A compound heterozygote for a novel missense mutation (G105R) in exon 3 and a missense mutation (D204E) in exon 5 of the lipoprotein lipase gene in a Japanese infant with hyperchylomicronaemia

We systematically investigated the molecular defects resulting in primary lipoprotein lipase (LPL) deficiency in a Japanese male infant (hereafter called 'the patient') with severe fasting hypertriglyceridaemia (type I hyperlipoproteinaemia). The primary LPL deficiency was diagnosed on the basis of the findings that no LPL activity was detected in post-heparin plasma (PHP) and that the immunoreactive LPL mass in PHP was less than 2% of the control level. The patient was a compound heterozygote for a novel missense mutation (G(568)GA-->AGA/Gly(105)-->Arg; G105R) in exon 3 and a missense mutation (GAC(867)-->GAG/Asp(204)-->Glu; D204E) in exon 5 of the LPL gene. The biological significance of both missense mutations was examined by an in vitro study of the expression of the mutant G105R LPL cDNA and D204E LPL cDNA in COS-1 cells. Both mutant LPLs were catalytically inactive and were barely released by heparin from the expressing COS-1 cells. These findings explain the failure to detect LPL activity and immunoreactive LPL mass in the patient's PHP. The G105R allele could be detected by digestion with the BsmAI restriction enzyme, and the D204E allele by digestion with HincII. The patient inherited the G105R allele from his mother and the D204E allele from his father. His parents were heterozygotes for the corresponding mutant allele, but normolipidaemic. The novel G105R missense mutation could not be detected by conventional analysis of single-strand conformation polymorphism, but it was identified by extensive sequencing of the entire exons and their flanking regions in the LPL gene.     

A novel mutation of the ceruloplasmin gene in a patient with heteroallelic ceruloplasmin gene mutation (HypoCPGM)

We found a novel missense mutation in the ceruloplasmin (Cp) gene in a patient with the heteroallelic Cp gene mutation (HypoCPGM). The patient was a 72-year-old woman who came to our hospital with a 1-year history of postural tremor of the hands. The diagnosis was made based on serum Cp and copper readings which were about half the normal levels, as well as MRI tests of her brain which showed characteristics for hereditary ceruloplasmin deficiency (HCD), known to be caused by the homoallelic Cp gene mutation. Polymerase chain reaction (PCR)-direct sequencing analysis of the Cp gene of the patient revealed a novel point mutation, A to T, at nucleotide position 82 in Exon 1. This mutation changes the Ile28 codon (ATT) to a Phe codon (TTT) (missense mutation). PCR-restriction analysis with restriction enzyme Tsp EI for the mutation revealed that both the patient and her son were heterozygotes for the mutation.     

C3 glomerulonephritis associated with a missense mutation in the factor H gene

The complement system, the major component of the innate immune functions resisting microbial infection, includes the classical complement pathway, the alternate pathway, and the mannose-binding lectin pathway. All of these merge at the level of complement component (C) 3. Complement factor H (CFH), a soluble complement mediator in blood, regulates alternate pathway activation; a conformational change of C3 molecules by C3 convertases leads to an enzyme complex formation resulting in opsonization and cell lysis. Clinical manifestations arising from CFH gene (CFH) abnormalities include hemolytic uremic syndrome and membranoproliferative glomerulonephritis. We encountered a 24-year-old woman initially diagnosed with C3 glomerulonephritis associated with persistently low circulating C3. Definitive diagnosis of C3 glomerulonephritis was made from immunohistologic demonstration of isolated mesangial C3 deposits. The biopsy specimen showed moderately increased mesangial proliferation, without thickening of the glomerular capillary walls. Genetic analysis disclosed a homozygous CFH missense mutation, a G-to-T transversion at nucleotide 3,048 in exon 18, resulting in substitution of Asp for Glu at position 936. A low serum CFH concentration (110 μg/mL) might reflect the consequences of this CFH mutation. C3 glomerulonephritis is associated with a CFH mutation, the mutation of which results in the unexpected activation of alternate pathway complement with clinical laboratory fluctuations, such as varying reduction of serum CFH and C3. The finding of a patient with a CFH mutation associated with C3 glomerulonephritis represents an opportunity to expand the phenotypic spectrum of the CFH mutations.     

A novel missense mutation (Asn5→Ile) in lecithin: cholesterol acyltransferase (LCAT) gene in a Japanese patient with LCAT deficiency

We identified a novel missense mutation in the lecithin: cholesterol acyltransferase gene in a new case of lecithin:cholesterol acyltransferase (LCAT) deficiency. The patient was a 64-year-old diabetic Japanese male who showed an extremely low level of serum high-density lipoprotein-cholesterol, corneal opacities, anemia, and proteinuria. Both the patient's LCAT activity and mass were markedly low. DNA sequence analysis of the LCAT gene showed an A-to-T transition at base 97 in exon 1, and predicted a change in asparagine to isoleucine at the 5th amino acid of the protein. Restriction analysis of polymerase chain reaction-amplified DNA usingAse I showed that the patient was homozygous for this mutation. Our results suggested that asparagine 5 was an important amino acid and substitution with isoleucine caused marked reduction of LCAT activity and mass, resulting in LCAT deficiency.     

Familial chylomicronemia (type I hyperlipoproteinemia) due to a single missense mutation in the lipoprotein lipase gene.

Complete deficiency of lipoprotein lipase (LPL) causes the chylomicronemia syndrome. To understand the molecular basis of LPL deficiency, two siblings with drastically reduced postheparin plasma lipolytic activities were selected for analysis of their LPL gene. We used the polymerase chain reaction to examine the nine coding LPL exons in the two affected siblings and three relatives. DNA sequence analysis revealed a single nucleotide change compared with the normal LPL cDNA: a G----A substitution at nucleotide position 680. This transition caused a replacement of glutamic acid for glycine at amino acid residue 142 of the mature LPL protein. Amino acid sequence comparisons of the region surrounding glycine-142 indicated that it is highly conserved among lipases from different species, suggesting a crucial role of this domain for the LPL structure. Expression studies of the mutant LPL cDNA in COS-7 cells produced normal amounts of enzyme mass. However, the mutated LPL was not catalytically active, nor was it efficiently secreted from the cells. This established that the Gly----Glu substitution at amino acid 142 is sufficient to abolish enzymatic activity and to result in the chylomicronemia syndrome observed in these patients.     

Expression of a novel missense mutation found in the A4GALT gene of Amish individuals with the p phenotype

BACKGROUND: The rare p phenotype is found at a higher frequency in Amish people than in other populations. Different mutations in the 4-alpha-galactosyltransferase gene (A4GALT), responsible for synthesis of P(k) (Gb(3)) antigen, have been found to cause the P(k)-deficient p phenotype. The aim of this study was to explore the molecular background of the p phenotype in people of Amish origin. STUDY DESIGN AND METHODS: Twenty blood samples with the p phenotype, 19 of them from Amish individuals and 1 Pakistani, were investigated. Amplification of genomic DNA by polymerase chain reaction (PCR) and sequencing by capillary electrophoresis were performed. Blood donors of different geographic origin were screened with PCR-allele-specific primer to investigate whether the novel mutation occurs among individuals with common phenotypes. The mutation was also cloned into an expression vector and transfected to Namalwa cells, which do not normally express P(k). P(k) expression on the transfected cells and P/P(k) on red blood cells (RBCs), both with p and with common phenotypes, were analyzed by flow cytometry. RESULTS: All 20 samples were homozygous for 299C>T changing serine to leucine in a region that is highly conserved in homologous genes across species borders. The mutation was not found in any of the 500 alleles of blood donors investigated. P(k) expression was neither observed by serology and flow cytometry on p RBCs from Amish individuals nor following transfection of cells with constructs containing the novel missense mutation. CONCLUSION: A novel A4GALT missense mutation causes the p phenotype in Amish individuals.     

A missense mutation in the fabB (beta-ketoacyl-acyl carrier protein synthase I) gene confers tiolactomycin resistance to Escherichia coli

Thiolactomycin (TLM) is an antibiotic that inhibits bacterial type II fatty acid synthesis at the condensing enzyme step, and beta-ketoacyl-acyl carrier protein synthase I (FabB) is the relevant target in Escherichia coli. TLM resistance is associated with the upregulation of efflux pumps. Therefore, a tolC knockout mutant (strain ANS1) was constructed to eliminate the contribution of type I secretion systems to TLM resistance. Six independent TLM-resistant clones of strain ANS1 were isolated, and all possessed the same missense mutation in the fabB gene (T1168G) that directed the expression of a mutant protein, FabB(F390V). FabB(F390V) was resistant to TLM in vitro. Leucine is the only other amino acid found at position 390 in nature, and the Staphylococcus aureus FabF protein, which contains this substitution, was sensitive to TLM. Structural modeling predicted that the CG2 methyl group of the valine side chain interfered with the positioning of the C11 methyl on the isoprenoid side chain of TLM in the binary complex, whereas the absence of a bulky methyl group on the leucine side chain permitted TLM binding. These data illustrate that missense mutations that introduce valine at position 390 confer TLM resistance while maintaining the vital catalytic properties of FabB.     

Homozygous protein C deficiency: identification of a novel missense mutation that causes impaired secretion of the mutant protein C.

We analyzed the promoter region and all the coding exons and exon-intron boundaries of the protein C gene in a Japanese patient with recurrent thromboembolism and complete protein C deficiency. By sequencing these fragments we identified a previously undescribed mutation. A guanine residue was replaced by an adenine residue converting Gly-292 (GGC) to Ser (AGC) in the last exon coding for the catalytic domain. Substitution of this key amino acid, invariably conserved in the serine protease superfamily to which protein C belongs, probably leads to destabilization of the tertiary structure. In a transient expression assay with COS 7 cells, the protein C level was extremely low in the culture medium of the cells transfected with the mutated protein C expression vector, as compared with the normal vector. In contrast, the cell extracts contained similar amounts of mutant and normal protein C, suggesting impaired secretion of the mutant protein C. Using mutagenic primers to introduce a new PvuII site into the mutant allele, we made a study of the family members in this patient's pedigree, revealing that the mutant allele had been inherited in the affected individuals in this pedigree.     

Prolonged sporadic hemiplegic migraine associated with a novel de novo missense ATP1A2 gene mutation

Hemiplegic migraine is a rare form of migraine characterized by periodic attacks of migraine with neurologic aura and transient hemiplegia. There are familial and sporadic cases, both on a genetic basis; we describe the case of a 6-year-old boy affected by sporadic hemiplegic migraine, showing a novel ATP1A2 gene missense mutation (p.Gly715Arg) in exon 16. Long-term treatment with flunarizine resulted in good clinical response and prevention of further attacks.     

一例凝血因子Ⅷ B区错义突变导致重型血友病A

目的：检测一例重型血友病Ａ患者（ＳＨ９）的基因突变。方法：用ＰＣＲ、变性梯度凝胶电泳（ＤＧＧＥ）和ＤＮＡ测序检测因子Ⅷ基因突变。先用Ｓｏｕｔｈｅｒｎｂｌｏｔｉｎｇ排除内含子２２倒位，然后应用ＰＣＲ对凝血因子Ⅷ基因进行扩增。扩增范围包括所有外显子及其侧翼内含子序列。结果：片段１４２在ＤＧＧＥ中泳动异常。ＤＮＡ测序证实Ｃ２５３５Ａ导致Ｂ区错义突变８２６Ａｓｐ（ＧＡＣ）→Ｇｌｕ（ＧＡＡ）。结论：该突变可能是导致重型血友病Ａ的原因，但有待进一步研究证实。     

A novel missense LIPA gene mutation, N98S, in a patient with cholesteryl ester storage disease

Lysosomal acid lipase plays an important role in maintaining cellular cholesterol homeostasis. Complete absence of lysosomal acid lipase activity results in Wolman disease and usually death in infancy, whereas partial deficiency of lysosomal acid lipase results in cholesteryl ester storage disease (CESD). We describe a 26 year-old female with CESD who presented with recurrent right upper quadrant abdominal pain. Abnormal liver function tests and a subsequent liver biopsy revealed features consistent with CESD. Sequencing of the LIPA gene revealed that she was a compound heterozygote for the previously reported exon 8 splice junction mutation and a novel missense mutation (N98S) in exon 4. The splice junction mutation allows some (approximately 3%) normal splicing to occur, and therefore gives rise to residual lysosomal acid lipase activity. Asn98 in lysosomal acid lipase is highly conserved among species and mutation of this residue could influence catalytic activity or accessibility to the active site. In summary, we describe a CESD patient compound heterozygous for the LIPA exon 8 splice junction mutation and a novel missense mutation, N98S.     

A novel missense mutation responsible for factor VII deficiency in research Beagle colonies

BACKGROUND: Canine factor VII (cFVII) deficiency, an autosomal recessive trait originally identified in research Beagles, is associated with a mild to moderate bleeding tendency. OBJECTIVE: Our aim was to identify and characterize the mutation causing cFVII deficiency. METHODS: In order to sequence the coding regions of the cFVII gene, we cloned the cFVII cDNA. Genomic DNA and plasma from FVII-deficient Beagles and obligate carriers were utilized. RESULTS: In all FVII-deficient dogs, we identified a single causative G to A missense mutation in exon 5, encoding the second epidermal growth factor-like domain, resulting in substitution of glycine 96 by glutamic acid, with plasma FVII coagulant activity of 相似文献