Level of RNA Deletions is Age-Independent.

<p>Intentional capture of mtRNA deletions by PCR amplification of extended cDNA regions.</p

Validation of Method.

<p>(A) Flow chart illustrating the procedure for detection of mtRNA integrity. (B) Comparison of RNA integrity in CoxI site obtained by using two different commercially available reverse transcriptases. (C) Estimation of errors introduced in the PCR amplication. The 12S rRNA region was amplified with increasing cycle number and 100 ng PCR product was either digested with TaqI or left untreated and subsequently analyzed for mutations. The mutation frequency is plotted as function of PCR cycle number. The figures show mean with SD from three independent experiments.</p

Stable Expression of Genes and Proteins with Age.

<p>(A) Expression of mitochondrial genes was analyzed by RT-qPCR normalized to <i>Gapdh</i> and presented relative to 1 month old mice. (B) Expression levels of proteins were evaluated by western analyses using antibodies as specified in Experimental Procedures. The relative increase in COXI and ND6 with age is indicated. Figure shows mean with SD. **p<0.01, *p< 0.05.</p

Site- and Age- Dependent Accumulation of Mutations in Brain mtDNA.

<p>(A) DNA from young (1 month, n = 8) and old (18 months, n = 8) mice were analyzed for mutation rate in 7 different sites in the mtDNA. DNA was isolated from whole brain. (B) DNA from heterozygous (mut/+, n = 3) and homozygous (mut/mut, n = 3) mutator mice (9 months) were analyzed in the same sites. Figures show mean with SD. **p<0.01, *p< 0.05.</p

RNA Error Frequency is Age-Independent in Controls but Elevated in Mutator Mice.

<p>(A) mtRNA errors were measured as described at the same 7 loci as for mtDNA from brains of young (1 months, n = 8) and old (18 months, n = 8). (B) mtRNA errors in heterozygous (mut/+, n = 3) and homozygous (mut/mut, n = 3) mice. Figures show mean with SD. p**<0.01 vs. 18 months, p*< 0.05 vs. 18 months.</p

Performance of Expanded Newborn Screening in Norway Supported by Post-Analytical Bioinformatics Tools and Rapid Second-Tier DNA Analyses

In 2012, the Norwegian newborn screening program (NBS) was expanded (eNBS) from screening for two diseases to that for 23 diseases (20 inborn errors of metabolism, IEMs) and again in 2018, to include a total of 25 conditions (21 IEMs). Between 1 March 2012 and 29 February 2020, 461,369 newborns were screened for 20 IEMs in addition to phenylketonuria (PKU). Excluding PKU, there were 75 true-positive (TP) (1:6151) and 107 (1:4311) false-positive IEM cases. Twenty-one percent of the TP cases were symptomatic at the time of the NBS results, but in two-thirds, the screening result directed the exact diagnosis. Eighty-two percent of the TP cases had good health outcomes, evaluated in 2020. The yearly positive predictive value was increased from 26% to 54% by the use of the Region 4 Stork post-analytical interpretive tool (R4S)/Collaborative Laboratory Integrated Reports 2.0 (CLIR), second-tier biochemical testing and genetic confirmation using DNA extracted from the original dried blood spots. The incidence of IEMs increased by 46% after eNBS was introduced, predominantly due to the finding of attenuated phenotypes. The next step is defining which newborns would truly benefit from screening at the milder end of the disease spectrum. This will require coordinated international collaboration, including proper case definitions and outcome studies