P.P.1 02 Combined approaches to diagnosis of congenital muscular dystrophies with α-dystroglycan hypoglycosylation

Congenital muscular dystrophies (CMD) with α-dystroglycan O-mannosylation abnormalities are autosomal recessive disorders often associated with mental retardation and structural brain and ocular malformations. They are caused by mutations in at least six genes encoding enzymes. The function of three of them are known: the protein O-mannosyltransferases, POMT1 and POMT2, which initiate protein O-mannosylation, and the protein O-mannosyl N-acetylglucosaminyltransferase 1, POMGnT1, in charge of the GlcNAcb 1–2Man linkage of O-mannosyl glycans. POMGnT1 and POMT activity testing was established in immortalized lymphoblastoid cell lines from patients in order to facilitate the molecular diagnosis, and to determine whether the mutations associated with the mildest phenotypes may induce detectable reductions in enzyme activities. POMGnT1 and POMT activities were studied in lymphoblastoid cell lines from controls and CMD patients with known and unknown mutations. New mutations were identified by sequencing. POMGnT1 activity measured in lymphocytes from two MEB patients carrying homozygous POMGnT1 mutations, respectively, IVS17 + 1G > A and R442H, and POMT activity in lymphocytes from patients with LGMD or CMD patients with moderate mental retardation harboring POMT1 mutations, respectively, G65R/W582C composite heterozygote and A200P homozygously. Activity testing revealed a highly significant decrease in all cases including the mildest case. The screening of other CMD patients with unknown molecular diagnosis showed a reduced POMGnT1 enzyme activity for one patient and POMT activity for two patients. The targeted sequencing led to identification of new mutations, POMGnT1 (p.S153X/p.del269C), POMT1 (p.A669T/p.722fs > 729 stop) and POMT2 (pY666C/5′UTR–46del CAGA). Our results indicate that the combined approach of POMGnT1 and POMT activity testing and gene sequencing is useful for the identification of congenital muscular dystrophies with α-dystroglycan abnormalities. In addition, this enzymatic testing allowed the biological validation of the identified mutation as pathogenic.