Sean Hanson Human Genetics

Mutant GlialCAM Causes Megalencephalic Leukoencephalopathy with Subcortical Cysts, Benign Familial Macrocephaly, and Macrocephaly with Retardation and Autism

Hypothesis: The authors knew from previous studies that MLC1 mutations are observed in 75% of MLC patients. The authors were trying to find other gene mutations besides MLC1 that cause megalencephalic leukoencephalopathy with subcortical cysts (MLC). Tests Performed: The authors first did a proteomic analysis of affinity-purified MLC1. They did this by creating plasma membrane-enriched protein fractions from pools of freshly isolated whole rat or mouse brains. They then prepared antibodies. Polyvinylidene fluoride membranes were probed with rabbit polyclonal antibodies a-N1 or a-NH, stained with goat anti-rabbit-HRP and developed with ECL+. Mass spectrometric analysis was first performed. They then used two protein quantification methods to evaluate the results. The relative quantity of a protein in affinity purification sample versus control was calculated and set as a median. Proteins were regarded as specifically purified if values were higher than 10. The diagnosis of MLC was established by MRI criteria. In MLC patients whom they found no MLC1 mutations by sequence analysis of genomic DNA, they analyzed HEPACAM in the individuals. By looking at the exon and intronic regions. The authors also prepared primary astrocytes from newborn rats and transduced andenoviruses expressing three copies of the epitope fused to wild-type human

GlialCAM or to recessive mutations p.Arg92Gln, p.Arg98Cys, and p.Ser196Tyr or the dominant mutations p.Arg92Trp and p.Gly89Asp. Results of Tests: The authors used quantitative proteomic analysis of affinity-purified MLC1 to see if there were other candidate genes for MLC. The results showed that three different antibodies were elevated all of which were directed against the peptides from MLC1 N terminus. The authors then looked at the quantitative mass spectrometry and they identified HepaCAM/GlialCAM as the protein that had the highest concentration in all of the purifications ran. Reverse affinity purification experiments confirmed that GlialCAM and MLC1 interact with each other. After the authors analyzed the exons and introns of HEPACAM in 40 patients from 34 different families around the world 10 of the patients from 8 families had HEPACAM mutations. After sequence analysis of this mutation in the families the authors determined the mutation inheritance to be mainly autosomal-recessive but autosomal-dominant inheritance can occur. The authors found that of the patients that have two HEPACAM mutations inherited in autosomal-recessive fashion had the classical phenotype of macrocephaly, delayed-onset motor deterioration, epilepsy, and cognitive decline. Patients that had one HEPACAM mutation had improving phenotype. The classical phenotype improved to almost normal or almost normal motor skills, and some had normal IQ. When the authors looked at the interaction between GlialCAM and MLC1 they noticed that the immunohistochemistry of human brain tissue showed mRNA of GlialCAM and protein detected in oligodendrocytes, astorcytes, and neurons. The MLC1 was not detected in oligodendrocytes. Looking at the astrocytes in the rats the MLC1 and

GlialCAM were located in cell-cell junction between astocytes.

Conclutions: The authors were trying to find what caused MLS in the 25% of people that don't have MLC1 mutations. Genetic-linkage studies failed to identify other genes responsible for MLS so authors created other tests to help identify other genes involved. They discovered HELPACAM as a gene disrupted in MLC patients. It is predominantly expressed in the CNS and can be referred to as GlialCAM. The HEPACAM mutations were either recessive or dominant. The recessive mutations were spread over the extracellular region of GlialCAM, and the dominant mutations were clustered in one of the first immunoglobulin-like domains. These mutations were both autosomal dominant inheritance and autosomal-recessive inheritance. The autosomal-domant inheritance showed improving phenotypes, while the autosomal-recessive inheritance showed consistent phenotypes. There were 12 MLS patients from ten families that had neither MLC1 or HEPACAM mutations. This draws questions on whether or not there are other genes involved, or the presence of hidden MLC1 and HEPACAM mutations.

Information not presented in introduction: There was no information about the types of test they were planning on performing to try to identify other genes involved. They stated that MLS is a autosomalrecessive inheritance but MLS can also be autosomal-dominant inheritance. They didn't tell us how they discovered that MLC1 mutations are involved with MLS. All in all they did a pretty good job describing the study in the introduction.

Information I wanted to see included I the Study: I wished the authors included how the patients with autosomal-dominant inheritance improved phenotypically. Or how the autosomal-recessive inherited individuals didn't improve. If the authors described any information on the function of MLC1 or GlialCAM it would help my understanding of the study immensely.

Tania Lpez-Hernndez Margreet C. Ridder, Marisol Montolio,Xavier CapdevilaNortes Emiel Polder Snia Sirisi, Anna Duarri Uwe Schulte Bernd Fakler, Virginia Nunes, Gert C. Scheper, Albert Martnez, Ral Estvez , and Marjo S. van der Knaap (2011) Mutant GlialCAM Causes Megalencephalic Leukoencephalopathy with Subcortical Cysts, Benign Familial Macrocephaly, and Macrocephaly with Retardation and Autis. American Journal of Human Genetics Volume 88, Issue 4, 422-432