Guilherme Lopes Yamamoto

Somatic mosaic activating mutations in PIK3CA cause CLOVES syndrome.

Congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies (CLOVES) is a sporadically occurring, nonhereditary disorder characterized by asymmetric somatic hypertrophy and anomalies in multiple organs. We hypothesized that CLOVES syndrome would be caused by a somatic mutation arising during early embryonic development. Therefore, we employed massively parallel sequencing to search for somatic mosaic mutations in fresh, frozen, or fixed archival tissue from six affected individuals. We identified mutations in PIK3CA in all six individuals, and mutant allele frequencies ranged from 3% to 30% in affected tissue from multiple embryonic lineages. Interestingly, these same mutations have been identified in cancer cells, in which they increase phosphoinositide-3-kinase activity. We conclude that CLOVES is caused by postzygotic activating mutations in PIK3CA. The application of similar sequencing strategies will probably identify additional genetic causes for sporadically occurring, nonheritable malformations.

Rare variants in SOS2 and LZTR1 are associated with Noonan syndrome

Background Noonan syndrome is an autosomal dominant, multisystemic disorder caused by dysregulation of the RAS/mitogen activated protein kinase (MAPK) pathway. Heterozygous, pathogenic variants in 11 known genes account for approximately 80% of cases. The identification of novel genes associated with Noonan syndrome has become increasingly challenging, since they might be responsible for very small fractions of the cases. Methods A cohort of 50 Brazilian probands negative for pathogenic variants in the known genes associated with Noonan syndrome was tested through whole-exome sequencing along with the relatives in the familial cases. Families from the USA and Poland with mutations in the newly identified genes were included subsequently. Results We identified rare, segregating or de novo missense variants in SOS2 and LZTR1 in 4% and 8%, respectively, of the 50 Brazilian probands. SOS2 and LZTR1 variants were also found to segregate in one American and one Polish family. Notably, SOS2 variants were identified in patients with marked ectodermal involvement, similar to patients with SOS1 mutations. Conclusions We identified two novel genes, SOS2 and LZTR1, associated with Noonan syndrome, thereby expanding the molecular spectrum of RASopathies. Mutations in these genes are responsible for approximately 3% of all patients with Noonan syndrome. While SOS2 is a natural candidate, because of its homology with SOS1, the functional role of LZTR1 in the RAS/MAPK pathway is not known, and it could not have been identified without the large pedigrees. Additional functional studies are needed to elucidate the role of LZTR1 in RAS/MAPK signalling and in the pathogenesis of Noonan syndrome.

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.

Exomic variants of an elderly cohort of Brazilians in the ABraOM database

Brazilians are highly admixed with ancestry from Europe, Africa, America, and Asia and yet still underrepresented in genomic databanks. We hereby present a collection of exomic variants from 609 elderly Brazilians in a census‐based cohort (SABE609) with comprehensive phenotyping. Variants were deposited in ABraOM (Online Archive of Brazilian Mutations), a Web‐based public database. Population representative phenotype and genotype repositories are essential for variant interpretation through allele frequency filtering; since elderly individuals are less likely to harbor pathogenic mutations for early‐ and adult‐onset diseases, such variant databases are of great interest. Among the over 2.3 million variants from the present cohort, 1,282,008 were high‐confidence calls. Importantly, 207,621 variants were absent from major public databases. We found 9,791 potential loss‐of‐function variants with about 300 mutations per individual. Pathogenic variants on clinically relevant genes (ACMG) were observed in 1.15% of the individuals and were correlated with clinical phenotype. We conducted incidence estimation for prevalent recessive disorders based upon heterozygous frequency and concluded that it relies on appropriate pathogenicity assertion. These observations illustrate the relevance of collecting demographic data from diverse, poorly characterized populations. Census‐based datasets of aged individuals with comprehensive phenotyping are an invaluable resource toward the improved understanding of variant pathogenicity.

Expansion of CD4+ CD25+ Foxp3+ T cells by bone marrow-derived dendritic cells

Dendritic cells (DCs) are the most important antigen‐presenting cells of the immune system and have a crucial role in T‐lymphocyte activation and adaptive immunity initiation. However, DCs have also been implicated in maintaining immunological tolerance. In this study, we evaluated changes in the CD4+ CD25+ Foxp3+ T‐cell population after co‐culture of lymph node cells from BALB/c mice with syngeneic bone marrow‐derived DCs. Our results showed an increase in CD4+ CD25+ Foxp3+ T cells after co‐culture which occurred regardless of the activation state of DCs and the presence of allogeneic apoptotic cells; however, it was greater when DCs were immature and were pulsed with the alloantigen. Interestingly, syngeneic apoptotic thymocytes were not as efficient as allogeneic apoptotic cells in expanding the CD4+ CD25+ Foxp3+ T‐cell population. In all experimental settings, DCs produced high amounts of transforming growth factor (TGF)‐β. The presence of allogeneic apoptotic cells induced interleukin (IL)‐2 production in immature and mature DC cultures. This cytokine was also detected in the supernatants under all experimental conditions and enhanced when immature DCs were pulsed with the alloantigen. CD4+ CD25+ Foxp3+ T‐cell expansion during co‐culture of lymph node cells with DCs strongly suggested that the presence of alloantigen enhanced the number of regulatory T cells (Tregs) in vitro. Our data also suggest a role for both TGF‐β and IL‐2 in the augmentation of the CD4+ CD25+ Foxp3+ population.

Milder course in Duchenne patients with nonsense mutations and no muscle dystrophin

Duchenne muscular dystrophy (DMD), a severe and lethal condition, is caused by the absence of muscle dystrophin. Therapeutic trials aiming at the amelioration of muscle function have been targeting the production of muscle dystrophin in affected Duchenne patients. However, how much dystrophin is required to rescue the DMD phenotype remains an open question. We have previously identified two exceptional golden retriever muscular dystrophy (GRMD) dogs with a milder course despite the total absence of muscle dystrophin. Here we report two unusual patients carrying nonsense mutations in the DMD gene and dystrophin deficiency but with an unexpectedly mild phenotype. Three reported polymorphisms, respectively in genes LTBP4, SPP1 and ACTN3 were excluded as possible DMD genetic modifiers in our patients. Finding the mechanisms that protect some rare patients and dogs from the deleterious effect of absent muscle dystrophin is of utmost importance and may lead to new avenues for treatment. Importantly, these observations indicate that it is possible to have a functional large muscle even without dystrophin.

Comprehensive assessment of the disputed RET Y791F variant shows no association with medullary thyroid carcinoma susceptibility

Accurate interpretation of germline mutations of the rearranged during transfection (RET) proto-oncogene is vital for the proper recommendation of preventive thyroidectomy in medullary thyroid carcinoma (MTC)-prone carriers. To gain information regarding the most disputed variant of RET, ATA-A Y791F, we sequenced blood DNA samples from a cohort of 2904 cancer-free elderly individuals (1261 via Sanger sequencing and 1643 via whole-exome/genome sequencing). We also accessed the exome sequences of an additional 8069 individuals from non-cancer-related laboratories and public databanks as well as genetic results from the Catalogue of Somatic Mutations in Cancer (COSMIC) project. The mean allelic frequency observed in the controls was 0.0031, with higher occurrences in Central European populations (0.006/0.008). The prevalence of RET Y791F in the control databases was extremely high compared with the 40 known RET pathogenic mutations (P=0.00003), while no somatic occurrence has been reported in tumours. In this study, we report new, unrelated Brazilian individuals with germline RET Y791F-only: two tumour-free elderly controls; two individuals with sporadic MTC whose Y791F-carrying relatives did not show any evidence of tumours; and a 74-year-old phaeochromocytoma patient without MTC. Furthermore, we showed that the co-occurrence of Y791F with the strong RET C634Y mutation explains the aggressive MTC phenotypes observed in a large affected family that was initially reported as Y791F-only. Our literature review revealed that limited analyses have led to the misclassification of RET Y791F as a probable pathogenic variant and, consequently, to the occurrence of unnecessary thyroidectomies. The current study will have a substantial clinical influence, as it reveals, in a comprehensive manner, that RET Y791F only shows no association with MTC susceptibility.

Mutations in PCYT1A Cause Spondylometaphyseal Dysplasia with Cone-Rod Dystrophy

Spondylometaphyseal dysplasia with cone-rod dystrophy is a rare autosomal-recessive disorder characterized by severe short stature, progressive lower-limb bowing, flattened vertebral bodies, metaphyseal involvement, and visual impairment caused by cone-rod dystrophy. Whole-exome sequencing of four individuals affected by this disorder from two Brazilian families identified two previously unreported homozygous mutations in PCYT1A. This gene encodes the alpha isoform of the phosphate cytidylyltransferase 1 choline enzyme, which is responsible for converting phosphocholine into cytidine diphosphate-choline, a key intermediate step in the phosphatidylcholine biosynthesis pathway. A different enzymatic defect in this pathway has been previously associated with a muscular dystrophy with mitochondrial structural abnormalities that does not have cartilage and/or bone or retinal involvement. Thus, the deregulation of the phosphatidylcholine pathway may play a role in multiple genetic diseases in humans, and further studies are necessary to uncover its precise pathogenic mechanisms and the entirety of its phenotypic spectrum.

Rare Variants in the Epithelial Cadherin Gene Underlying the Genetic Etiology of Nonsyndromic Cleft Lip with or without Cleft Palate

Nonsyndromic orofacial cleft (NSOFC) is a complex disease of still unclear genetic etiology. To investigate the contribution of rare epithelial cadherin (CDH1) gene variants to NSOFC, we target sequenced 221 probands. Candidate variants were evaluated via in vitro, in silico, or segregation analyses. Three probably pathogenic variants (c.760G>A [p.Asp254Asn], c.1023T>G [p.Tyr341*], and c.2351G>A [p.Arg784His]) segregated according to autosomal dominant inheritance in four nonsyndromic cleft lip with or without cleft palate (NSCL/P) families (Lod score: 5.8 at θ = 0; 47% penetrance). A fourth possibly pathogenic variant (c.387+5G>A) was also found, but further functional analyses are needed (overall prevalence of CDH1 candidate variants: 2%; 15.4% among familial cases). CDH1 mutational burden was higher among probands from familial cases when compared to that of controls (P = 0.002). We concluded that CDH1 contributes to NSCL/P with mainly rare, moderately penetrant variants, and CDH1 haploinsufficiency is the likely etiological mechanism.

Schinzel-Giedion Syndrome in Two Brazilian Patients: Report of a Novel Mutation in SETBP1 and Literature Review of the Clinical Features

Schinzel–Giedion syndrome is a rare autosomal dominant disorder comprising postnatal growth failure, profound developmental delay, seizures, facial dysmorphisms, genitourinary, skeletal, neurological, and cardiac defects. It was recently revealed that Schinzel–Giedion syndrome is caused by de novo mutations in SETBP1, but there are few reports of this syndrome with molecular confirmation. We describe two unrelated Brazilian patients with Schinzel–Giedion syndrome, one of them carrying a novel mutation. We also present a review of clinical manifestations of the syndrome, comparing our cases to patients reported in literature emphasizing the importance of the facial gestalt associated with neurological involvement for diagnostic suspicion of this syndrome.