Search

Our recent studies on the molecular basis of the autosomal recessive disorder congenital afibrinogenemia showed that the most common mutation is a donor splice mutation in FGA intron 4, IVS4 + 1 G-->T, accounting for approximately half of disease alleles. The effect of this mutation on messenger RNA (mRNA) splicing, however, remained unproven. COS-7 cells transfected with a normal plasmid construct produced 100% mRNA molecules with correct splicing, whereas cells transfected with a mutant construct produced multiple aberrant mRNAs, due to utilization of cryptic donor splice sites situated in exon 4 and intron 4. One particular site situated 4 base pairs (bp) downstream of the normal site was used in 85% of transcripts causing afibrinogenemia by a 4-bp insertion-frameshift, leading to premature alpha-chain truncation. Our results confirm the utility of transfecting COS-7 cells to study mRNA splice-site mutations and demonstrate that the common FGA IVS4 variant is a null mutation leading to afibrinogenemia.

Congenital afibrinogenemia is a rare, autosomal, recessive disorder characterized by the complete absence of detectable fibrinogen. We previously identified the first causative mutations in a nonconsanguineous Swiss family; the 4 affected persons have homozygous deletions of approximately 11 kb of the fibrinogen alpha (FGA) gene. Haplotype data implied that these deletions occurred on distinct ancestral chromosomes, suggesting that this region may be susceptible to deletion by a common mechanism. We subsequently showed that all the deletions were identical to the base pair and probably resulted from a nonhomologous recombination mediated by 7-bp direct repeats. In this study, we have collected data on 13 additional unrelated patients to identify the causative mutations and to determine the prevalence of the 11-kb deletion. A common recurrent mutation, at the donor splice site of FGA intron 4 (IVS4 + 1 G > T), accounted for 14 of the 26 (54%) alleles. One patient was heterozygous for the previously identified deletion. Three more frameshift mutations, 2 nonsense mutations, and a second splice site mutation were also identified. Consequently, 86% of afibrinogenemia alleles analyzed to date have truncating mutations of FGA, though mutations in all 3 fibrinogen genes, FGG, FGA, and FGB, might be predicted to cause congenital afibrinogenemia.

Combined factor V-factor VIII deficiency (F5F8D) is a rare, autosomal recessive coagulation disorder in which the levels of both coagulation factors V and VIII are diminished. The F5F8D locus was previously mapped to a 1-cM interval on chromosome 18q21. Mutations in a candidate gene in this region, ERGIC-53, were recently found to be associated with the coagulation defect in nine Jewish families. We performed single-strand conformation and sequence analysis of the ERGIC-53 gene in 35 F5F8D families of different ethnic origins. We identified 13 distinct mutations accounting for 52 of 70 mutant alleles. These were 3 splice site mutations, 6 insertions and deletions resulting in translational frameshifts, 3 nonsense codons, and elimination of the translation initiation codon. These mutations are predicted to result in synthesis of either a truncated protein product or no protein at all. This study revealed that F5F8D shows extensive allelic heterogeneity and all ERGIC-53 mutations resulting in F5F8D are "null." Approximately 26% of the mutations have not been identified, suggesting that lesions in regulatory elements or severe abnormalities within the introns may be responsible for the disease in these individuals. In two such families, ERGIC-53 protein was detectable at normal levels in patients' lymphocytes, raising the further possibility of defects at other genetic loci.

Twenty-two molecular diagnostic laboratories from 14 countries participated in a consortium study to estimate the impact of Factor VIII gene inversions in severe hemophilia A. A total of 2,093 patients with severe hemophilia A were studied; of those, 740 (35%) had a type 1 (distal) factor VIII inversion, and 140 (7%) showed a type 2 (proximal) inversion. In 25 cases, the molecular analysis showed additional abnormal or polymorphic patterns. Ninety-eight percent of 532 mothers of patients with inversions were carriers of the abnormal factor VIII gene; when only mothers of nonfamilial cases were studied, 9 de novo inversions in maternal germ cells were observed among 225 cases (approximately 1 de novo maternal origin of the inversion in 25 mothers of sporadic cases). When the maternal grandparental origin was examined, the inversions occurred de novo in male germ cells in 69 cases and female germ cells in 1 case. The presence of factor VIII inversions is not a major predisposing factor for the development of factor VIII inhibitors; however, slightly more patients with severe hemophilia A and factor VIII inversions develop inhibitors (130 of 642 [20%]) than patients with severe hemophilia A without inversions (131 of 821 [16%]).

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.

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.

Hemoglobin beta Knossos (beta 27Ala-Ser) is a cause of beta-thalassemia due to its reduced synthesis. To investigate the basis for this observation, we have isolated the beta Knossos gene and examined its expression in heterologous cells. We have found that some beta Knossos RNA transcripts are abnormally processed, utilizing a cryptic splice sequence that is enhanced by the Knossos substitution. This form of abnormal RNA processing is seen in two other mutations in this region (a silent substitution in codon 24 and the substitution in codon 26 that produces the beta E variant) and most likely contributes appreciably to the reduced synthesis of beta Knossos.

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.

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.

A G to T transversion at the fifth nucleotide of the first intervening sequence (IVS-1) of the beta-globin gene has been identified in cloned beta-thalassemia genes of two unrelated individuals, one of Mediterranean and the other of Anglo Saxon ancestry. In each patient the mutation was present in a different beta globin gene framework, defined by intragenic restriction site polymorphisms, thereby suggesting the occurrence of independent mutations. The study of the RNA products of one of these cloned genes, after transfer and transient expression in HeLa cells, showed partial inactivation of the normal donor splice site of IVS-1 and activation of two major and one minor cryptic splice sites. Only one of the two major cryptic sites was utilized in a cell-free splicing extract. The effects of this mutation on messenger RNA (mRNA) splicing are similar to that of another beta thalassemia gene with a G to C transition at the same position.

We have identified two novel RNA-splicing mutations affecting a critical nucleotide (nt) in the acceptor consensus sequences at both the IVS-1/exon 2 and IVS-2/exon 3 junctions of the human beta-globin gene. Both mutations are single nt substitutions, T to G and C to A, at position -3 adjacent to the invariant AG dinucleotide. For the IVS-2/exon 3 mutation abnormal splicing into the cryptic splice site at IVS-2 nt 579 is documented. Identification of these two mutations provides further support for the importance of the location of specific nucleotides within the consensus sequences in splice site selection and RNA processing.

Levels of fetal hemoglobin (HbF) bearing reticulocytes (F reticulocytes) range from 2% to 50% in patients with sickle cell (SS) anemia. To learn whether any portion of such variation in F cell production is regulated by loci genetically separable from the beta-globin gene cluster, percentages of F reticulocytes were compared in 59 sib pairs composed solely of SS members, including 40 pairs from Jamaica and 19 from the United States. We reasoned that differences in F reticulocyte levels might arise (1) from any of several kinds of artifact, (2) via half-sib status, or (3) because one or more genes regulating F cell production segregate separately from beta S. We minimized the role of artifact by assay of fresh samples from 84 SS individuals, including both members of 38 sib pairs. In 78 of the 84 subjects, serial values for percent F reticulocytes fell within 99.9% confidence limits or were alike by t test (P greater than or equal to .05). This left 32 sib pairs for which F reticulocyte levels in each member were reproducible. When sib-sib comparisons were limited to these 32 pairs, percentages of F reticulocytes were grossly dissimilar within 12 Jamaican and 3 American sibships. Within them, the probability that sibs were alike was always less than or equal to .005 and usually less than or equal to 10(-4). We next minimized the contribution of half-sibs among Jamaicans by a combination of paternity testing and sib-sib comparison of beta-globin region DNA restriction fragment length polymorphisms, especially among discordant pairs. We thereafter concluded that at least seven to eight Jamaican pairs were composed of reproducibly discordant full sibs. There is thus little doubt that there are genes regulating between-patient differences in F cell production that are separate from the beta-globin gene cluster. Still unanswered is (1) whether or not these genes are actually linked to beta S, (2) why F reticulocyte levels in Americans tend to be lower than in Jamaicans, and (3) whether or not differences in F cell production among SS patients are regulated by several major loci or by only one.