Kenneth D. Lanclos

βs haplotypes in various world populations

SummaryWe have determined the βs haplotypes in 709 patients with sickle cell anemia, 30 with SC disease, 91 with S-β-thalassemia, and in 322 Hb S heterozygotes from different countries. The methodology concerned the detection of mutations in the promoter sequences of the Gγ- and Aγ-globin genes through dot blot analysis of amplified DNA with 32P-labeled probes, and an analysis of isolated Hb F by reversed phase high performance liquid chromatography to detect the presence of the AγT chain [Aγ75 (E19) Ile→Thr] that is characteristic for haplotype 17 (Cameroon). The results support previously published data obtained with conventional methodology that indicates that the βs gene arose separately in different locations. The present methodology has the advantage of being relatively inexpensive and fast, allowing the collection of a vast body of data in a short period of time. It also offers the opportunity of identifying unusual βs haplotypes that may be associated with a milder expression of the disease. The numerous blood samples obtained from many SS patients living in different countries made it possible to compare their hematological data. Such information is included (as average values) for 395 SS patients with haplotype 19/19, for 2 with haplotype 17/17, for 50 with haplotype 20/20, for 2 with haplotype 3/3, and for 37 with haplotype 31/31. Some information on haplotype characteristics of normal βA chromosomes is also presented.

Haplotypes and α globin gene analyses in sickle cell anaemia patients from Kenya

Summary. Over 60 patients from the Luo and Luhya tribes of Western Kenya, aged 1–23 years. with severe sickle cell anaemia were evaluated through haematological and gene mapping analyses. Nearly all (56 of 58 tested) were homozygous for haplotype 20 (Antonarakis et al, 1984) which is also frequently present in SS patients of the Central African Republic. All patients had a severe haemolytic anaemia with low Hb F levels and low levels of Gγ chains. An α‐thalassae‐mia‐2 heterozygosity (‐α/αα; ‐ 3·7 kb deletion) was present in 26 of 53 patients tested: one patient was a homozygote [f(—α) = 0·255]. The α‐thal‐2 was type I in all but one subject with this deficiency; the one exception had an α‐thal‐2 heterozygosity, type II. Heterozygosity for the α‐thal‐2 did not affect the clinical condition nor the haematology; Hb F levels were somewhat lower in SS patients with —α/αα than in those with αα/αα. A high frequency was observed for the absence of an Xba I restriction site 5′ to the ξ globin gene; the frequency of this anomaly [f(Xba I‐)] was estimated at 0·39 for the chromosome with two α globin genes and at 0·74 for that with the α‐thal‐2 deletion. An Apa I restriction site polymorphism was observed in the IVS‐II of the α2 globin gene; 13 α2 genes of 53 normal (αα/) chromosomes had this restriction site which was absent in the hybrid α globin gene of the —α/ chromosome.

A (GATA)7 motif located in the 5′ boundary area of the human β-globin locus control region exhibits silencer activity in erythroid cells

A 40‐bp DNA, consisting of seven tandem GATA repeats, is located near the HS5 site in the 5′ boundary area of the locus control region (LCR) of human β‐globin gene. This (GATA)7 motif, named 5a, exhibits silencer activity in erythroid cells. In transfected, recombinant plasmids containing the chloramphenicol acetyltransferase (CAT) reporter gene, 5a repressed the activity of the cis‐linked housekeeping phosphoglycerate kinase (pgk) promoter; 5a also repressed the activity of the cis‐linked HS2 enhancer regardless of whether the CAT gene was driven by the pgk or the ϵ‐globin promoter. Repression by 5a was most severe when 5a was spliced upstream of HS2 at a distance of less than 200 bases from the HS2 enhancer core. The silencer activity of 5a was independent of whether the component GATA motifs were in head to tail orientation as in the wild type 5a or in head to head or tail to tail orientation as in a mutant 5a. Band shift experiments show that the GATA‐1 protein binds to both 5a and the mutant 5a and forms a large protein complex. Together, the results suggest that GATA‐1 bound at 5a is a strong, proximal repressor of HS2 enhancer activity. Am. J. Hematol. 65:14–24, 2000.

Characterization of chromosomes with hybrid genes for Hb Lepore-Washington, Hb Lepore-Baltimore, Hb P-Nilotic, and Hb Kenya.

SummaryThis study concerns the characterization of chromosomes with hybrid genes for Hb Lepore-Washington (44 chromosomes), for Hb Lepore-Baltimore (5 chromosomes), for Hb P-Nilotic (8 chromosomes), and for Hb Kenya (7 chromosomes) by determining a relatively large number of restriction enzyme polymorphism. Two, and possibly three, different Hb Lepore-Washington chromosomes were identified by specific haplotypes, while the haplotype of the Hb Lepore-Baltimore chromosome had its own characteristic pattern. A likely conclusion is that the crossovers leading to the formation of these chromosomes have occurred as independent events within the populations. Chromosomes with the δβ-Lepore-Washington hybrid gene maintained specific characteristies (such as increased Hb F levels in heterozygotes, and high or low Gγ values in this Hb F) which have been observed in normal individuals with chromosomes having comparable haplotypes. Only one haplotype was observed for each of the chromosomes carrying either the βδ-P-Nilotic hybrid gene or the Aγβ hybrid gene of Hb Kenya.

Evidence for the involvement of transglutaminase in the uptake of vitellogenin by Xenopuslaevis oöcytes

Abstract The yolk protein, vitellogenin, is sequestered by the developing oocyte by receptor-mediated endocytosis, the process by which cells bind and internalize extracellular proteins. Endocytosis of a variety of proteins follows a similar pathway, whereby internalization of receptor-bound ligand takes place over clathrin-coated regions of the cell membrane. The protein crosslinking enzyme, transglutaminase, has been reported to be essential for the receptor-mediated endocytosis of insulin and α2-macroglobulin. In this study, the presence of transglutaminase activity was demonstrated in the Xenopus laevis ovary and was effectively inhibited by poly L-lysine, an inhibitor of vitellogenin uptake, and dansylcadaverine, a known inhibitor of transglutaminase activity. Two other less poteint inhibitors of transglutaminase, methylamine and bacitracin produced partial inhibition of the ovarian enzyme. Furthermore, dansylcadaverine and methylamine were found to inhibit the appearance of vitellogenin in the yolk platelets of the oocyte.

Biochemical and Molecular Aspects of β-Thalassemia Types in Northern Sardinia

Forty-three patients with β-thalassemia from Northern Sardinia (31 severe and polytransfused, six follow-up babies, five adults with mild thalassemia who were not transfusion dependent, and a young transfused patient was also affected by a disease of intermediate severity) were studied in order to establish the fetal hemoglobin composition, restriction fragment 1ength polymorphisin haplotypes at the β-globin gene cluster, and the type(s) of mutation. Haplotype II was prevalent, [56/86 chromosomes (65%)], haplotype I was also fairly common, [22/86 chromosomes (25%)], while other types were relatively rare. The nonsense mutation at codon 39 was nearly exclusive, [76/80 chromosomes (95%)]. Other β-thalassemia mutations occurred on chromosomes with haplotypes 111, IX, X, and perhaps V, and a new type related to II. The mutated AγT gene was associated with type type II, X, and the new type. Type IX was linked to a βd` gene and to an Xmn I site 5′ to the Gγ gene, to a high Gγ globin level, and to a disease of m...

Absence of the testicular determining factor gene SRY in XX true hermaphrodites and presence of this locus in most subjects with gonadal dysgenesis caused by Y aneuploidy

OBJECTIVES The purpose of our study was to discover whether the testicular determining factor gene SRY (sex-determining region on Y) is present or absent in XX true hermaphrodites and in subjects with gonadal dysgenesis caused by Y aneuploidy. STUDY DESIGN We screened five XX true hermaphrodites and 24 subjects with gonadal dysgenesis caused by Y aneuploidy for the presence or absence of SRY. With the polymerase chain reaction technique, the sequence coding the 80 amino acid-conserved motif was amplified. The 0.9 kb Hincll pY53.3 subclone, which covers the open reading frame of SRY, serves as a probe for Southern blot analysis. RESULTS Test results for all five XX true hermaphrodites were negative for SRY. Conversely, 22 of the 24 individuals with 45,X/46,XY gonadal dysgenesis were positive for SRY, including the 10 subjects with only bilateral streak gonads. CONCLUSIONS The absence of SRY in XX true hemaphrodites and the presence of SRY in 10 subjects with 45,X/46,XY constitution who harbored only bilateral streak gonads seem to indicate that multiple genes are involved in gonadal differentiation.

Patients with Idiopathic Hypogonadotropic Hypogonadism Have Normal Gonadotropin-Releasing Hormone Gene Structure

Abstract Study Objective: To determine if the exons encoding the structural protein for gonadotropin-releasing hormone (GnRH) are present in patients with idiopathic hypogonadotropic hypogonadism (IHH), and whether individual exons possess smaller mutations not detected previously. Design: Study of patients with IHH by DNA analysis using polymerase chain reaction (PCR) and DNA sequencing. Setting: Laboratories of the Department of Obstetrics and Gynecology, Department of Oral Biology, Medical College of Georgia. Participants: Fifteen well-characterized patients (13 females and 2 males) presenting with delayed puberty due to IHH and two fertile controls. Interventions: DNA extraction from all individuals for the performance of PCR and DNA sequencing for exon II, the coding region for the GnRH decapeptide. Main Outcome Measures: For PCR analysis, fragment sizes were evaluated on agarose gels stained with ethidium bromide in the presence of a molecular weight marker. Specific nucleotide sequences were determined from radiographs of DNA sequencing gels. Results: Each of the fragments containing exons II–IV of the GnRH gene were present and of normal size in all patients with IHH and controls. Exon II and splice junction sequences were normal in four females with IHH. Conclusions: PCR of individual exons encoding the structural gene for GnRH is normal in 15 patients with IHH. DNA sequencing of exon II in four women with IHH is normal. PCR analysis and preliminary DNA sequencing fail to demonstrate causative mutations in the GnRH gene in our patients with IHH, which contributes additional information not provided by Southern analysis and previous studies.

Effect of calmodulin inhibitor, Stelazine, on the endocytosis of vitellogenin and transglutaminase activity in Xenopus laevis oöcytes.

SummaryThe specific endocytosis of vitellogenin was measured in the presence of various concentrations of Stelazine, a specific inhibitor of the calcium regulating protein, calmodulin. Stelazine (200 µ M) was found to inhibit the endocytosis of vitellogenin by 63% as determined by decreased uptake of vitellogenin into transitional yolk bodies and yolk platelets. The enzymatic activity of transglutaminase, a calcium-requiring enzyme implicated in receptor-mediated endocytosis, was not affected by these concentrations of Stelazine.

The β-δ crossover leading to the βδ hybrid gene of hemoglobin P-nilotic is located within 54 base-pairs of the 5' end of exon 2 or between codons 31 and 50

Abstract The crossover region of the βδ hybrid gene of the hemoglobin variant Hb P-Nilotic was defined in detail through cloning and sequencing of appropriate DNA segments. The crossover must have occurred without loss of bases within a 54 base-pair stretch of DNA between bases 275 and 330 (or between amino acid residues 31 and 50), indicating that the exon 1 and IVS-1 originate from β, and exon 2, IVS-2 and exon 3 from δ. The data support the speculation that the IVS-1, in contrast to IVS-2, has no effect on the expression of this hybrid gene.