Evolution of RH genes in hominoids: characterization of a gorilla RHCE-like gene

The human RH locus is responsible for the expression of the Rh blood group antigens. It consists of two closely linked genes, RHD and RHCE, that exhibit 92% similarity between coding regions. These observations suggest that they are derived from a relatively recent duplication event. Previously a study of nonhuman primate RH-like genes demonstrated that ancestral RH gene duplication occurred in the common ancestor of man, chimpanzees and gorillas. By amplification of intron 3 and intron 4 of gorilla RH-like genes, we have now shown that, like man, gorillas possess two types of RH intron 3 (RHCE intron 3 being 289 bp longer than the RHD intron 3) and two types of intron 4 (RHCE intron 4 being 654 bp longer than the RHD intron 4). Here we report the characterization of a cDNA encoded by a gorilla RH-like gene which possesses introns 3 and 4 of the RHCE type. A comparison of this gorilla RHCE-like coding sequence with previously characterized human and ape cDNA sequences suggests that RH genes experienced complex recombination events after duplication in the common ancestor of humans, chimpanzees and gorillas.     

An Alu-mediated large deletion of the FUT2 gene in individuals with the ABO-Bombay phenotype

Recently, we have found an allelic deletion of the secretor alpha(1,2)fucosyltransferase (FUT2) gene in individuals with the classical Bombay phenotype of the ABO system. The FUT2 gene consists of two exons separated by an intron that spans approximately 7 kb. The first exon is noncoding, whereas exon 2 contains the complete coding sequence. Since the 5' breakpoint of the deletion has previously been mapped to the single intron of FUT2, we have cloned the junction region of the deletion in a Bombay individual by cassette-mediated polymerase chain reaction. In addition, the region from the 3' untranslated region of FUT2 to the 3' breakpoint sequence has been amplified from a control individual. DNA sequence analysis of this region indicates that the 5' breakpoint is within a free left Alu monomer (FLAM-C) sequence that lies 1.3 kb downstream of exon 1, and that the 3' breakpoint is within a complete Alu element (AluSx) that is positioned 1.5 kb downstream of exon 2. The size of the deletion is estimated to be about 10 kb. There is a 25-bp sequence identity between the reference DNA sequences surrounding the 5' and 3' breakpoints. This demonstrates that an Alu-mediated large gene deletion generated by unequal crossover is responsible for secretor alpha(1,2)fucosyltransferase deficiency in Indian Bombay individuals.     

Rh gene evolution in primates: study of intron sequences

By amplification and sequencing of RH gene intron 4 of various primates we demonstrate that an Alu-Sx-like element has been inserted in the RH gene of the common ancestor of humans, apes, Old World monkeys, and New World monkeys. The study of mouse and lemur intron 4 sequences allowed us to precisely define the insertion point of the Alu-Sx element in intron 4 of the RH gene ancestor common to Anthropoidea. Like humans, chimpanzees and gorillas possess two types of RH intron 4, characterized by the presence (human RHCE and ape RHCE-like genes) or absence (human RHD and ape RHD-like genes) of the Alu-Sx element. This led us to conclude that in the RH common ancestor of humans, chimpanzees, and gorillas, a duplication of the common ancestor gene gave rise to two genes, one differing from the other by a 654-bp deletion encompassing an Alu-Sx element. Moreover, most of chimpanzees and some gorillas posses two types of RHD-like intron 4. The introns 4 of type 1 have a length similar to that of human RHD intron 4, whereas introns 4 of type 2 display an insertion of 12 bp. The latest insertion was not found in the human genome (72 individuals tested). The study of RH intron 3 length polymorphism confirmed that, like humans, chimpanzees and gorillas possess two types of intron 3, with the RHD-type intron 3 being 289 bases shorter than the RHCE intron 3. By amplification and sequencing of regions encompassing introns 3 and 4, we demonstrated that chimpanzee and gorilla RH-like genes displayed associations of introns 3 and 4 distinct to those found in man. Altogether, the results demonstrate that, as in humans, chimpanzee and gorilla RH genes experienced intergenic exchanges.     

The analysis of nucleotide substitutions, gaps, and recombination events between RHD and RHCE genes through complete sequencing

We determined the entire nucleotide sequences of all introns within the RHD and RHCE genes by amplifying genomic DNA using long PCR methods. The RHD and RHCE genes were 57,295 and 57,831 bp in length, respectively. Aligning both genes revealed 138 gaps (insertions and deletions) below 100 bp, 1116 substitutions in all introns and all exons (coding region), and 5 gaps of over 100 bp. Homologies (%) between the RH genes were 93.8% over all introns and coding exons and 91.7% over all exons and introns. Various short tandem repeats (STRs) and many interspersed nuclear elements were identified in both genes. The proportions of Alu sequences in the RHD and RHCE genes were 25.9 and 25.7%, respectively and these Alu sequences were concentrated in several regions. We confirmed multiple recombinations in introns 1 and 2. Such multiple recombination, which probably arose due to the concentrations of Alu sequences and the high level of the homology (%), is one of most important factors in the formation and evolution of RH gene. The variability of the Rh system may be generated because of these features of RH genes. Apparent mutational hotspots and regions with low of K values (the numbers of substitutions per nucleotide site) caused by recombinations as well as true mutational hotspots may be found in human genome. Accordingly, in searching for and identifying single nucleotide polymorphisms (SNPs) especially in noncoding regions, apparent mutational hotspots and areas of low K values by recombination should be noted since the unequal distribution of SNPs will reduce the power of SNPs as genetic maker. Combining the complete sequences' data of both RH genes with serological findings will provide beneficial information with which to elucidate the mechanism of recombination, mutation, polymorphism, and evolution of other genes containing the RH gene as well as to analyze Rh variants and develop new methods of Rh genotyping.     

Gene organization and rearrangements at the human Rhesus blood group locus revealed by fiber-FISH analysis

The human Rhesus (Rh) blood group locus is composed of two highly homologous genes, the RHD and RHCE genes on chromosome 1, encoding the D, C/c, and E/e antigens in common Rh-positive phenotypes. In general, the RHD gene is either absent or grossly deleted in Rh-negative individuals. In this study, gene organization at the RH locus of Japanese donors with different serological phenotypes was directly analyzed by two-color fluorescence in situ hybridization on DNA fibers released from their lymphocytes (fiber-FISH) and by using DNA probes of introns 3 and 7 of the RHCE and RHD genes. Six Rh-positive samples (two with the D+C-c+E+e-, two with the D+C+c-E-e+, and two with the D+C+c+E+e+ phenotype) showed the presence of two RH genes within a region of less than 200 kb on chromosome 1p36.1. Of great interest was the finding that the genes were arranged in the antidromic order of the telomere -RHCE (5'--> 3') -RHD (3'-->5') - centromere. On the other hand, two typical Rh-negative samples (D-C-c+E+e+) showed the presence of only one RHCE gene, as expected. Moreover, further analysis combined with a locus-specific assay of three Rh-negative samples (D-C+c+E+e+, D-C+c+E-e+, and D-C+c-E-e+) showed the possible presence of the RHD gene(s) and complex rearrangements, including partial deletion, duplication, and recombination, in this region; these could be responsible for the Rh-negative phenotype.     

Nonhomologous recombination in the human genome: deletions in the human factor VIII gene

Four deletions in the human factor VIII gene have been characterized at the sequence level in patients with hemophilia A. Deletion JH 1 extends 57 kb from IVS 10 to IVS 18. Intron 13 and exon 14 are partially deleted in patients JH 7 and JH 37, with a loss of 3.2 and 2.4 kb of DNA, respectively. The 3' deletion breakpoint of the JH 21 event resides in intron 3 and extends 5' into intron 1, resulting in the loss of exons 2 and 3. Seven of the eight breakpoints sequenced (5' and 3' for each of the four deletions) occur in nonrepetitive sequence, while the 3' breakpoint of the JH 1 resides in an Alu repetitive element. All of the deletions are the result of nonhomologous recombination. The 5' and 3' breakpoints of JH 1, JH 7, and JH 37 share 2- to 3-bp homologies at the deletion junctions. In contrast, two nucleotides have been inserted at the JH 21 deletion junction. Short sequence homologies may facilitate end-joining reactions in nonhomologous recombination events.     

An Alu-mediated deletion at Xp11.23 leading to Wiskott-Aldrich syndrome

Wiskott-Aldrich syndrome (WAS) is an X-linked disease characterized by thrombocytopenia, eczema and immunodeficiency of varying severity. The WASP gene, mutations of which are responsible for the phenotype, maps to Xp11.23. We describe here a patient with a large deletion in the Xp11.23 region. The deletion, which totals 15.8 kb, begins downstream of DXS1696 and encompasses 13 kb upstream of WASP and includes the distal and proximal promoters and exons 1-6. Analysis of the 5'-boundary region identified sequences missing in the Human Genome database and, as a result, the normal DNA sequence was revised to include 743 bp of novel sequence (AF466616). The patient's upstream breakpoint was localized to an AluSg element within a highly repetitive DNA region containing other Alu elements. A 26-bp recombinogenic element is located downstream of the 5' breakpoint. A 16-bp sequence just upstream of the 5' breakpoint shares close homology with the sequence that spans the 3' breakpoint in intron 6. A heptanucleotide of unknown origin, CAGGGGG, links the 5' and 3' breakpoints. To our knowledge this is the largest deletion in a WAS patient.     

A recombination hot spot in the Rh genes revealed by analysis of unrelated donors with the rare D-- phenotype.

We have studied the arrangement of Rh (rhesus) genes in donors who are completely null for the products of one of them, RHCE. We show that five of six homozygous individuals with the so-called Rh D-- phenotype, who express no red-cell antigens of the C/c and E/e series, have rearranged RHCE genes in which internal sequences have been replaced by the corresponding sequences from RHD. Moreover, although there is heterogeneity at the 3' end, the 5' boundary of this chimerism is within the same small interval around exon 2. This interval is characterized by an exceptionally high degree of sequence homology between RHCE and RHD, a high density of dispersed repetitive elements, and the presence of an alternating purine-pyrimidine copolymer tract. We suggest that these features may explain the mechanistic basis for the origin of the rearrangement.     

鉴定9个新的RHD基因mRNA可变剪接体

为了研究各种RHD基因mRNA可变剪接体的基因结构, 应用逆转录聚合酶链反应(RT-PCR)检测正常人脐血样本RHD mRNA, 对RHD cDNA进行TA克隆和序列分析, 对各可变剪接体的剪接位点进行DNA序列分析, 并将RHD mRNA进行表达序列标签(ESTs)分析。结果在28个阳性克隆中, 除全长RHD cDNA外, 共检测到12种(包括9种新的)RHD可变剪接体, 发现外显子遗漏、5′和3′剪接位点变异3种剪接形式, 涉及外显子2~9, 其中6种新的剪接体同时存在RHD和RHCE基因同源杂交现象。ESTs分析还检索到内含子保留形式的剪接体。研究表明, RHD基因mRNA存在复杂的可变剪接机制, 除已报道的剪接体外, 检测到9种新的RHD可变剪接体, 并发现了可变剪接和同源杂交并存现象。     

Gene arrangement at the Rhesus blood group locus of chimpanzees detected by fiber-FISH

The Rhesus (Rh) blood group system in humans is encoded by two genes with high sequence homology. These two genes, namely, RHCE and RHD, have been implied to be duplicated during evolution. However, the genomic organization of Rh genes in chimpanzees and other nonhuman primates has not been precisely studied. We analyzed the arrangement of the Rh genes of chimpanzees (Pan troglodytes) by two-color fluorescence in situ hybridization on chromatin DNA fibers (fiber-FISH) using two genomic DNA probes that respectively contain introns 3 and 7 of human RH genes. Among the five chimpanzees studied, three were found to be homozygous for the two-Rh-gene type, in an arrangement of Rh (5'-->3') - Rh (3'<--5'). Although a similar gene arrangement can be detected in the RH gene locus of typical Rh-positive humans, the distance between the two genes in chimpanzees was about 50 kb longer than that in humans. The remaining two chimpanzees were homozygous for a four-Rh-gene type, in an arrangement of Rh (5'-->3') - Rh (3'<--5') - Rh (3'<--5') - Rh (3'<--5') within a region spanning about 300 kb. This four-Rh-gene type has not been detected in humans. Further analysis of other great apes showed different gene arrangements: a bonobo was homozygous for the three-Rh-gene type; a gorilla was heterozygous for the one-Rh- and two-Rh-gene types; an orangutan was homozygous for the one-Rh-gene type. Our findings on the intra- and interspecific genomic variations in the Rh gene locus in Hominoids would shed further light on reconstructing the genomic pathways of Rh gene duplication during evolution.     

Hypobetalipoproteinemia due to an apolipoprotein B gene exon 21 deletion derived by Alu-Alu recombination

We report the molecular defect in an individual with homozygous hypobetalipoproteinemia. A unique TaqI restriction fragment length polymorphism was found in the midportion of the apolipoprotein B (apoB) gene using the genomic probe, pB51. The probe, which identifies TaqI fragments of 8.4 and 2.8 kilobases (kb) in normal individuals, hybridized to a single 11-kb fragment in the proband. The parents of the proband showed all three TaqI fragments, implying that they are heterozygotes for the mutant apoB allele. In this family, the mutant allele cosegregated with low total cholesterol levels and formal linkage analysis gave a decimal logarithm of the ratio score of 3.3 at a recombination frequency of 0. The polymorphic TaqI site was localized to an EcoRI fragment of 4 kb in normal individuals. The corresponding fragment in the proband was 3.4 kb, suggesting a 0.6-kb deletion in the mutant allele. Both the normal 4-kb EcoRI fragment and the mutant 3.4-kb EcoRI fragment were cloned and sequenced. In the normal allele, the 4-kb EcoRI fragment extends from intron 20 to 23. Exon 21 is flanked by Alu sequences that are in the same orientation. The mutant allele had a 694-bp deletion in this region which included a small part of the Alu sequence in intron 20, the entire exon 21, and most of the Alu sequence in intron 21. The polymorphic TaqI site, which lies within the Alu sequence in intron 21, was absent in the proband as a result of the deletion. The deletion of exon 21 results in a frame shift mutation and the introduction of a stop codon. Translation of the encoded mRNA would yield a prematurely terminated protein. This mutant apoB protein would be 1085 amino acids long with the 73 carboxyl-terminal residues out of frame. We postulate that the deletion of exon 21 is the consequence of a crossover event between the Alu sequences in introns 20 and 21 resulting in nonreciprocal exchange between two chromosomes.     

Nucleotide sequence of both reciprocal translocation junction regions in a patient with Ph positive acute lymphoblastic leukaemia, with a breakpoint within the first intron of the BCR gene.

Breakpoints on chromosome 22 in the translocation t(9;22) found in Philadelphia positive acute lymphoblastic leukaemia patients fall within two categories. In the first the breakpoint is localized within the breakpoint cluster region of the BCR gene, analogous to the chromosome 22 breakpoint in chronic myeloid leukaemia. The second category has a breakpoint 5' of this area, but still within the BCR gene. We have previously shown that these breakpoints occur within the first intron of the BCR gene and cloned the 9q+ junction from such a patient. We have now determined the sequences around the breakpoints on both translocation partners from this patient as well as the germline regions. The chromosome 9 ABL sequence around the breakpoint shows homology to the consensus Alu sequence whereas the chromosome 22 BCR sequence does not. At the junction there is a 6 bp duplication of the chromosome 22 sequence which is present both in the 9q+ and in the 22q- translocation products. Possible mechanisms for the generation of the translocation are discussed.     

Molecular genetics of human erythrocyte MiIII and MiVI glycophorins. Use of a pseudoexon in construction of two delta-alpha-delta hybrid genes resulting in antigenic diversification

Human glycophorins alpha and delta (or A and B) specify the MNSs blood group antigens; they exhibit considerable structural variation among populations. We show that two variant phenotypes of Miltenberger class III and VI are encoded by similar hybrid glycophorin genes in a delta-alpha-delta arrangement. Restriction mapping identified altered fragments unique to the MiIII and MiVI genes. Genomic sequences spanning exons 2 to 4 of the two genes were obtained by allele-specific polymerase chain reaction. Restriction analysis and direct sequencing of the amplified DNA revealed that MiIII and MiVI genes are identical to the delta gene except that, in both, an internal segment of the delta gene has been replaced by its homologous counterpart of the alpha gene, resulting in a delta-alpha-delta hybrid structure. In the process of hybrid formation a portion of alpha exon 3 and intron 3, that carries a functional 5' splicing signal, has been fused to an exon-like sequence in the delta gene that retains a 3' but lacks a 5' splicing signal. These rearrangements created a composite exon resulting in the expression of the ordinarily unexpressed delta gene sequence and conferred the hybrid proteins with new antigenic specificities. The expression of this sequence in MiIII glycophorin is directly demonstrated by protein sequencing. MiIII and MiVI genes differ in the location of upstream (delta-alpha) and downstream (alpha-delta) breakpoints and in the length of sequence replacement. The delta-alpha breakpoints of the two genes occur at different locations within a 35-base pair sequence of exon 3 that is clustered with multiple inverted repeats, whereas the alpha-delta breakpoints reside downstream in two dissimilar blocks of sequences of intron 3. The minimal length of the delta gene sequence that has been replaced by the alpha gene is 55 base pairs in the MiIII gene and 131 base pairs in the MiVI gene. Such segmental DNA transfers may have proceeded unidirectionally through the mechanisms of gene conversion.     

Chromosomal arrangement of the duck alpha-globin genes and primary structure of the embryonic alpha-globin gene pi

A recombinant lambda Charon 4A bacteriophage, D alpha G-1, carrying the genes coding for the duck embryonic (pi') and adult (alpha A, alpha D) alpha-like globins was isolated from a previously constructed duck DNA recombinant library. The three globin genes are transcribed from the same DNA strand and are arranged in the order of their expression during development: 5'-pi'-alpha D-alpha A-3'. We have determined the complete nucleotide sequence of the duck pi'-globin gene, including the flanking regions. Due to the unusual length of intron 1 (963 bp) and intron 2 (568 bp) the 2167-bp duck pi'-globin gene is by far the largest among all known mammalian or avian alpha- and beta-globin genes. For instance, the duck pi'-globin gene introns are almost twice as long as those of the chicken pi'-globin genes. A surprisingly high degree of nucleotide sequence homology (88%) has been found for the 5' flanking region (positions -1 to -223) of the duck and chicken pi'-globin gene.