Subacute tributyltin exposure alters the development and morphology of mammary glands in association with CYP19A1 expression in female rats.

Tributyltin (TBT) is an endocrine-disrupting chemical (EDC) related to reproductive dysfunctions. However, few studies have investigated the effects of TBT exposure on mammary gland development. Thus, we assessed whether subacute TBT exposure causes irregularities in mammary gland development. We administered TBT (100 and 1,000 ng/kg/day for 30 days) to female rats from postnatal day (PND) 25 to PND 55, and mammary gland development, morphology, inflammation, collagen deposition, and protein expression were evaluated. Abnormal mammary gland development was observed in both TBT groups. Specifically, TBT exposure reduced the number of terminal end buds (TEBs), type 1 (AB1) alveolar buds, and type 2 (AB2) alveolar buds. An increase in the lobule and differentiation (DF) 2 score was found in the mammary glands of TBT rats. TBT exposure increased mammary gland blood vessels, mast cell numbers, and collagen deposition. Additionally, both TBT rats exhibited intraductal hyperplasia and TEB-like structures. An increase in estrogen receptor alpha (ERα), progesterone receptor (PR), and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) - positive cells was observed in the mammary glands of TBT rats. A strong negative correlation was observed between CYP19A1- positive cells and TEB number. In addition, CYP19A1 - positive cells were positively correlated with mammary gland TEB-like structure, ductal hyperplasia, inflammation, and collagen deposition. Thus, these data suggest that TBT exposure impairs mammary gland development through the modulation of CYP19A1 signaling pathways in female rats.

Genetic Polymorphisms of P2RX7 but Not of ADORA2A Are Associated with the Severity of SARS-CoV-2 Infection.

SARS-CoV-2 infection ranges from mild to severe presentations, according to the intensity of the aberrant inflammatory response. Purinergic receptors dually control the inflammatory response: while adenosine A2A receptors (A2ARs) are anti-inflammatory, ATP P2X7 receptors (P2X7Rs) exert pro-inflammatory effects. The aim of this study was to assess if there were differences in allelic and genotypic frequencies of a loss-of-function SNP of ADORA2A (rs2298383) and a gain-of-function single nucleotide polymorphism (SNP) of P2RX7 (rs208294) in the severity of SARS-CoV-2-associated infection. Fifty-five individuals were enrolled and categorized according to the severity of the infection. Endpoint genotyping was performed in blood cells to screen for both SNPs. The TT genotype (vs. CT + CC) and the T allele (vs. C allele) of P2RX7 SNP were found to be associated with more severe forms of COVID-19, whereas the association between ADORA2A SNP and the severity of infection was not significantly different. The T allele of P2RX7 SNP was more frequent in people with more than one comorbidity and with cardiovascular conditions and was associated with colorectal cancer. Our findings suggest a more prominent role of P2X7R rather than of A2AR polymorphisms in SARS-CoV-2 infection, although larger population-based studies should be performed to validate our conclusions.

Portuguese Neonatal Screening Program: A Cohort Study of 18 Years Using MS/MS.

The Portuguese Neonatal Screening Program (PNSP) conducts nationwide screening for rare diseases, covering nearly 100% of neonates and screening for 28 disorders, including 24 inborn errors of metabolism (IEMs). The study's purpose is to assess the epidemiology of the screened metabolic diseases and to evaluate the impact of second-tier testing (2TT) within the PNSP. From 2004 to 2022, 1,764,830 neonates underwent screening using tandem mass spectrometry (MS/MS) to analyze amino acids and acylcarnitines in dried blood spot samples. 2TT was applied when necessary. Neonates with profiles indicating an IEM were reported to a reference treatment center, and subsequent biochemical and molecular studies were conducted for diagnostic confirmation. Among the screened neonates, 677 patients of IEM were identified, yielding an estimated birth prevalence of 1:2607 neonates. The introduction of 2TT significantly reduced false positives for various disorders, and 59 maternal cases were also detected. This study underscores the transformative role of MS/MS in neonatal screening, emphasizing the positive impact of 2TT in enhancing sensitivity, specificity, and positive predictive value. Our data highlight the efficiency and robustness of neonatal screening for IEM in Portugal, contributing to early and life-changing diagnoses.

Pharmacogenetic Variants Can Influence Optical Medication Use.

INTRODUCTION: Single Nucleotide Polymorphisms (SNPs) are used as drug susceptibility biomarkers in metabolic diseases. Alterations in the gene encoding triggers the enzyme flavin monooxygenase 3 (FMO3), involved in the Sulindac metabolization, which also is responsible for the inherited metabolic disorder. Trimethylaminuria (TMAu, OMIM: 602079). DPYD gene variants are associated with the enzyme dihydropyrimidine dehydrogenase deficiency (DPD; OMIM: 274270). This autosomal recessive metabolic disorder, ultimately leads to the inability to metabolize fluoropyrimidines, which causes severe toxicity in individuals treated with these drugs. METHODS: Variants in genes responsible for the expression of enzymes that encode transporters or receptors involved in the metabolization pathways of certain drugs may condition the individuals response to certain drugs, compromising the therapeutic response and clinical prognosis. Thus the sequencing and identification of variants become relevant, not only gain knowledge on effects of these variants' on disease causality but also in terms of its side effects resulting from the coding enzymes responsible for drug metabolization. RESULTS: It was found that patients with the c.472G>A (p.Glu158Lys) and c.923A>G (p.Glu308Gly) polymorphisms, in homozygosity, in FMO3 gene did not develop polyps, thus have a protective effect in the treatment of Familial Adenomatous Polyposis (PAF). However, in the case of the DPYD gene, c.1905+1G>A (IVS14+1G>A), c.1679T>G (p.Ile560Ser), c.2846A>T (p.Asp949Val) e c.1236G>A/HapB3 variants can be lethal in cancer patients indicated for fluoropyrimidine-based chemotherapy. CONCLUSION: Knowledge on the drug mechanisms will affect the therapeutic response of patients treated with a given drug. Thus, pharmacogenetics is an essential tool in personalized medicine, since molecular studies allows the clinician to predict the probability of efficacy and toxicity of certain drugs, resulting higher efficiency in individualizing treatment and also improving the safety of the patient. From a personalized medicine perspective, the study of the characteristics of the drug and its metabolization site, the genes involved in the encoding of enzymes responsible for its metabolization will be of great interest.

Methylmalonyl Coenzyme A (CoA) Epimerase Deficiency, an Ultra-Rare Cause of Isolated Methylmalonic Aciduria With Predominant Neurological Features.

Methylmalonyl coenzyme A (CoA) epimerase (MCE) converts D-methylmalonyl-CoA into L-methylmalonyl CoA in the final common degradation pathway of valine, isoleucine, methionine, threonine, odd-chain fatty acids, and cholesterol side chains. Methylmalonyl-CoA epimerase deficiency is an ultra-rare autosomal recessive disorder where methylmalonic acid, methylcitrate, 3-hydroxypropionate, and propionylcarnitine are accumulated. We describe two novel pediatric patients and review the previously reported cases of MCE deficiency. Including our two novel patients, at least 24 cases of MCE deficiency have been described, with a broad clinical spectrum ranging from asymptomatic to severely neurologically impaired patients. Our patients are siblings of Arabic origin who presented with metabolic decompensation with coma and epilepsy during infancy. Methylmalonic aciduria was disclosed, L-methylmalonyl-CoA mutase deficiency was assumed, and they were treated accordingly. When first seen in our country, aged 10 and four years, respectively, both presented severe intellectual disability and spasticity. The younger had an ataxic gait, and the older was wheelchair-ridden. The study of the MMUT, MMAA, MMAB, and MMADHC genes was normal. Subsequently, the pathogenic variant c.139C>T (p.Arg47*) in the MCEE gene was identified in homozygosity in both patients, leading to the diagnosis of MCE deficiency (Online Mendelian Inheritance in Man (OMIM®) 251120, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, MD, USA). Most patients were homozygous for that variant (83% of the alleles). Correct diagnosis allowed treatment adequacy and genetic counseling. Methylmalonyl-CoA epimerase deficiency shares a similar biochemical profile with other rare genetic disorders. Patients present with overlapping clinical features with predominant neurological manifestations; genetic testing is indispensable for diagnosis. We found no association between genotype and biochemical and clinical phenotypes.

Non-dipping circadian pattern as a predictor of incipient nephropathy in normotensive normoalbuminuric type 1 diabetics.

The objective of this study was to assess the value of the abnormal circadian blood pressure pattern by ambulatory blood pressure monitoring (ABPM) to predict the onset of abnormal albuminuria in normotensive and normoalbuminuric DM1 patients. The participators were submitted to ABPM and followed prospectively until the onset of albuminuria or the end of follow-up. The patients with normal circadian blood pressure pattern were compared with the non-dippers in regard of the time interval free of albuminuria. The survival curves were evaluated by the Kaplan-Meier method. Of 34 patients screened, 10 patients matched the exclusion criteria. Therefore, 24 patients were submitted to ABPM, aged 24 ± 8.3 y, 18 men, and all Caucasian. Elevated levels of albuminuria did not occurin any individual with normal systolic blood pressure dip (>10%) at 54 months of follow-up. Only 22% of patients among non-dippers were free of albuminuria (<30 mg/g maintained for 3 months) at the same time (p = 0.049). Patients that reached the outcome were homogeneous in regard to other clinical and ABPM data evaluated. Abnormal systolic blood pressure circadian pattern predicts the evolution to incipient nephropathy in normotensive normoalbuminuric DM1 patients.

SERAC1 Deficiency- A New Phenotype.

Introduction - SERAC1 deficiency phenotype range from MEGD(H)EL syndrome, the most severe, to juvenile complicated spastic paraplegia, to adult-onset dystonic features (in only one patient). The MEGD(H)EL syndrome is characterized by (3-methylglutaconic aciduria with deafness-dystonia, [hepatopathy], encephalopathy, and Leigh-like syndrome). Biochemical abnormalities: elevated urinary 3 - metilglutaconic and 3-metilglutaric acids, high lactate and alanine in serum. Diagnosis is confirmed when biallelic pathogenic variants in SERAC1 gene are found. Brain MRI: basal ganglia lesions and generalized atrophy. Results/Case report - A 30-year-old patient with a moderate intellectual disability, developed, since the age of 25, a progressive loss of previous capacities (hand dexterity, oral language), and later subacute generalized dystonic features. Currently he has spastic tetraparesis, dystonia, scoliosis and autistic behavior, with bilateral basal ganglia lesions on brain MRI. Genetic study revealed biallelic pathogenic variants in SERAC1 gene, confirm MEGD(H)EL. A 73 years old patient with cognitive impairment and progressive spastic tetraparesis had multiple periventricular T2 hyperintense lesions. She has a homozygotic SERAC1 variant NM_032861: exon4:c.T139A: p.F471 (rs112780453), considered benign. Biochemical study revealed elevated plasmatic alanine and urinary3-metilglutaconic and 3-metilglutaric acid. This profile is concordant with mitochondrial dysfunction and SERAC1 Deficit. Conclusion - The first patient has the clinical symptoms associated to the MEGD(H)EL syndrome, and the biochemical and genetic confirmation of the diagnosis, without reservations. However, in the second patient, the progressive paraparesis and cognitive impairment did not appear to be caused by multiple sclerosis nor subcortical vascular leukoencephalopathy (without vascular risk factors). The abnormal biochemical profile is suggestive of SERAC1 Deficiency, even without genetic confirmation. In what should we believe?

Portuguese Neonatal Screening Programme: A Retrospective Cohort Study of 18 Years of MS/MS.

INTRODUCTION: The Portuguese Neonatal Screening Programme (PNSP) identifies patients with rare diseases through nationwide screening. Currently, 27 diseases are diagnosed, amongst which are 24 Inborn Errors of Metabolism (IEM), covering approximately 100% of neonates (1). In 2004, the national laboratory implemented a new screening method, tandem mass spectrometry (MS/MS) to test for amino acids and acylcarnitines. This new protocol revolutionized the PNSP and allowed for the analysis of an increased number of IEM, with clear improvements in treatment timings and clinical outcomes (2). METHODS: From 2004 to 2022, 1 764 830 neonates were screened with MS/MS technology. Those who displayed biochemical profiles indicating an IEM were subjected to molecular characterization via genomic DNA extraction, PCR amplification, and direct Sanger sequencing method of dried blood spot samples. RESULTS/CASE REPORT: A cohort of 681 newborns were diagnosed with an IEM. MCAD deficiency is the most frequent, with 233 confirmed diagnoses, showing predominantly c.985A>G (p.K329E) mutation of the ACADM gene in homozygosity. Approximately 1/3 of the 33 confirmed cases of Glutaric Aciduria type I present homozygous for the c.1204C>T (p.Arg402Trp) mutation in GCDH. Around 60% of cases of MAT II/III deficiency display the dominant mutation of the MAT1A gene, c.791G>A (p.Arg264His). These genetic profiles and others were determined as diagnostic confirmation for 24 of the IEM screened. CONCLUSION: This data shows the molecular epidemiology of patients with confirmed IEM diagnosis identified by neonatal screening. Some diseases out of the scope of the PNSP were also detected as a differential diagnosis after biochemical suspicion in the dried blood spot sample. The retrospective analysis of the PNSP allows for an overview of 18 years of achievements accomplished by the national screening for IEM since MS/MS was implemented. For some pathologies with low incidence, it's difficult to trace a discernible pattern. However, presenting de novo mutations for these diseases might provide insights on how to approach different phenotypes. The aim of this work is to establish the molecular epidemiology of metabolic diseases screened.

Leigh Syndrome Spectrum: A Portuguese Population Cohort in an Evolutionary Genetic Era.

Mitochondrial diseases are the most common inherited inborn error of metabolism resulting in deficient ATP generation, due to failure in homeostasis and proper bioenergetics. The most frequent mitochondrial disease manifestation in children is Leigh syndrome (LS), encompassing clinical, neuroradiological, biochemical, and molecular features. It typically affects infants but occurs anytime in life. Considering recent updates, LS clinical presentation has been stretched, and is now named LS spectrum (LSS), including classical LS and Leigh-like presentations. Apart from clinical diagnosis challenges, the molecular characterization also progressed from Sanger techniques to NGS (next-generation sequencing), encompassing analysis of nuclear (nDNA) and mitochondrial DNA (mtDNA). This upgrade resumed steps and favored diagnosis. Hereby, our paper presents molecular and clinical data on a Portuguese cohort of 40 positive cases of LSS. A total of 28 patients presented mutation in mtDNA and 12 in nDNA, with novel mutations identified in a heterogeneous group of genes. The present results contribute to the better knowledge of the molecular basis of LS and expand the clinical spectrum associated with this syndrome.

Diagnosis across a cohort of "atypical" atypical and complex parkinsonism.

INTRODUCTION: The diagnostic approach for adulthood parkinsonism can be challenging when atypical features hamper its classification in one of the two main parkinsonian groups: Parkinson's disease or atypical parkinsonian syndromes (APS). Atypical features are usually associated with non-sporadic neurodegenerative causes. METHODS: Retrospective analysis of patients with a working clinical diagnosis of "atypical" APS and complex parkinsonism. "Atypical" APS were classified according to the diagnostic research criteria and the "4-step diagnostic approach" (Stamelou et al. 2013). When not indicated, the final aetiological diagnosis was prospectively assessed. Brain MRI of progressive supranuclear palsy (PSP) look-alikes was reviewed by a neuroradiologist. RESULTS: Among 18 patients enrolled, ten were assigned to the "atypical" APS and eight to the complex parkinsonism group. In the "atypical" APS group, nine patients had PSP and one had corticobasal degeneration. In the PSP group the median magnetic resonance parkinsonism index was 17.1. A final aetiological diagnosis was established for 11 patients, four from the complex parkinsonism (L-2-hidroxiglutaric aciduria and DiGeorge syndrome) and seven from the "atypical" APS (Perry syndrome, postencephalitic PSP, vascular PSP, and MTP-AT6 mitochondrial disease) group. CONCLUSIONS: In this study, the identification of atypical APS features, as proposed in the "4-step diagnostic approach", successfully guided the investigation of alternative diagnoses. Distinctive non-neurodegenerative etiologies causing "atypical" atypical and complex parkinsonism were uncovered, including acquired (post-encephalitis and vascular) and genetic (MTP-AT6 mitochondrial disease mimicking PSP, described for the first time) ones. In the future, accurate clinical identification and distinction between neurodegenerative and non-neurodegenerative parkinsonism etiologies will allow for refining clinical trials.

RNA-seq reveals that anti-obesity irisin and triiodothyronine (T3) hormones differentially affect the purinergic signaling transcriptomics in differentiated human adipocytes.

The anti-obesity thyroid hormone, triiodothyronine (T3), and irisin, an exercise- and/or cold-induced myokine, stimulate thermogenesis and energy consumption while decreasing lipid accumulation. The involvement of ATP signaling in adipocyte cell function and obesity has attracted increasing attention, but the crosstalk between the purinergic signaling cascade and anti-obesity hormones lacks experimental evidence. In this study, we investigated the effects of T3 and irisin in the transcriptomics of membrane-bound purinoceptors, ectonucleotidase enzymes and nucleoside transporters participating in the purinergic signaling in cultured human adipocytes. The RNA-seq analysis revealed that differentiated adipocytes express high amounts of ADORA1, P2RY11, P2RY12, and P2RX6 gene transcripts, along with abundant levels of transcriptional products encoding to purine metabolizing enzymes (ENPP2, ENPP1, NT5E, ADA and ADK) and transporters (SLC29A1, SCL29A2). The transcriptomics of purinergic signaling markers changed in parallel to the upsurge of "browning" adipocyte markers, like UCP1 and P2RX5, after treatment with T3 and irisin. Upregulation of ADORA1, ADORA2A and P2RX4 gene transcription was obtained with irisin, whereas T3 preferentially upregulated NT5E, SLC29A2 and P2RY11 genes. Irisin was more powerful than T3 towards inhibition of the leptin gene transcription, the SCL29A1 gene encoding for the ENT1 transporter, the E-NPP2 (autotaxin) gene, and genes that encode for two ADP-sensitive P2Y receptors, P2RY1 and P2RY12. These findings indicate that anti-obesity irisin and T3 hormones differentially affect the purinergic signaling transcriptomics, which might point towards new directions for the treatment of obesity and related metabolic disorders that are worth to be pursued in future functional studies.

Approach to adult patients with primary hypothyroidism in some special situations: a position statement from the Thyroid Department of the Brazilian Society of Endocrinology and Metabolism (SBEM)

ABSTRACT Primary hypothyroidism is a common disorder in clinical practice. The management of most cases of hypothyroidism is usually straightforward, but the best approach in some special situations may raise questions among physicians. This position statement was prepared by experts from the Brazilian Society of Endocrinology and Metabolism to guide the management of three special situations, namely, hypothyroidism in the elderly, subclinical hypothyroidism in patients with heart disease, and difficult-to-control hypothyroidism. The authors prepared the present statement after conducting a search on the databases MEDLINE/PubMed, LILACS, and SciELO and selecting articles with the best evidence quality addressing the selected situations. The statement presents information about the current approach to patients in these special situations.

Approach to adult patients with primary hypothyroidism in some special situations: a position statement from the Thyroid Department of the Brazilian Society of Endocrinology and Metabolism (SBEM).

Primary hypothyroidism is a common disorder in clinical practice. The management of most cases of hypothyroidism is usually straightforward, but the best approach in some special situations may raise questions among physicians. This position statement was prepared by experts from the Brazilian Society of Endocrinology and Metabolism to guide the management of three special situations, namely, hypothyroidism in the elderly, subclinical hypothyroidism in patients with heart disease, and difficult-to-control hypothyroidism. The authors prepared the present statement after conducting a search on the databases MEDLINE/PubMed, LILACS, and SciELO and selecting articles with the best evidence quality addressing the selected situations. The statement presents information about the current approach to patients in these special situations.

Effects of Triiodothyronine on Human Osteoblast-Like Cells: Novel Insights From a Global Transcriptome Analysis.

Background: Thyroid hormones play a significant role in bone development and maintenance, with triiodothyronine (T3) particularly being an important modulator of osteoblast differentiation, proliferation, and maintenance. However, details of the biological processes (BPs) and molecular pathways affected by T3 in osteoblasts remain unclear. Methods: To address this issue, primary cultures of human adipose-derived mesenchymal stem cells were subjected to our previously established osteoinduction protocol, and the resultant osteoblast-like cells were treated with 1 nm or 10 nm T3 for 72 h. RNA sequencing (RNA-Seq) was performed using the Illumina platform, and differentially expressed genes (DEGs) were identified from the raw data using Kallisto and DESeq2. Enrichment analysis of DEGs was performed against the Gene Ontology Consortium database for BP terms using the R package clusterProfiler and protein network analysis by STRING. Results: Approximately 16,300 genes were analyzed by RNA-Seq, with 343 DEGs regulated in the 1 nm T3 group and 467 upregulated in the 10 nm T3 group. Several independent BP terms related to bone metabolism were significantly enriched, with a number of genes shared among them (FGFR2, WNT5A, WNT3, ROR2, VEGFA, FBLN1, S1PR1, PRKCZ, TGFB3, and OSR1 for 1nM T3; and FZD1, SMAD6, NOG, NEO1, and ENG for 10 nm T3). An osteoblast-related search in the literature regarding this set of genes suggests that both T3 doses are unfavorable for osteoblast development, mainly hindering BMP and canonical and non-canonical WNT signaling. Conclusions: Therefore, this study provides new directions toward the elucidation of the mechanisms of T3 action on osteoblast metabolism, with potential future implications for the treatment of endocrine-related bone pathologies.

Placental model as an important tool to study maternal-fetal interface.

The placenta is a temporary organ that plays critical roles at the maternal-fetal interface. Normal development and function of the placenta is dependent on hormonal signaling pathways that make the placenta a target of endocrine disrupting chemical (EDC) action. Studies showing association between prenatal exposure, hormone disruption, and reproductive damage indicate that EDCs are developmentally toxic and can impact future generations. In this context, new placental models (trophoblast-derived cell lines, organotypic or 3D cell models, and physiologically based kinetic models) have been developed in order to create new approach methodology (NAM) to assess and even prevent such disastrous toxic harm in future generations. With the widespread discouragement of conducting animal studies, it has become irrefutable to develop in vitro models that can serve as a substitute for in vivo models. The goal of this review is to discuss the newest in vitro models to understand the maternal-fetal interface and predict placental development, physiology, and dysfunction generated by failures in molecular hormone control mechanisms, which, consequently, may change epigenetic programming to increase susceptibility to metabolic and other disorders in the offspring. We summarize the latest placental models for developmental toxicology studies, focusing mainly on three-dimensional (3D) culture models.

Prediction of Non-canonical Routes for SARS-CoV-2 Infection in Human Placenta Cells.

The SARS-CoV-2 is the causative agent of the COVID-19 pandemic. The data available about COVID-19 during pregnancy have demonstrated placental infection; however, the mechanisms associated with intrauterine transmission of SARS-CoV-2 is still debated. Intriguingly, while canonical SARS-CoV-2 cell entry mediators are expressed at low levels in placental cells, the receptors for viruses that cause congenital infections such as the cytomegalovirus and Zika virus are highly expressed in these cells. Here we analyzed the transcriptional profile (microarray and single-cell RNA-Seq) of proteins potentially interacting with coronaviruses to identify non- canonical mediators of SARS-CoV-2 infection and replication in the placenta. Despite low levels of the canonical cell entry mediators ACE2 and TMPRSS2, we show that cells of the syncytiotrophoblast, villous cytotrophoblast, and extravillous trophoblast co-express high levels of the potential non-canonical cell-entry mediators DPP4 and CTSL. We also found changes in the expression of DAAM1 and PAICS genes during pregnancy, which are translated into proteins also predicted to interact with coronaviruses proteins. These results provide new insight into the interaction between SARS-CoV-2 and host proteins that may act as non-canonical routes for SARS-CoV-2 infection and replication in the placenta cells.

MiR-125a-3p and MiR-320b Differentially Expressed in Patients with Chronic Myeloid Leukemia Treated with Allogeneic Hematopoietic Stem Cell Transplantation and Imatinib Mesylate.

Chronic myeloid leukemia (CML), a hematopoietic neoplasm arising from the fusion of BCR (breakpoint cluster region) gene on chromosome 22 to the ABL (Abelson leukemia virus) gene on chromosome 9 (BCR-ABL1 oncogene), originates from a small population of leukemic stem cells with extensive capacity for self-renewal and an inflammatory microenvironment. Currently, CML treatment is based on tyrosine kinase inhibitors (TKIs). However, allogeneic hematopoietic stem cell transplantation (HSCT-allo) is currently the only effective treatment of CML. The difficulty of finding a compatible donor and high rates of morbidity and mortality limit transplantation treatment. Despite the safety and efficacy of TKIs, patients can develop resistance. Thus, microRNAs (miRNAs) play a prominent role as biomarkers and post-transcriptional regulators of gene expression. The aim of this study was to analyze the miRNA profile in CML patients who achieved cytogenetic remission after treatment with both HSCT-allo and TKI. Expression analyses of the 758 miRNAs were performed using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Bioinformatics tools were used for data analysis. We detected miRNA profiles using their possible target genes and target pathways. MiR-125a-3p stood out among the downregulated miRNAs, showing an interaction network with 52 target genes. MiR-320b was the only upregulated miRNA, with an interaction network of 26 genes. The results are expected to aid future studies of miRNAs, residual leukemic cells, and prognosis in CML.

Role of RNA in Molecular Diagnosis of MADD Patients.

The electron-transfer flavoprotein dehydrogenase gene (ETFDH) encodes the ETF-ubiquinone oxidoreductase (ETF-QO) and has been reported to be the major cause of multiple acyl-CoA dehydrogenase deficiency (MADD). In this study, we present the clinical and molecular diagnostic challenges, at the DNA and RNA levels, involved in establishing the genotype of four MADD patients with novel ETFDH variants: a missense variant, two deep intronic variants and a gross deletion. RNA sequencing allowed the identification of the second causative allele in all studied patients. Simultaneous DNA and RNA investigation can increase the number of MADD patients that can be confirmed following the suggestive data results of an expanded newborn screening program. In clinical practice, accurate identification of pathogenic mutations is fundamental, particularly with regard to diagnostic, prognostic, therapeutic and ethical issues. Our study highlights the importance of RNA studies for a definitive molecular diagnosis of MADD patients, expands the background of ETFDH mutations and will be important in providing an accurate genetic counseling and a prenatal diagnosis for the affected families.