To evaluate the effectiveness of whole-genome array comparative genomic hybridization (aCGH) in prenatal diagnosis in Hong Kong.
Array CGH was performed on 220 samples recruited prospectively as the ...first-tier test study. In addition 150 prenatal samples with abnormal fetal ultrasound findings found to have normal karyotypes were analyzed as a 'further-test' study using NimbleGen CGX-135K oligonucleotide arrays.
Array CGH findings were concordant with conventional cytogenetic results with the exception of one case of triploidy. It was found in the first-tier test study that aCGH detected 20% (44/220) clinically significant copy number variants (CNV), of which 21 were common aneuploidies and 23 had other chromosomal imbalances. There were 3.2% (7/220) samples with CNVs detected by aCGH but not by conventional cytogenetics. In the 'further-test' study, the additional diagnostic yield of detecting chromosome imbalance was 6% (9/150). The overall detection for CNVs of unclear clinical significance was 2.7% (10/370) with 0.9% found to be de novo. Eleven loci of common CNVs were found in the local population.
Whole-genome aCGH offered a higher resolution diagnostic capacity than conventional karyotyping for prenatal diagnosis either as a first-tier test or as a 'further-test' for pregnancies with fetal ultrasound anomalies. We propose replacing conventional cytogenetics with aCGH for all pregnancies undergoing invasive diagnostic procedures after excluding common aneuploidies and triploidies by quantitative fluorescent PCR. Conventional cytogenetics can be reserved for visualization of clinically significant CNVs.
Abstract
Mosaicism refers to the presence of two or more populations of genetically distinct cells within an individual, all of which originate from a single zygote. Previous literature estimated the ...percentage of parental mosaicism ranged from 0.33 to 25.9%. In this study, parents whose children had previously been diagnosed with developmental disorders with an apparent de novo variant were recruited. Peripheral blood, buccal and semen samples were collected from these parents if available for the detection of potential parental mosaicism using droplet digital PCR, complemented with the method of blocker displacement amplification. Among the 20 families being analyzed, we report four families with parental mosaicism (4/20, 20%). Two families have maternal gonosomal mosaicism (
EYA1
and
EBF3
) and one family has paternal gonadal mosaicism (
CHD7
) with a pathogenic/ likely pathogenic variant. One family has a paternal gonosomal mosaicism with a variant of uncertain significance (
FLNC
) with high clinical relevance. The detectable variant allele frequency in our cohort ranged from 8.7–35.9%, limit of detection 0.08–0.16% based on our in-house
EBF3
assay. Detecting parental mosaicism not only informs family with a more accurate recurrence risk, but also facilitates medical teams to create appropriate plans for pregnancy and delivery, offering the most suitable care.
The maternal immune system needs to tolerate the semi-allogeneic fetus in pregnancy. The adaptation occurs locally at the maternal-fetal interface as well as systemically through the maternal ...circulation. Failure to tolerate the paternal antigens may result in pregnancy complications, such as pregnancy loss and pre-eclampsia. However, the mechanism that regulates maternal immune tolerance, especially at the systemic level, is still an enigma. Here we report that the first-trimester placenta-derived exosomes (pEXOs) contribute to maternal immune tolerance by reprogramming the circulating monocytes.
pEXOs predominantly target monocytes and pEXO-educated monocytes exhibit an immunosuppressive phenotype as demonstrated by reduced expression of marker genes for monocyte activation, T-cell activation and antigen-process/presentation at the transcriptomic level. They also have a greater propensity towards M2 polarization when compared to the monocytes without pEXO treatment. The inclusion of pEXOs in a monocyte-T-cell coculture model significantly reduces proliferation of the T helper cells and cytotoxic T cells and elevates the expansion of regulatory T cells. By integrating the microRNAome of pEXO and the transcriptomes of pEXO-educated monocytes as well as various immune cell functional assays, we demonstrate that the pEXO-derived microRNA miR-29a-3p promotes the expression of programmed cell death ligand-1, a well-known surface receptor that suppresses the adaptive immune system, by down-regulation of phosphatase and tensin homolog in monocytes.
This is the first report to show how human pEXO directly regulates monocyte functions and its molecular mechanism during early pregnancy. The results uncover the importance of pEXO in regulating the maternal systemic immune response during early pregnancy by reprogramming circulating monocytes. The study provides the basis for understanding the regulation of maternal immune tolerance to the fetal allograft.
To evaluate the clinical impact of chromosomal microarray (CMA) on the management of paediatric patients in Hong Kong.
We performed NimbleGen 135k oligonucleotide array on 327 children with ...intellectual disability (ID)/developmental delay (DD), autism spectrum disorders (ASD), and/or multiple congenital anomalies (MCAs) in a university-affiliated paediatric unit from January 2011 to May 2013. The medical records of patients were reviewed in September 2013, focusing on the pathogenic/likely pathogenic CMA findings and their "clinical actionability" based on established criteria.
Thirty-seven patients were reported to have pathogenic/likely pathogenic results, while 40 had findings of unknown significance. This gives a detection rate of 11% for clinically significant (pathogenic/likely pathogenic) findings. The significant findings have prompted clinical actions in 28 out of 37 patients (75.7%), while the findings with unknown significance have led to further management recommendation in only 1 patient (p < 0.001). Nineteen out of the 28 management recommendations are "evidence-based" on either practice guidelines endorsed by a professional society (n = 9, Level 1) or peer-reviewed publications making medical management recommendation (n = 10, Level 2). CMA results impact medical management by precipitating referral to a specialist (n = 24); diagnostic testing (n = 25), surveillance of complications (n = 19), interventional procedure (n = 7), medication (n = 15) or lifestyle modification (n = 12).
The application of CMA in children with ID/DD, ASD, and/or MCAs in Hong Kong results in a diagnostic yield of ∼ 11% for pathogenic/likely pathogenic results. Importantly the yield for clinically actionable results is 8.6%. We advocate using diagnostic yield of clinically actionable results to evaluate CMA as it provides information of both clinical validity and clinical utility. Furthermore, it incorporates evidence-based medicine into the practice of genomic medicine. The same framework can be applied to other genomic testing strategies enabled by next-generation sequencing.
RNA sequencing (RNA-seq) is emerging in genetic diagnoses as it provides functional support for the interpretation of variants of uncertain significance. However, the use of amniotic fluid (AF) cells ...for RNA-seq has not yet been explored. Here, we examined the expression of clinically relevant genes in AF cells (n = 48) compared with whole blood and fibroblasts. The number of well-expressed genes in AF cells was comparable to that in fibroblasts and much higher than that in blood across different disease categories. We found AF cells RNA-seq feasible and beneficial in prenatal diagnosis (n = 4) as transcriptomic data elucidated the molecular consequence leading to the pathogenicity upgrade of variants in CHD7 and COL1A2 and revising the in silico prediction of a variant in MYRF. AF cells RNA-seq could become a reasonable choice for postnatal patients with advantages over fibroblasts and blood as it prevents invasive procedures.
Whole-exome sequencing (WES) has become an invaluable tool for genetic diagnosis in paediatrics. However, it has not been widely adopted in the prenatal setting. This study evaluated the use of WES ...in prenatal genetic diagnosis in fetuses with structural congenital anomalies (SCAs) detected on prenatal ultrasound.
Thirty-three families with fetal SCAs on prenatal ultrasonography and normal chromosomal microarray results were recruited. Genomic DNA was extracted from various fetal samples including amniotic fluid, chorionic villi, and placental tissue. Parental DNA was extracted from peripheral blood when available. We used WES to sequence the coding regions of parental-fetal trios and to identify the causal variants based on the ultrasonographic features of the fetus.
Pathogenic mutations were identified in three families (n = 3/33, 9.1%), including mutations in DNAH11, RAF1 and CHD7, which were associated with primary ciliary dyskinesia, Noonan syndrome, and CHARGE syndrome, respectively. In addition, variants of unknown significance (VUSs) were detected in six families (18.2%), in which genetic changes only partly explained prenatal features.
WES identified pathogenic mutations in 9.1% of fetuses with SCAs and normal chromosomal microarray results. Databases for fetal genotype-phenotype correlations and standardized guidelines for variant interpretation in prenatal diagnosis need to be established to facilitate the use of WES for routine testing in prenatal diagnosis.
Chromosomal microarray (CMA) is recommended as a first tier investigation for patients with developmental delay (DD), intellectual disability (ID), autistic spectrum disorder (ASD), and multiple ...congenital anomalies (MCA). It is widely used in the prenatal and postnatal settings for detection of chromosomal aberrations. This is a retrospective review of all array comparative genomic hybridization (aCGH/ array CGH) findings ascertained in two major prenatal and postnatal genetic diagnostic centers in Hong Kong from June 2012 to December 2017. Medical records were reviewed for cases with pathogenic and variants of uncertain clinical significance (VUS). Classification of copy number variants (CNVs) was based on current knowledge and experience by August 2018. The aims of this review are to study the diagnostic yield of array CGH application in prenatal and postnatal settings in Hong Kong and to describe the spectrum of abnormalities found. Prenatal indications included abnormal ultrasound findings, positive Down syndrome screening, abnormal noninvasive prenatal test results, advanced maternal age and family history of chromosomal or genetic abnormalities. Postnatal indications included unexplained DD, ID, ASD, and MCA.
A total of 1,261 prenatal subjects and 3,096 postnatal patients were reviewed. The prenatal diagnostic yield of pathogenic CNV and VUS (excluding those detectable by karyotype) was 3.5%. The postnatal diagnostic yield of pathogenic CNV was 15.2%. The detection rates for well‐defined microdeletion and microduplication syndromes were 4.6% in prenatal and 6.1% (1 in 16 index patients) in postnatal cases, respectively. Chromosomes 15, 16, and 22 accounted for over 21 and 25% of pathogenic CNVs detected in prenatal and postnatal cohorts, respectively. This review provides the first large scale overview of genomic imbalance of mostly Chinese patients in prenatal and postnatal settings.
Hemophilia A is a rare bleeding disorder with variable expressivity and allelic heterogeneity. Despite the advancement of prenatal diagnostics and molecular studies, the number of studies reviewing ...the reproductive choices of hemophilia A carriers and affected individuals remains limited. Through this retrospective review, we hope to gain a deeper understanding of hemophilia A-affected individuals' clinical and molecular characteristics, as well as the reproductive choices of the at-risk couples. A total of 122 individuals harboring likely causative F8 gene alterations from 64 apparently unrelated families attending three centers between 3/2000 and 3/2023 were included in this study. Their clinical and molecular findings as well as reproductive choices were gathered in a clinical setting and verified through the electronic medical record database of the public health system. Forty-seven affected males and 75 female heterozygous carriers were included in the analysis. Among 64 apparently unrelated families, 36 distinct pathogenic/likely pathogenic variants were identified, of which 30.6% (11/36) of variants were novel. While the majority of clinical findings and genotype-phenotype correlations appear to be in accordance with existing literature, female carriers who had no fertility intention were significantly more likely to have affected sons than those who had fertility intention (5/19 vs. 4/5; p = 0.047). Through this retrospective review, we summarized the clinical and molecular characteristics of 122 individuals harboring pathogenic/likely pathogenic F8 variants, as well as their fertility intentions and reproductive outcomes. Further studies are required to look into the considerations involved in reproductive decision-making.
Bulleted Statements:
What's already known about this topic?
The incidence of discordant fetal sex was estimated to be 1 in 1500–2000. Comprehensive evaluation is required to investigate the ...underlying cause.
What does this study add?
Chimera is one of the possible causes of genotype–phenotype sex discordance. It could be detected prenatally with the combination of noninvasive prenatal screening and ultrasound.
Genetic counseling to prospective parents on chimera can be complex where early support and information from experienced professionals are important.
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