Screening for functional sequence variations and mutations in ABCA1

Mutations in the ATP-binding cassette 1 transporter gene (ABCA1) are responsible for the genetic HDL-deficiency syndromes, which are characterized by severely diminished plasma HDL-C levels and a predisposition to cardiovascular disease and splenomegaly. The ABCA1 gene contains 50 exons and codes for a 2261-amino acid long membrane protein that facilitates phospholipid and cholesterol transport. Several mutations have been identified so far as responsible either for Tangier disease or for reduced HDL levels. We have selectively looked for additional polymorphisms in functionally relevant regions of the gene in cohorts constituted of individuals with altered HDL levels as well as healthy blood donors and octogenarians, and screened for mutations in the complete coding region of selected individuals with extremely aberrant HDL levels. In the promoter region, which is important for regulation of gene expression, we have identified several polymorphisms including one VNTR polymorphism, located at a putative ZNF202 binding site, which displayed different binding of ZNF202 in an electromobility shift assay. Three novel SNPs were discovered in the promoter region (G1047C, C1152T and C1440T). The prevalence of exchange G1047C (G-395C) was found significantly increased in probands with low HDL compared to probands with high HDL. Exchanges C1152T (C-290T) and C1440T (C-7T) were significantly more frequent in the cohort with low HDL compared to healthy blood donors and octogenarians. In the C-terminal part of ABCA1, known to interact with other proteins, two novel sequence variations (F2163S and V2244I) have been found in one phenotype related to cardiovascular disease, but none in the aforementioned cohorts. In one individual with extremely high HDL levels, the V771M polymorphism was found in a homozygous state. In patients with HDL deficiency, three novel mutations have been identified (W590L, W840R and R1068C). To facilitate further research in ABCA1 sequence variations and expand our understanding of their effects, we are introducing a webpage archive (http://www.abca1-mutants.all.at) containing all sequence variations reported in ABCA1 so far. This webpage provides a more recent and detailed summary of sequence variations and mutations in ABCA1 than existing databases and should also be of interest for molecular diagnosis of ABCA1-related HDL deficiency.     

Identification of a de novo BRCA1 mutation in a woman with early onset bilateral breast cancer

De novo mutations are rarely reported in BRCA1 and BRCA2. We report a proven BRCA1 de novo mutation in a woman diagnosed with young onset bilateral breast cancer with a limited family history.     

人免疫缺陷病毒I型外膜糖蛋白gp^120和gp^160在重组痘苗…

人免疫缺陷病毒Ｉ型（ＨＩＶ－１）是严重威胁人类健康的病原体，其主要抗原是病毒外膜糖蛋白ｇｐ＾１６０（ｇｐ＾１２０＋ｇｐ＾４１）。本文报道运用遗传工程技术，在噬菌体Ｔ７启动子或痘苗病毒启动子ｐ７．５控制下，在重组痘苗病毒系统中构建和表达ＨＩＶ－１ｇｐ＾１２０和ｇｐ＾１６０。进一步研究表明，在此系统中表达的重组ＨＩＶ－１ｇｐ＾１２和ｇｐ＾１６０以糖蛋白形式存在，保留着病毒在自然状况下的抗原表位构象，可     

Expanding the neurodevelopmental phenotype of PURA syndrome

PURA syndrome is a recently described developmental encephalopathy presenting with neonatal hypotonia, feeding difficulties, global developmental delay, severe intellectual disability, and frequent apnea and epilepsy. We describe 18 new individuals with heterozygous sequence variations in PURA. A neuromotor disorder starting with neonatal hyptonia, but ultimately allowing delayed progression to walking, was present in nearly all individuals. Congenital apnea was present in 56% during infancy, but all cases in this cohort resolved during the first year of life. Feeding difficulties were frequently reported, with gastrostomy tube placement required in 28%. Epilepsy was present in 50% of the subjects, including infantile spasms and Lennox–Gastaut syndrome. Skeletal complications were found in 39%. Disorders of gastrointestinal motility and nystagmus were also recurrent features. Autism was diagnosed in one individual, potentially expanding the neurodevelopmental phenotype associated with this syndrome. However, we did not find additional PURA sequence variations in a cohort of 120 subjects with autism. We also present the first neuropathologic studies of PURA syndrome, and describe chronic inflammatory changes around the arterioles within the deep white matter. We did not find significant correlations between mutational class and severity, nor between location of the sequence variation in PUR repeat domains. Further studies are required in larger cohorts of subjects with PURA syndrome to clarify these genotype–phenotype associations.     

Hereditary eosinophil peroxidase deficiency: immunochemical and spectroscopic studies and evidence for a compound heterozygosity of the defect.

Hereditary eosinophil peroxidase (EPO; EC 1.11.1.7) deficiency is a rare abnormality of eosinophil granulocytes characterized by decreased or absent peroxidase activity and decreased volume of the granule matrix. The molecular basis of the defect is not known. We report here its molecular characterization in an EPO-deficient subject and his family members. The EPO-deficient eosinophils contained EPO-related material as determined immunochemically using either monoclonal or polyclonal anti-EPO antibodies but had no spectroscopic evidence of EPO. Eosinophil precursors from the EPO-deficient subject contained normally sized EPO mRNA, which was reverse transcribed into the corresponding cDNA clones encompassing the whole gene. Sequencing of these clones disclosed two mutations, a G-->A transition causing a nonconservative replacement of an arginine residue with a histidine and an insertion causing a shift in the reading frame with the appearance of a premature stop codon. The two mutations were located on different chromosomes indicating a compound heterozygosity for the defect. Both the son and the daughter of the proband inherited the G-->A transition, and their eosinophils contained a peroxidase activity intermediate between that of control subjects and the proband, suggesting that the transition is a deficiency-causing mutation. Eosinophil precursors from the EPO-deficient subject were found to actively synthesize an EPO that was apparently normal in terms of cytochemical reaction for peroxidase and immunoreactivity with monoclonal and polyclonal anti-EPO antibodies, but spectroscopically abnormal. The cytochemical reaction for peroxidase tended to decrease or disappear in the eosinophil precursors of the EPO-deficient subject but not of a normal subject as differentiation went on, suggesting that the Arg-->His substitution causes the production of an unstable EPO that undergoes progressive degradation as the cells mature.     

Variation in the apo AI/CIII/AIV gene complex: its association with hyperlipidemia

This study shows that 33.3% of English patients with primary hyperlipidemia (52/156) had the S2 allele of the apo AI/CIII/AIV complex compared to 6.1% of normolipidemic individuals (3/49). The increased frequency of the allele was statistically significant in each of the hyperlipidemic groups (type IIA, excluding patients with FH, type IIB and IV) examined and was not specifically related to hypertriglyceridemia. This finding may account for the result of several studies which showed groups of patients with CHD had a significantly higher prevalence of the S2 allele than control groups. Our data do not support the notion that the increased frequency of this allele in CHD patients is independent of variations in plasma lipid levels, since we find the frequency of the S2 allele in an apparently healthy hyperlipidemic group of patients is very similar to a hyperlipidemic group with symptomatic premature atherosclerotic disease. This study also shows the BMI of the type IIB and IV hyperlipidemic patients is significantly higher than the type IIA (no xanthomas) group. This may modulate the expression of the defect associated with the S2 allele. When the type IIB and IV hyperlipidemic groups were divided into 2 groups according to their apo AI/CIII/AIV genotype (i.e., S1S1, S1S2 (including S2S2] there was no significant difference in the mean plasma level of total cholesterol, HDL-cholesterol and triglycerides between the 2 groups. In contrast the S1S2 type IIA individuals had higher plasma cholesterol levels.     

Stimulation of Trypanosoma cruzi adenylyl cyclase by an alpha D-globin fragment from Triatoma hindgut: effect on differentiation of epimastigote to trypomastigote forms.

A peptide from hindguts of the Triatoma hematophagous Chagas insect vector activates adenylyl cyclase activity in Trypanosoma cruzi epimastigote membranes and stimulates the in vitro differentiation of epimastigotes to metacyclic trypomastigotes. Hindguts were obtained from insects fed 2 days earlier with chicken blood. Purification was performed by gel filtration and HPLC on C18 and C4 columns. SDS/PAGE of the purified peptide showed a single band of about 10 kDa. The following sequence was determined for the 20 amino-terminal residues of this peptide: H2N-Met-Leu-Thr-Ala-Glu-Asp-Lys-Lys-Leu-Ile-Gln- Gln-Ala-Trp-Glu-Lys-Ala-Ala-Ser-His. This sequence is identical to the amino terminus of chicken alpha D-globin. On a Western blot, the peptide immunoreacted with a polyclonal antibody against chicken globin D. A synthetic peptide corresponding to residues 1-40 of the alpha D-globin amino terminus also stimulated adenylyl cyclase activity and promoted differentiation. This 125I-labeled synthetic peptide bound specifically to T. cruzi epimastigote cells. Activation of epimastigote adenylyl cyclase by the hemoglobin-derived peptide may play an important role in T. cruzi differentiation and consequently in the transmission of Chagas disease.     

Endogenous Interferon-α Concentration and Outcome of Interferon Treatment in Patients with Chronic Hepatitis C

To verify its value with regard to the outcomeof therapy in chronic hepatitis C, seruminterferon- (IFN) was measured by ELISA in 70patients (43 male, 27 female) with chronic hepatitis C,treated with IFN 9 MU/week subcutaneously for up to oneyear. Serum IFN was detectable in 49/70 patients, 16 ofwhom had IFN values >125 pg/ml. Only 1/22 patientswho maintained a sustained response six months after the end of treatment had pretreatment serum IFN> 125 pg/ml, in comparison to 15/48 patients who didnot respond or who relapsed ( 6.1, P < 0.02). Atmultivariate analysis the predictive value of serum IFN was independent of age, sex, presence ofcirrhosis, infection by genotype 1b (improvement 7.1, P < 0.01). In patients with chronic hepatitis C,measurement of serum IFN at baseline might be useful for the selection of patients with higherprobability of long-term response.