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L1 elements constitute a highly repetitive human DNA family (50,000 to 100,000 copies) lacking long terminal repeats and ending in a poly(A) tail. Some L1 elements are capable of retrotransposition in the human genome (Kazazian, H. H., Jr., C. Wong, H. Youssoufian, A. F. Scott, D. G. Phillips, and S.E. Antonarakis, Nature (London) 332:164-166, 1988). Although most are 5' truncated, a consensus sequence of complete L1 elements is 6 kb long and contains two open reading frames (ORFs) (Scott, A. F., B. J. Schmeckpeper, M. Abdelrazik, C. T. Comey, B. O'Hara, J. P. Rossiter, T. Cooley, P. Health, K. D. Smith, and L. Margolet, Genomics 1:113-125, 1987). The protein encoded by ORF2 has reverse transcriptase (RT) activity in vitro (Mathias, S. L., A. F. Scott, H. H. Kazazian, Jr., J. D. Boeke, and A. Gabriel, Science 254:1808-1810, 1991). Because L1 elements are so numerous, efficient methods for identifying active copies are required. We have developed a simple in vivo assay for the activity of L1 RT based on the system developed by Derr et al. (Derr, L. K., J. N. Strathern, and D. J. Garfinkel, Cell 67:355-364, 1991) for yeast HIS3 pseudogene formation. L1 ORF2 displays an in vivo RT activity similar to that of yeast Ty1 RT in this system and generates pseudogenes with unusual structures. Like the HIS3 pseudogenes whose formation depends on Ty1 RT, the HIS3 pseudogenes generated by L1 RT are joined to Ty1 sequences and often are part of complex arrays of Ty1 elements, multiple HIS3 pseudogenes, and hybrid Ty1/L1 elements. These pseudogenes differ from those previously described in that there are base pairs of unknown origin inserted at several of the junctions. In two of three HIS3 pseudogenes studied, the L1 RT appears to have jumped from the 5' end of a Ty1/L1 transcript to the poly(A) tract of the HIS3 RNA.

Single nucleotide substitutions in the promoter regions of the A gamma- and G gamma-globin genes have been associated with increased fetal hemoglobin (HbF) production. We wished to determine whether these or other unrecognized substitutions in the gamma promoter regions are responsible for the 20-fold variation in HbF production in sickle cell patients or normal adults. From a random sampling of 250 sickle cell (SS) patients and 125 normal adults, 17 individuals representing the highest and lowest HbF producers were selected for study. All three common restriction fragment length polymorphism beta-globin region haplotypes (Benin, Central African Republic, and Senegal) were found in both the highest and lowest HbF producers with SS disease. Using the polymerase chain reaction amplification and direct sequencing of the amplified DNA product, we examined the promoter regions of both the A gamma and G gamma genes from -350 bp to +50 bp of the CAP site. No mutations were found in either gamma gene promoter region. We conclude that nucleotide substitutions in the promoter regions (-350 to +50 bp) of both gamma genes are not responsible for the marked variation in HbF production among SS or normal individuals.

Summary. A major obstacle for delivery of factor (F)VIII using adeno-associated virus (AAV) vectors is the large size of FVIII cDNA, which is well above the 5 kb packaging limit for AAV. Here we construct a

What are all of those retrotransposons doing buzzing about in our genome? According to Kazazian, in his Perspective this week, these mobile pieces of DNA are busy reshaping our genome, making it more diverse and enabling us to survive and thrive through the vagaries of evolution. And just how do they do this?...zip to page 1152 to find out.

To test the hypothesis that factor VIII expressed in the epidermis can correct hemophilia A, we generated transgenic mice in a factor VIII-deficient background that express human factor VIII under control of the involucrin promoter. Mice from 5 transgenic lines had both phenotypic correction and plasma factor VIII activity. In addition to the skin, however, some factor VIII expression was detected in other tissues that have stratified squamous epithelia. To determine whether an exclusively cutaneous source of factor VIII could correct factor VIII deficiency, we grafted skin explants from transgenic mice onto mice that are double knockouts for the factor VIII and RAG-1 genes. Two graft recipients had plasma factor VIII activity of 4% to 20% of normal and improved whole blood clotting compared with factor VIII-deficient mice. Thus, expression of factor VIII from the epidermis can correct hemophilia A mice, thereby supporting the feasibility of cutaneous gene therapy for systemic disease. (Blood. 2000;95:2799-2805)

Using chemical mismatch analysis or denaturing gradient gel electrophoresis followed by nucleotide sequencing, we have identified the same G6545A mutation leading to an Arg2163 His subsitution in the factor VIII gene of three haemophiliacs from unrelated families. One of the affected individuals has severe haemophilia, while the other two are moderately severe. While we cannot exclude the possibility that these differences in phenotype arise from differences in VIII:C assay methods, other studies have also identified different clinical phenotypes in individuals with the same mutations, and suggested that they may arise from extragenic factors that affect or modify gene expression or protein function. The G6545A mutation occurs at a CG dinucleotide which is a known mutation hotspot, and which may explain the independent occurrence in unrelated families.

Factor VIII (FVIII) is the protein defective in the bleeding disorder hemophilia A. Approximately 5% of hemophilia A patients have normal amounts of a dysfunctional FVIII protein and are termed cross-reacting material (CRM)-positive. The majority of genetic alterations that result in CRM-positive hemophilia A are missense mutations within the A2-domain. To determine the mechanistic basis of the genetic defects within the A2-domain for FVIII function we constructed six mutations within the FVIII cDNA that were previously found in five CRM-positive hemophilia A patients (R527W, S558F, I566T, V634A, and V634M) and one CRM-reduced hemophilia A patient (DeltaF652/3). The specific activity for each mutant secreted into the conditioned medium from transiently transfected COS-1 cells correlated with published data for the patients plasma-derived FVIII, confirming the basis of the genetic defect. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated FVIII protein radiolabeled in COS-1 cells showed that all CRM-positive mutant proteins were synthesized and secreted into the medium at rates similar to wild-type FVIII. The majority of the DeltaF652/3 mutant was defective in secretion and was degraded within the cell. All mutant FVIII proteins were susceptible to thrombin cleavage, and the A2-domain fragment from the I566T mutant had a reduced mobility because of use of an introduced potential N-linked glycosylation site that was confirmed by N-glycanase digestion. To evaluate interaction of FVIII with factor IXa, we performed an inhibition assay using a synthetic peptide corresponding to FVIII residues 558 to 565, previously shown to be a factor IXa interaction site. The concentration of peptide required for 50% inhibition of FVIII activity (IC50) was reduced for the I566T (800 mumol/L) and the S558F (960 mumol/L) mutants compared with wild-type FVIII (2,000 mumol/L). N-glycanase digestion increased I566T mutant FVIII activity and increased its IC50 for the peptide (1,400 mumol/L). In comparison to S558F, a more conservative mutant (S558A) had a sixfold increased specific activity that also correlated with an increased IC50 for the peptide. These results provided support that the defects in the I566T and S558F FVIII molecules are caused by steric hindrance for interaction with factor IXa.

The age of patients with homozygous beta-thalassemia is increasing because of better treatment and decreased births. A countering influence is immigration of ethnic groups with a high prevalence of thalassemia.a questionnaire sent to 48 North American centers requested information about current patients with homozygous beta-thalassemia: age, clinical severity, and ethnicity. An 83% response was obtained. Twelve reference hospitals that participated in similar surveys in 1972 and 1984 were included.Five hundred eighteen patients with homozygous beta-thalassemia represent most North American patients. Four hundred forty-three (86%) of these had transfusion-dependent thalassemia major (TM); 75 (14%) had thalassemia intermedia (TI). Sixty-two percent were of Greek and Italian ancestry. There were approximately equal numbers of patients with TM in 5-year intervals between 0 and 25 years of age. Thereafter, the numbers of patients fell sharply. The mean age (+/- SD) of the patients with TM was 16.1 +/- 9.2 years. Striking differences were seen in Italian and Greek patients compared with those of other ancestries. Sixty-six percent of the 271 Italian and Greek patients with TM were older than 16 years of age, whereas 77% of the 172 patients of other ethnic groups with TM were younger than 15 years of age. The mean age of the 75 patients with TI was greater than that of the patients with TM. Seventy-three percent of African-American patients had TI, compared with 0% of Southeastern Asian patients. Comparisons of patients with TM from the 12 reference hospitals for two decades show increasing mean ages of TM patients (1973, 11.4 +/- 6.7 years; 1985, 14/2 +/- 7.3 years; and 1993, 16.1 +/- 9.2 years).There are probably only 750 to 1000 patients with homozygous beta-thalassemia in North America. Only about 15 to 20 new cases are diagnosed each year. The increasing mean age and age distribution indicate that modern therapies are effective, but immigration of non-Mediterranean ethnic groups with thalassemia has resulted in more, younger patients. TM is increasingly becoming a disease of young adults.

We have previously described a unique type of delta beta-thalassemia in a Chinese family characterized by increased expression of the G gamma and A gamma fetal globin genes in the absence of a large deletion in the beta-globlin gene cluster. Our earlier study of the beta-globin gene on this delta beta-thalassemia chromosome showed a promoter mutation in the TATA box. In this report, we describe the results of our study of the fetal globin domain of this delta beta-thalassemia chromosome. We have cloned a 13-kb DNA fragment that includes the G gamma and the A gamma genes and the 3' A gamma enhancer element of this delta beta-thalassemia chromosome. DNA sequence analysis of the G gamma and A gamma-globin genes including their promoters did not show any mutations, but analysis of the putative enhancer element downstream from the A gamma-globin gene showed a C to T substitution 2,401 nucleotides downstream from the A gamma cap site. We performed DNA linkage analysis to determine if this mutation is unique to this chromosome or represents a common polymorphism. Our linkage analysis showed that this mutation is not a common polymorphism and that it is also not an intrinsic part of the haplotype of the chromosome on which it was found. We also studied the interaction of nuclear proteins from erythroid and nonerythroid cells with the DNA sequences surrounding this mutation. We have shown by in vitro DNase I footprinting that this mutation falls within a region that is occupied by a novel DNA-binding protein that binds to this site in nuclear extracts from erythroid, but not nonerythroid cells. The binding of this nuclear protein to DNA appears to be dependent on GATA-1 binding to an adjacent GATA-1 site. We have also developed a new functional assay to compare the activity of the normal and mutant A gamma enhancer elements in erythroid cells. Analysis of the activity of the mutant enhancer shows that the mutation completely eliminates all enhancer activity in this assay. These findings suggest that this mutation of the A gamma enhancer on a chromosome that carries a partially inactivated beta-globin gene may be responsible for the increased expression of both gamma-globin genes seen in this condition.

Mutations producing beta-thalassemia reach individual gene frequencies greater than .01 in malarial-endemic regions because beta-thalassemia trait individuals have increased genetic fitness over that of normal individuals. Exon 3 of the beta-globin gene has been relatively spared as a site of common beta-thalassemia mutations. Frameshifts caused by the loss of a single nucleotide and nonsense mutations produce beta-thalassemia trait when they occur in exons 1 and 2. In contrast, they usually produce chronic hemolytic anemia when present in exon 3. Certain missense mutations in exon 3 produce unstable globins and thalassemia intermedia with hemolysis in heterozygotes. Here we report two new mutations in exon 3 of the beta-globin gene. One is a single nucleotide deletion in codon 109 in a 78-year-old Lithuanian with chronic hemolytic anemia and features of thalassemia. It leads to an abnormal globin (beta Manhattan) that is elongated to 156 amino acids. The second is a CAG-CGG missense mutation at codon 127 that causes a Gln----Pro substitution (beta Houston) and a thalassemia intermedia with hemolysis in three generations of a British-American family. Although the clinical phenotypes of these two patients differed little, differences in globin-synthetic ratios were significant, presumably reflecting differences in the ability of each abnormal beta-globin to form alpha beta dimers. The paucity of high-frequency exon 3 mutations and their worldwide distribution is likely attributable to their phenotypic severity and loss of increased genetic fitness vis-a-vis malaria.

We studied the mechanism of ring chromosome 21 (r(21)) formation in 13 patients (11 unique r(21)s), consisting of 7 from five families with familial r(21) and 6 with de novo r(21). The copy number of chromosome 21 sequences in the rings of these patients was determined by quantitative dosage analyses for 13 loci on 21q. Nine of 11 r(21)s, including the 5 familial r(21)s, showed no evidence for duplication of 21q sequences but did show molecular evidence of partial deletion of 21q. These data were consistent with the breakage and reunion of short- and long-arm regions to form the r(21), resulting in deletion of varying amounts of 21q22.1 to 21qter. The data from one individual who had a Down syndrome phenotype were consistent with asymmetric breakage and reunion of 21q sequences from an intermediate isochromosome or Robertsonian translocation chromosome as reported by Wong et al. Another patient, who also exhibited Down syndrome, showed evidence of a third mechanism of ring formation. The likely initial event was breakage and reunion of the short and long arms, resulting in a small r(21), followed by a sister-chromatid exchange resulting in a double-sized and symmetrically dicentric r(21). The phenotype of patients correlated well with the extent of deletion or duplication of chromosome 21 sequences. These data demonstrate three mechanisms of r(21) formation and show that the phenotype of r(21) patients varies with the extent of chromosome 21 monosomy or trisomy.

Samples of genomic DNA from three unrelated American black infants having both biochemical and clinical features of classical infantile Tay-Sachs disease were sequenced following PCR amplification. A G----T transversion was observed in the AG acceptor splice site preceding exon 5 of the beta-hexosaminidase alpha-subunit gene in the first black family. This transversion changed the acceptor splice site from the consensus sequence, AG, to AT, thereby interfering with splicing at this intron 4/exon 5 junction. The proband was homozygous for this mutation; his mother and a brother are heterozygous. The same mutation was found in a second, apparently unrelated, black GM2-gangliosidosis patient. The second patient was a compound heterozygote, as only one allele carried this mutation. The mother and a brother in this second family are carriers for this mutation, while the father and a noncarrier sister are normal for this region of the gene. The third proband did not have this mutation; nor did the mother of a fourth black proband. Eight other independently ascertained non-black, non-Jewish, GM2-gangliosidosis families did not have this mutation. The observation of the same novel mutation in two unrelated black GM2-gangliosidosis patients indicates that the American black population has segregating within it at least one GM2-gangliosidosis mutation which may be specific to this population and not a result of migration.

In this review I have discussed the state of our knowledge of the molecular basis of beta-thalassemia and its prenatal diagnosis. Improved but more complicated genetic counselling is now available as a result of our increased knowledge of the effects of various defects in the beta-globin gene. Our knowledge of the heterogenous molecular basis of the thalassemia syndromes has become very impressive and it is hoped that effective therapy will soon follow. However, for the present, prevention of the birth of affected children is the most effective means of reducing the suffering associated with the thalassemia syndromes, and prevention of this type is succeeding in many parts of the world, including North America.

We have used molecular and cytogenetic methods to study the derivation of a ring chromosome 13 in the fetus of a woman mosaic for a translocation chromosome 13. DNA analysis showed that the translocation chromosome was a Robertsonian translocation, not an isochromosome. We suggest that the ring is derived from the translocation chromosome by breaks in both long arms and subsequent reunion, r(13) (q12q14).

In this review I have outlined the molecular basis and prenatal diagnosis of alpha-thalassemia and then concentrated on the state of our knowledge of the molecular basis of beta-thalassemia and its prenatal diagnosis. I discussed the improved but more complicated genetic counselling now available as a result of our increased knowledge of the effects of various defects in the beta-globin gene. Our knowledge of the heterogeneous molecular basis of the thalassemia syndromes has become very impressive and it is hoped that effective therapy will soon follow. For the present, however, prevention of the birth of affected children is the most effective means of reducing the suffering associated with the thalassemia syndromes, and prevention of this type is succeeding in many parts of the world, including North America.

The molecular defect responsible for moderate and severe hemophilia A has been identified for two unrelated patients with the CRM-positive form of this disorder (factor VIII activity of 0.02 and 0.05 U/mL with factor VIII antigen of 0.87 and 2.20 U/mL). In both cases, the immunopurified dysfunctional factor VIII protein is abnormal, in that the 80 Kd light chain is not cleaved by thrombin at arginine-1689. The basis for this failure was identified by polymerase chain reaction amplification of exon 14 of the variant factor VIII genes and direct sequencing of the amplified products. In both cases, a single base substitution (C to T) was identified that produces an arginine to cysteine substitution at amino acid residue 1689. These data identify the molecular defects of the two identical factor VIII variant proteins. The dysfunctional factor VIII has been designated "Factor VIII-East Hartford," the residence of the patient in whom the defect was first identified.

In 1989 we are continuing to move gene diagnosis over to the direct detection mode. We have sickle cell anemia, alpha-thalassemia, beta-thalassemia, Duchenne muscular dystrophy, Becker muscular dystrophy and cystic fibrosis moved to direct detection with hemophilia B and alpha-1-antitrypsin deficiency soon to be there. For indirect detection, we still have hemophilia A, and a comment on the genetics of hemophilia A is important. Remember that sickle cell anemia is caused by one mutation, while beta-thalassemia and cystic fibrosis have a finite number of alleles. Duchenne muscular dystrophy results from a different mutation for every affected individual, but most of these are deletions and can be directly detected. Hemophilia A is another X-linked disorder with almost every affected individual having a different mutation. That means that there probably are 100 ways to get beta-thalassemia and about 10,000 ways to get hemophilia A, so we need some really good novel techniques to detect these directly, and we are working hard on such techniques. I would not be surprised if hemophilia A moved into the direct detection category in the next year or so. We need to find the Huntington disease gene, and then it will move into the direct detection column. Neurofibromatosis is still in the indirect detection group but also may move very soon. Polycystic kidney disease is also still in the indirect detection column. This summarizes where prenatal and presymptomatic gene diagnosis stands in late 1989.

Gene probes can now be used to detect a variety of mutations that produce single-gene disorders. In present clinical practice, restriction endonuclease analysis is used for the prenatal diagnosis of sickle cell anemia, alpha-thalassemia, and beta-thalassemia. Direct detection of the mutation is possible in alpha-thalassemia, where a deletion has usually occurred, and in sickle cell anemia, where the mutation alters the recognition sequence of the restriction endonuclease, Mst II. Indirect detection of beta-thalassemia is based on using normal variations in DNA (DNA polymorphisms) to track normal and affected beta-globin genes in families. This latter kind of analysis is also useful in detecting the phenylalanine hydroxylase genes affected in phenylketonuria and will often be used in disorders where the mutations are unknown. In cases where the mutation is known, direct analysis by use of oligonucleotide probes is a new and important advance. An example of this type of gene detection in a family with classical hemophilia is presented. In addition, with chorion villus biopsy, detection of these inherited diseases is feasible by the 12th week of pregnancy.

To determine whether hemoglobin regulation is normal in diseases affecting beta-globin gene expression, globin synthesis was examined in members of a family of a patient with hereditary persistence of fetal hemoglobin/beta o-thalassemia (HPFH/beta o-thal). The HPFH defect is the Ghanian type II, with a deletion from psi beta 1 to at least 20 kb 3′ to beta. The beta o-thal gene has the haplotype II restriction enzyme pattern and has the beta 39 nonsense mutation. Erythroid colonies from blood BFU-E were radiolabeled, and globin chains were separated by gel electrophoresis. Colonies from the beta o-thal heterozygote had non-alpha/alpha ratios more balanced than in the reticulocytes. Gamma synthesis was 11% of non-alpha, which is higher than in reticulocytes, but within the range seen in normal adult colonies. Both HPFH heterozygotes produced 20%-30% gamma in erythroid colonies as well as reticulocytes, although non-alpha/alpha was more balanced in the colonies. The HPFH/beta o-thal patient produced 100% gamma in reticulocytes and in colonies. G gamma and gamma-synthetic proportions were not correlated at the individual colony level in the heterozygotes, suggesting that they had “adult” and not “fetal” progenitor cells. The Hb expression of these adult progenitors is presumably modulated normally in vivo in beta o-thal, but the normal decrease in HbF production does not occur in gene deletion HPFH.

Using DNA polymorphisms adjacent to single-copy genomic fragments derived from human chromosome 21, we initiated the construction of a linkage map of human chromosome 21. The probes were genomic EcoRI fragments pW228C, pW236B, pW231C and a portion of the superoxide dismutase gene (SOD-1). DNA polymorphisms adjacent to each of the probes were used as markers in informative families to perform classical linkage analysis. No crossing-over was observed between the polymorphic sites adjacent to genomic fragments pW228C and pW236B in 31 chances for recombination. Therefore, these fragments are closely linked to one another (theta = 0.00, lod score = 6.91, 95% confidence limits = 0-10 cM) and can be treated as one 'locus' with four high-frequency markers. There is a high degree of non-random association of markers adjacent to each of these two probes which suggests that they are physically very close to one another in the genome. The pW228C - pW236B 'locus' was also linked to the SOD-1 gene (theta = 0.07, lod score = 4.33, 95% confidence limits = 1-20 cM). On the other hand, no evidence for linkage was found between the pW228C-pW236B 'locus' and the genomic fragment pW231C (theta = 0.5, lod score = 0.00). Based on the fact that pW231C maps to 21q22.3 and SOD-1 to 21q22.1, we suggest that the pW228C-pW236B 'locus' lies in the proximal long arm of chromosome 21. These data provide the outline of a linkage map for the long arm of chromosome 21, and indicate that the pW228C-pW236B 'locus' is a useful marker system to differentiate various chromosome 21s in a population.

The 5'-terminal structures of murine alpha- and beta-globin mRNA were determined after incubating cells of the erythropoietic spleens of mice with [methyl-3H]methionine. Globin mRNA was obtained from total cellular RNA by oligo(dT)-cellulose chromatography followed by elution of mRNA from formamide gels after electrophoresis. The globin mRNA was then hydrolyzed with KOH or digested with a combination of RNase T2 and bacterial alkaline phosphatase, and 5'-terminal structures were isolated by DEAE-cellulose chromatography. The methylated nucleotides of these 5'-structures were determined following digestion with specific ribonucleases and bacterial alkaline phosphatase. Analyses of mRNA fractions enriched for either alpha- or beta-mRNA gave similar results. Our data indicate that murine alpha- and beta-globin mRNAs are identical through the first three nucleotides and that partial dimethylation exists at the second position: m7G(5')ppp(5') [m6Am/Am]pCmpNp.

The order of methylation of the 5'-terminus of globin mRNA of mice was studied by incubation of staged nucleated erythroid cells and peripheral reticulocytes with [methyl-3H] methionine. Methylation of the 5'-termini of alpha and beta- globin mRNAs in enucleated reticulocytes was demonstrated as follows: (a) [methyl-3H] incorporation into poly(A)+ RNA of reticulocytes co-migrated with the alpha- and beta- globin mRNAs on gel electrophoresis, and (b) following digestion of this RNA, radioactivity was localized to the four methyl sites at the 5'-capped structure of mouse globin mRNAs. However, this methylation is only 5 to 8% as efficient as in nucleated erythroid precursor cells, suggesting that most globin mRNA molecules are fully methylated prior to the reticulocyte stage. Incubations of early and late nucleated erythroid precursor cells and pulse-chase experiments with reticulocytes demonstrate that addition of the four 5'-terminal methyl groups follows an orderly sequence. In addition, the pulse-chase experiments suggest the turnover of the N7-methyl group on the 5'-terminal guanosine, but not of the other methyl groups in the 5'-terminus of globin mRNA. Thus, 5'-terminal methylation of globin mRNA is a nonrandom, dynamic process.

Relative amounts and rates of synthesis of alpha- and beta-globin mRNAs were determined during splenic erythropoiesis in mice. At times after injection of mice with phenylhydrazine, alpha- and beta-globin mRNAs were separated by gel electrophoresis and quantitated by densitometric scanning of stained gels. At 66 hr after injection, the ratio of beta to alpha mRNA is about 1.2. By 138 hr, total globin mRNA is 5-fold greater in spleen cells, and the beta to alpha mRNA ratio approaches 2. This ratio remains around 1 in reticulocytes throughout this period. Analyses of globin products directed by these mRNAs from spleen cells and reticulocytes in the ascites cell-free system reflect the beta to alpha mRNA ratio observed by electrophoresis. Relative rates of synthesis of globin mRNAs were estimated after incubation of spleen cells with either [3H] uridine or [3H] adenosine. Although synthesis of both mRNAs is maximal at 114 hr and then declines sharply, beta mRNA is synthesized at a greater rate than alpha mRNA at every developmental stage. In contrast to the excess accumulation of beta mRNA in spleen cells, synthesis of alpha- and beta-globin chains remains balanced throughout erythroid development. These data suggest that during erythropoiesis in this system, equal synthesis of alpha and beta globin involves regulation at both transcriptional and post-transcriptional levels.

We have characterized the structural rearrangements of a chromosome 21 that led to the de novo formation of a human ring chromosome 21 [r(21)]. Molecular cloning and chromosomal localization of the DNA regions flanking the ring junction provide evidence for a long arm to long arm fusion in formation of the r(21). In addition, the centromere and proximal long arm region of a maternal chromosome 21 are duplicated in the r(21). Therefore, the mechanism in formation of the r(21) was complex involving two sequential chromosomal rearrangements. (i) Duplication of the centromere and long arm of one maternal chromosome 21 occurred forming a rearranged intermediate. (ii) Chromosomal breaks in both the proximal and telomeric long arm regions on opposite arms of this rearranged chromosome occurred with subsequent reunion producing the r(21).

After incubating peripheral blood of various anemic individuals with [methyl-3H]methionine, 5'-terminal cap structures and the sequential methylation of human globin mRNAs were determined. Human alpha- and beta-globin mRNAs have the same methylated nucleotides at their 5' ends as the alpha- and beta-globin mRNAs of mouse and rabbit: m7G(5')ppp(5')[m6Am/Am]pCmp. The order of methyl group addition to human globin mRNA is also similar to that of mouse globin mRNA in that methylation of the 5'-terminal guanosine and the ribose of adenosine occurs earlier than that of the base of adenosine and the ribose of cytidine. Thus, both the cap structures and the order of methylation are conserved in the globin mRNAs of these species, suggesting that variation in the structure of the 5' terminus of globin mRNAs may alter the function of these molecules.

The six loci, beta-globin (HBBC), parathyroid hormone (PTH), oncogene c-Ha-ras-1 (HRAS1), insulin (INS), calcitonin (CAL) and catalase (CAT) loci, have been mapped to 11p in the order: CAT-CAL-PTH-HBBC-(HRAS1-INS). The purpose of the current study was to examine the linkage relationships, especially the multipoint relationships of these loci in detail. In the 18 families studied, a significant sex difference in recombination was found for the HBBC: HRAS1 linkage with more recombination in the male parent than the female parent (22 vs. 2%). The results of the multipoint analyses provided further evidence for the order CAT-CAL-PTH-HBBC-(HRAS1-INS); however, the order of the last two tightly linked loci is still not clear.

DETECTION of sickle hemoglobin in the human fetus was first accomplished nearly 10 years ago.1 , 2 This marked the beginning of a technology for prenatal diagnosis of the hemoglobinopathies. When m...

The cloned beta-globin gene of an Indian patient with beta-thalassemia revealed a 25-nucleotide deletion at the 3'-end of the first intervening sequence, including the acceptor RNA splicing site. RNA transcripts of this mutant gene produced following transfection into HeLa cells remained unspliced at both the first intervening sequence donor and acceptor sites. This beta-thalassemic gene is the first in which critical sequences of an acceptor splice junction are mutated and associated with abnormal RNA processing.

A base substitution (C----T) at position -88 relative to the cap site was identified in the beta-globin gene cloned from an individual with a mild form of beta-thalassemia. This nucleotide change lies in the sequence ACACCC proposed as a distal promoter element. Transient expression of the mutant gene in HeLa cells revealed a modest deficit in beta-globin mRNA production. RNA processing was normal. The -88 beta-thalassemia mutation lends further support for the in vivo role of the distal element in transcription.

In this chapter I have tried to relate the salient features of globin gene structure, genomic organization, normal variation, and mutations affecting gene expression. The lessons learned from the normal beta-globin gene cluster and mutations producing beta-thalassemia should be highly applicable to studies of other inherited diseases. As more and more gene probes become available, which have relevance to the study of human disease, the striking extent of genetic heterogeneity producing single gene disorders of man will be illuminated.

We have studied the spectrum of mutations producing beta-thalassemia (beta-thal) in South China and Southeast Asia in two groups of patients. In randomly selected patients with beta-thal major we characterized 78 beta-thal genes. In patients with beta-thal intermedia, 22 beta-thal genes were studied. The relevant mutation was characterized in all 78 genes of the first group, and 21 of 22 (96%) of mutant genes in the second group. Eight point mutations were found among the 100 genes studied. Of these eight alleles, four constituted 90% of the total. Prenatal diagnosis of beta-thalassemia in this region should be feasible by simplified techniques for direct detection of point mutations.

The cloned beta-globin gene of an Indian patient with beta-thalassemia revealed a 25-nucleotide deletion at the 3'-end of the first intervening sequence, including the acceptor RNA splicing site. RNA transcripts of this mutant gene produced following transfection into HeLa cells remained unspliced at both the first intervening sequence donor and acceptor sites. This beta-thalassemic gene is the first in which critical sequences of an acceptor splice junction are mutated and associated with abnormal RNA processing.

Strong linkage disequilibrium (LD) was found between DNA marker XV2c and the cystic fibrosis (CF) locus (delta = 0.46) and between DNA marker KM19 and CF (delta = 0.67) in 157 CF and 138 normal chromosomes from U.S. Caucasians. DNA haplotypes with nine polymorphic sites were created in 54 Caucasian families. There is a strong LD between the haplotypes and the presence of the mutant CF genes. This implies that the DNA polymorphisms examined are close to the CF gene and that one mutation of the CF gene predominates in the Caucasian population. Haplotype analysis can also be used to refine estimates of CF carrier risk in Caucasians. Data for XV2c and MET markers in 16 American black patients and their families revealed a different haplotype distribution and LD pattern with the CF locus. These data suggest that racial admixture alone does not explain the occurrence of CF in American blacks and that multiple alleles of the CF gene may exist in this population.

The molecular characterization of mutations producing beta-thalassemia in world populations is nearing completion. We expect that new rare alleles in thoroughly studied groups and other alleles in less studied groups, eg, inhabitants of New Guinea, Latin America, and certain Pacific Islands, will be found. Knowledge of the molecular basis of the disease and new technology that allows rapid detection of single nucleotide changes in genomic DNA have led to the reality of prenatal diagnosis by direct mutation detection even in the heterogeneous US population. Programs aimed at prevention of beta-thalassemia should be facilitated by these developments.

Hemophilia A is an X-linked disease of coagulation caused by deficiency of factor VIII. Using cloned cDNA and synthetic oligonucleotide probes, we have now screened 240 patients and found CG-to-TG transitions in an exon in nine. We have previously reported four of these patients; and here we report the remaining five, all of whom were severely affected. In one patient a TaqI site was lost in exon 23, and in the other four it was lost in exon 24. The novel exon 23 mutation is a CG-to-TG substitution at the codon for amino acid residue 2166, producing a nonsense codon in place of the normal codon for arginine. Similarly, the exon 24 mutations are also generated by CG-to-TG transitions, either on the sense strand producing nonsense mutations or on the antisense strand producing missense mutations (Arg to Gln) at position 2228. The novel missense mutations are the first such mutations observed in association with severe hemophilia A. These results provide further evidence that recurrent mutations are not uncommon in hemophilia A, and they also allow us to estimate that the extent of hypermutability of CG dinucleotides is 10-20 times greater than the average mutation rate for hemophilia A.