Expanding Access to Noninvasive Prenatal Diagnosis for Monogenic Conditions to Consanguineous Families.

BACKGROUND: Cell-free fetal DNA exists within the maternal bloodstream during pregnancy and provides a means for noninvasive prenatal diagnosis (NIPD). Our accredited clinical service offers definitive NIPD for several autosomal recessive (AR) and X-linked conditions using relative haplotype dosage analysis (RHDO). RHDO involves next-generation sequencing (NGS) of thousands of common single nucleotide polymorphism (SNPs) surrounding the gene of interest in the parents and an affected or unaffected offspring to conduct haplotype phasing of the high- and low-risk alleles. NGS is carried out in parallel on the maternal cell-free DNA, and fetal inheritance is predicted using sensitive dosage calculations performed at sites where the parental genotypes differ. RHDO is not currently offered to consanguineous couples owing to the shared haplotype between parents. Here we test the expansion of RHDO for AR monogenic conditions to include consanguineous couples. METHODS: The existing sequential probability ratio test analysis pipeline was modified to apply to SNPs where both parents are heterozygous for the same genotype. Quality control thresholds were developed using 33 nonconsanguineous cases. The performance of the adapted RHDO pipeline was tested on 8 consanguineous cases. RESULTS: The correct fetal genotype was predicted by our revised RHDO approach in all conclusive cases with known genotypes (n = 5). Haplotype block classification accuracies of 94.5% and 93.9% were obtained for the nonconsanguineous and consanguineous case cohorts, respectively. CONCLUSIONS: Our modified RHDO pipeline correctly predicts the genotype in fetuses from consanguineous families, allowing the potential to expand access to NIPD services for these families.

Non-invasive fetal genotyping for maternal alleles with droplet digital PCR: A comparative study of analytical approaches.

OBJECTIVES: To develop a flexible droplet digital PCR (ddPCR) workflow to perform non-invasive prenatal diagnosis via relative mutation dosage (RMD) for maternal pathogenic variants with a range of inheritance patterns, and to compare the accuracy of multiple analytical approaches. METHODS: Cell free DNA (cfDNA) was tested from 124 archived maternal plasma samples: 88 cases for sickle cell disease and 36 for rare Mendelian conditions. Three analytical methods were compared: sequential probability ratio testing (SPRT), Bayesian and z-score analyses. RESULTS: The SPRT, Bayesian and z-score analyses performed similarly well with correct prediction rates of 96%, 97% and 98%, respectively. However, there were high rates of inconclusive results for each cohort, particularly for z-score analysis which was 31% overall. Two samples were incorrectly classified by all three analytical methods; a false negative result predicted for a fetus affected with sickle cell disease and a false positive result predicting the presence of an X-linked IDS variant in an unaffected fetus. CONCLUSIONS: ddPCR can be applied to RMD for diverse conditions and inheritance patterns, but all methods carry a small risk of erroneous results. Further evaluation is required both to reduce the rate of inconclusive results and explore discordant results in more detail.

Acute neuromuscular and perceptual responses to U-18 English Premier League academy football match play.

We examined the sensitivity and time-course of recovery of neuromuscular and perceptual player monitoring measures to U-18 English Premier League academy football match play. Eighteen players performed maximal posterior chain, hip adductor and abductor isometric strength tests, countermovement jumps (CMJ) and provided self-report wellbeing scores around eight competitive league home games: 1 day before (MD-1), pre-match (MD-PRE), post-match (MD-POST) and two (MD+2) and three (MD+3) days post-match. A permutational multivariate analysis of variance and post hoc univariate analyses of variance were used to examine match-induced responses to monitoring variables. Between MD-1 and MD-POST, we observed small to moderate reductions to the adductor and abductor peak force and maximal impulse and IPCS peak force; small reductions to CMJ jump height (JH) (flight time), eccentric peak force and eccentric deceleration rate of force development and moderate to large reductions to perceived fatigue, muscle soreness and mood. No match-induced changes were observed for CMJ flight time: contraction time or eccentric duration. Posterior chain, abductor, CMJ and self-report measures normalised by MD+3 but adductor peak force remained compromised at MD+3 (ES = small). Posterior chain, adductor and abductor peak isometric force and maximal impulse; CMJ JH, eccentric peak force and eccentric deceleration rate of force development and perceived fatigue, muscle soreness and mood are sensitive to match-induced fatigue. Since adductor peak force remained compromised at MD+3, it is apparent that players might not achieve complete neuromuscular recovery within 3 days of match play, and that the adductor muscle group might be particularly vulnerable to match-induced fatigue.

Engineering triacylglycerol production from sugars in oleaginous yeasts.

Oleaginous yeasts natively produce surplus triacylglycerol lipid and can be engineered for higher yield and productivity. Most enzymatic steps of triacylglycerol production are characterized, but key parts of the pathway remain unknown. This introduces uncertainty to metabolic engineering strategy and the upper limit of achievable lipid yield. Here, we present our current understanding of the oleaginous yeast triacylglycerol biosynthesis pathway, review metabolic engineering strategies, and discuss bioprocess constraints on lipid production. We also present a simplified substrate allocation model capturing the interaction of percent lipid content on overall triacylglycerol yield, productivity, and fermentation cost, which should help frame achievable bioprocess metrics.

Advances in molecular-based diagnostics in meeting crop biosecurity and phytosanitary issues.

Awareness of crop biosecurity and phytosanitation has been heightened since 9/11 and the unresolved anthrax releases in October 2001. Crops are highly vulnerable to accidental or deliberate introductions of crop pathogens from outside U.S. borders. Strategic thinking about protection against deliberate or accidental release of a plant pathogen is an urgent priority. Rapid detection will be the key to success. This review summarizes recent progress in the development of rapid real-time PCR protocols and evaluates their effectiveness in a proposed nationwide network of diagnostic laboratories that will facilitate rapid diagnostics and improved communication.