Marissa A. LeBlanc

Mutations in origin recognition complex gene ORC4 cause Meier-Gorlin syndrome

Meier-Gorlin syndrome is a rare autosomal recessive genetic condition whose primary clinical hallmarks include small stature, small external ears and small or absent patellae. Using marker-assisted mapping in multiple families from a founder population and traditional coding exon sequencing of positional candidate genes, we identified three different mutations in the gene encoding ORC4, a component of the eukaryotic origin recognition complex, in five individuals with Meier-Gorlin syndrome. In two such individuals that were negative for mutations in ORC4, we found potential mutations in ORC1 and CDT1, two other genes involved in origin recognition. ORC4 is well conserved in eukaryotes, and the yeast equivalent of the human ORC4 missense mutation was shown to be pathogenic in functional assays of cell growth. This is the first report, to our knowledge, of a germline mutation in any gene of the origin recognition complex in a vertebrate organism.

Lipid Binding Requirements for Oxysterol-binding Protein Kes1 Inhibition of Autophagy and Endosome-trans-Golgi Trafficking Pathways

The Saccharomyces cerevisiae protein Kes1/Osh4 is a member of the enigmatic family of oxysterol-binding proteins found throughout Eukarya united by a β-barrel structure that binds sterols and oxysterols. In this study, we determined that phosphoinositides are the major determinant in membranes that facilitate Kes1 association both in vitro and in cells. Increased expression of Kes1 in yeast cells decreased the levels of both phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 3-phosphate (PI3P). Phosphoinositide and sterol bindings by Kes1 were necessary for Kes1 to decrease the level of PI4P but not PI3P. Kes1 inhibited vesicular trafficking between the trans-Golgi and plasma membrane as evidenced by accumulation of the vacuolar soluble NSF attachment protein receptors Snc1 in the cytoplasmic vesicles. Sterol and phosphoinositide binding by Kes1 both contributed to its regulation of Snc1 trafficking. This study also describes a previously unknown role for Kes1 in the regulation of the autophagy/cytoplasm to the vacuole trafficking pathway. The Kes1-mediated regulation of the autophagy/cytoplasm to the vacuole trafficking pathway was prevented by increasing expression of the PI3K Vps34, suggesting that it is the Kes1-mediated decrease in PI3P level that contributes to this regulation.

Germline mutations in MAP3K6 are associated with familial gastric cancer.

Gastric cancer is among the leading causes of cancer-related deaths worldwide. While heritable forms of gastric cancer are relatively rare, identifying the genes responsible for such cases can inform diagnosis and treatment for both hereditary and sporadic cases of gastric cancer. Mutations in the E-cadherin gene, CDH1, account for 40% of the most common form of familial gastric cancer (FGC), hereditary diffuse gastric cancer (HDGC). The genes responsible for the remaining forms of FGC are currently unknown. Here we examined a large family from Maritime Canada with FGC without CDH1 mutations, and identified a germline coding variant (p.P946L) in mitogen-activated protein kinase kinase kinase 6 (MAP3K6). Based on conservation, predicted pathogenicity and a known role of the gene in cancer predisposition, MAP3K6 was considered a strong candidate and was investigated further. Screening of an additional 115 unrelated individuals with non-CDH1 FGC identified the p.P946L MAP3K6 variant, as well as four additional coding variants in MAP3K6 (p.F849Sfs*142, p.P958T, p.D200Y and p.V207G). A somatic second-hit variant (p.H506Y) was present in DNA obtained from one of the tumor specimens, and evidence of DNA hypermethylation within the MAP3K6 gene was observed in DNA from the tumor of another affected individual. These findings, together with previous evidence from mouse models that MAP3K6 acts as a tumor suppressor, and studies showing the presence of somatic mutations in MAP3K6 in non-hereditary gastric cancers and gastric cancer cell lines, point towards MAP3K6 variants as a predisposing factor for FGC.

Focused chemical genomics using zebrafish xenotransplantation as a pre-clinical therapeutic platform for T-cell acute lymphoblastic leukemia

Cancer therapeutics is evolving to precision medicine, with the goal of matching targeted compounds with molecular aberrations underlying a patient’s cancer. While murine models offer a pre-clinical tool, associated costs and time are not compatible with actionable patient-directed interventions. Using the paradigm of T-cell acute lymphoblastic leukemia, a high-risk disease with defined molecular underpinnings, we developed a zebrafish human cancer xenotransplantation model to inform therapeutic decisions. Using a focused chemical genomic approach, we demonstrate that xenografted cell lines harboring mutations in the NOTCH1 and PI3K/AKT pathways respond concordantly to their targeted therapies, patient-derived T-cell acute lymphoblastic leukemia can be successfully engrafted in zebrafish and specific drug responses can be quantitatively determined. Using this approach, we identified a mutation sensitive to γ-secretase inhibition in a xenograft from a child with T-cell acute lymphoblastic leukemia, confirmed by Sanger sequencing and validated as a gain-of-function NOTCH1 mutation. The zebrafish xenotransplantation platform provides a novel cost-effective means of tailoring leukemia therapy in real time.

Phenotypic overlap between familial exudative vitreoretinopathy and microcephaly, lymphedema, and chorioretinal dysplasia caused by KIF11 mutations.

IMPORTANCE Retinal detachment with avascularity of the peripheral retina, typically associated with familial exudative vitreoretinopathy (FEVR), can result from mutations in KIF11, a gene recently identified to cause microcephaly, lymphedema, and chorioretinal dysplasia (MLCRD) as well as chorioretinal dysplasia, microcephaly, and mental retardation (CDMMR). Ophthalmologists should be aware of the range of presentations for mutations in KIF11 because the phenotypic distinction between FEVR and MLCRD/CDMMR portends management implications in patients with these conditions. OBJECTIVE To identify gene mutations in patients who present with a FEVR phenotype and explore the spectrum of ocular and systemic abnormalities caused by KIF11 mutations in a cohort of patients with FEVR or microcephaly in conjunction with chorioretinopathy or FEVR. DESIGN, SETTING, AND PARTICIPANTS Clinical data and DNA were collected from each participant between 1998 and 2013 from the clinical practices of ophthalmologists and clinical geneticists internationally. Twenty-eight FEVR probands with diagnoses made by the referring physician and without a known FEVR gene mutation, and 3 with microcephaly and chorioretinopathy, were included. At least 1 patient in each pedigree manifested 1 or more of the following: macular dragging, partial retinal detachment, falciform folds, or total retinal detachment. EXPOSURES Whole-exome sequencing was conducted on affected members in multiplex pedigrees, and Sanger sequencing of the 22 exons of the KIF11 gene was performed on singletons. Clinical data and history were collected and reviewed. MAIN OUTCOMES AND MEASURES Identification of mutations in KIF11. RESULTS Four novel heterozygous KIF11 mutations and 1 previously published mutation were identified in probands with FEVR: p.A218Gfs*15, p.E470X, p.R221G, c.790-1G>T, and the previously described heterozygous p.R47X. Documentation of peripheral avascular areas on intravenous fluorescein angiography was possible in 2 probands with fibrovascular proliferation demonstrating phenotypic overlap with FEVR. CONCLUSIONS AND RELEVANCE Mutations in KIF11 cause a broader spectrum of ocular disease than previously reported, including retinal detachment. The KIF11 gene likely plays a role in retinal vascular development and mutations in this gene can lead to clinical overlap with FEVR. Cases of FEVR should be carefully inspected for the presence of microcephaly as a marker for KIF11-related disease to enhance the accuracy of the prognosis and genetic counseling.

The Yeast Oxysterol Binding Protein Kes1 Maintains Sphingolipid Levels

The oxysterol binding protein family are amphitropic proteins that bind oxysterols, sterols, and possibly phosphoinositides, in a conserved binding pocket. The Saccharomyces cerevisiae oxysterol binding protein family member Kes1 (also known as Osh4) also binds phosphoinositides on a distinct surface of the protein from the conserved binding pocket. In this study, we determine that the oxysterol binding protein family member Kes1 is required to maintain the ratio of complex sphingolipids and levels of ceramide, sphingosine-phosphate and sphingosine. This inability to maintain normal sphingolipid homeostasis resulted in misdistribution of Pma1, a protein that requires normal sphingolipid synthesis to occur to partition into membrane rafts at the Golgi for its trafficking to the plasma membrane.

A novel rearrangement of occludin causes brain calcification and renal dysfunction

Pediatric intracranial calcification may be caused by inherited or acquired factors. We describe the identification of a novel rearrangement in which a downstream pseudogene translocates into exon 9 of OCLN, resulting in band-like brain calcification and advanced chronic kidney disease in early childhood. SNP genotyping and read-depth variation from whole exome sequencing initially pointed to a mutation in the OCLN gene. The high degree of identity between OCLN and two pseudogenes required a combination of multiplex ligation-dependent probe amplification, PCR, and Sanger sequencing to identify the genomic rearrangement that was the underlying genetic cause of the disease. Mutations in exon 3, or at the 5–6 intron splice site, of OCLN have been reported to cause brain calcification and polymicrogyria with no evidence of extra-cranial phenotypes. Of the OCLN splice variants described, all make use of exon 9, while OCLN variants that use exons 3, 5, and 6 are tissue specific. The genetic rearrangement we identified in exon 9 provides a plausible explanation for the expanded clinical phenotype observed in our individuals. Furthermore, the lack of polymicrogyria associated with the rearrangement of OCLN in our patients extends the range of cranial defects that can be observed due to OCLN mutations.

Localization of lipid raft proteins to the plasma membrane is a major function of the phospholipid transfer protein Sec14.

The Sec14 protein domain is a conserved tertiary structure that binds hydrophobic ligands. The Sec14 protein from Saccharomyces cerevisiae is essential with studies of S. cerevisiae Sec14 cellular function facilitated by a sole temperature sensitive allele, sec14ts. The sec14ts allele encodes a protein with a point mutation resulting in a single amino acid change, Sec14G266D. In this study results from a genome-wide genetic screen, and pharmacological data, provide evidence that the Sec14G266D protein is present at a reduced level compared to wild type Sec14 due to its being targeted to the proteosome. Increased expression of the sec14ts allele ameliorated growth arrest, but did not restore the defects in membrane accumulation or vesicular transport known to be defective in sec14ts cells. We determined that trafficking and localization of two well characterized lipid raft resident proteins, Pma1 and Fus-Mid-GFP, were aberrant in sec14ts cells. Localization of both lipid raft proteins was restored upon increased expression of the sec14ts allele. We suggest that a major function provided by Sec14 is trafficking and localization of lipid raft proteins.

Surprising roles for phospholipid binding proteins revealed by high throughput genetics

Saccharomyces cerevisiae remains an ideal organism for studying the cell biological roles of lipids in vivo, as yeast has phospholipid metabolic pathways similar to mammalian cells, is easy and economical to manipulate, and is genetically tractable. The availability of isogenic strains containing specific genetic inactivation of each non-essential gene allowed for the development of a high-throughput method, called synthetic genetic analysis (SGA), to identify and describe precise pathways or functions associated with specific genes. This review describes the use of SGA to aid in elucidating the function of two lipid-binding proteins that regulate vesicular transport, Sec14 and Kes1. Sec14 was first identified as a phosphatidylcholine (PC) - phosphatidylinositol (PI) transfer protein required for viability, with reduced Sec14 function resulting in diminished vesicular transport out of the trans-Golgi. Although Sec14 is required for cell viability, inactivating the KES1 gene that encodes for a member of the oxysterol binding protein family in cells lacking Sec14 function results in restoration of vesicular transport and cell growth. SGA analysis identified a role for Kes1 and Sec14 in regulating the level and function of Golgi PI-4-phosphate (PI-4-P). SGA also determined that Sec14 not only regulates vesicular transport out of the trans-Golgi, but also transport from endosomes to the trans-Golgi. Comparing SGA screens in databases, coupled with genetic and cell biological analyses, further determined that the PI-4-P pool affected by Kes1 is generated by the PI 4-kinase Pik1. An important biological role for Sec14 and Kes1 revealed by SGA is coordinate regulation of the Pik1-generated Golgi PI-4-P pool that in turn is essential for vesicular transport into and out of the trans-Golgi.

Study of Glycine and Folic Acid Supplementation to Ameliorate Transfusion Dependence in Congenital SLC25A38 Mutated Sideroblastic Anemia.

Congenital sideroblastic anemia (CSA) is a hematological disorder characterized by the presence of ringed sideroblasts in bone marrow erythroid precursors. Mutations in the erythroid‐specific glycine mitochondrial transporter gene SLC25A38 have been found in a subset of patients with transfusion‐dependent congenital CSA. Further studies in a zebrafish model identified a promising ameliorative strategy with combined supplementation with glycine and folate. We tested this combination in three individuals with SLC25A38 CSA, with a primary objective to decrease red blood cell transfusion requirements. No significant impact was observed on transfusion requirements or any hematologic parameters.