Genetic determination of exocrine pancreatic function in cystic fibrosis.

We showed elsewhere that the pancreatic function status of cystic fibrosis (CF) patients could be correlated to mutations in the CF transmembrane conductance regulator (CFTR) gene. Although the majority of CF mutations--including the most common, delta F508--strongly correlated with pancreatic insufficiency (PI), approximately 10% of the mutant alleles may confer pancreatic sufficiency (PS). To extend this observation, genomic DNA of 538 CF patients with well-documented pancreatic function status were analyzed for a series of known mutations in their CFTR genes. Only 20 of the 25 mutations tested were found in this population. They accounted for 84% of the CF chromosomes, with delta F508 being the most frequent (71%), and the other mutations accounted for less than 5% each. A total of 30 different, complete genotypes could be determined in 394 (73%) of the patients. The data showed that each genotype was associated only with PI or only with PS, but not with both. This result is thus consistent with the hypothesis that PI and PS in CF are predisposed by the genotype at the CFTR locus; the PS phenotype occurs in patients who have one or two mild CFTR mutations, such as R117H, R334W, R347P, A455E, and P574H, whereas the PI phenotype occurs in patients with two severe alleles, such as delta F508, delta I507, Q493X, G542X, R553X, W1282X, 621 + 1G----T, 1717-1G----A, 556delA, 3659delC, I148T, G480C, V520F, G551D, and R560T.     

Fine mapping of the Autosomal Dominant Split Hand/Split Foot Locus on Chromosome 7, Band q21.3-q22.1

Split hand/split foot (SHFD) is a human developmental defect characterized by missing digits, fusion of remaining digits, and a deep median cleft in the hands and feet. Cytogenetic studies of deletions and translocations associated with this disorder have indicated that an autosomal dominant split hand/split foot locus (gene SHFD1) maps to 7q21-q22. To characterize the SHFD1 locus, somatic cell hybrid lines were constructed from cytogenetically abnormal individuals with SHFD. Molecular analysis resulted in the localization of 93 DNA markers to one of 10 intervals surrounding the SHFD1 locus. The translocation breakpoints in four SHFD patients were encompassed by the smallest region of overlap among the SHFD-associated deletions. The order of DNA markers in the SHFD1 critical region has been defined as PON–D7S812–SHFD1–D7S811–ASNS. One DNA marker, D7S811, detected altered restriction enzyme fragments in three patients with translocations when examined by pulsed-field gel electro-phoresis (PFGE). These data map SHFD1, a gene that is crucial for human limb differentiation, to a small interval in the q21.3-q22.1 region of human chromosome 7.     

Modes of DAPI banding and simultaneous in situ hybridization

By controlling the degree of chromatin denaturation through formamide incubation, or by heat treatment and/or by high pH, three types of high quality 4,6-diamidino-2-phenylindole (DAPI) bands can be produced sequentially on the same set of 5-bromo-2-deoxyuridine (BrdU)-incorporated chromosomes: first DAPI multibanding (the equivalent of Q-banding), then partial C-banding including distamycin A (DA)/DAPI banding, and finally C-banding pattern. It is assumed that the different DAPI-chromatin interactions following these treatments reflect the different chromatin structures at the chromosomal sites. Since the DAPI banding protocol is compatible with in situ hybridization, the combination of fluorescent in situ hybridization (FISH) with DAPI banding allows the simultaneous detection of signals from the DNA probes and the identification of the chromosomal band location of the probe. We demonstrate this useful application with the localization of the cystic fibrosis and Duchenne muscular dystrophy gene probes to their appropriate bands.     

Molecular Characterization and Mapping of Murine Genes Encoding Three Members of the Stefin Family of Cysteine Proteinase Inhibitors

Stefins or Type 1 cystatins belong to a large, evolutionarily conserved protein superfamily, the members of which inhibit the papain-like cysteine proteinases. We report here on the molecular cloning and chromosomal localization of three newly identified members of the murine stefin gene family. These genes, designated herein as mouse stefins 1, 2, and 3, were isolated on the basis of their relatively increased expression in moth-eaten viable compared to normal congenic mouse bone marrow cells. The open reading frames of the stefin cDNAs encode proteins of approximately 11.5 kDa that show between 50 and 92% identity to sequences of stefins isolated from various other species. Data from Southern analysis suggest that the murine stefin gene family encompasses at least 6 and possibly 10-20 members, all of which appear to be clustered in the genome. Analysis of interspecific backcross mice indicates that the genes encoding the three mouse stefins all map to mouse chromosome 16, a localization that is consistent with the recent assignment of the human stefin A gene to a region of conserved homology between human chromosome 3q and the proximal region of mouse chromosome 16.     

Respiratory function of velopharyngeal constrictor muscles during wakefulness in normal adults

Launois, Sandrine H., Judy Tsui, and J. Woodrow Weiss.Respiratory function of velopharyngeal constrictor muscles during wakefulness in normal adults. J. Appl.Physiol. 82(2): 584-591, 1997.The levator velipalatini (LVP) and the superior pharyngeal constrictor (SPC) influencevelopharyngeal patency and soft palate position, but their behaviorduring respiration is incompletely characterized. To further clarifytheir respiratory function, we recorded electromyographic activity(EMG) in the LVP and the SPC in awake normal subjects breathing orally.EMG data were obtained in six subjects for the LVP and in nine subjectsfor the SPC. EMG activity and timing and ventilation were measuredduring isocapnic hypoxia and hyperoxic hypercapnia. Phasic EMG activitywas inconsistently present during unstimulated oral breathing. Timingof EMG phasic activity was variable for both muscles. Peak LVP activitywas mainly or exclusively expiratory in three of six subjects. Peak SPCactivity was mainly or exclusively expiratory in five of nine subjects.With chemostimulation, recruitment of phasic activity was observed inthe LVP in four of six subjects and in the SPC in five of ninesubjects. Tonic activity increased in four of six subjects for the LVPand in three of nine subjects for the SPC. However, the response wasalinear, and intersubject as well as breath-to-breath variability wassubstantial. In conclusion, LVP and SPC are characterized by the highinter- and intrasubject variability of EMG activity, timing ofactivation, and response to chemostimulation.