A Deep Intronic PKHD1 Variant Identified by SpliceAI in a Deceased Neonate With Autosomal Recessive Polycystic Kidney Disease.

The etiologies of newborn deaths in neonatal intensive care units usually remain unknown, even after genetic testing. Whole-genome sequencing, combined with artificial intelligence-based methods for predicting the effects of non-coding variants, provide an avenue for resolving these deaths. Using one such method, SpliceAI, we identified a maternally inherited deep intronic PKHD1 splice variant (chr6:52030169T>C), in trans with a pathogenic missense variant (p.Thr36Met), in a newborn who died of autosomal recessive polycystic kidney disease at age 2 days. We validated the deep intronic variant's impact in maternal urine-derived cells expressing PKHD1. Reverse transcription polymerase chain reaction followed by Sanger sequencing showed that the variant causes inclusion of 147bp of the canonical intron between exons 29 and 30 of PKHD1 into the mRNA, including a premature stop codon. Allele-specific expression analysis at a heterozygous site in the mother showed that the mutant allele completely suppresses canonical splicing. In an unrelated healthy control, there was no evidence of transcripts including the novel splice junction. We returned a diagnostic report to the parents, who underwent in vitro embryo selection.

A quality improvement initiative to reduce antibiotic use in transient tachypnea of the newborn.

OBJECTIVE: Evidence suggests that antibiotics are unnecessary in infants with transient tachypnea of the newborn (TTN) that are low-risk for early-onset sepsis. The aim was to reduce ampicillin and gentamicin days of therapy (DOT) in infants with suspected TTN by 10% within 12 months. STUDY DESIGN: We used the Model for Improvement to test interventions from August 2019 to September 2021 to decrease antibiotic utilization in low-risk infants with TTN. Interventions included the creation of an evidence-based clinical pathway, admission huddles, and prescriber audit and feedback. RESULTS: We reduced ampicillin and gentamicin use by 26% and 23%, respectively. In 123 infants with suspected TTN, we sequentially decreased starting antibiotics in this group from 71% to 41%, 13% and 0%. There were no cases of missed bacteremia. CONCLUSION: Creation of a multidisciplinary antimicrobial stewardship QI team and subsequent interventions were successful in safely reducing antibiotic use in infants with TTN.

Khdc3 Regulates Metabolism Across Generations in a DNA-Independent Manner.

Genetic variants can alter the profile of heritable molecules such as small RNAs in sperm and oocytes, and in this manner ancestral genetic variants can have a significant effect on offspring phenotypes even if they are not themselves inherited. Here we show that wild type female mice descended from ancestors with a mutation in the mammalian germ cell gene Khdc3 have hepatic metabolic defects that persist over multiple generations. We find that genetically wild type females descended from Khdc3 mutants have transcriptional dysregulation of critical hepatic metabolic genes, which persist over multiple generations and pass through both female and male lineages. This was associated with dysregulation of hepatically-metabolized molecules in the blood of these wild type mice with mutational ancestry. The oocytes of Khdc3-null females, as well as their wild type descendants, had dysregulation of multiple small RNAs, suggesting that these epigenetic changes in the gametes transmit the phenotype between generations. Our results demonstrate that ancestral mutation in Khdc3 can produce transgenerational inherited phenotypes, potentially indefinitely.

Enhancement of tumor immunotherapy by deletion of the A2A adenosine receptor.

The A(2A) adenosine receptor plays a critical and non-redundant role in suppressing inflammation at sites of hypoxia and tissue damage. The tumor microenvironment has high levels of adenosine as a result of hypoxia and ectopic expression of enzymes responsible for the generation of extracellular adenosine. Thus, we sought to determine the ability of A(2A) receptor null mice to immunologically reject tumors. We observed that mice lacking the A(2A) adenosine receptor showed significantly delayed growth of lymphoma cells when compared to WT mice. Furthermore, when immunized with a low dose of tumor or with an irradiated GM-CSF-secreting tumor vaccine, A(2A) receptor null mice showed significantly enhanced protection from a subsequent high-dose challenge from both immunogenic and poorly immunogenic tumor lines. This increase in protection was accompanied by an increase in the number of tumor-antigen-specific CD8 T cells at the vaccine-site draining lymph node. Finally, we found that A(2A) receptor null mice displayed more robust anti-tumor responses than WT mice when they were treated with a soluble B7-DC/Fc fusion protein designed to antagonize B7-H1-mediated co-inhibition. This combinatorial immunotherapy strategy could also be recapitulated with pharmacological A(2A) receptor blockade paired with B7-DC/Fc administration. In light of these data, we believe that blockade of the A(2A) adenosine receptor is an attractive target for tumor immunotherapy that synergizes with other immunomodulatory approaches currently in clinical trials.

Evidence for germline non-genetic inheritance of human phenotypes and diseases.

It is becoming increasingly apparent that certain phenotypes are inherited across generations independent of the information contained in the DNA sequence, by factors in germ cells that remain largely uncharacterized. As evidence for germline non-genetic inheritance of phenotypes and diseases continues to grow in model organisms, there are fewer reports of this phenomenon in humans, due to a variety of complications in evaluating this mechanism of inheritance in humans. This review summarizes the evidence for germline-based non-genetic inheritance in humans, as well as the significant challenges and important caveats that must be considered when evaluating this process in human populations. Most reports of this process evaluate the association of a lifetime exposure in ancestors with changes in DNA methylation or small RNA expression in germ cells, as well as the association between ancestral experiences and the inheritance of a phenotype in descendants, down to great-grandchildren in some cases. Collectively, these studies provide evidence that phenotypes can be inherited in a DNA-independent manner; the extent to which this process contributes to disease development, as well as the cellular and molecular regulation of this process, remain largely undefined.

Is ultrasound useful in the diagnosis of adolescents with polycystic ovary syndrome?

BACKGROUND: Diagnosis of adolescent polycystic ovary syndrome (PCOS) remains a challenge despite several existing criteria, and may be difficult to distinguish from pubertal changes. Different parameters to study ovarian function using ultrasonography have been proposed, but there is still no consensus about their diagnostic value. OBJECTIVE: To evaluate the role of ultrasonography in the diagnosis of adolescent PCOS by reviewing available studies that assessed the ovarian volume (OV) and other ovarian morphological features such as location and number of follicles, stromal area, and volume. METHODS: MEDLINE/PubMed database were searched to identify studies that assessed ovarian characteristics of adolescent PCOS patients by ultrasound. Studies on adults were also reviewed if study population included adolescents and stromal characteristics were assessed by three-dimensional (3D) sonogram. RESULTS: Five studies, including 262 PCOS adolescents (10-19 years of age) and two-dimensional (2D) ultrasound analysis, were identified. Mean OV was 9.29 cm³ for PCOS patients and 4.77 cm³ for controls. The morphology of ovarian follicles, when reported, showed multiple (>10) peripheral follicles in 83% of cases. Two studies, including 157 PCOS adolescents and young women (15-35 years of age) and 2D and 3D ultrasound analysis, were identified. Patients with PCOS patients had a MOV 13.1 cm³, multiple follicles (>15), and a statistically significant greater S/A ratio compared to controls. Stromal volume indices were positively correlated with hyperandrogenemia in PCOS patients. CONCLUSION: Pelvic ultrasound is an increasingly important aid in the diagnosis of PCOS in adolescents. Besides ovarian volume, ovarian morphology must be assessed with 2D ultrasound to look for peripherally located multiple follicles. Further studies are warranted to evaluate the utility of 3D ultrasonographic assessment in adolescents with PCOS.