Second-Tier Next Generation Sequencing Integrated in Nationwide Newborn Screening Provides Rapid Molecular Diagnostics of Severe Combined Immunodeficiency.

Severe combined immunodeficiency (SCID) and other T cell lymphopenias can be detected during newborn screening (NBS) by measuring T cell receptor excision circles (TRECs) in dried blood spot (DBS) DNA. Second tier next generation sequencing (NGS) with an amplicon based targeted gene panel using the same DBS DNA was introduced as part of our prospective pilot research project in 2015. With written parental consent, 21 000 newborns were TREC-tested in the pilot. Three newborns were identified with SCID, and disease-causing variants in IL2RG, RAG2, and RMRP were confirmed by NGS on the initial DBS DNA. The molecular findings directed follow-up and therapy: the IL2RG-SCID underwent early hematopoietic stem cell transplantation (HSCT) without any complications; the leaky RAG2-SCID received prophylactic antibiotics, antifungals, and immunoglobulin infusions, and underwent HSCT at 1 year of age. The child with RMRP-SCID had complete Hirschsprung disease and died at 1 month of age. Since January 2018, all newborns in Norway have been offered NBS for SCID using 1st tier TRECs and 2nd tier gene panel NGS on DBS DNA. During the first 20 months of nationwide SCID screening an additional 88 000 newborns were TREC tested, and four new SCID cases were identified. Disease-causing variants in DCLRE1C, JAK3, NBN, and IL2RG were molecularly confirmed on day 8, 15, 8 and 6, respectively after birth, using the initial NBS blood spot. Targeted gene panel NGS integrated into the NBS algorithm rapidly delineated the specific molecular diagnoses and provided information useful for management, targeted therapy and follow-up i.e., X rays and CT scans were avoided in the radiosensitive SCID. Second tier targeted NGS on the same DBS DNA as the TREC test provided instant confirmation or exclusion of SCID, and made it possible to use a less stringent TREC cut-off value. This allowed for the detection of leaky SCIDs, and simultaneously reduced the number of control samples, recalls and false positives. Mothers were instructed to stop breastfeeding until maternal cytomegalovirus (CMV) status was determined. Our limited data suggest that shorter time-interval from birth to intervention, may prevent breast milk transmitted CMV infection in classical SCID.

Association between umbilical cord artery pCO2 and the Apgar score; elevated levels of pCO2 may be beneficial for neonatal vitality after moderate acidemia.

OBJECTIVE: To determine the association between 5-min Apgar score and umbilical cord artery carbon dioxide tension (pCO2). DESIGN: Observational study. SETTING: European hospital labor wards. POPULATION: Data from 36,432 newborns ≥36 gestational weeks were obtained from three sources: two trials of monitoring with fetal electrocardiogram (the Swedish randomized controlled trial and the European Union Fetal ECG trial) and Mölndal Hospital data. After validation of the acid-base values, 25,806 5-min Apgar scores were available for analysis. METHODS: Validation of the umbilical cord acid-base values was performed to obtain reliable data. 5-min Apgar score was regressed against cord artery pCO2 in a polynomial multilevel model. MAIN OUTCOME MEASURES: Five-min Apgar score, umbilical cord pCO2, pH, and base deficit. RESULTS: Overall, a higher cord artery pCO2 was found to be associated with lower 5-min Apgar scores. However, among newborns with moderate acidemia, lower umbilical cord artery pCO2 (≤median pCO2 for the specific cord artery pH) was associated with lower 5-min Apgar scores, with a relative risk of 2.0 (95% confidence interval: 1.4-2.8) for 5-min Apgar scores 0-6. CONCLUSIONS: Metabolic acidosis affects the newborn's vitality more than respiratory acidosis. In addition, elevated levels of pCO2 may be beneficial for fetuses with moderate acidemia, and thus cord artery pCO2 is a factor that should be considered when assessing the compromised newborn.

Impaired diastolic function and disruption of the force-frequency relationship in the right ventricle of newborn pigs resuscitated with 100% oxygen.

BACKGROUND: Resuscitation with 100% oxygen increases oxidative stress and is detrimental for organ function. OBJECTIVE: To study the effects of resuscitation with 100% oxygen compared to room-air on myocardial function. METHODS: Twenty-eight newborn pigs underwent global hypoxia (8% oxygen/N2) until base excess reached -20 mmol/l. The animals were randomized into two groups and resuscitated with either 100% or room air for 30 min. Myocardial tissue Doppler velocities and acceleration of the mitral and tricuspid valve annuli during systole and diastole were assessed before global hypoxia and after resuscitation together with troponin I. RESULTS: Peak early diastolic velocity (E') and acceleration (pEac) in the septum and pEac in the lateral tricuspid valve annulus were lower after resuscitation with 100% oxygen, suggesting impaired diastolic relaxation in the right ventricle. Lower systolic velocities and acceleration in the right ventricle relative to heart rate indicate disruption of the right ventricular force-frequency relationship after resuscitation with 100% oxygen. Troponins were higher in the 100% oxygen group, suggesting increased myocardial damage in this group. CONCLUSION: Resuscitation with 100% oxygen compared to room air induces diastolic dysfunction, disrupts the systolic force-frequency relationship and increases myocardial damage in the newborn pig.

Atrioventricular valve annulus velocity and acceleration during global hypoxia in newborn pigs - assessment of myocardial function.

BACKGROUND: Global hypoxia may affect regions of the neonatal heart during systole and diastole differently. OBJECTIVE: In the first study, the aim was to assess longitudinal myocardial function in newborn pigs during global hypoxia and recovery. In the second study, invasive hemodynamic data were evaluated and compared with tissue velocities and acceleration. METHODS: Myocardial Doppler measurements of velocity and acceleration in the atrioventricular valve annuli during global hypoxia were made. RESULTS: In the first experiment, systolic velocity (S), early diastolic velocity (E') and peak systolic acceleration (pSac) decreased during hypoxia. S, pSac and E'(mva) (mitral valve annulus) recovered after reoxygenation, while E'(septal) and E'(tva) (tricuspid valve annulus) did not. Isovolumic acceleration (IVA) did not reflect systolic dysfunction. In the second experiment, S(mva) and pSac(mva) declined during hypoxia similarly to maximum dP/dT. E'(mva) and E'(tva) were correlated with tau and both changed parallel to minimum dP/dT. In the TVA maximum dP/dT and pSac(tva) did not change, while S(tva) declined. By correcting for heart rate the pattern of alteration in S and pSac became comparable to the changes in E'. CONCLUSION: Tissue Doppler velocity in systole, diastole and acceleration during rapid ejection reflect hemodynamic changes in the neonatal myocardium during global hypoxia and recovery. S and pSac reflect systolic function, while E' correlates with tau and mirrors diastolic function.