Louise C. Pyle

Cloning and Characterization of HB2, a Candidate High Density Lipoprotein Receptor SEQUENCE HOMOLOGY WITH MEMBERS OF THE IMMUNOGLOBULIN SUPERFAMILY OF MEMBRANE PROTEINS

The protection against coronary artery disease attributed to high density lipoprotein (HDL) may be associated with several functions, including its central role in reverse cholesterol transport, possible antioxidant and antithrombotic properties and others not yet identified which may depend on specific interactions between HDL and cell receptors. Several HDL-binding proteins have been identified including two candidate liver HDL receptors, HB1 and HB2 recently purified in this laboratory. We now report the cloning, sequencing, and some properties of HB2, the most abundant of the pair. It shows significant homology with the adhesion molecules ALCAM and BEN of the immunoglobulin superfamily and the cDNA, when transfected into HepG2 or COS cells, caused specific HDL3 binding to increase by 80–100%. Further, ligand blotting of glycoproteins isolated from phorbol 12-myristate 13-acetate-treated THP-1 cells or from transfected HepG2 and Chinese hamster ovary cells also provided evidence of increased binding of HDL3 to HB2. Differentiation of THP-1 cells into macrophages resulted in a striking increase in HB2 mRNA which was attenuated if cells were cholesterol-loaded by incubation with acetylated low density lipoprotein. If the interaction between HDL and HB2 reduces the adhesion-induced inflammatory cellular events that characterize arterial wall injury, thereby achieving the protection associated with higher plasma levels of HDL, these findings may provide a clue to one mitigating effect of HDL in heart disease.

ΔF508 CFTR processing correction and activity in polarized airway and non-airway cell monolayers

We examined the activity of DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) stably expressed in polarized cystic fibrosis bronchial epithelial cells (CFBE41o(-)) human airway cells and Fisher Rat Thyroid (FRT) cells following treatment with low temperature and a panel of small molecule correctors of DeltaF508 CFTR misprocessing. Corr-4a increased DeltaF508 CFTR-dependent Cl(-) conductance in both cell types, whereas treatment with VRT-325 or VRT-640 increased activity only in FRT cells. Total currents stimulated by forskolin and genistein demonstrated similar dose/response effects to Corr-4a treatment in each cell type. When examining the relative contribution of forskolin and genistein to total stimulated current, CFBE41o(-) cells had smaller forskolin-stimulated I(sc) following either low temperature or corr-4a treatment (10-30% of the total I(sc) produced by the combination of both CFTR agonists). In contrast, forskolin consistently contributed greater than 40% of total I(sc) in DeltaF508 CFTR-expressing FRT cells corrected with low temperature, and corr-4a treatment preferentially enhanced forskolin dependent currents only in FRT cells (60% of total I(sc)). DeltaF508 CFTR cDNA transcript levels, DeltaF508 CFTR C band levels, or cAMP signaling did not account for the reduced forskolin response in CFBE41o(-) cells. Treatment with non-specific inhibitors of phosphodiesterases (papaverine) or phosphatases (endothall) did not restore DeltaF508 CFTR activation by forskolin in CFBE41o(-) cells, indicating that the Cl(-) transport defect in airway cells is distal to cAMP or its metabolism. The results identify important differences in DeltaF508 CFTR activation in polarizing epithelial models of CF, and have important implications regarding detection of rescued of DeltaF508 CFTR in vivo.

Activation of the cystic fibrosis transmembrane conductance regulator by the flavonoid quercetin: potential use as a biomarker of ΔF508 cystic fibrosis transmembrane conductance regulator rescue.

Therapies to correct the ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) folding defect require sensitive methods to detect channel activity in vivo. The β₂ adrenergic receptor agonists, which provide the CFTR stimuli commonly used in nasal potential difference assays, may not overcome the channel gating defects seen in ΔF508 CFTR after plasma membrane localization. In this study, we identify an agent, quercetin, that enhances the detection of surface ΔF508 CFTR, and is suitable for nasal perfusion. A screen of flavonoids in CFBE41o⁻ cells stably transduced with ΔF508 CFTR, corrected to the cell surface with low temperature growth, revealed that quercetin stimulated an increase in the short-circuit current. This increase was dose-dependent in both Fisher rat thyroid and CFBE41o⁻ cells. High concentrations inhibited Cl⁻ conductance. In CFBE41o⁻ airway cells, quercetin (20 μg/ml) activated ΔF508 CFTR, whereas the β₂ adrenergic receptor agonist isoproterenol did not. Quercetin had limited effects on cAMP levels, but did not produce detectable phosphorylation of the isolated CFTR R-domain, suggesting an activation independent of channel phosphorylation. When perfused in the nares of Cftr(+) mice, quercetin (20 μg/ml) produced a hyperpolarization of the potential difference that was absent in Cftr(-/-) mice. Finally, quercetin-induced, dose-dependent hyperpolarization of the nasal potential difference was also seen in normal human subjects. Quercetin activates CFTR-mediated anion transport in respiratory epithelia in vitro and in vivo, and may be useful in studies intended to detect the rescue of ΔF508 CFTR by nasal potential difference.

Meta-analysis of five genome-wide association studies identifies multiple new loci associated with testicular germ cell tumor

The international Testicular Cancer Consortium (TECAC) combined five published genome-wide association studies of testicular germ cell tumor (TGCT; 3,558 cases and 13,970 controls) to identify new susceptibility loci. We conducted a fixed-effects meta-analysis, including, to our knowledge, the first analysis of the X chromosome. Eight new loci mapping to 2q14.2, 3q26.2, 4q35.2, 7q36.3, 10q26.13, 15q21.3, 15q22.31, and Xq28 achieved genome-wide significance (P < 5 × 10−8). Most loci harbor biologically plausible candidate genes. We refined previously reported associations at 9p24.3 and 19p12 by identifying one and three additional independent SNPs, respectively. In aggregate, the 39 independent markers identified to date explain 37% of father-to-son familial risk, 8% of which can be attributed to the 12 new signals reported here. Our findings substantially increase the number of known TGCT susceptibility alleles, move the field closer to a comprehensive understanding of the underlying genetic architecture of TGCT, and provide further clues to the etiology of TGCT.

Regulatory domain phosphorylation to distinguish the mechanistic basis underlying acute CFTR modulators.

Modulator compounds intended to overcome disease-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) show significant promise in clinical testing for cystic fibrosis. However, the mechanism(s) of action underlying these compounds are not fully understood. Activation of CFTR ion transport requires PKA-regulated phosphorylation of the regulatory domain (R-D) and dimerization of the nucleotide binding domains. Using a newly developed assay, we evaluated nine compounds including both CFTR potentatiators and activators discovered via various high-throughput screening strategies to acutely augment CFTR activity. We found considerable differences in the effects on R-D phosphorylation. Some (including UC(CF)-152) stimulated robust phosphorylation, and others had little effect (e.g., VRT-532 and VX-770). We then compared CFTR activation by UC(CF)-152 and VRT-532 in Ussing chamber studies using two epithelial models, CFBE41o(-) and Fischer rat thyroid cells, expressing various CFTR forms. UC(CF)-152 activated wild-type-, G551D-, and rescued F508del-CFTR currents but did not potentiate cAMP-mediated CFTR activation. In contrast, VRT-532 moderately activated CFTR short-circuit current and strongly potentiated forskolin-mediated current. Combined with the result that UC(CF)-152, but not VRT-532 or VX-770, acts by increasing CFTR R-D phosphorylation, these findings indicate that potentiation of endogenous cAMP-mediated activation of mutant CFTR is not due to a pathway involving augmented R-D phosphorylation. This study presents an assay useful to distinguish preclinical compounds by a crucial mechanism underlying CFTR activation, delineates two types of compound able to acutely augment CFTR activity (e.g., activators and potentiators), and demonstrates that a number of different mechanisms can be successfully employed to activate mutant CFTR.

Mandibulofacial Dysostosis with Microcephaly: Mutation and Database Update

Mandibulofacial dysostosis with microcephaly (MFDM) is a multiple malformation syndrome comprising microcephaly, craniofacial anomalies, hearing loss, dysmorphic features, and, in some cases, esophageal atresia. Haploinsufficiency of a spliceosomal GTPase, U5–116 kDa/EFTUD2, is responsible. Here, we review the molecular basis of MFDM in the 69 individuals described to date, and report mutations in 38 new individuals, bringing the total number of reported individuals to 107 individuals from 94 kindreds. Pathogenic EFTUD2 variants comprise 76 distinct mutations and seven microdeletions. Among point mutations, missense substitutions are infrequent (14 out of 76; 18%) relative to stop‐gain (29 out of 76; 38%), and splicing (33 out of 76; 43%) mutations. Where known, mutation origin was de novo in 48 out of 64 individuals (75%), dominantly inherited in 12 out of 64 (19%), and due to proven germline mosaicism in four out of 64 (6%). Highly penetrant clinical features include, microcephaly, first and second arch craniofacial malformations, and hearing loss; esophageal atresia is present in an estimated ∼27%. Microcephaly is virtually universal in childhood, with some adults exhibiting late “catch‐up” growth and normocephaly at maturity. Occasionally reported anomalies, include vestibular and ossicular malformations, reduced mouth opening, atrophy of cerebral white matter, structural brain malformations, and epibulbar dermoid. All reported EFTUD2 mutations can be found in the EFTUD2 mutation database (http://databases.lovd.nl/shared/genes/EFTUD2).

Pathologic Variants of the Mitochondrial Phosphate Carrier SLC25A3: Two New Patients and Expansion of the Cardiomyopathy/Skeletal Myopathy Phenotype With and Without Lactic Acidosis

Variants in the SLC25A3 gene, which codes for the mitochondrial phosphate transporter (PiC), lead to a failure of inorganic phosphate (Pi) transport across the mitochondrial membrane, which is required in the final step of oxidative phosphorylation. The literature described two affected sibships with variants in SLC25A3; all cases had skeletal myopathy and cardiomyopathy (OMIM 610773). We report here two new patients who had neonatal cardiomyopathy; one of whom did not have skeletal myopathy nor elevated lactate. Patient 1 had a homozygous splice site variant, c.158-9A>G, which has been previously reported in a Turkish family. Patient 2 was found to be a compound heterozygote for two novel variants, c.599T>G (p.Leu200Trp) and c. 886_898delGGTAGCAGTGCTTinsCAGATAC (p.Gly296_Ser300delinsGlnIlePro). Protein structure analysis indicated that both variants are likely to be pathogenic. Sequencing of SLC25A3 should be considered in patients with isolated cardiomyopathy, even those without generalized skeletal myopathy or lactic acidosis.

Genetic changes associated with testicular cancer susceptibility

Testicular germ cell tumor (TGCT) is a highly heritable cancer primarily affecting young white men. Genome-wide association studies (GWAS) have been particularly effective in identifying multiple common variants with strong contribution to TGCT risk. These loci identified through association studies have implicated multiple genes as associated with TGCT predisposition, many of which are unique among cancer types, and regulate processes such as pluripotency, sex specification, and microtubule assembly. Together these biologically plausible genes converge on pathways involved in male germ cell development and maturation, and suggest that perturbation of them confers susceptibility to TGCT, as a developmental defect of germ cell differentiation.

Channel Gating Regulation by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) First Cytosolic Loop.

Background: Elucidating the mechanochemistry of ABC transporters is essential for understanding several human disease states. Results: We show a novel intermolecular binding step that governs CFTR channel activity. Conclusion: CFTR gating and ATP hydrolysis require a properly configured transmembrane domain interface. Significance: These findings indicate a new approach for discovering therapeutic targets within ABC proteins that modulate function. In this study, we present data indicating a robust and specific domain interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) first cytosolic loop (CL1) and nucleotide binding domain 1 (NBD1) that allows ion transport to proceed in a regulated fashion. We used co-precipitation and ELISA to establish the molecular contact and showed that binding kinetics were not altered by the common clinical mutation F508del. Both intrinsic ATPase activity and CFTR channel gating were inhibited severely by CL1 peptide, suggesting that NBD1/CL1 binding is a crucial requirement for ATP hydrolysis and channel function. In addition to cystic fibrosis, CFTR dysregulation has been implicated in the pathogenesis of prevalent diseases such as chronic obstructive pulmonary disease, acquired rhinosinusitis, pancreatitis, and lethal secretory diarrhea (e.g. cholera). On the basis of clinical relevance of the CFTR as a therapeutic target, a cell-free drug screen was established to identify modulators of NBD1/CL1 channel activity independent of F508del CFTR and pharmacologic rescue. Our findings support a targetable mechanism of CFTR regulation in which conformational changes in the NBDs cause reorientation of transmembrane domains via interactions with CL1 and result in channel gating.

A practical guide for evaluating gonadal germ cell tumor predisposition in differences of sex development.

Differences of Sex Development (DSD) includes a wide spectrum of etiologies and phenotypes. A subset of individuals with DSDs are predisposed to gonadal germ cell tumor (GCT). In this setting, GCT risk varies widely, depending on the DSD molecular etiology and penetrance. Prognostication based on molecular diagnosis remains challenging, as natural history data specific to recently identified molecular causes of DSD is lacking. In this review, we provide a framework for the clinical geneticist to consider GCT tumor risk in the patient with DSD. We discuss germ cell development and etiology of GCT growth, along with parameters to consider when recommending prophylactic gonadectomy including fertility, hormonal output, and malignant GTC treatment outcomes. Shortly after the 2006 reorganization of DSD nomenclature, literature reviews of natural history publications stratified GCT risk by a chromosomal, pathological, and hormonal taxonomy. Our 2017 literature review reveals a larger body of publications. However, the broad DSD GCT risk stratification within the 2006 taxonomy remains stable. We discuss precise GCT risk assessment for specific diagnoses, including androgen insensitivity, Smith–Lemli–Opitz, and 46,XY with MAP3K1 mutations and gonadal dysgenesis, as examples. We also examine the GCT risk in non‐DSD syndromes, in addition to the cancer risks in DSD patients with dimorphic gonads and genitalia. This review is intended to provide a nuanced assessment of relative germ cell tumor risk in the DSD patient, including modern precise molecular diagnosis, for use by the clinical geneticist.