Kathleen McKie

Prevalence, prevention, and treatment of microalbuminuria and proteinuria in children with sickle cell disease

Microalbuminuria (MA) and proteinuria (P) are believed to be precursors of sickle cell nephropathy. We analyzed our longitudinal data on MA/P in children with sickle cell disease (SS) to define the age of onset, association with age, sex, and hemoglobin, and to explore the safety and efficacy of hydroxyurea and angiotensin converting enzyme inhibitor (ACEI) therapy. Data on 191 patients with SS (ages 3 to 20 y) with a mean follow up of 2.19 years±2.05 were available. Urine MA was measured yearly with follow-up testing if abnormal. Prevalence of MA/P was 19.4%. Increasing age and lower hemoglobin levels were related to MA/P but sex was not. Microalbumin excretion normalized in 44% of patients treated with hydroxyurea and 56% of patients treated with ACEI. Hyperkalemia developed in 4 ACEI patients resulting in discontinuation of treatment in 3 children. In summary, MA/P often develops in childhood and preventive and treatment strategies for sickle cell nephropathy should be a focus of pediatric programs. Our preliminary data suggest that although both hydroxyurea and ACEI therapy may be beneficial for MA/P, hyperkalemia may limit the utility of ACEI.

Serum ferritin level changes in children with sickle cell disease on chronic blood transfusion are nonlinear and are associated with iron load and liver injury

Chronic blood transfusion is increasingly indicated in patients with sickle cell disease. Measuring resulting iron overload remains a challenge. Children without viral hepatitis enrolled in 2 trials for stroke prevention were examined for iron overload (STOP and STOP2; n = 271). Most received desferrioxamine chelation. Serum ferritin (SF) changes appeared nonlinear compared with prechelation estimated transfusion iron load (TIL) or with liver iron concentrations (LICs). Averaged correlation coefficient between SF and TIL (patients/observations, 26 of 164) was r = 0.70; between SF and LIC (patients/observations, 33 of 47) was r = 0.55. In mixed models, SF was associated with LIC (P = .006), alanine transaminase (P = .025), and weight (P = .026). Most patients with SF between 750 and 1500 ng/mL had a TIL between 25 and 100 mg/kg (72.8% +/- 5.9%; patients/observations, 24 of 50) or an LIC between 2.5 and 10 mg/g dry liver weight (75% +/- 0%; patients/observations, 8 of 9). Most patients with SF of 3000 ng/mL or greater had a TIL of 100 mg/kg or greater (95.3% +/- 6.7%; patients/observations, 7 of 16) or an LIC of 10 mg/g dry liver weight or greater (87.7% +/- 4.3%; patients/observations, 11 of 18). Although SF changes are nonlinear, levels less than 1500 ng/mL indicated mostly acceptable iron overload; levels of 3000 ng/mL or greater were specific for significant iron overload and were associated with liver injury. However, to determine accurately iron overload in patients with intermediately elevated SF levels, other methods are required. These trials are registered at www.clinicaltrials.gov as #NCT00000592 and #NCT00006182.

Severe Hb S‐β°‐thalassaemia with a T → C substitution in the donor splice site of the first intron of the β‐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‐β°‐thalassaemia. The substitution involved a T → C replacement at the second position of the donor splice site of the first intervening sequence of the β‐globin gene. The clinical and haematological observations made in Black subjects with Hb S‐β°‐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.

Different ζ globin gene deletions among Black Americans

SummaryFour types of chromosomes with a deletion between the human embryonic ζ and ψζ globin genes were identified among 2.8% of 321 Black Americans from Georgia. Two deletions of approximately 11 kb which differed by about 300 bp occurred on chromosomes with or without a polymorphic Xba I site 5′ to the ζ globin gene [(X+) or (X-)]. The deletions are identifiable in Xba I digests of genomic DNA using an α or a ζ globin gene probe which yield fragments of 23 kb from (X+)−ζ*αα chromosomes or 27 kb from (X−)−ζ*αα chromosomes. Digestion with other enzymes and probing with both α and ζ probes gave fragments typical of the two ζ globin gene deletions previously identified in Polynesians. Among Black Americans, these ζ globin gene deletions have been found in combination with α globin gene deletions in trans but not in cis. Homozygotes have not been found. Hematologic data on carriers of the ζ globin gene deletions in association with Hb AS, SS, and SC suggest that these deletions have no effect on the function of the adult α globin genes.

The influence of uridine diphosphate glucuronosyl transferase 1A promoter polymorphisms, βS‐globin gene haplotype, co‐inherited α‐thalassemia trait and Hb F on steady‐state serum bilirubin levels in sickle cell anemia

Abstract:  Purpose: Homozygosity for the (AT)7 allele of uridine diphosphate glucuronosyl transferase 1A (UGT1A1) gene polymorphism is associated with increased bilirubin levels in sickle cell anemia (SCA). In the present study, in addition to UGT1A1 promoter genotype, serum bilirubin level was related to other genetic modifiers –βS‐globin gene haplotype, Hb F, co‐inherited α‐thal trait, age and gender. Methods: The patients were randomly selected from the sickle cell clinic, Medical College of Georgia. UGT1A1 promoter polymorphisms were determined using automated sequencing. Other investigations were with standard techniques. Results: There were 67 SCA patients (41 males and 26 females), aged 2–44 yr (mean of 20.6 ± 10.7). Ten (14.9%) patients were homozygous for the (AT)6 UGT1A1 allele, 35 (52.2%) were heterozygous for (AT)6 and (AT)7 alleles while 22 (32.8%) were homozygous for (AT)7. Serum bilirubin was significantly higher in the homozygous (AT)7 group (3.7 ± 1.5, 3.8 ± 2.3 and 5.6 ± 2.4 mg/dL, respectively). It was also significantly higher in males than females and in patients aged >10 yr. There was a significant negative linear correlation (r = −0.304, P = 0.016) of serum bilirubin with Hb F. The β‐globin haplotype and co‐existing α‐thal trait did not have any significant influence on serum bilirubin levels. Patients on hydroxyurea were older, had lower Hb F, but higher mean serum bilirubin. The latter also was signifcantly higher among those with UGT1A1 (AT)7 homozygosity. Conclusions: Apart from UGT1A1 (AT)7 homozygosity, Hb F, age and gender are the other factors that significantly influence serum bilirubin level in SCA.

Diagnostic complication and molecular characteristics of Hb SC-Chicago disease with α-thal-2 (-α3.7 deletion): Effects of multiple variant on patient's phenotype

Dear Editor, Co-inheritance of α and β chain variants are not uncommon and mostly influence the molecular interactions of hemoglobin (Hb) tetramers, create complex genotype/ phenotype syndromes or diseases with unpredictable phenotypes. Detection of Hb variants currently utilizes techniques such as electrophoresis, high-pressure liquid chromatography (HPLC), and DNA-based methods [1]. However, these methods sometimes have limitations, and protein-based methods alone create diagnostic difficulties by misrepresenting the true extent of genotypic variations. Here, we report a subject that simultaneously inherited four globin gene abnormalities with a genotype heterozygous for alpha thalassemia-2 (α-thal-2)-α deletion, alpha chain variant Hb Chicago (α2136 [H19] Leu→Met), and two beta chain variants Hb CS (β6 [A3] Glu→Lys/Glu→Val) leading to Hb SC-Chicago-α-thal-2 (-α deletion) disease and discuss molecular characteristics as well as genotype/ phenotype correlations. The patient, presented to the pediatric clinic at the Medical College of Georgia (MCG), was a 3-year-old African American male with known SC disease. Hb Chicago and α-thal-2 are not detectable with conventional screening methods, such as complete blood count (CBC) and electrophoreses, and may thus lead to misdiagnosis of phenotypic disorders [5, 9]. The patient genotypically inherited four different abnormalities while phenotypically presented Hb SC disease. Born without complications, the patient developed seizure-like episodes at 3 weeks of age and was evaluated by a pediatric neurologist. Subsequent electroencephalogram (EEG) and physical examinations revealed a well-developed, wellnourished, active child with no remarkable findings and no relapse of seizures after the initial episode. CBC lab results revealed Hb 11.3 g/dL, Hct 35.2%, mean corpuscular volume (MCV) 64.8 fl, mean corpuscular hemoglobin (MCH) 20.8 pg, mean corpuscular hemoglobin concentration (MCHC) 32.1 g/dl, and reticulocyte 1.7%. Family history was significant for a mother with Hb AC, a father with HbAS-Chicago, and a paternal grandmother with HbAChicago. Since family evaluation showed a complex picture, blood samples were collected (in a test tube with ethylenediaminetetraacetic acid (EDTA)) from available family members and patient and sent to the Titus HJ Huisman hemoglobinopathy laboratory at the MCG, Augusta, GA, USA, for further testing. The patient’s father was not available for testing. Informed consent was obtained from patient’s mother. Red cell lysates were analyzed by isoelectric focusing (IEF) and two types of HPLC (cation exchange and reverse phase), utilizing the methods reported earlier [1].A theoretical three-dimensionalmolecularmodel of Hb SC-Chicago has been designed by using research collaboratory for structural bioinformatics protein data bank code 1FHD (Hb molecule) as a template on RasWin Molecular Graphics Windows V2.7.4.2 molecular software. DNA was isolated from the patient and paternal grandmother’s peripheral blood to perform DNA analyses. Sequencing of β, α, and α globin genes were done utilizing the methods described earlier [2, 3]. Since MCV and MCH values of the patient were lower than expected for a typical SC disease and also incidence of α-thal-2 is very high in the African Ann Hematol (2009) 88:1151–1153 DOI 10.1007/s00277-009-0740-8

A new Variant, Hb Muscat [α2β232(B14)Leu→val] Observed in Association With Hb S in an Arabian Family

The silent Hb Muscat with a Leu→Val replacement at position β32 was discovered by reversed phase high performance liquid chromatography in two members of an Arabian family from Oman; in one person Hb Muscat occurred with Hb S and in the other with Hb A. Hb Muscat is slightly unstable but its presence has no apparent adverse effect on the health of its carriers. Additional hemoglobin abnormalities observed in this family were a common a-thal-assemia-2 (-3.7 kb) and Hb S. The βS haplotypes in the heterozygous carriers and the two sickle cell anemia patients were #19 (Benin) and #20 (Bantu); the latter likely originated from an East African population.

Three Sickle Cell Anemia Patients Each with a Different α Chain Variant. Diagnostic Complications

We have studied three sickle cell anemia patients who also carried a heterozygosity for one of the following a chain abnormalities: Hb G-Philadelphia [α68(E17)Asn→Lys], Hb Montgomery [α 48 (CE6)Leu→Arg], and Hb Chicago [αl36(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.

HEART DISEASE IN THALASSEMIA HETEROZYGOTES WITH SICKLE CELL ANEMIA

The purpose of this study was to ascertain whether thalassemia heterozygotes (α thal) who have sickle cell anemia (SCA) suffer fewer cardiac effects of their SCA due to an increased oxygen carrying capacity or decreased sickling. Echocardiograms and graded, maximal exercise tests were performed in 22 subjects withα thal and SCA, and in 22 age and sex matched controls (C) with SCA alone. The patients ranged in age from 8-32 years. None were ill within two weeks of study. No significant differences were found for any of the following measurements: age (α thal 19.0 years, C 18.5); hemoglobin (α thal 8.8 gm%, C 8.2); hemoglobin F (α thal 6.6%, C 7.8); body surface area (α thal 1.43 M2, C 1.45) heart rate (α thal 78.6, C 80.3); left ventricular (LV) dimension (α thal 5.38 cm., C 5.42); LV wall thickness (α thal 0.91 cm., C 0.92); right ventricular (RV) dimension (α thal 2.26 cm., C2.42); left atrial dimension (α thal 3.70 cm., C 3.91); shortening fraction (α thal 34.8%, C 36.5); and work capacity (α thal 7.3 kg-m/min/kg, C 8.4. Exercise induced S-T depression was noted in 8/44 (18%) of subjects. The “α thal group was more often affected 6/8 (NS). α thal was not protective in heterozygotes with SCA. Abnormalities attributable to increased viscosity at capillary level persist in α thal heterozygotes with SCA. α thal homozygotes would thus be an ideal group to examine these effects independent of the manifestations of severe anemia, since they tend to have higher hemoglobins than sickle cell patients without α thal.

76 DIGITIZED ECHOCARDIOGRAPHIC ASSESSMENT OF LEFT VENTRICULAR WALL MOTION IN INFANTS AND ADOLESCENTS WITH SICKLE CELL ANEMIA

To examine ventricular wall motion in sickle cell anemia (SS), two groups of SS patients (pts) underwent digitized echocardiographic analysis. Group I consisted of 30 SS infants (mean age 0.8 yrs). Group II consisted of 70 SS pts (mean age 13.8 yrs). When these groups were compared to nonanemic controls (C) of similar age, no differences were found in shortening fraction or systolic time intervals. Computer derived information included: R-R interval, maximum (Max) and minimum (Min) LV size, Max and Min septal and freewall thickness, Max rate of septal and freewall thickening and thinning. Timing of each event was normalized as a percentage of the total R-R interval. Mean value ± standard deviation and a p value for various groups are shown below:Adolescents with SS have abnormalities of LV dimension and wall motion which are not present in infancy. Systolic delay in achieving Min LV size may be secondary to increased stroke volume. The etiology of diastolic delay septal thinning and max cavity filling is unknown.