Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene.

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.

Evidence of a founder effect for four cathepsin C gene mutations in Papillon-Lefèvre syndrome patients.

We describe a mutation and haplotype analysis of Papillon-Lefèvre syndrome probands that provides evidence of a founder effect for four separate cathepsin C mutations. A total of 25 different cathepsin C mutations have been reported in 32 families with Papillon-Lefèvre syndrome (PLS) and associated conditions. A characteristic of these findings is the diversity of different cathepsin C mutations that have been identified. To evaluate the generality of cathepsin C mutations, PLS probands representative of five reportedly unrelated Saudi Arabian families were evaluated by mutational and haplotype analyses. Sequence analysis identified two cathepsin C gene mutations: a novel exon 7 G300D mutation was found in the proband from one family, while probands from four families shared a common R272P mutation in exon 6. The R272P mutation has been previously reported in two other non-Saudi families. The presence of the R272P mutation in probands from these four Saudi families makes this the most frequently reported cathepsin C mutation. To distinguish between the presence of a possible founder effect or a mutational hot spot for the R272P mutation, we performed haplotype analysis using six novel DNA polymorphisms that span a 165 kb interval containing the cathepsin C gene. Results of haplotype analysis for genetic polymorphisms within and flanking the cathepsin C gene are consistent with inheritance of the R272P mutation "identical by descent" from a common ancestor in these four Saudi families. Haplotype analysis of multiple PLS probands homozygous for other cathepsin C mutations (W249X, Q286X, and T153I) also supports inheritance of each of these mutations from common ancestors. These data suggest that four of the more frequently reported cathepsin C mutations have been inherited from common ancestors and provide the first direct evidence for a founder effect for cathepsin C gene mutations in PLS. Identification of these six short tandem repeat polymorphisms that span the cathepsin C gene will permit haplotype analyses to determine other founder haplotypes of cathepsin C mutations in additional PLS families.

Novel ENAM mutation responsible for autosomal recessive amelogenesis imperfecta and localised enamel defects.

The genetic basis of non-syndromic autosomal recessive forms of amelogenesis imperfecta (AI) is unknown. To evaluate five candidate genes for an aetiological role in AI. In this study 20 consanguineous families with AI were identified in whom probands suggested autosomal recessive transmission. Family members were genotyped for genetic markers spanning five candidate genes: AMBN and ENAM (4q13.3), TUFT1 (1q21), MMP20 (11q22.3-q23), and KLK4 (19q13). Genotype data were evaluated to identify homozygosity in affected individuals. Mutational analysis was by genomic sequencing. Homozygosity linkage studies were consistent for localisation of an AI locus in three families to the chromosome 4q region containing the ENAM gene. ENAM sequence analysis in families identified a 2 bp insertion mutation that introduced a premature stop codon in exon 10. All three probands were homozygous for the same g.13185_13186insAG mutation. These probands presented with a generalised hypoplastic AI phenotype and a class II openbite malocclusion. All heterozygous carriers of the g.13185_13186insAG mutation had localised hypoplastic enamel pitting defects, but none had AI or openbite. The phenotype associated with the g.13185_13186insAG ENAM mutation is dose dependent such that ARAI with openbite malocclusion segregates as a recessive trait, and enamel pitting as a dominant trait.

Mutations of the UMOD gene are responsible for medullary cystic kidney disease 2 and familial juvenile hyperuricaemic nephropathy.

INTRODUCTION: Medullary cystic kidney disease 2 (MCKD2) and familial juvenile hyperuricaemic nephropathy (FJHN) are both autosomal dominant renal diseases characterised by juvenile onset of hyperuricaemia, gout, and progressive renal failure. Clinical features of both conditions vary in presence and severity. Often definitive diagnosis is possible only after significant pathology has occurred. Genetic linkage studies have localised genes for both conditions to overlapping regions of chromosome 16p11-p13. These clinical and genetic findings suggest that these conditions may be allelic. AIM: To identify the gene and associated mutation(s) responsible for FJHN and MCKD2. METHODS: Two large, multigenerational families segregating FJHN were studied by genetic linkage and haplotype analyses to sublocalise the chromosome 16p FJHN gene locus. To permit refinement of the candidate interval and localisation of candidate genes, an integrated physical and genetic map of the candidate region was developed. DNA sequencing of candidate genes was performed to detect mutations in subjects affected with FJHN (three unrelated families) and MCKD2 (one family). RESULTS: We identified four novel uromodulin (UMOD) gene mutations that segregate with the disease phenotype in three families with FJHN and in one family with MCKD2. CONCLUSION: These data provide the first direct evidence that MCKD2 and FJHN arise from mutation of the UMOD gene and are allelic disorders. UMOD is a GPI anchored glycoprotein and the most abundant protein in normal urine. We postulate that mutation of UMOD disrupts the tertiary structure of UMOD and is responsible for the clinical changes of interstitial renal disease, polyuria, and hyperuricaemia found in MCKD2 and FJHN.

Genomic organization of the human fibroblast growth factor receptor 2 (FGFR2) gene and comparative analysis of the human FGFR gene family.

The human fibroblast growth factor receptor (FGFR) genes play important roles in normal vertebrate development. Mutations in the human FGFR2 gene have been associated with many craniosynostotic syndromes and malformations, including Crouzon, Pfeiffer, Apert, Jackson-Weiss, Beare-Stevenson cutis gyrata, and Antley-Bixler syndromes, and Kleeblaatschadel (cloverleaf skull) deformity. The mutations identified to date are concentrated in the previously characterized region of FGFR2 that codes for the extracellular IgIII domain of the receptor protein. The search for mutations in other regions of the gene, however, has been hindered by lack of knowledge of the genomic structure. Using a combination of genomic library screening, long-range PCR, and genomic walking, we have characterized the genomic structure of nearly the entire human FGFR2 gene, including a delineation of the organization and size of all introns and exons and determination of the DNA sequences at the intron/exon boundaries. Comparative analysis of the human FGFR gene family reveals that the genomic organization of the FGFRs is relatively conserved. Moreover, alignment of the amino acid sequences shows that the four corresponding proteins share 46% identity overall, with up to 70% identity between individual pairs of FGFR proteins. However, the FGFR2 gene contains an additional exon not found in other members of the family, and it also has much larger intronic sequences throughout the gene. Remarkable similarities in genomic organization, intron/exon boundaries, and intron sizes are found between the human and mouse FGFR2 genes. Knowledge gained from this study of the human FGFR2 gene structure may prove useful in future screening studies designed to find additional mutations associated with craniosynostotic syndromes, and in understanding the molecular and cell biology of this receptor family.

Lack of R117H mutation in the cationic trypsinogen gene in patients with tropical pancreatitis from Bangladesh.

The etiology of nonalcoholic chronic pancreatitis, occurring in tropical regions, is unknown. Although environmental factors may play a role in its pathogenesis, a specific genetic predisposition may be necessary. The genetic mutation responsible for hereditary pancreatitis was described recently. Unlike in patients with hereditary pancreatitis, we found a lack of the R117H mutation in the cationic trypsinogen gene in all patients with tropical pancreatitis from Bangladesh.

RNA differential display of scarless wound healing in fetal rabbit indicates downregulation of a CCT chaperonin subunit and upregulation of a glycophorin-like gene transcript.

BACKGROUND/PURPOSE: Scars form as wounds heal in adult organisms. In addition to disrupting cosmetic appearance, scar tissue can cause significant morbidity, and even death if it blocks vital organ function. Previous work has established that fetal wounds, especially in early to midgestation, can heal without scarring. Because such inherent physiological mechanisms ultimately are under genetic control, a study was initiated to elucidate the differences in gene expression that produce scarless wound healing in the mammalian fetus but scarring in postnatal wounds. Reverse transcription polymerase chain reaction (RT-PCR) differential display (DD) was used to detect differentially expressed mRNA transcripts in a rabbit model of wound healing. METHODS: Adult and 21-day fetal full-thickness rabbit skin specimens from wounded and unwounded sites were harvested 12 hours postwounding. RNA extracted from the tissue was used as a template in DD reactions using anchoring and random primers to generate tissue-specific gene expression fingerprints. The over 2,000 resulting amplimers (gene transcripts) were screened for differential expression among the 4 types of specimens: fetal control (unwounded), fetal wound, adult control, and adult wound. Selected bands distinctly upregulated or downregulated in fetal wound lanes on the DD gels were excised, and the cDNA was extracted, reamplified, cloned into vectors, and sequenced. DD results were confirmed by limiting-dilution RT-PCR using sequence-specific primers. RESULTS: Differential display (DD) showed 22 amplimers that were significantly upregulated in all fetal wound samples as compared with little or no expression in fetal control, adult control, or adult wound tissues. Conversely, 5 transcripts were downregulated in the fetal wound specimens but highly expressed in the 3 comparison tissues. Reamplification of selected transcripts by PCR, followed by cloning and DNA sequencing, yielded 7 distinct sequences, each representing a gene expressed differently in fetal wound than in the other 3 tissues. A transcript that was downregulated in fetal wound showed very high sequence homology to part of the human gene for the eta subunit of the hetero-oligomeric particle CCT (the chaperonin containing T-complex polypeptide 1 or TCP-1). An upregulated amplimer showed significant DNA sequence homology to glycophorins A and B. One sequence was identified as 28S rRNA. The remaining 4 candidate sequences showed no significant homology to known genes, but 1 had high homology to expressed sequence tags of unknown function. CONCLUSIONS: With careful experimental design and proper controls and verifications, differential display of RNA expression is a potentially powerful method of finding genes that specifically regulate a particular physiological process such as fetal wound healing. No a priori knowledge of what genes might be involved, or why, is necessary. This study indicates that downregulation of a gene that codes for a chaperonin subunit and upregulation of several other genes may be involved in the striking scarless character of wound healing in the mammalian fetus. Results suggest the hypothesis that downregulation of the CCT chaperonin in fetal wound may inhibit the formation of myofibroblasts, a cell type that correlates highly with scarring in postnatal wound healing, by preventing the folding of sufficient alpha-smooth muscle actin to form the stress fibers characteristic of these cells.

Oncolytic virotherapy for ovarian carcinomatosis using a replication-selective vaccinia virus armed with a yeast cytosine deaminase gene.

In this study, we assessed the ability of a highly tumor-selective oncolytic vaccinia virus armed with a yeast cytosine deaminase gene to infect and lyse human and murine ovarian tumors both in vitro and in vivo. The virus vvDD-CD could infect, replicate in and effectively lyse both human and mouse ovarian cancer cells in vitro. In two different treatment schedules involving either murine MOSEC or human A2780 ovarian carcinomatosis models, regional delivery of vvDD-CD selectively targeted tumor cells and ovarian tissue, effectively delaying the development of either tumor or ascites and leading to significant survival advantages. Oncolytic virotherapy using vvDD-CD in combination with the prodrug 5-fluorocytosine conferred an additional long-term survival advantage upon tumor-bearing immunocompetent mice. These findings demonstrate that a tumor-selective oncolytic vaccinia combined with gene-directed enzyme prodrug therapy is a highly effective strategy for treating advanced ovarian cancers in both syngeneic mouse and human xenograft models. Given the biological safety, tumor selectivity and oncolytic potency of this armed oncolytic virus, this dual therapy merits further investigation as a promising new treatment for metastatic ovarian cancer.

Aquaporin expression in developing human teeth and selected orofacial tissues.

The aquaporin (AQP) family of membrane channel proteins function as selective pores through which water, glycerol, and other small solutes cross the cell plasma membrane. To date, 11 members of this transporter family, designated AQP0-10, have been cloned and characterized in humans. The AQPs are differentially expressed in temporospatial patterns, where different AQPs demonstrate distinct tissue distributions that may reflect differing cell membrane transport functions. The purpose of this study was to evaluate AQP expression in the developing human teeth by RT-PCR and Western blot analysis. To access the generality of AQP expression, selected other orofacial tissues were studied by RT-PCR. The presence of all eleven human AQPs was screened in each tissue by RT-PCR. Positive amplification products were verified by direct DNA sequencing. AQPs 1, 3, 4, 5, 6, and 10 were identified by RT-PCR in developing teeth, and AQP1, 3, 5, and 6 were confirmed by Western blot analysis. AQP 4 was not detected by Western blot analysis, and we were unable to test for the recently identified AQP10 due to unavailability of antibodies. AQPs detected in other orofacial tissues by RT-PCR included gingiva (AQP3, 7, 10); Meckel's cartilage (AQP1, 3, 4, 5, 6); submandibular gland (AQP1, 3, 4, 5, 6, 7); masseter muscle (AQP1, 3, 4, 7, 8, 9,10); and infrahyoid muscle (AQP1, 3, 4,10). These results demonstrate that multiple aquaporins are expressed in developing teeth and in selected orofacial tissues.

Evidence of bacterial metabolic activity in culture-negative otitis media with effusion.

CONTEXT: Otitis media with effusion (OME) can lead to significant hearing loss in children. Although previous studies have shown that bacterial DNA is present in a significant percentage of effusions sterile by culture, whether the DNA represents viable organisms or "fossilized remains" is unknown. OBJECTIVE: To determine if bacterial messenger RNA (mRNA), as detected by a reverse transcriptase-polymerase chain reaction (RT-PCR)-based assay, is present in chronic pediatric middle ear effusions that contain bacterial DNA but are sterile by standard cultural methods. Bacterial mRNAs have a half-life measured in seconds to minutes; therefore, detection of bacteria-specific mRNAs would be evidence that metabolically active organisms are present. DESIGN: Blinded comparative study. PATIENTS: A total of 93 effusions from pediatric outpatients seen for myringotomy and tube placement for chronic (>3 months) OME (median age of children, 17 months). SETTING: Tertiary care pediatric hospital. MAIN OUTCOME MEASURES: Percentage of positive test results for RT-PCR-based assays compared with culture for Haemophilus influenzae and concordance between RT-PCR and PCR-based findings for bacterial nucleic acids. RESULTS: Eleven (11.8%) of the 93 specimens tested positive by culture, PCR, and RT-PCR for H influenzae. A total of 29 specimens (31.2%) were positive by PCR but negative by culture for H influenzae. All 29 specimens were positive by RT-PCR for H influenzae-specific mRNA. CONCLUSIONS: The RT-PCR-based assay system can detect the presence of bacterial mRNA in a significant percentage of culturally sterile middle ear effusions, establishing the presence of viable, metabolically active, intact organisms in some culture-negative OME.

Localization, genomic organization, and alternative transcription of a novel human SAM-dependent methyltransferase gene on chromosome 2p22-->p21.

As part of our studies to identify the gene responsible for hereditary gingival fibromatosis, GINGF (OMIM 135300), we have identified and cloned a novel human gene that contains the highly conserved methyltransferase domain characteristic of S-adenosylmethionine-dependent methyltransferases. We localized this gene (C2orf8 encoding 288L6 SAM-methyltransferase) to chromosome 2p22-->p21 by FISH, and sublocalized it to BAC RP11 288L6 flanked by D2S2238 and D2S2331. Computational analysis of aligned ESTs identified ten exons in the hypothetical C2orf8 gene. Results of RACE analyses in placenta identified multiple transcripts of this gene with heterogeneity at the 5'-UTR. Alternative transcription and tissue specific expression of C2orf8 were detected by RT-PCR and Northern blot analyses. C2orf8 is expressed in a variety of tissues including brain, colon, gingiva, heart, kidney, liver, lung, placenta, small intestine, spleen, and thymus. Open reading frame analysis of the alternative transcripts identified a shared coding region spanning exons 6-10. This ORF consists of 732 nucleotides encoding a putative 244 amino acid protein. Bioinformational searches of both C2orf8 and the putative protein product identified three methyltransferase motifs conserved across many prokaryotic and eukaryotic species. Sequence analyses of C2orf8 excluded coding region mutations as causative of GINGF.

Evolution of human immunodeficiency virus type 1 envelope sequences in infected individuals with differing disease progression profiles.

Sequence variation displayed by the human immunodeficiency virus type 1 (HIV-1) has been proposed to be linked to the pathogenesis of acquired immunodeficiency syndrome (AIDS). To assess viral evolution during the course of infection, we evaluated sequence variability in the env variable domains in four HIV-1-infected individuals exhibiting differing profiles of CD4+ T cell decline when followed from seroconversion until the development of AIDS or loss of followup. Proviral sequences encoding the V3-V5 region of gp 120 were obtained following PCR amplification of peripheral blood mononuclear cell DNA and cloning. Virus in each patient was relatively homogeneous early in infection and then diverged with time, more consistently at its nonsynonymous sites. Just prior to or coincident with a rapid decline in CD4+ T cell numbers, sequences were found with basic amino acid substitutions clustered within and downstream of the gp 120 V3 domain. Within the constraints of the current data set, we conclude that the virus appears to continually accumulate changes in its amino acid sequences well into the time of marked CD4+ T cell decline.

Clinical characteristics of hereditary pancreatitis in a large family, based on high-risk haplotype. The Midwest Multicenter Pancreatic Study Group (MMPSG)

OBJECTIVES: Because there are no markers for hereditary pancreatitis (HP), diagnosis has relied on clinical features and inferences. Identification of the HP disease gene locus on chromosome 7q35 provides the first genetic marker for HP, allowing an accurate comparison of the clinical diagnosis of HP with the presence of a high-risk HP haplotype. Our objectives were to compare the clinical diagnosis of HP with inheritance of the HP gene and to characterize the common clinical features. METHODS: A detailed questionnaire was administered to 102 study participants of a large HP kindred. Blood samples were taken for DNA extraction and high-risk haplotype determination. Clinical findings were compared with the presence of a high-risk haplotype. RESULTS: A family tree of more than 500 members and eight generations was constructed, and clinical features of the 102 participants were determined. HP occurred before the age of 5 yr in 58% of subjects, who presented with common symptoms of abdominal pain, nausea/vomiting, and frequent attacks. Thirty-five probands, of whom 80% had clinical symptoms, carried the high-risk haplotype, confirming previous estimates of 80% penetrance. Thirty-two of the study participants had been clinically diagnosed with HP, whereas 70 were clinically unaffected. With regard to the presence of the high-risk haplotype, 87.5% of the clinically diagnosed patients were affected by HP (true positive), whereas 12.5% did not carry the high-risk haplotype (false positive). Seven obligate carriers were identified through DNA analysis; three had previously been unrecognized because of lack of affected offspring. CONCLUSIONS: The diagnosis of hereditary pancreatitis on clinical grounds alone may be inaccurate in less severe cases, as is the exclusion of carrier status through family tree analysis. Therefore, a definitive diagnosis of hereditary pancreatitis in equivocal cases or exclusion of a carrier state should include analysis of genetic markers.

Mutations in the cationic trypsinogen gene are associated with recurrent acute and chronic pancreatitis.

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: The onset of symptoms was delayed and hospitalizations were fewer in HP2 compared with HP1 (P < 0.05). 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. CONCLUSIONS: 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.

Evaluation of human leukocyte N-formylpeptide receptor (FPR1) SNPs in aggressive periodontitis patients.

Polymorphonuclear neutrophils (PMNs) are attracted to sites of infection by N-formylpeptide (fMLP) chemoattractants. The high-affinity fMLP receptor (FPR1) of phagocytic cells interacts with bacterial fMLP and mediates chemotaxis, degranulation, and superoxide production. These cellular functions are disrupted in PMN from aggressive periodontitis (AP) patients. Two FPR1 gene single nucleotide polymorphisms (SNPs), c.329T>C and c.378C>G, have been associated with a localized form of AP in African-American patients. To evaluate the generality of these SNPs in AP patients, we sequenced a 363 bp interval of the FPR1 gene in an ethnically diverse group of patients (n=111) and controls (n=115). Neither c.329T>C nor c.378C>G were detected in the 452 alleles sequenced. Six SNPs were identified including two located in the FPR1 second extracellular loop that were significantly associated with the AP phenotype in African-American patients (p.R190W, P=0.0033; and p.N192K, P=0.0018). These two SNPs show three predominant haplotypes, each associated with a different disease risk in African-Americans. These data do not support the hypothesis that the FPR1 SNPs c.329T>C and c.378C>G play an etiologic role in aggressive periodontitis, but do suggest that SNPs in the second extracellular loop may be etiologically important.

Crouzon syndrome: mutations in two spliceoforms of FGFR2 and a common point mutation shared with Jackson-Weiss syndrome.

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.