Funding information Natera, Inc.Single-nucleotide polymorphism (SNP)-based non-invasive prenatal testing (NIPT) can currently predict a subset of submicroscopic abnormalities associated with severe clinical manifestations.We retrospectively analyzed the performance of SNP-based NIPT in 80 449 referrals for 22q11.2 deletion syndrome and 42 326 referrals for 1p36, cri-du-chat, Prader-Willi, and Angelman microdeletion syndromes over a 1-year period, and compared the original screening protocol with a revision that reflexively sequenced high-risk calls at a higher depth of read. The prevalence of these microdeletion syndromes was also estimated in the referral population.The positive predictive value of the original test was 15.7% for 22q11.2 deletion syndrome, and 5.2% for the other 4 disorders combined. With the revised protocol, these values increased to 44.2% for 22q11.2 and 31.7% for the others. The 0.33% false-positive rate (FPR) for 22q11.2 deletion syndrome decreased to 0.07% with the revised protocol. Similarly, the FPR for the other 4 disorders combined decreased from 0.56% to 0.07%. Minimal prevalences were estimated to be 1 in 1255 for 22q11.2 deletion syndrome and 1 in 1464 for 1p36, cri-du-chat, and Angelman syndromes combined. Our results show that these microdeletions are relatively common in the referral population, and that the performance of SNP-based NIPT is improved with high-depth resequencing.

Our results confirm the known inverse relationship between advanced maternal age (>35 years) and embryo euploidy, demonstrating that equal numbers of euploid embryos are available at day 3 and day 5.

Our findings that SNP-based PGT-A can mitigate the negative effects of maternal age on IVF outcomes in cycles with transfer, and that pregnancy outcomes from SET cycles are not significantly different from those of double-embryo transfer cycles, support the use of SET when transfers are combined with SNP-based PGT-A.

Likelihood estimates of local rates of evolution within proteins reveal that selective constraints on structure and function are quantitatively stable over billions of years of divergence. The stability of constraints produces an intramolecular clock that gives each protein a characteristic pattern of evolutionary rates along its sequence. This pattern allows the identification of constrained regions and, because the rate of evolution is a quantitative measure of the strength of the constraint, of their functional importance. We show that results from such analyses, which require only sequence alignments, are consistent with experimental and mutational data. The methodology has significant predictive power and may be used to guide structure-function studies for any protein represented by a modest number of homologs in sequence databases.T he principle that the rate of molecular evolution is inversely correlated with the strength of selective constraints has long been known (1, 2). The average evolutionary rate of a protein reflects the overall importance of the protein for organismal functions, whereas rate variation within the protein reflects intramolecular differences in structural and functional constraints. Intramolecular rate variation has been the subject of many studies focused on devising more realistic models of sequence evolution that do not assume rate constancy among sites (e.g., refs. 3-6). A more recent application of estimating rate variation within proteins has been the inference of structural and functional constraints (7-9).To identify evolutionarily constrained regions (ECRs) we devised a general approach to inferring rate variation within proteins. We construct a multiple sequence alignment of orthologs and͞or closely related paralogs and build the maximum likelihood tree. Holding the branching structure of the tree fixed, we then calculate the number of substitutions in each window of a fixed width over the entire alignment. The ''relative rate'' in the window is obtained by dividing the number of substitutions per site in the window by the average of all windows. Plotting the windows' relative rates as a function of their position generates a rate profile (RP), and a heuristic algorithm automatically identifies ECRs and ranks them by their rate of evolution. This approach allows us to infer both the existence of constrained regions in a protein and, because the rate of evolution is a quantitative measure of the strength of the constraint, the relative importance of the identified region.Our method requires only a multiple sequence alignment and is sufficiently powerful to allow analyses involving a relatively small number of fairly closely related sequences. It enables us (i) to use sequences for which the quality of the alignment over most its length is indisputably robust, and (ii) to use alignments of orthologs and closely related paralogs for which conservation of structural and functional constraints can be reasonably assumed. We show below that the method identifies known domains wit...

Dimers of CH3 domains from human IgG1 were used to study the effect of mutations constructed at a domain-domain interface upon domain dissociation and unfolding, "complex stability". Alanine replacement mutants were constructed on one side of the interface for each of the sixteen interdomain contact residues by using a single-chain CH3 dimer in which the carboxyl terminus of one domain was joined to the amino terminus of the second domain via a (G4S)4 linker. Single-chain variants were expressed in Escherichia coli grown in a fermentor and recovered in yields of 6-90 mg L-1 by immobilized metal affinity chromatography. Guanidine hydrochloride-induced denaturation was used to follow domain dissociation and unfolding. Surprisingly, the linker did not perturb the complex stability for either the wild type or two destabilizing mutants. The CH3 domain dissociation and unfolding energetics are dominated by six contact residues where corresponding alanine mutations each destabilize the complex by >2.0 kcal mol-1. Five of these residues (T366, L368, F405, Y407, and K409) form a patch at the center of the interface and are located on the two internal antiparallel beta-strands. These energetically key residues are surrounded by 10 residues on the two external beta-strands whose contribution to complex stability is small (three have a Delta DeltaG of 1.1-1.3 kcal mol-1) or very small (seven have a Delta DeltaG of

We demonstrate proof-of-concept for the use of massively multiplexed PCR and next-generation sequencing (mmPCR-NGS) to identify both clonal and subclonal copy-number variants (CNVs) in circulating tumor DNA. This is the first report of a targeted methodology for detection of CNVs in plasma.Using an in vitro model of cell-free DNA, we show that mmPCR-NGS can accurately detect CNVs with average allelic imbalances as low as 0.5%, an improvement over previously reported whole-genome sequencing approaches. Our method revealed differences in the spectrum of CNVs detected in tumor tissue subsections and matching plasma samples from 11 patients with stage II breast cancer. Moreover, we showed that liquid biopsies are able to detect subclonal mutations that may be missed in tumor tissue biopsies. We anticipate that this mmPCR-NGS methodology will have broad applicability for the characterization, diagnosis, and therapeutic monitoring of CNV-enriched cancers, such as breast, ovarian, and lung cancer.

Fetal movements (FM) may be spontaneous, originating in the fetus itself, or evoked by external stimuli. The relative rate of spontaneous and evoked FM has not yet been documented. As it is impossible to differentiate in a singleton pregnancy between these types of movements – the natural model of twin pregnancy was used. The FM of 10 normal twin pregnancies between 33 and 39 weeks of gestation were studied by two realtime ultrasounds, and were operated by two ultrasonographers over a period of 10 min. The mean number of independent FM of each fetus which occurred while the other fetus was resting was 7.9 FM and 7.7 FM, respectively (average 7.8 FM). A mean of 2.5 FM occurred simultaneously. The rate of independent FM was 75.7% and that of simultaneous FM was 24.3%. Vibroacoustic stimulation applied to the maternal abdomen evoked simultaneous movements of both fetuses in all cases. It is suggested that the independent FM may represent spontaneous FM and that the simultaneous FM may indicate the presence of an external stimuli and are therefore evoked responses.

No abstract