Linkage analysis of the family suffering from autosomal recessive atypical congenital myopathy.

<p>(<b>A</b>) Pedigree of the family. Filled and unfilled symbols represent affected and unaffected individuals, respectively. The arrows denote individuals whose DNA samples were analyzed by SNP250K. (<b>B</b>) Multipoint linkage analysis using SNP data showing LOD score <i>Z_max</i> = 3.86 at θ = 0.0 on chromosome 19q13. X-axis: genetic distance in cM., Y-axis: Lod score.</p

Effect of the pY3016C mutation on RyR1 protein expression on patient and control muscle biopsies.

<p>The western blot shows a dramatic decrease of the RyR1 protein and of the DHPRalpha 1.1 expression in the patient’s biopsy compared to control’s biopsy (P<0.05). Protein expression levels were quantified by densitometric analysis and normalized to the expression of myosin heavy chain. The bar plot on the right shows the mean % protein content (± SEM; of 3 different western blots) in biopsies from a control and patient Y3016C−/− (P<0.05 by the Student <i>t</i> test).</p

Clinical features and laboratory findings in the patients with myopathy.

<p>F: female; M: male; N: normal; FSV: fiber-size variation; IDN: internally-displaced nuclei; EF: endomysial fibrosis; Y:yes; No: No.</p

Histological analysis of patients muscle biopsies.

<p>Frozen sections of 3 cases (V28, V31 & V36) that were available for pathological review display non-specific dystrophic-like changes, consistent with muscular dystrophy. H&E stained sections (A–C) show marked variation in myofiber-diameter, in random distribution. The number of internally displaced nuclei (white arrows) is markedly increased. There are no clear-cut signs of necrosis, regeneration or any other specific structural change in the myofibers. There is focal endomysial fibrosis (black arrows). There is no inflammatory infiltrate. The blood vessels are unremarkable. NADH histochemical stain (D–F) is not showing significant changes in the cytoarchitecture, except for occasional moth-eaten-like fibers (red arrows) and overstaining of atrophic fibers (yellow fibers). (Original magnification ×40; Bars = 50 μm).</p

Analysis of <i>RYR1</i> at the DNA level in patients and controls.

<p>(<b>A</b>) Sequence of the <i>RYR1</i> gene revealed a homozygous A to G nucleotide substitution leading to an amino acid change (p.Y3016C) within exon 60 in patient (arrow). (<b>B</b>) Analysis of the mutation in family members and control. Left panel: PCR amplification products of <i>RYR1</i> from exon -60 to intron-60 using primers specific to the mutated allele (MUT Primers, product size = 210 bp). Right panel: PCR amplification products of <i>RYR1</i> from exon-60 to intron – 60 using primers specific to wild type allele (WT Primers, product size = 210 bp). This test confirms the cosegregation of the <i>RYR1</i> mutation with the phenotype and haplotypes in the family. C: control. Affected individuals are underlined.</p

Ca²⁺ homeostasis analysis in EBV immortalized cells from affected, non affected family members and controls.

<p>(<b>A</b>) Comparison of the resting [Ca²⁺]_i shows lower [Ca²⁺]_i in cells carrying the mutation compared with control cells. **P<0.001 ANOVA followed by the Bonferroni post hoc test (<b>B</b>) The thapsigargin-induced Ca²⁺ release in lymphoblastoid B cells are represented by the difference between the resting [Ca²⁺]_i and the [Ca²⁺]_i after addition of 400 nM thapsigargin. No significant differences between cells carrying the homozygous or heterozygous p.Y3016C mutation were found. (<b>C</b>) Dose-dependent 4-chloro-m-cresol induced Ca²⁺ release in lymphoblastoid B cells. No significant changes were evident at concentrations <750 µM though at higher concentrations cells carrying the heterozygous and homozygous p.Y3016C substitution showed significantly lower Ca²⁺ release compared to control cells ***P<0.05 (ANOVA and Bonferroni post hoc test, P<0.05).</p