Saturation-scale functional evidence supports clinical variant interpretation in Lynch syndrome.

BACKGROUND: Lynch syndrome (LS) is a cancer predisposition syndrome affecting more than 1 in every 300 individuals worldwide. Clinical genetic testing for LS can be life-saving but is complicated by the heavy burden of variants of uncertain significance (VUS), especially missense changes. RESULT: To address this challenge, we leverage a multiplexed analysis of variant effect (MAVE) map covering >94% of the 17,746 possible missense variants in the key LS gene MSH2. To establish this map's utility in large-scale variant reclassification, we overlay it on clinical databases of >15,000 individuals with LS gene variants uncovered during clinical genetic testing. We validate these functional measurements in a cohort of individuals with paired tumor-normal test results and find that MAVE-based function scores agree with the clinical interpretation for every one of the MSH2 missense variants with an available classification. We use these scores to attempt reclassification for 682 unique missense VUS, among which 34 scored as deleterious by our function map, in line with previously published rates for other cancer predisposition genes. Combining functional data and other evidence, ten missense VUS are reclassified as pathogenic/likely pathogenic, and another 497 could be moved to benign/likely benign. Finally, we apply these functional scores to paired tumor-normal genetic tests and identify a subset of patients with biallelic somatic loss of function, reflecting a sporadic Lynch-like Syndrome with distinct implications for treatment and relatives' risk. CONCLUSION: This study demonstrates how high-throughput functional assays can empower scalable VUS resolution and prospectively generate strong evidence for variant classification.

High-throughput splicing assays identify missense and silent splice-disruptive POU1F1 variants underlying pituitary hormone deficiency.

Pituitary hormone deficiency occurs in ∼1:4,000 live births. Approximately 3% of the cases are due to mutations in the alpha isoform of POU1F1, a pituitary-specific transcriptional activator. We found four separate heterozygous missense variants in unrelated individuals with hypopituitarism that were predicted to affect a minor isoform, POU1F1 beta, which can act as a transcriptional repressor. These variants retain repressor activity, but they shift splicing to favor the expression of the beta isoform, resulting in dominant-negative loss of function. Using a high-throughput splicing reporter assay, we tested 1,070 single-nucleotide variants in POU1F1. We identified 96 splice-disruptive variants, including 14 synonymous variants. In separate cohorts, we found two additional synonymous variants nominated by this screen that co-segregate with hypopituitarism. This study underlines the importance of evaluating the impact of variants on splicing and provides a catalog for interpretation of variants of unknown significance in POU1F1.

The effect of epinephrine on coronary flow in the setting of a systemic-to-pulmonary artery shunt.

BACKGROUND: Indirect clinical evidence suggests that coronary blood flow (CBF) is altered in patients palliated with systemic-to-pulmonary artery shunts (SPSs). The addition of epinephrine may exert additional effects. METHODS: A total of 11 newborn piglets underwent placement of a 3.5- to 4-mm graft between the innominate artery and the pulmonary artery. Doppler probes measured flow continuously in the aorta (aortic flow [AoF]), pulmonary artery and left coronary artery at baseline (SPS closed), SPS open, and during epinephrine administration (SPS closed and open). Each animal served as its own control. Systolic and diastolic CBF, resistance (coronary vascular resistance index [CVRI]), and myocardial oxygen supply demand ratio were calculated. RESULTS: Opening the SPS increased AoF and decreased systolic and diastolic pressure from baseline, with and without the presence of epinephrine. The CBF and CVRI decreased on opening the SPS in the presence of epinephrine. The decrease occurred only in diastole and was proportional to pulmonary-to-systemic flow ratio (Qp/Qs). Epinephrine infusion itself reduced CVRI with SPS closed, but there was little further decrease on opening SPS. Myocardial oxygen supply-demand ratio decreased on opening SPS at baseline and with epinephrine. CONCLUSIONS: This study suggests that SPS decreases CBF, especially in the presence of a higher Qp/Qs and epinephrine. The mechanism is largely due to the decrease in diastolic pressure and the inability of the coronary arteries to compensate with vasodilation.

Cardiopulmonary bypass in an infant with a recent history of malaria.

Severe malarial infection is associated with impaired cardiac function. We report a child who underwent repair of tetralogy of Fallot two weeks after being treated for malaria. The postoperative course was complicated by impaired left ventricular function. The pathogenesis of malaria is discussed as well as the potential complications of cardiopulmonary bypass (CPB).

Interstage feeding and weight gain in infants following the Norwood operation: can we change the outcome?

BACKGROUND: Poor weight gain is common in infants after Stage I Norwood operation and can negatively impact outcomes. OBJECTIVES: The purpose of this study was to examine the impact of feeding strategy on interstage weight gain. METHODS: In a multi-centre study, 158 infants discharged following the Norwood operation were enrolled prospectively. Weight and feeding data were obtained at 2-week intervals. Differences between feeding regimens in average daily weight gain and change in weight-for-age z-score between Stage I discharge and Stage II surgery were examined. RESULTS: Discharge feeding regimens were oral only in 52%, oral with tube supplementation in 33%, and by nasogastric/gastrostomy tube only in 15%. There were significant differences in the average daily interstage weight gain among the feeding groups - oral only 25.0 grams per day, oral/tube 21.4 grams per day, and tube only 22.3 grams per day - p = 0.019. Tube-only-fed infants were significantly older at Stage II (p = 0.004) and had a significantly greater change in weight-for-age z-score (p = 0.007). The overall rate of weight gain was 16-32 grams per day, similar to infant norms. The rate of weight gain declined over time, with earlier decline observed for oral- and oral/tube-fed infants (less than 15 grams per day at 5.4 months) in comparison with tube-only-fed infants (less than 15 grams per day at 8.6 months). CONCLUSION: Following Stage I Norwood, infants discharged on oral feeding had better average daily weight gain than infants with tube-assisted feeding. The overall weight gain was within the normal limits in all feeding groups, but the rate of weight gain decreased over time, with an earlier decline in infants fed orally.