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

通过对96个遗传性非球形细胞溶血性贫血家系的酶学分析，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。我们发现了一例患有严重贫血的胎儿，这是日本首例HK缺乏症。母亲在怀孕29周时因宫内发育迟缓而住院。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5.8 mg/dl。孕32周的红细胞酶分析表明，HK活性下降到15%左右。父母的红细胞HK活性也下降到正常平均值的50%左右。这个家庭没有血缘关系。受影响的…更多的胎儿在怀孕37周时死于宫内。为阐明先证者HK缺乏症的分子损害，采用长链聚合酶链式反应(Long-PCR)的方法，从网织红细胞RNA中扩增出I型HK(HK-I)的全长cDNA。根据相应的5‘和3’非编码序列，从网织红细胞丰富的对照RNA中扩增出2.8kb的HK-I基因，从先证者的网织红细胞中扩增出2.3kb的HK-I基因。测序结果显示，2.3kb的片段缺失外显子5~8。为了阐明导致这些外显子跳跃的基因异常，我们测定了人HK-L基因的基因组结构。HK-I基因全长约67kb，有19个编码外显子。基因组序列分析表明，该胎儿是一个9kb基因内缺失的纯合子。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族。一名日本男孩在8岁时因细小病毒感染导致急性再生障碍性疾病而住院。从一过性红系再生障碍性贫血恢复后，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1.0 mg/dl。红细胞酶测定显示先证者的烯醇酶活性降低：4.08IU/gHb(正常范围6.04-8.10)。双亲的烯醇酶活性也有所下降，父亲为4.93，母亲为2.83。为阐明烯醇化酶缺乏症的分子损伤，采用网织红细胞核糖核酸逆转录聚合酶链式反应扩增α烯醇化酶全长基因。我们阐明了先证者和他的母亲有错义突变(183位Arg到His)。较少

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