RESUMEN
Autosomal recessive hereditary inclusion body myopathy (HIBM or IBM2) is a progressive adult onset muscle wasting disorder characterized by sparing of the quadriceps. IBM2 is also known as distal myopathy with rimmed vacuoles or nonaka myopathy. IBM2 is associated with mutations in the UDP-GlcNAc 2-Epimerase/ManNAc Kinase gene (GNE). GNE is the rate-limiting enzyme of N-Acetylneuraminate (Neu5Ac, Sialic acid) biosynthesis. The GNE coding region of 64 symptomatic patients were sequenced. Twenty-eight patients were found to bear GNE mutations. Ten novel mutations were identified among nine patients, including four nonsense (p.R8X, p.W204X, p.Q436X, and p.S615X) and five missense (p.R71W, p.I142T, p.I298T, p.L556S, and p.E2G) variations spanning both the epimerase and kinase domains of GNE. Additionally, a synonymous variation (p.Y591Y, codon tac > tat) was seen in a patient bearing compound heterozygous nonsynonymous mutations (p.S615X and p.Y675H). Six of the nine are Caucasian, one patient is Taiwanese, one patient is Asian Indian, and one patient is of European descent. These findings further expand the clinical and genetic spectrum of IBM2.
Asunto(s)
Miopatías Distales/enzimología , Miopatías Distales/genética , Complejos Multienzimáticos/genética , Mutación , Adulto , Alelos , Sustitución de Aminoácidos , Codón sin Sentido , Análisis Mutacional de ADN , Miopatías Distales/patología , Miopatías Distales/fisiopatología , Etnicidad/genética , Femenino , Frecuencia de los Genes , Humanos , Cuerpos de Inclusión/patología , Masculino , Persona de Mediana Edad , Complejos Multienzimáticos/química , Mutación Missense , Penetrancia , Estructura Terciaria de ProteínaRESUMEN
The 5,10-methylenetetrahydrofolate reductase gene (MTHFR) 677C>T polymorphism produces an elevation in plasma homocysteine concentrations when present in the homozygous state. Increased homocysteine levels have been associated with a greater risk for vascular diseases, including cardiovascular disease and ischemic stroke. In this study, we genotyped 42 nucleic acid samples for the C677T allele from our database of Middle Eastern patients as routine validation of the MTHFR 677C>T assay. Our study is the first to evaluate MTHFR C677T genotype frequency in a population of Middle Eastern patients residing in the United States. Among the patients, 47.6% were wild type, 40.5% were heterozygous, and 11.9% were homozygous for the C677T variant. Although C677T genotype frequency in our patient population is slightly higher than that reported by Golbahar et al. (2005), statistical analysis showed no statistically significant difference beyond chance in genotype profiles (chi(2) = 1.54, df = 2, p = 0.1675). However, our findings implicate the need for a larger sample size to explore the need to implement standard clinical screening of MTHFR 677C>T. We also highlight the robust, reliable, and reproducible assay afforded by the use of anchor and sensor hybridization probes within the LightCycler platform to perform amplification and melting curve analysis protocols. Melting curve profiles that are produced display distinct and robust T(m) peaks based on the degree of anchor and sensor hybridization to amplicons produced from template DNA that is either wild-type, heterozygous, or a homozygous variant at the MTHFR 677C>T locus. A 10 degrees C gap between T(m) peaks allows for rapid and accurate qualitative identification of genotype.
Asunto(s)
Pueblo Asiatico , Frecuencia de los Genes , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Mutación , Reacción en Cadena de la Polimerasa/métodos , Temperatura de Transición , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/genética , ADN/análisis , ADN/aislamiento & purificación , Sondas de ADN , Predisposición Genética a la Enfermedad , Genotipo , Heterocigoto , Homocigoto , Humanos , Medio Oriente/etnología , Reproducibilidad de los Resultados , Estados Unidos/epidemiología , Estados Unidos/etnologíaRESUMEN
Hereditary inclusion body myopathy/distal myopathy with rimmed vacuoles is an adult onset autosomal recessive muscle-wasting disease common in people of Iranian-Jewish descent, due to the founder allelic variant GNE:p.M712T. High correlation of disease susceptibility with GNE:p.M712T allows its use as a molecular marker for diagnosis. In this study, we applied and validated the use of melting curve analysis using SimpleProbe technology for detection of this mutation using specimens obtained by mouthwash, buccal swab, and whole blood. The assay was then applied to 43 clinical specimens, and results were validated by additional methods. A probe spanning this mutation in exon 12 accurately discerns two Tm corresponding to its hybridization to wild-type and M712T-derived amplicons. A 10 degrees C divergence in Tm allowed rapid single-tube genotyping of reference and patient samples with 100% accuracy. Distal myopathy constitutes a large heterogeneous group of pathologies with similar physiological manifestations and little molecular markers for distinguishing subtypes. Application of SimpleProbes for detection of GNE:p.M712T on genomic DNA obtained from buccal epithelial cells allows accurate, rapid, and cost-effective identification of this allele in individuals at risk. This procedure is amenable to automated high-throughput applications and can be extended to both clinical and research applications.