aspiration samples, which is a promising method for early cancer diagnosis and treatment guideline.

A familial analysis of two brothers with azoospermia caused by maternal 46,Y, t(X; 1) (q28; q21) chromosomal abnormality.

Chromosomal abnormality is a primary genetic factor that lead to azoospermia and male infertility. Here, we report the cases of two brothers with primary infertility, whose chromosomes displayed a balanced translocation, and their karyotypes were 46,Y, t(X; 1) (q28; q21). Both presented an azoospermia phenotype without abnormal clinical symptoms. Their mother's karyotype was 46,X, t(X; 1) (q28; q21), and their father's chromosome karyotype was 46,XY. No abnormal changes were noted in the copy number of chromosome fragments in the whole genome. This study is the first to report showing that 46,Y, t(X; 1) (q28; q21) chromosomal abnormalities are associated with azoospermia.

[Prenatal diagnosis of a rare case with de novo partial 21q(21q22.1→ qter) trisomy syndrome and absent nasal bone].

OBJECTIVE: To carry out prenatal diagnosis for a fetus with absent nasal bone by using cytogenetic and molecular techniques. METHODS: Chromosomal karyotyping, single nucleotide polymorphism array (SNP-array) and fluorescence in situ hybridization (FISH) assays were applied for the diagnoses. Peripheral blood samples were also taken from the parents for chromosomal karyotyping and FISH analysis. RESULTS: The fetus was found to have a 46,XX,add(21)(p11.2) karyotype, and SNP-array has revealed a 11.3 Mb duplication at 21q22.12q22.3 (hg19: 36 762 648-48 093 361), which was confirmed by FISH. Both parents were found to be normal by chromosomal karyotyping and FISH analysis. The fetus was ultimately found to have a karyotype of 46,XX,der(21)t(21;21)(p11.2;q22.1), resulting a de novo partial trisomy of 21q22.1. CONCLUSION: Combined use of various techniques has enabled accurate prenatal diagnosis and genetic counseling for the fetus.

[Clinical and molecular genetic analysis of a patient with 3-M syndrome].

OBJECTIVE: To analyze the clinical features and molecular genetic etiology of a patient with 3-M (Miller McKusick Malvaux) syndrome from a consanguineous parentage family, and to explore the relationship between genotype and phenotype. METHODS: After the consent of the proband's guardian and the informed consent form was signed, DNA was extracted from peripheral blood samples of the proband and her parents for chromosome microarray analysis, medical exome sequencing and parental verification. RESULTS: A total of 247.1 Mb loss of heterozygosity was found in the proband with a CytoScan 750K array. Furthermore, a homozygous variant (c.458dupG) of the OBSL1 gene was found using high-throughput sequencing, which was inherited from her parents. Based on the criteria and guidelines of genetic variation of American College of Medical Genetics and Genomics, the variant is predicted to be pathogenic (PVS1+PM2+PP4), and only one case was reported previously. CONCLUSION: Spina bifida occulta and lower eyelid fat pad may be a special phenotype of c.458dupG variant of the OBSL1 gene. Our study may provide a useful reference for evaluating the relationship between genotype and phenotype of 3-M syndrome type 2.

Pedigree analysis of two brothers with severe oligozoospermia caused by maternal inv(X) (p22.3, q22) chromosome abnormality.

Sex chromosome abnormality (SCA) is one of the major causes of male spermatogenesis dysfunction. In our study, we sought to investigate the novel X chromosome inversion leading to severe oligozoospermia. Here, we report two brothers with severe oligozoospermia without any other abnormal clinical phenotype. The chromosome karyotypes in peripheral blood of both brothers were 46, Y, inv (X) (p22.3, q22), and no Y chromosome microdeletion was found. The karyotype of their mother was 46, X, inv (X) (p22.3, q22) and that of their father was 46, XY. This is the first report in China that X chromosomal inversion, 46, Y, inv (X) (p22.3, q22), is associated with severe oligozoospermia. This inversion may be a direct genetic risk factor for spermatogenesis.

[Prenatal diagnosis of two fetuses with de novo 46,X,psu dic(Y)/45,X mosaicism].

OBJECTIVE: To carry out prenatal diagnosis for a fetus with increased nuchal translucency (NT) and another fetus with non-invasive prenatal testing (NIPT) suggested reduced sex chromosomes by cytogenetic and molecular techniques. METHODS: Chromosomal karyotyping, single nucleotide polymorphism array (SNP-array) and fluorescence in situ hybridization (FISH) were applied for the diagnoses. Peripheral blood samples were also taken from their parents for chromosomal karyotyping and SNP-array analysis. RESULTS: Both fetuses showed a 46,X,+mar/45,X karyotype. SNP-array has detected a 22.0 Mb duplication at Yp11.31q11.223 and a 3.9 Mb microdeletion at Yq11.223q11.23 in fetus 1, and a 16.9 Mb duplication at Yp11.31q11.221 and a 8.1 Mb deletion at Yq11.222q11.23 in fetus 2. The results were confirmed by FISH. The parents of both fetuses were normal by chromosomal karyotyping and SNP-array. CONCLUSION: Combined use of various techniques can enable accurate prenatal diagnosis and genetic counseling.

[Prenatal genetic analysis of three fetuses with abnormalities of chromosome 22].

OBJECTIVE: To carry out genetic testing for 3 fetuses with abnormal prenatal screening. METHODS: Fetal ultrasound, karyotype analysis, single nucleotide polymorphism (SNP) array and fluorescence in situ hybridization were performed. RESULTS: Abnormalities of chromosome 22 were found with all 3 fetuses. Fetus 1 harbored a 7.1 Mb deletion in 22q13.2q13.33 region, which involved 54 OMIM genes including SHANK3 and FBLN1. Fetus 2 had a mosaicism karyotype, with 12% of cells harboring a 6.6 Mb deletion in 22q13.31q13.33, covering 48 OMIM genes such as SHANK3 and PPARA, and 5% of cells harboring a 26.1 Mb duplication in 22q11.1q13.2 involving 285 OMIM genes. Fetus 3 carried a tandem duplication of 1.7 Mb in 22q11.1q11.21, which involved 10 OMIM genes including CECR1, CECR2 and ATP6V1E1. No abnormality was found in the three couples by chromosomal karyotyping and SNP array analysis. CONCLUSION: The severity of diseases caused by chromosome 22 abnormalities not only depends on the range of the deletion or duplication, but is also closely related to chromosome structure, gene dose and genetic environment. Combined ultrasonography and various genetic testing techniques in prenatal diagnosis can greatly increase the detection rate of genetic diseases with substantial phenotypic variation.

[Prenatal genetic diagnosis of a partial 21 trisomy fetus with nasal bone dysplasia].

OBJECTIVE: To explore the nature of chromosomal abnormality in a fetus with nasal bone dysplasia and clarify its clinical effect. METHODS: Fetal chromosome karyotype was analyzed by G-banding. Single nucleotide polymorphism array (SNP-array) was used to detect the chromosomal copy number variations, and fluorescence in situ hybridization (FISH) was used to verify the result. RESULTS: Fetal karyotype analysis showed an unknown chromosomal fragment in 21q21 region. SNP-array discovered a 7.5 Mb duplication in the 21q22.12q22.3 region. FISH confirmed that the unknown fragment was derived from a 21q22.12q22.3 duplication. CONCLUSION: Combined use of karyotype analysis, SNP-array and FISH has clarified the nature of chromosomal abnormality in a fetus with nasal bone dysplasia, which has enabled more accurate prenatal diagnosis and genetic counseling.

Carrier screening for spinal muscular atrophy with a simple test based on melting analysis.

Carrier screening of spinal muscular atrophy (SMA) can provide reproductive options for carriers and prevent the birth defects. Here, we developed a simple screening test based on melting analysis. The test comprises a duplex PCR with two primer pairs and three probes to simultaneous amplify SMN1, SMN2, and CFTR. By analyzing the melting profiles, we were able to determine the SMN1/SMN2 ratio and SMN1 + SMN2 copy number to subsequently determine the copy number of SMN1. Samples with one copy of SMN1 were considered as "high risk for carrier," while samples with ≥2 copies of SMN1 were considered as "low risk for carrier." We evaluated the clinical performance of this test using 215 clinical samples with various genotypes that had been previously confirmed by multiplex ligation-dependent probe amplification (MLPA). The test showed high sensitivity (100%) and specificity (97.1%) as well as high positive (97.3%) and negative (100%) predictive value, and was in perfect agreement with the gold standard test, MLPA (k = 0.97). Moreover, it is rapid, inexpensive, and easy to perform and automate, with high reproducibility and capacity. Therefore, we expect this test will advance carrier screening for SMA.

[Prenatal diagnosis of a fetus with two small supernumerary marker chromosomes].

OBJECTIVE: To explore the clinical significance of a prenatal case with two small supernumerary marker chromosomes (sSMC) through identification of their origins. METHODS: G-banding chromosomal karyotyping analysis were carried out on fetal amniotic fluid sample and peripheral blood samples from both patients. Fluorescence in situ hybridization (FISH) and single nucleotide polymorphism-array (SNP-array) were used to analyze the component and size of the sSMCs. RESULTS: The karyotype of the fetus was determined as 47, XX, +mar[53]/48, XX, +2 mar[31]/46, XX[14]. SNP-array has revealed four copies of chromosome 2q11.1q11.2 with a size of 2.6 Mb and three copies of 10p11.23q11.23 with a size of 20.6 Mb. The results was confirmed by FISH. CONCLUSION: A rare chromosomal abnormality with two sSMCs was identified by combined karyotype analysis, SNP-array and FISH, which provided valuable information for prenatal diagnosis.

[Cytogenetic and molecular genetic analysis of the amniotic fluid cells of a fetus with pseudodicentric isochromosome 22 resulting in partial tetraploidy of 22q].

OBJECTIVE: To diagnose chromosomal abnormalities in amniotic fluid cells by combining karyotyping and single nucleotide polymorphism array (SNP-array) analysis, and to explore the application of SNP-array in routine clinical practice. METHODS: Conventional G banding was used to karyotype a fetal amniotic fluid sample and the corresponding peripheral blood samples from the parents, followed by SNP-array analysis of the fetal genomic DNA from the amniotic fluid. RESULTS: The karyotype of the amniocytes was 47, XX, +mar. The marker chromosome was further identified as psu idic (22) (q11.2) by SNP-array analysis, revealing tetraploidy of a 1.7 Mb fragment in 22q11.1-q11.2 interval that involves the critical region for Cat eye syndrome. CONCLUSION: A rare chromosomal abnormality was identified by combining conventional G banding and SNP-array. The high resolution SNP-array could provide more detailed information for determining the origin of chromosomal abnormalities.

Spray-painted human fibronectin coating as an effective strategy to enhance graft ligamentization of a polyethylene terephthalate artificial ligament.

To enhance graft ligamentization after anterior cruciate ligament (ACL) reconstruction, human fibronectin (FN) was coated on polyethylene terephthalate (PET) ligaments by spray painting. The FN-coated PET ligaments were investigated in vitro using rat mesenchymal stromal cells (MSCs). MSCs cultured on FN-coated grafts resulted in similar cell densities and amounts of proliferating cells with control grafts without coating. The FN-coated group not only gave rise to MSC-derived collagen-like tissues but also enhanced the expression of collagen-I gene. Furthermore, rat ACL reconstruction models were used to evaluate the effect of the FN coating in vivo. The FN coating significantly promoted new ligament tissue regeneration into the graft fibers. In conclusion, sprayed FN coating had a positive effect to enhance graft ligamentization of PET artificial ligament.

Retrospective study revealed integration of CNV-seq and karyotype analysis is an effective strategy for prenatal diagnosis of chromosomal abnormalities.

Fetal chromosomal abnormalities are the main cause of adverse pregnancy outcomes and are the focus of invasive prenatal diagnosis. Recent studies have demonstrated that various techniques have distinct advantages. Achieving high-resolution and effective prenatal chromosomal abnormality diagnosis requires a multi-technology integration strategy. Based on retrospective samples from a single center, we propose that integrating CNV-seq and karyotype analysis is an effective strategy for prenatal diagnosis of chromosomal abnormalities. In this study, 13.80% of the pregnant women (347/2514) were found to have likely pathogenic or pathogenic fetal chromosomal abnormalities using this integrated approach. Among these cases, 53.89% (187/347) had consistent chromosomal abnormalities detected by both CNV-seq and karyotyping analysis, while 19.02% (66/347) and 27.09% (94/347) of cases were diagnosed solely by CNV-seq or karyotyping, respectively. Fetal chromosomal abnormalities were identified in 18.39% of samples with abnormal ultrasound, which was significantly higher than the percentage found in samples with normal ultrasound (p < 0.001). Samples with multiple ultrasound abnormalities and single-indicator ultrasound abnormalities such as nasal bone dysplasia, renal dysplasia, or echogenic fetal bowel also had higher rates of chromosomal abnormalities (p < 0.05) compared to normal samples. Analyzing samples with Trio family data (N = 521) revealed that about 94% of variants of uncertain significance were inherited from parents and were non-pathogenic. Overall, integrating CNV-seq and karyotype analysis is an effective strategy for prenatal diagnosis of chromosomal abnormalities. This study provides valuable insights for correlating prenatal screening indicators with chromosomal abnormalities.

Epidemiology of human papillomavirus infection in women from Xiamen, China, 2013 to 2023.

Background: Cervical cancer is primarily caused by HPV infection. The epidemiology of HPV infection in specific areas is of great meaning of guide cervical cancer screening and formulating HPV vaccination strategies. Here, we evaluated the epidemiological characteristics of HPV infection in Xiamen population. Methods: In total, 159,049 cervical exfoliated cell samples collected from female outpatients in Women and Children's Hospital, School of Medicine, Xiamen between January 2013 and July 2023 were analyzed. HPV DNA detection was performed using HPV genotyping kits (Hybribio Limited Corp, China). An analysis was conducted on the prevalence of HPV infection, taking into account factors such as age, year, and multiple patterns of HPV infection. The differences in prevalence among age groups and years were compared using χ2 test. Results: The overall prevalence of any 21 HPV genotypes was 18.4%, of which the high-risk HPV (HR-HPV) positive rate was 14.6%. The age-specific prevalence of HPV infection showed a bimodal distribution, with two distinct peaks, one at <25 years (31.2%) and the other at 60-64 years (32.9%). There was a downward trend in the prevalence of HPV infection over time, decreasing from 26.2% in 2013 to 14.5% in 2021, and then increasing to 19.0% in 2023. The five most prevent HR-HPV genotypes were HPV52 (4.0%), 58 (2.6%), 16 (2.5%), 51 (1.8%), and 39 (1.7%). Among the positive cases, 76.7% were detected with only one genotype and 23.3% with multiple genotypes. The most common co-infection was HPV52 + HPV58 (0.24%), followed by HPV16 + HPV52 (0.24%), HPV52 + HPV53 (0.21%), HPV52 + HPV81 (0.21%), HPV51 + HPV52 (0.19%), HPV16 + HPV58 (0.18%), and HPV39 + HPV52 (0.17%). Conclusion: The study provided the largest scale information on the recent epidemiological characteristics of HPV infection in Xiamen, and even in Fujian Province, China, which would support making the prevention and control strategies for cervical cancer in the region.

[Identification of origins of marker chromosomes using fluorescence in situ hybridization].

OBJECTIVE: To assess the value of fluorescence in situ hybridization (FISH) and bacterial artificial chromosome FISH (BAC-FISH) for the diagnosis for patients with marker chromosomes. METHODS: Sixteen patients with marker chromosomes were analyzed with technologies including GTG-banding, Q-banding, multiplex FISH and BAC-FISH. RESULTS: The marker chromosomes in the 16 patients were verified as der(Y) (2 cases), psu dic(Y) (1 case), psu dic(15) (1 case), dic(15) (1 case), del(Y) (1 case), r(X) (5 cases), i(14 or 22) (2 cases), i(18) (1 case). CONCLUSION: FISH and BAC-FISH can both verify the origin of marker chromosomes and provide accurate information for the diagnosis and treatment of patients.

Investigation of the frequencies of prenatally diagnosed fetal chromosomal abnormalities at a single institution.

The objective of this study was to investigate, retrospectively, the frequencies of fetal chromosomal abnormalities identified in 4176 prenatal cytogenetic examinations at the Xiamen Maternity and Child Health Care Hospital over the 5-year period from October 2005 to September 2010. The frequency of abnormal fetal karyotypes was 4.6%. Numerical chromosome abnormalities were identified in 150 cases. The frequency of trisomy 21 was by far the highest, followed by trisomy 18. Structural aberrations of chromosomes were identified in 43 cases, including 21 cases with balanced and 22 cases with unbalanced chromosomal aberrations. In addition, 16 cases of apparently de novo chromosomal aberrations and 27 cases of familial inheritances were observed. Increased awareness of the frequencies of fetal chromosome abnormalities is important for the improvement of prenatal care and providing the options of termination or continuation of the pregnancy. Data obtained in this study provide the basis of a database for genetic counseling.

The value of NIPT combined with serum cell-free DNA, estriol, AFP, and b-HCG levels in the recognition of trisomy 21 and 18 in the second trimester.

Background: This study aimed to evaluate the clinical application value of noninvasive prenatal testing from DNA (NIPT) and serum screening for screening in detecting fetal trisomy 21 and 18. Methods: As a retrospective analysis, we collected data from 1383 women (singleton pregnancy) who underwent serum screening and noninvasive prenatal testing from DNA (NIPT) in our department from May 2015 to September 2017 and calculated the diagnostic value of the two methods.

Prevalence and genotype distribution of HPV infection among women in Xiamen, China.

Objective: This study aimed to evaluate the prevalence of HPV and genotype distribution among female populations in Xiamen, Fujian Province, China, which can be conducive for local governments to formulate cervical cancer screening and HPV vaccine strategies. Methods: Cervical swabs were collected from 47,926 participants aged 16-92 years at the Women and Children's Hospital, Xiamen University, from November 2019 to June 2020. HPV DNA was extracted and detected using conventional PCR, followed by HPV subtype-specific hybridisation. HPV infection rates based on different groups were compared using the χ2 test. HPV prevalence and the corresponding 95% confidence intervals (95% CI) were calculated using SPSS 19.0. Results: The overall HPV prevalence among the 47,926 cervical swabs that were analysed was 15.13%, of which single, double, and multiple infections accounted for 76.83, 16.70 and 6.47%, respectively. The age-specific prevalence of HPV infection presented a "U" curve with a HPV prevalence peak observed in women aged <20 years. The gynaecology clinic group had significantly higher HPV positive rates than the health examination group (p < 0.001). The five most common HR-HPV subtypes in Xiamen were HPV52, 58, 16, 51, and 39 (2.69, 1.63, 1.23, 1.05, and 0.98%, respectively). The five most common LR-HPV subtypes were HPV54, 61, 81, 70, 34, and 84 (0.92, 0.86, 0.71, 0.45 and 0.35%, respectively). Conclusion: Our findings demonstrate that the 9-valent HPV vaccine is recommended for regular immunisation in Xiamen. It is necessary for elderly women to participate in HPV screening to decrease the morbidity and mortality of cervical cancer.