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Additional file 1: Supplemental methods. Supplemental Table S1. Probit regression analysis for the propensity score predicted by the logistic regression model as the dose variable and the risk of 50-day in-hospital mortality as the response variable. Supplemental Table 2. Association between plasma procalcitonin level at admission and all-cause 50-day mortality in multivariable logistic regression analysis. Supplemental Table S3. Kaplan-Meier analysis reporting the probability of survival without in-hospital mortality according to the ROC-defined plasma procalcitonin threshold (> 4.24 ng/mL) at baseline. Supplemental Table S4. Hazard ratios and 95% confidence interval for the pairwise comparison of survival probabilities without in-hospital mortality according to the ROC-defined plasma procalcitonin threshold (> 4.24 ng/mL) at baseline. Supplemental Table S5. Association between plasma procalcitonin level at admission and all-cause in-hospital mortality in multivariable Cox proportional-hazards regression. Supplemental Table S6. Distribution of procalcitonin values according to procalcitonin quartiles. Supplemental Table S7. Kaplan-Meier analysis reporting the probability of survival without in-hospital mortality in the 452 studied patients according to baseline plasma procalcitonin quartiles. Supplemental Table S8. Hazard ratios and 95% confidence interval for the pairwise comparison of survival probabilities without in-hospital mortality according to baseline procalcitonin quartiles. Supplemental Figure S1. Flow diagram of patient selection. Supplemental Figure S2. (A) Kaplan-Meier analysis reporting the probability of survival without in-hospital mortality according to the ROC-defined plasma troponin threshold (> 0.05 ng/mL) at baseline; (B) Kaplan-Meier analysis reporting the probability of survival without in-hospital mortality according to the ROC-defined plasma lactates threshold (> 4.24 ng/mL) at baseline. Supplemental Figure S3. Probit sigmoid dose-response curve showing the propensity score predicted by the logistic regression model as the dose variable and the risk of 50-day in-hospital mortality as the response variable. The red line shows the probability and corresponding dose. The dashed curves represent the 95% confidence interval for the respective dose. The dose and 95% confidence interval corresponding with a particular probability are taken from a horizontal line at that probability level. Supplemental Figure S4. (A) Kaplan-Meier ainalysis reporting the probability of survival without in-hospital mortality in the 452 studied patients. The dashed lines represent the 95% confidence interval of the survival probabilities; (B) Kaplan-Meier analysis reporting the probability of survival without in-hospital mortality according to the ROC-defined plasma procalcitonin threshold (> 4.24 ng/mL) at baseline.

Additional file 2. Supplemental Figure S2. (A) 2-D plot using the two top eigenvectors (PC1, PC3) derived from the primary component analysis on the genome-wide methylome landscape of the studied patients and controls. (B) 2-D plot using the PC1 and PC2 eigenvectors derived from the primary component analysis on the methylome landscape of chromosome 1 of the studied patients and controls.

Additional file 3. Supplemental Table S1. Forward and reverse primers for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) of MMACHC, TESK2 transcripts and PRDX1 aberrant transcript.

Additional file 1. Table S1. Primers used for PRDX1 mutational analysis and multiplex RT-PCR of the MMACHC gene. Table S2. Clinical findings of patients with epi-cblC disease. Table S3. Metabolic findings at diagnosis of patients with epi-cblC disease. Table S4. PRDX1:c.515-1G>T homozygosity vs MMACHC:c.271dupA homozygosity: metabolic and clinical findings from the Tuscany/Umbria NBS program. Figure S1. Density distribution plot of the methylome profiles assayed by the Infinium MethylationEPIC BeadChip array in the analysed subjects. All the DNA methylome profiles had a density distribution that followed a beta distribution and were included in the analysis. Figure S2. Estimation of epi-cblC prevalence and PRDX1 mutant allele frequency. a Distribution of cblC diagnoses in the Tuscany-Umbria NBS-case cohort and birth prevalence of cblC and epi-cblC diseases. Three different subgroups of patients have been distinguished on the basis of gene/genes mutated. MMACHC and MMACHC/PRDX1 cases are coloured in blue and orange, respectively. The PRDX1-case with the bi-allelic PRDX1:c.515-1G>T variant, is in red. Values in the pie chart indicate the number and percentage of cases belonging to each subgroup. b Distribution of cblC diagnoses in the total cblC-case cohort referred for genetic test to our Unit since 2006. c Allele frequencies of the most common cblC disease-causing variants identified in the total cblC-case cohort. The MMACHC genetic variants are coloured in blue, whereas the PRDX1 epimutation is in red.

Transcobalamin (TC), a vitamin B12 (cobalamin, Cbl) binding protein in plasma, promotes the cellular uptake of the vitamin by receptor-mediated endocytosis. Inherited TC deficiency is an autosomal recessive disorder characterized by megaloblastic anaemia caused by cellular vitamin B12 depletion. It may be accompanied by neurological complications, including a delay in psychomotor and mental development. This report describes three sisters with inherited TC deficiency resulting from a splicing defect in the TC gene. A point mutation was identified in intron 3 splice site of the TC gene that activates a cryptic splice site in exon 3. The transcript generated has an in-frame deletion of 81 nucleotides and the resulting truncated protein is unstable and not secreted by the cells. Until now, genetic studies have been reported in only five patients with TC deficiency and the molecular defect was different in each of them, which gives evidence for a genetic heterogeneity of the disease.

Transcobalamin (TC), a vitamin B12 (cobalamin, Cbl) binding protein in plasma, promotes the cellular uptake of the vitamin by receptor-mediated endocytosis. Inherited TC deficiency is an autosomal recessive disorder characterized by megaloblastic anaemia caused by cellular vitamin B12 depletion. It may be accompanied by neurological complications, including a delay in psychomotor and mental development. This report describes three sisters with inherited TC deficiency resulting from a splicing defect in the TC gene. A point mutation was identified in intron 3 splice site of the TC gene that activates a cryptic splice site in exon 3. The transcript generated has an in-frame deletion of 81 nucleotides and the resulting truncated protein is unstable and not secreted by the cells. Until now, genetic studies have been reported in only five patients with TC deficiency and the molecular defect was different in each of them, which gives evidence for a genetic heterogeneity of the disease.

Hereditary spastic paraplegias (HSPs) are characterized by lower extremity spasticity and weakness. HSP is often caused by mutations in SPG genes, but it may also be produced by inborn errors of metabolism. We performed next-generation sequencing of 4813 genes in one adult twin pair with HSP and severe muscular weakness occurring at the same age. We found two pathogenic compound heterozygous variants in MTHFR, including a variant not referenced in international databases, c.197C>T (p.Pro66Leu) and a known variant, c.470G>A (p.Arg157Gln), and two heterozygous pathogenic variants in POLG, c.1760C>T (p.Pro587Leu) and c.752C>T (p.Thr251Ile). MTHFR and POLG mutations were consistent with the severe muscle weakness and the metabolic changes, including hyperhomocysteinemia and decreased activity of both N(5,10)methylenetetrahydrofolate reductase (MTHFR) and complexes I and II of the mitochondrial respiratory chain. These data suggest the potential role of MTHFR and POLG mutations through consequences on mitochondrial dysfunction in the occurrence of spastic paraparesis phenotype with combined metabolic, muscular, and neurological components.