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1.
Am J Hum Genet ; 108(1): 163-175, 2021 01 07.
Article in English | MEDLINE | ID: mdl-33357406

ABSTRACT

The lack of functional evidence for the majority of missense variants limits their clinical interpretability and poses a key barrier to the broad utility of carrier screening. In Lynch syndrome (LS), one of the most highly prevalent cancer syndromes, nearly 90% of clinically observed missense variants are deemed "variants of uncertain significance" (VUS). To systematically resolve their functional status, we performed a massively parallel screen in human cells to identify loss-of-function missense variants in the key DNA mismatch repair factor MSH2. The resulting functional effect map is substantially complete, covering 94% of the 17,746 possible variants, and is highly concordant (96%) with existing functional data and expert clinicians' interpretations. The large majority (89%) of missense variants were functionally neutral, perhaps unexpectedly in light of its evolutionary conservation. These data provide ready-to-use functional evidence to resolve the ∼1,300 extant missense VUSs in MSH2 and may facilitate the prospective classification of newly discovered variants in the clinic.


Subject(s)
Genetic Predisposition to Disease/genetics , MutS Homolog 2 Protein/genetics , Mutation, Missense/genetics , Colorectal Neoplasms, Hereditary Nonpolyposis/genetics , DNA Mismatch Repair/genetics , HEK293 Cells , Humans
2.
Sci Rep ; 9(1): 9609, 2019 07 03.
Article in English | MEDLINE | ID: mdl-31270356

ABSTRACT

Defective biosynthesis of the phospholipid PI(3,5)P2 underlies neurological disorders characterized by cytoplasmic accumulation of large lysosome-derived vacuoles. To identify novel genetic causes of lysosomal vacuolization, we developed an assay for enlargement of the lysosome compartment that is amenable to cell sorting and pooled screens. We first demonstrated that the enlarged vacuoles that accumulate in fibroblasts lacking FIG4, a PI(3,5)P2 biosynthetic factor, have a hyperacidic pH compared to normal cells'. We then carried out a genome-wide knockout screen in human HAP1 cells for accumulation of acidic vesicles by FACS sorting. A pilot screen captured fifteen genes, including VAC14, a previously identified cause of endolysosomal vacuolization. Three genes not previously associated with lysosome dysfunction were selected to validate the screen: C10orf35, LRRC8A, and MARCH7. We analyzed two clonal knockout cell lines for each gene. All of the knockout lines contained enlarged acidic vesicles that were positive for LAMP2, confirming their endolysosomal origin. This assay will be useful in the future for functional evaluation of patient variants in these genes, and for a more extensive genome-wide screen for genes required for endolysosome function. This approach may also be adapted for drug screens to identify small molecules that rescue endolysosomal vacuolization.


Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Gene Knockout Techniques , Genetic Association Studies , Genetic Testing , Lysosomes/metabolism , Animals , Base Sequence , Biomarkers , Cell Line , Cellular Microenvironment , Fibroblasts , Flavoproteins/genetics , Gene Expression , High-Throughput Screening Assays , Hydrogen-Ion Concentration , Immunophenotyping , Mice , Mutation , Phosphoinositide Phosphatases/genetics , Sequence Analysis, DNA
3.
Article in English | MEDLINE | ID: mdl-29739035

ABSTRACT

BACKGROUND: Congenital Hypopituitarism is caused by genetic and environmental factors. Over 30 genes have been implicated in isolated and/or combined pituitary hormone deficiency. The etiology remains unknown for up to 80% of the patients, but most cases have been analyzed by limited candidate gene screening. Mutations in the PROP1 gene are the most common known cause, and the frequency of mutations in this gene varies greatly by ethnicity. We designed a custom array to assess the frequency of mutations in known hypopituitarism genes and new candidates, using single molecule molecular inversion probes sequencing (smMIPS). METHODS: We used this panel for the first systematic screening for causes of hypopituitarism in children. Molecular inversion probes were designed to capture 693 coding exons of 30 known genes and 37 candidate genes. We captured genomic DNA from 51 pediatric patients with CPHD (n = 43) or isolated GH deficiency (IGHD) (n = 8) and their parents and conducted next generation sequencing. RESULTS: We obtained deep coverage over targeted regions and demonstrated accurate variant detection by comparison to whole-genome sequencing in a control individual. We found a dominant mutation GH1, p.R209H, in a three-generation pedigree with IGHD. CONCLUSIONS: smMIPS is an efficient and inexpensive method to detect mutations in patients with hypopituitarism, drastically limiting the need for screening individual genes by Sanger sequencing.

4.
Sci Transl Med ; 8(328): 328ra28, 2016 Mar 02.
Article in English | MEDLINE | ID: mdl-26936505

ABSTRACT

Recent work in human glioblastoma (GBM) has documented recurrent mutations in the histone chaperone protein ATRX. We developed an animal model of ATRX-deficient GBM and showed that loss of ATRX reduces median survival and increases genetic instability. Further, analysis of genome-wide data for human gliomas showed that ATRX mutation is associated with increased mutation rate at the single-nucleotide variant (SNV) level. In mouse tumors, ATRX deficiency impairs nonhomologous end joining and increases sensitivity to DNA-damaging agents that induce double-stranded DNA breaks. We propose that ATRX loss results in a genetically unstable tumor, which is more aggressive when left untreated but is more responsive to double-stranded DNA-damaging agents, resulting in improved overall survival.


Subject(s)
Brain Neoplasms/pathology , DNA End-Joining Repair , DNA Helicases/deficiency , Glioma/pathology , Nuclear Proteins/deficiency , Animals , Brain Neoplasms/genetics , Cell Proliferation , Chromosomes, Mammalian/genetics , DNA Copy Number Variations/genetics , DNA Damage , DNA Helicases/genetics , DNA Helicases/metabolism , Disease Models, Animal , Glioma/genetics , Humans , Mice , Microsatellite Instability , Mutation/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Polymorphism, Single Nucleotide/genetics , Survival Analysis , Telomere Homeostasis , Transposases/metabolism , X-linked Nuclear Protein
5.
Article in English | MEDLINE | ID: mdl-25068145

ABSTRACT

We developed a combined conditional cytotoxic, i.e., herpes simplex type 1-thymidine kinase (TK), plus immune-stimulatory, i.e., fms-like tyrosine kinase ligand-3-mediated gene therapy for glioblastoma multiforme (GBM). Therapeutic transgenes were encoded within high-capacity adenoviral vectors (HC-Ad); TK was expressed constitutively, while Flt3L was under the control of the TetOn regulatable promoter. We previously assessed efficacy and safety in intracranial GBM rodent models. But, since this approach involves expression of a cytokine within the brain, we chose the nonhuman primate, i.e., Callithrix jaccus (marmoset) as it has been established that its immune response shares similarities with man. We characterized the safety, cell-type specific expression, and doxycycline (DOX)-inducibility of HC-Ad-TetOn-Flt3L delivered within the striatum. We used allometrically scaled DOX doses delivered orally, twice daily for one month, mimicking the route and duration of DOX administration planned for the GBM trial. Flt3L was effectively expressed within astrocytes, microglia, oligodendrocytes, and neurons. No evidence of brain or systemic toxicities due to the treatment was encountered. Our data indicate that DOX doses equivalent to those used in humans to treat infections can be safely used "off-label" to turn "on" therapeutic gene expression from HC-Ad-TetOn-Flt3L; providing evidence for the safety of this approach in the clinic.

6.
Arch Neurol ; 61(7): 1025-9, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15262732

ABSTRACT

BACKGROUND: Paroxysmal dystonic choreoathetosis (PDC) is characterized by attacks of involuntary movements that occur spontaneously while at rest and following caffeine or alcohol consumption. Previously, we and others identified a locus for autosomal dominant PDC on chromosome 2q33-2q35. OBJECTIVE: To identify the PDC gene. DESIGN: Analysis of PDC positional candidate genes by exon sequencing and reverse transcription-polymerase chain reaction. SETTING: Outpatient clinical and molecular genetic laboratory at a university hospital. Patients Affected (n = 12) and unaffected (n = 26) subjects from 2 unrelated families with PDC and 105 unrelated control subjects. RESULTS: We identified missense mutations in the myofibrillogenesis regulator gene (MR-1) in affected subjects in 2 unrelated PDC kindreds. These mutations were absent in control subjects and caused substitutions of valine for alanine at amino acid positions 7 and 9. The substitutions disturb interspecies conserved residues and are predicted to alter the MR-1 gene's amino-terminal alpha helix. The MR-1 exon containing these mutations (exon 1) was expressed only in the brain, a finding that explains the brain-specific symptoms of subjects with these mutations. CONCLUSIONS: Although MR-1 gene function is unknown, the precedence of ion channel disturbance in other episodic neurologic disorders suggests that the pathophysiologic features of PDC also involve abnormal ion localization. The discovery that MR-1 mutations underlie PDC provides opportunities to explore this condition's pathophysiologic characteristics and may provide insight into the causes of other paroxysmal neurologic disorders as well as the neurophysiologic mechanisms of alcohol and caffeine, which frequently precipitate PDC attacks.


Subject(s)
Athetosis/genetics , Chorea/genetics , Dystonia/genetics , Genes, Regulator/physiology , Muscle Development/genetics , Mutation , Athetosis/physiopathology , Chorea/physiopathology , Chromosomes, Human, Pair 2/genetics , Dystonia/physiopathology , Female , Genes, Regulator/genetics , Genetic Linkage/genetics , Haplotypes/genetics , Humans , Male , Pedigree , Physical Chromosome Mapping
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