8 research outputs found

    Association and Mutation Analyses of 16p11.2 Autism Candidate Genes

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    Autism is a complex childhood neurodevelopmental disorder with a strong genetic basis. Microdeletion or duplication of a approximately 500-700-kb genomic rearrangement on 16p11.2 that contains 24 genes represents the second most frequent chromosomal disorder associated with autism. The role of common and rare 16p11.2 sequence variants in autism etiology is unknown.To identify common 16p11.2 variants with a potential role in autism, we performed association studies using existing data generated from three microarray platforms: Affymetrix 5.0 (777 families), Illumina 550 K (943 families), and Affymetrix 500 K (60 families). No common variants were identified that were significantly associated with autism. To look for rare variants, we performed resequencing of coding and promoter regions for eight candidate genes selected based on their known expression patterns and functions. In total, we identified 26 novel variants in autism: 13 exonic (nine non-synonymous, three synonymous, and one untranslated region) and 13 promoter variants. We found a significant association between autism and a coding variant in the seizure-related gene SEZ6L2 (12/1106 autism vs. 3/1161 controls; p = 0.018). Sez6l2 expression in mouse embryos was restricted to the spinal cord and brain. SEZ6L2 expression in human fetal brain was highest in post-mitotic cortical layers, hippocampus, amygdala, and thalamus. Association analysis of SEZ6L2 in an independent sample set failed to replicate our initial findings.We have identified sequence variation in at least one candidate gene in 16p11.2 that may represent a novel genetic risk factor for autism. However, further studies are required to substantiate these preliminary findings

    Defining Substrate Requirements for Cleavage of Prelamin A by the Zinc Metalloprotease ZMPSTE24

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    Proteases play important roles in diverse biological processes relevant to human health and disease and defining their substrate specificity is an essential component to understanding molecular mechanisms of proteolysis. ZMPSTE24 is a zinc metalloprotease that is critical to the proteolytic maturation of lamin A, an intermediate filament protein and component of the nuclear lamina. When ZMPSTE24-dependent processing of prelamin A is disrupted by mutations in either the substrate or the protease, accumulation of uncleaved prelamin A causes a spectrum of genetic disorders including the premature aging disease Hutchinson Gilford Progeria Syndrome (HGPS) and related progeroid disorders mandibuloacral dysplasia (MAD-B) and restrictive dermopathy (RD). Prelamin A is the only known mammalian substrate for ZMPSTE24, however, the basis of this specificity remains unclear. To begin to define the sequence requirements for ZMPSTE24 recognition, I have performed a comprehensive mutagenesis scan of the eight residues flanking the cleavage site, from amino acid residue T643 to N650 (TRSY↓LLGN). Mutants were tested in an in vivo humanized yeast assay that provides a sensitive measure of ZMPSTE24 processing efficiency. Substitutions C-terminal to the cleavage site were generally more disruptive than N-terminal substitutions, although R644 shows a modest bias for positively charged residues. Of particular note is L647 in the P1’ position C-terminal to the cleavage site, of which mutation to arginine has long been known to be “uncleavable.” Here I show that charged residues, aromatics, and proline disrupt cleavage, while hydrophobic residues are well-tolerated. L648 at P2’ shows a similar pattern with several key differences. I have generated a heat map of cleavage efficacy for ZMPSTE24 and positions P1’ and P2’ have emerged as critical for ZMPSTE24 substrate recognition, with a strong preference for hydrophobic residues. Hydrophobic residues at these two positions are evolutionarily conserved. I have shown that the 8 residues flanking the deduced ZMPSTE24 cleavage site in prelamin A sequences from amphibians, birds, fish, and mammals can promote cleavage when placed in the context of human lamin A, albeit with varying efficiency. Taken together, my results provide mechanistic insights into the recognition of prelamin A by ZMPSTE24 and begin to define a consensus motif with potential predictive value to identify other ZMPSTE24 substrates

    Defining Substrate Requirements for Cleavage of Prelamin A by the Zinc Metalloprotease ZMPSTE24

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    Proteases play important roles in diverse biological processes relevant to human health and disease and defining their substrate specificity is an essential component to understanding molecular mechanisms of proteolysis. ZMPSTE24 is a zinc metalloprotease that is critical to the proteolytic maturation of lamin A, an intermediate filament protein and component of the nuclear lamina. When ZMPSTE24-dependent processing of prelamin A is disrupted by mutations in either the substrate or the protease, accumulation of uncleaved prelamin A causes a spectrum of genetic disorders including the premature aging disease Hutchinson Gilford Progeria Syndrome (HGPS) and related progeroid disorders mandibuloacral dysplasia (MAD-B) and restrictive dermopathy (RD). Prelamin A is the only known mammalian substrate for ZMPSTE24, however, the basis of this specificity remains unclear. To begin to define the sequence requirements for ZMPSTE24 recognition, I have performed a comprehensive mutagenesis scan of the eight residues flanking the cleavage site, from amino acid residue T643 to N650 (TRSY↓LLGN). Mutants were tested in an in vivo humanized yeast assay that provides a sensitive measure of ZMPSTE24 processing efficiency. Substitutions C-terminal to the cleavage site were generally more disruptive than N-terminal substitutions, although R644 shows a modest bias for positively charged residues. Of particular note is L647 in the P1’ position C-terminal to the cleavage site, of which mutation to arginine has long been known to be “uncleavable.” Here I show that charged residues, aromatics, and proline disrupt cleavage, while hydrophobic residues are well-tolerated. L648 at P2’ shows a similar pattern with several key differences. I have generated a heat map of cleavage efficacy for ZMPSTE24 and positions P1’ and P2’ have emerged as critical for ZMPSTE24 substrate recognition, with a strong preference for hydrophobic residues. Hydrophobic residues at these two positions are evolutionarily conserved. I have shown that the 8 residues flanking the deduced ZMPSTE24 cleavage site in prelamin A sequences from amphibians, birds, fish, and mammals can promote cleavage when placed in the context of human lamin A, albeit with varying efficiency. Taken together, my results provide mechanistic insights into the recognition of prelamin A by ZMPSTE24 and begin to define a consensus motif with potential predictive value to identify other ZMPSTE24 substrates

    TUBA1A mutations cause wide spectrum lissencephaly (smooth brain) and suggest that multiple neuronal migration pathways converge on alpha tubulins

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    We previously showed that mutations in LIS1 and DCX account for ∼85% of patients with the classic form of lissencephaly (LIS). Some rare forms of LIS are associated with a disproportionately small cerebellum, referred to as lissencephaly with cerebellar hypoplasia (LCH). Tubulin alpha1A (TUBA1A), encoding a critical structural subunit of microtubules, has recently been implicated in LIS. Here, we screen the largest cohort of unexplained LIS patients examined to date to determine: (i) the frequency of TUBA1A mutations in patients with lissencephaly, (ii) the spectrum of phenotypes associated with TUBA1A mutations and (iii) the functional consequences of different TUBA1A mutations on microtubule function. We identified novel and recurrent TUBA1A mutations in ∼1% of children with classic LIS and in ∼30% of children with LCH, making this the first major gene associated with the rare LCH phenotype. We also unexpectedly found a TUBA1A mutation in one child with agenesis of the corpus callosum and cerebellar hypoplasia without LIS. Thus, our data demonstrate a wider spectrum of phenotypes than previously reported and allow us to propose new recommendations for clinical testing. We also provide cellular and structural data suggesting that LIS-associated mutations of TUBA1A operate via diverse mechanisms that include disruption of binding sites for microtubule-associated proteins (MAPs)

    Immunotherapy of Metastatic Colorectal Cancer: Prevailing Challenges and New Perspectives

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