Granulocyte and monocyte CD11b expression during plasma separation is dependent on complement factor 5 (C5) - an ex vivo study with blood from a C5-deficient individual.

The aim of the study was to investigate the role of complement factor 5 (C5) in reactions elicited by plasma separation using blood from a C5-deficient (C5D) individual, comparing it to C5-deficient blood reconstituted with C5 (C5DR) and blood from healthy donors. Blood was circulated through an ex vivo plasma separation model. Leukocyte CD11b expression and leukocyte-platelet conjugates were measured by flow cytometry during a 30-min period. Other markers were assessed during a 240-min period. Granulocyte and monocyte CD11b expression did not increase in C5D blood during plasma separation. In C5DR samples granulocytes CD11b expression, measured by mean fluorescence intensity (MFI), increased from 10481 ± 6022 (SD) to 62703 ± 4936, and monocytes CD11b expression changed from 13837 ± 7047 to 40063 ± 713. Granulocyte-platelet conjugates showed a 2.5-fold increase in the C5DR sample compared to the C5D sample. Monocyte-platelet conjugates increased independently of C5. In the C5D samples, platelet count decreased from 210 × 109 /L (201-219) (median and range) to 51 × 109 /L (50-51), and C3bc increased from 14 CAU/mL (21-7) to 198 CAU/mL (127-269), whereas TCC formation was blocked during plasma separation. In conclusion, up-regulation of granulocyte and monocyte CD11b during plasma separation was C5-dependent. The results also indicate C5 dependency in granulocyte-platelet conjugates formation.

Genetic characterization of antithrombin, protein C, and protein S deficiencies in Polish patients.

INTRODUCTION    Inherited deficiencies of natural anticoagulants such as antithrombin (AT; gene: SERPINC1), protein C (PC; PROC), and protein S (PS; PROS1), with the prevalence in the general European population of 0.02% to 0.17%, 0.2% to 0.3%, and 0.5%, respectively, are associated with increased risk of thromboembolic events. Only a few case reports of Polish deficient patients with known causal mutations have been published so far. OBJECTIVES    The aim of the study was to characterize the frequency of SERPINC1, PROC, and PROS1 mutations and their thromboembolic manifestations in patients with AT, PC, or PS deficiencies, inhabiting southern Poland. PATIENTS AND METHODS     Ninety unrelated patients (mean [SD] age, 40.1 [13.2] years) with AT (n = 35), PC (n = 28), or PS (n = 27) deficiencies, with a history of venous 73 (81%) or arterial 17 (19%) thromboembolism, were screened for mutations using the Sanger sequencing or multiplex ligation­dependent probe amplification. RESULTS    Twenty mutations (29%) described here were new, mostly in the SERPINC1 and PROC genes. Missense mutations accounted for 84% of all mutations in the PROC gene and approximately 50% of those in the SERPINC1 and PROS1 genes. In all 3 genes, the ratio of nonsense and splice-site mutations was 8% to 31% and 8% to 23%, respectively. The mutation detection rate was 90% for AT or PC when anticoagulant activity was below 70%, while for the PROS1 gene, the rate reached 80% at the free PS levels below 40%. CONCLUSIONS    To our knowledge, this is the largest cohort of Polish patients deficient in natural anticoagulants and evaluated for the causal genetic background. Several new Polish detrimental mutations were detected, mostly in AT- and PC­deficient patients.

Mutations and polymorphism in bottlenose dolphin (Tursiops truncatus, Montagu 1821) albumin gene: First identification of mutations responsible for inherited bisalbuminemia.

Hereditary bisalbuminemia is an asymptomatic and heterozygous condition in a range of species characterized by the presence of two serum albumin fractions with different electrophoretic mobility resulting in a bicuspid pattern on serum electrophoresis. Bisalbuminemia has been diagnosed by electrophoresis in two bottlenose dolphin (Tursiops truncatus) families, but causative mutations and the inheritance pattern have not been identified. The aims of this work are: to investigate polymorphisms of the bottlenose dolphin albumin gene and to identify mutations causative of bisalbuminemia; to identify the inheritance pattern in two bottlenose dolphin families. Coding regions of the albumin gene were screened for mutations in 15 bottlenose dolphins kept under human care from two distinct families. Eighteen albumin mutations (three synonymous and 15 non-synonymous) were identified. Two non-synonymous variations co-segregated with bisalbuminemic phenotype: p.Phe146Leu in exon 4 and p.Tyr163His in exon 5. The amino acid change in exon 5 was associated with the secondary and/or tertiary structure variation of the protein and has been reported as causative of bisalbuminemia in humans. Pedigree analysis of the dolphin families showed an autosomal codominant inheritance pattern. In this work, the mutations potentially responsible for bisalbuminemia were identified and confirmed the autosomal codominant trait in bottlenose dolphins.

Detection of hereditary bisalbuminemia in bottlenose dolphins (Tursiops truncatus, Montagu 1821): comparison between capillary zone and agarose gel electrophoresis.

BACKGROUND: Hereditary bisalbuminemia is a relatively rare anomaly characterized by the occurrence of two albumin fractions on serum protein separation by electrophoresis. In human medicine, it is usually revealed by chance, is not been clearly associated with a specific disease and the causative genetic alteration is a point mutation of human serum albumin gene inherited in an autosomal codominant pattern. This type of alteration is well recognizable by capillary zone electrophoresis (CZE), whilst agarose gel electrophoresis (AGE) not always produces a clear separation of albumin fractions. The aims of this study is to report the presence of this abnormality in two separate groups of related bottlenose dolphins and to compare the results obtained with capillary zone and agarose gel electrophoresis. RESULTS: Serum samples from 40 bottlenose dolphins kept under human care were analyzed. In 9 samples a double albumin peak was evident in CZE electrophoresis while no double peak was noted in AGE profile. Since only an apparently wider albumin peaks were noted in some AGE electrophoretic profiles, the ratio between base and height (b/h) of the albumin peak was calculated and each point-value recorded in the whole set of data was used to calculate a receiver operating characteristic curve: when the b/h ratio of albumin peak was equal or higher than 0.25, the sensitivity and specificity of AGE to detect bisalbuminemic samples were 87 and 63 %, respectively. The bisalbuminemic dolphins belong to two distinct families: in the first family, all the siblings derived from the same normal sire were bisalbuminemic, whereas in the second family bisalbuminemia was present in a sire and in two out of three siblings. CONCLUSIONS: We report for the first time the presence of hereditary bisalbuminemia in two groups of related bottlenose dolphins identified by means of CZE and we confirm that AGE could fail in the identification of this alteration.

Bisalbuminaemia due to novel mutation at a critical residue involved in recycling; Albumin Lyon (510His→Arg).

OBJECTIVES: To define the underlying cause of bisalbuminaemia in an individual presenting with spontaneous venous thrombosis. METHOD: Plasma was examined by electrospray time-of-flight mass spectrometry (TOF MS) to assess albumin mutations and to quantify variant expression level. Tryptic peptide mapping and DNA sequencing were used to precisely define the mutation. RESULTS: Whole protein MS indicated a 19Da increase in the mass of 50% of the albumin molecules suggesting a His→Arg substitution. A novel heterozygous 510His→Arg mutation was identified by peptide mass mapping and confirmed by DNA sequencing of exon 12 of the albumin gene. CONCLUSION: The nature and location of the mutation suggest it would have no direct influence on haemostasis through altered warfarin binding or increased fibrinogen attachment and it appears to be incidental to the thrombotic phenotype. However the highly conserved His510 residue is recognised as being of critical importance in albumin recycling through interaction with its savaging neonatal Fc receptor. The normal albumin level of 41.1g/l and the coequal expression of albumin Lyon demonstrate that the conservative 510His→Arg substitution does not interfere with the pH dependant capture and release of albumin by the receptor.

Complement regulators in human disease: lessons from modern genetics.

First identified in human serum in the late 19th century as a 'complement' to antibodies in mediating bacterial lysis, the complement system emerged more than a billion years ago probably as the first humoral immune system. The contemporary complement system consists of nearly 60 proteins in three activation pathways (classical, alternative and lectin) and a terminal cytolytic pathway common to all. Modern molecular biology and genetics have not only led to further elucidation of the structure of complement system components, but have also revealed function-altering rare variants and common polymorphisms, particularly in regulators of the alternative pathway, that predispose to human disease by creating 'hyperinflammatory complement phenotypes'. To treat these 'complementopathies', a monoclonal antibody against the initiator of the membrane attack complex, C5, has received approval for use. Additional therapeutic reagents are on the horizon.

Hyperphosphorylation of autoantigenic targets of paraproteins is due to inactivation of PP2A.

Paratarg-7, a frequent autoantigenic target, and all other autoantigenic targets of human paraproteins molecularly defined to date are hyperphosphorylated in the respective patients compared with healthy controls, suggesting that hyperphosphorylation of autoantigenic paraprotein targets is a general mechanism underlying the pathogenesis of these paraproteins. We now show that hyperphosphorylation of paratarg-7 occurs because of an additional phosphorylation of Ser17, which is located within the paraprotein-binding epitope. Coimmunoprecipitation identified phosphokinase C ζ (PKCζ) as the kinase responsible for the phosphorylation of most, and phosphatase 2A (PP2A) as the phosphatase responsible for the dephosphorylation of all hyperphosphorylated autoantigenic targets of paraproteins. Single-nucleotide polymorphisms (SNPs) or mutations of PKCζ and PP2A were excluded. However, PP2A was inactivated by phosphorylation of its catalytic subunit at Y307. Stimulation of T cells from healthy carriers of wild-type paratarg-7 induced a partial and transient hyperphosphorylation between days 4 and 18, which was maintained by incubation with inhibitors of PP2A, again indicating that an inactivation of PP2A is responsible for the hyperphosphorylation of autoantigenic paraprotein targets. We conclude that the genetic defect underlying the dominantly inherited hyperphosphorylation of autoantigenic paraprotein targets is not in the PP2A itself, but in genes or proteins controlling PP2A activity by phosphorylation of its catalytic subunit.

First reported case of hereditary bisalbuminaemia in Pakistan.

A 50 year old female patient demonstrated double peaks of albumin on serum protein electrophoresis (SPE). Patient's detailed medical history was taken and investigations were carried out to rule out systemic causes. Serum electrophoresis was also done on 5 of patient's children. Mixing studies were performed in gradually decreasing concentration of bisalbuminemic serum. Hereditary Bisalbuminaemia with the variant albumin showing slower mobility, was observed.

[Clinical characteristics and ALB gene mutation analysis of Korean patients with bisalbuminemia].

BACKGROUND: Bisalbuminemia is a hereditary or an acquired condition characterized by the presence of 2 albumin variants with different mobilities on serum protein electrophoresis (SPE). The clinical significance of bisalbuminemia has not been clearly established. However, some regions of the albumin variant may affect the biochemical analysis of biomolecules such as steroid or thyroid hormones by altering their albumin-binding affinities. In this study, we analyzed the clinical manifestations, genetic variations, and the albumin-binding characteristics in Korean patients with bisalbuminemia. METHODS: We performed SPE for samples from 580 Korean subjects and identified bisalbuminemia on the basis of the results of SPE. The clinical and biochemical characteristics, ALB gene mutations, and the structures of the albumin variants of patients with bisalbuminemia were analyzed. RESULTS: SPE showed bisalbuminemia in 2 patients. One patient showed a genetic variation known as Nagasaki-1 (Asp293Gly) and the other showed a hitherto unreported missense mutation (c.593A>T; Lys198Ile). In both cases, the serum concentrations of the substances with binding affinity for albumin were not affected, and the mutation sites of the albumin were not located with the protein-binding loci. CONCLUSIONS: The 2 Korean patients with bisalbuminemia showed genetic variations, including a novel missense mutation. The ALB gene analysis with 3D modeling is useful for determining the nature of bisalbuminemia and for predicting the effects on the albumin-binding affinity of other biochemical compounds.

Hereditary C1q deficiency: a new family with C1qA deficiency.

Hereditary deficiency of complement component C1q is a rare genetic disorder with susceptibility to recurrent infections with polysaccharide-containing encapsulated microorganisms and a high prevalence of autoimmune diseases, most often systemic lupus erythematosus (SLE). Here, we report a 29-month-old boy who presented with facial rash and history of early death of a sibling with infections, who was found to have a selective deficiency of C1q. The facial rash was composed of patchy erythematous plaques and centrally hypopigmented macules and desquamation. Two siblings had died of severe bacterial infections and his uncle had died of meningitis. Molecular study disclosed a homozygous point mutation in the C1qA chain gene. Five members of the family, including the parents and three healthy siblings, were heterozygous for this mutation.

A rare familiar case of hereditary bisalbuminemia and diabetic predisposition: a possible predictive link?

We present a case report of hereditary bisalbuminemia in an Italian family with three affected members. Bisalbuminemia represents a genetic variant of the albumin, it will then be permanent, or acquired and then be transient. It is characterized by the presence of two albumin bands in electrophoresis: the first band with the same mobility of the normal albumin, the second band with a fast variable or a slow variable. The double band of albumin was detected fortuitously on a routine analytical study of an adult woman who was referred to our laboratory with an increase of fasting glucose value, this originated the study of the rest of the members of the family. Finally, it is like the genetic peculiarity of this family core show a possible predictive link between bisalbuminemia on one hand and the predisposition to type II diabetes mellitus on the other hand. As a result of such high probability we are eager to continue further search at our medicine predictive centre.

Location of the mutation site in the first two reported cases of analbuminemia.

OBJECTIVE: To find the mutation causing the first two reported cases of analbuminemia. DESIGN AND METHODS: DNA was extracted from blocks of fixed embedded liver. Exons of the albumin gene were amplified and sequenced. RESULTS: A substitution of C>T in exon 12 was found in both subjects, changing the codon CGA for arginine (aa509) to TGA, a stop codon. CONCLUSIONS: The data suggest that analbuminemia is the result of widely scattered random mutations.

Analbuminemia Zonguldak: case report and mutational analysis.

OBJECTIVES: To document a new case of the rare disease analbuminemia and to study the molecular defect responsible for the trait. DESIGN AND METHODS: Single-strand conformational polymorphism (SSCP), heteroduplex analysis (HA), and DNA sequencing of the 14 exons and their flanking intron regions, as well as of the 5' and 3' UTR, of the albumin gene were conducted on DNA extracted from peripheral blood samples. RESULTS: DNA sequence analysis showed that the proband was homozygous, and his parents were both heterozygous, for a previously unreported 5180 T-->A transversion. This silent mutation creates at position 5180-81 a new AG dinucleotide, the invariant sequence encountered in all eukaryotic intron acceptor splice sites. This aberrant splice site near the 3'end of exon 5 might alter the normal splicing mechanism. No other mutation was found in the examined regions of the gene. CONCLUSIONS: Our results define a new molecular defect in the albumin gene.

Hereditary complement C7 deficiency in nine families: subtotal C7 deficiency revisited.

Deficiencies in terminal complement components, including the component C7, are uncommon and associated with an increased risk of recurrent systemic neisserial infection. A total of 22 molecular defects have been reported in the C7 gene with both complete (C7Q0) and subtotal (C7SD) C7 deficiencies. In this study we report the molecular basis of nine new cases of C7 deficiencies that were characterized by exon-specific sequence analysis. Seven different C7 gene mutations were identified corresponding to small deletions (n=2), splice site changes (n=1) and single base pair substitutions leading to nonsense (n=1) or missense (n=3) mutations. Altogether, three changes of the C7 gene (G357R, R499S and 5' splice donor site of intron 16) account for half of the molecular defects which emphasize that a restricted number of molecular abnormalities are involved in this deficiency. We identified two patients with combined C7Q0/C7SD(R499S) and established the C7SD(R499S) frequency at about 1% in normal Caucasian population. We demonstrated that C7(R499S) mutant protein is retained in the endoplasmic reticulum whereas the wild-type C7 is located in the Golgi apparatus. Our results provide evidence that R499S represents a loss-of-function polymorphism of C7 due to a defective folding of the protein.

Early onset insulin-dependent diabetes mellitus as an initial manifestation of aceruloplasminaemia.

BACKGROUND: Aceruloplasminaemia is an autosomal recessive disorder caused by specific mutations in the ceruloplasmin gene. Aceruloplasminaemia is clinically characterized by diabetes mellitus, pigment degeneration of the retina, and neurological abnormalities, such as cerebellar ataxia, extrapyramidal signs, and dementia. We present a patient with aceruloplasminaemia who, until progressive neurological abnormalities were noticed, had been treated for more than 30 years as having Type 1 diabetes mellitus requiring multiple insulin injection therapy. CASE REPORT: The patient was a 58-year-old man. At the age of 23 years, he developed diabetes that required multiple insulin injection therapy. At the age of 39 years, he was commenced on continuous subcutaneous insulin infusion (CSII) therapy. Despite CSII therapy, the patient's blood glucose levels were poorly controlled (HbA(1c), approximately 9.5%). He was diagnosed as having aceruloplasminaemia at 58 years of age when he presented with progressive cerebellar ataxia, extrapyramidal signs of recent onset and pigment degeneration of the retina. CONCLUSIONS: It is possible that some diabetic patients with aceruloplasminaemia are mistakenly diagnosed as having Type 1 diabetes mellitus, as they have reduced insulin secretion and develop diabetes at a younger age, before neurological abnormalities associated with aceruloplasminaemia are apparent. Therefore, aceruloplasminaemia should be considered in patients with insulin-dependent diabetes mellitus who develop progressive neurological abnormalities of unknown aetiology along with a microcytic hypochromic anaemia and retinal degeneration.