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A report of a case of prolonged apnoea after succicurarium in a female patient with abnormal pseudo-cholinesterases is followed by a presentation of a study of pseudo-cholinesterases in 12 members of the family. The value of the use of 4 tests of inhibition of cholinesterase activity in determining the genotype of an individual is emphasised.
Type I familial amyloid polyneuropathy (FAP) is an autosomal dominant hereditary generalized amyloidosis characterized by polyneuropathy and autonomic nerve failure. The main component of the amyloid fibril protein in this disorder has been shown to be a variant prealbumin with a single substitution of a methionine residue for valine at position 30. In the present study we have investigated 19 patients with FAP aged 31 to 67 and an asymptomatic family member using gene analysis with primer-directed enzymatic amplification (PCR) of DNA, isolation of plasma variant prealbumin and immunohistochemical identification of tissue amyloid protein. All patients and a symptom-free boy in the affected family had the mutant prealbumin gene showing abnormal DNA fragments by treatment with restriction endonuclease Bal I, and plasma variant prealbumin was also detected in all of them by reverse-phase high performance liquid chromatography. Rectum biopsies obtained from 9 patients showed amyloid deposits which were specifically immunostained by anti-human prealbumin antiserum. However, an asymptomatic carrier at the age of 16 showed no rectal amyloid deposition. Recent studies of FAP have disclosed that the expression of type I FAP is closely associated with the gene mutation of prealbumin. Accordingly, a simple and rapid method to detect this gene abnormality using PCR technique is considered to be very useful for diagnosis of type I FAP and can also provide valuable information for the genetic counselling of the family members at risk.
The t(9;11)(p22;q23) is a recurring abnormality in acute nonlymphocytic leukemia. The analysis of complex 9;11 translocations will aid in the identification of the conserved chromosomal junction or the critical genetic alteration created by the rearrangement; however, variant translocations involving chromosomes #9 and #11 have not been reported. We have identified such variants in two patients who had acute myelomonocytic leukemia and acute monocytic leukemia, characterized by a t(9;11;18)(p22;q23;q12) and a t(9;11;13)(p22;q23;q34), respectively. The conserved junction resulting from these rearrangements is created by the translocation of chromosomal material from 9p to 11q.
Thyroxine-binding globulin (TBG) is the major thyroid hormone transport protein. Several inherited TBG variants resulting in partial or complete TBG deficiencies have been shown to be caused by either one or two nucleotide substitutions, or one nucleotide deletion in the coding regions of the TBG gene. In this report, a Japanese female patient (proband) with hyperthyroid state, whose lower TBG levels did not return to normal under the euthyroid state after treatment was examined. Genomic DNA samples from the proband with thyroxine-binding globulin deficiency (termed TBG-Kumamoto) and her family were subjected to the polymerase chain reaction, and the generated DNA fragments were sequenced. A single nucleotide substitution in the codon for the amino acid 363 of native TBG molecule (CCT to CTT) was found, resulting in the replacement of proline by leucine. It was revealed that the proband was a heterozygote and her father was a hemizygote. The mutation was confirmed by the allele-specific amplification of genomic DNAs from the proband and her father using oligonucleotide primers of normal or mutant residues at the 3' position in the polymerase chain reaction. These results indicate that the abnormality of TBG-Kumamoto is the consequence of this mutation. Genetically, this point mutation observed in TBG-Kumamoto might be classified as a new type of TBG deficiency.
Identification of the serological variants according to G. Wauters et al. involves examination of one strain in the agglutination test with 32 sera, and to specify a number of serological variants, investigations with monosera are necessary. Six group sera are suggested for the determination of the minimal set of monovalent sera necessary for the identification of the tested strain serological variant. Employment of group sera considerably simplifies the serologic identification of the strains. The selected serum titers help obtain a clear-cut agglutination on the glass within 1 min. A set of 6 group and 31 monoreceptor sera has been prepared. Since serovariants O10 and O34 have been found antigenically identical, serum O34 has not been included in the set. A table for the determination of the serological variant from the results of the agglutination test is offered.
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.
Human leukocyte transketolase of fresh cell extracts has been analyzed by isoelectrofocusing on agarose gels (pH 3-10). The enzyme was then transblotted on nitrocellulose and detected with specific anti-transketolase IgG coupled to an avidin/biotin-immunoperoxidase system. Each sample yielded multiple enzyme forms, within a pI range of about 7.4-8.4. Transketolase profile, however, was not identical in all extracts. There are two mainly distinct patterns, showing qualitative and quantitative differences: a standard profile, which is predominant, and a variant, found in three unrelated subjects out of the two hundred and twenty. Standard and variant enzyme have similar Km values for ribose 5-P and xylulose 5-P and the same mobility on SDS-PAGE.
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The primary structure of an HLA class I genomic clone isolated from a homozygous HLA-B35 Caucasian individual of hispanic origin was determined. The nucleotide sequence of exons 1, 2, 4, 5, 6, and 7 is identical to that of the HLA-B35 allele of Oriental origin reported previously. Exon 3 differs in only three nucleotides present in a stretch of 23 bp. These changes introduce three amino acid substitutions in residues 109 (Leu----Phe), 114 (Asp----Asn), and 116 (Ser----Tyr), two of which (114 and 116) are located in one of the beta sheets at the bottom of the peptide binding site. The nature of these replacements in this HLA-B35 variant is likely to affect peptide binding and recognition by T lymphocytes. Introns 1, 2, 3, 4, 5, and 6 from this genomic clone are identical to those present in HLA-Bw58, further confirming the evolutionary origin of HLA-B35.
OBJECTIVE: To characterize the clinical phenotypes and identify the genetic etiology in four unrelated families affected by Turner-type X-linked intellectual disability (XLID). METHODS: Peripheral blood samples were collected from four probands and their parents. Genomic DNA was extracted, and a comprehensive genetic analysis was performed using trio-based Whole Exome Sequencing (WES) combined with low-pass Copy Number Variation sequencing (CNV-seq). Candidate variants were subsequently validated via Sanger sequencing. RESULTS: Genetic analysis identified distinct variants in the HUWE1 across the four families. Specifically, four distinct HUWE1 variants were identified across the families: a hemizygous c.10034 > T (p.Lys3345Met) in Family 1; a heterozygous c.9209G > A (p.Arg3070His) in Family 2; a heterozygous c.12688T > C (p.Phe4230Leu) in Family 3; and a hemizygous c.9070G > A (p.Ala3024Thr) in Family 4. In accordance with ACMG guidelines, the novel variants in Families 1, 3, and 4 were classified as "Likely Pathogenic" (PS2 + PM2_Supporting + PP2 + PP3). In contrast, the previously reported variant in Family 2 was categorized as "Pathogenic" based on the criteria PS2 + PM2_Supporting + PM5 + PP2 + PP3_Moderate. All probands were clinically diagnosed with Turner-type XLID. CONCLUSIONS: This study expands the pathogenic variant spectrum of HUWE1 and provides novel molecular evidence for the clinical diagnosis of Turner-type XLID. These findings are of significant value for genetic counseling, carrier screening, and prenatal diagnosis for the affected families.
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High-pressure liquid chromatography on a reversed-phase column has been used to separate the tryptic peptides of a human hemoglobin variant which was then identified as hemoglobin E.
Restriction endonuclease digestion of genomic DNA from 24 lymphoblastoid cell lines homozygous for the HLA class II specificity DQw3, followed by hybridization with a DQ beta-chain cDNA probe, identified a genomic polymorphism with variable BamHI and HindIII recognition sites. This restriction fragment pattern was found for several haplotypes associated with the DQw3 specificity, including some haplotypes positive for the HLA-DR specificities DR4, DR5, DRw8, and DRw12. The variant fragment pattern corresponds precisely with the reactivity of a monoclonal antibody, A-10-83, previously shown to define a serologic split of DQw3. Serologic detection of the specific DQw3.1 genomic polymorphism indicated that the corresponding DQ beta-chain variants are expressed. This polymorphic restriction fragment pattern, then, represents a selective marker for DQ beta-chain genes that appear to define a DQ beta-chain-associated specificity, here called DQw3.1.
Using Epstein-Barr virus (EBV) transformation of B cells isolated from peripheral blood of two asymptomatic human immunodeficiency virus type 1-(HIV-1) infected subjects, we have produced four IgG1 human monoclonal antibodies (HMAbs) that bind to HIV-1 gp120, as determined by Western blot analysis. Two of these HMAbs, designated N70-1.5e and N70-2.3a, react with epitopes of gp120 expressed by all strains tested thus far, and therefore, appear to identify conserved epitopes. The other two HMAbs, K24-3b and N70-1.9b, identify variant epitopes; K24-3b binds to an epitope which is absent from two strains but heterogeneously expressed in eight other strains; N70-1.9b binds to an epitope that is found in relatively few strains. We also describe a novel immunoassay in which viral glycoproteins, produced by HIV-1-infected cells grown in serum-free medium, are affinity immobilized in Concanavalin A-coated wells of enzyme-linked immunosorbent assay (ELISA) plates. This method greatly facilitates the preparation of solid-phase HIV envelope glycoproteins from multiple virus strains and screening immunoassays based on this method are highly sensitive and effective in detecting antibodies to gp120.
A variant CYP2D6(C) P450 protein was found in a liver characterized by deficient microsomal metabolism of bufuralol and sparteine, prototypical substrates for the debrisoquine-sparteine drug oxidation polymorphism. This protein was present at decreased levels in liver and had a slightly different relative mobility on SDS-polyacrylamide gels. The cDNA cloning and sequencing of the variant, designated CYP2D6(C), revealed that its mRNA lacked a single codon resulting in deletion of Lys281. This was the result of a three base pair deletion at the 3' end of CYP2D6 exon 5. The CYP2D6(C) P450, produced in HepG2 cells using vaccinia virus mediated cDNA expression displayed Km values toward bufuralol, debrisoquine and sparteine that were not significantly different from wild type CYP2D6. These data suggest that the poor metabolizer phenotype in livers expressing CYP2D6(C) is not due to a catalytically defective enzyme but perhaps due to decreased levels of the P450 protein in microsomal membranes. Low microsomal CYP2D6(C) contents could result from deficient membrane insertion or decreased stability of the P450 protein. A polymerase chain reaction-based procedure, developed to detect CYP2D6(C) alleles, indicates that this variant probably represents less than 1.5% of all CYP2D6 alleles.
Variants of a cloned laboratory stock of the trypanosomatid parasite Crithidia luciliae have been distinguished from "parental type" organisms. These variants accumulated spontaneously over time as the protozoan was maintained by continuous passage in a chemically defined medium. Cloned lines of these variants have been isolated by plating on nutrient agar and partially characterized on the basis of their growth characteristics in culture, their colony and cellular morphology as well as their surface protein expression. One cloned line consisted of motile, flagellated forms which, unlike "parental type" organisms, did not adhere to the surface of culture flasks. Another cloned line was composed of non-adherent, nonmotile, amastigote-like forms which were further distinguished from "parental type" cells by virtue of their constitutive expression, in nutrient-replete medium, of high levels of a surface membrane associated 3'-nucleotidase/nuclease (3'-N'ase) activity. Both the motile, flagellated and amastigote-like variants, like the "parental type" organisms, exhibited elevated levels of the 3'-N'ase activity upon exposure to purine starvation conditions. The variants described are of potential importance in elucidating the mechanism of induction of the highly regulated 3'-N'ase activity as well as for understanding the cytoskeletal systems and the surface properties of these protozoa.
Liquid chromatography techniques were used to separate and identify human growth hormone (hGH) variants. N-terminal modified forms, such as des-Phe (2-191) and methionyl-hGH (met-1-191), were separated from recombinant human growth hormone (rhGH (1-191] by hydrophobic interaction chromatography (HIC). A proteolytically cleaved ('clip') form of rhGH which has a break in the polypeptide chain between Thr(142) and Tyr(143), also proved to be separable from rhGH by HIC. In addition, a mutated form of rhGH with only two amino acid substitutions, Glu(65) to Val(65) and Glu(66) to Lys(66), on a random coil domain of the molecule, was separated from rhGH by HIC, indicating that these substitutions altered the hydrophobicity of the molecule. Treatment of rhGH with hydrogen peroxide led to sulphoxide formation in two methionine residues Met(14) and Met(125); it was not possible to oxidize Met(170). The oxidized forms of rhGH were readily separated from rhGH(1-191) by reversed-phase chromatography. Analyses of rhGH batches showed very low levels (less than 0.3%) of oxidized rhGH, indicating that rhGH is highly resistant to oxidative reactions. Deamidations were induced in rhGH by heat treatment. The primary deamidation site was found to be Asn(149). Monodesamido rhGH and didesamido rhGH were efficiently separated from rhGH(1-191) by anion-exchange chromatography.
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