Olfaction in Parkin single and compound heterozygotes in a cohort of young onset Parkinson's disease patients.

BACKGROUND: Parkin related Parkinson's disease (PD) is differentiated from idiopathic PD by absent or sparse Lewy bodies, and preserved olfaction. The significance of single Parkin mutations in the pathogenesis of PD is debated. OBJECTIVES: To assess olfaction results according to Parkin mutation status. To compare the prevalence of Parkin single heterozygous mutations in patients diagnosed with PD to the rate in healthy controls in order to establish whether these single mutations could be a risk factor for developing PD. METHODS: Parkin gene mutation testing was performed in young onset PD (diagnosed <50 years old) to identify three groups: Parkin homozygous or compound heterozygote mutation carriers, Parkin single heterozygote mutation carriers, and non-carriers of Parkin mutations. Olfaction was tested using the 40-item British version of the University of Pennsylvania smell identification test (UPSIT). RESULTS: Of 344 young onset PD cases tested, 8 (2.3%) were Parkin compound heterozygotes and 13 (3.8%) were Parkin single heterozygotes. Olfaction results were available in 282 cases (eight compound heterozygotes, nine single heterozygotes, and 265 non-carriers). In Parkin compound heterozygotes, the median UPSIT score was 33, interquartile range (IQR) 28.5-36.5, which was significantly better than in single Parkin heterozygotes (median 19, IQR 18-28) and non-carriers (median score 22, IQR 16-28) (ANOVA P < 0.001). These differences persisted after adjusting for age, disease duration, gender, and smoking (P < 0.001). There was no significant difference in UPSIT scores between single heterozygotes and non-carriers (P = 0.90). CONCLUSIONS: Patients with Parkin compound heterozygous mutations have relatively preserved olfaction compared to Parkin single heterozygotes and non-carriers. The prevalence of Parkin single heterozygosity is similar to the 3.7% rate reported in healthy controls.

Repeatability of cerebrospinal fluid constant rate infusion study.

OBJECTIVE: Infusion tests are important tools to assess cerebrospinal fluid (CSF)dynamics used in the preoperative selection of patients for shunt surgery, or to predict the scope of improvement from shunt revision. The aim of this study was to assess the repeatability of the key quantitative parameters describing CSF dynamics that are determined with infusion testing. MATERIALS AND METHODS: Eighteen patients in whom a constant infusion test was repeated within 102 days, without any intermediate surgical intervention, were studied. From each test baseline ICP, baseline pulse amplitude, outflow resistance, elastance coefficient and slope of the amplitude-pressure line were calculated and investigated with a regression and Bland-Altman analysis. RESULTS: Significant correlations (P < 0.01) were found for the outflow resistance (R = 0.96), the elastance coefficient (R = 0.778) and the slope of the amplitude-pressure line (R = 0.876). The estimated 95% confidence level for outflow resistance was 3 mmHg/ml min. Likewise, the elastance coefficient lay within a range of 0.16/ml and the slope of the amplitude-pressure line within 0.25. The most inconsistent parameter found were baseline ICP (R = 0.272) and baseline pulse amplitude (R = 0.171). CONCLUSION: The results of this study imply that the parameters resulting from an infusion study have to be considered within a range rather than as an absolute value.

Apical MUC1 expression revealed on the foveolar epithelium in H. pylori gastritis.

BACKGROUND: The membrane mucin MUC1 is altered in its pattern of expression in cancer, and also in other pathological situations, including Helicobacter pylori gastritis. Here we investigate the basis for the loss of apical staining of the gastric foveolar epithelium in H. pylori gastritis. METHODS: MUC1 was examined in the gastric antrum from cases of H. pylori gastritis and normal controls. We used tissue sections that were either treated or not treated with periodate to effect deglycosylation, and the monoclonal antibodies LICRLonM8, MUSE-11, CT2 and BC2. RESULTS: We show that the epitopes on the TR domain of MUC1 are partially cryptic due to glycosylation and that MUC1 is present on the apical surface of the gastric foveolar epithelium of gastritis patients. CONCLUSION: This observation suggests that there is no substantial loss of the mucin domain of MUC1 from the apical surface in gastritis, as suggested by others, but rather the H. pylori influences the glycosylation of MUC1. This paper highlights the issue of epitope specificity of monoclonal antibodies directed against disease-associated markers, specifically when they are glycoproteins, as is the case for many cancer markers.

Contribution of functional variation in the IL13 gene to allergy, hay fever and asthma in the NSHD longitudinal 1946 birth cohort.

BACKGROUND: Genetic variants of the two adjacent genes, IL13 and IL4 have frequently been reported as being associated with susceptibility to atopy and asthma, both in adults and children, and some studies also suggest association with lung function and chronic obstructive pulmonary disease. METHODS: In this study, we examined for the first time the effect of these variants in 2918 adults in a longitudinal birth cohort, the British National Survey of Health and Development, where there are extensive life style, developmental and environmental data. We examine two IL13 single nucleotide polymorphisms (SNPs) IL13 rs20541 (R110Q) and rs1800925 (-1024C>T) and one IL4 SNP, rs2070874 (-33C>T) with likely function. RESULTS: We show that IL13 rs20541 and rs1800925 are each significantly associated with self-reported asthma and allergy, and that this association is not confounded by any of the known developmental and environmental risk factors for asthma and atopy, including in particular place of birth. IL13 rs20541 does however act as a confounder for the IL13 rs1800925 associations, meaning that there is no statistical support for rs1800925 having an independent effect. There is nevertheless evidence for interaction between smoking and rs1800925, with allergy as outcome. None of the SNPs showed association with measures of lung function, nor any interaction with the effect of smoking on lung function. CONCLUSION: In a longitudinal population cohort we have established a role for polymorphism of IL13 in determining susceptibility to both atopy and asthma.

MUC4 and MUC5AC are highly specific tumour-associated mucins in biliary tract cancer.

Alterations in epithelial mucin expression are associated with carcinogenesis, but there are few data in biliary tract cancer (BTC). In pancreatic malignancy, MUC4 is a diagnostic and prognostic tumour marker, whereas MUC5AC has been proposed as a sensitive serological marker for BTC. We assessed MUC4 and MUC5AC expression in (i) prospectively collected bile and serum specimens from 72 patients with biliary obstruction (39 BTC) by real-time reverse transcriptase-PCR (qPCR) and western blot analysis, and (ii) 79 archived biliary tissues (69 BTC) by immunohistochemistry. In bile, MUC4 protein was detected in 27% of BTC and 29% of primary sclerosing cholangitis (PSC) cases, but not in other benign and malignant biliary diseases (P<0.01 and P=0.06). qPCR revealed a 1.9-fold increased MUC4 mRNA expression in BTC patients' bile compared with benign disease. In archived tissues, MUC4 protein was detected in 37% of BTC but in none of the benign samples (P=0.03). In serum, MUC5AC was found exclusively in BTC and PSC sera (44% and 13%, respectively; P<0.001 for BTC vs non-BTC) and correlated negatively with BTC survival. Biliary MUC4 and serum MUC5AC are highly specific tumour-associated mucins that may be useful in the diagnosis and formulation of therapeutic strategies in BTC.

Common polymorphism in a highly variable region upstream of the human lactase gene affects DNA-protein interactions.

In most mammals lactase activity declines after weaning when lactose is no longer part of the diet, but in many humans lactase activity persists into adult life. The difference responsible for this phenotypic polymorphism has been shown to be cis-acting to the lactase gene. The causal sequence difference has not been found so far, but a number of polymorphic sites have been found within and near to the lactase gene. We have shown previously that in Europeans there are two polymorphic sites in a small region between 974 bp and 852 bp upstream from the start of transcription, which are detectable by denaturing gradient gel electrophoresis (DGGE). In this study, analysis of individuals from five other population groups by the same DGGE method reveals four new alleles resulting from three additional nucleotide changes within this very small region. Analysis of sequence in four primate species and comparison with the published pig sequence shows that the overall sequence of this highly variable human region is conserved in pigs as well as primates, and that it lies within a 1kb region which has been shown to control lactase downregulation in pigs. Electrophoretic mobility shift assay (EMSA) studies were carried out to determine whether common variation affected protein-DNA binding and several binding activities were found using this technique. A novel two base-pair deletion that is common in most populations tested, but is not present in Europeans, caused no change in binding activity. However, a previously published C to T transition at -958bp dramatically reduced binding activity, although the functional significance of this is not clear.

DNA polymorphisms in the lactase gene. Linkage disequilibrium across the 70-kb region.

The enzyme lactase, which is responsible for the digestion of dietary lactose, is present in the intestine of some adults but not others. As a means of providing a platform to explore the molecular basis of this nutritionally relevant genetic variation we have screened for polymorphism in several regions of the lactase gene. In each case simple polymerase chain reaction-based procedures (including single-strand conformation analysis and denaturing gradient gel electrophoresis) were used, combined with silver staining as a method of detection. Allelic variation was found at 6 different sites. One previously published polymorphism was also tested. The frequencies of the alleles were determined in more than 100 unrelated individuals of the Centre d'Etude du Polymorphisme Humain (CEPH) panel, and the haplotypes were deduced. A region of linkage disequilibrium was observed, which spans the whole coding region of the lactase gene (approximately 60-70 kb); there were only 3 common haplotypes in this population. When the CEPH sample was subdivided according to the population of origin (France or Utah) the haplotype frequencies were shown to be markedly different.

Genetic polymorphism of MUC7: allele frequencies and association with asthma.

MUC7 encodes a small salivary mucin, previously called MG2, a glycoprotein with a putative role in facilitating the clearance of oral bacteria. The central domain of this glycoprotein was previously shown to comprise five or six tandemly repeated units of 23 amino-acids which carry most of the O-linked glycans. The polymorphism of these two allelic forms (MUC7*5 or MUC7*6) has been confirmed in this study in which we have analysed a large cohort of subjects (n = 375) of various ethnic origins. We have also identified a novel rare allele with eight tandem repeats (MUC7*8). MUC7*6 was the most common allele (0.78-0.95) in all the populations tested. The tandem repeat arrays of 22 MUC7*5 alleles and 34 MUC7*6 alleles were sequenced. No sequence differences were detected in any of the MUC7*6 alleles. Twenty-one MUC7*5 alleles sequenced lacked the 4th tandem repeat (structure TR12356), while one showed the structure TR12127. The structure of the MUC7*8 allele was TR12343456. Because of the known role of MUC7 in bacterial binding, and thus its potential involvement in susceptibility to chest disease we also tested MUC7 in our previously described series of Northern European atopic individuals with and without associated asthma. The MUC7*5 allele was rarer in the atopic asthmatics than in the atopic non-asthmatics (P = 0.014, OR for no asthma in atopic individuals 3.13, CI 1.01-6.10), and the difference in frequency between all asthmatics and all non-asthmatics was statistically significant (P = 0.009) while there was no difference between atopy and non-atopy (P = 0.199). In this study we also report the electrophoretic analysis of the MUC7 glycoprotein in saliva from individuals of different MUC7 genotype.

MUC1 gene polymorphism in the gastric carcinogenesis pathway.

MUC1 like most mucin genes shows extensive length polymorphism in the central core region. In a previous study it was shown that individuals with small MUC1 alleles/genotypes have an increased risk for development of gastric carcinoma. Our aim was to see if MUC1 gene polymorphism was involved in susceptibility for the development of conditions that precede gastric carcinoma: chronic atrophic gastritis (CAG) and intestinal metaplasia (IM). We evaluated MUC1 polymorphism in a population of 174 individuals with chronic gastritis (CG) displaying (CAG) and/or intestinal metaplasia (IM). The population of patients with CG shows MUC1 allele frequencies significantly different from the gastric carcinoma patients and blood donors population. A significantly lower frequency of CAG and IM was observed in MUC1 VNTR heterozygotic patients. Within the group of patients with IM, MUC1 large VNTR homozygotes show a significantly higher frequency of complete IM while small VNTR homozygotes show a significantly higher frequency of incomplete IM. These findings show that MUC1 polymorphism may define different susceptibility backgrounds for the development of conditions that precede gastric carcinoma: chronic atrophic gastritis (CAG) and intestinal metaplasia (IM).

Characterisation of a human homologue of a yeast cell division cycle gene, MCM6, located adjacent to the 5' end of the lactase gene on chromosome 2q21.

Four exons of a human homologue of a yeast cell division cycle gene (MCM6/mis5, which is thought to encode a DNA replication licensing factor) have been identified 3.3 kb upstream from the start of transcription of the intestinal lactase gene on human chromosome 2q21, initially by similarity to a rat 'intestinal crypt-cell replication factor'. RT-PCR analysis shows, that unlike lactase, MCM6 is not restricted in its tissue distribution and does not show person-to-person variation in the level of expression in adult intestine.

Characterisation of an epitope recognised by a monoclonal antibody against horse alcohol dehydrogenase using peptides synthesised on solid support.

Immunological analysis, using the Pepscan technique, of the tetradecapeptide, Pro344-Glu357 (PLITHVLPFEKINE), from horse liver alcohol dehydrogenase has identified a five amino acid sequence, HVLPF, which binds a monoclonal antibody. The epitope seems to be rather flexible with only two of the amino acids, Pro and Phe, having the characteristics of contact residues. However, the presence of the adjacent glutamic acid residue as part of the Pepscan peptide has a dramatic negative neighbourhood effect and inhibits binding. This highlights the potential risk of missing an epitope altogether when using the Pepscan procedure for epitope mapping.

Preparation of a monoclonal antibody against human lactase.

A simple procedure for screening for anti-enzyme monoclonal antibodies is described. The properties of our first antibody identified this way, directed against human lactase, are reported.

Deletion mapping: further evidence for the location of acid phosphatase (ACP1) within 2p23.

The human red cell acid phosphatase (ACP1) locus was assigned to region 2p23 leads to 2pter by Ferguson-Smith et al [3], more specifically to 2p23 by Hamerton et al [5]. We describe two unrelated patients with deletion of chromosome 2, with similar breakpoints in the distal portion of band p23 (del(2) (p23)). ACP1 typing in both patients revealed heterozygous BA phenotypes. Thus, we assign the locus for ACP1 to the distal portion of 2p23.

Detection of the urinary 'PUM' polymorphism by the tumour-binding monoclonal antibodies Ca1, Ca2, Ca3, HMFG1, and HMFG2.

A series of human urinary mucin-like glycoproteins, previously detected using lectins to stain gels after electrophoresis, and showing genetic polymorphism (Karlsson et al., 1983) can also be detected using the tumour-binding monoclonal antibodies, Ca1, Ca2, Ca3, HMFG1, and HMFG2. The evidence from immunoprecipitation and immunoadsorbant chromatography experiments is that the epitopes recognized by these antibodies are carried on the same molecules as the lectin-binding determinants. The discovery that the antibodies bind specifically to a family of molecules which show genetic polymorphism provides a powerful new tool for the analysis of the material expressed aberrantly in cancer.

Expression of the hypervariable PUM locus in normal and malignant lung: the tumor-associated epitopes are present but masked in normal tissue.

A single highly polymorphic gene locus PUM codes for a family of mucin-type glycoproteins present in human urine. These glycoproteins can be detected after electrophoresis using a group of monoclonal antibodies which show marked tumour specificity on immunohistology and include the HMFG and Ca antibodies (Swallow et al., 1986, 1987). Here we show by electrophoretic analysis of lung specimens and urine samples from nine individuals, that the PUM locus is expressed both in malignant and in normal lung. In contrast immunohistology of frozen sections of normal lung showed very little staining using the same antibodies, occasional reactive type 2 pneumocytes alone staining, whilst the carcinoma material showed strong staining in each case. However, after formalin fixation much more staining was observed in normal lung, all type 1 and 2 pneumocytes being stained. These observations suggest a difference in accessibility of the epitopes in normal and malignant lung, rather than a difference in expression of the PUM gene.

The breast tumour-associated epithelial mucins and the peanut lectin binding urinary mucins are coded by a single highly polymorphic gene locus 'PUM'.

A family of mucin-type glycoproteins, present in human urine, is coded by a single highly polymorphic gene locus PUM. We have previously shown that these glycoproteins carry epitopes recognized by a series of monoclonal antibodies, many of which were raised to the human milk-fat globule membrane, and which bind to a wide variety of carcinomas and certain normal epithelia. Here we show that in the normal human mammary gland, and in breast cancers the epitopes are present on the same family of molecules as that found in urine. Thus the genetically determined variation at the PUM locus accounts for much of the electrophoretic heterogeneity of the mucin-type glycoproteins present in breast cancer and serum from breast cancer patients that has been reported previously. Knowledge of this normal inherited polymorphism is essential to the interpretation of possible changes to these molecules in malignancy.

A microsatellite within the MUC1 locus at 1q21 is altered in the neoplastic cells of breast cancer patients.

Paired DNA samples from the neoplastic and nonneoplastic cells of 118 patients with the sporadic, nonfamilial form of breast cancer were analyzed for evidence of genetic alteration at a polymorphic microsatellite mapped to intron 6 within the MUC1 gene at 1q21. Two other microsatellite loci, D1S104 and APO-A2, which also map to 1q21, were analyzed as well. The frequency of alteration at the microsatellite within the MUC1 locus was significantly higher than D1S104 or APO-A2 (P < 0.001). Analysis by Southern blotting of the VNTR region of the MUC1 gene revealed an amplification of one allele in 34 of 54 informative cases (63%). There was no significant association between these alterations and alterations of the microsatellite within the same locus, suggesting independent mechanisms were responsible for the genetic changes. Microsatellite loci D17S579 at 17q21, the site of the BRCA1 gene, and D18S34 at 18q21-qter, the deleted in colorectal cancer locus, were also analyzed by PCR. Alterations at D17S579 and D18S34 were detected in 18.8% and 6.2% of patients, respectively (P < 0.001, and P < 0.1 relative to the frequency of alteration at D1S104 or APO-A2). A previously described polymorphism of hMSH2 was altered in 16.4% of cases.

Expression of a peptide epitope of the colonic mucin MUC2 in precursor lesions to gastric carcinoma.

Previous histochemical studies have shown that changes occur in the composition of mucins both in preneoplastic and neoplastic lesions of the gastric mucosa. Since monoclonal antibodies are now available which recognize the protein product of distinct mucin genes, they are likely to provide useful tools for evaluating these changes. Thus, a monoclonal antibody 996/1 raised against a peptide epitope of the colonic mucin MUC2 was examined for its potential as a prognostic indicator in gastric cancer. 996/1 works well on formalin-fixed paraffin sections and shows good staining of the colonic goblet cells in the region of the golgi, while there is no staining of normal control gastric mucosa. The epitope was detected in all cases of intestinal metaplasia (44 samples) and some but not all cases of dysplasia (26 samples) and gastric carcinoma (74 samples). There was no significant difference between the positivity of the tumours according to their classification, stage and lymph node status. These results unfortunately gave little indication that this antibody would be a useful prognostic tool in gastric cancer. However, the pattern of 996/1 staining provides useful information about the molecular changes in mucin expression that occur in gastric carcinogenesis.

Direct evidence of milk consumption from ancient human dental calculus.

Milk is a major food of global economic importance, and its consumption is regarded as a classic example of gene-culture evolution. Humans have exploited animal milk as a food resource for at least 8500 years, but the origins, spread, and scale of dairying remain poorly understood. Indirect lines of evidence, such as lipid isotopic ratios of pottery residues, faunal mortality profiles, and lactase persistence allele frequencies, provide a partial picture of this process; however, in order to understand how, where, and when humans consumed milk products, it is necessary to link evidence of consumption directly to individuals and their dairy livestock. Here we report the first direct evidence of milk consumption, the whey protein ß-lactoglobulin (BLG), preserved in human dental calculus from the Bronze Age (ca. 3000 BCE) to the present day. Using protein tandem mass spectrometry, we demonstrate that BLG is a species-specific biomarker of dairy consumption, and we identify individuals consuming cattle, sheep, and goat milk products in the archaeological record. We then apply this method to human dental calculus from Greenland's medieval Norse colonies, and report a decline of this biomarker leading up to the abandonment of the Norse Greenland colonies in the 15(th) century CE.