Two chimaeric transcription units result from an inversion breaking intron 1 of the factor VIII gene and a region reportedly affected by reciprocal translocations in T-cell leukaemia
Two chimaeric transcription units result from an inversion breaking intron 1 of the factor VIII gene and a region reportedly affected by reciprocal translocations in T-cell leukaemiaAstrid Brinke1,, Luigina Tagliavacca1,+,, Jennifer Naylor1, Peter Green1, Paul Giangrande2 and Francesco Giannelli1,*
1Division of Medical and Molecular Genetics, UMDS, Guy's Hospital, London, UK and 2Oxford Haemophilia Centre, Churchill Hospital, Oxford, UK
Received July 15 1996;Revised and Accepted September 23, 1996
Analysis of mRNA in two haemophilic monozygotic twins offers novel information on the organisation of expressed sequences distal to the coagulation factor VIII gene. These patients show an inversion that, in contrast to the common inversions responsible for 1/5 of all haemophilia A, affects the first rather than intron 22 of the gene. This displaces the most telomeric of the factor VIII exons (exon 1) by ~100 kb towards the telomere, and close to the region of the C6.1A gene. This novel inversion creates two hybrid transcription units: one formed by the promoter and first exon of the factor VIII gene followed by a widely expressed sequence; the other by the promoter and coding region of the C6.1A gene plus most of the factor VIII gene (part of intron 1 and exons 2-26). Investigation of this transcription unit reveals that the C6.1A gene has an unsuspected intron in the region coding for the previously described 3'-untranslated tail of the message. Furthermore, exons located beyond the known C6.1A sequence and present in normal transcripts precede exons 2-26 of the factor VIII gene in the hybrid mRNA of the haemophilic twins. The factor VIII sequences in this hybrid mRNA are not expected to be expressed because they lack the first exon, encoding the prepeptide, and follow a translation stop in the C6.1A gene. Leukaemia-related translocations in the C6.1A region suggest that this region may be somewhat unstable.
Haemophilia A, an X-linked bleeding disorder affecting 1/5000 males, is due to mutation of the large and complex factor VIII (FVIII) gene (186 kb, 26 exons) (1 ). This disease has been maintained in the population by an equilibrium between mutation and selection that, at least until the introduction of modern therapy, resulted in the renewal of the population pool of haemophilia A mutations at a rate of ~1/6 per generation (2 ). Unrelated patients are therefore expected to carry mutations of independent origin and, accordingly, a great variety of gene mutations has been detected by the analysis of haemophilia A patients (3 ). Recently and unexpectedly, however, it has been found that 45% of patients with severe haemophilia or 20% of all patients have large inversions that break the FVIII gene in intron 22 (4 ,5 ). These inversions are caused by intra-chromosome (-chromatid) homologous recombination between a 9.5 kb region (int22h) in the intron 22 of the FVIII gene and either one of two extra copies of the same sequence that are both in inverted orientation relative to the former and ~400 and 500 kb telomeric to the FVIII gene (6 ,7 ). The high incidence of these inversions is due to their repeated occurrence, and a mutation rate of 4-7.2 * 10-6 per gene per gamete per generation has been proposed (5 ,8 ).
In this study, we report the molecular characterisation of a novel inversion in two monozygotic twins severely affected by haemophilia A. The inversion involves the first intron of the FVIII gene and more distal sequences mapped ~70 kb telomeric and upstream of the FVIII gene. This mutation results in the production of two chimaeric transcription units that we characterise.
The FVIII gene of the monozygotic twins in this study was examined by the procedure of Naylor et al. (9 ). This amplifies, from RNA and genomic DNA, the essential sequences of the FVIII gene (i.e. the putative promoter, coding and the polyadenylation signal regions) in eight overlapping sections. These are then screened by chemical mismatch.
All amplified sections of the patients' gene showed no abnormalities, but the first mRNA segment extending from exon 1 to exon 8 consistently failed to amplify (Fig. 1 A and B). RT-PCR of this region was attempted, therefore, using combinations of nested primers spanning different, smaller segments. This revealed that amplification across the exon 1-2 boundary was impossible, while a segment containing exons 2-8 amplified readily using the nested primers 2Q, 1B, 1E and 1D (Fig. 1 A and B) and contained no mutation. Furthermore, amplification (with primers 7A and 1L) and sequencing of a region of genomic DNA containing the putative promoter, exon 1 and the first donor splice site (Fig. 1 C) showed no abnormality.
In order to investigate whether the first intron of the patients' FVIII gene was involved in a gross rearrangement, we carried out a pulsed-field gel electrophoresis (PFGE) analysis using the NruI restriction enzyme. As shown diagrammatically in Figure 2 , the FVIII gene is normally contained within a ~270 kb NruI fragment that lies proximal to an ~1 Mb segment (7 ,10 ), but when the patients' DNA was hybridised to a cDNA probe consisting of exons 1 and 2, two bands were observed: one of ~270 kb and one of ~1 Mb. The latter band was also seen in the patients' mother but not, as expected, in the normal control (not shown). This suggested the possibility that the first FVIII exon in the patient was placed distally to the NruI site that normally flanks the telomeric (5') end of the FVIII gene and that helps define the telomeric 1 Mb NruI fragment. An inversion delimited by a break in intron 1 and one telomeric to the factor VIII gene (Fig. 2 ) could therefore be present in the patients and their mother.
If an inversion had occurred, the first exon of the FVIII gene would be separated from and in opposite orientation to the rest of the gene (Fig. 2 ). The FVIII promoter could then allow the expression of a transcript containing the first FVIII exon and novel sequences. In order to isolate these sequences, we used the 3'RACE/vectorette procedure developed for the analysis of the common int22h-related inversions mentioned in the Introduction (5 ). After 3' RACE with the FVIII-specific primer 1A and amplification of the ClaI vectorette library with the FVIII primer 1C (see Fig. 1 A for the location of primers 1A and 1C), and the vectorette 224 primer a product of ~900 bp was obtained. Sequence analysis performed using the FVIII 1C primer revealed that exon 1 of the FVIII gene splices onto a new sequence of unknown origin. A primer derived from this sequence (5'A) and the vectorette 224 primer served to generate a probe containing the new sequence. This was used to screen a panel of yeast artificial chromosomes (YACs) covering the Xq28 region surrounding the FVIII gene. This mapped the novel sequence between markers C6 and 924R-C (5 ) at ~70-200 kb 5' or telomeric to the FVIII gene (see Fig. 2 ). This clearly demonstrates the presence of the inversion proposed above.
The novel sequence spliced to exon 1 of the FVIII gene was hybridised to a northern blot containing human mRNA from brain, heart, kidney, liver, lung, pancreas, placenta and skeletal muscle and showed (Fig. 3 ) a band of ~1.7 kb in all tissues, with the strongest signal in heart, kidney and skeletal muscle. In addition to this, a larger transcript of ~6.2 kb is present in skeletal muscle only.
In the patients' DNA, exons 2-26 are no longer downstream of the normal FVIII gene promoter. Their expression in the patients' mRNA therefore indicates the presence of a new promoter 5' of exon 2 of the FVIII gene. In order to identify this new promoter and characterise the new transcription unit created by the inversion, we considered the location of the distal boundary of the inversion in relation to known expressed sequences. A CpG island associated with two transcripts called C6.1A and C6.1B (11 ) maps close to the inversion boundary (see C6 in Fig. 2 ). Thus, if the inversion break had occurred distal to the CpG island, the inversion would have brought this promoter close to exon 2 of the FVIII gene as shown in Figure 2 . Furthermore, the C6.1A gene, normally in opposite orientation to that for factor VIII, would now be oriented as the FVIII gene, and a hybrid transcription unit consisting of part of C6.1A at the 5' end and exons 2-26 of the FVIII gene at the 3' end would be plausible.
RT-PCR of normal and patients' RNA directed by forward primers in C6.1A (C6.1A/F1 and C6.1A/F2) and reverse primers in exon 2 of the FVIII gene (2R and 2L) only succeeded in the patients' RNA, where four specific bands were obtained (Fig. 4 A). Three of these products were fully sequenced and they demonstrated that the patients' C6.1A mRNA, which lacked the known most 3' sequences, is joined to the FVIII exons 2-26 by a stretch of novel sequence made up of different modules (Fig. 4 B) which are not adjacent to each other in genomic DNA and, therefore, are exon-like.
We describe the first inversion found in haemophilia A not related to the int22h sequence in intron 22 of the FVIII gene and its intra-chromosomal recombination with int22h regions 400-500 kb more telomeric. This novel inversion, that breaks the FVIII gene in intron 1 and involves ~100 kb of DNA 5' or telomeric of the FVIII gene, is present in a pair of monozygotic twins with severe haemophilia A and their heterozygous mother. Since this mutation has not been observed previously, it may represent a rare event. Nevertheless, inversion mutations cannot be detected by procedures that examine all the FVIII exons in genomic DNA, and even the common inversions that break the FVIII gene in intron 22, and that account for 45% of severely affected patients went undetected for 8 years from the cloning of the FVIII gene until the screening of haemophilia A mutations was based on mRNA analysis (12 ). So far, only a small number of patients has been examined by mRNA-based procedures capable of detecting novel inversions. We have examined 44 families, mostly with severe disease. Besides the family that is the object of this work, 26 showed different non-inversion mutations, and 17 inversions breaking the FVIII gene in intron 22 (13 ,14 and unpublished observations).
The inversion described here extends from intron 1 of the FVIII gene to a region beyond the known sequence of the C6.1A gene, and results in the formation of two hybrid transcription units. One of these is formed by both the promoter plus first exon of the FVIII gene and sequences normally expressed in a wide variety of tissues (see Fig. 3 ). These sequences are mapped by our work telomeric to the known C6.1A sequence. The other hybrid transcription unit contains in its 5' section the CpG island and all the known sequence of the C6.1A gene, while the 3' section consists of most of the FVIII gene (part of intron 1 followed by exons 2-26). The transcript of this unit is spliced so that the penultimate exon of the known sequence of the C6.1A gene (11 ) is joined to a variable number of exons distal to the published C6.1A sequence (11 ) and then to exons 2-26 of the FVIII gene. This hybrid transcription unit should allow the expression of the C6.1A gene unless the hybrid mRNA were unduly unstable. By contrast, the transcribed factor VIII sequence should not be translated because it is preceded by at least the translation stop signal at the start of the 3'-untranslated tail of the published C6.1A message (11 ). This mutation, therefore, easily explains the severe haemophilia affecting both twins.
Unexpectedly, we have found an intron in the region of the C6.1A gene specifying the 3'-untranslated tail of the message, and we have revealed that transcripts may normally extend beyond the known C6.1A sequence to include novel exons alternatively spliced. Such longer transcripts are part of the patients' hybrid message. Further work is required to locate the inversion breakpoints and to define in detail both the transcription map of the region immediately distal to the known C6.1A sequences and the role of the novel exons found in the patients' hybrid mRNAs.
Interestingly, three translocations affecting the C6.1A gene and the T-cell receptor [alpha]/[delta] locus have been described in the lymphocytes of three patients with T-cell leukaemia. One of these was found in a patient with ataxia telangiectasia (AT) and chronic prolymphocytic leukaemia, and two in non-AT patients with the same type of leukaemia (15 -17 ). The mechanism by which these translocations may favour the development of T-cell leukaemia is not yet clear, but it has been suggested that they may act by affecting the expression of the gene C6.1B that originates from the same CpG island as C6.1A but extends in the opposite direction towards the FVIII gene (15 -17 ). Whatever the relevance of these translocations in chronic T-cell leukaemia, they suggest some instability of Xq28 in the region of the C6.1A gene.
The patients, monozygotic twins with severe haemophilia A, have <1% plasma factor VIII activity. Inhibitors have not been reported in either patient. No other members of the family are affected. Samples of blood in EDTA anticoagulant were used to obtain RNA and DNA.
A 10 ml sample of blood was used for extracting DNA by a standard technique (18 ). RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction (19 ) from peripheral lymphocytes purified by centrifugation through a layer of Histopaque 1077 (Sigma) from 10 ml of blood (9 ).
DNA from the patient or YAC clones was digested with restriction enzymes according to the manufacturer's instructions, then blotted on Hybond-N membrane and hybridised to 32P-labelled probes according to standard procedures (18 ).
Between 200 and 500 ng of total lymphocyte RNA was incubated at 65oC for 10 min in a volume of 9.5 [mu]l with 50 ng of the appropriate 3' oligonucleotide primer for cDNA synthesis as already described (9 ,21 ). After two nested PCR reactions, the products were purified by electrophoretic separation on a 1% agarose gel and by absorption to Geneclean II (Bio 101). Genomic DNA was amplified for a single round of 30 cycles (93oC 1 min, 58-61oC 30 s, 72oC 3 min). The primers newly developed for the amplification reactions used in this work are listed in Table 1 .
The combination of 3' RACE and vectorette amplification introduced by Naylor et al. (5 ) for the amplification of normal 3' ends of rare transcripts was used as previously described (5 ). 3' RACE used a gene-specific primer, as specified in Results, and the 3' RACE T17 + adapter primer (22 ). Following ClaI digestion of 3' RACE products and ligation to a vectorette bubble (23 ), 5 [mu]l of the DNA mixture were used in a PCR amplification of 35 cycles at 93oC 1 min; 60oC 1 min, 72oC 5 min with a nested gene-specific primer and the 224 primer (23 ).
The amplified sequences from the patients' FVIII gene and cDNA were denatured and annealed in a 10:1 ratio with homologous wild-type 32P-end-labelled probes. The hybrids were divided into two aliquots and treated with hydroxylamine solution (3 M hydroxylaminehydrochloride, 1.75 M diethylamine, 37oC for 2 h) or osmium tetroxide solution (0.025% OsO4, 3% pyridine, 37oC for 2 h) to modify mispaired cytosine (C) and thymine (T) residues respectively. These were then cleaved by 1 M piperidine (90oC for 30 min) and sized by 4% acrylamide gel electrophoresis followed by autoradiography (24 ,25 ).
A contig of YACs and cosmids representing the Xq28 region surrounding the FVIII gene was screened by hybridisation with a patient-specific probe derived from sequences obtained by 3' RACE/vectorette or RT-PCR.
Approximately 3 [mu]g of genomic DNA from the patient were digested with restriction enzymes MseI, MboI and TaqI followed by an ethanol precipitation. The DNA was resuspended in Tris EDTA buffer and ligated to 50 pmol of the appropriate vectorette cassette (23 ) in a final volume of 100 [mu]l. Vectorette PCR was performed for 30 cycles at 94oC for 1 min, 65oC 1 min, 72oC 5 min on 5 [mu]l of vectorette library with primers C6.1A/F1 or C6.1A/R3 (see Fig. 4 C) in combination with vectorette primer 224 (23 ). Subsequently, 2 [mu]l of a 1:100 dilution of first round product was used in a heminested amplification using primers C6.1A/F2 and C6.1A/R2 respectively under the conditions specified above. Amplification products were purified from agarose gel slices and sequenced as described above.
We thank Sheila Hassock for YAC and cosmid clones and Adrienne Knight for secretarial help. This work was supported by the Medical Research Council and the Wellcome Trust. We also acknowledge the support of the Generation Trust.
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*To whom correspondence should be addressed
+Present address: Department of Biological Chemistry, The University of Michigan Medical School, Ann Arbor, MI, USA
These authors contributed equally to this work
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