Large genomic rearrangements of both BRCA2 and BRCA1 are a feature of the inherited breast/ovarian cancer phenotype in selected families.

INTRODUCTION: A strong family history of breast and/or ovarian cancer can often be explained by small insertions, deletions, or substitutions in BRCA1 or BRCA2 and large genomic rearrangements in BRCA1. However, there is little evidence that genomic rearrangements are a major factor in BRCA2 associated breast cancer and the frequencies of rearrangements in BRCA1 in large clinic based populations are unknown. OBJECTIVE: To investigate the frequency of large genomic rearrangements in BRCA1 and BRCA2 in a large clinic based population at high risk of developing breast and/or ovarian cancer. METHODS: Multiplex ligation dependent probe amplification was used to comprehensively screen BRCA1 and/or BRCA2 in 312 index cases. RESULTS: Three novel deletions detected in BRCA2 were found exclusively in families with at least one case of male breast cancer. Novel rearrangements in BRCA1 were detected mostly in families with both breast and ovarian cancer. Families with these mutations were significantly younger at average age of cancer diagnosis. CONCLUSION: Screening for large genomic rearrangements in both BRCA1 and BRCA2 is strongly supported by this study, in particular in multiple case breast/ovarian families with a young age of onset (BRCA1) and families containing at least one case of male breast cancer (BRCA2).

A strategy for the determination of enzyme kinetics using electrospray ionization with an ion trap mass spectrometer.

A simple and rapid means of enzyme kinetic analysis was achieved using electrospray ionization mass spectrometry and a one-point normalization factor. The model system used, glutathione S-transferase from porcine liver, is a two-substrate enzyme catalyzing the conjugation of glutathione with a variety of compounds containing an electrophilic center. An internal standard that is structurally similar to the product was added to the reaction quench solution, and a single-point normalization factor was used to determine the product concentration without the need of a calibration curve. Kinetic parameters, such as Km, Vmax and Ki (for thyroxine), obtained by electrospray mass spectrometry agreed with those obtained from traditional UV-vis spectroscopy, and competitive vs noncompetitive inhibition reactions could be delineated via mass spectrometry. These results suggest that our method can be applied to enzymatic processes in which spectrophotometric or spectrofluorometric assays are not feasible or when the relevant substrates do not incorporate chromophores or fluorophores. This new method is competitive with traditional UV assays in that it is facile and it involves very little analysis time.

Biosynthesis of sialylated lipooligosaccharides in Haemophilus ducreyi is dependent on exogenous sialic acid and not mannosamine. Incorporation studies using N-acylmannosamine analogues, N-glycolylneuraminic acid, and 13C-labeled N-acetylneuraminic acid.

Haemophilus ducreyi is a Gram-negative bacterium that causes chancroid, a sexually transmitted disease. Cell surface lipooligosaccharides (LOS) of H. ducreyi are thought to play important biological roles in host infection. The vast majority of H. ducreyi strains contain high levels of sialic acid (N-acetylneuraminic acid, NeuAc) in their LOS. Here we investigate the biosynthetic origin of H. ducreyi sialosides by metabolic incorporation studies using a panel of N-acylmannosamine and sialic acid analogues. Incorporation of sialosides into LOS was assessed by matrix-assisted laser desorption and electrospray ionization mass spectrometry. A Fourier transform ion cyclotron resonance mass spectrometer provided accurate mass measurements, and a quadrupole time-of-flight instrument was used to obtain characteristic fragment ions and partial carbohydrate sequences. Exogenously supplied N-acetylmannosamine analogues were not converted to LOS-associated sialosides at a detectable level. In contrast, exogenous (13)C-labeled N-acetylneuraminic acid ([(13)C]NeuAc) and N-glycolylneuraminic acid (NeuGc) were efficiently incorporated into LOS in a dose-dependent fashion. Moreover, approximately 1.3 microM total exogenous sialic acid was sufficient to obtain about 50% of the maximum production of sialic acid-containing glycoforms observed under in vitro growth conditions. Together, these data suggest that the expressed levels of sialylated LOS glycoforms observed in H. ducreyi are in large part controlled by the exogenous concentrations of sialic acid and at levels one might expect in vivo. Moreover, these studies show that to properly exploit the sialic acid biosynthetic pathway for metabolic oligosaccharide engineering in H. ducreyi and possibly other prokaryotes that share similar pathways, precursors based on sialic acid and not mannosamine must be used.

Design, formation and properties of tetrahedral M(4)L(4) and M(4)L(6) supramolecular clusters.

The rigid tris- and bis(catecholamide) ligands H(6)A, H(4)B and H(4)C form tetrahedral clusters of the type M(4)L(4) and M(4)L(6) through self-assembly reactions with tri- and tetravalent metal ions such as Ga(III), Fe(III), Ti(IV) and Sn(IV). General design principles for the synthesis of such clusters are presented with an emphasis on geometric requirements and kinetic and thermodynamic considerations. The solution and solid-state characterization of these complexes is presented, and their dynamic solution behavior is described. The tris-catecholamide H(6)A forms M(4)L(4) tetrahedra with Ga(III), Ti(IV), and Sn(IV); (Et(3)N)(8)[Ti(4)A(4)] crystallizes in R3(-)c (No. 167), with a = 22.6143(5) A, c = 106.038(2) A. The cluster is a racemic mixture of homoconfigurational tetrahedra (all Delta or all Lambda at the metal centers within a given cluster). Though the synthetic procedure for synthesis of the cluster is markedly metal-dependent, extensive electrospray mass spectrometry investigations show that the M(4)A(4) (M = Ga(III), Ti(IV), and Sn(IV)) clusters are remarkably stable once formed. Two approaches are presented for the formation of M(4)L(6) tetrahedral clusters. Of the bis(catecholamide) ligands, H(4)B forms an M(4)L(6) tetrahedron (M = Ga(III)) based on an "edge-on" design, while H(4)C forms an M(4)L(6) tetrahedron (M = Ga(III), Fe(III)) based on a "face-on" strategy. K(5)[Et(4)N](7)[Fe(4)C(6)] crystallizes in I43(-)d (No. 220) with a = 43.706(8) A. This M(4)L(6) tetrahedral cluster is also a racemic mixture of homoconfigurational tetrahedra and has a cavity large enough to encapsulate a molecule of Et(4)N(+). This host-guest interaction is maintained in solution as revealed by NMR investigations of the Ga(III) complex.

Evidence of block and randomly sequenced chondroitin polysaccharides: sequential enzymatic digestion and quantification using ion trap tandem mass spectrometry.

A method for determining the sequence type of the disaccharide repeat region of cartilage samples is introduced. The samples are sequentially subjected to selective and nonselective enzymatic digestion, and the isomeric products from each step are quantified using tandem mass spectrometry. The two-step digestion/quantification protocol identifies whether the global makeup of the polymer is "alternating", "random", or "blocked" with respect to the two main components of the cartilage, 4- and 6-sulfated disaccharides. Using this procedure, the sequence type of two biologically isolated chondroitin polysaccharides was identified. The results for chondroitin sulfate A, isolated from bovine trachea, are consistent with the 4- and 6-sulfated disaccharides randomly distributed throughout the repeat region of the polysaccharide. For chondroitin sulfate C, shark cartilage, the 6-sulfated disaccharides are adjacent to each other to a larger extent than one would expect for a randomly distributed polymer, indicating that "blocks" of repeating disaccharides with the same sulfation site are present.

A library approach to the generation of bisubstrate analogue sulfotransferase inhibitors.

[reaction: see text]. A library of potential bisubstrate analogue inhibitors (1) targeting sulfotransferase enzymes was generated by the chemoselective ligation of the PAPS mimic 2 with a panel of 447 aldehydes. Preliminary screening has identified compounds that inhibit estrogen sulfotransferase (EST), an enzyme relevant to breast cancer.

Molecular orbital considerations in probing the stereoselective dissociations of cobalt-coordinated hexosamine monosaccharides.

The mechanisms for the stereoselective dissociation pathways of isomeric [CoIII(diaminopropane)2(hexosamine-2H)]+ complexes are studied by ion trap and Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS). The exact masses of product ions were measured in order to determine the composition of each loss, and isotopic labeling experiments were used to determine which atoms were lost during dissociation. MS3 studies were used to probe the structures of the product ions from MS2 experiments. Based on the experimental evidence obtained, mechanisms explaining the dissociations are postulated. In deciphering the mechanisms, careful attention was paid to the molecular orbital alignment of the reacting bonds, and based on the molecular orbitals, transition state conformations were postulated. These transition states suggest how the observed stereoselectivity occurs. In each case, the carbohydrate/metal interaction was crucial in the dissociation processes.

Stabilization and linkage analysis of metal-ligated sialic acid containing oligosaccharides.

The dissociation of metal-ligated sialyllactose and sialyl-N-acetyllactosamine was investigated. Metal-ligand derivatization of the carbohydrate samples with the diethylenetriamine ligand and one of four transition metals [Co(II), Ni(II), Cu(II), Zn(II)] suppressed sialic acid loss in the collision-induced dissociation process. Suppression of sialic acid loss allows sialic acid linkage information to be gained through tandem mass spectrometry. Sialic acid stabilization is postulated to occur due to the doubly charged metal ion which allows for deprotonation of the sialic acid moiety. Furthermore, a connection between the metal center and the amount of sialic acid loss was found. These results were rationalized using the Irving-Williams series and a competition between different sites of deprotonation. Analysis of the product ion spectra showed a clear differentiation of sialic acid linkage. Linkage determination is proposed to be effective due to the available conformations allowed by the different linkages. A more flexible linkage will allow more coordination of the sialic acid residue with the metal center, whereas a less flexible linkage will make this interaction unlikely.

The relationship between diagnosis and outcome in arthroscopy of the hip.

PURPOSE: The purpose of this study was to compare the results of arthroscopy of the hip for osteonecrosis (ON) with those obtained for other diagnoses by presenting a cohort of patients with diagnosis and symptoms as dependant variables in a consecutive series of 86 cases of hip arthroscopy. TYPE OF STUDY: Retrospective review of outcomes. METHODS: There were 83 patients (86 hips) who underwent arthroscopy. Indications included ON (43%), labral injuries (20%), osteoarthritis (degenerative joint disease, DJD) (10%), Legg-Calvé-Perthes (LCP) disease (10%), and loose bodies (10%). All but 2 patients had had symptoms for at least 6 months. Symptoms were pain (100%), mechanical problems (78%), and loss of motion (56%). Arthroscopy was performed in the supine position, using a standard traction table, 30 degrees and/or 70 degrees arthroscopes, and the anterior and peritrochanteric portals. Data were collected longitudinally, retrospectively reviewed, and statistically analyzed. RESULTS: No complications were seen; 60% of the patients had significant improvement over an average follow-up of 30 months. Better results were with labral tears (91%, P <.003) or LCP disease (89%, P <.05). ON and DJD did worse with only 40% and 44% improvement, respectively. After free-vascularized fibular graft (FVFG), 34% of patients showed improvement at follow-up (P =.003). Eighteen patients (21%) underwent total hip arthroplasty at an average of 8.4 months after arthroscopy. Mechanical symptoms were a significant favorable prognostic factor (P =.0019), with 85% having a good result. Patients with ON and mechanical symptoms had a significantly lower conversion rate to total hip arthroplasty than those with only pain or pain and decreased range of motion (P =.0043). CONCLUSIONS: Arthroscopy of the hip is useful for diagnosis and therapy of loose bodies, labral injuries, focal chondral lesions, or the late sequellae of LCP disease. We conclude that the presence of mechanical symptoms is a favorable prognostic factor for any diagnosis except degenerative arthritis. Furthermore, the identification of mechanical symptoms is a specific indication for arthroscopy in ON before or after FVFG.

Utilization of MS3 spectra for the multicomponent quantification of diastereomeric N-acetylhexosamines.

A rapid and accurate means of quantifying mixtures of diastereomeric N-acetylhexosamine monosaccharides using MS3 product ions is introduced. The method involves derivatizing the monosaccharides with [Co(DAP)2Cl2]Cl (where DAP is diaminopropane), and subjecting the derivatized products to collision-induced dissociation (CID) in a quadrupole ion trap mass spectrometer. Each diastereomer provides unique MS3 product ion abundances. The abundances for the pure monosaccharide standards are used in a system of equations in order to quantify mixtures of these diastereomers. Using the system of equations is quite advantageous, as it is the only mass spectrometric method that has been shown to successfully quantify mixtures of more than two isomers. The utility of the method is demonstrated by successfully quantifying various two and three component mixtures of the diastereomeric monosaccharides. Furthermore, the method is used to quantify the recovery of a single diastereomeric monosaccharide from an acidic resin. Although the multicomponent quantification method described herein is used to quantify mixtures of N-acetylhexosamine diastereomers, it could be applied to any group of isomers, provided distinguishing CID spectra are obtained. This is the first known report of utilizing MS3 product ions for quantification of structural isomeric mixtures.

Detection and quantification of the sulfated disaccharides in chondroitin sulfate by electrospray tandem mass spectrometry.

A new method of identifying and quantifying the disaccharide building blocks of glycosaminoglycans is introduced. The polysaccharides are subjected to an enzymatic digestion that releases the sulfated disaccharides. The disaccharides are then identified using a combination of electrospray ionization mass spectrometry and tandem mass spectrometry. Quantification of the isomeric disaccharides is also achieved by tandem mass spectrometry, using a recently developed methodology which quantifies mixtures of isomers without the use of chromatography or prior separation. Using mass spectrometry to characterize the components of glycosaminoglycans significantly reduces both sample consumption and analysis time of traditional methods.

Mass spectral characterization of lipooligosaccharides from Haemophilus influenzae 2019.

Lipooligosaccharide (LOS) glycoforms from Haemophilus influenzae 2019 were profiled using the high-resolution and accurate mass capabilities of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. Sequence and linkage for two previously unknown LOS glycoforms were subsequently obtained through MSn analyses on FT-ICR and quadrupole ion trap (qIT) instruments. MSn analysis of negative ion precursors confirmed structural details within the lipid moiety, while CID spectra of sodiated precursor ions provided monosaccharide sequence and linkage for the oligosaccharide portion of the molecule. Results obtained in this study indicate that extensive heterogeneity exists within the oligosaccharide moieties in LOS from H. influenzae 2019. More importantly, the data suggest that additional hexose moieties, which are added onto the LOS, are not simple extensions of one particular core structure but rather that structural isomers with different connectivities are present within the heterogeneous mixture.

Mass spectrometry and immobilized enzymes for the screening of inhibitor libraries.

A technique has been developed to rapidly screen enzyme inhibitor candidates from complex mixtures, such as those created by combinatorial synthesis. Inhibitor libraries are screened by using immobilized enzyme technologies and electrospray ionization ion cyclotron resonance mass spectrometry. The library mixture is first sprayed into the mass spectrometer, and compounds are identified. The library is subsequently incubated with the immobilized enzyme of interest under the correct conditions (buffer, pH, temperature) by using an excess of enzyme to ensure a surplus of sites for ligand binding. The immobilized enzyme/inhibitor mixture is centrifuged, and an aliquot of supernatant is again analyzed by electrospray ionization mass spectrometry. Potential inhibitors are quickly identified by comparison of the spectra before and after incubation with the immobilized enzyme. Non-inhibitors show no change in ion intensity after incubation, whereas weak inhibitors exhibit a visible decrease in ion abundance. Once inhibitor candidates have been identified, the library is reinjected into the mass spectrometer, and tandem mass spectrometry is used to determine the structure of the inhibitor candidates as needed. This method has been successfully demonstrated by identifying inhibitors of the enzymes pepsin and glutathione S-transferase from a 19- and 17-component library, respectively. It is further shown that the immobilized enzyme can be recycled and reused for continuous screening of additional new libraries without adding additional enzyme.

Subungual melanoma. A review of 93 cases with identification of prognostic variables.

From 1970 to 1996, 93 patients received diagnoses of subungual melanoma. Followup data were complete on all patients and reviewed with a median duration of followup of 5.2 years. This study identifies significant clinicopathologic variables that affect survival and provides the orthopaedic surgeon assistance in the early diagnosis and treatment of this lesion. Eight-three percent of patients presented with Stage I disease, whereas 17% had nodal or distant disease. Fifty-three percent had locally advanced disease at presentation. Twelve percent of the patients were African-American. Fifty-five percent of the lesions arose on the hands with thumb involvement predominating in more than half of these cases. Operative therapy consisted of amputation. Elective lymph node dissection was performed in 34 patients (37%) for Stage I tumors of intermediate thickness. Therapeutic node dissection was required in 16 patients (17%) for positive nodes. Five-year survival was 74% for patients with Stage I disease and 40% for patients with Stage II disease. Statistical analysis identified stage at diagnosis, Clark and Wihm's level, the patient's race, and the presence of ulceration as prognostic variables affecting survival. The diagnosis of subungual melanoma carries a grave prognosis and often is misdiagnosed in the early stages. The treatment of choice is amputation at the appropriate level.

Combined partial acid hydrolysis and electrospray ionization-mass spectrometry for the structural determination of oligosaccharides.

A general oligosaccharide acid hydrolysis method, amenable to electrospray ionization mass spectrometry (ESI-MS), is described that allows for hydrolysis of glycosidic bonds for both hexose- and N-acetylhexosamine-containing oligosaccharides. The partial acid hydrolysis of oligosaccharides is obtained by using an acid-exchange resin as the acid catalyst. A ladder sequence of the glycan is produced in solution that is directly analyzed by ESI tandem mass spectrometry, employing both ion trap and Fourier transform ion cyclotron resonance mass spectrometers, to provide sequence and linkage information. Unlike traditional acid hydrolysis procedures, there is minimal degradation of monosaccharide residues or deacetylation of N-acetylhexosamines by employing this technique. It is further demonstrated that the stereochemistry of the released monosaccharides and the anomeric configuration within disaccharides is determined by direct derivatization of the hydrolysate with Zn(dien)-Cl2 followed by ESI-MS/MS.

STAT: a saccharide topology analysis tool used in combination with tandem mass spectrometry.

Sequential stages of mass spectrometry (MSn) have the potential to provide a great deal of structural information in glycan analysis. The saccharide topology analysis tool (STAT) presented here is a Web-based computational program that can quickly extract sequence information from a set of MSn spectra for an oligosaccharide of up to 10 residues. After information such as precursor ion mass, possible monosaccharide moieties, charge carrier, and product ion mass has been input, all possible connectivities are generated and evaluated against the MSn data. The list of possible structures is given a rating based on the likelihood that it is the correct sequence. Examples are given to demonstrate the feasibility of applying STAT to MSn data generated from bacterial lipooligosaccharides and an N-linked glycan. The major advantage of STAT is that the list of possible structures is generated quickly and the rating system pushes the more likely structures to the top of the list. Combining the data generated by STAT with data on the branching patterns of the glycan serves to eliminate all but a handful of structures. These remaining structures could then be used to guide further structural analysis.

Multicomponent quantification of diastereomeric hexosamine monosaccharides using ion trap tandem mass spectrometry.

A rapid means of stereochemical differentiation and quantification for the hexosamine monosaccharides was achieved using electrospray ionization quadrupole ion trap mass spectrometry. The hexosamine monosaccharides, glucosamine, galactosamine, and mannosamine, were derivatized with [Co(DAP)2Cl2]Cl, and the complex [Co(DAP)2(HexNH2)]Cl was generated. Subjecting this complex to collision-induced dissociation provided a unique product ion spectrum for each of the diastereomeric monosaccharide complexes, thus differentiating the stereoisomers. Furthermore, the stereoisomers were quantified. This was achieved by using the relative abundances of product ions from pure standards and using these values to determine the ratio of isomeric products in a mixture. The utility of this quantification method was demonstrated by successfully determining the composition of two- and three-component mixtures of the hexosamines.

Differentiation of diastereomeric N-acetylhexosamine monosaccharides using ion trap tandem mass spectrometry.

A quadrupole ion trap mass spectrometer equipped with electrospray ionization was used to distinguish three diastereomeric monosaccharides, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine. The saccharides were derivatized to form the metal complex [CoIII(DAP)2HexNAc]Cl3 which, when collisionally activated, produced dramatically different product ion spectra. The product ion spectra generated for the three monosaccharide diastereomers were then used to confirm the stereochemistry of N-acetylhexosamines from a hydrolyzed oligosaccharide. Finally, the origin of each product ion was determined through isotopic labeling studies, and mechanisms were proposed which explain each resulting dissociation.