OBJECTIVES: Sequencing of cell-free fetal DNA from maternal plasma, also known as non-invasive prenatal testing (NIPT), has enabled accurate prenatal diagnosis of aneuploidy. This approach is gaining clinical acceptance, as it significantly reduces the necessity of invasive diagnostic procedures such as amniocentesis that carry a significant risk of fetal loss. Recent studies have demonstrated the potential for NIPT to detect subchromosomal abnormalities. We investigated whether NIPT using semiconductor sequencing could reliably detect subchromosomal deletions and duplications in a large population of women carrying high-risk fetuses. METHODS: We applied a sliding window method to reduce required sequencing depth. First, we demonstrated that increasing concentration of abnormal DNA as well as increasing number of sequencing reads improved detection of chromosomal abnormalities. We then analyzed plasma from 1456 pregnant women to develop a method to predict the fraction of fetal DNA based on the size distribution of DNA fragments (r = 0.818, p<2.2e À 16, linear regression model). Finally, we collected plasma from 938 of pregnant women having a fetus with structural abnormalities detected on ultrasound who also underwent amniocentesis, chorionic villous sampling, or umbilical cord blood sampling. We sequenced these samples using from 3M up to 15M reads to detect subchromosomal abnormalities, in parallel with array comparative genomic hybridization performed on each invasively-derived sample as a comparison. RESULTS: In total, 100% (57/57) instances of aneuploidy and 36/40 (90%) of samples with subchromosomal abnormalities greater than 5MB in size were detected using only 3 million (3 M) reads, while 5M reads was required to detect two more samples subchromosomal abnormalities greater than 5MB in size and 12/19 samples with chromosomal abnormalities between 1.5 and 5 MB. While increasing sequencing depth up to 15M reads increased accuracy for larger deletions/duplications, 88.1% (52/59) abnormalities larger than 1.5M could be detected using this method, while 5 abnormalities under 1.5MB were not reliably detected. CONCLUSIONS: This study demonstrates the viability of NIPT using semiconductor sequencing to accurately detect subchromosomal abnormalities greater than 1.5MB in a high-risk population.

BACKGROUND:Noninvasive prenatal testing based on massively parallel sequencing (MPS) of cell-free DNA in maternal plasma has become rapidly integrated into clinical practice for detecting fetal chromosomal aneuploidy. We directly determined the fetal fraction (FF) from results obtained with MPS tag counting and examined the relationships of FF to such biological parameters as fetal karyotype and maternal demographics.

We present the data and the technology, a combination of which allows us to determine the identity of proprotein convertases (PCs) related to the processing of specific protein targets including viral and bacterial pathogens. Our results, which support and extend the data of other laboratories, are required for the design of effective inhibitors of PCs because, in general, an inhibitor design starts with a specific substrate. Seven proteinases of the human PC family cleave the multibasic motifs R-X-(R/K/X)-R2 and, as a result, transform proproteins, including those from pathogens, into biologically active proteins and peptides. The precise cleavage preferences of PCs have not been known in sufficient detail; hence we were unable to determine the relative importance of the individual PCs in infectious diseases, thus making the design of specific inhibitors exceedingly difficult. To determine the cleavage preferences of PCs in more detail, we evaluated the relative efficiency of furin, PC2, PC4, PC5/6, PC7, and PACE4 in cleaving over 100 decapeptide sequences representing the R-X-(R/K/X)-R2 motifs of human, bacterial, and viral proteins. Our computer analysis of the data and the follow-on cleavage analysis of the selected full-length proteins corroborated our initial results thus allowing us to determine the cleavage preferences of the PCs and to suggest which PCs are promising drug targets in infectious diseases. Our results also suggest that pathogens, including anthrax PA83 and the avian influenza A H5N1 (bird flu) hemagglutinin precursor, evolved to be as sensitive to PC proteolysis as the most sensitive normal human proteins.

-EJB 91 0543 aB-crystallin, a polypeptide of molecular mass 22 kDa, is considered to be one of two subunits (aA and aB) of the multimeric lens-specific protein, a-crystallin. Recent demonstrations of the extralenticular presence of aB-crystallin have suggested that outside of the lens, this polypeptide may have functions independent of aA. Within the lens however, as part of the protein a-crystallin, its function is assumed to be structural. In an effort to investigate the functional status of aB-crystallin in the lens, we have characterized this polypeptide in the rat heart and the human lens.Unequivocal identity of aB-crystallin in the rat heart and the rat lens was established by the sequence analyses of the respective cDNA clones. Size exclusion chromatography (FPLC) and immunoblotting showed that in the rat heart, aB-crystallin exists as an aggregate of 300-400 kDa average molecular mass, similar to that of purified ixB-crystallin isolated from bovine lens. Interestingly, analysis of the human lens proteins by iinmunoblotting showed that, with age, unlike aAcrystallin, the aB subunit remains detectable in the soluble fractions derived from normal lenses as old as 82 years. Importantly, the average molecular mass of the aB subunit in the soluble fractions prepared from 60-80-year-old human lens nuclei was also found to be 300-400 kDa. These data lead to the conclusion that aB-crystallin may exist as an independent protein not only in non-lens tissues (e.g. heart) but in the lens as well a-Crystallin is one of the predominant multimeric structural proteins of the mammalian lens, known to be composed of two primary gene products, aA and aB [l, 21. It is the first crystallin to appear during mammalian lens development and is known to undergo a number of gerontological changes in its structure [3 -71. Its concentration in the lens reaches up to 30% of the total soluble protein and is, therefore, one of the chief contributory factors for the maintenance of transparency [8], the main functional attribute of the ocular lens.a-Crystallin is involved in the formation of high-molecular-mass protein aggregates during aging and is eventually lost from the soluble pool of the adult lens proteins. It is also one of the main components of very-high-molecular-mass insoluble aggregates present in opaque lenses [7, 9-121. The two polypeptides (aA and a s ) have been historically treated as the two integral subunits of one structural protein, cc-crystallin [l]. However, most of the studies conducted with acrystallin have been restricted to aA because of its predominance in the lens (aA: aB, 3 : 1) [I]. Observations made primarily with aA have been extrapolated to aB, including the presumed structural function for this polypeptide [13]. This tentative status has been further fortified by the fact that a A and aB share 57% sequence similarity at the polypeptide level [14].Both of these proteins also share significant sequence similarity with small heat-shock proteins and are, therefore, re- garded as members of the small heat-...