Lafora disease (LD) is a fatal progressive epilepsy essentially caused by loss‐of‐function mutations in the glycogen phosphatase laforin or the ubiquitin E3 ligase malin. Glycogen in LD is hyperphosphorylated and poorly hydrosoluble. It precipitates and accumulates into neurotoxic Lafora bodies (LBs). The leading LD hypothesis that hyperphosphorylation causes the insolubility was recently challenged by the observation that phosphatase‐inactive laforin rescues the laforin‐deficient LD mouse model, apparently through correction of a general autophagy impairment. We were for the first time able to quantify brain glycogen phosphate. We also measured glycogen content and chain lengths, LBs, and autophagy markers in several laforin‐ or malin‐deficient mouse lines expressing phosphatase‐inactive laforin. We find that: (i) in laforin‐deficient mice, phosphatase‐inactive laforin corrects glycogen chain lengths, and not hyperphosphorylation, which leads to correction of glycogen amounts and prevention of LBs; (ii) in malin‐deficient mice, phosphatase‐inactive laforin confers no correction; (iii) general impairment of autophagy is not necessary in LD. We conclude that laforin's principle function is to control glycogen chain lengths, in a malin‐dependent fashion, and that loss of this control underlies LD.

SUMMARY Lafora disease (LD) and adult polyglucosan body disease (APBD) are glycogen storage diseases characterized by a pathogenic buildup of insoluble glycogen. Mechanisms causing glycogen insolubility are poorly understood. Here, in two mouse models of LD (Epm2a−/− and Epm2b−/−) and one of APBD (Gbe1ys/ys), the separation of soluble and insoluble muscle glycogen is described, enabling separate analysis of each fraction. Total glycogen is increased in LD and APBD mice, which, together with abnormal chain length and molecule size distributions, is largely if not fully attributed to insoluble glycogen. Soluble glycogen consists of molecules with distinct chain length distributions and differential corresponding solubility, providing a mechanistic link between soluble and insoluble glycogen in vivo. Phosphorylation states differ across glycogen fractions and mouse models, demonstrating that hyperphosphorylation is not a basic feature of insoluble glycogen. Lastly, model-specific variances in protein and activity levels of key glycogen synthesis enzymes suggest uninvestigated regulatory mechanisms.

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BRCA1 and BRCA2 testing for hereditary breast and ovarian cancer (HBOC) does not identify all pathogenic variants. Sequencing of 20 complete genes in HBOC patients with uninformative test results (N = 287), including noncoding and flanking sequences of ATM, BARD1, BRCA1, BRCA2, CDH1, CHEK2, EPCAM, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD51B, STK11, TP53, and XRCC2, identified 38,372 unique variants. We apply information theory (IT) to predict and prioritize noncoding variants of uncertain significance in regulatory, coding, and intronic regions based on changes in binding sites in these genes. Besides mRNA splicing, IT provides a common framework to evaluate potential affinity changes in transcription factor (TFBSs), splicing regulatory (SRBSs), and RNA-binding protein (RBBSs) binding sites following mutation. We prioritized variants affecting the strengths of 10 splice sites (four natural, six cryptic), 148 SRBS, 36 TFBS, and 31 RBBS. Three variants were also prioritized based on their predicted effects on mRNA secondary (2°) structure and 17 for pseudoexon activation. Additionally, four frameshift, two in-frame deletions, and five stop-gain mutations were identified. When combined with pedigree information, complete gene sequence analysis can focus attention on a limited set of variants in a wide spectrum of functional mutation types for downstream functional and co-segregation analysis.