RESUMO
Neonatal diabetes mellitus (NDM) is defined as the occurrence of severe hyperglycemia in infants under 6 months old and may be permanent (PNDM) or transient (TNDM). When diabetes is diagnosed at 6-12 months of age (early onset diabetes [EOD]), the etiology may be monogenic; however, most cases consist of type 1 diabetes mellitus (T1DM). Molecular diagnosis was determined in a cohort of 35 unrelated Brazilian patients with NDM or EOD based on targeted next-generation sequencing panel and/or chromosome 6q24 abnormalities. The impact of genetic testing on treatment and follow-up was evaluated. Overall, 24 patients had NDM: with 18 (75.0%) having PNDM, 5 TNDM (20.8%) and 1 case in which this information was unknown. Eleven patients had EOD. Genetic testing was positive in 20/24 patients with NDM (83.3%) and in 18.2% of cases of EOD. The commonest causes were ATP-sensitive potassium (KATP) channel genes, and GCK and IPEX mutations (37.1%, 11.4% and 5.7%, respectively). Patients with PNDM due to KCNJ11 and ABCC8 mutations transitioned successfully to sulfonylureas in almost 60% of cases, reinforcing the benefit of performing genetic testing in NDM as early as possible. This report refers to the largest series of cases of NDM (TNDM and PNDM) and EOD in Brazil in which patients were submitted to molecular investigation and in which the clinical impact of genetic diagnosis was also evaluated.
Assuntos
Diabetes Mellitus Tipo 1 , Diabetes Mellitus , Doenças do Recém-Nascido , Canais de Potássio Corretores do Fluxo de Internalização , Lactente , Recém-Nascido , Humanos , Brasil , Canais de Potássio Corretores do Fluxo de Internalização/genética , Diabetes Mellitus Tipo 1/genética , Mutação , Testes Genéticos , Doenças do Recém-Nascido/genética , Diabetes Mellitus/genéticaRESUMO
BACKGROUND: Thyrotoxic Hypokalemic Periodic Paralysis (THPP) is a rare neuromuscular disease characterized by recurrent episodes of skeletal muscle weakness associated with hypokalemia. Alterations in protein-encoding genes that are part of ion channels seem to be related to the development of this disease. However, the pathogenic potential of some variants in these genomic regions is not yet fully understood. The aim of this study was to screen genetic alterations in regions coding for calcium (cav1.1), sodium (nav1.4), and potassium (Kir2.6) channels, evaluating its impact on the phenotype of patients with THPP. METHODS: Four patients with a diagnosis of THPP followed by the Endocrinology Service of the University Hospital of the Federal University of Maranhão (Brazil) were investigated for the presence of molecular abnormalities in CACNA1S, SCN4A, and KCNJ18 genes. RESULTS: The KCNJ18 analysis revealed at least one polymorphic variant in each patient. Considering the haplotypic classification of R39Q, R40H, A56E, and I249V variants, two cases were named Kir2.6_RRAI and the other two patients were named Kir2.6_QHEV. No patient had point mutations in the regions evaluated for CACNA1S and SCN4A genes. CONCLUSION: The identification of the Kir2.6_RRAI and Kir2.6_QHEV haplotypes reinforces the existence of two main haplotypes involving these four loci of the KCNJ18gene. On the other hand, point mutations in CACNA1S, SCN4A, and KCNJ18 genes do not seem to be the main mechanism of pathogenesis of THPP, indicating that many questions about this topic still remain unclear. So, the diagnosis of this rare disorder should still be based on clinical and biochemical aspects presented by the patient.
Assuntos
Paralisia Periódica Hipopotassêmica , Canais de Potássio Corretores do Fluxo de Internalização , Brasil/epidemiologia , Testes Genéticos , Humanos , Paralisia Periódica Hipopotassêmica/diagnóstico , Paralisia Periódica Hipopotassêmica/epidemiologia , Paralisia Periódica Hipopotassêmica/genética , Mutação , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Centros de Atenção TerciáriaRESUMO
Andersen-Tawil syndrome (ATS) is a very rare orphan genetic multisystem channelopathy without structural heart disease (with rare exceptions). ATS type 1 is inherited in an autosomal dominant fashion and is caused by mutations in the KCNJ2 gene, which encodes the α subunit of the K+ channel protein Kir2.1 (in ≈ 50-60% of cases). ATS type 2 is in turn linked to a rare mutation in the KCNJ5-GIRK4 gene that encodes the G protein-sensitive-activated inwardly rectifying K+ channel Kir3.4 (15%), which carries the acetylcholine-induced potassium current. About 30% of cases are de novo/sporadic, suggesting that additional as-yet unidentified genes also cause the disorder. A triad of periodic muscle paralysis, repolarization changes in the electrocardiogram, and structural body changes characterize ATS. The typical muscular change is episodic flaccid muscle weakness. Prolongation of the QU/QUc intervals and normal or minimally prolonged QT/QTc intervals with a tendency to ventricular arrhythmias are typical repolarization changes. Bidirectional ventricular tachycardia is the hallmark ventricular arrhythmia, but also premature ventricular contractions, and rarely, polymorphic ventricular tachycardia of torsade de pointes type may be present. Patients with ATS have characteristic physical developmental dysmorphisms that affect the face, skull, limbs, thorax, and stature. Mild learning difficulties and a distinct neurocognitive phenotype (deficits in executive function and abstract reasoning) have been described. About 60% of affected individuals have all features of the major triad. The purpose of this review is to present historical aspects, nomenclature (observations/criticisms), epidemiology, genetics, electrocardiography, arrhythmias, electrophysiological mechanisms, diagnostic criteria/clues of periodic paralysis, prognosis, and management of ATS.
Assuntos
Síndrome de Andersen , Síndrome do QT Longo , Canais de Potássio Corretores do Fluxo de Internalização , Taquicardia Ventricular , Síndrome de Andersen/diagnóstico , Síndrome de Andersen/epidemiologia , Síndrome de Andersen/genética , Humanos , Mutação , Paralisia , Fenótipo , Canais de Potássio Corretores do Fluxo de Internalização/genéticaRESUMO
BACKGROUND AND PURPOSE: Aminoglycoside antibiotics are positively charged molecules that are known to inhibit several ion channels. In this study, we have shown that aminoglycosides also inhibit the activity of Kir4.1 channels. Aminoglycosides inhibit Kir4.1 channels by a pore-blocking mechanism, plugging the central vestibule of the channel. EXPERIMENTAL APPROACH: Patch-clamp recordings were made in HEK-293 cells transiently expressing Kir4.1 channels to analyse the effects of gentamicin, neomycin and kanamycin. In silico modelling followed by mutagenesis were realized to identify the residues critical for aminoglycosides binding to Kir4.1. KEY RESULTS: Aminoglycoside antibiotics block Kir4.1 channels in a concentration- and voltage-dependent manner, getting access to the protein from the intracellular side of the plasma membrane. Aminoglycosides block Ki4.1 with a rank order of potency as follows: gentamicin Ë neomycin Ë kanamycin. The residues T128 and principally E158, facing the central cavity of Kir4.1, are important structural determinants for aminoglycosides binding to the channel, as determined by our in silico modelling and confirmed by mutagenesis experiments. CONCLUSION AND IMPLICATIONS: Kir4.1 channels are also target of aminoglycoside antibiotics, which could affect potassium transport in several tissues.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Simulação por Computador , Células HEK293 , Humanos , Canais de Potássio Corretores do Fluxo de Internalização/genéticaRESUMO
Kir7.1 is an inwardly rectifying K+ channel present in epithelia where it shares membrane localization with the Na+/K+-pump. In the present communication we report the presence of a novel splice variant of Kir7.1 in mouse tissues including kidney, lung, choroid plexus and retinal pigment epithelium (RPE). The variant named mKir7.1-SV2 lacks most of the C-terminus domain but is predicted to have the two transmembrane domains and permeation pathway unaffected. Similarly truncated predicted proteins, Kir7.1-R166X and Kir7.1-Q219X, would arise from mutations associated with Leber Congenital Amaurosis, a rare recessive hereditary retinal disease that results in vision loss at early age. We found that mKir7.1-SV2 and the pathological variants do not produce any channel activity when expressed alone in HEK-293â¯cells due to their scarce presence in the plasma membrane. Simultaneous expression with the full length Kir7.1 however leads to a reduction in activity of the wild-type channel that might be due to partial proteasome degradation of WT-mutant channel heteromers.
Assuntos
Amaurose Congênita de Leber/genética , Mutação/genética , Especificidade de Órgãos , Canais de Potássio Corretores do Fluxo de Internalização/genética , Splicing de RNA/genética , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Proteínas Mutantes/metabolismo , Especificidade de Órgãos/efeitos dos fármacos , Peptídeos/genética , Potássio/metabolismo , Inibidores de Proteassoma/farmacologia , Splicing de RNA/efeitos dos fármacosRESUMO
Insulin secretion is regulated by ATP-sensitive potassium channels (KATP). The potassium inwardly-rectifying channel, subfamily J, member 11 (KCNJ11) gene, located on chromosome 11p15.1, encodes the subunit Kir6.2 that forms the pore region of KATP channels in pancreatic ß-cells. Among the single nucleotide polymorphisms (SNPs) associated with KCNJ11, the E23K polymorphism (rs5219) promotes a substitution (G > A) of a glutamic acid residue for lysine at position 23. The E23K SNP has been associated with diabetes in several populations, although with controversial results. The aim of this study was to evaluate the association of the E23K SNP with type 1 and 2 diabetes in a case-control study approved by the Ethics Committee. We genotyped 458 Euro-Brazilian individuals, classified as healthy (control group, CTRL, N = 217), patients with type 1 diabetes mellitus (T1D, N = 102), and patients with type 2 diabetes mellitus (T2D, N = 139). Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using BanII restriction digestion. The restriction fragments were separated by polyacrylamide gel electrophoresis and visualized by ethidium bromide staining. The genotype (EE/EK/KK) frequencies (%) for the CTRL group (38.2/50.2/11.6), T1D (34.3/52.0/13.7), and T2D (38.2/48.9/12.9) were in Hardy-Weinberg equilibrium and there were no significant differences (CRTL vs T1D, P = 0.771; CRTL vs T2D, P = 0.937; T1D vs T2D, P = 0.831). The minor allele frequencies (MAF; K) for CTRL (37.0%), T1D (39.7%), and T2D (37.4%) were not different among the groups (P > 0.05). The MAF value for healthy subjects was similar to other Caucasian populations (34.5-37.5%). In summary, the E23K polymorphism (rs5219) was not associated with type 1 or 2 diabetes mellitus in the studied population.
Assuntos
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Adulto , Idoso , Brasil , Estudos de Casos e Controles , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Células Secretoras de Insulina/metabolismo , Canais KATP/genética , Canais KATP/metabolismo , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , População Branca/genéticaRESUMO
In contrast to the Mendelian inheritance model, parental alleles can contribute unequally to gene expression, which may result in phenotypic variance among individuals and bias in the predicted additive effect of molecular markers associated with production traits. Given the need to understand the effects of allelic variation and parent-of-origin effects on the expression of genes with a commercial interest in cattle, we analyzed the expression of KCNJ11 (potassium inwardly rectifying channel, subfamily J, member 11), which was previously described as a functional candidate gene for meat tenderness. Allele-specific and parent-of-origin-dependent expression of this gene were assessed in bovine muscle using the rs379610823 single nucleotide polymorphism as a reference. Biallelic expression was observed; however, the T allele was expressed at significantly higher levels than the C allele. Furthermore, increased expression of KCNJ11 was found in animals harboring the maternal T allele. This study is the first to describe the differential allelic expression of bovine KCNJ11. Our findings are important for understanding the mechanisms that underlie the pattern of KCNJ11 expression and its potential impact on the phenotypic variation of meat tenderness in Nelore beef cattle. This reinforces the need for further investigation of allelic- and parent-of-origin expression deviation in genetic markers eligible for the selection of target traits.
Assuntos
Marcadores Genéticos , Padrões de Herança , Carne/análise , Canais de Potássio Corretores do Fluxo de Internalização/genética , Característica Quantitativa Herdável , Alelos , Animais , Bovinos , Feminino , Expressão Gênica , Genótipo , Masculino , Músculo Esquelético/metabolismo , Fenótipo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
AIMS: Neonatal diabetes mellitus (NDM) is a rare monogenic disorder, reported to affect less than 2 cases per 100,000 infants. There are two types, permanent (PNDM) and transient (TNDM). We describe our clinical experience in determining and comparing the genetic basis of diabetes in children with onset before 6months versus those diagnosed between 6 and 12months of age. METHODS: We reviewed medical records of children with diabetes diagnosed before 12months of age. Genetic testing was performed in all cases. RESULTS: 12 patients were diagnosed with diabetes before 6months of age (PNDM=6; TNDM=6), and 11 patients between 6 and 12months (all with permanent diabetes). Among children with PNDM, we identified three different KCNJ11 mutations in 5 patients, and one novel ABCC8 mutation in a single patient. Among children with TNDM, we detected a KCNJ11 and ABCC8 mutation each in a single patient and methylation abnormalities at chromosome 6q24 in 4 patients. Among children with diabetes diagnosed between 6 and 12months, 1 patient had an INS mutation and one patient was homozygous for an SLC19A2 mutation which confirmed a diagnosis of thiamine-responsive megaloblastic anaemia syndrome. Five of the patients with an ABCC8 or KCNJ11 mutation have successfully transferred from insulin to glibenclamide whist 1 child demonstrated a partial response to sulfonylurea treatment. CONCLUSIONS: Investigating the underlying genetic basis of diabetes in children with onset before 1year is useful for choosing the most efficient treatment, the basis of Personalized Medicine.
Assuntos
Diabetes Mellitus/tratamento farmacológico , Hipoglicemiantes/administração & dosagem , Doenças do Recém-Nascido/tratamento farmacológico , Insulina/administração & dosagem , Administração Oral , Diabetes Mellitus/genética , Feminino , Testes Genéticos , Humanos , Lactente , Recém-Nascido , Doenças do Recém-Nascido/genética , Insulina/genética , Masculino , Mutação/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Compostos de Sulfonilureia/administração & dosagem , Receptores de Sulfonilureias/genéticaRESUMO
Next-generation sequencing (NGS) has enriched the understanding of the human genome. However, homologous or repetitive sequences shared among genes frequently produce dubious alignments and can puzzle NGS mutation analysis, especially for paralogous potassium channels. Potassium inward rectifier (Kir) channels are important to establish the resting membrane potential and regulating the muscle excitability. Mutations in Kir channels cause disorders affecting the heart and skeletal muscle, such as arrhythmia and periodic paralysis. Recently, a susceptibility muscle channelopathy-thyrotoxic periodic paralysis (TPP)-has been related to Kir2.6 channel (KCNJ18 gene). Due to their high nucleotide sequence homology, variants found in the potassium channels Kir2.6 and Kir2.5 have been mistakenly attributable to Kir2.2 polymorphisms or mutations. We aimed at elucidating nucleotide misalignments by performing realignment of whole exome sequencing (WES) and whole genome sequencing (WGS) reads to specific Kir2.2, Kir2.5, and Kir2.6 cDNA sequences using BWA-MEM/GATK pipeline. WES/WGS reads correctly aligned 26.9/43.2, 37.6/31.0, and 35.4/25.8 % to Kir2.2, Kir2.5, and Kir2.6, respectively. Realignment was able to reduce over 94 % of misalignments. No putative mutations of Kir2.6 were identified for the three TPP patients included in the cohort of 36 healthy controls using either WES or WGS. We also distinguished sequences for a single Kir2.2, a single Kir2.5 sequence, and two Kir2.6 isoforms, which haplotypes were named RRAI and QHEV, based on changes at 39, 40, 56, and 249 residues. Electrophysiology records on both Kir2.6_RRAI and _QHEV showed typical rectifying currents. In our study, the reduction of misalignments allowed the elucidation of paralogous gene sequences and two distinct Kir2.6 haplotypes, and pointed the need for checking the frequency of these polymorphisms in other populations with different genetic background.
Assuntos
Canalopatias/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Análise de Sequência de DNA/métodos , Mapeamento Cromossômico/métodos , Exoma , Predisposição Genética para Doença , Genoma Humano , Células HEK293 , Humanos , Polimorfismo de Nucleotídeo Único , Isoformas de Proteínas/genética , Alinhamento de SequênciaRESUMO
Permanent neonatal diabetes (PNDM) can result from activating heterozygous mutations in KCNJ11 gene, encoding the Kir6.2 subunit of the pancreatic ATP-sensitive potassium channels (KATP). Sulfonylureas promote KATP closure and stimulate insulin secretion, being an alternative therapy in PNDM, instead of insulin. Male, 20 years old, diagnosed with diabetes at 3 months of age. The genetic study identified a novel heterozygous mutation in exon 1 of the KCNJ11 gene – KCNJ11:c1001G>7 (p.Gly334Val) – and confirmed the diagnosis of PNDM. Therefore it was attempted to switch from insulin therapy to sulfonylurea. During glibenclamide institution C-peptide levels increased, however the suboptimal glycemic control lead us to restart an intensive insulin scheme. This new variant of KCNJ11 mutation had a phenotypic lack of response to sulfonylurea therapy. Age, prior poor metabolic control and functional change of KATP channel induced by this specific mutation may explain the observed unsuccessful switch to sulfonylurea. Interestingly, C-peptide levels raise during glibenclamide administration support some degree of improvement in insulin secretory capacity induced by the treatment. Understanding the response to sulfonylurea is crucial as successful treatment may be life-changing in these patients.