RASopathy Cohort of Patients Enrolled in a Brazilian Reference Center for Rare Diseases: A Novel Familial LZTR1 Variant and Recurrent Mutations.

Purpose: Noonan syndrome and related disorders are genetic conditions affecting 1:1000-2000 individuals. Variants causing hyperactivation of the RAS/MAPK pathway lead to phenotypic overlap between syndromes, in addition to an increased risk of pediatric tumors. DNA sequencing methods have been optimized to provide a molecular diagnosis for clinical and genetic heterogeneity conditions. This work aimed to investigate the genetic basis in RASopathy patients through Next Generation Sequencing in a Reference Center for Rare Diseases (IFF/Fiocruz) and implement the precision medicine at a public health institute in Brazil. Patients and Methods: This study comprises 26 cases with clinical suspicion of RASopathies. Sanger sequencing was used to screen variants in exons usually affected in the PTPN11 and HRAS genes for cases with clinical features of Noonan and Costello syndrome, respectively. Posteriorly, negative and new cases with clinical suspicion of RASopathy were analyzed by clinical or whole-exome sequencing. Results: Molecular analysis revealed recurrent variants and a novel LZTR1 missense variant: 24 unrelated individuals with pathogenic variants [PTPN11(11), NF1(2), SOS1(2), SHOC2(2), HRAS(1), BRAF(1), LZTR (1), RAF1(1), KRAS(1), RIT1(1), a patient with co-occurrence of PTPN11 and NF1 mutations (1)]; familial cases carrying a known pathogenic variant in PTPN11 (mother-two children), and a previously undescribed paternally inherited variant in LZTR1. The comparative modeling analysis of the novel LZTR1 variant p.Pro225Leu showed local and global changes in the secondary and tertiary structures, showing a decrease of about 1% in the ß-sheet content. Furthermore, evolutionary conservation indicated that Pro225 is in a highly conserved region, as observed for known dominant pathogenic variants in this protein. Conclusion: Bringing precision medicine through NGS towards congenital syndromes promotes a better understanding of complex clinical and/or undiagnosed cases. The National Policy for Rare Diseases in Brazil emphasizes the importance of incorporating and optimizing diagnostic methodologies in the Unified Brazilian Health System (SUS). Therefore, this work is an important step for the NGS inclusion in diagnostic genetic routine in the public health system.

High-Resolution Melting Analysis for Rapid Detection of Mutations in Patients with FGFR3-Related Skeletal Dysplasias.

Background: Mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are related to skeletal dysplasias (SDs): acondroplasia (ACH), hypochodroplasia (HCH) and type I (TDI) and II (TDII) tanatophoric dysplasias. This study was designed to standardize and implement a high-resolution melting (HRM) technique to identify mutations in patients with these phenotypes. Methods: Initially, FGFR3 gene segments from 84 patients were PCR amplified and subjected to Sanger sequencing. Samples from 29 patients positive for mutations were analyzed by HRM. Results: Twelve of the patients FGFR3 mutations had ACH (six g.16081 G > A, three g.16081 G > C and three g.16081 G > A + g.16002 C > T); thirteen of patients with HCH had FGFR3 mutations (eight g.17333 C > A, five g.17333 C > G and five were negative); and four patients with DTI had FGFR3 mutations (three g.13526 C > T and one g.16051G > T and two patients with DTII (presented mutation g.17852 A > G). When analyzing the four SDs altogether, an overlap of the dissociation curves was observed, making genotyping difficult. When analyzed separately, however, the HRM analysis method proved to be efficient for discriminating among the mutations for each SD type, except for those patients carrying additional polymorphism concomitant to the recurrent mutation. Conclusion: We conclude that for recurrent mutations in the FGFR3 gene, that the HRM technique can be used as a faster, reliable and less expensive genotyping routine for the diagnosis of these pathologies than Sanger sequencing.

Identification of Novel and Recurrent RMRP Variants in a Series of Brazilian Patients with Cartilage-Hair Hypoplasia: McKusick Syndrome.

Cartilage-hair hypoplasia syndrome (CHH) is an autosomal recessive disorder caused by pathogenic variants of the RMRP gene and characterized by metaphyseal bone dysplasia associated with hypotrichosis, immunodeficiency, and predisposition to malignancy. However, the genotype-phenotype correlation in CHH is not well understood. Here, we report a single country cohort of 23 Brazilian patients with clinical and radiological features consistent with CHH. We found 23 different pathogenic variants in the RMRP gene - 12 novel and 11 previously described in the literature. Interestingly, the most frequent Finnish pathogenic variant related to CHH (g.71A>G) was not found in our cohort. In contrast, more than 50% of the patients carried the rare g.196C>T variant suggesting a possible founder effect in the Brazilian population. In silico analysis showed that pathogenic variants occurred either in the regions conserved in mammalian species or within essential domains for the ribonucleoprotein complex. Pathogenicity prediction studies can improve the understanding of how these variants affect RNA.

Pregnancy-induced hypertension, preterm birth, and cord blood adipokine levels.

Maternal hypertension may alter physiological parameters, dysregulating the release of hormones such as adipokines, thus influencing the fetal growth course. This study investigated whether hypertensive disorders of pregnancy alter cord blood adipokine levels and correlate these with anthropometric parameters in preterm infants. This is a prospective cohort study with pregnant women < 37-week gestation with and without hypertension and their offspring. Cord blood leptin, adiponectin, and ghrelin were analyzed by LUMINEX®. These adipokines were compared between the groups exposed or not to gestational hypertension using non-parametric statistical tests. The hypertensive pregnancies had significantly higher cord blood leptin (1.00 (IQR 0.67-1.20 ng/mL)) and adiponectin (18.52 (IQR 17.52-25.13 µg/mL)) levels than those without hypertension (0.07 (IQR 0.06-0.08 ng/mL) and 8.13 (IQR 6.50-8.68 µg/mL), respectively, p < 0.0001). The adipokine levels were higher in AGA and SGA infants in the exposed group for both moderate and late preterm. SGA had significantly higher ghrelin levels than the AGA infants. Ghrelin levels were negatively correlated with birth weight (r = - 0.613, p < 0.001), birth length (r = - 0.510, p < 0.001), head circumference (- 0.346, p < 0.002), and gestational age (r = - 0.612, p < 0.001).Conclusions: Our findings demonstrate an increase in adipokine levels in the cord blood of preterm newborn infants exposed to maternal hypertension. What is Known: • Clinical evidence suggests that concentration of the serum adipokines may be affected by risk of hypertension in both adults and pregnant women. • Maternal profile as hypertension alters intrauterine environment and could affect the function of fetal metabolism, impairing fetal growth. What is New: • Gestational hypertension modifies the adipokine profile, with higher rates already present at birth in cord blood samples. • Within the hypertensive group and stratifying for gestation age, ghrelin concentrations were higher in SGA newborns, both in the moderate and late preterm, compared with AGA newborns.

miRNA Genetic Variants Alter Their Secondary Structure and Expression in Patients With RASopathies Syndromes.

RASopathies are a group of rare genetic diseases caused by germline mutations in genes involved in the RAS-mitogen-activated protein kinase (RAS-MAPK) pathway. Whole-exome sequencing (WES) is a powerful approach for identifying new variants in coding and noncoding DNA sequences, including miRNAs. miRNAs are fine-tuning negative regulators of gene expression. The presence of variants in miRNAs could lead to malfunctions of regulation, resulting in diseases. Here, we identified 41 variants in mature miRNAs through WES analysis in five patients with previous clinical diagnosis of RASopathies syndromes. The pathways, biological processes, and diseases that were over-represented among the target genes of the mature miRNAs harboring variants included the RAS, MAPK, RAP1, and PIK3-Akt signaling pathways, neuronal differentiation, neurogenesis and nervous system development, congenital cardiac defects (hypertrophic cardiomyopathy, dilated cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy), and the phenotypes and syndromes of RASopathies (Noonan syndrome, Legius syndrome, Costello syndrome, Cafe au lait spots multiple, subaortic stenosis, pulmonary valve stenosis, and LEOPARD syndrome). Furthermore, eight selected variants in nine mature miRNAs (hsa-miR-1304, hsa-miR-146a, hsa-miR-196a2, hsa-miR-499a/hsa-miR-499b, hsa-miR-449b, hsa-miR-548l, hsa-miR-575, and hsa-miR-593) may have caused alterations in the secondary structures of miRNA precursor. Selected miRNAs containing variants such as hsa-miR-146a-3p, hsa-miR-196a-3p, hsa-miR-548l, hsa-miR-449b-5p, hsa-miR-575, and hsa-miR499a-3p could regulate classical genes associated with Rasopathies and RAS-MAPK pathways, contributing to modify the expression pattern of miRNAs in patients. RT-qPCR expression analysis revealed four differentially expressed miRNAs that were downregulated: miRNA-146a-3p in P1, P2, P3, P4, and P5, miR-1304-3p in P2, P3, P4, and P5, miR-196a2-3p in P3, and miR-499b-5p in P1. miR-499a-3p was upregulated in P1, P3, and P5. These results indicate that miRNAs show different expression patterns when these variants are present in patients. Therefore, this study characterized the role of miRNAs harboring variants related to RASopathies for the first time and indicated the possible implications of these variants for phenotypes of RASopathies such as congenital cardiac defects and cardio-cerebrovascular diseases. The expression and existence of miRNA variants may be used in the study of biomarkers of the RASopathies.

Novel and Recurrent Mutations in the FGFR3 Gene and Double Heterozygosity Cases in a Cohort of Brazilian Patients with Skeletal Dysplasia.

Mutations in the fibroblast growth factor receptor 3 gene (FGFR3) cause achondroplasia (ACH), hypochondroplasia (HCH), and thanatophoric dysplasia types I and II (TDI/TDII). In this study, we performed a genetic study of 123 Brazilian patients with these phenotypes. Mutation hotspots of the FGFR3 gene were PCR amplified and sequenced. All cases had recurrent mutations related to ACH, HCH, TDI or TDII, except for 2 patients. One of them had a classical TDI phenotype but a typical ACH mutation (c.1138G>A) in combination with a novel c.1130T>C mutation predicted as being pathogenic. The presence of the second c.1130T>C mutation likely explained the more severe phenotype. Another atypical patient presented with a compound phenotype that resulted from a combination of ACH and X-linked spondyloepiphyseal dysplasia tarda (OMIM 313400). Next-generation sequencing of this patient's DNA showed double heterozygosity for a typical de novo ACH c.1138G>A mutation and a maternally inherited TRAPPC2 c.6del mutation. All mutations were confirmed by Sanger sequencing. A pilot study using high-resolution melting (HRM) technique was also performed to confirm several mutations identified through sequencing. We concluded that for recurrent FGFR3 mutations, HRM can be used as a faster, reliable, and less expensive genotyping test than Sanger sequencing.

Conserved transcription factor binding sites suggest an activator basal promoter and a distal inhibitor in the galanin gene promoter in mouse ES cells.

Galanin and its receptors have been shown to be expressed in undifferentiated mouse embryonic stem (ES) cells through transcriptome and proteomic analyses. Although transcriptional regulation of galanin has been extensively studied, the regulatory proteins that mediate galanin expression in mouse ES cells have not yet been determined. Through sequence alignments, we have found a high degree of similarity between mouse and human galanin upstream sequences at -146 bp/+69 bp (proximal region) and -2,408 bp/-2,186 bp (distal region). These regions could be recognized by ES cell nuclear proteins, and EMSA analysis suggests a specific functionality. Analysis of the proximal region (PR) using EMSA and ChIP assays showed that the CREB protein interacts with the galanin promoter both in vitro and in vivo. Additional EMSA analysis revealed that an SP1 consensus site mediated protein-DNA complex formation. Reporter assays showed that CREB is an activator of galanin expression and works cooperatively with SP1. Furthermore, analysis of the distal region (DR) using EMSA assays demonstrated that both HOX-F and PAX 4/6 consensus sites mediated protein-DNA complex formation, and both sites inhibited luciferase activity in reporter assays. These data together suggest that CRE and SP1 act as activators at the basal promoter, while HOX-F and PAX 4/6 act as silencers of transcription. The interplay of these transcription factors (TF) may drive regulated galanin expression in mouse ES cells.

Characterization of a Smad motif similar to Drosophila mad in the mouse Msx 1 promoter.

Mouse Msx 1 gene, orthologous of the Drosophila msh, is involved in several developmental processes. BMP family members are major proteins in the regulation of Msx 1 expression. BMP signaling activates Smad 1/5/8 proteins, which associate to Smad 4 before translocating to the nucleus. Analysis of Msx 1 promoter revealed the presence of three elements similar to the consensus established for Mad, the Smad 1 Drosophila counterpart. Notably, such an element was identified in an enhancer important for Msx 1 regulation. Gel shift analysis demonstrated that proteins from 13.5 dpc embryo associate to this enhancer. Remarkably, supershift assays showed that Smad proteins are present in the complex. Purified Smad 1 and 4 also bind to this fragment. We demonstrate that functional binding sites in this enhancer are confined to the Mad motif and flanking region. Our data suggest that this Mad motif may be functional in response to BMP signaling.