A novel variant in Serpina7 gene in a family with thyroxine-binding globulin deficiency

Thyroxine-binding globulin (TBG) carries approximately 75% of serum T4 and T3. This protein is encoded by serpina7 gene, formerly known as TBG gene, localized on X-chromosome (Xq22.2). A deficiency in TBG is suspected when abnormally low serum total T4 and T3 are encountered in clinically euthyroid subjects in the presence of normal serum TSH. This condition has been associated with different serpina7 gene mutations resulting in amino acid substitutions or truncations in the mature protein. Herein, we report a new serpina7 gene variant in three members of the same family. It results in the replacement of the normal asparagine 233 by isoleucine and, subsequently, in disruption of a glycosylation site. Co-segregation of this new variant with undetectable levels of TBG in the hemizygous man studied and failure to recognize the same variant in 100 alleles at random, made us to consider it as the underlying cause of the TBG deficiency.     

Humoral autoreactivity to an alternatively spliced variant of ICA512/IA-2 in Type I diabetes

Aims/hypothesis. The receptor tyrosine phosphatase like-protein ICA512/IA-2 occurs as a proteolytically-processed 65,000 M_r type 1 transmembrane glycoprotein in beta cells and is a major autoantigen of Type I (insulin-dependent) diabetes mellitus. We investigated whether alternative splicing could affect humoral autoreactivity to the molecule.¶Methods. Genomic and cDNA sequence analysis showed the presence of a ICA512 variant in islets and lymphoid tissues with an in-frame deletion of exon 13 which produces a secreted form lacking aa 557–629 including the transmembrane domain (aa 577 to 600). The alternatively spliced protein is detectable by western blotting in normal islets and translated into a protein that is processed to a series of soluble forms of 25,000–35,000 M_r. Radioimmunoprecipitation assays for anti-ICA512 autoantibodies were developed with the widely used ICA512.bdc construct (which has exon 13 deleted) and a series of full-length and modified ICA512/IA-2 molecules. ¶Results. The assays showed that ICA512.bdc and ICA512_604–979 gave the best discrimination between diabetic and control sera. With ICA512_604–979 a somewhat greater proportion of patients expressing antibodies were detected than with ICA512.bdc in the groups studied (70.5 % vs 63.2 % of prediabetic/new-onset and 25.0 vs 13.9 % in patients with diabetes > 20 years). Conversely, a small proportion (3 % recent-onset and 6 % > 20 years) had antibodies to ICA512.bdc but not ICA512_604–979.¶Conclusion/interpretation. Important epitopes lie within the exon 13 region and others can be generated by the alternative splicing. As the Δexon 13 variant is probably secreted by the beta cell, it could be recognized by the cellular and humoral arm of the immune system in the absence of cellular damage. [Diabetologia (2000) 43: 1293–1301]     

Progressive ataxia and myoclonic epilepsy in a patient with a homozygous mutation in the FOLR1 gene

Several unrelated disorders can lead to 5-methyltetrahydrofolate (5MTHF) depletion in the cerobrospinal fluid (CSF), including primary genetic disorders in folate-related pathways or those causing defective transport across the blood-CSF barrier. We report a case of cerebral folate transport deficiency due to a novel homozygous mutation in the FOLR1 gene, in an effort to clarify phenotype–genotype correlation in this newly identified neurometabolic disorder. A previously healthy infant developed an ataxic syndrome in the second year of life, followed by choreic movements and progressive myoclonic epilepsy. At the age of 26 months, we analyzed CSF 5MTHF by HPLC with fluorescence detection and conducted magnetic resonance (MR) imaging and spectroscopy studies. Finally, we performed mutational screening in the coding region of the FOLR1 gene. MR showed a diffuse abnormal signal of the cerebral white matter, cerebellar atrophy and a reduced peak of choline in spectroscopy. A profound deficiency of CSF 5MTHF (2 nmol/L; NV 48-127) with reduced CSF/plasma folate ratio (0.4; NV 1.5-3.5) was highly suggestive of defective brain folate-specific transport across the blood-CSF/brain barrier. Mutation screening of FOLR1 revealed a new homozygous missense mutation (p.Cys105Arg) that is predicted to abolish a disulfide bond, probably necessary for the correct folding of the protein. Both parents were heterozygous carriers of the same variant. Mutation screening in the FOLR1 gene is advisable in children with profound 5MTHF deficiency and decreased CSF/serum folate ratio. Progressive ataxia and myoclonic epilepsy, together with impaired brain myelination, are clinical hallmarks of the disease.     

A common genetic variant in WFS1 determines impaired glucagon-like peptide-1-induced insulin secretion

Aims/hypothesis

WFS1 type 2 diabetes risk variants appear to be associated with impaired beta cell function, although it is unclear whether insulin secretion is affected directly or secondarily via alteration of insulin sensitivity. We aimed to investigate the effect of a common WFS1 single-nucleotide polymorphism on several aspects of insulin secretion.

Methods

A total of 1,578 non-diabetic individuals (534 men and 1,044 women, aged 40?±?13 years, BMI 28.9?±?8.2 kg/m² [mean ± SD]) at increased risk of type 2 diabetes were genotyped for rs10010131 within the WFS1 gene. All participants underwent an OGTT (and a subset additionally an IVGTT [n?=?319]) and a hyperglycaemic clamp combined with glucagon-like peptide-1 (GLP-1) and arginine stimuli (n?=?102).

Results

rs10010131 was associated with reduced OGTT-derived insulin secretion (p?=?0.03). In contrast, insulin secretion induced by an i.v. glucose challenge in the IVGTT and hyperglycaemic clamp was not different between the genotypes. GLP-1 infusion combined with a hyperglycaemic clamp showed a significant reduction of the insulin secretion rate during the first and second phases of GLP-1-induced insulin secretion in carriers of the risk allele (reduction of 36% and 26%, respectively; p?=?0.007 and p?=?0.04, respectively).

Conclusions/interpretation

A common genetic variant in WFS1 specifically impairs GLP-1-induced insulin secretion independently of insulin sensitivity. This defect might explain the impaired insulin secretion in carriers of the risk allele and confer the increased risk of type 2 diabetes.     

Two genetic defects in a patient with complete deficiency of the b- subunit for coagulation factor XIII

A genomic DNA obtained from a female patient with complete b-subunit deficiency was examined by Southern blotting analysis and in vitro amplification. Nucleotide sequence analysis showed that adenosine-4161 at the acceptor splice junction of intron A/exon II was deleted in half of the amplified DNAs, resulting in a loss of the obligatory AG splicing sequence. The absence of adenosine-4161 was confirmed by cleavage with TaqI endonuclease of the amplified DNAs. Moreover, sequence analysis showed that guanosine-11499 coding for Cys 430 (TGC) in exon VIII was replaced by thymidine in half of the amplified DNAs, resulting in an amino acid change to Phe (TTC) and the destruction of a disulfide bond in the seventh Sushi domain. This mutation was also confirmed by cleavage with MboII endonuclease. Thus, the proband turned out to be a compound heterozygote of two separate defective alleles. Although half of the amplified DNAs for exon VIII of her daughter or son were cleaved by MboII, those for intron A were not cleaved by TaqI. The replacement of guanosine-11499 by thymidine in their exon VIII has also been confirmed by sequence analysis, indicating that they are heterozygous for one normal and one defective allele.     

TAT gene mutation analysis in three Palestinian kindreds with oculocutaneous tyrosinaemia type II; characterization of a silent exonic transversion that causes complete missplicing by exon 11 skipping

Summary Deficiency of the hepatic cytosolic enzyme tyrosine aminotransferase (TAT) causes marked hypertyrosinaemia leading to painful palmoplantar hyperkeratoses, pseudodendritic keratitis and variable mental retardation (oculocutaneous tyrosinaemia type II or Richner–Hanhart syndrome). Parents may therefore seek prenatal diagnosis, but this is not possible by biochemical assays as tyrosine does not accumulate in amniotic fluid and TAT is not expressed in chorionic villi or amniocytes. Molecular analysis is therefore the only possible approach for prenatal diagnosis and carrier detection. To this end, we sought TAT gene mutations in 9 tyrosinaemia II patients from three consanguineous Palestinian kindreds. In two kindreds (7 patients), the only potential abnormality identified after sequencing all 12 exons and exon–intron boundaries was homozygosity for a silent, single-nucleotide transversion c.1224G > T (p.T408T) at the last base of exon 11. This was predicted to disrupt the 5′ donor splice site of exon 11 and result in missplicing. However, as TAT is expressed exclusively in liver, patient mRNA could not be obtained for splicing analysis. A minigene approach was therefore used to assess the effect of c.1224G > T on exon 11 splicing. Transfection experiments with wild-type and c.1224G > T mutant minigene constructs demonstrated that c.1224G > T results in complete exon 11 skipping, illustrating the utility of this approach for confirming a putative splicing defect when cDNA is unavailable. Homozygosity for a c.1249C > T (R417X) exon 12 nonsense mutation (previously reported in a French patient) was identified in both patients from the third kindred, enabling successful prenatal diagnosis of an unaffected fetus using chorionic villous tissue. Competing interests: None declared References to electronic databases: tyrosinaemia type II (OMIM +276600); hepatic cytosolic tyrosine aminotransferase (EC 2.6.1.5); GenBank: accession number for TAT mRNA, NM_000353; accession number for TAT genomic DNA, NC_000016.     

Molecular analysis of delta-aminolevulinate dehydratase deficiency in a patient with an unusual late-onset porphyria

Cloning, expression, and genotype studies of the defective gene for delta-aminolevulinate dehydratase (ALAD) in a patient with an unusual late onset of ALAD deficiency porphyria (ADP) were carried out. This patient was unique in that he developed the inherited disease, together with polycythemia, at the age of 63. ALAD activity in erythrocytes of the patient was less than 1% of the normal control level. ALAD complementary DNA (cDNA) isolated from the patient's Epstein-Barr virus (EBV)-transformed lymphoblastoid cells had 2 base transitions in the same allele, G(177) to C and G(397) to A, resulting in amino acid substitutions K59N and G133R, respectively. It has been verified that the patient had no other ALAD mutations in this and in the other allele. By restriction fragment length polymorphism (RFLP) analysis, all family members of the proband who had one-half ALAD activity compared with the ALAD activity of the healthy control were shown to have the same set of base transitions. Expression of ALAD cDNA in CHO cells revealed that K59N cDNA produced a protein with normal ALAD activity, while G133R and K59N/G133R cDNA produced proteins with 8% and 16% ALAD activity, respectively, compared with that expressed by the wild type cDNA. These findings indicate that while the proband was heterozygous for ALAD deficiency, the G(397) to A transition resulting in the G133R substitution is responsible for ADP, and the clinical porphyria developed presumably due to an expansion of the polycythemic clone in erythrocytes that carried the mutant alad allele.     

Organization of the gene for human platelet/endothelial cell adhesion molecule-1 shows alternatively spliced isoforms and a functionally complex cytoplasmic domain

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell-cell adhesion molecule that is expressed on circulating platelets, on leukocytes, and at the intercellular junctions of vascular endothelial cells and mediates the interactions of these cells during the process of transendothelial cell migration. The cDNA for PECAM-1 encodes an open reading frame of 738 amino acids (aa) that is organized into a 27- aa signal peptide, a 574-aa extracellular domain composed of 6 Ig homology units, and a relatively long cytoplasmic tail of 118 aa containing multiple sites for posttranslational modification and postreceptor signal transduction. To provide a molecular basis for the precise evaluation of the structure and function of this transmembrane glycoprotein, we have determined the organization of the human PECAM-1 gene. The PECAM-1 gene, which has been localized to human chromosome 17, is a single-copy gene of approximately 65 kb in length and is broken into 16 exons by introns ranging in size from 86 to greater than 12,000 bp in length. Typical of other members of the Ig superfamily, each of the extracellular Ig homology domains is encoded by a separate exon, consistent with PECAM-1 having arisen by gene duplication and exon shuffling of ancestral Ig superfamily genes. However, the cytoplasmic domain was found to be surprisingly complex, being encoded by seven short exons that may represent discrete functional entities. Alternative splicing of the cytoplasmic tail appears to generate multiple PECAM-1 isoforms that may regulate phosphorylation, cytoskeletal association, and affinity modulation of the mature protein. Finally, a processed pseudogene having 76% identity with PECAM- 1 cDNA was identified and localized to human chromosome 3. These findings should have important implications for structure/function analysis of PECAM-1 and its role in vascular adhesive interactions.     

A thymidine to cytosine substitution for codon 26 of exon 3 of apolipoprotein C-II gene in a patient with apolipoprotein C-II deficiency

A 52-year-old Japanese woman was evaluated for severe hypertriglyceridemia and recurrent acute pancreatitis. This hypertriglyceridemia was found to be due to the absence of serum apolipoprotein C-II (apo C-II) which was identified by Western blotting using polyclonal anti-apo C-II antiserum. DNA sequence analysis of the apo C-II gene from the patient revealed a homozygous nucleotide change: a thymidine (T) to cytosine (C) substitution in codon 26 (TGG->CGG) at the third exon of the apo C-II gene, that resulted in a Trp26 to Arg substitution. The mutation was also confirmed by restriction fragment length polymorphism (RFLP) analysis with the restriction enzyme Hpa II. The same mutation has been found in a case previously reported in Japan, and was named apo C-II Wakayama. However, the case in Wakayama prefecture showed two concomitant point mutations at the 5'-flanking region upstream from the first exon, which were not identified in our case by RFLP analysis with the restriction enzyme BstXI. Considering that the prefectures of these two cases, Nara and Wakayama, are next to each other, the mutation in our case may be a genetic forebear of apo C-II Wakayama. However, no familial relationship between the two cases has been documented.     

An unusual cause of biliary stricture in a patient with neurofibromatosis type 1

The majority of biliary strictures occur as a consequence of iatrogenic injury to the extrahepatic biliary tract, with more than 80% following cholecystectomy. The laparoscopic era has led to heightened awareness of this problem. The occurrence of an iatrogenic stricture can be particularly devastating to both patient and surgeon. The literature highlights a number of factors involved in the aetiology of such traumatic stricture formation. We report an unusual case of a Bismuth 2 stricture of the proximal common hepatic duct,occurring in a patient with type 1 neurofibromatosis, following an iatrogenic bile duct injury that occurred during a laparoscopic cholecystectomy. Histological examination of the strictured region of bile duct removed at surgery demonstrated multiple neurofibromas of varying sizes present in the submucosa. Neurofibromatosis type 1 (von Recklinghausen disease) affects the gastrointestinal tract in up to 25% of cases, and in such cases is characterized by multiple submucosal neurofibromas. We believe this is the first reported case of a biliary stricture in a patient with neurofibromatosis type 1, which appeared to be as a consequence of neurofibromas in the submucosa of the bile duct.     

A point mutation in an invariant splice donor site leads to exon skipping in two unrelated Dutch patients with dihydropyrimidine dehydrogenase deficiency

Summary Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disease characterized by thymine-uraciluria and associated with a variable clinical phenotype. In order to identify the molecular defect underlying complete DPD deficiency in a Dutch patient previously shown to have a 165 base pair deletion in the mature DPD mRNA, we cloned the genomic region encompassing the skipped exon and its flanking intron sequences. Sequence analysis revealed that the patient was homozygous for a single GA point mutation in the invariant GT dinucleotide splice donor site downstream of the skipped exon. The same mutation was identified in another, unrelated, Dutch patient. Because this mutation destroys a uniqueMaeII restriction site, rapid screening using restriction enzyme cleavage of the amplified genomic region encompassing this mutation is possible. Analysis of 50 controls revealed no individuals heterozygous for this mutation     

A patient with pseudohypoaldosteronism type II caused by a novel mutation in WNK4 gene

Pseudohypoaldosteronism Type II (PHAII) is a very rare disorder characterized by hyperkalemia, hypertension, and slight hyper-chloremic metabolic acidosis. The index patient showed typical features of PHAII, including elevated blood pressure (140–150/90–100 mmHg), hyperkalemia in the range of 5.30–5.60 mmol/l (normal range is 3.50–5.10 mmol/l), accompanied by hyperchloremia of 109.5–112.0 mmol/l (normal 95.0–108.0 mmol/l) and acidosis with bicarbonate levels of 19.5–20.1 mmol/l (normal 22.0–27.0), GFR was 98.95 ml/min (normal > 90). However, these features were absent in his parents. Sequencing analysis found the patient with a WNK4 gene mutation, 1682 C > T in Exon 7, which resulted a missense mutation at codon 561 (P561L). The variation in codon 561 was not found in his parents and 100 unrelated control subjects. The identified WNK4 mutation which has not been described previously is the probable cause of PHAII.     

Bak基因诱导p27KIP1基因的过表达使HCC-9204细胞在G1期停滞

目的Bak基因的过表达能够使HCC-9204细胞的G1期延长并导致细胞凋亡,探讨p27KIP1基因是否在这一过程中起重要作用。方法建立Bak诱导HCC-9204细胞凋亡和细胞周期停滞的模型。从Bak诱导的凋亡模型中获得p27KIP1基因并测序。构建pMD-KIP1可诱导型表达系统并转染HCC-9204细胞,获得稳定转染子。进一步研究p27KIP1对细胞生长和细胞周期的调控作用。Westernblot证实在这一过程中该基因的表达水平上调。结果在诱导48h后p27KIP1基因稳定转染细胞的活力降低35%。在诱导后24h时p27KIP1能够使HCC-9204细胞在G1期停滞,累积的细胞增加40%。结论Bak使HCC-9204细胞的G1期延长是通过上调p27KIP1的表达;可诱导型p27KIP1基因的过表达能够诱导G1期停滞。