A rapid screening with direct sequencing from blood samples for the diagnosis of Leigh syndrome

Large numbers of genes are responsible for Leigh syndrome (LS), making genetic confirmation of LS difficult. We screened our patients with LS using a limited set of 21 primers encompassing the frequently reported gene for the respiratory chain complexes I (ND1–ND6, and ND4L), IV(SURF1), and V(ATP6) and the pyruvate dehydrogenase E1α-subunit. Of 18 LS patients, we identified mutations in 11 patients, including 7 in mDNA (two with ATP6), 4 in nuclear (three with SURF1). Overall, we identified mutations in 61% of LS patients (11/18 individuals) in this cohort. Sanger sequencing with our limited set of primers allowed us a rapid genetic confirmation of more than half of the LS patients and it appears to be efficient as a primary genetic screening in this cohort

Reduced expression of sarcospan in muscles of Fukuyama congenital muscular dystrophy

Summary. Expression profiles of sarcospan in muscles with muscular dystrophies are scarcely reported. To examine this, we studied five Fukuyama congenital muscular dystrophy (FCMD) muscles, five Duchenne muscular dystrophy (DMD) muscles, five disease control and five normal control muscles. Immunoblot showed reactions of sarcospan markedly decreased in FCMD and DMD muscle extracts. Immunohistochemistry of FCMD muscles showed that most large diameter myofibers expressed sarcospan discontinuously at their surface membranes. Immature small diameter FCMD myofibers usually did not express sarcospan. Immunoreactivity of sarcospan in DMD muscles was similarly reduced. With regard to dystroglycans and sarcoglycans, immunohistochemistry of FCMD muscles showed selective deficiency of glycosylated α- dystroglycan, together with reduced expression of ß- dystroglycan and α-, ß-, γ-, δ-sarcoglycans. Although the expression of glycosylated α-dystroglycan was lost, scattered FCMD myofibers showed positive immunoreaction with an antibody against the core protein of α-dystroglycan. The group mean ratios of sarcospan mRNA copy number versus GAPDH mRNA copy number by real-time RT-PCR showed that the ratios between FCMD and normal control groups were not significantly different (P>0.1 by the two-tailed t test). This study implied either O-linked glycosylation defects of α-dystroglycan in the Golgi apparatus of FCMD muscles may lead to decreased expression of sarcoglycan and sarcospan molecules, or selective deficiency of glycosylated α-dystroglycan due to impaired glycosylation in FCMD muscles may affect the molecular integrity of the basal lamina of myofibers. This, in turn, leads to decreased expression of sarcoglycans, and finally of sarcospan at the FCMD myofiber surfaces

Identification of <em>ATP1A3</em> Mutations by Exome Sequencing as the Cause of Alternating Hemiplegia of Childhood in Japanese Patients

<div><h3>Background</h3><p>Alternating hemiplegia of childhood (AHC) is a rare disorder characterized by transient repeated attacks of paresis and cognitive impairment. Recent studies from the U.S. and Europe have described <em>ATP1A3</em> mutations in AHC. However, the genotype-phenotype relationship remains unclear. The purpose of this study was to identify the genetic abnormality in a Japanese cohort of AHC using exome analysis.</p> <h3>Principal Findings</h3><p>A total of 712,558 genetic single nucleotide variations in 8 patients with sporadic AHC were found. After a series of exclusions, mutations of three genes were regarded as candidate causes of AHC. Each patient harbored a heterozygous missense mutation of <em>ATP1A3</em>, which included G755C, E815K, C927Y and D801N. All mutations were at highly conserved amino acid residues and deduced to affect ATPase activity of the corresponding ATP pump, the product of <em>ATP1A3</em>. They were <em>de novo</em> mutations and not identified in 96 healthy volunteers. Using Sanger sequencing, E815K was found in two other sporadic cases of AHC. In this study, E815K was found in 5 of 10 patients (50%), a prevalence higher than that reported in two recent studies [19 of 82 (23%) and 7 of 24 (29%)]. Furthermore, the clinical data of the affected individuals indicated that E815K resulted in a severer phenotype compared with other <em>ATP1A3</em> mutations.</p> <h3>Interpretation</h3><p>Heterozygous <em>de novo</em> mutations of <em>ATP1A3</em> were identified in all Japanese patients with AHC examined in this study, confirming that <em>ATP1A3</em> mutation is the cause of AHC.</p> </div

Chromatograms of four <i>de novo</i> mutations identified in <i>ATP1A3</i>.

<p>Data were obtained by Sanger sequencing during the confirmation process. In trio of each pedigree, black shadow represents the proband. In the chromatograms, <i>Black letters</i> show exonic nucleotide sequences, <i>gray letters</i> show intronic nucleotide sequences. Amino acids are shown in a single letter notation. Nucleotides and amino acids in red indicate mutations. (A) G755C was identified only in Patient I-1. (B) E815K was identified in Patients II-1, III-1, IV-1, IX-1 and X-1. (C) C927Y was identified in Patient V-1 only. (D) D801N was identified in Patients VI-1, VII-1 and VIII-1. None of the mutations was detected in the father or mother except for Patient IX-1, whose parents refused to undergo genetic analysis.</p

Pipeline for detection of novel <i>de nov</i>o mutations.

<p>The pipeline was used to identify pathogenic mutations of alternating hemiplegia of childhood (AHC). All genetic variants detected by exome sequencing are sequentially filtered through the pipeline. First, variations are screened according to databases of registered single nucleotide polymorphisms (SNP) and only non-registered SNP undergo the next selection as “Novel variants”. In the next step, non-synonymous novel variants of genes expressed in the central nervous system are selected. When variations of the same gene are found in the patient, the impact of such variation is evaluated <i>in silico</i> using Grantham score and PolyPhen-2. Mutations identified at this stage are reconfirmed by Sanger sequence. <i>De novo</i> mutation is validated by analyzing samples from parents. Mutations considered pathogenic are sought in other patients with AHC if necessary.</p