RESUMO
Thyroglobulin (TG), the predominant glycoprotein of the thyroid gland, functions as matrix protein in thyroid hormonegenesis. TG deficiency results in thyroid dyshormonogenesis. These variants produce a heterogeneous spectrum of congenital goitre, with an autosomal recessive mode of inheritance. The purpose of this study was to identify and functionally characterize new variants in the TG gene in order to increase the understanding of the molecular mechanisms responsible for thyroid dyshormonogenesis. A total of four patients from two non-consanguineous families with marked alteration of TG synthesis were studied. The two families were previously analysed in our laboratory, only one deleterious allele, in each one, was detected after sequencing the TG gene (c.2359 C > T [p.Arg787*], c.5560 G > T [p.Glu1854*]). These findings were confirmed in the present studies by Next-Generation Sequencing. The single nucleotide coding variants of the TG gene were then analyzed to predict the possible variant causing the disease. The p.Pro2232Leu (c.6695 C > T), identified in both families, showing a low frequency population in gnomAD v2.1.1 database and protein homology, amino acid prediction, and 3D modeling analysis predict a potential pathogenic effect of this variant. We also transiently express p.Pro2232Leu in a full-length rat TG cDNA clone and confirmed that this point variant was sufficient to cause intracellular retention of mutant TG in HEK293T cells. Consequently, each family carried a compound heterozygous for p.Arg787*/p.Pro2232Leu or p.Glu1854*/p.Pro2232Leu variants. In conclusion, our results confirm the pathophysiological importance of altered TG folding as a consequence of missense variants located in the ChEL domain of TG.
Assuntos
Hipotireoidismo Congênito , Bócio , Animais , Humanos , Ratos , Hipotireoidismo Congênito/genética , Células HEK293 , Tireoglobulina/genética , Tireoglobulina/metabolismo , Transporte Proteico/genéticaRESUMO
Las talasemias son desórdenes autosómicos recesivos de las cadenas de hemoglobina que poseen expresión clínica variable según el tipo de mutación o deleción. Presentamos el caso de dos jóvenes mujeres costarricenses no relacionadas entre sí y ambas diagnosticadas con la mutación común en el codón 39 (C>T) (β0) en combinación con la deleción siciliana (δβ0) 13.4 kb. La caracterización de doble heterocigota no había sido descrita antes en la literatura médica, y discutimos el significado de este genotipo que causa un defecto tipo β0 talasemia transfusión dependiente.
Thalassemia are autosomal recessive disorders of hemoglobin chains with variable clinical expression depending on the type of mutation or deletion present. We present the common codon 39(C>T) (β0) in combination with the δβ0 13.4 kb Sicilian deletion in two non-related young women from Costa Rica. We report the characterization of the compound heterozygous not previously described phenotype, and discuss the significance of this genotype combination with a transfusion dependent β0 defect Thalassemia.
Assuntos
Humanos , Feminino , Adolescente , Talassemia beta/diagnóstico , Anemia/diagnóstico , Costa RicaRESUMO
BACKGROUND: Friedreich's ataxia (FRDA) is caused by homozygous GAA repeat expansions or compound heterozygous (CH) mutations in FXN gene. Its broad clinical spectrum makes it difficult to identify, thus an accurate diagnosis can only be made by genetic testing. OBJECTIVE: This study aims to present data on FXN variants observed in patients with sporadic or recessive ataxia, including detailed data of the first CH Mexican patients. MATERIALS AND METHODS: One hundred and eight patients with recessive or sporadic cerebellar ataxia were referred to our institution between 2009 and 2019 for FXN molecular testing. This was achieved using a combined methodology of triplet repeat-primed PCR (polymerase chain reaction), long PCR, FXN sequencing and multiplex-ligation probe-amplification. RESULTS: Eighteen patients had a homozygous FXN genotype; whereas five were CH patients with a slow progression and phenotypic variability, including a late-onset case with spastic paraparesis, and a Charcot-Marie-Tooth-like case. CONCLUSIONS: These first Mexican CH patients pose important implications for genetic counseling and FRDA management.
Assuntos
Ataxia de Friedreich , Proteínas de Ligação ao Ferro/genética , Ataxia de Friedreich/genética , Testes Genéticos , Humanos , México , Mutação , Repetições de Trinucleotídeos , FrataxinaRESUMO
Compound heterozygous mutations, where two distinct mutated alleles are present within a particular gene, can give rise to the Bardet-Biedl syndrome (BBS). There is limited evidence suggesting that some compound heterozygotes can present with milder phenotypic characteristics than homozygotes. We report on the clinical characteristics of a 22-year-old Puerto Rican male who was compound heterozygous for the Bardet-Biedl syndrome type 1. Our patient had deteriorating visual acuity since early childhood. Clinical and ophthalmic examination revealed retinal dystrophy, polydactyly, and very mild learning disabilities. No additional systemic complications commonly observed in patients with the BBS were present. Allele-specific testing and DNA sequencing revealed compound heterozygous mutations (M390R and E549X) in the BBS1 gene. Our findings could suggest that patients who are compound heterozygotes for these specific BBS mutations can exhibit milder clinical signs than homozygous patients.
Assuntos
Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/diagnóstico , Análise Mutacional de DNA , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/genética , Mutação , Adulto JovemRESUMO
Thyroglobulin (TG) is a homodimeric glycoprotein synthesized by the thyroid gland. To date, two hundred twenty-seven variations of the TG gene have been identified in humans. Thyroid dyshormonogenesis due to TG gene mutations have an estimated incidence of approximately 1 in 100,000 newborns. The clinical spectrum ranges from euthyroid to mild or severe hypothyroidism. The purpose of the present study was to identify and characterize new variants in the TG gene. We report an Argentine patient with congenital hypothyroidism, enlarged thyroid gland and low levels of serum TG. Sequencing of DNA, expression of chimeric minigenes as well as bioinformatics analysis were performed. DNA sequencing identified the presence of compound heterozygous mutations in the TG gene: the maternal mutation consists of a c.3001+5G > A, whereas the paternal mutation consists of p.Arg296*. Minigen analysis of the variant c.3001+5A performed in HeLa, CV1 and Hek293T cell lines, showed a total lack of transcript expression. So, in order to validate that the loss of expression was caused by such variation, site-directed mutagenesis was performed on the mutated clone, which previously had a pSPL3 vector change, to give rise to a wild-type clone c.3001+5G, endorsing that the mutation c.3001+5G > A is the cause of the total lack of expression. In conclusion, we demonstrate that the c.3001+5G > A mutation causes a rare genotype, altering the splicing of the pre-mRNA. This work contributes to elucidating the molecular bases of TG defects associated with congenital hypothyroidism and expands our knowledge in relation to the pathologic roles of the position 5 in the donor splice site.
Assuntos
Biologia Computacional , Íntrons/genética , Mutação/genética , Precursores de RNA/genética , Sítios de Splice de RNA/genética , Splicing de RNA/genética , Tireoglobulina/genética , Sequência de Bases , Genótipo , Células HEK293 , Células HeLa , Humanos , Recém-Nascido , Masculino , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Precursores de RNA/metabolismo , Tireoglobulina/químicaRESUMO
Resumo A hipercolesterolemia familiar (HF) é uma doença genética causada por um defeito primário no gene que codifica o receptor da LDL. Mutações diferentes no mesmo gene caracterizam um heterozigoto composto, mas pouco se sabe sobre o fenótipo dos portadores. Portanto, neste estudo, descrevemos o rastreamento em cascata de uma família brasileira com essa característica. O caso-índice é um homem de 36 anos, com colesterol total (CT) de 360 mg/dL (9,3 mmol/L) e concentração de LDL-c de 259 mg/dL (6,7 mmol/L), além de xantomas de tendão de Aquiles, obesidade e pré-hipertensão. A genotipagem identificou as mutações 661G>A, 670G>A e 682G>A, no exon 4, e 919G>A, no exon 6. A mesma mutação no exon 4 foi observada no filho do caso-índice (7 anos), que também tem hipercolesterolemia e xantomas tendinosos, ao passo que a filha do caso-índice (9 anos) apresenta mutação no exon 6 e hiperlipidemia, sem xantomas. Em suma, este relato permite uma melhor compreensão acerca da base molecular da HF no Brasil, um país multirracial, onde é esperada uma população heterogênea.
Abstract Familial hypercholesterolemia (FH) is a genetic disease caused by a primary defect in the LDL-receptor gene. Distinct variants in the same gene characterize a compound heterozygote, but little is known about the phenotypes of the carriers. Therefore, herein, we describe the cascade screening of a Brazilian family with this characteristic. The index case, a 36-year-old male, had a total cholesterol level of 360 mg/dL (9.3 mmol/L) and LDL-c value of 259 mg/dL (6.7 mmol/L), in addition to Achilles tendon xanthomas, obesity and prehypertension. Genotyping identified the variants 661G>A, 670G>A, 682G>A in exon 4 and 919G>A in exon 6. The same variant in exon 4 was found in the index case's son (7-y), who also had hypercholesterolemia and xanthomas, while the index case's daughter (9-y) had the variant in exon 6 and hyperlipidemia, without xanthomas. In summary, this report allows for a better insight into the molecular basis of FH in Brazil, a multi-racial country where a heterogeneous population is expected.
Assuntos
Humanos , Masculino , Adulto , Hiperlipoproteinemia Tipo II/genética , Fenótipo , Brasil , Receptores de LDL/genética , HeterozigotoRESUMO
Friedreich ataxia (FRDA) is the most common autosomal recessive ataxia in Caucasian populations. It is caused by a homozygous GAA expansion in the first intron of the frataxin gene (FXN) (OMIM: 606829) in 96% of the affected individuals. The remaining patients have a GAA expansion in one allele and a point mutation in the other. Little is known about compound heterozygous patients outside Europe and North America. We have thus designed a study to determine the frequency and mutational profile of these patients in Brazil. To accomplish that, we recruited all patients with ataxia and at least one expanded GAA allele at FXN from 3 national reference centers. We identified those subjects with a single expansion and proceeded with further genetic testing (Sanger sequencing and CGH arrays) for those. There were 143 unrelated patients (128 families), five of which had a single expanded allele. We identified point mutations in three out of these five (3/128 = 2.34%). Two patients had the c.157delC variant, whereas one individual had the novel variant c.482+1G>T. These results indicate that FXN point mutations are rare, but exist in Brazilian patients with FRDA. This has obvious implications for diagnostic testing and genetic counseling.
Assuntos
Ataxia de Friedreich/epidemiologia , Ataxia de Friedreich/genética , Perfil Genético , Testes Genéticos , Adolescente , Adulto , Brasil/epidemiologia , Criança , Feminino , Ataxia de Friedreich/diagnóstico , Testes Genéticos/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Proteins expressed by recombination activating genes 1 and 2 (RAG1/2) are essential in the process of V(D)J recombination that leads to generation of the T and B cell repertoires. Clinical and immunological phenotypes of patients with RAG deficiencies correlate well to the degree of impaired RAG activity and this has been expanding to variants of combined immunodeficiency (CID) or even milder antibody deficiency syndromes. Pathogenic variants that severely impair recombinase activity of RAG1/2 determine a severe combined immunodeficiency (SCID) phenotype, whereas hypomorphic variants result in leaky (partial) SCID and other immunodeficiencies. We report a patient with novel pathogenic compound heterozygous RAG2 variants that result in a CID phenotype with two distinctive characteristics: late-onset progressive hypogammaglobulinemia and highly elevated B cell count. In addition, the patient had early onset of infections, T cell lymphopenia and expansion of lymphocytes after exposure to herpes family viruses. This case highlights the importance of considering pathogenic RAG variants among patients with preserved B cell count and CID phenotype.
RESUMO
PURPOSE: CARD9 deficiency is an inborn error of immunity that predisposes otherwise healthy humans to mucocutaneous and invasive fungal infections, mostly caused by Candida, but also by dermatophytes, Aspergillus, and other fungi. Phaeohyphomycosis are an emerging group of fungal infections caused by dematiaceous fungi (phaeohyphomycetes) and are being increasingly identified in patients with CARD9 deficiency. The Corynespora genus belongs to phaeohyphomycetes and only one adult patient with CARD9 deficiency has been reported to suffer from invasive disease caused by C. cassiicola. We identified a Colombian child with an early-onset, deep, and destructive mucocutaneous infection due to C. cassiicola and we searched for mutations in CARD9. METHODS: We reviewed the medical records and immunological findings in the patient. Microbiologic tests and biopsies were performed. Whole-exome sequencing (WES) was made and Sanger sequencing was used to confirm the CARD9 mutations in the patient and her family. Finally, CARD9 protein expression was evaluated in peripheral blood mononuclear cells (PBMC) by western blotting. RESULTS: The patient was affected by a large, indurated, foul-smelling, and verrucous ulcerated lesion on the left side of the face with extensive necrosis and crusting, due to a C. cassiicola infectious disease. WES led to the identification of compound heterozygous mutations in the patient consisting of the previously reported p.Q289* nonsense (c.865C > T, exon 6) mutation, and a novel deletion (c.23_29del; p.Asp8Alafs10*) leading to a frameshift and a premature stop codon in exon 2. CARD9 protein expression was absent in peripheral blood mononuclear cells from the patient. CONCLUSION: We describe here compound heterozygous loss-of-expression mutations in CARD9 leading to severe deep and destructive mucocutaneous phaeohyphomycosis due to C. cassiicola in a Colombian child.
Assuntos
Ascomicetos , Proteínas Adaptadoras de Sinalização CARD/genética , Predisposição Genética para Doença , Heterozigoto , Infecções Fúngicas Invasivas , Mutação , Feoifomicose/epidemiologia , Feoifomicose/etiologia , Fatores Etários , Idade de Início , Ascomicetos/genética , Ascomicetos/imunologia , Biomarcadores , Pré-Escolar , Colômbia/epidemiologia , Biologia Computacional/métodos , Análise Mutacional de DNA , Feminino , Humanos , Imuno-Histoquímica , Imunofenotipagem , Imageamento por Ressonância Magnética , Linhagem , Feoifomicose/diagnóstico , Feoifomicose/imunologia , Fenótipo , Tomografia Computadorizada por Raios X , Sequenciamento do ExomaRESUMO
Abstract Anophthalmia is a rare eye development anomaly resulting in absent ocular globes or tissue in the orbit since birth. Here, we investigated a newborn with bilateral anophthalmia in a Chinese family. Exome sequencing revealed that compound heterozygous mutations c.287G > A (p.(Arg96His)) and c.709G > A (p.(Gly237Arg)) of the ALDH1A3 gene were present in the affected newborn. Both mutations were absent in all of the searched databases, including 10,000 in-house Chinese exome sequences, and these mutations were confirmed as having been transmitted from the parents. Comparative amino acid sequence analysis across distantly related species revealed that the residues at positions 96 and 234 were evolutionarily highly conserved. In silico analysis predicted these changes to be damaging, and in vitro expression analysis revealed that the mutated alleles were associated with decreased protein production and impaired tetrameric protein formation. This study firstly reported that compound heterozygous mutations of the ALDH1A3 gene can result in anophthalmia in humans, thus highlighting those heterozygous mutations in ALDH1A3 should be considered for molecular screening in anophthalmia, particularly in cases from families without consanguineous relationships.
RESUMO
Iodide Organification defects (IOD) represent 10% of cases of congenital hypothyroidism (CH) being the main genes affected that of TPO (thyroid peroxidase) and DUOX2 (dual oxidasa 2). From a patient with clinical and biochemical criteria suggestive with CH associated with IOD, TPO and DUOX2 genes were analyzed by means of PCR-Single Strand Conformation Polymorphism analysis and sequencing. A novel heterozygous compound to the mutations c.2335-1G>C (paternal mutation, intron 17) and c.3264_3267delCAGC (maternal mutation, exon 24) was identified in the DUOX2 gene. Ex-vivo splicing assays and subsequent RT-PCR and sequencing analyses were performed on mRNA isolated from the HeLa cells transfected with wild-type and mutant pSPL3 expression vectors. The wild-type and c.2335-1G>C mutant alleles result in the complete inclusion or exclusion of exon 18, or in the activation of an exonic cryptic 5' ss with the consequent deletion of 169 bp at the end of this exon. However, we observed only a band of the expected size in normal thyroid tissue by RT-PCR. Additionally, the c.2335-1G>C mutation activates an unusual cryptic donor splice site in intron 17, located at position -14 of the authentic intron 17/exon 18 junction site, with an insertion of the last 14 nucleotides of the intron 17 in mutant transcripts with complete and partial inclusion of exon 18. The theoretical consequences of splice site mutation, predicted with the bioinformatics NNSplice, Fsplice, SPL, SPLM and MaxEntScan programs were investigated and evaluated in relation with the experimental evidence. These analyses confirm that c.2335-1G>C mutant allele would result in the abolition of the authentic splice acceptor site. The results suggest the coexistence in our patient of four putative truncated proteins of 786, 805, 806 and 1105 amino acids, with conservation of peroxidase-like domain and loss of gp91(phox)/NOX2-like domain. In conclusion a novel heterozygous compound was identified being responsible of IOD. Cryptic splicing sites have been characterized in DUOX2 gene for the first time. The use of molecular biology techniques is a valuable tool for understanding the molecular pathophysiology of this type of thyroid defects.
Assuntos
Hipotireoidismo Congênito/genética , Mutação , NADPH Oxidases/genética , Sítios de Splice de RNA , Criança , Hipotireoidismo Congênito/metabolismo , Oxidases Duais , Células HeLa , Heterozigoto , Humanos , Masculino , NADPH Oxidases/metabolismo , Linhagem , Polimorfismo Conformacional de Fita Simples , Análise de Sequência de DNA , Glândula Tireoide/metabolismoRESUMO
Several patients were identified with dyshormonogenesis caused by mutations in the thyroglobulin (TG) gene. These defects are inherited in an autosomal recessive manner and affected individuals are either homozygous or compound heterozygous for the mutations. The aim of the present study was to identify new TG mutations in a patient of Vietnamese origin affected by congenital hypothyroidism, goiter and low levels of serum TG. DNA sequencing identified the presence of compound heterozygous mutations in the TG gene: the maternal mutation consists of a novel c.745+1G>A (g.IVS6 + 1G>A), whereas the hypothetical paternal mutation consists of a novel c.7036+2T>A (g.IVS40 + 2T>A). The father was not available for segregation analysis. Ex-vivo splicing assays and subsequent RT-PCR analyses were performed on mRNA isolated from the eukaryotic-cells transfected with normal and mutant expression vectors. Minigene analysis of the c.745+1G>A mutant showed that the exon 6 is skipped during pre-mRNA splicing or partially included by use of a cryptic 5' splice site located to 55 nucleotides upstream of the authentic exon 6/intron 6 junction site. The functional analysis of c.7036+2T>A mutation showed a complete skipping of exon 40. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool NNSplice, Fsplice, SPL, SPLM and MaxEntScan programs were investigated and evaluated in relation with the experimental evidence. These analyses predicted that both mutant alleles would result in the abolition of the authentic splice donor sites. The c.745+1G>A mutation originates two putative truncated proteins of 200 and 1142 amino acids, whereas c.7036+2T>A mutation results in a putative truncated protein of 2277 amino acids. In conclusion, we show that the c.745+1G>A mutation promotes the activation of a new cryptic donor splice site in the exon 6 of the TG gene. The functional consequences of these mutations could be structural changes in the protein molecule that alter the biosynthesis of thyroid hormones.
Assuntos
Povo Asiático/genética , Hipotireoidismo Congênito/genética , Bócio/congênito , Bócio/genética , Polimorfismo de Nucleotídeo Único , Sítios de Splice de RNA , Tireoglobulina/genética , Adolescente , Animais , Células COS , Chlorocebus aethiops , Hipotireoidismo Congênito/patologia , Éxons , Feminino , Bócio/patologia , Células HeLa , Heterozigoto , Humanos , Masculino , Linhagem , Análise de Sequência de DNA , Tireoglobulina/sangue , VietnãRESUMO
Hypophosphatasia (HPP) is an inherited disorder of mineral metabolism caused by mutations in ALPL, encoding tissue non-specific alkaline phosphatase (TNAP). Here, we report the molecular findings from monozygotic twins, clinically diagnosed with tooth-specific odontohypophosphatasia (odonto-HPP). Sequencing of ALPL identified two genetic alterations in the probands, including a heterozygous missense mutation c.454C>T, leading to change of arginine 152 to cysteine (p.R152C), and a novel heterozygous gene deletion c.1318_1320delAAC, leading to the loss of an asparagine residue at codon 440 (p.N440del). Clinical identification of low serum TNAP activity, dental abnormalities, and pedigree data strongly suggests a genotype-phenotype correlation between p.N440del and odonto-HPP in this family. Computational analysis of the p.N440del protein structure revealed an alteration in the tertiary structure affecting the collagen-binding site (loop 422-452), which could potentially impair the mineralization process. Nevertheless, the probands (compound heterozygous: p.[N440del];[R152C]) feature early-onset and severe odonto-HPP phenotype, whereas the father (p.[N440del];[=]) has only moderate symptoms, suggesting p.R152C may contribute or predispose to a more severe dental phenotype in combination with the deletion. These results assist in defining the genotype-phenotype associations for odonto-HPP, and further identify the collagen-binding site as a region of potential structural importance for TNAP function in the biomineralization.