BACKGROUND: Cell-free fetal DNA (cffDNA) constitutes approximately 10% of the cell-free DNA in maternal plasma and is a suitable source of fetal genetic material for noninvasive prenatal diagnosis (NIPD). The objective of this study was to determine the feasibility of using digital PCR for NIPD in pregnancies at risk of sickle cell anemia.

Aortic arch artery patterning defects account for approximately 20% of congenital cardiovascular malformations and are observed frequently in velocardiofacial syndrome (VCFS). In the current study, we screened for chromosome rearrangements in patients suspected of VCFS, but who lacked a 22q11 deletion or TBX1 mutation. One individual displayed hemizygous CHD7, which encodes a chromodomain protein. CHD7 haploinsufficiency is the major cause of coloboma, heart defect, atresia choanae, retarded growth and development, genital hypoplasia, and ear anomalies/deafness (CHARGE) syndrome, but this patient lacked the major diagnostic features of coloboma and choanal atresia. Because a subset of CHARGE cases also display 22q11 deletions, we explored the embryological relationship between CHARGE and VCSF using mouse models. The hallmark of Tbx1 haploinsufficiency is hypo/aplasia of the fourth pharyngeal arch artery (PAA) at E10.5. Identical malformations were observed in Chd7 heterozygotes, with resulting aortic arch interruption at later stages. Other than Tbx1, Chd7 is the only gene reported to affect fourth PAA development by haploinsufficiency. Moreover, Tbx1 +/-;Chd7 +/-double heterozygotes demonstrated a synergistic interaction during fourth PAA, thymus, and ear morphogenesis. We could not rescue PAA morphogenesis by restoring neural crest Chd7 expression. Rather, biallelic expression of Chd7 and Tbx1 in the pharyngeal ectoderm was required for normal PAA development.

ObjectiveAccurate prenatal diagnosis of genetic conditions can be challenging and usually requires invasive testing. Here, we demonstrate the potential of next-generation sequencing (NGS) for the analysis of cell-free DNA in maternal blood to transform prenatal diagnosis of monogenic disorders.MethodsAnalysis of cell-free DNA using a PCR and restriction enzyme digest (PCR–RED) was compared with a novel NGS assay in pregnancies at risk of achondroplasia and thanatophoric dysplasia.ResultsPCR–RED was performed in 72 cases and was correct in 88.6%, inconclusive in 7% with one false negative. NGS was performed in 47 cases and was accurate in 96.2% with no inconclusives. Both approaches were used in 27 cases, with NGS giving the correct result in the two cases inconclusive with PCR–RED.ConclusionNGS provides an accurate, flexible approach to non-invasive prenatal diagnosis of de novo and paternally inherited mutations. It is more sensitive than PCR–RED and is ideal when screening a gene with multiple potential pathogenic mutations. These findings highlight the value of NGS in the development of non-invasive prenatal diagnosis for other monogenic disorders. © 2015 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.What's already known about this topic?Non-invasive prenatal diagnosis (NIPD) using PCR-based methods has been reported for the detection or exclusion of individual paternally inherited or de novo alleles in maternal plasma.What does this study add?NIPD using next generation sequencing provides an accurate, more sensitive approach which can be used to detect multiple mutations in a single assay and so is ideal when screening a gene with multiple potential pathogenic mutations. Next generation sequencing thus provides a flexible approach to non-invasive prenatal diagnosis ideal for use in a busy service laboratory.

The PLU-1 gene is expressed at the level of message in breast cancers and breast cancer cell lines and shows restricted expression in normal adult tissues with the exception of testis. The predicted protein sequence contains several domains, including the PLU domain, which is shared by other proteins involved in transcription and/or development. We have developed a polyclonal antiserum to a C-terminal fragment of the PLU-1 protein, which shows little homology to other family members. Immunohistochemic analysis with the antiserum ␣-PLU-1C confirmed the nuclear localisation of PLU-1. ␣-PLU-1C also reacted with the mouse homologue of PLU-1 (mPlu-1) but not with the closest family member, RBP2. Using Western blot analysis, PLU-1 was shown to be well expressed in breast cancers and breast cancer cell lines, while it was not detected in a range of normal adult tissues. Our results suggest that the PLU-1 protein may belong to the class of testis/cancer antigens. © 2002 Wiley-Liss, Inc. Key words: breast cancer: PLU-1 protein; testis/cancer antigenThe PLU-1 gene was identified as being downregulated in a human mammary epithelial cell line (MTSV1-7) transfected with HER2/c-erbB2, 1 after treatment with the 4D5/HERCEPTIN antibody, which inhibits c-erbB2 signaling. 2 However, in situ hybridisation and Northern blot analyses showed that PLU-1 mRNA is expressed in most breast cancers and breast cancer cell lines, regardless of the level of c-erbB2. 3 However, Northern blot analysis of total mRNA from normal human adult tissues showed high levels of expression in testis and detectable levels in placenta, but levels were undetectable in the other tissues examined.Following our own report on the identification and characterisation of PLU-1, other investigators reported on the cloning of cDNAs of splice and transcriptional variants of the PLU-1 gene. These were referred to as RBP2-H1 4 and RBBP2H1A, 5 respectively. The nomenclature of the splice and transcriptional variants of PLU-1 reflects the high homology between the predicted protein sequence of PLU-1 and the RBP2 6 in conserved domains, particularly in the novel PLU domain. 3,7 PLU-1 binds to a conserved consensus sequence in 2 transcriptions factors through the novel PLU domain. 8 Additionally, PLU-1 was a potent transcriptional repressor and may be involved in gene regulation in breast cancer. We also defined the sequence of mPlu-1 cDNA, which shows at the amino acid level an overall homology with human PLU-1 of 94% and almost 100% identity within the conserved domains. 9 The very conserved sequence of PLU-1 from human to mouse in combination with an almost identical expression pattern in adult tissues and breast cancers in both species suggests a conserved function in breast cancer.The later studies by Vogt et al. 4 and Kashuba et al. 5 on the RNA variants of PLU-1 examined mRNA expression using polyAϩ RNA and probes that would pick up all 3 transcripts. Expression was reported to be less restricted in normal tissues than we observed using total RNA. However, as we repo...