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The distributed genome hypothesis (DGH) states that each strain within a bacterial species receives a unique distribution of genes from a population-based supragenome that is many times larger than the genome of any given strain. The observations that natural infecting populations are often polyclonal and that most chronic bacterial pathogens have highly developed mechanisms for horizontal gene transfer suggested the DGH and provided the means and the mechanisms to explain how chronic infections persist in the face of a mammalian host's adaptive defense mechanisms. Having previously established the validity of the DGH for obligate pathogens, we wished to evaluate its applicability to an opportunistic bacterial pathogen. This was accomplished by construction and analysis of a highly redundant pooled genomic library containing approximately 216,000 functional clones that was constructed from 12 low-passage clinical isolates of Pseudomonas aeruginosa , 6 otorrheic isolates and 6 from other body sites. Sequence analysis of 3,214 randomly picked clones (mean insert size, ∼1.4 kb) from this library demonstrated that 348 (10.8%) of the clones were unique with respect to all genomic sequences of the P. aeruginosa prototype strain, PAO1. Hypothetical translations of the open reading frames within these unique sequences demonstrated protein homologies to a number of bacterial virulence factors and other proteins not previously identified in P. aeruginosa . PCR and reverse transcription-PCR-based assays were performed to analyze the distribution and expression patterns of a 70-open reading frame subset of these sequences among 11 of the clinical strains. These sequences were unevenly distributed among the clinical isolates, with nearly half (34/70) of the novel sequences being present in only one or two of the individual strains. Expression profiling revealed that a vast majority of these sequences are expressed, strongly suggesting they encode functional proteins.

We previously mapped a gene for severe pediatric gastroesophageal reflux disease ( GERD1) to a 9-cM interval on chromosome 13q14. In this report, we present the results of DNA sequencing and allelic association analyses that were done in an attempt to clone the GERD1 gene. Using a candidate transcript approach, we screened affected individuals for mutations in all transcribed regions of all genes, putative genes, and ESTs identified within the 6.2-Mb GERD1 locus based on alignments with the GenBank cDNA databases. From a total of 50 identifiable genes and 99 EST clusters in the GERD1 locus, we identified 163 polymorphisms (143 SNPs and 20 INDELs) in 21 genes and 37 ESTs. The patterns of inheritance and/or the high population frequencies of all polymorphic alleles identified in this study argued against causative relationships between any of the alleles and the GERD phenotype. Using a subset of 51 SNPs distributed throughout the GERD1 locus, we performed case-control and family (TDT) allelic association analyses on two sets of samples. The case-control study was performed with 73 GERD cases and 93 controls, and the family study was performed using 22 small families. SNP 160 (position 38,925,329 Mb, UCSChg15 map) gave a significant P value prior to multiple test correction in both the case control and family studies, while SNP168 (at 40,442,903 Mb) showed significant association after multiple test correction in the case-control sample, but was uninformative in the family sample. The results suggest that the GERD1 gene might be located near SNP160 or SNP168.

Summary Genetic epidemiological studies have shown that genetic factors are important in the pathogenesis of the idiopathic inflammatory bowel diseases (IBD), Crohn disease (CD), and ulcerative colitis (UC). A genome screen in the United Kingdom found linkage of IBD to a 41-cM region of chromosome 12, surrounding D12S83. We aimed to replicate this linkage and to narrow the region of interest. Nonparametric linkage analyses at microsatellites surrounding D12S83 were performed in 122 North American Caucasian families containing 208 genotyped IBD-affected relative pairs. Transmission/disequilibrium tests (TDTs) were also performed. We confirmed that IBD is linked to chromosome 12 (peak GENEHUNTER-PLUS LOD* score 2.76 [ P = .00016] between D12S1724 and D12S90). The evidence for linkage is contributed by both the group of CD-affected relative pairs (peak GENEHUNTER-PLUS LOD* score 1.79 [ P = .0021] between D12S1724 and D12S90) and the group of UC-affected relative pairs (peak GENEHUNTER-PLUS LOD* score 1.82 [ P = .0019] at D12S335). The TDT is positive at the D12S83 locus (global χ 2 = 16.41, 6 df, P = .012). In conclusion, we have independently confirmed linkage of IBD to the chromosome 12 region that we investigated. A positive TDT at D12S83 suggests that we have greatly narrowed the chromosome 12 region that contains an IBD locus.

Hereditary pancreatitis (HP) is a rare, early-onset genetic disorder characterized by epigastric pain and often more serious complications. We now report that an Arg-His substitution at residue 117 of the cationic trypsinogen gene is associated with the HP phenotype. This mutation was observed in all HP affected individuals and obligate carriers from five kindreds, but not in individuals who married into the families nor in 140 unrelated individuals. X-ray crystal structure analysis, molecular modelling, and protein digest data indicate that the Arg 117 residue is a trypsin-sensitive site. Cleavage at this site is probably part of a fail-safe mechanism by which trypsin, which is activated within the pancreas, may be inactivated; loss of this cleavage site would permit autodigestion resulting in pancreatitis.

Dominant mutations in the fibroblast growth factor receptor 2 (FGFR2) gene have been recently identified as causes of four phenotypically distinct craniosynostosis syndromes, including Crouzon, Jackson-Weiss, Pfeiffer, and Apert syndromes. These data suggest that the genetics of the craniosynostosis syndromes is more complex than would be expected from their simple autosomal-dominant inheritance pattern. Identical mutations in the FGFR2 gene have been reported to cause both Pfeiffer and Crouzon syndrome phenotypes. We now report the finding of a mutation in exon IIIc of the FGFR2 gene in a kindred affected with Crouzon syndrome (C1043 to G; Ala344Gly) that is identical to the mutation previously associated with Jackson-Weiss syndrome. We also report finding in a Crouzon kindred a mutation in the 3' end of exon IIIu (formerly referred to as exon 5, exon 7, or exon U) (A878 to C; Gln289Pro) which encodes the amino terminal portion of the Ig-like III domain of the FGFR2 protein. This exon is common to both the FGFR2 and the KGFR spliceoforms of the FGFR2 gene, unlike all previously reported Crouzon mutations, which have been found only in the FGFR2 spliceoform. These findings reveal further unexpected complexity in the molecular genetics of these craniosynostosis syndromes. The data implies that second-site mutations in FGFR2 itself (outside of exon IIIc) or in other genes may determine specific aspects of the phenotypes of craniosynostosis syndromes.

Eight low-passage-number Streptococcus pneumoniae clinical isolates, each of a different serotype and a different multilocus sequence type, were obtained from pediatric participants in a pneumococcal vaccine trial. Comparative genomic analyses were performed with these strains and two S. pneumoniae reference strains. Individual genomic libraries were constructed for each of the eight clinical isolates, with an average insert size of ∼1 kb. A total of 73,728 clones were picked for arraying, providing more than four times genomic coverage per strain. A subset of 4,793 clones were sequenced, for which homology searches revealed that 750 (15.6%) of the sequences were unique with respect to the TIGR4 reference genome and 263 (5.5%) clones were unrelated to any available streptococcal sequence. Hypothetical translations of the open reading frames identified within these novel sequences showed homologies to a variety of proteins, including bacterial virulence factors not previously identified in S. pneumoniae . The distribution and expression patterns of 58 of these novel sequences among the eight clinical isolates were analyzed by PCR- and reverse transcriptase PCR-based analyses, respectively. These unique sequences were nonuniformly distributed among the eight isolates, and transcription of these genes in planktonic cultures was detected in 81% (172/212) of their genic occurrences. All 58 novel sequences were transcribed in one or more of the clinical strains, suggesting that they all correspond to functional genes. Sixty-five percent (38/58) of these sequences were found in 50% or less of the clinical strains, indicating a significant degree of genomic plasticity among natural isolates.

We have analysed HALCA and global VLBI data for 2021+614, the first of eleven GPS quasars and galaxies to be observed by HALCA. We show that 2021+614 is a compact symmetric object of overall size similar to 40 pc, and confirm that the speed of separation of the two dominant lobes is approximately one tenth of the speed of light. Its apparent age of less than 1000 years provides additional support for the contention that compact symmetric radio objects associated with galaxies are young radio sources and the possible precursors of the classical FR1 or FR2 radio galaxies. (C) 2000 COSPAR. Published by Elsevier Science Ltd.

We recently identified a single R117H mutation in the cationic trypsinogen gene in several kindreds with an inherited form of acute and chronic pancreatitis (HP1), providing strong evidence that trypsin plays a central role in premature zymogen activation and pancreatitis. However, not all families studied have this mutation. The aim of this study was to determine the disease-causing mutation in kindreds with hereditary pancreatitis that lack the previously identified mutation.Clinical features of the HP1 kindreds were compared with those of the new kindreds (HP2), and genetic linkage analysis, screening for mutations through DNA sequencing, and screening an unaffected population were performed.The onset of symptoms was delayed and hospitalizations were fewer in HP2 compared with HP1 (P0.05). Linkage of the disease gene to chromosome 7q35 was established (logarithm of the odds, 3.73). Mutational screening identified a single A to T mutation resulting in an asparagine to isoleucine transition mutation at position 21 (N21I) in cationic trypsinogen. The mutation was absent in 94 unrelated individuals, representing 188 unique chromosomes.The identification of a second mutation in the cationic trypsinogen gene (HP2) suggests a dominant role of trypsin in premature protease activation-mediated forms of acute pancreatitis. The pathogenesis of hereditary pancreatitis also suggests that chronic pancreatitis may result from recurrent acute pancreatitis.

BACKGROUND & AIMS: Hereditary pancreatitis (HP) is an autosomal- dominant disorder with incomplete penetrance characterized by recurrent bouts of severe epigastric pain with onset usually at 5-10 years of age. A genetic linkage study was designed to identify the HP gene. METHODS: A 500-member pedigree was constructed from a U.S. kindred centered in eastern Kentucky and western Virginia. A genome-wide search strategy was employed using a 36-member subset of this family to determine the genetic locus for HP. Testing for linkage to microsatellite loci was performed at 20-cM intervals. RESULTS: Linkage was established between the HP phenotype and chromosome 7q in this subset of the family. Modeled as an autosomal dominant disorder with 80% penetrance, a maximal multipoint logarithm of the odds score of 4.3 was obtained using a four-point analysis consisting of markers D7S684, D7S661, D7S505, and the HP locus. Two microsatellite markers, D7S661 and D7S505, that correspond to the 7q35 region of chromosome 7 spanning a 6-cM region did not evidence obligate recombinations with HP. The centromeric and telomeric limits are defined by recombinations at D7S684 and D7S483, respectively, which generates a 19-cM locus for HP. Utilizing family members from the extended pedigree, a break in the high-risk haplotype between D7S684 and D7S661 was observed, which suggests it may be possible to exclude an additional 8 cM from the HP locus. A maximal pairwise logarithm of the odds score of 4.73 at a recombination fraction of theta at D7S684 was obtained with the addition of these extended family members. CONCLUSIONS: Linkage of HP to 7q35 represents a major advancement in our understanding of the genetic basis of this disorder. (Gastroenterology 1996 Jun;110(6):1975-80)

Crouzon craniofacial dysostosis (CFD) is an autosomal dominant craniofacial disorder characterized by premature craniosynostosis, shallow orbits and hypoplastic maxilla. To map the gene responsible, we have used a mapping strategy of testing for linkage to known developmental genes. Analysis of a large kindred established linkage between CFD and three loci (D10S190, D10S209 and D10S216) that span a 13 cM region on chromosome 10q. A maximum pairwise lod score of 4.42 (theta = 0) at D10S190 was obtained and the addition of a second kindred produced a combined pairwise lod score of 5.32 (theta = 0) at the same locus. The developmental gene, PAX2, located within this region, is an attractive candidate gene.

ContextGastroesophageal reflux (GER) has not previously been widely regarded as a hereditary disease. A few reports have suggested, however, that a genetic component may contribute to the incidence of GER, especially in its severe or chronic forms.ObjectiveTo identify a genetic locus that cosegregates with a severe pediatric GER phenotype in families with multiple affected members.DesignA genome-wide scan of families affected by severe pediatric GER using polymorphic microsatellite markers spaced at an average of 8 centimorgans (cM), followed by haplotyping and by pairwise and multipoint linkage analyses.SettingGeneral US community, with research performed in a university tertiary care hospital.SubjectsAffected and unaffected family members from 5 families having multiple individuals affected by severe pediatric GER, identified through a patient support group.Main Outcome MeasuresDetermination of inheritance patterns and linkage of a genetic locus with the severe pediatric GER phenotype by logarithm-of-odds (lod) score analysis, considering a lod score of 3 or greater as evidence of linkage.ResultsIn these families, severe pediatric GER followed an autosomal dominant hereditary pattern with high penetrance. A gene for severe pediatric GER was mapped to a 13-cM region on chromosome 13q between microsatellite markers D13S171 and D13S263. A maximum multifamily 2-point lod score of 5.58 and a maximum multifamily multipoint lod score of 7.15 were obtained for marker D13S1253 at map position 35 cM when presumptively affected persons were modeled as unknown (a maximum multipoint score of 4.88 was obtained when presumptively affected persons were modeled as unaffected).ConclusionThese data suggest that a gene for severe pediatric GER maps to chromosome 13q14.

Objective. —To determine if the polymerase chain reaction (PCR) can detect bacterial DNA in pediatric middle ear effusions that are sterile by standard cultural methods. Design. —Single-center, blinded, comparative study of diagnostic assays. The PCR-based detection systems for Moraxella catarrhalis, Haemophilus influenzae , and Streptococcus pneumoniae were designed and validated using a battery of DNAs obtained from cultured bacteria. Chronic middle ear effusion specimens were collected and comparatively analyzed by culture and the PCR. Setting. —Tertiary care pediatric hospital. Patients. —A total of 97 middle ear effusions were collected from pediatric outpatients at Children's Hospital of Pittsburgh (Pa) during myringotomy and tube placement for chronic otitis media with effusion (duration >3 months). All patients had failed multiple courses of antimicrobial therapy and were diagnosed by a combination of validated otoscopy and tympanograms. Main Outcome Measure. —Differences in the percentage of positive test results between PCR-based assays and culture for M catarrhalis, H influenzae , and S pneumoniae . Results. —Of the 97 specimens of otitis media with effusion, 28 (28.9%) tested positive by both culture and PCR for M catarrhalis, H influenzae , or S pneumoniae . An additional 47 specimens (48%) were PCR positive/culture negative for these three bacterial species. Thus, 75 (77.3%) of the 97 specimens tested PCR positive for one or more of the three test organisms. The minimum number of bacterial genomic equivalents present in the average culture-negative ear was estimated to be greater than 10 4 based on dilutional experiments. Conclusions. —The PCR-based assay systems can detect the presence of bacterial DNA in a significant percentage of culturally sterile middle ear effusions. While this finding is not proof of an active bacterial infectious process, the large number of bacterial genomic equivalents present in the ears is suggestive of an active process. ( JAMA . 1995;273:1598-1604)

Lack of major involvement of human uroplakin genes in vesicoureteral reflux: Implications for disease heterogeneity. background primary vesicoureteral reflux (vur) is a hereditary disorder characterized by the retrograde flow of urine into the ureters and kidneys. it affects about 1% of the young children and is thus one of the most common hereditary diseases. its associated nephropathy is an important cause of end-stage renal failure in children and adults. recent studies indicate that genetic ablation of mouse uroplakin (up) iii gene, which encodes a 47 kd urothelial-specific integral membrane protein forming urothelial plaques, causes vur and hydronephrosis. methods to begin to determine whether mutations in up genes might play a role in human vur, we genotyped all four up genes in 76 patients with radiologically proven primary vur by polymerase chain reaction (pcr) amplification and sequencing of all their exons plus 50 to 150 bp of flanking intronic sequences. results eighteen single nucleotide polymorphisms (snps) were identified, seven of which were missense, with no truncation or frame shift mutations. since healthy relatives of the vur probands are not reliable negative controls for vur, we used a population of 90 race-matched, healthy individuals, unrelated to the vur patients, as controls to perform an association study. most of the snps were not found to be significantly associated with vur. however, snp1 of up ia gene affecting a c to t conversion and an ala7val change, and snp7 of up iii affecting a c to g conversion and a pro154ala change, were marginally associated with vur (both p = 0.08). studies of additional cases yielded a second set of data that, in combination with the first set, confirmed a weak association of up iii snp7 in vur ( p = 0.036 adjusted for both subsets of cases vs. controls). conclusion such a weak association and the lack of families with simple dominant Mendelian inheritance suggest that missense changes of uroplakin genes cannot play a dominant role in causing VUR in humans, although they may be weak risk factors contributing to a complex polygenic disease. The fact that no truncation or frame shift mutations have been found in any of the VUR patients, coupled with our recent finding that some breeding pairs of UP III knockout mice yield litters that show not only VUR, but also severe hydronephrosis and neonatal death, raises the possibility that major uroplakin mutations could be embryonically or postnatally lethal in humans.

Thermal structure measurements obtained by the two Vega balloons show the Venus atmosphere in the middle cloud layer to be near-adiabatic, on the whole; but discrete air masses are present that differ slightly from one another in potential temperature and entropy. The Vega 1 temperatures are 6.5 K warmer than measured by Vega 2 at given pressures. Measurements taken by the Vega 2 lander on descent through these levels agree with the Vega 2 balloon data.

The typical 1-2 m/sec vertical winds encountered by the Vega balloons probably result from thermal convection. The consistent 6.5-kelvin differential between the Vega 1 and Vega 2 temperatures is attributable to disturbances of synoptic or planetary scale. According to the Doppler tracking the winds were stronger than on earlier missions, perhaps because of solar thermal tides. The motions of Vega 2 may have been affected by waves from mountainous terrain.