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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4361-4367
A Large Deletion Within the Protein 4.1 Gene Associated With a Stable
Truncated mRNA and an Unaltered Tissue-Specific Alternative Splicing
By
N. Dalla Venezia,
P. Maillet,
L. Morlé,
L. Roda,
J. Delaunay, and
F. Baklouti
From CNRS URA 1171, Institut Pasteur de Lyon, Lyon, France; the
Centre Hospitalier Territorial, Papeete, Tahiti; and the Centre de
Génétique Moléculaire et Cellulaire,
Université Lyon, Villeurbanne, France.
 |
ABSTRACT |
Protein 4.1 is a major protein of the red blood cell skeleton. It
binds to the membrane through its 30-kD N-terminal domain and
to the spectrin-actin lattice through its 10-kD domain. We describe
here the molecular basis of a heterozygous hereditary elliptocytosis
(HE) associated with protein 4.1 partial deficiency. The responsible
allele displayed a greater than 70-kb genomic deletion, beginning
within intron 1 and ending within a 1.3-kb region upstream from exon
13. This deletion encompassed both erythroid and nonerythroid
translation initiation sites. It accounts for the largest deletion
known in genes encoding proteins of the red blood cell membrane.
The corresponding mRNA was shortened by 1727 bases, due to the absence
of exons 2 to 12. Nevertheless, this mRNA was stable. It showed a
similar pattern in lymphoblastoid cells as in reticulocytes.
Differential splicing of exons within the undeleted region remained
regulated in a tissue-specific manner. Exons 14, 15, and 17a were
absent from both reticulocyte and lymphocyte mRNAs, whereas exon 16 was
present in reticulocytes but absent from lymphocytes. Thus,
differential splicing on a local scale was not dependent on the overall
structure of protein 4.1 mRNA in this particular instance.
| |
INTRODUCTION |
PROTEIN 4.1 IS A MAJOR component of the
red blood cell (RBC) skeleton that laminates the inner surface of the
plasma membrane (for review, see Lux and Palek,1
Delaunay,2 and Conboy3). It contributes to the
skeleton anchoring to the membrane through interactions of its 30-kD
domain with band 34,5 and mainly with glycophorin C and
p55.5-7 Functional studies have shown that protein 4.1 tightens the interaction of spectrin and actin via its 10-kD internal
domain.8-12
Protein 4.1 arises from a single gene that maps to
1p33-p34.2.13 Multiple isoforms derive from alternative
splicing.14,15 Erythroid 80-kD protein 4.1 results from two
major splicing events. (1) A 17-bp motif, located in exon 2 and
containing the upstream translation initiation codon, is spliced out,
and (2) a 63-bp exon (exon 16) of the spectrin-actin binding domain is
retained16,17 (Fig 1). Erythroid protein 4.1 is translated from the downstream initiation codon in exon 4. In
nonerythroid tissues, the 17-bp motif is present and the resulting mRNA
encodes a 135-kD protein 4.1, thus converting 618 bp of the erythroid
5 untranslated region into an in-frame translatable sequence.
Immunologically related forms of protein 4.1 are present in a number of
tissues.18 A protein sharing a 70% homology with protein
4.1 and encoded by a distinct gene has been recently
described.19
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| Fig 1.
Schematic representations of protein 4.1 (A), cDNA (B),
primers (C), and cDNA probes (D). (A) Structural domains of the
erythroid protein 4.1, derived from limited chymotryptic cleavage. (B)
( ) Constitutive coding sequences; ( ) alternative coding
sequences; ( ) the 5 end of exon 2 and exon 16 are the only
motifs where alternative splicing is endowed with known physiological
significance; ( ) alternative noncoding sequence; ( ) untranslated
regions. ATG* and ATG** indicate upstream and downstream translation
initiation codons, respectively. (C) Schematic representation of the
primers used in 5 RACE experiments (see also Table 1). (D)
Schematic localization of the cDNA and genomic DNA probes used in
Southern blot analysis. Probes encompassing exonic (thick bar) and
intronic (thin bar) regions.
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4.1( ) hereditary elliptocytosis (HE) designates a
condition in which the RBC membrane contains a reduced (heterozygous
state)20 or a usually null (homozygous state) quantity of
erythroid protein 4.1. The homozygous state described so far produces a
severe hemolytic picture and is exceptional.21,22
In a family with heterozygous 4.1() HE, we report a deletion
greater than 70-kb long encompassing 10 coding exons,23 the largest deletion reported so far within genes encoding proteins of the
RBC membrane. This deleted allele was designated allele 4.1 Annecy.
However, the corresponding mRNA exhibited no quantitative defect,
although it was truncated by 1727 bases and lacked both translation
initiation codons. Lymphoblastoid cells, established from the
patient's peripheral blood, contained the same mRNA truncation as
reticulocytes. Differential splicing of the spared exons remained regulated in a tissue-specific manner. Thus, differential splicing on a
local scale was not dependent on the overall structure of protein 4.1 mRNA in this particular instance.
| |
MATERIALS AND METHODS |
Genomic DNA studies.
The present family was screened upon during a survey of 4.1() HE
in a small area of the French Northern Alps.24 Their HE members (mother, son, and daughter), who were typical 4.1() HE heterozygotes, failed to exhibit the mutation commonly encountered in
their region.12 Northern blots allowed us to perceive a
shortened protein 4.1 mRNA, but no further investigations were then
performed.
DNA was extracted from peripheral blood leukocytes,25
digested with several restriction endonucleases, and subjected to Southern blot analysis. Fragments were separated by electrophoresis through 0.8% agarose gels and transfered onto a positively charged nylon membranes (Boehringer Mannheim Corp, Indianapolis, IN). Hybridizations and washing were performed at 68°C (or 60°C for short probes) according to standard conditions suggested by the manufacturer. Among the random primed probes used, three 4.1 cDNA fragments (pLym 5 [1.46 kb], pLym 3 [1.28 kb], and
pA2.1 [0.72 kb]) have previously been described24 (Fig
1). Five polymerase chain reaction (PCR)-amplified probes (3EX, EX13,
EX11/12, EX2, and NTER; Fig 1) were obtained using the following pairs
of primers: VI/IX, VIII/IX, VI/VII, IV/V, and II/III, respectively
(Table 1). They were cloned into pCRII
(Invitrogen, San Diego, CA) and labeled by a second round of PCR with
DIG-11-dUTP (Boehringer Mannheim Corp). Hybridized probes were detected
with DIG Luminescent Detection kit (Boehringer Mannheim Corp) according
to the manufacturer's instruction.
Amplification of up to 20-kb DNA fragments was performed by PCR using
the Advantage Tth polymerase mix/Genomic PCR kit (Clontech Laboratories Inc, Palo Alto, CA). One or two rounds of 10 to 35 cycles
were performed as follows: denaturation at 95°C for 20 seconds and
annealing-extension at 70°C for 20 minutes.
mRNA studies.
Total reticulocyte RNA was obtained from peripheral blood as previously
described.24 Lymphoblastoid cell lines were kindly established by Prof G. Lenoir (International Agency for Research on
Cancer, Lyon, France) from blood sample of proband I.2. Total RNA was
extracted from cultured cells according to Chomczynski and
Sacchi.26
Reverse transcription (RT) and PCR were performed essentially as
previously referred to22 using sequence specific primers (Table 1). The 30 to 35 cycles of amplification were performed as
follows: denaturation at 94°C for 45 seconds, annealing at 56°C
for 30 seconds or at 60°C for 45 seconds, extension at 72°C for
60 seconds. 5 Rapid amplification of cDNA ends (5RACE)
was performed with the 5-Amplifinder Race kit (Clontech)
following the manufacturer's protocol. The PCR products were analyzed
by electrophoresis on 3% or 4% NuSieve GTG agarose gels (FMC
BioProducts, Rockland, ME) and cloned into pUC 18 EcoRI/BAP
(Pharmacia, Uppsala, Sweden) or pCRII (Invitrogen) vector. Nucleotide
sequencing was performed on double-strand DNAs by the dideoxynucleotide
method, with the Sequenase kit (US Biochemical, Cleveland, OH), using universal M13/pUC primers.
Northern blot analysis was performed on reticulocyte and lymphoblastoid
cell RNA using random primed pLym 5 and pLym 3 probes, as
previously described.24
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RESULTS |
A shortened protein 4.1 mRNA.
5RACE was performed to characterize the shortened protein 4.1 mRNA revealed by Northern blot analyses.24 Either random hexamers or primer XI in RT experiments and two pairs of primers (I/XII) or (I/X) in PCR experiments (Table 1 and Fig 1) were used. The
reactions yielded a major 522-bp fragment (not shown) and a 418-bp
fragment, respectively, corresponding to the abnormal 4.1 allele
(Fig 2A). Because the size of the amplified
fragment would exceed 1 kb (maximum size recommended by the
manufacturer) in the normal allele, no PCR products were expected from
these amplifications in control mRNA. Indeed, no PCR products were
obtained from the control (Fig 2A). 5RACE using an upstream
reverse primer within exon 13 (primer IX, Fig 1 and Table 1) showed a
similar pattern, with a major 244-bp band in the proposita (Fig 2A).
The amplified product suggested the presence of the 3 region of
exon 13, encompassing primer IX sequence, on the abnormal mRNA.
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| Fig 2.
Reticulocyte mRNA analysis. (A) 5RACE on
reticulocyte mRNA, using the pairs of primers I/IX (left side) and I/X
(right side). C, control; I.2, proposita. The PCR products are
schematically represented and their sizes in basepairs are shown.
For schematic representation of exons, see Fig 1. (B) Use of internal
cryptic splicing sites within exon 13. Sequencing analysis
performed on RT-PCR products derived from normal12 or 4.1 Annecy (this report) mRNAs showed discrete use of cryptic acceptor
sites within exon 13. The several sequences obtained from 4.1 Annecy tend to suggest a different pattern of use of these sites.
() Major site; ( ) secondary
site; (---) site not used. Intronic and exonic
sequences are represented in lowercase and capital bolded letters,
respectively.
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The 418-bp and 244-bp fragments were cloned and 8 positive clones (4 of
each size) were sequenced. They all showed an abnormal sequence with
exon 1 3 end joined to exon 13 5 end, suggesting a large
deletion of about 1727 bp (Fig 2B). This deletion encompasses 10 coding
exons (exons 2 through 12, exon 3 being a non coding sequence23) and therefore removes both upstream and
downstream translation initiation codons (Fig 1). To confirm the
5RACE results, and knowing that the deletion spares exon 1 sequences, we performed a standard RT-PCR experiment using primers IV
and IX, designed within exon 1 and exon 13, respectively. Again, a
specific PCR product was obtained from the patient's RNA only.
Nucleotide sequencing of the 167-bp corresponding band confirmed the
large deletion observed in 5RACE experiments (not shown).
In addition to the large deletion, nucleotide sequencing of the
5RACE clones showed two discrete changes at the new exon 1/exon
13 boundary (Fig 2B). None of the 8 clones actually showed an intact
junction with three repeated CAG codons. Instead, 5 clones showed two
of these repeats, and the three remaining clones contained only one CAG
codon. Exon 1/intron 1 boundary sequence does not display an
alternative donor splicing site with a consensus context. Therefore, it
most likely participates to the splicing reaction with the wildtype
splicing site. However, the succession of three AG dinucleotides at the
intron 13/exon 13 junction offers three potential acceptor splicing
sites. It is therefore plausible that the skipping of CAG codons must
derive from the use of internal cryptic acceptor sites within exon 13 (Fig 2B). In a previous work, we brought up the use of the first
internal cryptic site in intron 12 splicing.12 Sequencing
analysis performed here suggests that splicing of the chimeric intron
1/12 preferentially uses the first internal cryptic site, and
secondarily the very downstream cryptic site, which is virtually not
used in normal intron 12 splicing.
Deleted protein 4.1 mRNA in lymphoblastoid cells.
Lymphoblastoid cell mRNAs originating from the proposita and from a
control were investigated. Northern blot analysis using pLym 5
showed a single 6.4-kb band in both the control and the patient. As in
reticulocyte 4.1 mRNA, pLym 3 showed a second major 4.4-kb band,
in the proposita (Fig 3). Thus, the mutant mRNA in lymphoblastoid cells was also stable and presented a similar 2-kb deletion.
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| Fig 3.
mRNA deletion in lymphoblastoid cells. Northern blot
analysis using cDNA probes pLym5 and pLym3. The band
sizes are given in kilobases. *Additional band present in the
proposita. C, control; I.2, proposita.
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RT-PCR experiments using primers IV and IX showed a 167-bp fragment
similar to that found in reticulocyte mRNA; the Taq I digestion
provided a rapid mean to probe the nature of this fragment. These data
suggest that lymphoblastoid cells also presented the same 1727-bp mRNA
deletion, which connected exon 1 to exon 13.
A large genomic deletion encompassing 11 exons.
To test the hypothesis of a gene rearrangement responsible for the mRNA
deletion, we first performed a Southern blot analysis of genomic DNA
from patients I.2 and II.1 and from the unaffected parent I.1 using a
pair of restriction endonucleases Msp I and Xba I. Hybridization with either pLym 3 or pA2.1 (Fig 1) showed an
additional band in both patients occurring at the expense of a normal
fragment; pLym 5 probe did not detect this particular fragment
(not shown). These findings suggested that the responsible genomic
abnormality would involve the 5 half of the gene and would end
within a genomic region encompassed by pA2.1 sequence.
Genomic DNAs were then further investigated by Southern blot analysis
using pA2.1 probe and EcoRI, Xba I, Bgl II, or
Msp I restriction enzymes. In 4.1() HE patients, several
bands presented a diminished signal. On Xba I, Bgl II,
and Msp I restriction patterns, additional abnormal-sized bands
were obtained (Fig 4A). These experiments,
together with the mRNA studies, strongly suggested that the
4.1() allele bears a large deletion at its 5 half.
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| Fig 4.
Southern blot analysis of the 3 endpoint genomic
deletion. (A) Southern blot analysis of genomic DNA from control (C)
and proposita (I.2), using pA2.1 cDNA probe (see also Fig 1).
Restriction enzymes used are indicated. Band sizes (in kilobases) are
given. Underlined sizes correspond to half intensity bands; *abnormal bands;  polymorphic site c.24 (B) Partial restriction
map, around exon 13, of normal 4.1 gene (top) and shortened allele 4.1 Annecy (bottom). X, B, M, and E represent restriction sites of
Xba I, Bgl II, Msp I, and EcoRI
enzymes, respectively. Underlined sites had been previously localized
(Baklouti et al23 and unpublished data).
Localization of Msp I site on the normal gene stemmed from results shown on (A). *Msp I site originating from intron 1 region. () The approximately 1-kb region containing the 3
endpoint of the genomic deletion.
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The 3 endpoint of the deletion was delineated by Southern blot
analysis using shorter cDNA probes: 3EX, EX11/12, and EX13 (Fig 1). The
abnormal-sized fragments detected with pA2.1 were consistently shown by
EX13 probe, which is an exon 13-specific probe, suggesting that the
3 endpoint must be at the vicinity of this exon. These data,
together with our previous genomic clone mapping (Baklouti et
al23 and unpublished data), allowed us to
locate the 3 endpoint within a 1-kb intronic region situated 250 bp upstream from exon 13 (Fig 4B).
To define the size and the 5 endpoint of the genomic deletion,
we analyzed the genomic region encompassing exons 1 and 2. Southern
blot using EX2 cDNA probe (Fig 1) on genomic DNA digested with
Xba I, Bgl II, Apa I, or Spe I
(Fig 5) showed half intensity signals in
4.1() HE carriers. The comparable signal intensity between the
control and the proposita, using pLym3 cDNA probe, argues
against a DNA loading difference and strongly suggests that the reduced
signal observed with EX2 probe is related to the gene deletion. These
results indicated that the genomic deletion encompassed all the coding
exons absent from the mRNA (exons 2 to 12). We further investigated the
genomic region surrounding exon 1 using NTER cDNA probe (Fig 1) and
fairly rare cutters (BamHI, EcoRV, and Kpn I).
The patterns obtained did not show any specific differences between the
controls and 4.1() HE patients (not shown), suggesting that no
additional event such as an inversion or a translocation had occurred
during the deletional event.
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| Fig 5.
Southern blot analysis of the 5 endpoint genomic
deletion. (Top) Southern blot analysis of genomic DNA from control (C)
and proposita (I.2), using EX2 cDNA probe (see also Fig 1). Notice the
reduced signal intensity (underlined sizes bands) consistently found in
the patient with EX2 probe. Restriction enzymes used are indicated.
Band sizes (in kilobases) are given. (Bottom) Hybridization of the same
Xba I/Bgl II membrane with pLym3 probe that
covers the nondeleted region of 4.1 Annecy gene.
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We finally attempted to capture the genomic region containing both the
5 and the 3 deletion endpoints using a long range PCR and
primers IV and IX (Table 1). The PCR experiments, using conditions for
up to the 20-kb long fragment, failed to amplify the deleted 4.1 gene.
These data, together with the absence of additional bands on Southern
blots using either EX2 or NTER probes, as explained above, suggested
that the 5 endpoint must lie within intron 1, several kilobases
away from both exon 1 and exon 2. Moreover, the exon-intron
organization of protein 4.1 gene study23 presented a gap in
the delineation of some intronic sequences: among others, intron 1 appears to be much larger than 25 kb. Therefore, exons 1 and 13 would
be separated by at least 70 kb.
In conclusion, 4.1 Annecy allele most likely results from a genomic
deletion that spans over 70 kb (Fig 6).
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| Fig 6.
Schematic representation of the genomic deletion. (Top)
Schematic out-of-scale representation of protein 4.1 gene. Tentative boundaries of the genomic deletion are depicted. ( ) Genomic deletion encompassing exons 2 to 12. () 5 and 3 endpoints
located downstream from exon 1 and in a 1-kb stretch upstream from exon
13, respectively. (Middle) Schematic representation of 4.1 Annecy gene.
(Bottom) Representation of the shortened 4.1 Annecy mRNA. For schematic representation of exons, see Fig 1.
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Erythroid and lymphoid expression of alternatively spliced exons.
The RT-PCR and 5RACE experiments described above clearly showed
that the chimeric intron, made of the 5 part of intron 1 and the
3 part of intron 12, is correctly spliced leading to a junction
of exon 1 and exon 13. We next asked whether the large genomic deletion
affects the tissue-specific splicing of the remaining exons. Two
overlapping RT-PCR experiments were performed on reticulocyte and
lymphoblastoid cell RNAs using primers designed within the nondeleted
cDNA sequence (Fig 7). These experiments showed that exons 14 and 15, tandemly present in brain,23,27 were
absent from normal and shortened mRNAs in both cell types. Exon 17a, recently described as a muscle specific exon,23 was
likewise absent in all cases. Similarly, exon 16 splicing still follows a tissue-specific regulation, regardless of the presence or absence of
the upstream region (exons 2 to 12) on the pre-mRNA: Exon 16 was indeed
present in reticulocyte mRNA, but absent from lymphocyte mRNA (Fig 7).
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| Fig 7.
Tissue-specific splicing of exons downstream from the
deletion. (Top) Schematic representation of the PCR strategy; the
expected PCR products and their sizes in basepairs are shown. (Bottom) RT-PCR on reticulocyte (R) or lymphoblastoid cell (L) mRNA, using primers XIII/XIV (A) or primers XV/XVI (B). C, control; I.2, proposita; M, size marker.
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DISCUSSION |
The present protein 4.1 allele, allele Annecy, provides the first known
instance of a very large deletion affecting a gene encoding an RBC
membrane protein (for review, see Delaunay2). It carries a
deletion longer than 70 kb. The 5 endpoint of the deletion is
situated in the intronic sequence 3 of exon 1. The 3
endpoint is located within a 1-kb region of the intronic sequence finishing 250 bp 5 upstream to exon 13. The extent of this
deletion cannot be assessed with greater accuracy, because the sizes
and sequences of the introns of protein 4.1 gene are still to be
defined.23 The mRNA that arises from this allele 4.1 Annecy
in erythroid cells carries a 1727-bp deletion, corresponding to 10 coding exons (exons 2 to 12; exon 3 is a noncoding sequence). This
deletion includes both the upstream and the downstream translation
initiation codons.
Following alleles 4.1 Madrid,22 4.1 Aravis,12
and 4.1 Lille,28 allele 4.1 Annecy is the fourth
4.1() HE allele the molecular basis of which is defined at the
gene level. The scarcity of protein 4.1 gene mutations elucidated so
far contrasts with the profusion of HE generating mutations in
SPTA1 and SPTB genes, encoding  - and  -spectrin
chains, respectively. This is presumably accounted for by the lack of
information at the protein level (protein 4.1 is just manifested by its
absence) and the knowledge, only recently acquired,23 of
the protein 4.1 gene structure.
The nucleotide sequence of mutated 4.1 mRNA Annecy is defined by a
deletion spanning exon 2 to exon 12 region and removing 1727 bases.
Exon 1 and exon 13 are joined, implying that the splicing had normally
occurred between these 2 exons. However, 3 nucleotides were missing in
the mutated sequence (exon 1)GCCCAGCAGCTG(exon 13) as
compared with the expected joined sequences (exon
1)GCCCAG/CAGCAGCTG(exon13). This stemed from the
use of a cryptic site within exon 13. Such an alternative splicing has
already been observed, although as a minor event, in normal
mRNA.12 Sequencing analysis showed still another minor
species generated, presumably by the skipping of a second CAG triplet
(exon 1)GCCCAGCT(exon 13) (Fig 2B), and the use of the
remaining AG cryptic site of the repetitive CAG sequence. These data
suggest that the sequence of exon 1 is identical to that in the normal
4.1 mRNA sequence and that the 5 end of exon 13 is submitted to
extra splicing events due to repetitive CAG sequences.
Among all 4.1() alleles described to date, only those
alterations whose expression is confined in the RBCs have been found at
the homozygous state; this is the case for allele 4.1 Madrid, mutated
in the erythroid translation initiation codon22 and the
Algerian 4.1 allele lacking three exons, including the one that carries
the downstream codon.29 Use of the upstream AUG codon
allows protein 4.1 expression in nonerythroid tissues. However, allele
4.1 Annecy is the only variant lacking both translation initiation
sites. One would speculate that such a variant would not exist at the
homozygous state.
Large deletions within functional genes usually jeopardize the
cytoplasmic mRNA accumulation either because of an altered transcription initiation, defective splicing, RNA transport, or in-frame premature stop codon. We and others have shown that the alteration of the translation initiation site often destabilizes mRNA22,30-32 (for review, see Cooper33).
Shortened mRNA 4.1 Annecy, although it potentially encodes for
nonfunctional protein(s), remained stable in the cytoplasm. It did so
not only in nucleated cells (lymphoblastoid cells), but also in
enucleated cells (reticulocytes), long after the nucleus to cytoplasm
transport.
We and others have shown that protein 4.1 pre-mRNA is endowed with
complex alternative splicing in a tissue and developmental stage-specific manners.14-17 In mRNA 4.1 Annecy, the
splicing pattern of exons downstream from the deletion is similar to
splicing in normal protein 4.1 mRNA. These findings, consistent with
our recent functional studies (Baklouti et al34 and
unpublished data), suggest that cis-elements
involved in exon recognition must lie within targeted exons and their
close vicinity intronic sequences.
In this work, we have defined the alteration of allele 4.1 Annecy,
which is responsible for 4.1() HE. We accurately defined the
1727-bp deletion at the mRNA level. This mRNA was stable and presented
the same feature in lymphoblastoid cells. At the gene level, we
delineated the first very large genomic deletion encountered in RBC
membrane protein genes, extending over 70 kb, starting and ending quite
far from the corresponding exons within introns 1 and 12, respectively.
Miniallele 4.1 Annecy nevertheless allowed the occurrence of a splicing
that connected exons 1 and 13 into a stable and shortened mRNA.
| |
FOOTNOTES |
Submitted May 5, 1997;
accepted January 16, 1998.
Supported by the Association Française contre les Myopathies, the
Centre National de la Recherche Scientifique (URA 1171), the
Université Claude Bernard Lyon I, the Conseil de la Région Rhône-Alpes, the Institut National de la Santé et de la
Recherche Médicale (CRE 930405), the Programme Hospitalier de
Recherche Clinique (1995-1997), and the Institut Pasteur de Lyon.
Address reprint requests to N. Dalla Venezia, PhD, CNRS UMR 5641, Laboratoire de Génétique, Domaine Rockefeller, 8 Avenue Rockefeller, 69373 Lyon Cedex 08, France.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank family PE for their kind cooperation. We thank Prof
G. Lenoir and M. Vuillaume (International Agency for Research on
Cancer) for obtaining the lymphoblastoid cell line and Dr J.P. Magaud
for growing the cells.
| |
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Abstract
Introduction
Abstract