Pooling Saliva Sample as an Effective Strategy for the Systematic CMV Screening of Newborns-A Multicentric Prospective Study.

BACKGROUND: Cytomegalovirus is the most common cause of congenital infections worldwide. Screening all newborns in the first 2 weeks of life is the only way to detect all cases of congenital infection, allowing the monitoring of children with asymptomatic infection at birth and early intervention. AIM: In this multicenter study, we aimed to evaluate the feasibility of using a saliva pool strategy for mass screening in 7 Portuguese hospitals, and to estimate the current prevalence of this congenital infection in these hospitals. METHODS: A total of 7033 newborns were screened between June 2020 and June 2022, and 704 pools of 10 saliva samples were analyzed by polymerase chain reaction (PCR). RESULTS: Of the 704 pools analyzed, 685 were negative and 19 had positive PCR results for cytomegalovirus. After individual PCR testing, 26 newborns had positive saliva results, of which 15 were confirmed by urine testing. Thus, this study's prevalence of congenital infection was 0.21% (95% confidence interval: 0.12%-0.35%). CONCLUSIONS: In this study, the pooling strategy proved to be effective for the systematic screening of newborns, although this low prevalence raises questions regarding the cost-effectiveness of implementing universal screening. However, this prevalence is probably the result of the control measures taken during the pandemic; therefore, the rates are expected to return to prepandemic values, but only a new study after the pandemic will be able to confirm this.

Lower prevalence of congenital cytomegalovirus infection in Portugal: possible impact of COVID-19 lockdown?

Cytomegalovirus (CMV) is the most frequent cause of congenital infection all over the world. Its prevalence ranges from 0.2 to 2.2%. Transmission from children to their pregnant mothers is a well-known risk factor, particularly if they attend a childcare centre. This study aims to compare the prevalence of CMV congenital infection (CMV_CI) in Portugal (Lisbon) between two studies, performed respectively in 2019 and 2020. In the 2019 study, performed in two hospitals, we found a 0.67% CMV_CI prevalence, using a pool strategy previously tested with saliva samples. In the 2020 study, using the same pool approach in four hospitals (the previous and two additional), and based on 1277 samples, the prevalence was 0.078%.Conclusion: The close temporal coincidence with COVID-19 lockdown suggests that these measures may have had a significant impact on this reduction, although other explanations cannot be ruled-out. What is Known: • Cytomegalovirus is the leading cause of congenital infection. • Behavioural measures decrease cytomegalovirus seroconversion in pregnant women. What is New: • From 2019 to 2020 there was a significant reduction in the prevalence of congenital CMV infection.

Saliva pools for screening of human cytomegalovirus using real-time PCR.

Human cytomegalovirus (HCMV) is the leading congenital infection agent in the world. The importance of screening this infection has been debated, as 10-15% of the asymptomatic newborns with HCMV at birth will present late sequelae. The aim of this study was to test the feasibility of using saliva pools from newborns in a screening program for congenital HCMV infection, in two Portuguese hospitals. The screening was based on the use of pools of 10 saliva samples for detection of viral DNA by real-time PCR. Whenever there was a positive pool, the samples were tested individually, and for each positive sample the result was confirmed with a urine sample collected in the first 2 weeks of life. The study involved 1492 newborns. One hundred and fifty pools were screened, with 14 positive results in saliva, but only 10 were confirmed in urine samples, giving a prevalence of congenital HCMV infection in both hospitals of 0.67% (CI95% 0.36 to 1.23%).Conclusion: The overall prevalence of congenital HCMV infection in both hospitals was 0.67%. The use of saliva pools proved to be effective for the screening of this congenital infection, allowing timely screening and confirmation in a large population, with associated cost reduction. What is Known: • Newborn screening for HCMV is desirable. • Saliva is a good and practical sample. What is New: • The feasibility of using saliva pools for a large-scale screening. • The cost reduction of this strategy.

Disclosing the functional changes of two genetic alterations in a patient with Chronic Progressive External Ophthalmoplegia: Report of the novel mtDNA m.7486G>A variant.

Chronic Progressive External Ophthalmoplegia (CPEO) is characterized by ptosis and ophthalmoplegia and is usually caused by mitochondrial DNA (mtDNA) deletions or mt-tRNA mutations. The aim of the present work was to clarify the genetic defect in a patient presenting with CPEO and elucidate the underlying pathogenic mechanism. This 62-year-old female first developed ptosis of the right eye at the age of 12 and subsequently the left eye at 45 years, and was found to have external ophthalmoplegia at the age of 55 years. Histopathological abnormalities were detected in the patient's muscle, including ragged-red fibres, a mosaic pattern of COX-deficient muscle fibres and combined deficiency of respiratory chain complexes I and IV. Genetic investigation revealed the "common deletion" in the patient's muscle and fibroblasts. Moreover, a novel, heteroplasmic mt-tRNASer(UCN) variant (m.7486G>A) in the anticodon loop was detected in muscle homogenate (50%), fibroblasts (11%) and blood (4%). Single-fibre analysis showed segregation with COX-deficient fibres for both genetic alterations. Assembly defects of mtDNA-encoded complexes were demonstrated in fibroblasts. Functional analyses showed significant bioenergetic dysfunction, reduction in respiration rate and ATP production and mitochondrial depolarization. Multilamellar bodies were detected by electron microscopy, suggesting disturbance in autophagy. In conclusion, we report a CPEO patient with two possible genetic origins, both segregating with biochemical and histochemical defect. The "common mtDNA deletion" is the most likely cause, yet the potential pathogenic effect of a novel mt-tRNASer(UCN) variant cannot be fully excluded.

In silico analysis for predicting pathogenicity of five unclassified mitochondrial DNA mutations associated with mitochondrial cytopathies' phenotypes.

Mitochondrial DNA (mtDNA) mutations have been assigned as a major cause of genetic disease. When a novel sequence variation is found, it is necessary to evaluate its functional impact, usually requiring functional molecular studies. Given the fact that this approach is difficult to put in practice in a routine basis, it is possible to take advantage of the in silico tools available and predict protein/RNA structure changes and therefore pathogenicity. Here, we describe the characterization of five undescribed mtDNA variants, upon detection of 23 unclassified alterations at Laboratory of Biochemical Genetics, from 2004 to 2014. Those five sequence variations are located in protein-coding genes, in five patients with a diverse range of mitochondrial respiratory chain disease phenotypes including encephalopathy, optic neuropathy, developmental delay, deafness and epilepsy. According to the prediction established by in silico analysis using tools to predict structure and function changes (ClustalW2®, PolyPhen-2®, SIFT®, MutationAssessor®, PredictProtein®, Provean®, I-TASSER®, Haplogrep®), from the 23 variants analyzed, the five described are potentially pathogenic. This approach is inexpensive and compatible with a rapid first line response to clinical demanding, contributing to a more rationale genetic diagnosis concerning novel mutations and to clarify the mtDNA involvement in these pathologies.

Identification of a novel deletion in SURF1 gene: Heterogeneity in Leigh syndrome with COX deficiency.

Leigh syndrome (LS) is a rare, progressive neurodegenerative mitochondrial disorder of infancy. It is a genetically heterogeneous disease. The mutations in SURF1 gene are the most frequently known cause. Here two cases of LS likely caused by SURF1 gene variants are reported: a 39-year-old male patient with a novel homozygous deletion (c.-11_13del), and a case of a 6-year-old boy with the same deletion and a nonsense mutation (c.868dupT), both in heterozygosity. Blue native PAGE showed absence of assembled complex IV. This is the first report of a variant that may abolish the SURF1 gene initiation codon in two LS patients.

Genetic Variation of MT-ND Genes in Frontotemporal Lobar Degeneration: Biochemical Phenotype-Genotype Correlation.

BACKGROUND: Frontotemporal lobar degeneration (FTLD) is the second most common early-onset dementia. Over the last few decades, a growing number of evidence suggests mitochondrial involvement in neurodegeneration, namely modifications of mitochondrial DNA (mtDNA) contributing to energy impairment. OBJECTIVE: To sequence the 7 mitochondrially encoded complex I (MT-ND) genes in 70 FTLD patients and investigate mitochondrial respiratory chain (MRC) complex I activity. METHODS: A sample of 70 patients was studied (39 females and 31 males; age range: 38-82 years, mean ± SD: 63 ± 11 years) with a probable diagnosis of FTLD. Total DNA was extracted from peripheral blood, and sequencing analysis of 7 MT-NDn (1, 2, 3, 4L, 4, 5, 6) genes was performed. Variants identified were submitted to in silico study. Spectrophotometric evaluation of MRC activity in lymphocytes was performed, and results were compared with age-matched controls. RESULTS: A total of 358 (161 different) alterations were found in 92.9% of patients. According to in silico analysis of nonsynonymous variants, only 5 variations are possibly or probably damaging. Complex I activity is significantly decreased in patients. CONCLUSION: To our knowledge, this is the first report of the complete sequence of the MT-ND genes in FTLD patients and correlation with MRC activity. The high number of mtDNA variations identified and a significant decrease in complex I activity suggest a possible involvement of mtDNA alterations in FTLD. Although the majority of these alterations are not primarily pathogenic, an interaction with other mutations may occur, leading to the disease, worsening its expression or influencing age of onset.

Metabolic effects of hypoxia in colorectal cancer by 13C NMR isotopomer analysis.

(13)C NMR isotopomer analysis was used to characterize intermediary metabolism in three colorectal cancer cell lines (WiDr, LS1034, and C2BBe1) and determine the "metabolic remodeling" that occurs under hypoxia. Under normoxia, the three colorectal cancer cell lines present high rates of lactate production and can be seen as "Warburg" like cancer cells independently of substrate availability, since such profile was dominant at both high and low glucose media contents. The LS1034 was the less glycolytic of the three cell lines and was the most affected by the event of hypoxia, raising abruptly glucose consumption and lactate production. The other two colorectal cell lines, WiDr and C2BBe1, adapted better to hypoxia and were able to maintain their oxidative fluxes even at the very low levels of oxygen. These differential metabolic behaviors of the three colorectal cell lines show how important an adequate knowledge of the "metabolic remodeling" that follows a given cancer treatment is towards the correct (re)design of therapeutic strategies against cancer.

Validation of control of allergic rhinitis and asthma test for children (CARATKids)--a prospective multicenter study.

BACKGROUND: Control of Allergic Rhinitis and Asthma Test for Children (CARATKids) is the first questionnaire that assesses simultaneously allergic rhinitis and asthma control in children. It was recently developed, but redundancy of questions and its psychometric properties were not assessed. This study aimed to (i) establish the final version of the CARATKids questionnaire and (ii) evaluate its reliability, responsiveness, cross-sectional validity, and longitudinal validity. METHODS: A prospective observational study was conducted in 11 Portuguese centers. During two visits separated by 6 wk, CARATKids, visual analog scale scales and childhood asthma control test were completed, and participant's asthma and rhinitis were evaluated by his/her physician without knowing the questionnaires' results. Data-driven item reduction was conducted, and internal consistency, responsiveness analysis, and associations with external measures of disease status were assessed. RESULTS: Of the 113 children included, 101 completed both visits. After item reduction, the final version of the questionnaire has 13 items, eight to be answered by the child and five by the caregiver. Its Cronbach's alpha was 0.80, the Guyatt's responsiveness index was -1.51, and a significant (p < 0.001) within-patient change of CARATKids score in clinical unstable patients was observed. Regarding cross-sectional validity, correlation coefficients of CARATKids with the external measures of control were between 0.45 and -0.69 and met the a priori predictions. In the longitudinal validity assessment, the correlation coefficients between the score changes of CARATKids and those of external measures of control ranged from 0.34 to 0.46. CONCLUSION: CARATKids showed adequate psychometric properties and is ready to be used in clinical practice.

Antenatal manifestations of mitochondrial disorders.

Mitochondrial respiratory chain diseases are a heterogeneous group of pathologies caused by genetic alterations affecting mitochondrial energy production. Theoretically, this deficiency may lead to any symptoms, in any organ or tissue, at any age even before birth. The aim of our study was to identify the frequency and characterize antenatal manifestations identifying possible associations between mitochondrial disease and more specific and earlier manifestation. We retrospectively review the files of 44 paediatric subjects with genetic and biochemical alterations of respiratory chain identified in the first decade of life and compare data with a control group (n = 88). Our results show that maternal age was similar in both groups. The female gender was predominant in patients group. Gestational age at delivery was similar in both groups. Concerning birth weight, it was significantly lower (p = 0.001) in patients (2899.9 ± 538.3 vs. 3246.6 ± 460.2 g). Fifteen pregnancies of the patients group were considered abnormal. Our findings show that intrauterine growth restriction was the most frequent antenatal feature observed. Neonatal morbidity was significantly higher (fivefold) in patients (p < 0.001). The clinical findings are independent of the molecular defect type. Our results are preliminary and more studies are needed, in order to learn more about mitochondrial physiology and activity in embryological development for the assessment of mitochondrial disease progress in fetal life. However, the present work is a significant contribution, given the scarcity of information in this field.

Frontotemporal dementia and mitochondrial DNA transitions.

Frontotemporal dementia (FTD) is the second most common type of primary degenerative dementia. Some patients present an overlap between Alzheimer's disease (AD) and FTD both in neuropathological and clinical aspects. This may suggest a similar overlap in physiopathology, namely an involvement of mitochondrial DNA (mtDNA) in FTD, as it has been associated to AD. To determine if mtDNA is involved in FTD, we performed a Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis, specific to mtDNA NADH Dehydrogenase subunit 1 (ND1) nucleotides 3337-3340, searching for mutations previously described in Parkinson's and AD patients. We could identify one FTD patient with two mtDNA transitions: one already known (3316 G-to-A) and another unreported (3337 G-to-A). Additionally, mitochondrial respiratory chain complex I activity was reduced in leukocytes of this patient (36% of the control mean activity). To our knowledge, this is the first report of mtDNA variants in FTD patients.