High positive predictive value of CNVs detected by clinical exome sequencing in suspected genetic diseases.

BACKGROUND: Genetic disorders often manifest as abnormal fetal or childhood development. Copy number variations (CNVs) represent a significant genetic mechanism underlying such disorders. Despite their importance, the effectiveness of clinical exome sequencing (CES) in detecting CNVs, particularly small ones, remains incompletely understood. We aimed to evaluate the detection of both large and small CNVs using CES in a substantial clinical cohort, including parent-offspring trios and proband only analysis. METHODS: We conducted a retrospective analysis of CES data from 2428 families, collected from 2018 to 2021. Detected CNV were categorized as large or small, and various validation techniques including chromosome microarray (CMA), Multiplex ligation-dependent probe amplification assay (MLPA), and/or PCR-based methods, were employed for cross-validation. RESULTS: Our CNV discovery pipeline identified 171 CNV events in 154 cases, resulting in an overall detection rate of 6.3%. Validation was performed on 113 CNVs from 103 cases to assess CES reliability. The overall concordance rate between CES and other validation methods was 88.49% (100/113). Specifically, CES demonstrated complete consistency in detecting large CNV. However, for small CNVs, consistency rates were 81.08% (30/37) for deletions and 73.91% (17/23) for duplications. CONCLUSION: CES demonstrated high sensitivity and reliability in CNV detection. It emerges as an economical and dependable option for the clinical CNV detection in cases of developmental abnormalities, especially fetal structural abnormalities.

Preliminary mechanism in fetal alloimmune thrombocytopenia associated with anti-HPA 15b antibodies.

OBJECTIVE: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a bleeding disease that can cause fetal hydrops, a rare but life-threatening condition in which abnormal amounts of fluid accumulate in one or two areas of the fetus's body. A case of FNAIT with fetal hydrops caused by anti-HPA-15b antibodies was involved in this study, as we investigated whether or not anti-HPA-15b antibodies can induce endothelial angiogenesis and apoptosis. METHODS: The monoclonal antibody immobilization of platelet antigens assay (MAIPA) was used to identify anti-HPA-15b antibodies. The three groups in Tube formation and apoptosis assays were the PBS group, the AB serum IgG group, and the anti-HPA-15b serum IgG group, all reacted with HPA-15bb HUVEC. RESULTS: The presence of anti-HPA-15b antibodies was found in this case by MAIPA assay. The OD values are 0.33 and 0.21, reacted with HPA-15bb and HPA-15ab platelets, respectively (cutoff OD value = 0.2). Quantitative analysis revealed that the length of capillary-like tube induced by anti-HPA-15b antibodies was significantly decreased over that of AB serum IgG (*p = 0.0005), but weaker than when incubated with thrombin (**p = 0.0009). The apoptosis results show a significantly increased number of apoptotic endothelial cells in the anti-HPA-15b antibody IgG group when compared with the PBS and AB serum IgG groups (*p < 0.0001, **p < 0.0001). In addition, there is no statistical difference between the PBS and AB serum groups. CONCLUSION: Anti-HPA-15b antibodies can inhibit angiogenesis and induce apoptosis. This may associate with hydrops fetalis (HF), or fetal hydrops of FNAIT.

Hydrops fetalis associated with anti-CD36 antibodies in fetal and neonatal alloimmune thrombocytopenia: Possible underlying mechanism.

OBJECTIVES: In the present study, we asked whether anti-CD36 antibodies impair the maturation of erythropoietic stem cells to mature red blood cells (RBCs), leading to anaemia and hydrops fetalis (HF). BACKGROUND: Recent studies have shown the importance of anti-CD36 antibodies in the development of Fetal/Neonatal Alloimmune Thrombocytopenia (FNAIT). In comparison to other types of antibody-mediated FNAIT, anti-CD36 antibodies are frequently associated with anaemia and HF. As mature RBCs do not express CD36, the reason for this phenomenon is currently not fully understood. MATERIAL AND METHODS: A case of FNAIT with signs of HF was characterised in this study. Maternal anti-CD36 antibodies were isolated by an absorption/elution approach. We cultured haematopoietic stem cells (HSCs) with purified anti-CD36 antibodies, and the formation of burst-forming unit-erythroid and colony-forming unit-erythroid (CFU-E/BFU-E) cells was analysed. Apoptosis of HSCs was also investigated. RESULTS: Analysis of the mother showed type-1 CD36 deficiency. Anti-CD36 antibodies were found in maternal serum, as well as on fetal platelets, by ELISA, and the specificity of these antibodies was further substantiated by flow cytometry. In comparison to control IgG, incubation of HSCs with purified anti-CD36 antibodies led to a significant reduction in CFU-E/BFU-E cell formation, and this result was associated with an increased number of apoptotic CD34+ erythroid/myeloid precursor cells. Administration of intra-uterine transfusion with washed RBCs was effective in improving fetal anaemia. CONCLUSIONS: Anti-CD36 antibodies may cause anaemia and trigger HF through apoptosis of CD34+ erythroid/myeloid precursor cells. However, the contribution of other cells must also be taken into account.

Noninvasive detection of fetal subchromosomal abnormalities by semiconductor sequencing of maternal plasma DNA.

Noninvasive prenatal testing (NIPT) using sequencing of fetal cell-free DNA from maternal plasma has enabled accurate prenatal diagnosis of aneuploidy and become increasingly accepted in clinical practice. We investigated whether NIPT using semiconductor sequencing platform (SSP) could reliably detect subchromosomal deletions/duplications in women carrying high-risk fetuses. We first showed that increasing concentration of abnormal DNA and sequencing depth improved detection. Subsequently, we analyzed plasma from 1,456 pregnant women to develop a method for estimating fetal DNA concentration based on the size distribution of DNA fragments. Finally, we collected plasma from 1,476 pregnant women with fetal structural abnormalities detected on ultrasound who also underwent an invasive diagnostic procedure. We used SSP of maternal plasma DNA to detect subchromosomal abnormalities and validated our results with array comparative genomic hybridization (aCGH). With 3.5 million reads, SSP detected 56 of 78 (71.8%) subchromosomal abnormalities detected by aCGH. With increased sequencing depth up to 10 million reads and restriction of the size of abnormalities to more than 1 Mb, sensitivity improved to 69 of 73 (94.5%). Of 55 false-positive samples, 35 were caused by deletions/duplications present in maternal DNA, indicating the necessity of a validation test to exclude maternal karyotype abnormalities. This study shows that detection of fetal subchromosomal abnormalities is a viable extension of NIPT based on SSP. Although we focused on the application of cell-free DNA sequencing for NIPT, we believe that this method has broader applications for genetic diagnosis, such as analysis of circulating tumor DNA for detection of cancer.

Genetic counseling and prenatal diagnosis for hereditary hearing loss in high-risk families.

OBJECTIVE: Genetic counseling and prenatal diagnosis are very necessary and accurate to detect hereditary hearing loss, especially in high-risk families. Prenatal diagnosis is testing for diseases or conditions in fetuses before born, which gives parents the chance to prepare psychologically, financially and medically for the probable health and educational needs of the affected neonates. METHODS: 54 unrelated families with children affected with non-syndromic sensorineural hearing loss were enrolled in the study and received genetic analysis with microarray and DNA sequencing technologies. Genetic counseling was provided to each participating families, and prenatal diagnosis was given to those at risk and would like to know their fetuses' genotypes and probable hearing statuses. RESULTS: Half the cases in the present study were diagnosed with confirmed pathogenic mutations and clear inheritance patterns. After receiving genetic counseling, 24 carrier couples with pathogenic mutations chose to proceed prenatal diagnosis, the results of which were in accordance with the pregnancy outcomes. Infants prenatally detected to be monoallelic mutation carriers and those harbored neither deafness-causing mutations form their parents passed newborn hearing screening and six-month follow-ups, while neonates prenatally detected to be carriers of diallelic or compound heterozygous mutations developed hearing loss after birth. CONCLUSIONS: With appropriate genetic counseling and support services provided, the genetic testing and the prenatal diagnosis of hearing loss were valued by carrier couples for the information provided for future family planning and probably the preparation for the health and educational needs of the affected neonates.

Noninvasive prenatal diagnosis of common aneuploidies by semiconductor sequencing.

Massively parallel sequencing (MPS) of cell-free fetal DNA from maternal plasma has revolutionized our ability to perform noninvasive prenatal diagnosis. This approach avoids the risk of fetal loss associated with more invasive diagnostic procedures. The present study developed an effective method for noninvasive prenatal diagnosis of common chromosomal aneuploidies using a benchtop semiconductor sequencing platform (SSP), which relies on the MPS platform but offers advantages over existing noninvasive screening techniques. A total of 2,275 pregnant subjects was included in the study; of these, 515 subjects who had full karyotyping results were used in a retrospective analysis, and 1,760 subjects without karyotyping were analyzed in a prospective study. In the retrospective study, all 55 fetal trisomy 21 cases were identified using the SSP with a sensitivity and specificity of 99.94% and 99.46%, respectively. The SSP also detected 16 trisomy 18 cases with 100% sensitivity and 99.24% specificity and 3 trisomy 13 cases with 100% sensitivity and 100% specificity. Furthermore, 15 fetuses with sex chromosome aneuploidies (10 45,X, 2 47,XYY, 2 47,XXX, and 1 47,XXY) were detected. In the prospective study, nine fetuses with trisomy 21, three with trisomy 18, three with trisomy 13, and one with 45,X were detected. To our knowledge, this is the first large-scale clinical study to systematically identify chromosomal aneuploidies based on cell-free fetal DNA using the SSP and provides an effective strategy for large-scale noninvasive screening for chromosomal aneuploidies in a clinical setting.

A prenatal missed diagnosed case of submicroscopic chromosomal abnormalities by karyotyping: the clinical utility of array-based CGH in prenatal diagnostics.

BACKGROUND: Array-based comparative genomic hybridization possesses a number of significant advantages over conventional cytogenetic and other molecular cytogenetic techniques, providing a sensitive and comprehensive detection platform for unexpected imbalances in the genome wide. CASE PRESENTATION: The newborn proband, demonstrated with craniofacial dysmorphism and multiple malformations, was born to a family with spontaneous abortions. This pregnancy was uneventful, except the prenatal ultrasound examination showed an increased nuchal translucency at 12(+) weeks of gestation. Cytogenetics revealed an apparently normal karyotype, and the couple decided to continue the pregnancy. Array-based CGH analysis was applied to the affected infant, identified a combination of 18p deletion and 7q duplication. Further study indicates that the unbalanced translocation was inherited from a balanced translocation carrier parent. CONCLUSIONS: In review of the case, several overlooked points leading to the missed diagnosis should be discussed and certain quality control strategies should be adopted to avoid similar problems in the future. Array-based CGH and karyotyping techniques are complemented by diverse detection spectrum and resolutions, and a combination of these methods could help providing optimal genetic diagnosis. Given that the array-CGH analysis will not introduce additional risk to patients, it is reasonable to recommend those already undergoing invasive testing should take array-based CGH as an adjunct to conventional cytogenetic tests and other molecular cytogenetic analysis.

Reference values of fetal serum ß2-microglobulin in the Chinese: evaluation of its clinical usefulness.

BACKGROUND: The level of ß2-microglobulin was generally used to evaluate the renal function in adults. Elevated levels of ß2-microglobulin were also applied to assess the perinatal situations in neonates and fetuses. The aim of our study was to establish and determine the reference values of fetal serum ß2-microglobulin in the Chinese and to assess its clinical benefits in abnormal fetuses. METHODS: Data from 308 normal cord blood samples were obtained to calculate the normal reference values of fetal serum ß2-microglobulin. According to the equations we obtained, we analyzed the level of ß2-microglobulin in four case groups: renal malformation, hydrops, cytomegalovirus (CMV) infection and rubella virus (RV) infection. RESULTS: In the normal group, the concentration of ß2-microglobulin decreased with the gestational age, with a mean value of 4.35±0.59 mg/L. The upper limit of ß2-microglobulin was calculated as 7.55-0.074×gestational age in weeks. The levels of ß2-microglobulin were significantly higher in the four case groups than the normal group. For the four groups, the sensitivity is 72.7%, 69.6%, 86.7% and 100%, respectively. CONCLUSIONS: Fetal serum ß2-microglobulin may be used as a predictor to evaluate the situations of fetal diseases.

The carrier rate and mutation spectrum of genes associated with hearing loss in South China hearing female population of childbearing age.

BACKGROUND: Given that hearing loss occurs in 1 to 3 of 1,000 live births and approximately 90 to 95 percent of them are born into hearing families, it is of importance and necessity to get better understanding about the carrier rate and mutation spectrum of genes associated with hearing impairment in the general population. METHODS: 7,263 unrelated women of childbearing age with normal hearing and without family history of hearing loss were tested with allele-specific PCR-based universal array. Further genetic testing were provided to the spouses of the screened carriers. For those couples at risk, multiple choices were provided, including prenatal diagnosis. RESULTS: Among the 7,263 normal hearing participants, 303 subjects carried pathogenic mutations included in the screening chip, which made the carrier rate 4.17%. Of the 303 screened carriers, 282 harbored heterozygous mutated genes associated with autosomal recessive hearing loss, and 95 spouses took further genetic tests. 8 out of the 9 couples harbored deafness-causing mutations in the same gene received prenatal diagnosis. CONCLUSIONS: Given that nearly 90 to 95 percent of deaf and hard-of-hearing babies are born into hearing families, better understanding about the carrier rate and mutation spectrum of genes associated with hearing impairment in the female population of childbearing age may be of importance in carrier screening and genetic counseling.