Molecular analysis of hereditary hemolytic anemia due to red cell enzyme anomalies

对96个遗传性非球形红细胞溶血性贫血家系进行酶分析，发现葡萄糖-6-磷酸脱氢酶缺乏症12例，丙酮酸激酶缺乏症3例，磷酸果糖激酶缺乏症1例。我们发现了一个严重贫血的胎儿，这是日本首例HK缺乏症。母亲在妊娠29周时因宫内发育迟缓住院。超声检查显示胎儿脑室周围白质软化。血液学检查显示胎儿有严重的溶血性贫血：血红蛋白3.7g/dl ;网织红细胞42.1% ;间接胆红素5.8mg/dl。孕32周红细胞酶分析显示HK活性下降至约15%。双亲红细胞HK活性也降至正常值的50%左右。这家人没有血缘关系。受影响 ...更多信息 胎儿在妊娠37周时死于子宫内。为阐明先证者HK缺乏症的分子病变，采用长链PCR方法，从网织红细胞RNA中扩增出HK-1基因的全长cDNA。用相应的5 ′-和3 ′-非编码序列引物，从网织红细胞丰富的对照中扩增出2.8kb的HK-1 cDNA，而从先证者的网织红细胞中扩增出2.3kb的HK-1 cDNA。对这些短cDNA序列的测定表明，2.3kb的cDNA缺少外显子5至8。为了阐明导致这些外显子跳跃的基因异常，我们确定了人HK-1基因的基因组结构。HK-I基因全长约67 kb，有19个编码外显子。基因组序列分析表明，该胎儿为9 kb基因内缺失纯合子，我们发现了一个与遗传性溶血性贫血相关的红细胞烯醇化酶缺乏症家系。一名日本男孩在8岁时因细小病毒感染后出现急性再生障碍性危象而住院。短暂性红细胞再生障碍恢复后，慢性溶血性贫血变得明显：血红蛋白，9.1g/dl ;网织红细胞，7.7% ;间接胆红素，1.0mg/dl。红细胞酶测定显示先证者烯醇化酶活性降低：4.08IU/gHb（正常范围6.04-8.10）。烯醇化酶活性的父母也下降：父亲，4.93 ;母亲，2.83。为阐明烯醇化酶缺乏症的分子病变，以网织红细胞RNA为模板，通过RT-PCR扩增α烯醇化酶全长cDNA。我们阐明了先证者及其母亲有错义突变（183位Arg至His）。少

We discovered 12 cases of glucose-6-hosphate dehydrogenase deficiency, 3 cases of pyruvate kinase deficiency and 1 case of phosphofructokinase deficiency by the enzymatic analysis of 96 families associated with hereditary non-spherocytic hemolytic anemia.Hexokinase (HK) deficiency is the rare red cell enzymopathy associated with hereditary hemolytic anemia. We discovered a fetus with severe anemia as a first Japanese case of HK deficiency. Mother was hospitalized at 29 weeks of gestation due to intrauterine growth retardation. Ultrasonography showed periventricular leucomalacia of the fetus. Hematological examination showed that the fetus had severe hemolytic anemia : hemoglobin, 3.7g/dl ; reticulocyte, 42.1% ; indirect bilirubin, 5.8mg/dl. Red cel enzyme analysis at 32 weeks of gestation showed that HK activity was decreased to about 15%. Red cell HK activities of the parents were also decreased to about 50% of normal mean value. There was no consanguinity in the family. The affected … More fetus died in utero at 37 weeks of gestation. To elucidated molecular lesion of HK deficiency of the proband, reticulocyte RNA was used for amplify the full-length cDNA for type-I HK (HK-I) by long PCR method. By use of the primer corresponding 5'- and 3'-noncoding sequence, 2.8kb HK-I cDNA was amplified by using RNA from a reticulocyte-rich control ; whereas 2.3kb was obtained from the proband's reticulocyte. Sequence of these short cDNA showed that 2.3kb cDNA lacked exon 5 through 8. To clarify gene abnormalities responsible for these exon skipping, we determined genomic structure of human HK-l gene. The HK-I gene spans about 67kb and had 19 coding exons. Analysis of the genomic sequence showed that the fetus was a homozygote of 9kb intragenic deletion.We discovered a family of red cell enolase deficiency associated with hereditary hemolytic anemia. A Japanese boy was hospitalized due to acute aplastic crisis following parvovirus infection at the age of 8 years. After the recovery from transient erythroid aplasia, chronic hemolytic anemia became evident : hemoglobin, 9.1g/dl ; reticulocyte, 7.7% ; indirect bilirubin, 1.0mg/dl. Red cell enzyme assay demonstrated that the proband had decreased enolase activity : 4.08IU/gHb (normal range 6.04-8.10). The enolase activities of the parents were also decreased : the father, 4.93 ; the mother, 2.83. To elucidated molecular lesion of enolase deficiency, reticulocyte RNA was used for the amplification of the full-length α enolase cDNA by RT-PCR. We elucidated that the proband and his mother had a missense mutation (Arg to His at position 183). Less