Biomedical subjects
Publications and source records attributed to F S Collins.
As we continue to localize, sequence, and characterize more and more genes and as we increase our understanding of the biologic function of the gene products--and the nature of the disruptions that mutations can cause--we will come closer to the goals of diagnosis, prevention, and therapy. Progress in the field is proceeding at a pace that no one could have envisioned a decade ago.
LIV-I, a high-affinity system that transports neutral, branched-chain amino acids into Escherichia coli, has two components, LivG and LivF, that are homologous to the cystic fibrosis (CF) transmembrane conductance regulator (CFTR). CF-associated mutations of human CFTR were introduced into corresponding regions of LivG, and their effects on leucine transport could be grouped into three classes. Mutations were found that (i) abolished LIV-I--directed transport, (ii) retained about a quarter of wild-type activity at the Michaelis-Menten constant (KM), and (iii) had minimal activity at the KM. A mutation equivalent to a benign polymorphism had no effect on transport. The correlation of these mutational phenotypes in LivG and CFTR suggests that the LIV-I prokaryotic transporter is functionally similar to the CF protein and that this similarity can be exploited to clarify the properties of the nucleotide-binding fold in this superfamily of proteins.
We have analyzed the binding of Sp1, a ubiquitously expressed transactivator, to the promoter region of the gamma genes. Low-affinity Sp1 sites were found at -50 and -200. A high-affinity site was detected at -140, over the CACCC sequence. To analyze the function of these sites, Drosophila SL-2 cells, which lack Sp1, were cotransfected with an Sp1 expression plasmid and gamma globin promoter-CAT constructs. In these assays, the gamma promoter was significantly stronger in the presence than in the absence of Sp1. Thus, the three Sp1 sites in the gamma promoter allow binding as well as transactivation of the promoter. The majority of this transactivation was due to the strong binding site at -140 because introduction of a point mutation at -144 (CACCC----AACCC) reduced Sp1-dependent promoter strength by 57%. Analysis of the -200 region suggested that in the wild-type promoter, Sp1 binding at this site contributes little to promoter strength. However, a point mutation (-198 T----C) associated with hereditary persistence of fetal hemoglobin (HPFH) dramatically increased the affinity of this site for Sp1 and significantly increased Sp1 dependent promoter strength in SL-2 cells. Three other point mutations associated with HPFH did not significantly affect the interaction of Sp1 with the -200 region.
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Von Recklinghausen neurofibromatosis, or type 1 neurofibromatosis (NF1), is a common autosomal dominant disorder characterized by abnormalities in multiple tissues derived from the embryonic neural crest. Portions of the gene have been recently identified by positional cloning, and sequence analysis has shown homology to the GTPase activating protein (GAP) family. In this report we present the results of an extensive cDNA walk resulting in the cloning of the complete coding region of the NF1 transcript. Analysis of the sequences reveals an open reading frame of 2818 amino acids, although alternatively spliced products may code for different protein isoforms. The gene extends for approximately 300 kb on chromosome 17, with its promoter in a CpG-rich island.
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Reports of families with members affected with both von Willebrand disease (vWD) and hereditary hemorrhagic telangiectasia (HHT) suggest a possible relationship between these two disorders. vWD, the most common inherited bleeding disorder in humans, is due to either a quantitative or qualitative defect in von Willebrand factor (vWF). The gene for vWF has been cloned and mapped to chromosome 12 (12p12----12pter). HHT, an uncommon inherited bleeding disorder, is characterized by malformed, dilated, fragile blood vessels. The chromosomal location of the gene for HHT is unknown. We studied two families by RFLP analysis to determine whether there is a molecular basis for the association of vWD and HHT. Family A is affected with both type IIA vWD and HHT; family B is affected with HHT alone. Linkage of HHT to the vWF gene was not detected, and vWF was ruled out as a candidate gene for HHT. The vWF gene was found to be tightly linked to type IIA vWD in family A (lod score 3.61 at recombination fraction .00). By PCR and DNA sequence analysis of vWF exon 28, a single T----C transition resulting in the substitution of Thr for Ile865 was identified. This substitution is located immediately adjacent to two previously identified type IIA vWD mutations.
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The neurofibromatosis type 1 (NF1) gene has been localized to the pericentromeric region of the long arm of chromosome 17. A chronology of events leading to the cloning of the NF1 gene is offered as a summary of modern gene hunting techniques. The gene was identified by its location rather than its function using positional cloning. Linkage analysis, based on DNA polymorphisms, is already available for prenatal and presymptomatic testing. This technique works only in cases when affected family members are available and is useless in spontaneous cases. The complete NF1 gene is huge, has a large number of exons, and is approximately 240,000 base pairs long. Its large size is consistent with the very high spontaneous mutation rate. The NF1 gene is evolutionarily conserved and expressed ubiquitously, not just in neural crest derivatives. Functional and structural homology with GAP (GTP [guanosine triphosphate]ase-activating protein) has been described. The GAP controls (or is controlled by) the ras oncogene. Aberration of ras function, which plays a fundamental role in growth, development, and differentiation, may play a role in the NF1 phenotype. Direct DNA diagnosis of mutations in the NF1 gene in clinical practice is premature. Current data suggest that the NF1 gene product may act as a tumor suppressor.
Cystic fibrosis (CF) is a recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We have identified in exon 7 two frameshift mutations, one caused by a two-nucleotide insertion and the other caused by a one-nucleotide deletion; these mutations--CF1154insTC and CF1213delT, respectively, are predicted to shift the reading frame of the protein and to introduce UAA(ochre) termination codons at residues 369 and 368.
The identification and cloning of the CF gene, which codes for a membrane protein that appears to regulate transmembrane ion transport, may lead to an understanding of the basic defect in the disease and to more effective treatment. More broadly, the cloning of the CF gene provides a fast start in the international effort to clone and map the entire human genome.
We have used retrovirus-mediated gene transfer to demonstrate complementation of the cystic fibrosis (CF) defect in vitro. Amphotropic retroviruses were used to transduce a functional cystic fibrosis transmembrane conductance regulator (CFTR) cDNA into CFPAC-1, a pancreatic adenocarcinoma cell line derived from a patient with CF that stably expresses the chloride transport abnormalities characteristic of CF. CFPAC-1 cells were exposed to control virus (PLJ) and CFTR-expressing virus (PLJ-CFTR); viral-transduced clones were isolated and subjected to molecular and physiologic analysis. RNA analysis detected a viral-derived CFTR transcript in all of the PLJ-CFTR clones that contained unrearranged proviral sequences. Agents that increase intracellular cAMP stimulated 125I efflux in PLJ-CFTR clones but not PLJ clones. Whole-cell patch-clamp performed on three responding clones showed that the anion efflux responses were due to cAMP stimulation of Cl conductance. Our findings indicate that expression of the normal CFTR gene confers cAMP-dependent Cl channel regulation on CF epithelial cells.
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