Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1839-1840
CORRESPONDENCE
The RhD
Trait in a White Patient With the
RhCCee Phenotype Attributed to a Four-Nucleotide Deletion in the
RHD Gene
 |
LETTER |
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 D
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 D 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 D serotypes. This appears
valid generally for D ce haplotypes of white origin
(frequency [f] = .39) who
account for the vast majority of D phenotypes. However,
exceptions have been reported from whites with the less frequent Ce and
cE haplotypes (f = .0098 and .0119, respectively) and
amongst nonwhites.5-10
We described two D CCee+ 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identified using multiplex PCR assays (unpublished observations, April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 5
untranslated region (nucleotide 
19) to
the 3 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 5 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 (5-TGC TGG TGG
AGG TGA CAG-3) and antisense primer (5-GAA CAC GTA GAT GTG CAT
CAT-3) specific to the RHD gene. PCR products included a band
at approximately 670 bp not present in D 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
(5-CAC CTC CTA AGT GAA GCT CTG-3) 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.
Three distinct banding patterns were observed. A single PCR product was
obtained for B1 and his two brothers who were also serologically
D and Ce+ 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
D+ 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 D+ 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 antibody-induced 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 D trait.
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
| |
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.
| |
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