Predicted mechanistic impacts of human protein missense variants.

Genome sequencing efforts have led to the discovery of tens of millions of protein missense variants found in the human population with the majority of these having no annotated role and some likely contributing to trait variation and disease. Sequence-based artificial intelligence approaches have become highly accurate at predicting variants that are detrimental to the function of proteins but they do not inform on mechanisms of disruption. Here we combined sequence and structure-based methods to perform proteome-wide prediction of deleterious variants with information on their impact on protein stability, protein-protein interactions and small-molecule binding pockets. AlphaFold2 structures were used to predict approximately 100,000 small-molecule binding pockets and stability changes for over 200 million variants. To inform on protein-protein interfaces we used AlphaFold2 to predict structures for nearly 500,000 protein complexes. We illustrate the value of mechanism-aware variant effect predictions to study the relation between protein stability and abundance and the structural properties of interfaces underlying trans protein quantitative trait loci (pQTLs). We characterised the distribution of mechanistic impacts of protein variants found in patients and experimentally studied example disease linked variants in FGFR1.

Promoting advanced medical services in the framework of 3PM-a proof-of-concept by the "Centro" Region of Portugal.

Multidisciplinary team from three universities based in the "Centro" Region of Portugal developed diverse approaches as parts of a project dedicated to enhancing and expanding Predictive, Preventive, and Personalized Medicine (3PM) in the Region. In a sense, outcomes acted as a proof-of-concept, in that they demonstrated the feasibility, but also the relevance of the approaches. The accomplishments comprise defining a new regional strategy for implementing 3PM within the Region, training of human resources in genomic sequencing, and generating good practices handbooks dedicated to diagnostic testing via next-generation sequencing, to legal and ethical concerns, and to knowledge transfer and entrepreneurship, aimed at increasing literacy on 3PM approaches. Further approaches also included support for entrepreneurship development and start-ups, and diverse and relevant initiatives aimed at increasing literacy relevant to 3PM. Efforts to enhance literacy encompassed citizens across the board, from patients and high school students to health professionals and health students. This focus on empowerment through literacy involved a variety of initiatives, including the creation of an illustrated book on genomics and the production of two theater plays centered on genetics. Additionally, authors stressed that genomic tools are relevant, but they are not the only resources 3PM is based on. Thus, they defend that other initiatives intended to enable citizens to take 3PM should include multi-omics and, having in mind the socio-economic burden of chronic diseases, suboptimal health status approaches in the 3PM framework should also be considered, in order to anticipate medical intervention in the subclinical phase. Supplementary Information: The online version contains supplementary material available at 10.1007/s13167-024-00353-9.

Maternal Urinary Iodine Concentration during Pregnancy and Its Impact on Child Growth and Neurodevelopment: An 11-Year Follow-Up Study.

Mild-to-moderate iodine deficiency during pregnancy is prevalent worldwide, but its consequences for maternal and child health are not clear. We aimed to investigate the impact of maternal iodine intake during pregnancy on the child's growth and neurodevelopment. This study involved a cohort of 11-year-old children (n = 70) whose mothers had participated in an iodine intake survey during pregnancy. Gestational, neonatal, anthropometric, intelligence quotient (IQ), and socioeconomic parameters were analyzed according to maternal urinary iodine concentration (UIC). There was a positive linear trend of current height Z-score, full-scale IQ, verbal IQ, family income, maternal education, and a negative trend of neonatal TSH levels with increasing maternal UIC levels. However, regression analysis indicated that maternal UIC was not an independent predictor of any gestational, neonatal, or childhood development parameter. Only maternal school education was positively associated with child height and IQ. In conclusion, we did not find any evidence of a direct effect of maternal iodine intake during pregnancy on the long-term growth and neurodevelopment of children. The results suggest that socioeconomic factors are important confounding factors that affect both maternal iodine intake and child development and must be considered when investigating the association between maternal iodine intake and child outcomes.

The impact of biological clock and sex hormones on the risk of disease.

Molecular clocks are responsible for defining 24-h cycles of behaviour and physiology that are called circadian rhythms. Several structures and tissues are responsible for generating these circadian rhythms and are named circadian clocks. The suprachiasmatic nucleus of the hypothalamus is believed to be the master circadian clock receiving light input via the optic nerve and aligning internal rhythms with environmental cues. Studies using both in vivo and in vitro methodologies have reported the relationship between the molecular clock and sex hormones. The circadian system is directly responsible for controlling the synthesis of sex hormones and this synthesis varies according to the time of day and phase of the estrous cycle. Sex hormones also directly interact with the circadian system to regulate circadian gene expression, adjust biological processes, and even adjust their own synthesis. Several diseases have been linked with alterations in either the sex hormone background or the molecular clock. So, in this chapter we aim to summarize the current understanding of the relationship between the circadian system and sex hormones and their combined role in the onset of several related diseases.

Hypoparathyroidism, deafness and renal dysplasia syndrome caused by a GATA3 splice site mutation leading to the activation of a cryptic splice site.

The HDR syndrome is a rare autosomal dominant disorder characterised by Hypoparathyroidism, Deafness, and Renal dysplasia, and is caused by inactivating heterozygous germline mutations in the GATA3 gene. We report an 11-year-old girl with HDR syndrome caused by a heterozygous mutation located at the splice acceptor site of exon 5 of the GATA3 gene (NM_001002295.2: c.925-1G>T). Functional studies using a minigene assay showed that this splice site mutation abolished the normal splicing of the GATA3 pre-mRNA and led to the use of a cryptic splice acceptor site, resulting in the loss of the first seven nucleotides (TCTGCAG) of exon 5 in the GATA3 mRNA. These findings increase the understanding of the mechanisms by which GATA3 splicing mutations can cause HDR syndrome.

A Common Variant in the CDK8 Gene Is Associated with Sporadic Pituitary Adenomas in the Portuguese Population: A Case-Control Study.

The majority of pituitary adenomas occur in a sporadic context, and in the absence of known genetic predisposition. Three common variants at the NEBL (rs2359536), PCDH15 (rs10763170) and CDK8 (rs17083838) loci were previously associated with sporadic pituitary adenomas in the Han Chinese population, but these findings have not yet been replicated in any other population. The aim of this case-control study was to assess if these variants are associated with susceptibility to sporadic pituitary adenomas in the Portuguese population. Genotype and allele frequencies were determined in 570 cases and in 546 controls. The CDK8 rs17083838 minor allele (A allele) was significantly associated with sporadic pituitary adenomas, under an additive (odds ratio (OR) 1.73, 95% confidence interval (CI) 1.19-2.50, p = 0.004) and dominant (OR 1.82, 95% CI 1.24-2.68, p = 0.002) inheritance model. The NEBL rs2359536 and PCDH15 rs10763170 variants were not associated with the overall risk for the disease, although a borderline significant association was observed between the PCDH15 rs10763170 minor allele (T allele) and somatotrophinomas (dominant model, OR 1.55, 95% CI 1.02-2.35, p = 0.035). These findings suggest that the CDK8 rs17083838 variant, and possibly the PCDH15 rs10763170 variant, may increase susceptibility to sporadic pituitary adenomas in the Portuguese population.

Disorder of Sex Development Due to 17-Beta-Hydroxysteroid Dehydrogenase Type 3 Deficiency: A Case Report and Review of 70 Different HSD17B3 Mutations Reported in 239 Patients.

The 17-beta-hydroxysteroid dehydrogenase type 3 (17-ß-HSD3) enzyme converts androstenedione to testosterone and is encoded by the HSD17B3 gene. Homozygous or compound heterozygous HSD17B3 mutations block the synthesis of testosterone in the fetal testis, resulting in a Disorder of Sex Development (DSD). We describe a child raised as a female in whom the discovery of testes in the inguinal canals led to a genetic study by whole exome sequencing (WES) and to the identification of a compound heterozygous mutation of the HSD17B3 gene (c.608C>T, p.Ala203Val, and c.645A>T, p.Glu215Asp). Furthermore, we review all HSD17B3 mutations published so far in cases of 17-ß-HSD3 deficiency. A total of 70 different HSD17B3 mutations have so far been reported in 239 patients from 187 families. A total of 118 families had homozygous mutations, 63 had compound heterozygous mutations and six had undetermined genotypes. Mutations occurred in all 11 exons and were missense (55%), splice-site (29%), small deletions and insertions (7%), nonsense (5%), and multiple exon deletions and duplications (2%). Several mutations were recurrent and missense mutations at codon 80 and the splice-site mutation c.277+4A>T each represented 17% of all mutated alleles. These findings may be useful to those involved in the clinical management and genetic diagnosis of this disorder.

A Novel FGFR1 Missense Mutation in a Portuguese Family with Congenital Hypogonadotropic Hypogonadism.

Congenital hypogonadotropic hypogonadism (CHH) is a rare reproductive endocrine disorder characterized by complete or partial failure of pubertal development and infertility due to deficiency of the gonadotropin-releasing hormone (GnRH). CHH has a significant clinical heterogeneity and can be caused by mutations in over 30 genes. The aim of this study was to investigate the genetic defect in two siblings with CHH. A woman with CHH associated with anosmia and her brother with normosmic CHH were investigated by whole exome sequencing. The genetic studies revealed a novel heterozygous missense mutation in the Fibroblast Growth Factor Receptor 1 (FGFR1) gene (NM_023110.3: c.242T>C, p.Ile81Thr) in the affected siblings and in their unaffected father. The mutation affected a conserved amino acid within the first Ig-like domain (D1) of the protein, was predicted to be pathogenic by structure and sequence-based prediction methods, and was absent in ethnically matched controls. These were consistent with a critical role for the identified missense mutation in the activity of the FGFR1 protein. In conclusion, our identification of a novel missense mutation of the FGFR1 gene associated with a variable expression and incomplete penetrance of CHH extends the known mutational spectrum of this gene and may contribute to the understanding of the pathogenesis of CHH.

Iodine Supplementation in Pregnancy in an Iodine-Deficient Region: A Cross-Sectional Survey.

Iodine deficiency is a common problem in pregnant women and may have implications for maternal and child health. Iodine supplementation during pregnancy has been recommended by several scientific societies. We undertook a cross-sectional survey to assess the efficacy of these recommendations in a European iodine-deficient region. Urinary iodine concentrations (UIC) were determined in pregnant women before (n = 203) and after (n = 136) the implementation of guidelines for iodine supplementation in pregnancy. Iodine supplementation (200 µg/day) reduced the proportion of pregnant women with severe iodine deficiency (37.4% to 18.0%, p = 0.0002). The median UIC increased from 67.6 µg/L to 106.8 µg/L but remained below the recommended target level (>150 µg/L) for pregnant women. In conclusion, iodine supplementation in pregnant women improved iodine status in this iodine-deficient region but was insufficient to achieve recommended iodine levels in pregnancy. Additional measures, such as the adjustment of the dose or timing of supplementation, or universal salt iodization, may be needed.

Clinical, Genetic and Functional Characterization of a Novel AVPR2 Missense Mutation in a Woman with X-Linked Recessive Nephrogenic Diabetes Insipidus.

Nephrogenic diabetes insipidus (NDI) is a rare disorder characterized by renal unresponsiveness to the hormone vasopressin, leading to excretion of large volumes of diluted urine. Mutations in the arginine vasopressin receptor-2 (AVPR2) gene cause congenital NDI and have an X-linked recessive inheritance. The disorder affects almost exclusively male family members, but female carriers occasionally present partial phenotypes due to skewed inactivation of the X-chromosome. Here, we report a rare case of a woman affected with X-linked recessive NDI, presenting an average urinary output of 12 L/day. Clinical and biochemical studies showed incomplete responses to water deprivation and vasopressin stimulation tests. Genetic analyses revealed a novel heterozygous missense mutation (c.493G > C, p.Ala165Pro) in the AVPR2 gene. Using a combination of in-silico protein modeling with human cellular models and molecular phenotyping, we provide functional evidence for phenotypic effects. The mutation destabilizes the helical structure of the AVPR2 transmembrane domains and disrupts its plasma membrane localization and downstream intracellular signaling pathways upon activation with its agonist vasopressin. These defects lead to deficient aquaporin 2 (AQP2) membrane translocation, explaining the inability to concentrate urine in this patient.

Promoter Demethylation Upregulates STEAP1 Gene Expression in Human Prostate Cancer: In Vitro and In Silico Analysis.

The Six Transmembrane Epithelial Antigen of the Prostate (STEAP1) is an oncogene overexpressed in several human tumors, particularly in prostate cancer (PCa). However, the mechanisms involved in its overexpression remain unknown. It is well known that epigenetic modifications may result in abnormal gene expression patterns, contributing to tumor initiation and progression. Therefore, this study aimed to analyze the methylation pattern of the STEAP1 gene in PCa versus non-neoplastic cells. Bisulfite amplicon sequencing of the CpG island at the STEAP1 gene promoter showed a higher methylation level in non-neoplastic PNT1A prostate cells than in human PCa samples. Bioinformatic analysis of the GEO datasets also showed the STEAP1 gene promoter as being demethylated in human PCa, and a negative association with STEAP1 mRNA expression was observed. These results are supported by the treatment of non-neoplastic PNT1A cells with DNMT and HDAC inhibitors, which induced a significant increase in STEAP1 mRNA expression. In addition, the involvement of HDAC in the regulation of STEAP1 mRNA expression was corroborated by a negative association between STEAP1 mRNA expression and HDAC4,5,7 and 9 in human PCa. In conclusion, our work indicates that STEAP1 overexpression in PCa can be driven by the hypomethylation of STEAP1 gene promoter.

The Glucocorticoid Receptor Gene (NR3C1) 9ß SNP Is Associated with Posttraumatic Stress Disorder.

Posttraumatic stress disorder (PTSD) has been associated with glucocorticoid (GC) hypersensitivity. Although genetic factors account for 30-46% of the variance in PTSD, no associations have been found between single nucleotide polymorphisms (SNPs) of the GC receptor (GR) gene (NR3C1) and risk for this disorder. We studied the association of five SNPs in the GR gene (rs10052957, rs6189/rs6190, rs6195, rs41423247, and rs6198) and haplotypes with PTSD, in a group of Portuguese male war veterans (33 with lifetime PTSD, 28 without). To determine whether the 9ß SNP (rs6198) was associated with chronically altered cortisol levels, we evaluated hair cortisol concentrations (HCC) in a sample of 69 veterans' offspring. The 9ß variant (G allele) was significantly associated with lifetime PTSD under a dominant model of inheritance. The 9ß variant was also significantly associated with severity of current PTSD symptoms. The haplotype analysis revealed an association between a common haplotype comprising the 9ß risk allele and lifetime PTSD. Carriers of the 9ß risk allele had significantly lower HCC than non-carriers. We found the 9ß risk allele and a haplotype comprising the 9ß risk allele of the GR gene to be associated with PTSD in veterans. This 9ß risk allele was also associated with lower HCC in their offspring.

The Choroid Plexus Is an Alternative Source of Prolactin to the Rat Brain.

Among the more than 300 functions attributed to prolactin (PRL), this hormone has been associated with the induction of neurogenesis and differentiation of olfactory neurons especially during pregnancy, which are essential for maternal behavior. Despite the original hypothesis that PRL enters the central nervous system through a process mediated by PRL receptors (PRLR) at the choroid plexus (CP), recent data suggested that PRL transport into the brain is independent of its receptors. Based on transcriptomic data suggesting that PRL could be expressed in the CP, this work aimed to confirm PRL synthesis and secretion by CP epithelial cells (CPEC). The secretion of PRL and the distribution of PRLR in CPEC were further characterized using an in vitro model of the rat blood-cerebrospinal fluid barrier. RT-PCR analysis of PRL transcripts showed its presence in pregnant rat CP, in CPEC, and in the rat immortalized CP cell line, Z310. These observations were reinforced by immunocytochemistry staining of PRL in CPEC and Z310 cell cytoplasm. A 63-kDa immunoreactive PRL protein was detected by Western blot in CP protein extracts as well as in culture medium incubated with rat pituitary and samples of rat cerebrospinal fluid and serum. Positive immunocytochemistry staining of PRLR was present throughout the CPEC cytoplasm and in the apical and basal membrane of these cells. Altogether, our evidences suggest that CP is an alternative source of PRL to the brain, which might impact neurogenesis of olfactory neurons at the subventricular zone, given its proximity to the CP.

Age, Sex Hormones, and Circadian Rhythm Regulate the Expression of Amyloid-Beta Scavengers at the Choroid Plexus.

Accumulation of amyloid-beta (Aß) in the brain is thought to derive from the impairment of Aß clearance mechanisms rather than from its overproduction, which consequently contributes to the development of Alzheimer's disease. The choroid plexus epithelial cells constitute an important clearance route for Aß, either by facilitating its transport from the cerebrospinal fluid to the blood, or by synthesizing and secreting various proteins involved in Aß degradation. Impaired choroid plexus synthesis, secretion, and transport of these Aß-metabolizing enzymes have been therefore associated with the disruption of Aß homeostasis and amyloid load. Factors such as aging, female gender, and circadian rhythm disturbances are related to the decline of choroid plexus functions that may be involved in the modulation of Aß-clearance mechanisms. In this study, we investigated the impact of age, sex hormones, and circadian rhythm on the expression of Aß scavengers such as apolipoprotein J, gelsolin, and transthyretin at the rat choroid plexus. Our results demonstrated that mRNA expression and both intracellular and secreted protein levels of the studied Aß scavengers are age-, sex-, and circadian-dependent. These data suggest that the Aß-degradation and clearance pathways at the choroid plexus, mediated by the presence of Aß scavengers, might be compromised as a consequence of aging and circadian disturbances. These are important findings that enhance the understanding of Aß-clearance-regulating mechanisms at the blood-cerebrospinal fluid barrier.

Vitamin D Pathway Genetic Variation and Type 1 Diabetes: A Case-Control Association Study.

Vitamin D has immunomodulatory effects, and its deficiency has been implicated in the autoimmune process of type 1 diabetes. Serum vitamin D levels are influenced by variants in genes involved in the synthesis, transport, hydroxylation and degradation of vitamin D. The aim of this study was to assess if single nucleotide polymorphisms (SNPs) at the DHCR7 (rs12785878), GC (rs2282679), CYP2R1 (rs2060793) and CYP24A1 (rs6013897) loci are associated with type 1 diabetes in the Portuguese population. Genotype and allele frequencies were determined in 350 cases of type 1 diabetes and in 490 controls. The frequency of each SNP alone was not significantly different between patients and controls. However, the combined analysis of the four SNPs showed that minor alleles of these variants clustered more frequently in patients. The proportion of individuals with three or more minor alleles was significantly higher in patients than in controls (56.3% vs. 48.5; odds ratio (OR) 1.37; 95% confidence interval (CI) 1.04-1.81; p-value 0.027). These results suggest a cumulative effect of SNPs at the DHCR7, GC, CYP2R1 and CYP24A1 loci on the susceptibility to type 1 diabetes, due to the roles of these genes in the vitamin D metabolic pathway.

The Rhythmicity of Clock Genes is Disrupted in the Choroid Plexus of the APP/PS1 Mouse Model of Alzheimer's Disease.

BACKGROUND: The choroid plexus (CP), which constitutes the blood-cerebrospinal fluid barrier, was recently identified as an important component of the circadian clock system. OBJECTIVE: The fact that circadian rhythm disruption is closely associated to Alzheimer's disease (AD) led us to investigate whether AD pathology can contribute to disturbances of the circadian clock in the CP. METHODS: For this purpose, we evaluated the expression of core-clock genes at different time points, in 6- and 12-month-old female and male APP/PS1 mouse models of AD. In addition, we also assessed the effect of melatonin pre-treatment in vitro before amyloid-ß stimulus in the daily pattern of brain and muscle Arnt-like protein 1 (Bmal1) expression. RESULTS: Our results showed a dysregulation of circadian rhythmicity of Bmal1 expression in female and male APP/PS1 transgenic 12-month-old mice and of Period 2 (Per2) expression in male mice. In addition, a significant circadian pattern of Bmal1 was measured the intermittent melatonin pre-treatment group, showing that melatonin can reset the CP circadian clock. CONCLUSION: These results demonstrated a connection between AD and the disruption of circadian rhythm in the CP, representing an attractive target for disease prevention and/or treatment.

Hypoparathyroidism, deafness, and renal dysplasia syndrome: 20 Years after the identification of the first GATA3 mutations.

The hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome is an autosomal dominant disorder caused by heterozygous mutations of the GATA3 gene. In the last 20 years, since the identification of the genetic cause of the HDR syndrome, GATA3 mutations have been reported in 124 families (177 patients). The clinical aspects and molecular genetics of the HDR syndrome are reviewed here together with the reported mutations and phenotypes. Reported mutations consist of 40% frameshift deletions or insertions, 23% missense mutations, 14% nonsense mutations, 6% splice-site mutations, 1% in-frame deletions or insertions, 15% whole-gene deletions, and 1% whole-gene duplication. Missense mutations were found to cluster in the regions encoding the two GATA3 zinc-finger domains. Patients showed great clinical variability and the penetrance of each HDR defect increased with age. The most frequently observed abnormality was deafness (93%), followed by hypoparathyroidism (87%) and renal defects (61%). The mean age of diagnosis of HDR was 15.3, 7.5, and 14.0 years, respectively. However, patients with whole-gene deletions and protein-truncating mutations were diagnosed earlier than patients with missense mutations.

Maturity-Onset Diabetes of the Young (MODY) in Portugal: Novel GCK, HNFA1 and HNFA4 Mutations.

Maturity-onset diabetes of the young (MODY) is a frequently misdiagnosed type of diabetes, which is characterized by early onset, autosomal dominant inheritance, and absence of insulin dependence. The most frequent subtypes are due to mutations of the GCK (MODY 2), HNF1A (MODY 3), and HNF4A (MODY 1) genes. We undertook the first multicenter genetic study of MODY in the Portuguese population. The GCK, HNF1A, and HNF4A genes were sequenced in 46 unrelated patients that had at least two of the three classical clinical criteria for MODY (age at diagnosis, family history, and clinical presentation). The functional consequences of the mutations were predicted by bioinformatics analysis. Mutations were identified in 23 (50%) families. Twelve families had mutations in the GCK gene, eight in the HNF1A gene, and three in the HNF4A gene. These included seven novel mutations (GCK c.494T>C, GCK c.563C>G, HNF1A c.1623G>A, HNF1A c.1729C>G, HNF4A c.68delG, HNF4A c.422G>C, HNF4A c.602A>C). Mutation-positive patients were younger at the time of diagnosis when compared to mutation-negative patients (14.3 vs. 23.0 years, p = 0.011). This study further expands the spectrum of known mutations associated with MODY, and may contribute to a better understanding of this type of diabetes and a more personalized clinical management of affected individuals.

Vascular mechanisms of testosterone: The non-genomic point of view.

Testosterone (T) is the predominant endogenous androgen in the bloodstream. At the vascular level, T presents genomic and non-genomic effects, and both effects may overlap. The genomic actions assume that androgens can freely cross the plasma membrane of target cells and bind to nuclear androgen receptors, inducing gene transcription and protein synthesis. The non-genomic effects have a more rapid onset and may be related to the interaction with protein/receptor/ion channels of the plasma membrane. The key T effect at the vascular level is vasorelaxation, which is primarily due to its rapid effect. Thus, the main purpose of this review is to discuss the T non-genomic effects at the vascular level and the molecular pathways involved in its vasodilator effect observed in in vivo and in vitro studies. In this sense, the nuclear receptor activation, the influence of vascular endothelium and the activation or inhibition of ion channels (potassium and calcium channels, respectively) will be reviewed regarding all the data that corroborated or not. Moreover, this review also provides a brief update on the association of T with the risk factors for cardiovascular diseases, namely metabolic syndrome, type 2 diabetes mellitus, obesity, atherosclerosis, dyslipidaemia, and hypertension. In summary, in this paper we consider the non-genomic vascular mode of action of androgen in physiological conditions and the main risk factors for cardiovascular diseases.

Vascular Pathways of Testosterone: Clinical Implications.

Cardiovascular diseases (CVD) are one of the leading causes of death worldwide. Testosterone (T) is an important sex hormone that triggers several genomic and non-genomic pathways, leading to improvements of several cardiovascular risk factors and quality of life in men. At the vascular level, the key effect of T is the vasorelaxation. This review discusses the molecular pathways and clinical implications of T in the vascular system. Firstly, the mechanisms involved in the T vasodilator effect will be presented. Then, it will be discussed the association of T with the main risks for CVD, namely metabolic syndrome, type 2 diabetes mellitus, obesity, atherosclerosis, dyslipidaemia and hypertension. Several studies have shown a correlation between low T levels and an increased prevalence of several CVD. These observations suggest that T has beneficial effects on the cardiovascular system and that testosterone replacement therapy may become a therapeutic reality for some of these disorders. Graphical abstract .