Download The RhD Trait in a White Patient With the RhCCee

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CORRESPONDENCE
Nineteen Budd-Chiari cases (age ranging from 13 to 45 years; males,
8; females, 11), in whom hepatic doppler ultrasonography showed
occluded hepatic veins followed by subsequent histological confirmation, were screened for the presence of factor V Leiden mutations by
polymerase chain reaction (PCR). Except for three, all the remaining
patients had a negative family history of thrombosis.
The DNA extracted from blood leukocytes was amplified using a set
of primers (Cybersyn, Lenni, PA), followed by Mnl 1 digestion. The
digested fragments were run in a 3% low-melting agarose gel for 2
hours. Five were heterozygous for factor V Leiden mutations (26%)
(Fig 1). None were found to be homozygous in this group. All other tests
such as protein C, protein S, and antithrombin III were normal in these
cases.
Thus, approximately one fourth of our Budd-Chiari cases were
shown to have this genetic defect, ie, CGA = CAA(Arg506Gln) in the
factor V gene. However, it is possible theoretically that the mutation
may not always be Arg506Gln. The information about the prevalence of
this defect in our population was inadequate, but a preliminary study on
north Indian population had a factor V Leiden allele frequency of 1.2%.5
When compared with this, it was found that the defect had a high
frequency in cases of Budd-Chiari, suggesting the causal relationship
between the two.
Because Budd-Chiari cases show severe hepatocellular insufficiency,
the coagulation tests for factor V Leiden may not have been informative
in determining the thrombophilia status. Nevertheless, the detection of
factor V Leiden mutation by a simple PCR followed by enzyme
digestion is economical and less time consuming, with 100% specificity. With this high frequency of factor V Leiden mutations and
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comparatively low cost of DNA analysis, it may be suggested that this
mutation be examined in all the Budd-Chiari cases.
Dipika Mohanty
Shrimati Shetty
Institute of Immunohaematology (ICMR)
KEM Hospital Campus
Parel, Mumbal, India
T.S. Narayanan
Philip Abraham
Department of Gastroenterology
KEM Hospital
Parel, Mumbai, India
REFERENCES
1. Boughton BJ: Hepatic and portal vein thrombosis is closely associated
with chronic myeloproliferative disorders. Br Med J 302:192, 1991
2. Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ, De
Ronde H, Van der Veiden PA, Reitsma PH: Mutation in blood coagulation
factor V associated with resistence to activated protein C. Nature 36:64, 1994
3. Zoller B, Dahlback B: Inherited resistence to activated protein C
as a pathogenic risk factor for venous thrombosis. Recent Adv Blood
Coagul 7. Churchill Livingstone, UK, 1997, p 49
4. Denninger MH, Beidjord K, Durand F, Denie C, Valla D, Guillin
MC: Budd-Chiari syndrome and factor V Leiden mutation. Lancet
345:525, 1995
5. Rees DC, Cox M, Clegg JB: World distribution of factor V Leiden.
Lancet 346:1133, 1995
The RhD2 Trait in a White Patient With the RhCCee Phenotype Attributed
to a Four-Nucleotide Deletion in the RHD Gene
To the Editor:
The Rh blood group locus comprises two closely linked genes,
designated RHCE and RHD, encoding integral membrane proteins that
carry the Cc/Ee and D antigens, respectively. The D2 phenotype is
usually due to the complete deletion of the RHD gene from the Rh
locus.1 The D antigen is extremely immunogenic and is associated with
hemolytic disease of the newborn (HDN). HDN occurs when antibodies
from D2 women, who have been sensitized to the D antigen, cross the
placenta and react with antigens on fetal red blood cells. Polymerase
chain reaction (PCR) assays to detect the RHD gene have been
developed for determining fetal RHD gene type2-4 and paternal RHD
gene dosage3 with potential clinical value in the management of
pregnancies at risk for HDN. Such assays assume the RHD gene will be
completely absent in D2 serotypes. This appears valid generally for D2
ce haplotypes of white origin (frequency [ f] 5 .39) who account for the
vast majority of D2 phenotypes. However, exceptions have been
reported from whites with the less frequent Ce and cE haplotypes
( f 5 .0098 and .0119, respectively) and amongst nonwhites.5-10
We described two D2 CCee1 white blood donors where the RHD
gene was present in some form.5 One lacks RHD gene exons between 2
and 9 and would be correctly identified using multiplex PCR assays
(unpublished observations, April 1996). We report here that the other
donor, designated B1,5 carries a four-nucleotide deletion at a splice
junction along an otherwise normal RHD gene that would prevent
expression of the D antigen.
Total RNA extracted from whole blood buffy coat preparations was
reverse transcribed into cDNA. This was used as template in PCR
reactions to amplify four overlapping products, spanning the entire
RHD gene, from the 58 untranslated region (nucleotide 219) to the 38
untranslated region (nucleotide 1536). Sequencing cDNA-derived PCR
products showed a 4-base deletion between nucleotide positions 487
and 492 compared with two previously published RHD gene sequences11,12 (GenBank accession no. AF 037626). This corresponds to
the intron 3/exon 4 boundary. Where the sequence ACAGACT was
expected commencing at the 58 region of exon 4 the sequence ACT was
observed. Although the gene is still transcribed into a full-length mRNA
transcript and the remainder of the sequence is normal, the reading
frame is altered from nucleotide 488 and a premature stop codon
introduced at positions 496-498.
To confirm that the four-nucleotide deletion was not a splicing error
and was indeed present at the genomic level, genomic DNA from B1
was used to amplify across intron 3 into exon 4. The sense primer was
common to both the RHCE and RHD genes (58-TGC TGG TGG AGG
TGA CAG-38) and antisense primer (58-GAA CAC GTA GAT GTG
CAT CAT-38) specific to the RHD gene. PCR products included a band
at approximately 670 bp not present in D2 controls. Sequencing with
the antisense primer showed this band spanned the intron 3/exon 4
junction and confirmed that B1’s sequence lacked the four nucleotides
corresponding to positions 488 to 491. It also provided an additional
intron 3 sequence from which a new sense primer (58-CAC CTC CTA
AGT GAA GCT CTG-38) was designed and used with the above
antisense primer to amplify across the intron 3/exon 4 junction region in
genomic DNA studies of family members. PCR products of 150 or 146
bp were expected for the normal and abnormal RHD genes, respectively.
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1840
CORRESPONDENCE
Three distinct banding patterns were observed. A single PCR product
was obtained for B1 and his two brothers who were also serologically
D2 and Ce1 with genotypes (D)Ce/Ce [where (D) is the phenotypically
silent D gene]. This product migrated slightly faster than the single PCR
product observed from D1 family members, including two with DCe/Ce
genotypes and two genetically unrelated members with DCe/DcE and
DCe/DCe genotypes. Unexpectedly, a third pattern was observed for the
two heterozygote family members, who were the daughter of B1 and his
brother, respectively. Both were D1 with the DCe/(D)Ce genotype.
These showed a doublet corresponding to the expected 150 and 146 bp
fragments, and a third slower migrating band shown to be a heteroduplex comprising the combined 150- and 146-bp bands. The genotype of
each band was confirmed by sequencing.
Avent et al6 recently described a CCee individual with a single
nucleotide substitution in the RHD gene at nucleotide 121 in exon 1,
which results in an in frame stop codon. As for the mutation we describe
above, the RHD gene was associated with the Ce haplotype and in both
cases would have typed as RHD gene positive by current multiplex PCR
assays potentially available for determining the risk of HDN. However,
these rare occurrences would have little clinical significance. A fetus
carrying these deletions would be typed as RHD gene positive and
presumably the full precautions for monitoring potential antibodyinduced red blood cell destruction would be used. However, these
findings do add to the small but diverse array of genetic variations, other
than complete D gene deletion, which can generate the D2 trait.
ACKNOWLEDGMENT
Supported by the National Health and Medical Research Council of
Australia, the Alexander Steele Young Memorial Lions Foundation, and
the Brisbane North Regional Authority Liver Transplant Unit.
K.T. Andrews
L.C. Wolter
A. Saul
C.A. Hyland
Australian Red Cross Blood Service—Queensland
The Queensland Institute of Medical Research
Queensland, Australia
REFERENCES
1. Cartron JP: Defining the Rh blood group antigen. Blood Rev
8:199, 1994
2. Bennett PR, Le Van Kim C, Colin Y, Warwick RM, Chérif-Zahar
B, Fisk NM, Cartron JP: Prenatal determination of fetal RhD type by
DNA amplification. N Engl J Med 329:607, 1993
3. Wolter LC, Hyland CA, Saul A: Rhesus D genotyping using
polymerase chain reaction. Blood 85:1682, 1993
4. Simsek S, Bleeker PMM, Von Dem Borne AEG: Prenatal
determination of fetal RhD type. N Engl J Med 330:795, 1994
5. Hyland CA, Wolter LC, Saul A: Three Unrelated RhD gene
polymorphisms identified among blood donors with Rhesus CCee (r8r8)
phenotypes. Blood 84:321, 1994
6. Avent ND, Martin PG, Armstrong-Fisher SS, Liu W, Finning MK,
Maddocks D, Urbaniak SJ: Evidence of genetic diversity underlying
RhD-, weak D (Du), and partial D phenotypes as determined by
multiplex polymerase chain reaction analysis of the RHD gene. Blood
89:2568, 1997
7. Aubin J-T, Le Van Kim C, Mouro I, Colin Y, Bignozzi C, Brossard
Y, Cartron J-P: Specificity and sensitivity of RHD genotyping methods
by PCR-based amplification. Br J Haematol 98:356, 1997
8. Carritt B, Steers FJ, Avent ND: Prenatal determination of fetal
RhD type. Lancet 344:205, 1994
9. Huang CH: Alteration of RH gene structure and expression in
human dCCee and DCw-red blood cells: Phenotypic homozygosity
versus genotypic heterozygosity. Blood 88:2326, 1996
10. Okuda H, Kawano M, Iwamoto S, Tanaka M, Seno T, Okubo Y,
Kajii E: The RHD gene is highly detectable in RhD-negative Japanese
donors. J Clin Invest 100:373, 1997
11. Le Van Kim C, Mouro I, Chérif-Zahar B, Raynal V, Cherrier C,
Cartron JP, Colin Y: Molecular cloning and primary structure of the
human blood group RhD polypeptide. Proc Natl Acad Sci USA
89:10925, 1992
12. Arce MA, Thompson ES, Wagner S, Coyne KE, Ferdman BA,
Lublin DM: Molecular cloning of RhD cDNA derived from a gene
present in RhD-positive, but not RhD-negative individuals. Blood
82:651, 1993
The 20210A Allele of the Prothrombin Gene Is Frequent in Young Women
With Unexplained Spinal Cord Infarction
To the Editor:
Spinal cord infarction is a severe event: because almost the entire
motor output and sensory input systems are concentrated in a small
cross-sectional area, small localized vascular lesions can produce
devastating consequences far beyond the damage. Its rarity is classically
explained by the fact that the anterior and posterior spinal arteries are
not usually involved by atherosclerosis. Most infarctions are caused by
ischemia secondary to distant vascular occlusions, and only occasionally by angeiitis or emboli. They are facilitated by the unique arterial
system that irrigates the spinal cord and typically occur in a vascular
watershed region between the artery of Adamkiewicz arising from the
lower aorta and the anterior spinal artery arising from the vertebral
arteries.
We studied nine women, ages 22 to 41 years, who had developed an
acute anterior spinal artery syndrome. Computed tomography scanning
and magnetic resonance imaging showed aspects of spinal cord
infarction. A vascular malformation of the spinal cord had been
secondarily ruled out by selective spinal angiography in six of them. No
criteria for systemic arteritis, aortic thrombosis/dissection, or emboli
could be found. Seven of the women were current smokers (15 to 25
cigarettes per day), all were oral-contraceptive users (35 to 50 µg ethinyl
estradiol; second generation compounds in five, third generation in
three), and they were nonobese women (body mass indexes ranging
from 22.3 to 26.4). The patients had no personal history of thromboembolic disease despite pregnancies in six of them. One of the women had
known familial history of phlebitis.
After obtaining informed consent, screening for the prothrombin
variant caused by a G to A transition at nucleotide 20210 in the 38
untranslated region of the prothrombin gene (PT 20210A allele) was
performed by HindIII cleavage of a 345-bp fragment amplified by
polymerase chain reaction (PCR) using a mutagenic primer as previously described by Poort et al.1 Genetic analysis of the FV Leiden Arg
506 to Gln mutation (1691 G = A) was determined by PCR and Mnl I
restriction analysis of PCR-amplified genomic factor V DNA fragments
according to Bertina et al.2 The common homozygous 677C = T
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1998 92: 1839-1840
The RhD− Trait in a White Patient With the RhCCee Phenotype Attributed to
a Four-Nucleotide Deletion in the RHD Gene
K.T. Andrews, L.C. Wolter, A. Saul and C.A. Hyland
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