Analysis of the mechanism of action of non-deletion hereditary persistence of fetal hemoglobin mutants in transgenic mice

Transgenic mice carrying an (A)gamma gene construct containing a -382 5' truncation of the (A)gamma gene promoter have a phenotype of hereditary persistence of fetal hemoglobin (HPFH) but, when the CACCC box of the -382(A)gamma promoter is deleted, there is no gamma gene expression in the adult mice. We used this system to investigate the mechanism whereby human HPFH mutations result in gamma gene expression in the adult. Introduction of the -198 T-->C HPFH mutation into the CACCC-less (A)gamma gene construct re-established the HPFH phenotype, indicating that this mutation increases promoter strength, most probably by establishing a novel CACCC box sequence in the -198(A)gamma region. The HPFH phenotype was also re-established when the -117 C-->T HPFH mutation was introduced into a -141(A)gamma promoter with a destroyed CACCC box, indicating that this mutation increases gamma promoter strength in the absence of the CACCC motif. The T-->A -175 HPFH mutation failed to re-establish the HPFH phenotype when the CACCC box was deleted, indicating that gamma gene expression in this mutation is CACCC box dependent. These results provide the first in vivo experimental evidence in support of mechanistic heterogeneity of the non-deletion HPFH mutants.     

ApoC-IIParis2: a premature termination mutation in the signal peptide of apoC-II resulting in the familial chylomicronemia syndrome.

The chemical mismatch method has been utilized to screen for mutations in the apoC-II gene of a patient with familial chylomicronemia and apoC-II deficiency. Cleavage of heteroduplexes formed between normal and patient DNA strands with hydroxylamine and osmium tetroxide readily localized a mutation near base 2660 of the mutant apoC-II. Sequence analysis of PCR amplified patient DNA in the mismatched region localized by this method identified the substitution of a thymidine (T) for a cytosine (C) at base 2668 in exon 2 of the patient's gene within a CpG dinucleotide. The C to T transition in the apoC-IIParis2 gene leads to the introduction of a premature termination codon (TGA) at a position corresponding to amino acid-19 of the signal peptide of apoC-II and the formation of a new Nla III restriction enzyme site absent in the normal apoC-II gene. Consistent with the history of consanguinity in this kindred, amplification of DNA isolated from the proband's parents by the polymerase chain reaction and digestion with Nla III established that the proband is a true homozygote for this genetic defect. Analysis of the patient's plasma by two-dimensional gel electrophoresis and immunoblotting failed to detect any plasma apoC-II. Thus, we have identified a novel mutation in the apoC-II gene of a patient with apoC-II deficiency from a Paris kindred presenting with severe hypertriglyceridemia and chylomicronemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

一个中国Gilbert综合征家系的遗传学分析

对一个中国汉族Gilbert综合征遗传家系致病基因突变位点进行鉴定,以期了解该病的分子遗传学基础。首先提取先证者基因组DNA,PCR扩增尿苷二磷酸葡萄糖醛酸转移酶UGT1A1基因的5个外显子,以琼脂糖电泳鉴定PCR产物,纯化后直接测序鉴定。基因扫描显示,与血清胆红素水平密切相关的UGT1A1基因在第1和第5外显子存在纯合突变,而 UGT1A1基因启动子区域和内含子/外显子剪接边界位点序列未检测到突变。进一步对其他家系成员该基因的相应位点进行突变检测,结果显示他们在第1和第5外显子也存在杂合突变,其中还有两个成员在启动子区域检测到(TA)插入突变。对家系成员未抗凝新鲜血液进行生化检测证实了基因突变分析的结果。综合以上结果发现该家系三种突变并存,致病因素为第1和/或第5外显子突变,为显性遗传,两种突变位点纯合导致先证者出现严重胆红素代谢功能障碍。该家系因此成为Gilbert综合征突变位点及其致病机理研究的一个典型临床病例。     

A deletion mutation in the ApoC-II gene (ApoC-II Nijmegen) of a patient with a deficiency of apolipoprotein C-II

The apolipoprotein C-II gene from a patient with a deficiency of apoC-II was cloned and sequenced. A single base deletion of a guanosine at position 2943 in exon three of the gene of the proband was identified by sequence analysis. This point mutation results in a shift of the reading frame and introduces a premature termination codon (TGA) at a position in the gene immediately following amino acid 17 of the mature C-II apolipoprotein. This single base deletion results in the loss of a normally occurring HphI restriction enzyme site in the apoC-II gene. Amplification of the mutant DNA sequence by the polymerase chain reaction and restriction enzyme digestion with HphI established that the patient is a homozygote for the base deletion. No apoC-II was detectable in the patient's plasma by two-dimensional gel electrophoresis and immunoblotting. We propose that the guanosine deletion is the primary genetic defect in this kindred leading to premature termination and formation of a nonfunctional truncated 17-amino acid C-II apolipoprotein which ultimately results in apoC-II deficiency.     

Transcription efficiency of human apolipoprotein A-I promoter varies with naturally occurring A to G transition.

In human, the gene coding for apolipoprotein A-I (apo A-I), a protein of the plasma lipid transport system, is expressed only in the liver and the intestine. A naturally occurring A to G substitution in the promoter at position -78 was shown to be associated with high density lipoproteins (HDL) in females. We have studied the effect of this mutation on promoter activity using various lengths of promoter sequences and the CAT reporter gene system. Transient expression studies after introduction of these constructs into Hep 3B cells revealed that in the region spanning -330 to +1 of the promoter an A to G substitution increases the activity approximately twofold. On the other hand, when further upstream region (-1469 to +397) is also included, the promoter activity seems comparable in both alleles. Our results show how minimal sequence variations can affect the in vitro analysis of promoter activity.     

Molecular analysis of three novel G6PD variants: G6PD Pedoplis-Ckaro, G6PD Piotrkow and G6PD Krakow

We present three novel mutations in the G6PD gene and discuss the changes they cause in the 3-dimensional structure of the enzyme: 573C-->G substitution that predicts Phe to Leu at position 191 in the C-terminus of helix alphae, 851T-->C mutation which results in the substitution 284Val--> -->Ala in the beta+alpha domain close to the C-terminal part of helix alphaj, and 1175T-->C substitution that predicts Ile to Thr change at position 392.     

An initiation codon mutation in the apoC-II gene (apoC-II Paris) of a patient with a deficiency of apolipoprotein C-II

We have identified the genetic defect that leads to a deficiency of apoC-II in the proband from the Paris kindred. Analysis of the apoC-IIParis DNA by Southern blot hybridization revealed no major gene rearrangements, but sequencing of polymerase chain reaction-amplified apoC-IIParis DNA revealed an A to G transition that changed the initiation AUG (methionine) codon to GUG (valine). Potential initiation of translation at the closest inframe methionine codon eliminates the entire signal peptide and the first 8 amino-terminal residues of apoC-II which would prevent apoC-II secretion into plasma. In agreement with this, no apoC-II was detected in the patient's plasma by radioimmunoassay or by two-dimensional gel electrophoresis and immunoblotting. Direct sequencing of amplified patient DNA from 12 different polymerase chain reaction samples demonstrated the presence of the A to G substitution in all, indicating that the proband is a homozygote for the defect. We propose that in the apoC-IIParis gene, a mutation in the initiation methionine codon prevents the normal initiation of apolipoprotein synthesis and leads to a deficiency of apoC-II. This initiation methionine mutation represents a new type of molecular defect that can result in Type I hyperlipoproteinemia.