Hb Hammersmith [beta 42(CD1) Phe-->Ser]: occurrence as a de novo mutation in black monozygotic twins with multiple congenital anomalies.

PURPOSE: To present the occurrence of Hb Hammersmith as a de novo mutation in African-American twins with multiple congenital anomalies. METHODS: Standard hematologic methods were used. The presence of an unstable Hb variant was confirmed by brilliant cresyl blue staining and an isopropanol stability test. Hb Hammersmith was confirmed by the sequencing of polymerase chain reaction-amplified beta-globin gene. RESULTS: The presence of Hb Hammersmith was confirmed in female monozygotic twins of African-American origin with congenital Heinz body hemolytic anemia and multiple congenital anomalies. The variant occurred as a de novo mutation in the twins. CONCLUSION: This report describes the occurrence of Hb Hammersmith [B42(CD1)Phe-->Ser] in African-American twins. As with the other reported cases, both twins were female. In addition to Heinz body hemolytic anemia, a low arterial O2 saturation in the proposita was shown by pulse oximetry. Multiple congenital anomalies involving various systems were also found in both twins.

Prevention of a first stroke by transfusions in children with sickle cell anemia and abnormal results on transcranial Doppler ultrasonography.

BACKGROUND: Blood transfusions prevent recurrent stroke in children with sickle cell anemia, but the value of transfusions in preventing a first stroke is unknown. We used transcranial Doppler ultrasonography to identify children with sickle cell anemia who were at high risk for stroke and then randomly assigned them to receive standard care or transfusions to prevent a first stroke. METHODS: To enter the study, children with sickle cell anemia and no history of stroke had to have undergone two transcranial Doppler studies that showed that the time-averaged mean blood-flow velocity in the internal carotid or middle cerebral artery was 200 cm per second or higher. The patients were randomly assigned to receive standard care or transfusions to reduce the hemoglobin S concentration to less than 30 percent of the total hemoglobin concentration. The incidence of stroke (cerebral infarction or intracranial hemorrhage) was compared between the two groups. RESULTS: A total of 130 children (mean [+/-SD] age, 8.3+/-3.3 years) were enrolled; 63 were randomly assigned to receive transfusions and 67 to receive standard care. At base line, the transfusion group had a slightly lower mean hemoglobin concentration (7.2 vs. 7.6 g per deciliter, P=0.001) and hematocrit (20.4 vs. 21.7 percent, P=0.002). Ten patients dropped out of the transfusion group, and two patients crossed over from the standard-care group to the transfusion group. There were 10 cerebral infarctions and 1 intracerebral hematoma in the standard-care group, as compared with 1 infarction in the transfusion group -- a 92 percent difference in the risk of stroke (P<0.001). This result led to the early termination of the trial. CONCLUSIONS: Transfusion greatly reduces the risk of a first stroke in children with sickle cell anemia who have abnormal results on transcranial Doppler ultrasonography.

Stroke prevention trial in sickle cell anemia.

Stroke occurs in 7-8% of children with Sickle Cell Disease (Hb SS) and is a major cause of morbidity. Rates of recurrence have been reduced from 46-90% to less than 10% through chronic blood transfusions. Prevention of first stroke, however, would be preferable because even one stroke can cause irreversible brain injury. Transcranial Doppler (TCD) ultrasound can detect arterial blood flow rates associated with subsequent stroke risk. By combining TCD screening and a potentially effective treatment, first stroke may be prevented. The Stroke Prevention Trial in Sickle Cell Anemia (STOP) is the first stroke prevention trial in Hb SS and the first randomized, controlled use of transfusion in Hb SS. This multi-center trial is designed to test whether reducing sickle hemoglobin to 30% or less with periodic blood transfusions will reduce first-time stroke by at least 70% compared to standard care. Primary endpoints will be clinically evident symptoms of cerebral infarction with consistent findings on Magnetic Resonance Imaging and Angiography (MRI/MRA) or symptomatic intracranial hemorrhage. Secondary endpoints will be asymptomatic brain lesions detected by MRI in brain areas not involved in primary endpoints. The design calls for a 6-month start-up interval, 18 months of TCD screening and randomization, and observation for stroke from entry through month 54. Key features of the trial are standardized TCD and MRI/MRA protocols interpreted blindly, and blinded adjudication of endpoints. The sample size (60 per treatment group) is based on prospective data relating TCD velocity to risk of stroke. A time-averaged mean velocity of > or = 200 cm/sec is associated with a 46% risk of cerebral infarction over 39 months. The sample size is sufficient to detect 70% reduction in the primary endpoint at 90% power. This trial will determine if transfusion is effective in the primary prevention of stroke. Secondary aims may further the understanding of the effects of transfusion on the brain and guide future research into cerebrovascular disease in Hb SS.

Long-term stroke risk in children with sickle cell disease screened with transcranial Doppler.

Stroke is an important complication of sickle cell disease. Stroke prediction is clinically important because it offers the possibility of primary prevention. In 1992, transcranial Doppler (TCD) evidence of elevated intracranial internal carotid or middle cerebral artery velocity was demonstrated to be associated strongly with an increased risk of ischemic stroke. This study extends the original study and includes 125 more children, longer follow-up, and intracranial hemorrhage in the stroke-risk model. Elevated time averaged mean maximum blood flow velocity, especially when velocity is 200 cm/sec or greater by TCD, was associated strongly with stroke risk. The cases not predicted by TCD point to the need for more information on the optimal timing of TCD surveillance for stroke risk.

Neuropsychological impairment in children with sickle cell anemia and cerebrovascular accidents.

Neuropsychological functioning of children with sickle cell anemia (HbSS) who have experienced a single stroke has not been extensively investigated. In this study, the neuropsychological functioning of 10 children with HbSS who were receiving transfusion therapy following stroke with no identifiable recurrence was examined. The patients were subgrouped into children with only left hemisphere stroke (LCI), N = 4, and those with only right hemisphere stroke (RCI), N = 6. Results indicated that these youngsters experienced significant impairments of cognitive functioning following stroke. It was found that the LCI and RCI children tended to perform more like adult stroke patients than what has been typically reported in children with infantile hemiplegia. These findings support the need for periodic neuropsychological evaluation following stroke in order to identify patterns of higher cortical dysfunction and assist in the development of appropriate rehabilitation and special education programs. Further, pediatricians, child neurologists, and psychologists who care for these children must act as strong advocates on their behalf in order to ensure that they receive appropriate rehabilitation and the special education services necessary for maximal recovery and future educational success.

Three sickle cell anemia patients each with a different alpha chain variant. Diagnostic complications.

We have studied three sickle cell anemia patients who also carried a heterozygosity for one of the following alpha chain abnormalities: Hb G-Philadelphia [alpha 68(E17)Asn-->Lys], Hb Montgomery [alpha 48 (CE6)Leu-->Arg], and Hb Chicago [alpha 136(H19)Leu-->Met]. Electrophoretic analyses alone may result in incomplete and incorrect information. Confirmation of the diagnosis of Hb SS or Hb SC disease by one of the fast high performance liquid chromatographic procedures is recommended.

Spleen in sickle cell anemia: comparative studies of Nigerian and U.S. patients.

Anecdotal reports have attributed persistent splenomegaly in African sickle cell anemia (SS) patients to the effects of malaria. However, no comparative studies of patients in malarial and nonmalarial regions have been conducted, and few studies of malaria antibody titers have been reported. In the present study, age- and sex-matched Nigerian patients (n = 310), while it was found only in 8% of U.S. patients (n = 100) from Georgia. There was significant linear correlation between spleen size and Hb levels and with serum immunoglobulins in the Nigerian group. However, serum complement levels (C3 and C4) were not affected by spleen size. In both groups, patients with splenomegaly had fewer circulating pitted red cells than their counterparts without splenomegaly. The mean +/- SE of IgG-specific malaria antibody titer among the Nigerian patients without palpable spleens was 9,386 +/- 2,036; 9,334 +/- 2,980 in those with spleens between 1 and 5 cm, 16,201 +/- 4,502 in those with spleens between 6 and 10 cm, and 22,445 +/- 8,456 in those with spleens above 10 cm. Coexistent alpha-thalassemia did not influence the prevalence of splenomegaly among the Nigerian SS patients. This study provides additional evidence that malaria plays a significant role in the persistence of splenomegaly in African patients.

Molecular characterization of Hb S(C) beta-thalassemia in American blacks.

An extension of previous reports describing the molecular defects and hematological abnormalities in black patients with Hb S(C) beta-thalassemia living in the Southeastern United States is presented. As many as 58 patients with Hb S-beta(+)-thalassemia, 16 with Hb C-beta(+)-thalassemia and 12 with Hb S-beta(0) -thalassemia have been studied. Patients with Hb S(C) beta(+)-thalassemia type 2 (high Hb A values) were most common; the thalassemia was due to mutations in the promoter of the beta-globin gene [-88 (C----T) and -29 (A----G)] or at the polyadenylation signal (T----C). Two patients with lower Hb A values (type 1) carried a mutation in the first intron of the beta-globin gene (IVS-1-5: G----T). The simultaneous presence of an alpha-thalassemia -2(-alpha/) resulted in some modifications of the hematological parameters, but had a minimal effect on the clinical condition. Patients with Hb S-beta (0) thalassemia had lower hemoglobin values, lower number of red blood cells, and lower MCHC values and suffered more frequently from complications than the patients with Hb S-beta(+)-thalassemia. A total of 17 different beta-thalassemia mutations were observed in 128 chromosomes; two mild beta(+)-thalassemia mutations [-88(C----T) and -29(A----G)] account for more than 80% of the thalassemic chromosomes.

Cerebral vessel stenosis in sickle cell disease: criteria for detection by transcranial Doppler.

Ischemic stroke is a common and disabling complication of sickle cell disease (Hb SS). Most infarctions occur in the presence of intracranial stenotic lesions of the large vessels of the circle of Willis. Transcranial Doppler (TCD), by measuring flow velocity in these arterial segments, can detect focal stenosis on the basis of elevated flow velocity. We report the preliminary results of a prospective study to develop criteria for detection of stenotic lesions based on TCD and identification of patients with Hb SS at risk for stroke. Comparing the TCD findings from six patients with lesions demonstrated by angiography to those from 115 Hb SS children without stroke, we conclude: (a) middle cerebral (MCA), anterior cerebral (ACA), or internal carotid (ICA) artery mean velocities greater than 190 cm/s strongly suggest focal stenosis; (b) MCA or ACA mean velocities of 150 to 190 cm/s suggest abnormality but at present cannot be considered diagnostic of stenosis; (c) mean velocities up to 150 cm/s are possibly due to the effects of low hematocrit and/or young age, and cannot as yet be distinguished from velocity elevations due to vessel stenosis.

Neuropsychologic impairment in children with sickle cell anemia.

In this study, the neuropsychologic functioning of 21 children with sickle cell anemia and 21 sibling controls, age range 7 through 16 years, with no history of neurologic disease, was examined. Outcome measures included tests of intelligence, constructional praxis, memory, and academic learning. On the Wechsler Intelligence Scale for Children--Revised, the sickle cell group had a mean Full Scale IQ of 77.7 (SD 12.4) compared with 94.3 (SD 11.0) for the control group. The profile of test scores was similar for the two groups, with the sickle cell group scoring significantly lower than the control group on almost all cognitive measures. Both groups showed academic achievement to be commensurate with their measured intellectual ability. These results suggest that subtle but significant and widespread neuropsychologic deficits are associated with sickle cell anemia even in the absence of neurologic complications. When and by what process this neuropsychologic impairment is caused needs to be determined.

Quantitation of erythropoietin stimulatory activity using [3H]thymidine uptake by K562 cells.

A microassay for erythropoietin (Ep) activity in serum using [3H]thymidine uptake by K562 cells is presented. The method is similar to that of Krystal except that cells of the K562 human pluripotent leukemia cell line replace spleen cells from phenylhydrazine-treated anemic mice. Response to the hormone by K562 cells and spleen cells was colinear. Using the Krystal bioassay, 14 young hemoglobin S homozygotes had Ep activity levels of 17.9-113.8 mU/ml serum, whereas the new method with K562 cells gave a range of 19.2-115.3 mU/ml. The correlation coefficient between the two sets of data (r) was 0.999 (p less than 0.001). With the modified technique we have assayed 34 sickle cell patients, whose sera ranged from 19.2 to 1400 mU of Ep/ml with corresponding hemoglobin concentrations of 10.7 g % to 3.0 g %. Values for normal subjects were 22.1 +/- 2.1 mU/ml (n = 7). The stimulation of [3H]thymidine uptake is significantly inhibited by an anti-Ep antiserum. The assay permits quantification of stimulatory activities in a large number of samples with relative ease and is also suitable to explore the interactions of erythropoietic factors with their appropriate receptors on stem cells.

Hb Evans or alpha 262(E11)Val----Met beta 2; an unstable hemoglobin causing a mild hemolytic anemia.

Structural analysis of the alpha chain of the hemoglobin from a Caucasian female with a mild hemolytic anemia showed the presence of a variant with a Val----Met substitution at position alpha 62. The valine at this position forms one of the contacts with heme and its replacement by methionine will likely decrease heme binding and cause a distortion of the heme crevice and a decreased stability of the abnormal protein. Dot-blot analysis of amplified DNA with 32P-labeled synthetic oligonucleotide probes confirmed the suspected G----A mutation in the first position of codon 62, and also located the mutation in the alpha 2-globin gene. The mutation was found in the proposita and one of her daughters but was most probably absent in her parents.

Severe Hb S-beta zero-thalassaemia with a T----C substitution in the donor splice site of the first intron of the beta-globin gene.

Through direct sequencing and dot-blot analyses with synthetic oligonucleotide probes of amplified DNA, a new nucleotide substitution was discovered in a Black teenager with severe Hb S-beta zero-thalassaemia. The substitution involved a T----C replacement at the second position of the donor splice site of the first intervening sequence of the beta-globin gene. The clinical and haematological observations made in Black subjects with Hb S-beta zero-thalassaemia, with different types of thalassaemia, suggest severe disease resembling sickle cell anaemia. Only an occasional patient had a milder clinical course, perhaps because of a greatly increased production of fetal haemoglobin.

Clinical and genetic heterogeneity in black patients with homozygous beta-thalassemia from the southeastern United States.

The presence of various substitutions and deletions resulting in beta-thalassemia was studied in 19 black patients with homozygous beta-thalassemia and in numerous relatives; all patients were from Georgia, South Carolina, and Alabama. Methodology included gene mapping, amplification of genomic DNA with Taq polymerase, identification of known nucleotide substitutions or a single nucleotide deletion through hybridization with synthetic oligonucleotides, cloning and sequencing of a beta-globin gene, and sequencing of amplified genomic DNA. Of the 38 chromosomes tested, 21 (55%) had the A----G substitution at nt -29, eight (21%) had the C----T substitution at nt -88, three (8%) had the substitution at codon 24, while one each of the following abnormalities were also detected: frameshift at codon 6, a C----A mutation at nt 848 of the beta IVS-II (new), an A----T mutation at codon 61 (new), a deletion of 1.35 kilobases including the 5' end of beta, a Ggamma(Agamma delta beta)(0)-thalassemia, and one thalassemia determinant that remained unidentified. The C----A mutation at nt 848 of IVS-II occurred at a position 3 nucleotides 5' to the third exon, adjacent to the invariant AG dinucleotide of the acceptor sequence. The A----T mutation in codon 61 (AAG----TAG) resulted in the creation of a stop codon and thus in beta(0)-thalassemia. The various mutations occurred on chromosomes with different haplotypes; however, chromosomes with a specific mutation but with different haplotypes belonged to one specific framework, which suggested that crossovers were responsible for these different types. Hemoglobin (Hb) F levels were generally high (55% to 75% with 98.5% in one patient with beta(0)/beta(0)); a few patients with specific haplotypes and an alpha-thalassemia-2 heterozygosity had a lower Hb F level. The Ggamma in the Hb F was consistently high when the C----T mutation occurred at nt -158 to the Cap site of the Ggamma-globin gene; seven patients with +/+ at this site had an average Ggamma of 73.8%, eight patients with +/- had 64.8%, and one patient with -/- had 34.2%. Variations in hematologic values and in Hb F, Ggamma, and Hb A2 levels of relatives with a beta-thalassemia heterozygosity depended to some extent on the types of mutations or deletions and on the haplotypes of the chromosomes with the beta-thalassemia determinant.

Down's syndrome and acute myelofibrosis. Time study of DNA content during the progression to leukemia.

Flow cytometry (FCM) for the determination of cellular DNA content was performed on multiple bone marrow biopsy specimens from a 3-year-old boy with Down's syndrome and myelofibrosis. A rapidly fatal acute nonlymphocytic leukemia developed within 3 months after initial bone marrow evaluation. The clinical and morphologic changes corresponded to the development of aneuploidy as determined by FCM and cytogenetic analysis. These findings support the clinical observations of the premalignant potential of myelofibrosis in Down's syndrome.