Homozygosity for a mutation in the lipoprotein lipase gene (Gly139→Ser) causes chylomicronaemia in a boy of Spanish descent

The enzyme lipoprotein lipase (LPL) plays a crucial role in triglyceride metabolism through catalysis of triglyceride-rich chylomicrons and very low density lipoproteins. Primary LPL deficiency manifests with chylomicronaemia and is caused by mutations in the LPL gene. In this paper we report a novel molecular defect (G₆₇₀A) in exon 4 of the LPL gene, resulting in a substitution of serine for glycine at position 139 in the mature protein. We identified homozygosity for this mutation in a boy of Spanish descent. In vitro mutagenesis provided formal proof that this missense mutation completely abolishes LPL function and therefore is the cause of LPL deficiency.     

Molecular cloning of a pair of human pepsinogen A genes which differ by a Glu→Lys mutation in the activation peptide

Summary Three human cosmid clones containing pepsinogen A (PGA) encoding sequences were isolated from a genomic bank derived from a single individual. One cosmid contains two PGA genes in tandem in a head-to-tail orientation, while the other two cosmids each contain a single PGA gene. The three cosmids were characterized by restriction mapping and sequence analysis (exons 1 and 2 and flanking regions). As judged from these data, three of the four PGA genes isolated appear to be nearly identical, but one of the tandem genes is clearly different from the other genes. The first exon of all four genes codes for the same amino acid sequence. However, in the second exon of one of the tandem genes we found a nucleotide substitution giving rise to a GluLys substitution of the 43rd amino acid residue of the activation peptide, leading to a charge difference of the corresponding isozymogens. The presence of two distinct PGA genes in the isolated gene pair conclusively proves the multigene structure of the PGA system. These genes might be responsible for at least part of the electrophoretic polymorphism at the protein level.     

Normalization of Epigenetic Change in the Genome by Peptide Bioregulator (Ala–Glu–Asp–Gly) in Pulmonary Tuberculosis

International Journal of Peptide Research and Therapeutics - The aim of this study was to evaluate genetic and epigenetic variation of the genome in patients with sensitive pulmonary tuberculosis...     

Heterozygosity for the IVS-I-5(G→C) mutation with a G→A change at codon 18 (Val→Met; Hb Baden) in cis and a T→G mutation at codon 126 (Val→Gly; Hb Djonburi) in trans resulting in a thalassemia intermedia

We have analyzed the hemoglobins of a young German patient with β-thalassemia intermedia and of his immediate family and included in these studies an evaluation of possible nucleotide changes in the β-globin through sequencing of amplified DNA. One chromosome of the propositus and one of his father's carried the $\text{G}\text{T}\text{G}\text{→}\text{G}\text{G}\text{G}$ mutation at codon 126 leading to the synthesis of Hb Dhoburi or α₂β₂126(H₄)Val→Gly; this variant is slightly unstable and is associated with mild thalassemic features. His second chromosome and one of his mother's had the common IVS-I-5 (G→C) mutation that leads to a rather severe β⁺-thalassemia and the $\text{G}\text{TG}\text{→}\text{A}\text{TG}$ mutation at codon 18, resulting in the replacement of a valine residue by a methionine residue. This newly discovered β-chain variant, named Hb Baden, was present for only 2–3% in both the patient and his mother. This low amount results from a decreased splicing of RNA at the donor splice-site of the first intron that is nearly completely deactivated by the IVS-I-5 (G→C) thalassemic mutation. The chromosome with the codon 18 ($\text{G}\text{TG}\text{→}\text{A}\text{TG}$) and the IVS-I-5 (G→C) mutations has thus far been found only in this German family; analysis of 51 chromosomes from patients with the IVS-I-5 (G→C) mutation living in different countries failed to detect the codon 18 ($\text{G}\text{TG}\text{→}\text{A}\text{TG}$) change.     

Acute intermittent porphyria caused by a C→T mutation that produces a stop codon in the porphobilinogen deaminase gene

Summary A mutation of the porphobilinogen (PBG) deaminase gene that produces the cross-reacting immunological material (CRIM)-negative type of acute intermittent porphyria (AIP) has been identified in one of 43 unrelated patients with this form of the disorder. The mutation is a CT transition that abolishes a PstI recognition site in exon 9 of the gene and converts a codon for glutamine to a stop codon.     

Identification of a single base-pair mutation of TAA (Stop codon) → GAA (Glu) that causes light chain extension in a CHO cell derived IgG1

We describe here the identification of a stop codon TAA (Stop) → GAA (Glu) = Stop221E mutation on the light chain of a recombinant IgG1 antibody expressed in a Chinese hamster ovary (CHO) cell line. The extended light chain variants, which were caused by translation beyond the mutated stop codon to the next alternative in-frame stop codon, were observed by mass spectra analysis. The abnormal peptide peaks present in tryptic and chymotryptic LC–MS peptide mapping were confirmed by N-terminal sequencing as C-terminal light chain extension peptides. Furthermore, LC-MS/MS of Glu-C peptide mapping confirmed the stop221E mutation, which is consistent with a single base-pair mutation in TAA (stop codon) to GAA (Glu). The light chain variants were approximately 13.6% of wild type light chain as estimated by RP-HPLC analysis. DNA sequencing techniques determined a single base pair stop codon mutation, instead of a stop codon read-through, as the cause of this light chain extension. To our knowledge, the stop codon mutation has not been reported for IgGs expressed in CHO cells. These results demonstrate orthogonal techniques should be implemented to characterize recombinant proteins and select appropriate cell lines for production of therapeutic proteins because modifications could occur at unexpected locations.     

Effect of mutation Arg91Gly on the thermal stability of β-tropomyosin

The mutation Arg91Gly (R91G) in β-tropomyosin (β-TM) is known to cause distal arthrogryposis, a severe congenital disorder of muscle tissues. The influence of this mutation in β-TM on its structure and thermal denaturation was demonstrated. It was shown by the differential scanning calorimetry and circular dichroism that this point mutation dramatically decreased the thermal stability of the significant part of the β-TM (about a half of the molecule). This part of the β-TM molecule carrying R91G mutation unfolds at ～28°C, i.e., at a much lower temperature than the other part of the molecule, which melts at ～40°C. The data of the differential scanning calorimetry were compared with the results of temperature dependence of pyrene eximer fluorescence, which decreased upon the dissociation of two β-TM chains in the region of pyrene-labeled Cys-36. This comparison allowed one to conclude that this thermal transition reflected the thermal unfolding of the whole N-terminal part of β-TM. Interestingly, the destabilizing effect of Arg91Gly mutation spread for a rather long distance along the tropomyosin coiled-coil indicating a high cooperativity of the thermal denaturation within this part of β-TM.     

Second-site mutation at M43 (Asn→Asp) compensates for the loss of two acidic residues in the QB site of the reaction center

Two acidic residues, L212Glu and L213Asp, in the Q_B binding sites of the photosynthetic reaction centers of Rhodobacter capsulatus and Rhodobacter sphaeroides are thought to play central roles in the transfer of protons to the quinone anion(s) generated by photoinduced electron transfer. We constructed the site-specific double mutant L212Ala-L213Ala in R. capsulatus, that is incapable of growth under photosynthetic conditions. A photocompetent derivative of that strain has been isolated that carries the original L212Ala-L213Ala double mutation and a second-site suppressor mutation at residue M43 (AsnAsp), outside of the Q_B binding site, that is solely responsible for restoring the photosynthetic phenotype. The Asp,Asn combination of residues at the L213 and M43 positions is conserved in the five species of photosynthetic bacteria whose reaction center sequences are known. In R. capsulatus and R. sphaeroides, the pair is L213Asp-M43Asn. But, the reaction centers of Rhodopseudomonas viridis, Rhodospirillum rubrum and Chloroflexus aurantiacus reverse the combination to L213Asn-M43Asp. In this respect, the Q_B site of the suppressor strain resembles that of the latter three species in that it couples an uncharged residue at L213 with an acidic residue at M43. These reaction centers, in which L213 is an amide, must employ an alternative proton transfer pathway. The observation that the M43AsnAsp mutation in R. capsulatus compensates for the loss of both acidic residues at L212 and L213 suggests that M43Asp is involved in a new proton transfer route in this species that resembles the one normally used in reaction centers of Rps. virddis, Rsp. rubrum and C. aurantiacus.     

A missense mutation (G197→A) in theα-l-fucosidase gene of fucosidosis patients leads to loss ofα-l-fucosidase

Fucosidosis is an autosomal recessive lysosomal storage disease resulting from the absence of -l-fucosidase activity. Two natural missense mutations (G¹⁹⁷A) and (A⁸⁶⁰G) within the -l-fucosidase gene have been reported to be homozygous in four patients with fucosidosis. Expression of wild-type and mutated -l-fucosidase cDNAs in COS-1 cells revealed complete deficiency of -l-fucosidase for the G¹⁹⁷A transition and a normal level of enzyme for A⁸⁶⁰G. We therefore conclude that the change of G¹⁹⁷A is responsible for fucosidosis in the patients while A⁸⁶⁰G is a normal polymorphic variant of -l-fucosidase.     

Prevalence and familial patterns of night eating in the Québec adipose and lifestyle investigation in youth (QUALITY) study

The prevalence and familial patterns of night eating syndrome (NES) in families enrolled in the Québec Adipose and Lifestyle InvesTigation in Youth (QUALITY) study was examined. Families (n = 395; one child, mother, and father for whom at least one parent was obese or had abdominal obesity) completed the Night Eating Questionnaire (NEQ) as part of a longitudinal study on the development of metabolic disease in children at risk for obesity. Responses on the NEQ were used to establish a diagnosis of NES and to determine the correlation and heritability of NES symptoms in families. Using comprehensive research diagnostic criteria, full threshold NES was rare: 0% of children, 0.5% of mothers, and 0.3% of fathers met criteria. When controlling for age, sex, and BMI, NEQ scores of spouses were not significantly correlated, but mothers' NEQ scores were significantly correlated with the scores of both sons (r = 0.19, P < 0.001) and daughters (r = 0.15, P = 0.05). The heritability of NEQ scores was 0.24 when controlling for age, sex, and BMI. These findings replicate previous research suggesting a low prevalence of night eating behavior in children and the aggregation of NES in families.     

Prevalence of Comorbidities and Baseline Characteristics of LAP-BAND AP? Subjects in the Helping Evaluate Reduction in Obesity (HERO) Study

Objective

To describe the baseline characteristics in patients who chose placement of a LAP-BAND AP® System (LBAP) and participated in the Helping Evaluate Reduction in Obesity (HERO) Study across regions.

Patients and Methods

HERO is a five- year, prospective, multicenter, international study of patients with LBAP placement between July 22, 2009 and January 31, 2011. In addition to baseline and peri-surgery clinical data, seven follow up visits are scheduled at 3, 6 and 12 months, and annually through year five. Data collection included family and medical history, clinical outcomes, laboratory data, health-related quality of life (HRQoL), productivity, healthcare resource utilization, and adverse events.

Results

LBAP were placed in 1106 enrolled patients; 56.6% from the US, 26.3% from Europe, 7.1% from Canada, and 10.0% from Australia. The majority were female (n = 877 (79.3%)) with a mean age of 43 years (s.d. = 11.4) and mean body mass index of 45.1 kg/m² (s.d. = 6.9). The most common comorbidities were hypertension (HTN) (overall  = 42.9%) and diabetes (overall 22.2%, with 27% from the US and 14% from Europe). Overall, less than 5% had a history of cardiovascular disease. The prevalence rates of HTN, diabetes and cardiovascular disease were significantly (p<0.001) higher in men than in women across all regions. Overall HRQoL also worsened with increasing BMI.

Conclusions

The HERO study is the first large, multinational and long-term registry with the LBAP. This study will provide real-world outcomes data on LAGB that will help inform patient choice, clinician treatment strategies, and payer reimbursement decisions.     

A new missense mutation (Cys297→Phe) of the low density lipoprotein receptor in Italian patients with familial hypercholesterolemia (FHTrieste)

During a survey of the mutations of the low density lipoprotein receptor (LDL-R) gene in Italian patients with familial hypercholesterolemia (FH), we identified a novel point mutation, that creates a new EcoRI site at the 5 end of exon 7, in a heterozygous FH subject (FH-100). The sequence of a cDNA fragment encompassing exon 7 showed the presence of a GT transversion in codon 297; this created a new EcoRI site and produced a missense mutation, leading to a Cys₂₉₇Phe substitution in repeat A of the epidermal growth factor (EGF) precursor homology domain of LDL-R. Since the substitution of Cys₂₉₇ disrupts the intracellular transport of the LDL-R protein, as previously demonstrated by site-directed mutagenesis, we suggest that this mutation is the cause of FH in the FH-100 proband. We screened the DNA of 303 Italian FH patients by amplification of exon 7 from genomic DNA followed by digestion with EcoRI or by Southern blotting. Two individuals (FH-64 and FH-127) were found to be carriers of the Cys₂₉₇Phe mutation. Restriction fragment length polymorphism analysis demonstrated that, in two kindreds (FH-64 and FH-100), the haplotype in linkage with the Cys₂₉₇Phe mutation was the same, suggesting the presence of a common ancestor. The Cys₂₉₇Phe mutation has been designated FH_Trieste after the name of the city in Northern Italy from which probands FH-100 and FH-127 originate.     

Polymerization of deoxyhemoglobin CHarlem (β6 Glu → Val, β73 Asp → Asn): The effect of β73 asparagine on the gelation and crystallization of hemoglobin

The kinetics of aggregation and the solubility of deoxy Hb2 C_Harlem (α₂β₂ 6 Val, 73 Asn) in concentrated phosphate buffers were studied in comparison with those of deoxy Hb S and deoxy Hb A. Deoxy Hb C_Harlem aggregated with a clear exhibition of a delay time. The length of the delay and aggregation times and the degree of the aggregation depended upon the initial hemoglobin concentration.The initial hemoglobin concentration required for the aggregation of deoxy Hb C_Harlem was approximately 200% of its solubility, a value much higher than that required for the aggregation of deoxy Hb S (120%). With the same hemoglobin concentration, the delay time for the aggregation of deoxy Hb C_Harlem was approximately 100 times longer than that of deoxy Hb S. The logarithmic plotting of the delay time versus hemoglobin concentration in 1.8 m-phosphate buffer (pH 7.4) showed linear lines with a slope (n) of 4.0 for deoxy Hb C_Harlem. In contrast to the results for the aggregation of deoxy Hb S, n values for deoxy Hb C_Harlem were unchanged with phosphate concentrations varying from 1.2 m to 2.0 m. The solubilities of deoxy Hb S and deoxy Hb C_Harlem were increased exponentially by lowering the pH of the medium, with the increase being more conspicuous for Hb C_Harlem. The gels (or aggregates) of Hb C_Harlem were converted to crystals at a rate much faster than were those of Hb A and Hb S. The kinetics for gelation and crystallization of deoxy Hb C_Harlem can be explained by the following scheme, where nuclei G and nuclei C are formed before gelation and crystallization, respectively. Monomenc deoxy Hb

The hemoglobin concentration required for the crystallization of deoxy Hb C_Harlem was about ten times lower than that required for deoxy Hb A. The solubility of deoxy Hb C_Harlem after aggregation was about twice that of deoxy Hb S, suggesting that the substitution of Asn for Asp at the β73 residue inhibits the formation of nuclei G and accelerates the formation of nuclei C.     

A novel missense mutation (Thr176→IIe) at the putative hinge of the neo N-terminus of activated protein C

We describe the detection of a novel missense mutation (Thr176Ile) that is located at the neo N-terminus of activated protein C. The Thr176Ile substitution leads to a type 1 deficiency state. Evidence is presented suggesting that this residue plays a role in pivoting the N-terminus of protein C to fold into the oxyanion hole.     

A rare polyadenylation signal mutation of the FOXP3 gene (AAUAAA→AAUGAA) leads to the IPEX syndrome

The mouse scurfy gene, Foxp3, and its human orthologue, FOXP3, which maps to Xp11.23-Xq13.3, were recently identified by positional cloning. Point mutations and microdeletions of the FOXP3 gene were found in the affected members of eight of nine families with IPEX (immune dysfunction, polyendocrinopathy, enteropathy, X-linked; OMIM 304930). We evaluated a pedigree with clinically typical IPEX in which mutations of the coding exons of FOXP3 were not detected. Our reevaluation of this pedigree identified an A-->G transition within the first polyadenylation signal (AAUAAA-->AAUGAA) after the stop codon. The next polyadenylation signal is not encountered for a further 5.1 kb. This transition was not detected in over 212 normal individuals (approximately 318 X chromosomes), excluding the possibility of a rare polymorphism. We suggest that this mutation is causal of IPEX in this family by a mechanism of nonspecific degradation of the FOXP3 gene message.     

Association between nasal allergy and a coding variant of the Fc ε RI β gene Glu237Gly in a Japanese population

The gene for the beta-chain of the high-affinity receptor for IgE (Fc epsilon RI beta) has been proposed as a candidate gene for atopy. A coding variant Glu237Gly has been studied in various populations with asthma and atopy, and the results were controversial for association of the variant with atopy/asthma. Because nasal allergy is a more common atopic disease and shows less remission than asthma, we analyzed whether the Glu237Gly variant is correlated with nasal allergy. The study enrolled 233 patients with nasal allergy and 100 control subjects. Further, three subgroups were selected: patients with perennial nasal allergy (n=149), Japanese cedar pollinosis (n=189), and allergy to multiple allergens (n=45). The allele frequency of Gly237 in the controls and patients was 0.14 and 0.20, and the frequency of Gly237-positive subjects was 0.23 and 0.356, respectively. There was a significant association between Gly237-positivity and nasal allergy, perennial nasal allergy, Japanese cedar pollinosis, and allergy to multiple allergens. Among all 333 subjects we observed a significant relationship between Gly237 and elevated levels of serum total IgE (>250 IU/ml) and very high IgE (>1000 IU/ml). Among patients positive for a specific IgE, Gly237 was significantly associated with high IgE for house dust, mite, and Japanese cedar pollen. These results suggest that the Glu237Gly variant of the Fc epsilon RI beta gene is involved in the development of nasal allergy through the process for the production of both specific and nonspecific IgE antibodies.     

Immunolocalization of β-(1→4) and β-(1→6)-D-galactan epitopes in the cell wall and Golgi stacks of developing flax root tissues

Summary Most cell wall components are carbohydrate including the major matrix polysaccharides, pectins and hemicelluloses, and the arabinogalactan-protein proteoglycans. Both types of molecules are assembled in the Golgi apparatus and transported in secretory vesicles to the cell surface. We have employed antibodies specific to -(16) and -(14)-D-galactans, present in plant cell wall polysaccharides, in conjunction with immunofluorescence and electron microscopy to determine the location of the galactan-containing components in the cell wall and Golgi stacks of flax root tip tissues. Immunofluorescence data show that -(14)-D-galactan epitopes are restricted to peripheral cells of the root cap. These epitopes are not expressed in meristematic and columella cells. In contrast, -(16)-D-galactan epitopes are found in all cell types of flax roots. Immunogold labeling experiments show that both epitopes are specifically located within the wall immediately adjacent to the plasma membrane. They are also detected in Golgi cisternae and secretory vesicles, which indicates the involvement of the Golgi apparatus in their synthesis and transport. These findings demonstrate that the synthesis and localization of -(14)-D-galactan epitopes are highly regulated in developing flax roots and that different -linked D-galactans associated with cell wall polysaccharides are expressed in a cell type-specific manner.     

Increased activities of antioxidant enzymes and decreased ATP concentration in cultured myoblasts with the 3243A→G mutation in mitochondrial DNA

The MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is most commonly caused by the 3243A→G mutation in mitochondrial DNA, resulting in impaired mitochondrial protein synthesis and decreased activities of the respiratory chain complexes. These defects may cause a reduced capacity for ATP synthesis and an increased rate of production of reactive oxygen species. Myoblasts cultured from controls and patients carrying the 3243A→G mutation were used to measure ATP, ADP, catalase and superoxide dismutase, which was also measured from blood samples. ATP and ADP concentrations were decreased in myoblasts with the 3243A→G mutation, but the ATP/ADP ratio remained constant, suggesting a decrease in the adenylate pool. The superoxide dismutase and catalase activities were higher than in control cells, and superoxide dismutase activity was slightly, but not significantly higher in the blood of patients with the mutation than in controls. We conclude that impairment of mitochondrial ATP production in myoblasts carrying the 3243A→G mutation results in adenylate catabolism, causing a decrease in the total adenylate pool. The increase in superoxide dismutase and catalase activities could be an adaptive response to increased production of reactive oxygen species due to dysfunction of the mitochondrial respiratory chain.     

Association of an Exon 3 Mutation (Trp66 → Gly) of the LDL Receptor with Variable Expression of Familial Hypercholesterolemia in a French Canadian Family

The ligand-binding domain of low-density lipoprotein (LDL) is composed of seven 40-amino-acid repeats encoded by exons 2–6. Previous studies identified a missense mutation in codon 66 of exon 3, which resulted in the production of LDL receptor protein that is not processed to its mature form. In the current investigation, we documented the presence of two identical mutant LDL receptor alleles (Trp₆₆→ Gly) in two familial hypercholesterolemia (FH) probands, II-1 and II-2, associated with markedly elevated plasma LDL cholesterol (17.22 ± 0.78 and 11.95 ± 0.24 mmol/liter, respectively). Functional assays of their fibroblast LDL receptor showed inefficient binding (39 and 50%), internalization (33 and 37%), and degradation (32 and 37%) compared with controls. The contribution of the apo B gene to variation in LDL levels was virtually eliminated given the normal ligand interaction with cell surface receptors and the absence of the mutation occurring in codon 3500 of the apo B gene. Similarly, the homozygous apo E₃/E₃wildtype phenotype excluded any genetic contribution of apo E to the lipoprotein abnormalities. Furthermore, the LPL mutations commonly observed in French Canadians could not account for the observed lipid alterations. Several alterations in lipoprotein composition characterized VLDL, IDL, LDL, HDL₂, and HDL₃fractions. Moreover, defective intestinal fat transport was observed in both probands (II-1 and II-2). Thus, the disturbance of lipoprotein concentration, composition, size, and metabolism may in part be related to the exon 3 mutation (Trp₆₆→ Gly) of the LDL receptor gene. The biochemical phenotype was more severe in the father (I-1) than in the mother (I-2), and in the younger homozygous proband (II-1) than in the older (II-2). The greater severity was associated with a higher LDL cholesterol/HDL cholesterol ratio. Whether the differences between the two probands are due to polygenic factors or to a metabolic consequence of a major nonallelic trait is unknown. Nevertheless, the present biochemical findings stress the extent of the lipid abnormalities associated with homozygous FH and the importance of the phenotypic variability encountered even among subjects carrying the same mutation.     

Evidence for association and linkage between atopy, airway hyper-responsiveness, and the β subunit Glu237Gly variant of the high-affinity receptor for immunoglobulin E in the French-Canadian population

Following detection of linkage between atopy and chromosome 11q13 markers, association between this disorder and variants of the beta subunit of the high-affinity receptor for immunoglobulin E (FcepsilonRI-beta, a candidate gene for asthma-related conditions co-localizing within the same region) was reported in Australian, British and Japanese populations. Investigations in several other ethnic groups failed to replicate these observations. Due to the complexity of defining intermediate phenotypes related to asthma, detection of such associations may have been hampered by clinical misclassifications. To assess whether the FcepsilonRI-beta gene was involved in atopy and/or airway hyperresponsiveness (AHR) in the French-Canadian population, we conducted a case-control study in 200 subjects using strict criteria for asthma and related conditions. The Ile181Leu and Glu237Gly FcepsilonRI-beta sequence variants were tested exploiting two amplification refractory mutation systems. No association was detected between atopy or AHR and the Ile181Leu FcepsilonRI-beta variant. However, a strong association was observed between atopy and the Glu237Gly FcepsilonRI-beta variant (odds ratio=12.25). Four large Eastern Québec families (n=106 subjects) were also recruited to perform a genetic linkage study. We observed suggestive evidence of linkage between atopy and the Glu237Gly FcepsilonRI-beta variant (Zmax=2.30). This study is the first to detect the presence of an association between atopy and the Glu237Gly FcepsilonRI-beta variant in French-Canadians. Our data suggest that a susceptibility locus for atopy is located on chromosome 11q13 in this population.