Novel COL6A1 splicing mutation in a family affected by mild Bethlem myopathy.

Bethlem myopathy is an early-onset benign myopathy characterized by proximal muscular weakness and multiple flexion contractures. It is a dominantly inherited disorder associated with mutations in the three COL6 genes encoding type VI collagen. We detected a g-->a substitution at +1 position of COL6A1 intron 3 in a four-generation Italian family affected by a mild form of Bethlem myopathy. The mutation results in the activation of a cryptic splice donor site at the 3' end of exon 3, leading to the loss of 66 nucleotides and an "in-frame" deletion of 22 amino acids in the NH2-domain. Molecular analysis on fibroblasts of the propositus showed that the mutated mRNA was present and stable, but the mutated protein could not be detected. Western blot and immunofluorescence analyses showed a decreased level of collagen VI synthesis and deposition in fibroblasts of the propositus. Together, the results suggest that the mutated protein was highly unstable and rapidly degraded, and that the mild phenotype was caused by a reduced amount of normal collagen VI microfibrils. In addition, we demonstrated that lymphocytes can be used for the first mutation screening analysis of patients with Bethlem myopathy.

Maternally transmitted severe glucose 6-phosphate dehydrogenase deficiency is an embryonic lethal.

Mouse chimeras from embryonic stem cells in which the X-linked glucose 6-phosphate dehydrogenase (G6PD) gene had been targeted were crossed with normal females. First-generation (F(1)) G6PD(+/-) heterozygotes born from this cross were essentially normal; analysis of their tissues demonstrated strong selection for cells with the targeted G6PD allele on the inactive X chromosome. When these F(1) G6PD(+/-) females were bred to normal males, only normal G6PD mice were born, because: (i) hemizygous G6PD(-) male embryos died by E10.5 and their development was arrested from E7.5, the time of onset of blood circulation; (ii) heterozygous G6PD(+/-) females showed abnormalities from E8.5, and died by E11.5; and (iii) severe pathological changes were present in the placenta of both G6PD(-) and G6PD(+/-) embryos. Thus, G6PD is not indispensable for early embryo development; however, severe G6PD deficiency in the extraembryonic tissues (consequent on selective inactivation of the normal paternal G6PD allele) impairs the development of the placenta and causes death of the embryo. Most importantly, G6PD is indispensable for survival when the embryo is exposed to oxygen through its blood supply.