Comparative assessment of gene-specific variant distribution in prenatal and postnatal cohorts tested for Noonan syndrome and related conditions.

PURPOSE: To compare the pattern of gene-specific involvement and the spectrum of variants observed in prenatal and postnatal (mean ± SD, 8.9 ± 9.4 years) cohorts tested for Noonan syndrome and related conditions. METHODS: Outcomes of sequencing panel testing were compared between prenatal (n = 845) and postnatal (n = 409) cohorts. RESULTS: PTPN11 and SOS1 harbored the majority of observed variants in both prenatal and postnatal cohorts, and BRAF, HRAS, KRAS, MAP2K1, MAP2K2, RAF1, and SHOC2 had similarities in their pattern of involvement in both cohorts. PTPN11 was the largest contributor of pathogenic variants and had the lowest frequency of variants of uncertain significance (VUS). SOS1 had the highest VUS frequency in both cohorts. The overall VUS frequency was twice as high in prenatal specimens (58.1 vs. 29.3%). PTPN11 and SOS1 had a 1.5-fold higher VUS frequency in the prenatal cohort (10.7 vs. 7.4% and 95 vs. 61.1%, respectively). The diagnostic yield was 3.7% for prenatal samples, with a higher yield of 12.3% in fetuses with cystic hygroma as a sole finding, and 21.3% for postnatal. CONCLUSION: Comparison of prenatal versus postnatal specimens demonstrates that the pattern of specific gene involvement is similar, whereas the classification spectrum of observed variants differs considerably.

Correction: Comparative assessment of gene-specific variant distribution in prenatal and postnatal cohorts tested for Noonan syndrome and related conditions.

The original version of this Article contained an error in the spelling of the author N. T. Leach, which was incorrectly given as N. L. Leach. This has been corrected in both the PDF and HTML versions of the Article.

Correlating cell cycle with apoptosis in a cell line expressing a tandem green fluorescent protein substrate specific for group II caspases.

BACKGROUND: We describe a rapid flow cytometric assay that correlates cell cycle with apoptotic cell death in a cell line expressing a tandem green fluorescent protein (GFP). METHODS: A Jurkat cell line was transfected with a gene construct coding for constitutive expression of a tandem GFP molecule carrying a consensus cleavage site (DEVD) for group II caspases (C-2-Y). Cells were treated with CD95 antibody (Ab), then incubated with annexin V-phycoerythrin (PE), propidium iodide (PI), and Hoechst 33342. RESULTS: After CD95 treatment, the C-2-Y cell line had twice the number of nonapoptotic cells compared with both control cell lines. This proportion of viable, nonapoptotic cells after treatment was unaffected by the level of GFP (DEVD) expression in the cells, as confirmed by sorted populations. The early apoptotic cells in the C-2-Y cell line had an increased G0-G1 phase population compared with the control cell lines. CONCLUSIONS: Apoptosis is delayed in the C-2-Y cell line and the early apoptotic cells have a higher G0-G1 cell cycle frequency. The artificial substrate competes with the natural substrate(s), thereby slowing the apoptotic process. The expression level of DEVD-GFP does not alter the delayed induction of apoptosis. Caspase activation occurs prior to phosphatidylserine translocation.

Improved detection of CFTR mutations in Southern California Hispanic CF patients.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), a common autosomal recessive disease in Caucasians. The broad mutation spectrum varies among different patient groups. Current molecular diagnoses are designed to detect 80-97% of CF chromosomes in Caucasians and Ashkenazi Jews but have a much lower detection rate in Hispanic CF patients. Grebe et al. [1994] reported a 58% detection rate in Hispanic patients. Since then, there has been no large-scale, complete mutational analysis of Hispanic CF patients. In this study, the mutations in 62 Hispanic patients from southern California were investigated. The entire coding and flanking intronic regions of the CFTR gene were analyzed by temporal temperature gradient gel electrophoresis (TTGE) followed by sequencing to identify the mutations. Eleven novel mutations were discovered in this patient group: 3876delA, 406-1G>A, 935delA, 663delT, 3271delGG, 2105-2117del13insAGAAA, 3199del6, Q179K, 2108delA, 3171delC, and 3500-2A>T. Among the mutations, seven were out-of-frame insertions and deletions that result in truncated proteins, two were splice-site mutations, one was an in-frame 6 bp deletion, and one was a missense mutation that involved the non-conservative change of glutamine-179 to lysine. All patients presented severe classical clinical course with pancreatic insufficiency and poor growth, consistent with the nature of truncation mutation. The results indicate that TTGE screening following the analysis of recurrent mutations will substantially improve the mutation detection rate for Hispanic CF patients from southern California.

Improved detection of cystic fibrosis mutations in the heterogeneous U.S. population using an expanded, pan-ethnic mutation panel.

PURPOSE: To determine the comparative frequency of 93 CFTR mutations in U.S. individuals with a clinical diagnosis of cystic fibrosis (CF). METHODS: A total of 5,840 CF chromosomes from Caucasians, Ashkenazi Jews, Hispanics, African Americans, Native Americans, Asians, and individuals of mixed race were analyzed using a pooled ASO hybridization strategy. RESULTS: Sixty-four mutations provided a sensitivity of 70% to 95% in all ethnic groups except Asians, and at least 81% when the U.S. population was considered as a whole. CONCLUSIONS: For population-based carrier screening for CF in the heterogeneous U.S. population, which is characterized by increasing admixture, a pan-ethnic mutation panel of 50 to 70 CFTR mutations may provide a practical test that maximizes sensitivity.

Development and application of a GFP-FRET intracellular caspase assay for drug screening.

Apoptosis is a crucial biological process, and activation of caspase endoproteases is essential for proper regulation and execution of apoptosis. Because caspases also appear to be central players in several pathological states, there is a practical need within the biopharmaceutical research community for facile, noninvasive cellular assays for the discovery of compounds that modulate caspase activity. Tandem molecules of green fluorescent protein (GFP) stably expressed within cells can serve as a genetically encoded sensor of protease activity. Using this technology, we have developed a stable cellular system for the screening of agents that modulate activation of the caspase cascade. This assay technology allows for the real-time monitoring of apoptosis in situ, using conventional fluorescent plate reader detection. By applying this assay system to an actual compound screen, small-molecule inducers of cell apoptosis were reliably identified. Follow-up pharmacology confirmed that the rank-order potency of primary hits using the intracellular GFP assay corresponded to that found using a conventional, cell lysis-based assay method.

Dynamic redistribution of calmodulin in HeLa cells during cell division as revealed by a GFP-calmodulin fusion protein technique.

It has been suggested by many studies that Ca2+ signaling plays an important role in regulating key steps in cell division. In order to study the down stream components of calcium signaling, we have fused the gene of calmodulin (CaM) with that of green fluorescent protein (GFP) and expressed it in HeLa cells. The GFP-CaM protein was found to have similar biochemical properties as the wild-type CaM, and its distribution was also similar to that of the endogenous CaM. Using this GFP-tagged CaM as a probe, we have conducted a detailed examination of the spatial- and temporal-dependent redistribution of calmodulin in living mammalian cells during cell division. Our major findings are: (1) high density of CaM was found to distribute in two sub-cellular locations during mitosis; one fraction was concentrated in the spindle poles, while the other was concentrated in the sub-membrane region around the cell. (2) The sub-membrane fraction of CaM became aggregated at the equatorial region where the cleavage furrow was about to form. The timing of this localized aggregation of CaM was closely associated with the onset of cytokinesis. (3) Using a TA-CaM probe, we found that the sub-membrane fraction of CaM near the cleavage furrow was selectively activated during cell division. (4) When we injected a CaM-specific inhibitory peptide into early anaphase cells, cytokinesis was either blocked or severely delayed. These findings suggest that, in addition to Ca2+ ion, CaM may represent a second signal that can also play an active role in determining the positioning and timing of the cleavage furrow formation.

Using GFP in FRET-based applications.

The use of green fluorescent protein (GFP) is a powerful technology that has recently enabled investigators to study dynamic molecular events within living cells. One method for detecting molecular interactions involves fluorescence resonance energy transfer (FRET) between two GFPs or between GFP and a second fluorophore. This review summarizes the use of GFP for FRET and illustrates the theme with specific examples on how GFP has been employed as an intracellular molecular sensor.

Dynamic and quantitative Ca2+ measurements using improved cameleons.

Cameleons are genetically-encoded fluorescent indicators for Ca2+ based on green fluorescent protein variants and calmodulin (CaM). Because cameleons can be targeted genetically and imaged by one- or two-photon excitation microscopy, they offer great promise for monitoring Ca2+ in whole organisms, tissues, organelles, and submicroscopic environments in which measurements were previously impossible. However, the original cameleons suffered from significant pH interference, and their Ca2+-buffering and cross-reactivity with endogenous CaM signaling pathways was uncharacterized. We have now greatly reduced the pH-sensitivity of the cameleons by introducing mutations V68L and Q69K into the acceptor yellow green fluorescent protein. The resulting new cameleons permit Ca2+ measurements despite significant cytosolic acidification. When Ca2+ is elevated, the CaM and CaM-binding peptide fused together in a cameleon predominantly interact with each other rather than with free CaM and CaM-dependent enzymes. Therefore, if cameleons are overexpressed, the primary effect is likely to be the unavoidable increase in Ca2+ buffering rather than specific perturbation of CaM-dependent signaling.

Bcl-2 and Bax interactions in mitochondria probed with green fluorescent protein and fluorescence resonance energy transfer.

It has been hypothesized that interaction of Bcl-2 and Bax may regulate apoptosis. The spatial and temporal interaction of Bcl-2 and Bax at the single cell level has not, however, been demonstrated. To achieve this goal, we have developed two-fusion FRET (fluorescence resonance energy transfer). Using green fluorescent protein (GFP)-Bax and blue fluorescent protein (BFP)-Bcl-2 fusion proteins coexpressed in the same cell, we demonstrate a direct interaction between Bcl-2 and Bax in individual mitochondria. Mitochondrially localized cytochrome c-GFP and BFP-Bcl-2 showed little or no FRET, while nuclear-localized GFP-human papillomavirus E6 and BFP-Bcl-2 did not interact when coexpressed in the same cell. These findings indicate that two-fusion FRET provides an opportunity to examine the interaction between two different proteins coexpressed in single intact mammalian cells.

Crystal structure and photodynamic behavior of the blue emission variant Y66H/Y145F of green fluorescent protein.

The crystal structure of a blue emission variant (Y66H/Y145F) of the Aequorea victoria green fluorescent protein has been determined by molecular replacement and the model refined. The crystallographic R-factor is 18.1% for all data from 20 to 2.1 A, and the model geometry is excellent. The chromophore is non-native and is autocatalytically generated from the internal tripeptide Ser65-His66-Gly67. The final electron density maps indicate that the formation of the chromophore is complete, including 1,2 dehydration of His66 as indicated by the planarity of the chromophore. The chromophore is in the cis conformation, with no evidence for any substantial fraction of the trans configuration or uncyclized apoprotein, and is well-shielded from bulk solvent by the folded protein. These characteristics indicate that the machinery for production of the chromophore from a buried tripeptide unit is not only intact but also highly efficient in spite of a major change in chromophore chemical structure. Nevertheless, there are significant rearrangements in the hydrogen bond configuration around the chromophore as compared to wild-type, indicating flexibility of the active site. pH titration of the intact protein and the chromopeptide (pKa1 = 4.9 +/- 0.1, pKa2 = 12.0 +/- 0.1) suggests that the predominant form of the chromophore in the intact protein is electrically neutral. In contrast to the wild-type protein [Chattoraj, M., King, B. A., Bublitz, G. U., & Boxer, S. G. (1996) Proc. Natl. Acad. Sci. U.S.A., 8362-8367], femtosecond fluorescence up-conversion spectroscopy of the intact protein and a partially deuterated form strongly suggests that excited-state proton transfer is not coupled to fluorescence emission.

Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.

Important Ca2+ signals in the cytosol and organelles are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations. We have dubbed these fluorescent indicators 'cameleons'. They consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs. Calmodulin mutations can tune the Ca2+ affinities to measure free Ca2+ concentrations in the range 10(-8) to 10(-2) M. We have visualized free Ca2+ dynamics in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca2+ concentration in the endoplasmic reticulum of individual cells ranged from 60 to 400 microM at rest, and 1 to 50 microM after Ca2+ mobilization. FRET is also an indicator of the reversible intermolecular association of cyan-GFP-labelled calmodulin with yellow-GFP-labelled M13. Thus FRET between GFP mutants can monitor localized Ca2+ signals and protein heterodimerization in individual live cells.

Rapid characterization of the variable length polythymidine tract in the cystic fibrosis (CFTR) gene: association of the 5T allele with selected CFTR mutations and its incidence in atypical sinopulmonary disease.

The CFTR intron 8 variable length polythymidine tract modulates the cystic fibrosis (CF) phenotype associated with the mutation R117H. To explore whether other mutations reside on multiple intron 8 backgrounds with discernible impacts on phenotype, we developed an allele-specific PCR assay to characterize this locus. Our approach types samples rapidly without the use or radioisotopes. Polythymidine alleles were identified for mutations either associated with a wide range of clinical phenotypes (R117H, R347P, G85E, D1152H, R334W, 2789 + 5 G > A, 3849 + 10kb C > T), and/or located at hypermutable CpG loci (R117H, 3845 + 10kb C > T, R553X, R334W, S945L and R75Q). R117H was detected in cis with each of three alleles (5T, 7T, 9T) at the intron 8 locus. The novel R117H-9T association was detected in a 10-month African-American male with borderline-to-mildly elevated sweat chloride values (approximately 50-66 mEq/L). All other mutations studied were associated with 7T except 3849 + 10kb C > T, which was detected on both 7T and 9T backgrounds, but not 5T. Three individuals with a delta F508/3849 + 10kb C > T genotype were 9T,9T and had pancreatic sufficiency and normal sweat chloride values, whereas 15 others who carried 3849 + 10kb C > T on a 7T background had variable pancreatic function (sufficient, n = 12, insufficient, n = 3), and variable sweat chloride values (normal, n = 12, elevated, n = 3). Surprisingly, when not associated with known CFTR mutations, 5T was detected with elevated frequency among individuals with sinopulmonary disease of ill-defined etiology, but with some characteristics of variant CF. In summary, the 5T allele was not found in cis with CF-causing mutations besides R117H, but an elevated 5T allele frequency in variant CF patients suggests 5T may be associated with disease in some situations.

Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer.

BACKGROUND: Variants of the green fluorescent protein (GFP) with different colors would be very useful for simultaneous comparisons of multiple protein fates, developmental lineages and gene expression levels. The simplest way to shift the emission color of GFP is to substitute histidine or tryptophan for the tyrosine in the chromophore, but such blue-shifted point mutants are only dimly fluorescent. The longest wavelengths previously reported for the excitation and emission peaks of GFP mutants are 488 and 511 nm, respectively. RESULTS: Additional substitutions, mainly in residues 145-163, have improved the brightness of the blue-shifted GFP mutants with histidine and tryptophan in place of tyrosine 66. Separate mutations have pushed the excitation and emission peaks of the most red-shifted mutant to 504 and 514 nm, respectively. At least three different colors of GFP mutants can now be cleanly distinguished from each other under the microscope, using appropriate filter sets. A fusion protein consisting of linked blue- and green-fluorescent proteins exhibits fluorescence resonance energy transfer, which is disrupted by proteolytic cleavage of the linker between the two domains. CONCLUSIONS: Our results demonstrate that the production of more and better GFP variants is possible and worthwhile. The production of such variants facilitates multicolor imaging of differential gene expression, protein localization or cell fate. Fusions between mutants of different colors may be useful substrates for the continuous in situ assay of proteases. Demonstration of energy transfer between GFP variants is an important step towards a general method for monitoring the mutual association of fusion proteins.

Double labelling of subcellular structures with organelle-targeted GFP mutants in vivo.

BACKGROUND: The green fluorescent protein (GFP) of Aequorea victoria is emerging as a unique tool for monitoring complex phenomena such as gene expression and organelle structure and dynamics in living cells. The recent description of GFP mutants with modified spectral properties opens numerous new applications in cell biology. However, the expression and the characteristics of these GFP mutants in living eukaryotic cells have not been verified yet. RESULTS: Here, we demonstrate the usefulness of the GFP mutants for cell biology studies in vivo, by the use of wild-type GFP, a 'bright' GFP mutant (S65T) and a mutant with blue-shifted excitation and emission spectra (Y66H/Y145F). We have constructed two GFP chimeras targeted to mitochondria, mtGFP(S65T) and mtGFP(Y66H/Y145F), with the same strategy used previously for mtGFP. In addition, two GFP chimeras targeted to the nucleus, nuGFP and nuGFP(S65T), were constructed by fusing the wild-type GFP or the (S65T) mutant to the rat glucocorticoid receptor. By co-transfecting mtGFP(Y66H/Y145F) and nuGFP, the nucleus and the mitochondria were visualized simultaneously in living cells. Similarly, mtGFP and mtGFP(Y66H/Y145F) were transfected into different populations of cells, and the events of cellular fusion, and mitochondrial intermixing and/or fusion, were directly monitored. CONCLUSIONS: The successful expression of organelle-targeted GFP mutants in live eukaryotes expands the uses of this fluorescent protein in cell biology, allowing direct access to key biological issues, such as the study of the interactions of different organelles in vivo. These results also open the way to other exciting applications, such as the direct study of protein redistribution and protein-protein interactions in living cells.

Understanding, improving and using green fluorescent proteins.

Green fluorescent proteins (GFPs) are presently attracting tremendous interest as the first general method to create strong visible fluorescence by purely molecular biological means. So far, they have been used as reporters of gene expression, tracers of cell lineage, and as fusion tags to monitor protein localization within living cells. However, the GFP originally cloned from the jellyfish Aequorea victoria has several nonoptimal properties including low brightness, a significant delay between protein synthesis and fluorescence development, and complex photoisomerization. Fortunately, the protein can be re-engineered by mutagenesis to ameliorate these deficiencies and shift the excitation and emission wavelengths, creating different colors and new applications.

Distribution of 13 truncating mutations in the neurofibromatosis 1 gene.

Neurofibromatosis 1 (NF1) is a common genetic disorder characterized by abnormalities of tissues derived from the neural crest. To define germ-line mutations in the NF1 gene, we studied 20 patients with familial or sporadic cases of NF1 diagnosed clinically and one patient with only café-au-lait spots and no other diagnostic criteria. A protein truncation assay identified abnormal polypeptides synthesized in vitro from five RT-PCR products that represented the entire NF1 coding region. Truncated polypeptides were observed in 14 individuals. The mutations responsible for the generation of abnormal polypeptides were characterized by DNA sequencing. Thirteen previously unpublished mutations were characterized in the 14 individuals. The mutation 2027insC was observed in two unrelated individuals; the other 12 mutations were unique. The sequence changes included seven nonsense and four frameshift mutations that created premature translation termination signals, and two large in-frame deletions that led to the synthesis of truncated polypeptides. One of the mutations was found in the child with a single clinical diagnostic criterion, providing her with a presumptive diagnosis of NF1. Our results confirm that truncating mutations are frequent in both familial and sporadic NF1 cases. The identification of mutations in 14 of 21 individuals studied (67%) suggests that the use of protein truncation assays will rapidly accelerate the rate of identification of NF1 mutations. Because we scanned the entire NF1 coding region in each individual, the distribution of NF1 truncating mutations was discerned for the first time. The mutations were relatively evenly distributed throughout the coding region with no evidence for clustering.