RESUMEN
The risk of severe adverse events related to thiopurine therapy can be reduced by personalizing dosing based on TPMT and NUDT15 genetic polymorphisms. However, the optimal genetic testing platform has not yet been established. In this study, we report on the TPMT and NUDT15 genotypes and phenotypes generated from 320 patients from a multicenter pediatric healthcare system using both Sanger sequencing and polymerase chain reaction genotyping (hereafter: genotyping) methods to determine the appropriateness of genotyping in our patient population. Sanger sequencing identified variant TPMT alleles including *3A (8, 3.2% of alleles), *3C (4, 1.6%), and *2 (1, 0.4%), and NUDT15 alleles including *2 (5, 3.6%) and *3 (1, 0.7%). For genotyped patients, variants identified in TPMT included *3A (12, 3.1%), *3C (4, 1%), *2 (2, 0.5%), and *8 (1, 0.25%), whereas NUDT15 included *4 (2, 1.9%) and *2 or *3 (1, 1%). Between Sanger sequencing and genotyping, no significant difference in allele, genotype, or phenotype frequency was identified for either TPMT or NUDT15. All patients who were tested using Sanger sequencing would have been accurately phenotyped for either TPMT (124/124), NUDT15 (69/69), or both genes (68/68) if they were assayed using the genotyping method. Considering 193 total TPMT and NUDT15 Sanger Sequencing tests reviewed, all tests would have resulted in an appropriate clinical recommendation if the test had instead been conducted using the comparison genotyping platforms. These results suggest that, in this study population, genotyping would be sufficient to provide accurate phenotype calls and clinical recommendations.
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Azatioprina , Polimorfismo Genético , Humanos , Azatioprina/efectos adversos , Pruebas Genéticas , Genotipo , Técnicas de GenotipajeRESUMEN
Chronic kidney disease (CKD) has major morbidity and mortality for children and adults. While in adults CKD often is associated with diabetic complications, genetic variants can be the underlying cause in both populations. Beginning in 2016 with the emergence of more affordable next-generation sequencing (NGS) technologies, the Molecular Diagnostics Lab at Nemours Children's Hospital-Delaware developed the first clinically actionable pediatric NGS kidney panel comprised of 46 genes including APOL1. Apolipoprotein L1 (APOL1) associated nephropathy is reported along a spectrum of non-diabetic kidney disease. It is significantly associated with two "risk alleles" defined as G1 and G2 and typically found in individuals of African descent. In early 2020, as COVID-19 spread across the globe, reports of patients with kidney failure began to emerge. A collapsing glomerulopathy in Black patients with COVID-19 was found to be associated with the APOL1 predisposition of the known G1 and/or G2 risk variants. We identified genetic variants in 11 genes (NPHS1; NPHS2; LAMB2; WT1; COL4A4; COL4A5; COQ8B; CUBN; MEFV; PMM2; SMARCAL1) known to be associated with pediatric onset nephrotic syndrome, or detection of the high-risk haplotype of APOL1, in the majority (78%) of patients tested. These clinically actionable results guided medical care and improved patient outcomes.
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Genetic white matter disorders have heterogeneous etiologies and overlapping clinical presentations. We performed a study of the diagnostic efficacy of genome sequencing in 41 unsolved cases with prior exome sequencing, resolving an additional 14 from an historical cohort (n = 191). Reanalysis in the context of novel disease-associated genes and improved variant curation and annotation resolved 64% of cases. The remaining diagnoses were directly attributable to genome sequencing, including cases with small and large copy number variants (CNVs) and variants in deep intronic and technically difficult regions. Genome sequencing, in combination with other methodologies, achieved a diagnostic yield of 85% in this retrospective cohort.
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Leucoencefalopatías/diagnóstico , Leucoencefalopatías/genética , Sistema de Registros , Secuenciación Completa del Genoma , Adolescente , Niño , Preescolar , Femenino , Humanos , Leucoencefalopatías/patología , Masculino , LinajeRESUMEN
Transient receptor potential cation channel, subfamily V, member 4 (TRPV4) is a polymodal modulated non-selective cation channel required for normal development and maintenance of bone and cartilage. Heterozygous mutations of this channel cause a variety of channelopathies, including metatropic dysplasia (MD). We analyzed the effect of a novel TRPV4 mutation c.2398G>A, p.Gly800Asp on intracellular calcium ([Ca(2+) ]i ) regulation in chondrocytes and compared this response to chondrocytes with a frequently observed mutation, c.2396C>T, p.Pro799Leu. We observed temperature-dependent [Ca(2+) ]i oscillations in both intact and MD chondrocytes however, MD mutations exhibited increased peak magnitudes of [Ca(2+) ]i during oscillations. We also found increased baseline [Ca(2+) ]i in MD primary cells, as well as increased [Ca(2+) ]i response to either hypotonic swelling or the TRVP4-specific agonist, GSK1016790A. Oscillations and stimulation responses were blocked with the TRPV4-specific antagonist, GSK205. Analysis of [Ca(2+) ]i response kinetics showed that MD chondrocytes had increased frequency of temperature-sensitive oscillations, and the magnitude and duration of [Ca(2+) ]i responses to given stimuli. Duration of the response of the p.Gly800Asp mutation to stimulation was greater than for the p.Pro799Leu mutation. These experiments show that this region of the channel is essential for proper [Ca(2+) ]i regulation. These studies of primary cells from patients show how both mutant and WT TRPV4 channels regulate cartilage and bone development. © 2015 Wiley Periodicals, Inc.
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Señalización del Calcio , Condrocitos/metabolismo , Enanismo/genética , Mutación , Osteocondrodisplasias/genética , Canales Catiónicos TRPV/genética , Huesos/metabolismo , Huesos/patología , Agonistas de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Cartílago/metabolismo , Cartílago/patología , Preescolar , Condrocitos/efectos de los fármacos , Condrocitos/patología , Enanismo/metabolismo , Enanismo/patología , Femenino , Expresión Génica , Humanos , Leucina/análogos & derivados , Leucina/farmacología , Presión Osmótica , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/patología , Fenotipo , Cultivo Primario de Células , Índice de Severidad de la Enfermedad , Sulfonamidas/farmacología , Canales Catiónicos TRPV/agonistas , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/metabolismoRESUMEN
Barth syndrome is caused by mutations in the TAZ (tafazzin) gene on human chromosome Xq28. The human tafazzin gene produces four major mRNA splice variants; two of which have been shown to be functional (TAZ lacking exon 5 and full-length) in complementation studies with yeast and Drosophila. This study characterizes the multiple alternative splice variants of TAZ mRNA and their proportions in blood samples from a cohort of individuals with Barth syndrome (BTHS). Because it has been reported that collection and processing methods can affect the expression of various genes, we tested and chose a stabilizing medium for collecting, shipping and processing of the blood samples of these individuals. In both healthy controls and in BTHS individuals, we found a greater variety of alternatively spliced forms than previously described, with a sizeable proportion of minor splice variants besides the four dominant isoforms. Individuals with certain exonic and intronic splice mutations produce additional mutant mRNAs that could be translated into two or more proteins with different amino acid substitutions in a single individual. A fraction of the minor splice variants is predicted to be non-productive.
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Empalme Alternativo , Síndrome de Barth/genética , Isoformas de ARN/metabolismo , ARN Mensajero/metabolismo , Factores de Transcripción/genética , Aciltransferasas , Sustitución de Aminoácidos , Recolección de Muestras de Sangre , Cromosomas Humanos X , Exones , Femenino , Humanos , Intrones , Masculino , Mutación Missense , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The objective of our study was to characterize the influence of multiple mutations in the MECP2 gene in a cohort of individuals with Rett syndrome. Further analysis demonstrated that nearly all resulted from de novo in cis mutations, where the disease severity was indistinguishable from single mutations. Our methods involved enrolling participants in the RTT Natural History Study (NHS). After providing informed consent through their parents or principal caretakers, additional molecular assessments were performed in the participants and their parents to assess the presence and location of more than one mutation in each. Clinical severity was assessed at each visit in those participants in the NHS. Non-contiguous MECP2 gene variations were detected in 12 participants and contiguous mutations involving a deletion and insertion in three participants. Thirteen of 15 participants had mutations that were in cis; four (of 13) had three MECP2 mutations; two (of 15) had mutations that were both in cis and in trans (i.e., on different alleles). Clinical severity did not appear different from NHS participants with a single similar mutation. Mutations in cis were identified in most participants; two individuals had mutations both in cis and in trans. The presence of multiple mutations was not associated with greater severity. Nevertheless, multiple mutations will require greater thought in the future, if genetic assignment to drug treatment protocols is considered.
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Proteína 2 de Unión a Metil-CpG/genética , Mutación , Síndrome de Rett/genética , Femenino , Humanos , Masculino , Padres , Síndrome de Rett/etiologíaRESUMEN
Spinal muscular atrophy (SMA), the most common autosomal recessive cause of infant death, is typically diagnosed by determination of SMN1 copy number. Approximately 3-5% of patients with SMA retain at least one copy of the SMN1 gene carrying pathogenic insertions, deletions, or point mutations. We report a patient with SMA who is homozygous for two mutations carried in cis: an 8 bp duplication (c.48_55dupGGATTCCG; p.Val19fs*24) and a point mutation (c.662C>T; p.Pro221Leu). The consanguineous parents carry the same two mutations within one SMN1 gene copy. We demonstrate that a more accurate diagnosis of the disease is obtained through a novel diagnostic assay and development of a capillary electrophoresis method to determine the copy number of their mutant alleles. This illustrates the complexity of SMN mutations and suggests additional testing (gene sequencing) may be appropriate when based on family lines.
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OBJECTIVE: To investigate the GCM2 gene in three siblings with congenital hypoparathyroidism and perform functional analysis. MATERIALS AND METHODS: We sequenced the GCM2 gene by PCR and analyzed the functional consequence of the mutation by transient transfection studies. Haplotype analysis was performed. RESULTS: We identified a nucleotide change, c.408C>A, in exon 3 that is predicted to truncate the Gcm2 protein (p.Tyr136Ter). All three affected siblings were homozygous and both parents were heterozygous for the mutation. Transfection studies revealed the mutant mRNA but not expression of the Gcm2 protein. Haplotype analysis revealed that the two mutant GCM2 alleles shared genotypes on chromosome 6p24.2. CONCLUSIONS: We describe the first GCM2 mutation in exon 3 in patients with severe congenital hypoparathyroidism. Informative genetic markers could not exclude identity by descent for the mutant alleles. Gcm2 protein was not detected after transfection, suggesting that complete lack of Gcm2 action accounts for severe hypoparathyroidism.
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Hipoparatiroidismo/congénito , Hipoparatiroidismo/genética , Mutación Missense , Proteínas Nucleares/genética , Factores de Transcripción/genética , Adulto , Secuencia de Bases , Niño , Familia , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Mutación Missense/fisiología , Linaje , Índice de Severidad de la EnfermedadRESUMEN
Mutations in the NK2 homeobox 1 gene (NKX2-1) cause a rare syndrome known as choreoathetosis, congenital hypothyroidism, and neonatal respiratory distress syndrome (OMIM 610978). Here we present the first reported patient with this condition caused by a 14q13.3 deletion which is adjacent to but does not interrupt NKX2-1, and review the literature on this condition. The infant presented at 23 months with a history of developmental delay, hyperkinesia, recurrent respiratory infections, neonatal respiratory distress, and hypothyroidism. Choreiform movements and delayed motor milestones were first noted at 6-8 months of age. TSH levels had been consistently elevated from 8 months of age. The clinical presentation was suggestive of an NKX2-1 mutation. Sequencing of all exons and splice site junctions of NKX2-1 was performed but was normal. Array CGH was then performed and a 3.29 Mb interstitial deletion at 14q13.1-q13.3 was detected. The distal region of loss of the deletion disrupted the surfactant associated 3 (SFTA3) gene but did disrupt NKX2-1. Findings were confirmed on high resolution SNP array and multiplex semiquanitative PCR. NKX2-1 encodes transcriptional factors involved in the developmental pathways for thyroid, lung, and brain. We hypothesize that the region centromeric to NKX2-1 is important for the normal functioning of this gene and when interrupted produces a phenotype that is typical of the choreoathetosis, congenital hypothyroidism, and neonatal respiratory distress syndrome, as seen in our patient. We conclude that deletions at 14q13.3 adjacent to but not involving NKX2-1 can cause choreoathetosis, congenital hypothyroidism, and neonatal respiratory distress syndrome.
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Atetosis/genética , Corea/genética , Hipotiroidismo Congénito/genética , Proteínas Nucleares/genética , Síndrome de Dificultad Respiratoria del Recién Nacido/genética , Factores de Transcripción/genética , Deleción Cromosómica , Cromosomas Humanos Par 14 , Femenino , Humanos , Lactante , Recién Nacido , Factor Nuclear Tiroideo 1RESUMEN
Brain-lung-thyroid disease is a rare familial disorder caused by mutations in thyroid transcription factor 1, a gene that regulates neuronal migration. We report the clinical features of ten patients from a single family with a novel gene mutation, including observations regarding treatment. Neurologic features of the kindred included developmental delay, learning difficulties, psychosis, chorea, and dystonia. Three patients had a history of seizure, which has not been previously reported in genetically confirmed cases. Low-dose dopamine-receptor blocking drugs were poorly tolerated in 2 patients who received this therapy, levodopa improved chorea in 3 of 4 children, and diazepam was markedly effective in a single adult patient. Chorea related to brain-lung-thyroid disease appears to respond paradoxically to antidopaminergic drugs. The unusual therapeutic response seen in our patients and others may help elucidate how disease-related migratory deficits affect neural pathways associated with motor control.
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Encefalopatías/genética , Predisposición Genética a la Enfermedad/genética , Enfermedades Pulmonares/genética , Mutación/genética , Proteínas Nucleares/genética , Enfermedades de la Tiroides/genética , Factores de Transcripción/genética , Adolescente , Adulto , Encefalopatías/complicaciones , Niño , Preescolar , Salud de la Familia , Femenino , Pruebas Genéticas , Humanos , Lactante , Enfermedades Pulmonares/complicaciones , Masculino , Índice de Severidad de la Enfermedad , Enfermedades de la Tiroides/complicaciones , Factor Nuclear Tiroideo 1RESUMEN
Infant respiratory distress syndrome (IRDS) can lead to impaired alveolarization and dysmorphic vascularization of bronchopulmonary dysplasia. Clara cell secretory protein (CC10) has anti-inflammatory properties but is deficient in the premature infant. Because surfactant and vascular endothelial growth factor (VEGF) profiles are impaired by inflammation and CC10 inhibits lung inflammation, we hypothesized that CC10 may up-regulate surfactant protein (SP) and VEGF expression. Preterm lambs ( N = 24; 126 +/- 3 days [standard error] gestation) with IRDS were randomized to receive 100 mg/kg surfactant, 100 mg/kg surfactant followed by intratracheal 0.5, 1.5, or 5 mg/kg rhCC10 and studied for 4 hours. Gas exchange and lung mechanics were monitored; surfactant protein and VEGF mRNA profiles in lung were assessed. There was a significant rhCC10 dose-dependent increase in respiratory compliance and ventilation efficiency index; both parameters were significantly greater in animals treated with 5 mg/kg rhCC10 than those treated with surfactant alone. Similarly, there was a significant rhCC10 dose and protein-dependent increase in surfactant protein (SP-B > SP-C > SP-A) and dose- and isoform-dependent increase in VEGF (VEGF189 > VEGF165 > VEGF121). These data demonstrate that early intervention with rhCC10 up-regulates surfactant protein and VEGF expression, supporting the role of CC10 to protect against hyperoxia and mechanical ventilation in the immature lung.
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Proteínas Asociadas a Surfactante Pulmonar/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes/farmacología , Síndrome de Dificultad Respiratoria del Recién Nacido/tratamiento farmacológico , Uteroglobina/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Animales Recién Nacidos , Productos Biológicos/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Recién Nacido , Pulmón/metabolismo , Proteínas Asociadas a Surfactante Pulmonar/farmacología , Surfactantes Pulmonares/farmacología , Distribución Aleatoria , Respiración Artificial , Síndrome de Dificultad Respiratoria del Recién Nacido/metabolismo , Pruebas de Función Respiratoria , Ovinos , Regulación hacia ArribaRESUMEN
OBJECTIVE: To report recurrent transmissions of Barth syndrome through a single oocyte donor carrying a de novo TAZ mutation. DESIGN: Case report. SETTING: Clinical molecular diagnostics laboratory. PATIENT(S): Oocyte donor and individuals conceived with her oocytes. INTERVENTION(S): Molecular testing. MAIN OUTCOME MEASURE(S): Detection of TAZ mutation. RESULT(S): Multiple individuals affected with Barth syndrome conceived from a single oocyte donor who is a carrier of a de novo TAZ mutation. CONCLUSION(S): We report multiple transmissions of Barth syndrome through a single oocyte donor with a de novo TAZ mutation.
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Cardiomiopatía Dilatada/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Mutación , Donación de Oocito/efectos adversos , Proteínas/genética , Factores de Transcripción/genética , Aciltransferasas , Cardiomiopatía Dilatada/etiología , Preescolar , Femenino , Enfermedades Genéticas Ligadas al Cromosoma X/etiología , Humanos , Masculino , SíndromeRESUMEN
Pulmonary alveolar type II cells synthesize and secrete phospholipids and surfactant proteins. In most mammalian species, the synthesis of phospholipids and proteins of lung surfactant increases with fetal lung maturation, which occurs late in gestation. Factors that may promote lung maturation and surfactant production include the placental hormone, leptin, whose expression increases with advancing gestational age. We demonstrate that physiologic concentrations of leptin (1 and 10 ng/mL) increase the levels of surfactant proteins (SP) A, B, and C mRNA as well as SP-A and SP-B protein in d-17 fetal rat lung explants in vitro. To determine whether leptin exerts similar effects in vivo, we administered leptin antenatally to pregnant rats and compared its effects to that of dexamethasone, a known mediator of fetal lung development. Antenatal treatment with leptin for 2 d significantly increased the average weight of the fetal lungs in relation to their body weight. Histologic analysis revealed that the increase in fetal lung weight was accompanied by an increase in the number and maturation of type II alveolar cells and the expression of surfactant proteins B and C in these cells. Collectively, these results suggest that leptin is a cytokine regulator of rat fetal lung maturity.