Genetic Variation of MARCKS & MLP in Neural Tube Defects
Gene Environmental Interactions Group
The myristoylated alanine-rich C kinase substrate, MARCKS, and its relative, the MARCKS-like protein (MLP) are well characterized acidic, amphiphilic protein kinase C (PKC) substrates that bind calmodulin and actin. MARCKS and MLP are believed to modulate rearrangement of the cortical actin cytoskeleton for various cell functions (cell spreading, migration, adhesion, neurosecretion, and endo-, exo-, and phagocytosis) and have been implicated in development of the central nervous system (CNS), neurulation, and postnatal survival.
A role for MARCKS and MLP in neurulation is supported by their patterns of expression during murine embryogenesis and results from knockout studies. MARCKS and MLP mRNAs are detected in the neural folds by embryonic day 7.5. As neurulation begins, MARCKS and MLP mRNA and protein levels increase markedly in the tips of the cranial neural folds and subsequently in the caudal region. These high levels are maintained as the neural folds approach each other and fuse. Levels of MARCKS and MLP mRNA remain elevated in the developing brain, spinal chord and cranial and peripheral nerves long after neural tube fusion, in the later stages of fetal life. Targeted disruption of Marcks and MLP in mice leads to mixed NTD phenotypes and universal perinatal death, but exencephaly (the muring equivelant to anencephaly) is the predominant finding. The exencephalic phenotype is eliminated when Marcks null mice are inter bred with a transgenic line expressing an epitope-tagged human MARCKS driven by 3.4 kb of human MARCKS promoter.
Study Design
The objective of this study is to identify genetic variants in MARCKS and MLP and test them for associations with the anencephalic phenotype using a case-control association study design. DNA collections being used in this study were obtained from two major NTD programs. The first collection was assembled by the Texas Department of Health from a famous cluster of NTD cases that occurred in 14 Texas counties along the US-Mexico border between January 1995 and February 1999. The controls are full term, normal infants matched for estimated date of birth and geographic region. All cases and controls were of Hispanic descent. The second collection of anencephalic DNAs and their matched controls were assembled by the Greenwood Genetics Center (Greenwood, SC). The majority of these were of Caucasian descent.
The 5' regulatory, protein coding regions, and intron-exon splice sites of both genes were sequenced to identify genetic variants. Each variant was tested for Hardy-Weinberg equilibrium (using standard goodness of fit analyses), allele frequencies and association (Fischer's exact), haplotype reconstruction and association (PHASE 2.0, exact permutation), and nucleotide diversity (ARLEQUIN). Genetic analyses are not yet complete but we have detected several interesting and possibly statistically significant variants that suggest that MARCKS and MLP are potential candidate genes for anencephaly, and require further study.
A role for MARCKS and MLP in neurulation is supported by their patterns of expression during murine embryogenesis and results from knockout studies. MARCKS and MLP mRNAs are detected in the neural folds by embryonic day 7.5. As neurulation begins, MARCKS and MLP mRNA and protein levels increase markedly in the tips of the cranial neural folds and subsequently in the caudal region. These high levels are maintained as the neural folds approach each other and fuse. Levels of MARCKS and MLP mRNA remain elevated in the developing brain, spinal chord and cranial and peripheral nerves long after neural tube fusion, in the later stages of fetal life. Targeted disruption of Marcks and MLP in mice leads to mixed NTD phenotypes and universal perinatal death, but exencephaly (the muring equivelant to anencephaly) is the predominant finding. The exencephalic phenotype is eliminated when Marcks null mice are inter bred with a transgenic line expressing an epitope-tagged human MARCKS driven by 3.4 kb of human MARCKS promoter.
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