Outcomes of endoscopic ultrasound-guided pancreatic FNAC diagnosis for solid and cystic lesions at Manchester Royal Infirmary based upon the Papanicolaou Society of Cytopathology pancreaticobiliary terminology classification scheme.

OBJECTIVE: To compare endoscopic ultrasound (EUS)-FNAC diagnosis of pancreatic lesions with patient outcome based upon the Papanicolaou Society of Cytopathology pancreaticobiliary terminology classification scheme diagnostic categories: Panc 1 (non-diagnostic); Panc 2 (negative for malignancy/neoplasia); Panc 3 (atypical); Panc 4B (neoplastic, benign); Panc 4O (neoplastic, other); Panc 5 (suspicious of malignancy); and Panc 6 (positive/malignant). METHODS: All EUS-FNA pancreas specimens taken at Manchester Royal Infirmary in 2015 were prospectively classified according to the above scheme at the time of cytology reporting and data recorded prospectively. Subsequently, outcomes based on clinical follow-up or histopathology diagnosis were compared with the cytology diagnosis. RESULTS: 120 EUS-FNA pancreas specimens from 111 patients were received, of which 112 (93.3%) specimens had follow-up data. There were 79 and 41 EUS-FNA pancreas specimens from solid and cystic lesions, respectively. Based on the cytology diagnosis the specimens were classified as Panc 1 (7.5%), Panc 2 (33.3%), Panc 3 (2.5%), Panc 4B (2.5%), Panc 4O (15.0%), Panc 5 (3.3%) and Panc 6 (35.9%). The performance indicators for diagnosis of malignancy or neoplasia with malignant potential, included sensitivity (95.4%), specificity (100%), positive predictive value (100%), negative predictive value (92.3%), false positive rate (0%) and false negative rate (4.6%). CONCLUSIONS: The Papanicolaou Society of Cytopathology pancreaticobiliary terminology classification scheme is a logical system that can easily be introduced in a diagnostic cytopathology service. This classification scheme acts as an aid to diagnostic reporting, clear communication of significant results including risk of neoplasia/malignancy to clinicians, clinical audit and comparison of results with other centres.

A high-resolution genetic map of the familial Mediterranean fever candidate region allows identification of haplotype-sharing among ethnic groups.

Familial Mediterranean fever (FMF) is a recessive disorder of inflammation caused by mutations in a gene (designated MEFV) on chromosome 16p13.3. We have recently constructed a 1-Mb cosmid contig that includes the FMF critical region. Here we show genotype data for 12 markers from our physical map, including 5 newly identified microsatellites, in FMF families. Intrafamilial recombinations placed MEFV in the approximately 285 kb between D16S468/D16S3070 and D16S3376. We observed significant linkage disequilibrium in the North African Jewish population, and historical recombinants in the founder haplotype placed MEFV between D16S3082 and D16S3373 (approximately 200 kb). In smaller panels of Iraqi Jewish, Arab, and Armenian families, there were significant allelic associations only for D16S3370 and D16S2617 among the Armenians. A sizable minority of Iraqi Jewish and Armenian carrier chromosomes appeared to be derived from the North African Jewish ancestral haplotype. We observed a unique FMF haplotype common to Iraqi Jews, Arabs, and Armenians and two other haplotypes restricted to either the Iraqi Jewish or the Armenian population. These data support the view that a few major mutations account for a large percentage of the cases of FMF and suggest that some of these mutations arose before the affected Middle Eastern populations diverged from one another.

Familial Mediterranean fever and hyperimmunoglobulinemia D syndrome: two diseases with distinct clinical, serologic, and genetic features.

OBJECTIVE: To determine whether the 2 periodic febrile syndromes familial Mediterranean fever (FMF) and hyperimmunoglobulinemia D syndrome (HIDS) are distinct diseases. METHODS: Clinical manifestations of the diseases were analyzed by physicians experienced with FMF and HIDS. Serum immunoglobulin (Ig) levels were studied in 70 patients with FMF using nephelometry or ELISA and compared with Ig levels in 50 patients with HIDS. Genetic linkage of HIDS with the chromosome 16 polymorphic locus RT70, currently used for refined localization of the FMF susceptibility gene (MEFV), was studied in 9 HIDS families (18 patients) using polymerase chain reaction amplification and gel electrophoresis. RESULTS: The main clinical features distinguishing FMF from HIDS were lymphadenectomy, skin eruption, and symmetrical oligoarthritis in HIDS, and monoarthritis, peritonitis, and pleuritis in FMF. Increased IgG levels were found in 12 patients with FMF (17%), IgA in 16 (23%), IgM in 9 (13%), and IgD in 9 (13%), significantly lower than the prevalence reported for HIDS. We found no evidence for genetic linkage between HIDS and the chromosome 16 marker RT70. CONCLUSION: HIDS and FMF are different entities, clinically, immunologically, and genetically.

Construction of a 1-Mb restriction-mapped cosmid contig containing the candidate region for the familial Mediterranean fever locus (MEFV) on chromosome 16p 13.3.

In this paper we describe the assembly and restriction map of a 1.05-Mb cosmid contig spanning the candidate region for familial Mediterranean fever (FMF), a recessively inherited disorder of inflammation localized to 16p13.3. Using a combination of cosmid walking and screening for P1, PAC, BAC, and YAC clones, we have generated a contig of genomic clones spanning approximately 1050 kb that contains the FMF critical region. The map consists of 179 cosmid, 15 P1, 10 PAC, 3 BAC, and 17 YAC clones, anchored by 27 STS markers. Eight additional STSs have been developed from the approximately 700 kb immediately centromeric to this genomic region. Five of the 35 STSs are microsatellites that have not been previously reported. NotI and EcoRI mapping of the overlapping cosmids, hybridization of restriction fragments from cosmids to one another, and STS analyses have been used to validate the assembly of the contig. Our contig totally subsumes the 250-kb interval recently reported, by founder haplotype analysis, to contain the FMF gene. Thus, our high-resolution clone map provides an ideal resource for transcriptional mapping toward the eventual identification of this disease gene.

Phylogenetic footprinting of hypersensitive site 3 of the beta-globin locus control region.

Hypersensitive site 3 (HS3) of the beta-like globin locus control region has been implicated as an important regulator of the beta-like globin genes, but the trans factors that bind HS3 have only been partially characterized. Using a five-species alignment (human, galago, rabbit, goat, and mouse) that represents 370 million years of evolution, we have identified 24 phylogenetic footprints in the HS3 core and surrounding regions. Probes corresponding to the human sequence at each footprint have been used in binding studies to identify the nuclear factors that bind within and near these conserved sequence elements. Among the high-affinity interactions observed were several binding sites for proteins with repressor activity, including YY1, CCAAT displacement protein, and G1/G2 complexes (uncharacterized putative repressors) and several binding sites for the stage selector protein. To complement this analysis, orthologous galago sequences were also used to derive probes and the pattern of proteins binding to human and galago probes was compared. Binding interactions differing between these two species could be responsible for the different expression patterns shown by the two gamma genes (galago gamma is embryonic; human gamma is fetal). Alternatively, binding interactions that are conserved in the two species may be important in the regulation of common expression patterns (eg, repression of gamma in adult life).

Linkage disequilibrium mapping places the gene causing familial Mediterranean fever close to D16S246.

This report presents refined genetic mapping data for the gene causing familial Mediterranean fever (FMF), a recessively inherited disorder of inflammation. We sampled 65 Jewish, Armenian, and Arab families and typed them for eight markers from chromosome 16p. Using a new algorithm that permits multipoint calculations for a dense map of markers in consanguineous families, we obtained a maximal LOD score of 49.2 at a location 1.6 cM centromeric to D16S246. A specific haplotype at D16S283-D16S94-D16S246 was found in 76% of Moroccan and 32% of non-Moroccan Jewish carrier chromosomes, but this haplotype was not overrepresented in Armenian or Arab FMF carriers. Moreover, the 2.5-kb allele at D16S246 was significantly associated with FMF in Moroccan and non-Moroccan Jews but not in Armenians or Arabs. Since the Moroccan Jewish community represents a relatively recently established and genetically isolated founder population, we analyzed the Moroccan linkage-disequilibrium data by using Luria-Delbrück formulas and simulations based on a Poisson branching process. These methods place the FMF susceptibility gene within 0.305 cM of D16S246 (2-LOD-unit range 0.02-0.64 cM).

Evolutionary strategies for the elucidation of cis and trans factors that regulate the developmental switching programs of the beta-like globin genes.

We describe three strategies for the identification of specific cis and trans factors that regulate globin gene expression, all three of which are based on the evolution of the globin genes and their expression patterns. The first approach, phylogenetic footprinting, relies on a search for sequence similarities and is designed to elucidate the factors that control those expression patterns which are shared by orthologous globin genes of all eutherian mammals (e.g., the expression of the epsilon globin genes in the embryonic yolk sac and its repression in fetal and adult hematopoietic tissues). The second approach, differential phylogenetic footprinting, relies on a search for sequence differences. This approach may be of value in identifying the mechanisms underlying the generation of novel expression patterns in specific lineages (e.g., the expression of gamma as a fetal gene in the simian primates in contrast with the embryonic expression of gamma in all other mammals). Finally, motif-based phylogenetic analysis takes into consideration the fact that many transcription factors are quite flexible in the recognition of their cognate sites. The approach allows the detection of functionally conserved binding sites despite their sequence variation.

Rural mental health coverage under health care reform.

Efforts to integrate services and financing under health care reform hold benefits for provision of services to rural mentally ill persons. Remote areas pose a particular challenge as the unique characteristics of rural America are even more evident. The model for managed care in remote rural areas will differ from those used in urban and their adjacent rural areas. Universal coverage would remove the barriers to accessing care for this population, but does not assure availability of adequate mental health services are or providers in rural areas. Characteristics of currently available rural mental health services are presented and obstacles to expanded delivery under health care reform are outlined.

Differential phylogenetic footprinting as a means to identify base changes responsible for recruitment of the anthropoid gamma gene to a fetal expression pattern.

Expression of the anthropoid (simian) gamma gene in fetal life contrasts with the exclusively embryonic expression pattern of the gamma-like genes of other eutherian mammals. To elucidate the factors responsible for this change in expression pattern, we utilized a strategy called differential phylogenetic footprinting (DPF). This strategy entails the following: (a) identification, within regulatory regions, of the gamma promoter, of individual nucleotides that differ between human (fetal expression), and galago (embryonic expression) gamma genes, (b) analysis of the effect of these nucleotide differences on the binding of nuclear proteins to human and galago sequences, and (c) assessment of the functional consequences of these binding changes in expression assays. The DPF analysis revealed several proteins that bind upstream from the CCAAT motif in the galago gamma promoter but do not bind to the corresponding region of the human gamma promoter. In transfection assays, binding of these proteins is associated with erythroid-specific repression of promoter strength. Binding sites for these proteins also occur near the CCAAT box of other embryonically expressed genes, including rabbit, mouse, and dwarf lemur gamma genes and the human epsilon globin gene. These data are consistent with the hypothesis that sequence changes near the proximal CCAAT box in the ancestral simian gamma gene may have facilitated a novel expression pattern by reducing the binding of repressors that act in the fetal stage.

Phylogenetic footprinting reveals unexpected complexity in trans factor binding upstream from the epsilon-globin gene.

The human epsilon-globin gene undergoes dramatic changes in transcriptional activity during development, but the molecular factors that control its high expression in the embryo and its complete repression at 6-8 weeks of gestation are unknown. Although a putative silencer has been identified, the action of this silencer appears to be necessary but not sufficient for complete repression of epsilon gene expression, suggesting that multiple control elements may be required. Phylogenetic footprinting is a strategy that uses evolution to aid in the elucidation of these multiple control points. The strategy is based on the observation that the characteristic developmental expression pattern of the epsilon gene is conserved in all placental mammals. By aligning epsilon genomic sequences (from -2.0 kb upstream to the epsilon polyadenylylation signal), conserved sequence elements that are likely binding sites for trans factors can be identified against the background of neutral DNA. Twenty-one such conserved elements (phylogenetic footprints) were found upstream of the epsilon gene. Oligonucleotides spanning these conserved elements were used in a gel-shift assay to reveal 47 nuclear binding sites. Among these were 8 binding sites for YY1 (yin and yang 1), a protein with dual (activator or repressor) activity; 5 binding sites for the putative stage selector protein, SSP; and 7 binding sites for an as yet unidentified protein. The large number of high-affinity interactions detected in this analysis further supports the notion that the epsilon gene is regulated by multiple redundant elements.

Phylogenetic footprinting reveals a nuclear protein which binds to silencer sequences in the human gamma and epsilon globin genes.

Tissue- and developmental stage-specific expression of the human beta-like globin genes is regulated by a combination of ubiquitous and erythroid-restricted trans factors that bind to cis elements near each of the five active genes. Additional interactions of these cis and trans factors with sequences located in the far 5' end of the cluster occur by as yet obscure mechanisms. Because of the complexity of this regulatory puzzle, precise identification of the determinants that control hemoglobin switching has proven difficult. Phylogenetic footprinting is an evolutionary approach to this problem which is based on the supposition that the basic mechanisms of switching are conserved throughout mammalian phylogeny. Alignment of the 5' flanking regions of the gamma genes of several species allows the identification of footprints of 100% conserved sequence. We have now tested oligomers spanning 13 such phylogenetic footprints and find that 12 are bound by nuclear proteins. One conserved element located at -1086 from the gamma genes exhibits repressor activity in transient transfection studies. The protein that binds this element, CSBP-1 (conserved sequence-binding protein 1), also binds at three sites within a silencer element upstream from the epsilon globin gene. Further analysis reveals that the CSBP-1 binding activity is identical to that of a recently cloned zinc finger protein that has been shown to act as a repressor in other systems. The binding of CSPB-1 to silencer sequences in the epsilon and gamma globin genes may be important in the stage-specific silencing of these genes.

Divergent patterns of incorporation of bromodeoxyuridine and iododeoxyuridine in human colorectal tumor cell lines.

Using a panel of four human colorectal tumor (HCT) cell lines, we have quantitatively characterized the incorporation of bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd) into DNA, both as individual agents and in combination with fluoropyrimidines. The intrinsic ability of these cell lines to incorporate BrdUrd, as reflected by the concentration required to achieve half-maximal incorporation, varied almost 4-fold across this panel, from 1.6 microM for HuTu80 cells to 6.1 microM for HT29 cells. Three of the four cell lines (HT29, SW480, SW620) responded to fluoropyrimidines as expected, displaying 100-150% increases in BrdUrd incorporation when combined with growth inhibitory concentrations of fluorouracil (FUra). In contrast, neither FUra nor fluorodeoxyuridine (FdUrd) was able to increase BrdUrd incorporation in HuTu80 cells by more than 25%, even in the presence of 100 microM leucovorin. IdUrd incorporation was modulated to a substantially higher degree in both HT29 and HuTu80 cell lines. Finally we demonstrate the feasibility of a technique for evaluating the net effect of fluoropyrimidine treatments on de novo thymidine nucleotide production in a single specimen, using a combination of normotopic and stable-isotope labeled BrdUrd. We propose that this approach may be useful in evaluating the response of an individual tumor to fluoropyrimidines in vivo.