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PubMed2026

Optical genome mapping improves structural variant detection and characterization in syndromic and neurogenetic disorders.

Optical Genome Mapping (OGM) offers superior resolution compared to standard diagnostic methods such as karyotyping and FISH, enabling the detection of nearly all types of chromosomal aberrations with non-centromeric breakpoints. This study evaluated OGM's potential to enhance the genetic findings in unsolved cases of neurogenetic and syndromic disease requiring further investigation after standard genetic testing. In 10 patients with various neurogenetic diagnoses, OGM confirmed all structural findings previously detected by karyotyping, chromosomal microarray (CMA), and/or NGS. Moreover, OGM provided additional structural insights in five cases, such as identifying a novel candidate gene in a patient with a balanced translocation, redefining of breakpoint regions in familial translocations, characterization of complex rearrangements, and revising of initial diagnostic interpretations. Most importantly, we present OGM results for three individuals with ring chromosomes 18, 20, and 22, highlighting the need to adjust filter settings and to incorporate the rare variant pipeline for accurate detection. Based on our experiences, we propose a strategic approach for identifying ring chromosomes using OGM. On the other hand, OGM did not identify causative variants in three unsolved cases with strong clinical suspicion of hereditary neuropathy. In summary, while OGM did not yield new insights for hereditary neuropathy, it provided additional or refined information in 6 out of 10 cases with other syndromic diseases. These findings underscore the value of OGM in increasing the diagnostic yield and precision of genetic testing.

Humans↗
PubMed1998

Structural variants of the vitamin D analogue EB1089 reduce its ligand sensitivity and promoter selectivity.

The nuclear hormone 1alpha,25-dihydroxyvitamin D3 (VD) has important cell-regulatory functions but also a strong calcemic effect. Therefore, various VD analogues have been synthesized and screened for their biological profile. In order to gain more insight into the molecular basis of the high antiproliferative but low calcemic action of the VD analogue EB1089, we characterized this compound in comparison to five structurally related VD analogues. The activities of the six VD analogues in in vitro assays (limited protease digestion assays for determining interaction with monomeric vitamin D receptor (VDR), ligand-dependent gel shift assays for showing the increase of DNA binding of VDR-retinoid X receptor (RXR) heterodimers, and reporter gene assays on different types of VD response elements for demonstrating the efficacy in nuclear VD signalling) were found to represent their biological potency (antiproliferative effect on different malignant cell lines). In this series, EB1089 proved to be the most potent VD analogue; that is, every structural modification (20-epi configuration, cis-configuration at position C24, or changes at the ethyl groups at position C25) appeared to reduce the determined activities mediated through the VDR of these analogues. Moreover, the modifications of EB1089 resulted in a loss of VD response element selectivity, suggesting that this parameter is very critical for the biological profile of this VD analogue.

Calcitriol↗
PubMed1997

Expression of HLA class I molecules assembled with structural variants of human beta2-microglobulin.

Mouse and human beta2-microglobulin (beta2m), which differ by 30% in their primary sequence, give rise to disparate levels of HLA class I heavy chain expression in transfectants of the beta2m-null FO-1 human melanoma cell line, i.e., mouse beta2m directs expression of HLA class I heavy chains that is only approximately 20%-30% of that observed for heavy chains assembled with human beta2m. In this report we describe our efforts to better understand the structural basis of this regulatory phenomenon. Initial insight into the importance of the N-terminus of beta2m on HLA expression came from studies with FO-1 cells transfected with chimeric (human X mouse) B2m genes. Chimeric beta2m molecules containing residues 1-69 from human beta2m and residues 70-99 from mouse beta2m (designated HM- beta2m) induced expression of HLA heavy chains to a significantly greater extent ( approximately 80% of level observed with cognate beta2m) than the reverse chimeric construct (designated MH- beta2m) (10%-15% of level observed with cognate beta2m). These data are consistent with the view that the major determinants of HLA class I heavy chain expression map to the portion of the beta2m molecule which forms the four-stranded beta-pleated sheet, comprised of S1, S2, S4, and S5, and one strand of the three-stranded sheet (S3). The mapping of class I regulatory sites to the portion of beta2m containing the four-stranded beta-pleated sheet supports the interpretation that the heavy chain contact residues on beta2m play the major role in regulating major histocompatibility (MHC) class I expression. To further dissect beta2m-mediated regulation of HLA class I expression, site-directed mutants of beta2m were prepared by conversion of human beta2m to the mouse sequence at individual amino acid positions within the four-stranded and three-stranded beta-pleated sheets. Human to mouse amino acid substitutions were made in each divergent residue between positions 1-66, and as controls for COOH-terminal modification, several residues between positions 75 and 94. Cytofluorometry with HLA class I-specific antibodies indicated that cell surface expression of HLA class I heavy chains was largely insensitive to each of the individual substitutions. It is concluded that a combination of divergent residues mapping to positions of heavy chain contact are responsible for the differences observed in MHC class I expression by heterologous forms of beta2m.

Amino Acid Sequence↗
PubMed1988

Apolipoprotein C-II deficiency: identification of a structural variant ApoC-II Padova.

Apolipoprotein(apo) C-II DNA, RNA and protein from a patient with a familial deficiency of apoC-II were evaluated and compared to normal individuals. No major defect of the apoC-II gene could be detected by Southern blot hybridization. Northern and slot blot analyses of total liver RNA documented normal levels of a normal sized apoC-II mRNA. Immunohistochemical studies of the liver of the apoC-II deficient patient revealed a normal to slightly elevated intracellular content of the C-II apolipoprotein. Plasma apoC-II was 3 to 5% of normal apoC-II levels and exhibited abnormal electrophoretic mobility on two dimensional gel electrophoresis and immunoblotting. We postulate that at the molecular level, the deficiency of apoC-II in the plasma of this patient results from a structural defect in the coding portion of the apoC-II gene leading to either defective secretion of cellular apoC-II or increased catabolism of a structurally defective apoC-II in plasma.

Apolipoprotein C-II↗
PubMed2002

A structurally variant form of the 2B4 antigen is expressed on the cell surface of mouse mast cells.

2B4 (CD244) is a 66-kDa CD2 family protein expressed on natural killer (NK) cells. Mouse NK cells express two isoforms of 2B4, termed 2B4L and 2B4S, whose molecular masses are 42 kDa and 36 kDa, respectively. In this study, we biochemically characterize the 2B4 antigen that was newly found on mouse bone marrow-derived mast cells (BMMC). Anti-2B4 mAb immunoprecipitated glycoproteins with a molecular mass of 60 kDa from BMMC. Removal of N-linked sugars from the antigen by N-glycosidase F treatment yielded two protein backbones of 35 kDa and 25 kDa, indicating that BMMC express the 2B4S isoform, but not 2B4L. Nucleotide sequence analyses confirmed that BMMC transcribe 2B4S mRNA. The preferential expression of the 2B4S isoform and the detection of an additional 25-kDa glycoprotein on BMMC indicate that differences in the structure of 2B4 antigen exist between BMMC and NK cells.

Animals↗
PubMed1981

Amino acid and carbohydrate structural variants of glycoprotein products (M-N glycoproteins) of the M-N allelic locus.

Major glycoprotein of MgM, MM Miltenberger III (MiIII), and M-N erythrocyte membranes from individual donors were cleaved with CNBr and their amino-terminal octapeptides were examined with respect to amino acid and carbohydrate composition. The amino-terminal octapeptides from the heterozygous MgM donor were resolved into two types, A and A'. MgM A was identical to octapeptide A from MM glycoproteins in carbohydrate and amino acid compositions. MgM A' exhibited amino acid composition similar to NN peptide A except for a single substitution of an Asx for a Thr and, as a result, was not glycosylated. MM(MiIII) octapeptide A was identical to M peptide A in amino acid composition, but differed in carbohydrate content. This glycopeptide contained three O-glycosidically linked carbohydrate units, one of which contained GlcNAc bound to a core of NeuAc, Gal, and GalNAc. About two such units were also present in the CNBr glycopeptide B of the glycoprotein, and on the basis of studies with alkaline borohydride and alkaline sulfite degradations, these units are believed to have the following structure: (formula see text) The Mg is an allelomorph of the M-N locus, likely evolved from a single base substitution in the N gene. The resulting single amino acid substitution effects the posttranslational carbohydration of neighboring Ser and Thr residues. The MM(MiIII) appears to be a product of the M gene that undergoes sequences of posttranslational glycosylations different from those of the M-N glycoproteins.

Alleles↗
PubMed1998

Factor VII deficiency caused by a structural variant N57D of the first epidermal growth factor domain.

We have previously described a kindred with factor VII (FVII) deficiency whose members exhibited reduced procoagulant activity relative to FVII antigen concentration. In this report, the molecular genetic basis of the FVII defect has been determined to be a heterozygous substitution of Asp for Asn at position 57 in the first epidermal growth factor (EGF) domain. Recombinant FVII (N57D) cDNA was created by site-directed mutagenesis and transiently expressed in human 293 cells. The transfected cells synthesized an immunoprecipitable protein with an apparent molecular weight of 50 kD. Quantitation of expression by FVII enzyme-linked immunosorbent assay indicated that mutant protein yields were consistently low, typically 10% to 30% of wild-type FVII. FVII (N57D) protein did not accumulate intracellularly, and Northern blot analysis indicated equivalent FVII mRNA levels in 293 cells expressing either wild-type FVII or FVII (N57D). Secreted FVII (N57D) protein did not bind tissue factor, exhibited no procoagulant activity, and failed to bind a conformation-dependent monoclonal antibody specific for the first EGF domain of FVII. Molecular modeling of the first EGF domain of FVII predicted that the N57D amino acid substitution would disrupt tertiary bonding structure. We conclude that the N57D mutation affects folding of the first EGF domain of FVII resulting in decreased cellular secretion of a mutant FVII molecule, which is unable to bind tissue factor and is therefore biologically inactive.

Animals↗
PubMed2003

Monitoring manufacturing process yields, purity and stability of structural variants of PEGylated staphylokinase mutant SY161 by quantitative reverse-phase chromatography.

Staphylokinase variant SY161 is a recombinant mutant of the Staphylococcus aureus polypeptide staphylokinase (Sak), and is currently in human clinical trials as a thrombolytic agent. The 15 kDa single chain SY161 protein is expressed as a soluble cytoplasmic product in E. coli with a single cysteine inserted near the N-terminus. The protein as extracted from E. coli is a mixture of both monomeric and intermolecularly disulfide crosslinked species. To improve protein purification yields SY161 is sulfitolyzed during the early stages of production, preventing disulfide formation. The protein is later modified during manufacturing to incorporate a single 5 kDa polyethylene glycol group on the single sulfhydryl sidechain. We have developed and qualified a reverse-phase chromatographic method to quantitate SY161 during product manufacturing. We discuss the use of the assay during manufacturing development to monitor fermentation yields, the SY161 PEGylation reaction, and as an in-process manufacturing control assay. The assay has been applied as a product purity and identity release assay and is suitable for use in assessing product structural integrity during stability testing. The assay has a linear range of quantitation for SY161 from at least 0.15 to 16 micro g, and is-in addition capable of detecting and quantitating protein de-PEGylation events and host cell-derived protein contaminants.

Amino Acid Sequence↗
PubMed1988

Supracrystallographic resolution of interactions contributing to enzyme catalysis by use of natural structural variants and reactivity-probe kinetics.

1. The influence on the reactivities of the catalytic sites of papain (EC 3.4.22.2) and actinidin (3.4.22.14) of providing for interactions involving the S1-S2 intersubsite regions of the enzymes was evaluated by using as a series of thiol-specific two-hydronic-state reactivity probes: n-propyl 2-pyridyl disulphide (I) (a 'featureless' probe), 2-(acetamido)ethyl 2'-pyridyl disulphide (II) (containing a P1-P2 amide bond), 2-(acetoxy)ethyl 2'-pyridyl disulphide (III) [the ester analogue of probe (II)] and 2-carboxyethyl 2'-pyridyl disulphide N-methylamide (IV) [the retroamide analogue of probe (II)]. Syntheses of compounds (I), (III) and (IV) are reported. 2. The reactivities of the two enzymes towards the four reactivity probes (I)-(IV) and also that of papain towards 2-(N'-acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide (VII) (containing both a P1-P2 amide bond and an L-phenylalanyl side chain as an occupant for the S2 subsite), in up to four hydronic (previously called protonic) states, were evaluated by analysis of pH-dependent stopped-flow kinetic data (for the release of pyridine-2-thione) by using an eight-parameter rate equation [described in the Appendix: Brocklehurst & Brocklehurst (1988) Biochem. J. 256, 556-558] to provide pH-independent rate constants and macroscopic pKa values. The analysis reveals the various ways in which the two enzymes respond very differently to the binding of ligands in the S1-S2 intersubsite regions despite the virtually superimposable crystal structures in these regions of the molecules. 3. Particularly striking differences between the behaviour of papain and that of actinidin are that (a) only papain responds to the presence of a P1-P2 amide bond in the probe such that a rate maximum at pH 6-7 is produced in the pH-k profile in place of the rate minimum, (b) only in the papain reactions does the pKa value of the alkaline limb of the pH-k profile change from 9.5 to approx. 8.2 [the value characteristic of a pH-(kcat./Km) profile] when the probe contains a P1-P2 amide bond, (c) only papain reactivity is affected by two positively co-operative hydronic dissociations with pKI congruent to pKII congruent to 4 and (d) modulation of the reactivity of the common -S(-)-ImH+ catalytic-site ion-pair (Cys-25/His-159 in papain and Cys-25/His-162 in actinidin) by hydronic dissociation with pKa approx. 5 is more marked and occurs more generally in reactions of actinidin than is the case for papain reactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Catalysis↗
PubMed1996

Truncated structural variants of lipoarabinomannan in ethambutol drug-resistant strains of Mycobacterium smegmatis. Inhibition of arabinan biosynthesis by ethambutol.

The anti-tuberculosis drug, ethambutol (Emb), was previously shown to inhibit the synthesis of arabinans of both the cell wall arabinogalactan (AG) and lipoarabinomannan (LAM) of Mycobacterium tuberculosis and other mycobacteria. However, an Emb-resistant mutant, isolated by consecutive passage of the Mycobacterium smegmatis parent strain in media containing increasing concentrations of Emb, while synthesizing a normal version of AG, produced truncated forms of LAM when maintained on 10 microg/ml Emb (Mikusová, K., Slayden, R. A., Besra, G. S., and Brennan, P. J. (1995) Antimicrob. Agents Chemother. 39, 2482-2489). We have now isolated and characterized the truncated LAMs made by both the resistant mutant and a recombinant strain transfected with a plasmid containing the emb region from Mycobacterium avium which encodes for Emb resistance. By chemical analysis, endoarabinanase digestion, high pH anion exchange chromatography, and mass spectrometry analyses, truncation was demonstrated as primarily a consequence of selective and partial inhibition of the synthesis of the linear arabinan terminal motif, which constitutes a substantial portion of the arabinan termini in LAM but not of AG. However, at higher concentrations, Emb also affected the general biosynthesis of arabinan destined for both AG and LAM, resulting in severely truncated LAM as well as AG with a reduced Ara:Gal ratio. The results suggested that Emb exerts its antimycobacterial effect by inhibiting an array of arabinosyltransferases involved in the biosynthesis of arabinans unique to the mycobacterial cell wall. It was further concluded that the uniquely branched terminal Ara6 motif common to both AG and LAM is an essential structural entity for a functional cell wall and, consequently, that the biosynthetic machinery responsible for its synthesis is the effective target of Emb in its role as a potent anti-tuberculosis drug.

Antigens, Bacterial↗
PubMed1988

Spectrin-alpha I/61: a new structural variant of alpha-spectrin in a double-heterozygous form of hereditary pyropoikilocytosis.

Recent biochemical studies have led to the identification of abnormal spectrins in the erythrocytes of patients with hereditary pyropoikilocytosis (HPP) and hereditary elliptocytosis (HE). In this report we describe the biochemical characterization of the erythrocytes from a proband with severe HPP who is doubly heterozygous for two mutant spectrins (Sp): Sp alpha I/74 and a new, previously undetected, mutant of alpha-spectrin designated Sp alpha I/61. The proband's erythrocytes are unstable when exposed to 45 degrees C, and her membrane skeletons exhibit instability to shear stress. The content of spectrin in the proband's erythrocyte membranes is decreased to 75% of control values. The amount of spectrin dimers in crude 4 degrees C spectrin extracts is increased (58%) as compared with control values (6% +/- 4%). Limited tryptic digestion reveals a marked decrease in the normal 80,000-dalton alpha I domain, an increase in the 74,000-dalton fragment that is characteristic of Sp alpha I/74, and an increase in a series of new fragments of 61,000, 55,000, 21,000, and 16,000 daltons. Both parents are asymptomatic, but they have increased amounts of spectrin dimers (17% to 25%). Limited tryptic digestion of the father's spectrin demonstrates the presence of a previously identified abnormal spectrin (Sp alpha I/74) that is characterized by a decrease in content of the 80,000-dalton peptide and an increase in concentration of the 74,000-dalton peptide. The mother's spectrin digests show a decrease in the amount of 80,000-dalton peptide and the formation of new peptides of 61,000, 55,000, 21,000, and 16,000 daltons. The data indicate that this severe form of HPP is due to the inheritance of two distinct abnormal spectrins, Sp alpha I/74 and a new spectrin mutant, Sp alpha I/61.

Anemia, Hemolytic, Congenital↗
PubMed2002

Natural structural variants of the nuclear receptor farnesoid X receptor affect transcriptional activation.

The Farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily that has been shown to play an important role in bile acid and cholesterol homeostasis. Here we identify four murine FXR transcripts, derived from a single gene, that encode four isoforms, FXRalpha1, FXRalpha2, FXRbeta1, and FXRbeta2. FXRalpha and FXRbeta differ at their amino terminus, and FXRalpha1 and FXRbeta1 have a four-amino acid residue insertion in the hinge region immediately adjacent to the DNA binding domain. Real time PCR and 5'-rapid amplification of cDNA ends followed by Southern blotting reveal that these four transcripts are expressed differentially in liver, intestine, kidney, adrenals, stomach, fat, and heart. Electrophoretic mobility shift assays demonstrate that FXRalpha2 and FXRbeta2 bind to FXR response elements with a higher affinity as compared with FXRalpha1 and FXRbeta1, suggesting that the four-amino acid insert may affect FXR function. Consistent with this idea, the results of transient transfection experiments demonstrate that the four FXR isoforms differentially transactivated a number of promoter-reporter genes; activation of an ileal bile acid-binding protein promoter-reporter gene varied 20-fold depending on the FXR isoform; the rank order of activation was FXRbeta2 > FXRalpha2 FXRalpha1 = FXRbeta1. In contrast, SHP reporter or BSEP reporter genes were activated to similar degrees by each of the FXR isoforms. Finally, NIH3T3 cells were stably infected with individual murine FXR isoforms, and the cells were treated with FXR ligands. The endogenous ileal bile acid-binding protein gene was activated by the four FXR isoforms with the same rank order as seen in transfections. This effect was gene-specific, since induction of bile salt export pump mRNA was independent of the FXR isoform. These observations suggest that there are four distinct murine FXR isoforms that differentially regulate gene expression in numerous tissues in vivo.

Animals↗
PubMed1996

Mass allele detection (MAD) of rare 5-HT1A structural variants with allele-specific amplification and electrochemiluminescent detection.

A strategy is described that exploits allele-specific amplification (ASA-PCR) and electrochemiluminescence (ECL) detection technology to rapidly and cheaply screen large numbers of DNAs arranged in pooled matrices in order to identify individual nucleotide sequence variants. To demonstrate this strategy, a large genomic DNA collection was screened for two nucleotide variants in the 5-HT1A serotonin receptor gene and individual heterozygotes were identified. Conversion of two SSCP variants to allele-specific PCR polymorphisms was accomplished, and PCR product capture and ECL detection were enabled by the covalent addition of biotin to allele-specific PCR primers and ruthenium to the nonspecific PCR primer. A two-level DNA pooling strategy was used to reduce the number of individual PCR reactions required. Pooling experiments established that ASA-PCR with ECL detection is sufficiently sensitive to reproducibly detect a single specific allele in the presence of a 40-fold excess of genomic DNA from individuals negative for the specific allele. The detection sensitivity of the ECL device and the design of the pooled DNA arrays reduced the number of PCRs required to detect the rare individuals with the variant sequences by approximately 90%. This strategy is called mass allele detection (MAD).

Alleles↗