RESUMO
Due to the GC-rich, repetitive nature of C9orf72 hexanucleotide repeat expansions, PCR based detection methods are challenging. Several limitations of PCR have been reported and overcoming these could help to define the pathogenic range. There is also a need to develop improved repeat-primed PCR assays which allow detection even in the presence of genomic variation around the repeat region. We have optimised PCR conditions for the C9orf72 hexanucleotide repeat expansion, using betaine as a co-solvent and specific cycling conditions, including slow ramping and a high denaturation temperature. We have developed a flanking assay, and repeat-primed PCR assays for both 3' and 5' ends of the repeat expansion, which when used together provide a robust strategy for detecting the presence or absence of expansions greater than â¼100 repeats, even in the presence of genomic variability at the 3' end of the repeat. Using our assays, we have detected repeat expansions in 47/442 Scottish ALS patients. Furthermore, we recommend the combined use of these assays in a clinical diagnostic setting.
Assuntos
Expansão das Repetições de DNA/genética , Reação em Cadeia da Polimerase/métodos , Proteínas/genética , Alelos , Esclerose Lateral Amiotrófica/genética , Artefatos , Sequência de Bases , Proteína C9orf72 , Primers do DNA/metabolismo , Humanos , Mosaicismo , Mutação/genética , Escócia , Sensibilidade e EspecificidadeRESUMO
The sodium leak channel nonselective protein (NALCN) is a regulator of the pacemaker neurons that are responsible for rhythmic behavior (including respiration), maintaining the resting membrane potential, and are required for action potential production. NALCN-null mice show early death associated with disrupted respiratory rhythms, characterized by frequent and profound apneas. We report 3 children (2 siblings) with compound heterozygous mutations in NALCN associated with developmental impairment, hypotonia, and central sleep-disordered breathing causing apneas. Supplemental oxygen normalized the respiratory rhythm. NALCN mutations have been previously reported to cause severe hypotonia, speech impairment, and cognitive delay as well as infantile neuroaxonal dystrophy and facial dysmorphism. Nonsynonymous changes in the 2 affected extracellular loops may be responsible for the deleterious effect on the stability of the respiratory rhythm. Although oxygen is known to be a stabilizer of respiratory rhythm in central apnea in children, its role in NALCN dysfunction requires further investigation.