Sodium Channel Gene Variants in Fetuses with Abnormal Sonographic Findings: Expanding the Prenatal Phenotypic Spectrum of Sodium Channelopathies.

Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in the brain and muscle. Pathogenic variants in genes encoding VGSCs have been associated with severe disorders including epileptic encephalopathies and congenital myopathies. In this study, we identified pathogenic variants in genes encoding the α subunit of VGSCs in the fetuses of two unrelated families with the use of trio-based whole exome sequencing, as part of a larger cohort study. Sanger sequencing was performed for variant confirmation as well as parental phasing. The fetus of the first family carried a known de novo heterozygous missense variant in the SCN2A gene (NM_001040143.2:c.751G>A p.(Val251Ile)) and presented intrauterine growth retardation, hand clenching and ventriculomegaly. Neonatally, the proband also exhibited refractory epilepsy, spasms and MRI abnormalities. The fetus of the second family was a compound heterozygote for two parentally inherited novel missense variants in the SCN4A gene (NM_000334.4:c.4340T>C, p.(Phe1447Ser), NM_000334.4:c.3798G>C, p.(Glu1266Asp)) and presented a severe prenatal phenotype including talipes, fetal hypokinesia, hypoplastic lungs, polyhydramnios, ear abnormalities and others. Both probands died soon after birth. In a subsequent pregnancy of the latter family, the fetus was also a compound heterozygote for the same parentally inherited variants. This pregnancy was terminated due to multiple ultrasound abnormalities similar to the first pregnancy. Our results suggest a potentially crucial role of the VGSC gene family in fetal development and early lethality.

Fast Temperature-Gradient COLD PCR for the enrichment of the paternally inherited SNPs in cell free fetal DNA; an application to non-invasive prenatal diagnosis of ß-thalassaemia.

OBJECTIVE: To develop a sensitive, specific, simple, cost-effective and reproducible platform for the non-invasive prenatal detection of paternally inherited alleles for ß-thalassaemia. The development of such an assay is of major significance in order to replace currently-applied invasive methods containing inherent fetal loss risks. METHODS: We present a fast Temperature-Gradient Co-amplification at Lower Denaturation Temperature Polymerase Chain Reaction (fast TG COLD PCR) methodology for the detection of the paternally-inherited fetal alleles in maternal plasma. Two single-nucleotide polymorphisms (SNPs), rs7480526 (G/T) and rs968857 (G/A) that are located on the ß-globin gene cluster and exhibit a high degree of heterozygosity in the Cypriot population were selected for evaluation. Seventeen maternal plasma samples from pregnancies at risk for ß-thalassemia were analysed for the selected SNPs using the novel fast TG COLD PCR assay. RESULTS: Using fast TG COLD PCR, the paternally inherited allele in cell free fetal DNA was correctly determined for all the 17 maternal plasma samples tested, showing full agreement with the Chorionic Villus Sampling (CVS) analysis. CONCLUSIONS: Our findings are encouraging and demonstrate the efficiency and sensitivity of fast TG COLD PCR in detecting the minor paternally-inherited fetal alleles in maternal plasma for the development of a NIPD assay for ß-thalassaemia.

Cell-Free DNA Analysis of Targeted Genomic Regions in Maternal Plasma for Non-Invasive Prenatal Testing of Trisomy 21, Trisomy 18, Trisomy 13, and Fetal Sex.

BACKGROUND: There is great need for the development of highly accurate cost effective technologies that could facilitate the widespread adoption of noninvasive prenatal testing (NIPT). METHODS: We developed an assay based on the targeted analysis of cell-free DNA for the detection of fetal aneuploidies of chromosomes 21, 18, and 13. This method enabled the capture and analysis of selected genomic regions of interest. An advanced fetal fraction estimation and aneuploidy determination algorithm was also developed. This assay allowed for accurate counting and assessment of chromosomal regions of interest. The analytical performance of the assay was evaluated in a blind study of 631 samples derived from pregnancies of at least 10 weeks of gestation that had also undergone invasive testing. RESULTS: Our blind study exhibited 100% diagnostic sensitivity and specificity and correctly classified 52/52 (95% CI, 93.2%-100%) cases of trisomy 21, 16/16 (95% CI, 79.4%-100%) cases of trisomy 18, 5/5 (95% CI, 47.8%-100%) cases of trisomy 13, and 538/538 (95% CI, 99.3%-100%) normal cases. The test also correctly identified fetal sex in all cases (95% CI, 99.4%-100%). One sample failed prespecified assay quality control criteria, and 19 samples were nonreportable because of low fetal fraction. CONCLUSIONS: The extent to which free fetal DNA testing can be applied as a universal screening tool for trisomy 21, 18, and 13 depends mainly on assay accuracy and cost. Cell-free DNA analysis of targeted genomic regions in maternal plasma enables accurate and cost-effective noninvasive fetal aneuploidy detection, which is critical for widespread adoption of NIPT.

Implementation of high resolution whole genome array CGH in the prenatal clinical setting: advantages, challenges, and review of the literature.

Array Comparative Genomic Hybridization analysis is replacing postnatal chromosomal analysis in cases of intellectual disabilities, and it has been postulated that it might also become the first-tier test in prenatal diagnosis. In this study, array CGH was applied in 64 prenatal samples with whole genome oligonucleotide arrays (BlueGnome, Ltd.) on DNA extracted from chorionic villi, amniotic fluid, foetal blood, and skin samples. Results were confirmed with Fluorescence In Situ Hybridization or Real-Time PCR. Fifty-three cases had normal karyotype and abnormal ultrasound findings, and seven samples had balanced rearrangements, five of which also had ultrasound findings. The value of array CGH in the characterization of previously known aberrations in five samples is also presented. Seventeen out of 64 samples carried copy number alterations giving a detection rate of 26.5%. Ten of these represent benign or variables of unknown significance, giving a diagnostic capacity of the method to be 10.9%. If karyotype is performed the additional diagnostic capacity of the method is 5.1% (3/59). This study indicates the ability of array CGH to identify chromosomal abnormalities which cannot be detected during routine prenatal cytogenetic analysis, therefore increasing the overall detection rate. In addition a thorough review of the literature is presented.

Clinical application of whole-genome array CGH during prenatal diagnosis: Study of 25 selected pregnancies with abnormal ultrasound findings or apparently balanced structural aberrations.

BACKGROUND: The purpose of the study was the application and evaluation of array Comparative Genomic Hybridization (array CGH) in selected cases during prenatal diagnosis. Array CGH was applied in 25 fetal samples out of which 15 had normal karyotypes and abnormal ultrasound findings and 10 had apparently balanced structural aberrations with or without abnormal ultrasound findings. DNA was extracted from peripheral blood, chorionic villi samples (CV) and amniotic fluid. Bacterial Artificial Chromosome (BAC) array CGH (Cytochip, BlueGnome Ltd.) of 1 Mb was applied and results were confirmed with either Fluorescence In Situ Hybridization (FISH), Multiplex Ligation-dependant Probe Amplification (MLPA) or Real-Time PCR. RESULTS: Three out of 25 samples (12%), referred for prenatal array CGH, were found to carry copy number alterations. The number of cases with clinically significant alterations was 2/25 (8%), while one (4%) was of uncertain clinical significance. Two benign Copy Number Variations (CNVs) were also found in 1/25 cases (4%). CONCLUSIONS: The outcome of this study indicates the ability of array CGH to identify chromosomal abnormalities which cannot be detected during routine prenatal cytogenetic analysis, therefore increasing the overall detection rate.