TThe DR15 allele at the HLA DRB1 locus is a marker for immune-mediated bone marrow failure syndromes. We hypothesized that HLA DR15 plays a role in T-cell interactions with hematopoiesis and investigated the role of HLA DR15 on graft-versus-host disease (GVHD) and graft-versus-leukemia effects in HLA-matched allogeneic blood or marrow transplants (BMT) performed for myeloid malignancies. We performed a retrospective analysis of 119 consecutive related and 48 consecutive unrelated allogeneic BMTs for myeloid malignancies treated between 1991 and 2005 to investigate the influence of HLA DR15 on overall survival (OS), progression-free survival (PFS) and incidence of grade II-IV acute GVHD. HLA DR15 was determined by either molecular (n=108) or serologic (n=59) methods. The incidence of HLA DR15 was similar to the general Caucasian population (35/167=21%). There were no significant differences in transplant characteristics between the HLA DR15 positive and negative groups. There was no significant difference in chronic GVHD, OS or PFS between the HLA DR15 positive vs. negative groups in any disease or donor relation subgroups. The HLA DR15 positive group experienced a significantly lower incidence of acute GVHD grade II-IV: 23% vs. 42%, p=0.041. These results suggest that HLA DR15 reduces the risk of acute GVHD.

The human globin genes are among the most extensively characterized in the human genome, yet the details of the molecular events regulating normal human hemoglobin switching and the potential reactivation of fetal hemoglobin in adult hematopoietic cells remain elusive. Recent discoveries demonstrate physical interactions between the beta locus control region and the downstream structural gamma- and beta-globin genes, and with transcription factors and chromatin remodeling complexes. These interactions all play roles in globin gene expression and globin switching at the human beta-globin locus. If the molecular events in hemoglobin switching were better understood and fetal hemoglobin could be more fully reactivated in adult cells, the insights obtained might lead to new approaches to the therapy of sickle cell disease and beta thalassemia by identifying specific new targets for molecular therapies.

The analysis of a number of cases of beta-globin thalassemia and hereditary persistence of fetal hemoglobin (HPFH) due to large deletions in the beta-globin locus has led to the identification of several DNA elements that have been implicated in the switch from human fetal gamma- to adult beta-globin gene expression. We have tested this hypothesis for an element that covers the minimal distance between the thalassemia and HPFH deletions and is thought to be responsible for the difference between a deletion HPFH and deltabeta-thalassemia, located 5' of the delta-globin gene. This element has been deleted from a yeast artificial chromosome (YAC) containing the complete human beta-globin locus. Analysis of this modified YAC in transgenic mice shows that early embryonic expression is unaffected, but in the fetal liver it is subject to position effects. In addition, the efficiency of transcription of the beta-globin gene is decreased, but the developmental silencing of the gamma-globin genes is unaffected by the deletion. These results show that the deleted element is involved in the activation of the beta-globin gene perhaps through the loss of a structural function required for gene activation by long-range interactions.

The barley Rar1 gene is an essential component of the race-specific, Mla-12-specified powdery mildew resistance reaction. As part of a map-based cloning strategy designed to isolate Rar1, five barley yeast artificial chromosomes (YACs) have been identified, ranging in size from 300 to 1100 kb. PCR-based YAC end-specific markers have been established and were employed to construct a local YAC contig. Four out of five YAC clones were found to be non-colinear with the source DNA. High-resolution genetic mapping of the YAC ends demonstrated that the set of five overlapping YAC clones encompasses the barley Rar1 gene.

The alpha-thalassemia-2 (alpha-thal-2) genotype or mild alpha-thalassemia gene consists of a single structural alpha-globin gene on the chromosome that normally bears two alpha-globin genes. We used blot hybridization to investigate variation in the molecular organization of this genotype and to determine the distributions of these variations in the world population. Two different patterns of gene organization responsible for the alpha-thal-2 genotype were found: the first was the result of a 4.2-kilobase pair deletion involving the normal 5' alpha-globin gene (leftward deletion alpha-thal-2 genotype), and the second probably the result of a crossover deletion of a DNA fragment bridging the two normal alpha-globin genes (rightward deletion alpha-thal-2- genotype). The rightward deletion was found in all 9 Black subjects, all 8 Mediterranean subjects, and 4 of 13 Chinese subjects. The leftward deletion was found in four and the nondeletion alpha-thalassemia lesion was found in five of the nine remaining Chinese subjects. It is likely that these deletions are related to specific DNA sequences that determine DNA recombinational events.

The site of crossover between the delta- and beta-globin gene sequences resulting in Lepore Boston globin gene formation has been localized at the DNA Level. Probes specific for detecting the intervening sequences (IVS) of the delta- and beta-globin genes were used to map the origin of cellular DNA fragments of a patient homozygous for hemoglobin Lepore Boston. Restriction endonuclease analysis and hybridization of this DNA to IVS specific probes show that most, if not all, of the large intervening sequences (IVS 2) in Lepore DNA are derived from the beta-globin gene IVS 2. In addition, the crossover occurs in a region of DNA in which the delta- and beta-genes have almost complete nucleotide homology, and might be expected to pair most tightly during meiosis.

The tuf gene which encodes peptide chain elongation factor Tu was found to be duplicated in nine enteric and four nonenteric gram-negative bacteria, but present only in one copy in two gram-positive genera. In two of the nonenteric gram-negative genera, Pseudomonas and Caulobacter, the duplicate tuf genes were found to be very close together on the chromosome, which contrasts with the situation in Escherichia coli, where they are more than 660 kilobases apart.

We have isolated and characterized a clone of human DNA from a patient with beta+ thalassemia containing the entire delta and beta structural genes and their flanking sequences. Partial Eco RI digestion of spleen DNA was used to obtain 15 to 20 kilobase (kb) pieces of human DNA that were then ligated to charon 4A lambda phage DNA. The 8 x 10(5) recombinants obtained were grown and screened for their content of beta gene sequences. Four positive clones were found, and one (beta T1-1) has been extensively analyzed. Subclones containing the entire beta gene and the large beta intervening sequence (IVS 2) have been isolated in the plasmid pBR 322. The fragments generated by restriction enzyme digestion in these subclones have been compared to those in similar subclones from normal beta genes. No differences have been found indication no significant rearrangements of deletions of the delta and beta genes. With the enzymes used, 11.2% of IVS 2 have been compared, and thus far no differences between the thalassemic and normal genes have been detected. The 24 enzymes used include Hph I, which recognizes the 5' end of IVS 2, and AIu I that cleaves at the 3' end. Thus, there appears to be conservation of nucleotide sequences at the ends of IVS 2 in this beta + thalassemia patient, although RNA metabolism studies suggest a possible defect in RNA processing.

We have analyzed human cellular DNA for its delta--and beta-globin gene sequence content by separation of restriction enzyme fragments by agarose gel electrophoresis; transfer of the DNA fragments to nitrocellulose filters; hybridization of filters with 32P--beta-globin cDNA; and analysis by autoradiography. A short cDNA has been used to identify specifically the 3' end of the genes and to orient the fragments. A comparison of the globin gene fragments generated by normal and Lepore DNA has been used to distinguish fragments representing DNA sequences between the delta and beta genes and those containing sequences flanking either 5' to the delta gene or 3' to the beta gene. The results indicate that unique restriction fragments are presented in normal DNA and absent in Lepore DNA, and allow preliminary ordering of these fragments on a restriction enzyme map. In addition, the Lepore, delta--and beta-globin genes have been found to contain at least one inserted nucleotide sequence of about 1000 bases which is not represented in mature globin mRNA.

Studies of the human hemoglobin system have provided new insights into the regulation of expression of a group of linked human genes, the gamma-delta-beta-globin gene complex in man. In particular, the thalassemia syndromes and related disorders of man are inherited anemias that provide mutations for the study of the regulation of globin gene expression. New methods, including restriction enzyme analysis and cloning of cellular DNA, have made it feasible to define more precisely the structure and organization of the globin genes in cellular DNA. Deletions of specific globin gene fragments have already been found in certain of these disorders and have been applied in prenatal diagnosis.

Restriction mapping of the globin genes from a homozygous delta beta thalassemia patient from Israel indicates that at least a 10-kilobase deletion is present extending 3' from within the large intervening sequence (IVS 2) of the delta globin gene and including the entire beta globin gene. Unique bands are seen when cellular DNA from this patient is digested with a variety of restriction endonucleases and hybridized with a probe specific for the delta IVS 2. Extensive analysis of the Israeli delta beta thalassemia DNA as well as material from an Italian delta beta thalassemia homozygote with enzymes which cleave more frequently in delta IVS 2 has localized the 5' end of the deletion to a 107-base pair region within delta IVS 2. This region contains a unique repetitive sequence (TG)4 which has been reported to be a specific recognition signal for recombination and may be involved in the formation of these mutant genes. Two homozygous delta(0) thalassemia DNA samples from Japan were also analyzed for gene rearrangements or other changes by restriction enzyme mapping. No changes from normal were seen using 14 different enzymes indicating the absence of large deletions in the region around the delta globin gene. More specifically, both the 5' and 3' splice junctions of the IVS 2 appear to be normal from hybridization of restriction fragments generated by HphI and AluI, respectively, with a delta IVS 2 specific probe. We have also shown that point mutations which could lead to termination codons are not present at codons 35, 37, 43, 61, and 121, since restriction enzymes which recognize these sites produce normal patterns. The delta(0) thalassemia phenotype in these two subjects is most likely due to a point mutation either at one of the other 24 potential termination codons not accessible to restriction analysis or to other single nucleotide changes which could either decrease delta globin gene transcription or lead to abnormal processing or transport of delta globin mRNA.

The structure and organization of the human globin genes at the nucleotide level has been established by restriction endonuclease digestion of cellular DNA, and by the isolation and purification of these genes in phage vectors. With this approach it has been possible to define alterations at the DNA level resulting in a group of inherited diseases of man known as the thalassemia syndromes, and related disorders. Combined with other known genetic and biochemical data, these studies provide a framework for understanding the pathogenesis of these disorders at the molecular level.

Restriction enzyme digestion analysis and direct human globin gene cloning have permitted analysis of the physical arrangement of nucleotide sequences within and surrounding the human globin genes. With these methods it has been shown that the linear arrangement 5' to 3' of the globin genes is G gamma-A gamma-delta-beta. The G gamma and A gamma genes are separated by about 3.5 kilobases (kb), while the A gamma and delta genes are 15 kb apart, and the delta and beta 6.5 kb apart. Each of these genes contains a large intervening sequence (IVS) of approximately 1 kb in precisely the same position between condons 104 and 105. In addition, each of these genes has a small IVS between codons 30 and 31. In homozygous delta beta thalassemia DNA, there is deletion of all of the normal delta and beta gene fragments. However, a new fragment 4.2 kb in size containing the 5' end of the delta globin gene is retained. Retention of this fragment in delta beta thalassemia, but not in HPFH is consistent with a role for sequences in this region for limiting gamma globin gene expression. Studies to date suggest that the beta + and beta 0 thalassemias will be due to a heterogeneous group of DNA defects affecting either beta globin gene transcription or beta mRNA processing. In most cases of beta + and beta 0 thalassemia DNA analyzed, there is no detectable deletion of beta or delta genes. In three India beta 0 patients, deletion of the 3' end of the beta gene has been found. Analysis of cloned beta globin genes from a patient with beta + thalasseia shows differences from normal in the fragments generated by restriction enzymes which cut frequently. Whether these differences are responsible for the defect in thalassemia or are polymorphisms unrelated to thalassemia remains to be determined.

Hemoglobin Miyada, an anti-Lepore hemoglobin, represents the protein product of a nonhomologous crossover between beta and delta genes. The mutant globin is beta-like from the N terminus to amino acid 12, and delta-like from amino acid 22 through the C terminus, thus predicting a crossover site in the first coding region of the gene. DNA analysis, using multiple restriction endonucleases and hybridization to delta and beta globin gene-specific probes, confirms that the beta delta hybrid gene 1) is located on a single chromosomal fragment between normal delta and beta genes, and 2) has 5' beta promoter sequences, delta IVS 1 and 2, and 3' coding and flanking sequences.

INTRODUCTION: After laminoplasty, difficulties with neck mobility often interfere with patients' activities of daily living (ADL). Although it has been reported that the flexion-extension range of motion significantly decreased after laminoplasty, in many studies using radiographs there were few patient-based outcomes. The purpose of this study was to reveal the frequency, severity and factors related to limitations of ADL accompanying neck mobility after laminoplasty. MATERIALS AND METHODS: A total of 58 patients were evaluated after laminoplasty to determine the frequency, severity and pre- and postoperative related factors of postoperative limitations of ADL accompanying each of three neck movements:(1) extension,(2) flexion and (3) rotation. The severity of limitations of each ADL was assessed using a questionnaire that was completed by the patient. RESULTS: Difficulties in neck movement, such as rotation (41%), extension (34%) and flexion (17%), in that order (P = 0.001), caused limitations of ADL. The most relevant factor of limitations of ADL accompanying extension, flexion, and rotation were small postoperative O-C7 range of motion (P = 0.0001), small preoperative O-C7 range of motion (P = 0.001), and small postoperative rotation range of motion (P = 0.0005), respectively. CONCLUSION: There were more than a few patients with limitations of ADL accompanying reduced neck mobility after laminoplasty. This knowledge may be useful in the clinical outcomes of cervical laminoplasty.

Studies of the human hemoglobin system have provided new insights into the regulation of expression of a group of linked human genes, the gamma-delta-beta-globin gene complex in man. In particular, the thalassemia syndromes and related disorders of man are inherited anemias that provide mutations for the study of the regulation of globin gene expression. New methods, including restriction enzyme analysis and cloning of cellular DNA, have made it feasible to define more precisely the structure and organization of the globin genes in cellular DNA. Deletions of specific globin gene fragments have already been found in certain of these disorders and have been applied in prenatal diagnosis.

Nucleotide sequence analysis of a cloned deletion beta-globin gene from a patient with beta(0)-thalassemia demonstrates a 619 nucleotide deletion extending from the 3' third of the second intervening sequence through 209 bases of 3' flanking DNA. However, an additional novel heptanucleotide was identified between the deletion endpoints, suggesting a complex etiology for this rearrangement.

We used a restriction endonuclease to analyze the beta-thalassemia gene in Sardinia. When we digested human DNA with the restriction enzyme Bam HI, the beta-globin gene split into a 5' portion contained in a fragment of DNA 1.8 kb in length and a 3' portion in a fragment 9.3 kb in length. In some subjects, a variation in the nucleotide sequence affecting the site recognized by this enzyme on the 3' side of the beta-globin gene resulted in a different fragment, 22 kb in length, which contained the 3' portion of the beta-globin gene. In Sardinians without beta-thalassemia, the frequency of the 9.3-kb fragment was 0.67, and that of the 22-kb fragment was 0.33. In contrast, all the beta 0-thalassemia genes were associated exclusively with the 9.3-kb fragment. Thus, the beta 0-thalassemia lesion in Sardinians apparently arose on a chromosome that had the 9.3-kb Bam HI fragment. This observation can be used in prenatal diagnosis of beta 0-thalassemia in Sardina, since demonstration of the 22.0-kb fragment would indicate the normal beta-globin genotype and exclude the beta 0-thalassemia lesion on that chromosome.(N Engl J Med 302:185-188, 1980).

We have used restriction endonuclease mapping to study a deletion involving the beta-globin gene cluster in a Mexican-American family with gamma delta beta-thalassemia. Analysis of DNA polymorphisms demonstrated deletion of the beta-globin gene from the affected chromosome. Using a DNA fragment that maps greater than 40 kilobases (kb) 5' to the epsilon-gene as a probe, reduced amounts of normal fragments were found in the DNA of affected family members. Similar analysis using radiolabeled DNA fragments located 3' to the beta-globin cluster has shown that the deletion extends more than 17 kb 3' to the beta-gene, but terminates before the 3' endpoint of the Ghanian HPFH deletion. Hence, this gamma delta beta-thalassemia deletion eliminates over 105 kb of DNA and is the first report of a deletion of the entire beta-globin gene cluster.

In this study we have characterized by DNA analysis the molecular basis of an alpha-thalassemia condition found in an infant, with 16% Hb Bart's at birth, who developed an hematologic picture similar to the alpha-thalassemia carrier state. Restriction endonuclease analysis and hybridization with alpha and zeta specific probes have provided strong evidence that this patient carries a genetic compound of deletion alpha-thalassemia-2 lesion (-alpha) and a non-deletion defect [(alpha alpha)th] with both alpha-structural genes intact on chromosome 16. He inherited the deletion alpha-thalassemia-2 chromosome (-alpha) from the father and the chromosome with non-deletion alpha-thalassemia defect from the mother. Because the deletion of one, two, or three alpha-globin structural genes is associated with 1-2%, 5-6%, or 25%, Hb Bart's respectively, these findings suggest that the non-deletion chromosome [(alpha alpha)th] contains two alpha-globin structural genes that are less active than a single alpha gene (-alpha).

A unique beta 0-thalassaemia in a Dutch family results in fetal haemoglobin expression comparable to that of delta 0 beta 0-thalassaemia. Haemoglobin analysis and restriction endonuclease mapping studies of DNA suggest that the beta-globin gene is entirely deleted, but that the delta-globin gene is intact. The 5' break point of the deletion is 3-4 kilobases 3' to the delta-globin gene, while the 3' break point is 6-7 kilobases 3' to the beta-globin gene (relative to the normal DNA restriction map). The result is a approximately 10 kilobase deletion of DNA whose 3' end point may lie very close to that for one delta 0 beta 0-thalassaemia, within a cluster of Kpn I-family repetitive sequences. The Dutch beta 0-thalassaemia deletion is thus the shortest one which, in the absence of additional chromosomal rearrangements, results in enhancement of gamma-chain synthesis above that seen for haemoglobin Lepore. These data support the hypothesis that the region of DNA 3' to the beta-globin gene may be important to the developmental regulation of fetal gamma versus adult beta chain production.

The beta-globin gene present on the deletion locus in a Dutch gamma beta-thalassaemic patient was found to be identical to the normal beta-globin gene with respect to DNA sequence and its transcription in HeLa cells. DNase I sensitivity and methylation experiments show that the affected beta-globin gene is present in an inactive configuration in vivo. This is the result of a translocation of a normally inactive locus next to the beta-globin gene on the affected chromosome, or the deletion of sequences which are normally required for the maintenance of the active state.