The impact of lesion length and vessel size on outcomes after sirolimus-eluting stent implantation for in-stent restenosis.

OBJECTIVES: We evaluated the predictors of recurrent restenosis and the impact of lesion length and vessel size on outcomes in patients treated with routine sirolimus-eluting stent (SES) implantation for in-stent restenosis (ISR) of bare-metal stent (BMS). METHODS: In this study, 250 consecutive patients with 275 lesions after SES implantation for ISR of BMS were enrolled. Follow-up angiogram was obtained in 239 patients with 258 lesions eight months after implantation (follow-up rate: 95.6%). We compared characteristics of patients and lesions between the two groups (the recurrent restenosis group and the no-restenosis group). RESULTS: Recurrent restenosis was angiographically documented in 43 lesions (16.7%). Recurrent restenosis was found in 30.4% with small vessel lesions (reference diameter of less than 2.5 mm, 92 lesions) and 23% with the diffuse type lesions (106 lesions). Seventy-two per cent of patients had a focal pattern of recurrent restenosis. Previously recurrent ISR lesions (odds ratio (OR) 1.94, 95% confidence interval (CI) 0.94 to 4.06, p = 0.05), reference diameter of less than 2.5 mm (OR 2.41, CI 1.05 to 5.41, p = 0.03), diffuse type restenosis (OR 4.48, CI 2.12 to 9.94, p = 0.0001) and dialysis patients (OR 4.72, CI 1.42 to 15.7, p = 0.01) were independent predictors of recurrent restenosis. CONCLUSIONS: Small vessels, diffuse type restenosis and dialysis patients were still the predictors of recurrent restenosis in patients treated with SES for ISR of BMS.

A new glucose-6-phosphate dehydrogenase variant G6PD Sugao (826C-->T) exhibiting chronic hemolytic anemia with episodes of hemolytic crisis immediately after birth.

A case of glucose-6-phosphate dehydrogenase (G6PD) deficiency associated with chronic hemolysis with episodes of hemolytic crisis immediately after birth is reported. The propositus was a 1-month-old Japanese male infant. Molecular analysis of the G6PD gene revealed a novel missense mutation (826C-->4T) in exon 8 predicting a single amino acid substitution, Pro276Ser. The mother was confirmed to be heterozygous for this mutation. We designated this novel class 1 variant as G6PD Sugao. Pro276 is a phylogenetically conserved residue that may play a significant role in dimer formation.

[A case of cervical myeloradiculopathy with positive serum anti-GT1a antibody].

A 19-year-old man with cervical myeloradiculopathy is reported. He was admitted to our hospital because of acute muscle weakness of upper limbs, which developed two weeks after respiratory tract infection. Neurologic examination revealed prominent muscular weakness of upper limbs. Deep tendon reflexes showed hyporeflexia in upper limbs and hyperreflexia in lower limbs. Serum IgG anti-GT1a antibody was detected by thin-layer chromatography and immunoblotting. Neck MRI revealed T2-weighted high intensity legions and swelling in spinal cord at third to sixth cervical segment. The muscular weakness and the cervical legion in MRI improved two weeks after steroid treatment. These findings indicate the involvement of cervical pyramidal tract as well as that of cervical roots in the patient. Neurological symptoms of the present case differed from those of pharyngeal-cervical-brachial variant (PCB) of Guillain-Barré syndrome, although serum anti-GT1a antibody was positive.

Distribution of glucose-6-phosphate dehydrogenase mutations in Southeast Asia.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a heterogeneous enzyme abnormality with high frequency in tropical areas. We performed population screening and molecular studies of G6PD variants to clarify their distribution and features in Southeast Asia. A total of 4317 participants (2019 males, 2298 females) from 16 ethnic groups in Myanmar, Lao in Laos, and Amboinese in Indonesia were screened with a single-step screening method. The prevalence of G6PD-deficient males ranged from 0% (the Akha) to 10.8% (the Shan). These G6PD-deficient individuals and 12 G6PD-deficient patients who had been diagnosed at hospitals in Indonesia and Malaysia were subjected to molecular analysis by a combination of polymerase-chain-reaction-based single-strand conformation polymorphism analysis and direct sequencing. Ten different missense mutations were identified in 63 G6PD-deficient individuals (50 hemizygotes, 11 heterozygotes, and 2 homozygotes) from 14 ethnic groups. One missense mutation (1291 G-->A) found in an Indonesian Chinese, viz., G6PD Surabaya, was previously unknown. The 487 G-->A (G6PD Mahidol) mutation was widely seen in Myanmar, 383 T-->C (G6PD Vanua Lava) was specifically found among Amboinese, 871 G-->A (G6PD Viangchan) was observed mainly in Lao, and 592 C-->T (G6PD Coimbra) was found in Malaysian aborigines (Orang Asli). The other five mutations, 95 A-->G (G6PD Gaohe), 1003 G-->A (G6PD Chatham), 1360 C-->T (G6PD Union), 1376 G-->T (G6PD Canton), and 1388 G-->A (G6PD Kaiping) were identified mostly in accordance with distributions reported previously.

Two novel glucose-6-phosphate dehydrogenase variants found in newborn mass-screening for galactosaemia.

UNLABELLED: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked recessive disorder in which haemolytic anaemia is the major symptom. The Beutler spot test employed in mass-screening for galactosaemia in newborns requires several intrinsic erythrocyte enzymes such as G6PD for its reaction and can theoretically detect G6PD deficiency apart from galactose-1-phosphate uridyltransferase deficiency. In this study, we detected two patients with G6PD deficiency using the quantitative Beutler test which was recently developed in our laboratory. Both patients lacked erythrocyte G6PD activity but exhibited no clinical symptoms. Molecular analysis in patients 1 and 2 revealed two novel missense mutations of C853T causing R285C and A1220C causing K407T, respectively. Molecular rather than enzymatic analysis was required in familial studies to detect and diagnose the carrier state. To date these patients have avoided oxidant stress and haemolytic diatheses have not been induced. CONCLUSION: Our results indicate that the quantitative Beutler test can detect glucose-6-phosphate dehydrogenase deficiency of class 1 and 2 and is therefore useful for early intervention and prevention of haemolytic diathesis in patients with this disorder.

Erythrocyte enzyme activities in cord blood of extremely low-birth-weight infants.

To investigate the features of erythrocyte metabolism in extremely immature infants, we assayed 21 enzyme activities and glutathione level in cord erythrocytes from 28 extremely low-birth-weight infants (ELBWI; defined as birth weight <1,000 g). The results were compared with those from normal adults and non-neonatal reticulocyte-rich controls. Statistical analysis revealed that activities of six enzymes (glucosephosphate isomerase, phosphoglycerate kinase, monophosphoglycerate mutase, enolase, glucose-6-phosphate dehydrogenase (G6PD), and glutathione reductase) were significantly higher, and those of eight other enzymes (phosphofructokinase, 6-phosphogluconate dehydrogenase (6PGD), glutathione peroxidase, adenylate kinase, adenosine deaminase, acetylcholinesterase, NADH methemoglobin reductase, and catalase) were lower in ELBWI taking their marked reticulocytosis into consideration. The 6PGD/G6PD ratio, which is consistently unchanged under various physiological and pathological conditions, was markedly reduced in ELBWI. Our results support the previous reports that neonatal erythrocytes have a unique metabolic pattern which is different from that of adult erythrocytes, and also suggest that the 6PGD/G6PD ratio might be an index for the developmental immaturity of fetal erythrocytes. This is the first report describing the pattern of erythrocyte enzyme activities in ELBWI.

Hemolytic crisis after excessive ingestion of fava beans in a male infant with G6PD Canton.

After ingesting fava beans, a 26-month-old Chinese-Japanese male infant showed a sickly complexion and yellowish-brownish skin and was hospitalized. Severe hemolytic anemia was observed on admission, and transfusion of 200 ml of packed red cells was required. Red cell enzyme assay revealed that the patient and the mother were deficient in glucose-6-phosphate dehydrogenase (G6PD). Subsequent molecular analysis showed that the patient had a missense mutation 1376 G to T (G6PD Canton) and his mother was a homozygote for the mutation. The patient was a son of a Chinese (Taiwanese) mother and a Japanese father. Although G6PD deficiency is rare in the original Japanese population, the number of "imported" cases could be rising rapidly. This is the first reported Japanese case of G6PD deficiency with G6PD Canton.

Frame shift mutation, exon skipping, and a two-codon deletion caused by splice site mutations account for pyruvate kinase deficiency.

Three novel splice site mutations and two novel missense mutations were identified by molecular analysis of pyruvate kinase (PK) deficiency associated with hereditary nonspherocytic hemolytic anemia. A Nepalese PK variant, PK Kowloon, was found to have a homozygous transversion at the 5'-splice site of the seventh intervening sequence (IVS) of the L-type PK gene (Ivs7[+1]gt --> tt). Using a reverse transcription polymerase chain reaction (RT-PCR) assay, we showed that the R-type PK mRNA in the proband's reticulocytes included the seventh IVS between the seventh and eighth exon, introducing a stop codon 3 nucleotides downstream of the mutated site. Consequently, the translational product may lack 44% of the R-PK polypeptide. A transition at the last nucleotide of exon 9 (1269GCG --> GCA) was found in a Japanese PK variant, PK 'Kamata.' The mutation did not alter the amino acid sequence, but caused skipping of the ninth exonic sequence in the R-PK transcripts. As a result, the affected R-type PK lost 51 amino acid residues (373Met-423Ala del). A transversion at the splice acceptor site of the third IVS (Ivs 3[-2]ag --> tg) was identified in PK 'Aomori.' The mutation resulted in aberrant splicing at a cryptic splice site within exon 4, causing deletion of two codons in the aberrant R-PK transcript (95 Gly-96 Pro --> del). Both PK 'Kamata' and PK 'Aomori' had a missense mutation on the other allele, 1044AAG --> AAT (348Lys --> Asn) and 1075CGC --> TGC (359Arg --> Cys), respectively. Although both 348Lys and 359Arg were located in the sixth loop of A domain (beta/alpha)8 barrel, which has been shown to contain the substrate and cation binding sites, the degree of anemia was much more severe in PK 'Kamata' than PK 'Aomori,' possibly because the 51 amino acid deletion of PK 'Kamata' but the 2 amino-acid deletion of PK 'Aomori' may abolish PK catalytic activity.

Molecular analysis of eight biochemically unique glucose-6-phosphate dehydrogenase variants found in Japan.

We analyzed the molecular mutations of eight known Japanese glucose-6-phosphate dehydrogenase (G6PD) variants with unique biochemical properties. Three of them were caused by novel missense mutations: G6PD Musashino by 185 C-->T, G6PD Asahikawa by 695 G-->A, and G6PD Kamiube by 1387 C-->T. Predicted amino acid substitutions causing asymptomatic variants G6PD Musashino (62 Pro-->Phe) and G6PD Kamiube (463 Arg-->Cys) were located in regions near the amino or carboxyl end of the polypeptide chain, whereas an amino acid change 232 Cys-->Tyr causing a class 1 variant G6PD Asahikawa was located in the region where amino acid alterations in some class 1 variants were clustered. The other five variants had known missense mutations, namely, G6PD Fukushima, 1246 G-->A, G6PD Morioka, 1339 G-->A, and G6PD Iwate, G6PD Niigata and G6PD Yamaguchi, 1160 G-->A, which cause variants, G6PD Tokyo, G6PD Santiago de Cuba, and G6PD Beverly Hills, respectively.

Molecular analysis of glucose phosphate isomerase deficiency associated with hereditary hemolytic anemia.

We report here two new cases of glucose phosphate isomerase (GPI) deficiency associated with hemolytic anemia and present the results of molecular analysis of the five Japanese GPI variants. A Japanese girl (GPI Fukuoka) had an episode of prolonged neonatal jaundice and at 3 years of age was admitted due to acute hemolytic crisis occurring with upper respiratory tract infection. Red blood cell (RBC) GPI activity was decreased to 11.8% of normal and the reduced glutathione (GSH) level of RBCs was slightly decreased. A 54-year-old Japanese man (GPI Iwate) was hospitalized due to chronic active hepatitis, and compensated hemolysis was noted. RBC GPI activity of the proband was decreased to 18.8%, and the GSH content was about half of the normal mean value. Sequencing of the reticulocyte GPIcDNA showed homozygous missense mutations 1028CAG-->CGG (343Gln-->Arg), 14ACC-->A7C (5Thr-->lle), 671ACG-->A7G (224Thr-->Met), and 1615GAC-->AAC (539Asp-->Asn) in GPI Narita, GPI Matsumoto, GPI Iwate, and GPI Fukuoka, respectively. We also identified GPI Kinki as a compound heterozygote of 1124ACA-->AGA(375Thr-->Arg)/ 1615GAC-->AAC(539Asp-->Asn). Our findings, together with the previous results of other investigators, showed that the GPI gene mutations so far identified were heterogeneous, although most GPI variants had common biochemical characteristics such as heat instability and normal kinetics. Several amino acid substitutions were identified in the proximity of the catalytically important amino acid residues such as Ser/Asp 159/160, Asp341, and Lys518, which have been identified in the structural analysis of the pig GPI. The molecular characterization of human GPI variants, therefore, may provide new insights into the genotype-phenotype correlation of GPI deficiency as well as the structure-function relationship of this enzyme.

[Human red cell metabolism and its pathophysiology].

The erythrocyte is a highly differentiated cell which has simple but active metabolic pathways. It lacks a nucleus, mitochondria and ribosomes, while glycolysis, hexose monophosphate pathway and glutathione synthesis maintain a high activity. Glycolysis is the only energy producing pathway crucial for red cell function and survival. The main function of red cell hexose monophosphate pathway is to generate NADPH, which is indispensable for maintaining high levels of a potent antioxidant reduced glutathione. Rapoport-Luebering pathway and pyrimidine 5'-nucleotide system are unique metabolic pathways specific for red cells. The former produces 2,3-diphosphoglycerate and the latter dephosphorylates useless pyrimidine nucleotides derived from the degenerated RNA.

[Glutathione synthetase deficiency].

CASE REPORT: The patient was a boy born in June, 1990. The proband's father had a history of nonspherocytic hemolytic anemia. The patient was anemic at birth (Hb 11.9 g/dl) and had a hemolytic attack on postnatal day 2. His hemolysis became well compensated, and his second hemolytic episode occurred at three years of age. CLINICAL AND LABORATORY FINDINGS: The patient's mental development had so far been normal and he has no neurological symptoms. His only clinical manifestation has been compensated hemolytic anemia with a hemoglobin concentration of about 11.0 g/dl and a reticulocyte count of 3-6%. He was positive on the Heinz body formation test, and target cells were seen on his peripheral blood smear. The osmotic fragility test yielded slightly increased value. Decreased reduced glutathione (GSH) was observed (4.4 mg/dlRBC) (normal range: 63.9 +/- 9.6), and he also had decreased glutathione synthetase (GS) activity of 0.03 U/gHb (0.38 +/- 0.08 U/gHb). A diagnosis of GS deficiency was made. Decreased glutathione S-transferase (GST) activity was also found (0.57 U/gHb) (normal range: 6.65 +/- 1.20). DISCUSSION: GS deficiency has been reported in about 30 families all over the world. This patient was the first Japanese patient with red cell GS deficiency.

Three cases of hereditary nonspherocytic hemolytic anemia associated with red blood cell glutathione deficiency.

Three unrelated Japanese patients with chronic nonspherocytic hemolytic anemia wer found to have marked deficiency of red blood cell (RBC) reduced glutathoine (GSH) (4.4%, 13.1%, and 6.9% of normal, respectively). A panel of RBC enzyme assays showed that one patient had decreased glutathione synthetase activity and the other two were moderately deficient in gamma-glutamylcystine synthetase. Some family members of each patient showed mild deficiency of the respective enzymes. RBCs of these patients also showed a decreased level of glutathione-S-transferase as in previously described GSH-deficient cases. Hemolytic anemia was their only manifestation, and neither 5-oxoprolinemia nor 5-oxoprolinuria, which are usually associated with to generalized type of glutathione synthetase deficiency, was noted in our patients.

Pyruvate kinase deficiency of mice associated with nonspherocytic hemolytic anemia and cure of the anemia by marrow transplantation without host irradiation.

Mutant mice with splenomegaly and nonspherocytic hemolytic anemia were found in an inbred colony of the CBA/N (hereafter CBA) strain maintained in the Japan SLC Haruno farm (Shuchi-gun, Shizuoka, Japan). The activity of pyruvate kinase (PK) in red blood cells (RBCs) of the anemic mutants decreased to 16.2% of normal (+/+) CBA mice. Because the mutant CBA mice showed a remarkable reticulocytosis (41.6%) and because the PK activity of reticulocytes is much higher than that of mature RBCs, the PK activity in mature RBCs of the mutant CBA mice was calculated to be 2.8% that of mature RBCs of CBA-(+/+) mice. Because RBC type PK is encoded by the Pk-1 locus of the mouse (chromosome 3), we designated the mutant locus as Pk-1slc. The anemia and PK deficiency of CBA-Pk-1slc/Pk-1slc mice were cured by bone marrow transplantation (BMT) from CBA-(+/+) mice. Prior irradiation was not necessary for the curative BMT. On the other hand, the BMT from CBA-Pk-1slc/Pk-1slc mice to nonirradiated CBA-(+/+) mice did not result in the decrease of RBCs and the reduction of PK activity. The present results indicate that CBA-Pk-1slc/Pk-1slc mice are a potentially useful animal model for studying pathophysiology of PK deficiency and for developing new therapeutic methods to correct PK deficiency.

Hereditary spherocytic anemia with deletion of the short arm of chromosome 8.

We describe a 30-month-old boy with multiple anomalies and mental retardation with hereditary spherocytic anemia. His karyotype was 46,XY,del(8)(p11.23p21.1). Genes for ankyrin and glutathione reductase (GSR) were localized to chromosome areas 8p11.2 and 8p21.1, respectively. Six patients with spherocytic anemia and interstitial deletion of 8p- have been reported. In these patients, severe mental retardation and multiple anomalies are common findings. This is a new contiguous gene syndrome. Lux et al. [1990: Nature 345:736-739] established that ankyrin deficiency and associated deficiencies of spectrin and protein 4.2 were responsible for spherocytosis in this syndrome. We reviewed the manifestations of this syndrome. Patients with spherocytic anemia and multiple congenital anomalies should be investigated by high-resolution chromosomal means to differentiate this syndrome.

Identification of two novel deletion mutations in glucose-6-phosphate dehydrogenase gene causing hemolytic anemia.

Among over 50 distinct mutations causing glucose-6-phosphate dehydrogenase (G6PD) deficiency, only two deletion mutations have so far been reported. Using nonradioisotopic single-strand conformation polymorphism analysis, we found two additional deletion mutations in two Japanese G6PD-deficient patients with nonspherocytic hemolytic anemia. Case no. 1 had a 3-nucleotide deletion in exon 6 predicting a deletion of a serine at amino acid 188 or 189, which caused a class 1 variant G6PD Tsukui. Case no. 2 had a 3-nucleotide deletion in exon 5 predicting a deletion of a lysine at residue 95, which caused a class 2 variant G6PD Urayasu. The 188th serine, which might be deleted in G6PD Tsukui, is located close to the putative G6P binding site. The 188th serine is also involved in the amino acid substitution in G6PD Mediterranean, but the kinetics of these two variants are totally different. The residue with an amino acid deletion in G6PD Urayasu was distant from the substrate binding sites and was located in a region with low sequence homology among species. The different properties of variants having mutations in exons 5 and 6 suggest that these two exons code distinct functional domains of the enzyme.

Haptoglobin therapy for acute favism: a Japanese boy with glucose-6-phosphate dehydrogenase Guadalajara.

We report the case of a 2-year-old Japanese boy with acute favism who was treated with human haptoglobin products. He had been exhibiting chronic nonspherocytic haemolytic anaemia until the diagnosis of glucose-6-phosphate dehydrogenase (G6PD) deficiency when 14 months old. He suffered a favic crisis at 24 months of age, when the administration of haptoglobin was effective for relieving bilirubinaemia and haemoglobinuria. Serum-free Hb rapidly decreased to normal levels despite the sustained level of serum lactate dehydrogenase. His G6PD gene was G6PD Guadalajara. This is the first application of haptoglobin therapy for acute favism and the first reported case of Japanese G6PD deficiency with typical favic crisis. Haptoglobin treatment might be helpful for managing the haemolytic crisis in the disease.