Molecular characterization of Chinese patients with small bowel adenocarcinoma.

PURPOSE: Small bowel adenocarcinoma (SBA) is a rare malignancy of the gastrointestinal tract, and its unique location within the small intestine presents difficulties in obtaining tissue samples from the lesions. This limitation hinders the research and development of effective clinical treatment methods. Circulating tumor DNA (ctDNA) analysis holds promise as an alternative approach for investigating SBA and guiding treatment decisions, thereby improving the prognosis of SBA. METHODS: Between January 2017 and August 2021, a total of 336 tissue or plasma samples were obtained and the corresponding mutation status in tissue or blood was evaluated with NGS. RESULTS AND CONCLUSIONS: The study found that in SBA tissues, the most commonly alternated genes were TP53, KRAS, and APC, and the most frequently affected pathways were RTK-RAS-MAPK, TP53, and WNT. Notably, the RTK-RAS-MAPK pathway was identified as a potential biomarker that could be targeted for treatment. Then, we validated the gene mutation profiling of ctDNA extracted from SBA patients exhibited the same characteristics as tissue samples for the first time. Subsequently, we applied ctDNA analysis on a terminal-stage patient who had shown no response to previous chemotherapy. After detecting alterations in the RTK-RAS-MAPK pathway in the ctDNA, the patient was treated with MEK + EGFR inhibitors and achieved a tumor shrinkage rate of 76.33%. Our study utilized the largest Chinese SBA cohort to uncover the molecular characteristics of this disease, which might facilitate clinical decision making for SBA patients.

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.

The recurrent homozygous translation start site variant in CCDC134 in an individual with severe osteogenesis imperfecta of non-Morrocan ancestry.

Osteogenesis imperfecta (OI) is a rare low-bone mass skeletal Mendelian disorder characterized by bone fragility leading to bone fractures, with deformities and stunted growth in the more severe phenotypes. Other common, nonskeletal findings include blue sclerae and dentinogenesis imperfecta. It is caused mainly by quantitative or structural defects in type I collagen, although dysregulation of different signaling pathways that play a role in bone morphogenesis has been described to be associated with a small fraction of individuals with OI. Recently, a homozygous variant in the translation start site of CCDC134, showing increased activation of the RAS/MAPK signaling pathway, has been reported in three families of Moroccan origin with a severe, deforming form of OI. We report on a 9-year-old Brazilian boy, harboring the same homozygous variant in CCDC134, also presenting severe bone involvement. This report contributes to the phenotypic delineation of this novel autosomal recessive form of OI, which presents with high prevalence of nonunion fractures considered rare events in OI in general. In addition, it expands the phenotype to include base skull anomalies, potentially leading to serious complications, as seen in severe forms of OI. A poor response to bisphosphonate therapy was observed in these individuals. As the variant in CCDC134 leads to dysregulation of the RAS/MAPK signaling pathway, drugs targeted to this pathway could be an alternative to achieve a better management of these individuals.

Etiology and Treatment of Growth Delay in Noonan Syndrome.

Noonan syndrome is characterized by multiple phenotypic features, including growth retardation, which represents the main cause of consultation to the clinician. Longitudinal growth during childhood and adolescence depends on several factors, among them an intact somatotrophic axis, which is characterized by an adequate growth hormone (GH) secretion by the pituitary, subsequent binding to its receptor, proper function of the post-receptor signaling pathway for this hormone (JAK-STAT5b and RAS/MAPK), and ultimately by the production of its main effector, insulin like growth factor 1 (IGF-1). Several studies regarding the function of the somatotrophic axis in patients with Noonan syndrome and data from murine models, suggest that partial GH insensitivity at a post-receptor level, as well as possible derangements in the RAS/MAPK pathway, are the most likely causes for the growth failure in these patients. Treatment with recombinant human growth hormone (rhGH) has been used extensively to promote linear growth in these patients. Numerous treatment protocols have been employed so far, but the published studies are quite heterogeneous regarding patient selection, length of treatment, and dose of rhGH utilized, so the true benefit of GH therapy is somewhat difficult to establish. This review will discuss the possible etiologies for the growth delay, as well as the outcomes following rhGH treatment in patients with Noonan syndrome.

Phenotype-genotype analysis of 242 individuals with RASopathies: 18-year experience of a tertiary center in Brazil.

We report the clinical and molecular data of a large cohort comprising 242 individuals with RASopathies, from a single Tertiary Center in Brazil, the largest study from Latin America. Noonan syndrome represented 76% of the subjects, with heterozygous variants in nine different genes, mainly PTPN11, SOS1, RAF1, LZTR1, and RIT1, detected by Sanger and next-generation sequencing. The latter was applied to 126 individuals, with a positive yield of 63% in genes of the RAS/MAPK cascade. We present evidence that there are some allelic differences in PTPN11 across distinct populations. We highlight the clinical aspects that pose more medical concerns, such as the cardiac anomalies, bleeding diathesis and proliferative lesions. The genotype-phenotype analysis between the RASopathies showed statistically significant differences in some cardinal features, such as craniofacial and cardiac anomalies, the latter also statistically significant for different genes in Noonan syndrome. We present two individuals with a Noonan syndrome phenotype, one with an atypical, structural cardiac defect, harboring variants in genes mainly associated with isolated hypertrophic cardiomyopathy and discuss the role of these variants in their phenotype.

Noonan syndrome with multiple Giant cell lesions, management and treatment with surgery and interferon alpha-2a therapy: Case report.

We report the case of a 14-year-old girl that was referred to the maxillo facial surgery unit at age 11 years because she exhibited swelling in the right side of her maxilla and right mandible. After a conservative surgery, she started with interferon alpha-2a to avoid recurrence. She has remained in treatment with successful results during her follow up. Considerable reduction of both maxilla and mandible lesions and bone fill have been documented. In addition, her clinical history and phenotype were suggestive of Noonan syndrome. She has short stature, broad and short neck; hypertelorism (increased distance between the eyes); downslanting palpebral fissures; sparse eyebrows and eyelashes; posteriorly rotated ears with fleshy lobes; follicular keratosis over the face, and developmental delay. Her karyotype was 46, XX. Molecular analysis of RAS/MAPK pathway genes showed a SOS1 amino acid substitution of arginine to lysine at position 552 (p.R552K). This case presents the infrequent condition of Noonan syndrome with multiple giant cell lesions (NS/MGCL) that would be the first patient as far as we know treated with surgery and interferon alpha-2a for her giant cell lesions.

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.

Copy number variants of Ras/MAPK pathway genes in patients with isolated cryptorchidism.

Cryptorchidism is the most common congenital disorder in boys, but the cause for most cases remains unknown. Patients with Noonan Syndrome are characterized by a typical face, growth retardation, congenital heart defects, learning disabilities and cryptorchidism. Copy number variations of Ras/MAPK pathway genes are unusual in patients with several clinical features of Noonan Syndrome; however, they have not been studied in patients with only one feature of this condition, such as cryptorchidism. Our aim was to determine whether patients with isolated cryptorchidism exhibit Ras/MAPK pathway gene copy number variations (CNVs). Fifty-nine patients with isolated cryptorchidism and negative for mutations in genes associated with Noonan Syndrome were recruited. Determination of Ras/MAPK pathway gene CNVs was performed by Comparative Genome Hybridization array. A CNV was identified in two individuals, a ~175 kb microduplication at 3p25.2, partially including RAF1. A similar RAF1 microduplication has been observed in a patient with testicular aplasia. This suggests that some patients with isolated cryptorchidism may harbor Ras/MAPK pathway gene CNVs.

Nutritional aspects of Noonan syndrome and Noonan-related disorders.

Rasopathies are a group of rare disorders characterized by neurocardiofaciocutaneous involvement, and caused by mutations in several genes of the RAS/MAPK pathway. In the present study, we characterized growth parameters, body composition, and nutritional aspects of children and adults (n = 62) affected by these disorders, mainly Noonan syndrome, using an indirect method-anthropometry-and a 24-hr recall questionnaire. The growth parameters in our cohort showed short stature, especially in individuals with RAF1 and SHOC2 mutations, lower obesity rates compared to the control population, and BMI scores highest in individuals with BRAF mutations and lowest in individuals with SHOC2. Body composition showed a compromise in the upper arm muscle circumference, with a statistically significant difference in the z-score of triceps skinfold (P = 0.0204) and upper arm fat area (P = 0.0388) between BRAF and SHOC2 groups and in the z-score of triceps skinfold between RAF1 and SHOC2 (P = 0.0218). The pattern of macronutrient consumption was similar to the control population. Our study is the first to address body composition in RASopathy individuals and the data indicate a compromise not only in adipose tissue, but also in muscle mass. Studies using different techniques, such as dual-energy X-ray absorptiometry or imaging studies, which give a more precise delineation of fat and non-fat mass, are required to confirm our results, ultimately causing an impact on management strategies. © 2016 Wiley Periodicals, Inc.

Further evidence of the importance of RIT1 in Noonan syndrome.

Noonan syndrome (NS) is an autosomal dominant disorder consisting of short stature, short and/or webbed neck, distinctive facial features, cardiac abnormalities, cryptorchidism, and coagulation defects. NS exhibits genetic heterogeneity, associated with mutated genes that participate in RAS-mitogen-activated protein kinase signal transduction. Recently, a new gene (RIT1) was discovered as the causative gene in 17 of 180 Japanese individuals who were negative for the previously known genes for NS and were studied using exome sequencing (four patients), followed by Sanger sequencing (13 patients). The present study used the same technique in 70 Brazilian patients with NS and identified six with RIT1 missense mutations. Thus, we confirm that RIT1 is responsible for approximately 10% of the patients negative for mutations in the previously known genes. The phenotype includes a high frequency of high birth weight, relative macrocephaly, left ventricular hypertrophy, and ectodermal findings, such as curly hair, hyperpigmentation, and wrinkled palms and soles. Short stature and pectus deformity were less frequent. The majority of patients with a RIT1 mutation did not show apparent intellectual disability. Because of the relatively high frequency of mutations in RIT1 among patients with NS and its occurrence in different populations, we suggest that it should be added to the list of genes included in panels for the molecular diagnosis of NS through targeted next-generation sequencing.