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From www.bloodjournal.org by guest on November 19, 2014. For personal use only.
1994 84: 2056
A second allele of spectrin alpha-gene associated with the alpha I/65
phenotype (allele alpha Ponte de Sor) [letter]
L Boulanger, D Dhermy, M Garbarz, C Silva, J Randon, R Wilmotte and J Delaunay
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CORRESPONDENCE
2056
Phenotype (Allele aPonle
de
1
ASecondAllele of Spedrin a-Gene Associated With the
Family SV
To the Editor:
Many mutations of spectrin a-gene result in hereditary elliptocytosis (HE) and are manifested by various peptide map abnormalities. A given alteration may derive from several mutations located
abnormalclose to one another. Inan opposite way, the so-called
ity has long been found to be associated only with the duplication
of TTG codon 154 (Leu).'.' defining allele a""ply,
as hereafter designated. This allele has a mild expression and appears in people from
Black Africa. Northern Africa, and Southern Italy. We describe here
two unrelated kindreds with a symptomless a"65change. Unexpectedly, the underlying mutation was a distinct and novel change: 151
GGT
GAT:Gly
Asp. The mutated allele was termed allele
a ~ ~P ~mW ~
+
Family RN
_"
It
1
l
Fig 1. Partial pedigrees. ( / ) Probands; (M) allele urmnd.
=; (0)
allele uLELv.
-+
Unrelated families SV and RN were Portuguese and French, respectively (Fig I ) . All members were symptomless and disclosed
normal red bloodcell indices. Elliptocytosis was observed in individuals SV 11.1 (40%) and RN 1.2 (20%). The methods used in protein
chemistry have been described or referred to before.'," Segments of
genomic DNA encompassing exon 2, 3, and 4 were amplified by
polymerase chain reaction andsequenced directly or after subcloning
in plasmid vector PGEM3Z. In some experiments, amplified DNA
was digested using BstEll or Fok I.
The percentage of spectrin dimers in crude Sp extracts (4°C) was
normal in three members of family SV (it was not investigated in
family RN). In individuals SV 1.2 and SV 11.1. two-dimensional
maps of spectrin disclosed the presence of a spot defining the a''"
phenotype (data not shown). Partial amino acid sequencing showed
that this fragment arose from an abnormal cleavage after Arg 137,
as is observed in the presence ofthe aDup allele, and disclosed
the 151 Glu Asp substitution. Nucleotide sequencing (individual
SV 11.1 and RN 11.1) displayed the 151 GGT -+ GAT change and
ruled out the duplication of TTG codon 154 (Fig 2). Whenever they
were looked for, a RsrEll site was abolished and a Fok I site was
created due IO the a151 mutation (data not shown). Nucleotide sequences of exons 2 and 3 (individual 11.1) did not show any additional alteration. Quantification of the a V 41-kD fragment" and assessment of exon 40 mutation' established heterozygosity for allele
al.l;l.Y
, a low expression allele, in four individuals (Fig l).
Taken together, two unrelated kindreds displaying the
pheno'sl
mutation but bore a novel
type failed to carry the common anuP
mutation at position I5 1 . The latter created the same abnormal trypsin cleavage site after Arg 137. A limited elliptocytosis accompanied
the al.lil.Y/aPa,ntC de SOr diplotype, but not the a""""dc '"/a diplotype.
The modulation by allele
assigned the primary alteration to
spectrin a-gene. The normal sequence of exons 2 and 3 ruled out
the possibility of any other mutation in the same conformational unit
(helices 2 or 3) or in helix I of repeat a2.Finally, the resemblance of
I" andthe fact that
the picture yielded by alleles aP""" si,' and
the latter is held responsible for elliptocytosis led us to consider that
the former yields elliptocytosis in a like fashion.
+
ACKNOWLEDGMENT
Supported in part by the "Association Franqaise contre les Myopathies." the "Conseil Scientifique de I'UFR Xavier-Bichat, UniversitC Paris VII," the "Centre National de la Recherche Scientifique," and the "lnstitut Pasteur de Lyon." We thank Dr L. Denoroy
(CNRS URA 1195. Lyon, France and Service Central d'Analyse.
CNRS, Vernaison, France) for having performed partial amino acid
sequencing.
Laurent Boulanger
Didier Dhermy
Michel Garbarz
INSERM U409
Faculte X . Bichat
Paris, France
Candido Silva
Lahorarorie de Hernarologia
Hospital de Santa Cruz
Camaxide, Portugal
Jacques Randon
Rick Wilmotte
Jean Delaunay
CNRS LIRA I171
Institut Pasteur de Lyon
L.yon. France
REFERENCES
I . Roux AF, Mode L, Guetarni D, Colonna P, Sahr K, Forget
BG, Delaunay J. Godet J: Molecular basis of Spa'"' hereditary elliptocytosis in North Africa: Insertion of a T G triplet between codon
147 and 149 in the a-spectrin gene from five unrelated families.
Blood 73:2196.1989
2. Sahr KE. Garbarz M, Dhermy D, Lecomte MC, Boivin P. Agre
P, Laughinghouse K, Scarpa A, Coetzer T, Palek J, Marchesi SL,
Forget BC: Use of the polymerase chain reaction for the detection
and characterization of mutations causing hereditary elliptocytosis,
in Cohen CM, Palek J(eds): Cellular and Molecular Biologyof
Normal and Abnormal Erythroid Membranes. New York, NY, Wiley-Liss. 1990, p 201
3. Lecomte MC, Gautero H, Garbarz M,BoivinP,Dhermy D:
Abnormal tryptic peptide from the spectrin a-chain resulting from
a-or P-chain mutations: Two genetically distinct forms of the Spa"
variant. Br J Haematol 76:406. 1990
4. Baklouti F, Marechal J, Wilmotte R, Alloisio N. Morle L,
Ducluzeau MT. Denoroy L, Mrad A, Ben Aribia MH, Kastally R,
Delaunay J: Elliptocytogenic a'"' spectrin Sfax lacks 9 amino acids
in helix 3 of repeat 4. Evidence for the activation of a cryptic S'
splice site in exon 8 of spectrin a-gene. Blood 79:2464, 1992
5. Wilmotte R, Marechal J, Mode L, Baklouti F, Philippe N.
Kastally R, Kotula L, Delaunay J, Alloisio N: Low expression allele
al.El.Y of red cell spectrin is associated with mutations in exon 40
polymorphism) andintron 45 andwithpartial
exon 46. J Clin Invest 91:2091, 1993
skipping of
From www.bloodjournal.org by guest on November 19, 2014. For personal use only.
2057
CORRESPONDENCE
3'
Leu
Leu
Y
NORMAL
MUTANT
"
1
ASP
Glu
Fig 2. Nucleotidesequencing
of part of exon 4 (individual SV
11.1).
Leu
Q
c
(3
r
C
Q
T
T
A
T
Q
C
Gin
3'
7
Q
G
A
C
C
Q
A
Q
T
Q
0
T
A
Q
v
C
5'
Gln
C
C
A
Q
T
Leu
"..
. .c -
,.,.
.I I
A C G T
ACGT
\ \a
Q
A
LYS
Giu
,C"
L
5'
Leu