J Hematol

Journal of Hematology, ISSN 1927-1212 print, 1927-1220 online, Open Access

Article copyright, the authors; Journal compilation copyright, J Hematol and Elmer Press Inc

Journal website http://www.thejh.org

Original Article

Volume 6, Number 4, October 2017, pages 73-80

The DAU Allele and Anti-D Alloimmunization Present With High Frequency in Brazilian Sickle Cell Disease Patients

Figure 1. Distribution of 1025T>C, which is related to the DAR allele, and its association with a high risk of hospitalization.

**Table 1.** Synthetic Oligonucleotides Used to Amplify Exons 3, 5, 7 and 8 of the *RHD* and *HBB* Genes
Gene	Sequence (5′→3′)	Exon RHD/mutation and HBB gene	Reference
F: forward; R: reverse. HBB: internal control beta globin gene.
RHDI4’	F: TAAGCACTTCACAGAGCGG	Exon 5/667T>G	[8]
EX5R	R: TCTTGCTGATCTTCCCTTGG
RHDEX3F	F: TATTCGGCTGGCCACCATGA	Exon 3/455A>C	[8]
RHDI3R	R: TTCTAATTTTGCCTGTAATC
RHD16F	F: TCATAGTGTGGTCCGTAAAC	Exon 7/1025T>C	[9]
RHDEX7R	R: CAATCATGCCCATTGCC
DAU 1B	F: TTGGCCATCGTGATAGCTCACAT	Exon 8/1136C>T	[10]
DAUB	R: GGAGATGGGGCACATAGACATC
HBB108	F: CAAGGACAGGTACGGCTGTCGTCATCAC	Promoter region	[11]
HBB109	R: TTTTCCCAAGGTTTGAACTAGCTCTTC	IVS-2 region

Table 1. Synthetic Oligonucleotides Used to Amplify Exons 3, 5, 7 and 8 of the RHD and HBB Genes

Gene

Sequence (5′→3′)

Exon RHD/mutation and HBB gene

Reference

F: forward; R: reverse. HBB: internal control beta globin gene.

RHDI4’

F: TAAGCACTTCACAGAGCGG

Exon 5/667T>G

[8]

EX5R

R: TCTTGCTGATCTTCCCTTGG

RHDEX3F

F: TATTCGGCTGGCCACCATGA

Exon 3/455A>C

[8]

RHDI3R

R: TTCTAATTTTGCCTGTAATC

RHD16F

F: TCATAGTGTGGTCCGTAAAC

Exon 7/1025T>C

[9]

RHDEX7R

R: CAATCATGCCCATTGCC

DAU 1B

F: TTGGCCATCGTGATAGCTCACAT

Exon 8/1136C>T

[10]

DAUB

R: GGAGATGGGGCACATAGACATC

HBB108

F: CAAGGACAGGTACGGCTGTCGTCATCAC

Promoter region

[11]

HBB109

R: TTTTCCCAAGGTTTGAACTAGCTCTTC

IVS-2 region

**Table 2.** *RHD* Gene Mutation Frequencies Among the SCD Patients and Healthy Individuals
Genotype	SCD (N = 193) (%)	Healthy group (N = 220) (%)	OR (95% CI); P
^§Chi-squared Yates corrected. ^§§Fisher’s exact test. SCD: sickle cell disease; OR: odds ratio; CI: confidence interval.
667T>G
T/T	174 (90.1)	211 (95.9)	2.56 (1.07 - 6.29); 0.034^§
T/G	15 (7.8)	6 (2.7)
G/D-	4 (2.1)	3 (1.4)
455A>C (DIIIa)
A/A	189 (97.9)	220 (100)	0.047^§§
A/C	3 (1.5)	0
C/D-	1 (0.6)	0
1025T>C (DAR)
T/T	178 (92.2)	211 (95.9)	1.99 (0.8 - 5.05); 0.16^§
T/C	12 (6.2)	6 (2.7)
C/D-	3 (1.6)	3 (1.4)
1136C>T (DAU)
Absence	150 (77.7)	212 (96.4)	7.65 (3.34 - 18.18); < 0.000^§
Presence	43 (22.3)	8 (3.6)
DIIIa/DAR or 1136C>T (DAU)
Absence	114 (78.6)**	203 (92.3)	3.25 (1.65 - 6.43); 0.002^§
Presence	31 (21.4)**	17 (7.7)

Table 2. RHD Gene Mutation Frequencies Among the SCD Patients and Healthy Individuals

Genotype

SCD (N = 193) (%)

Healthy group (N = 220) (%)

OR (95% CI); P

^§Chi-squared Yates corrected. ^§§Fisher’s exact test. SCD: sickle cell disease; OR: odds ratio; CI: confidence interval.

667T>G

T/T

174 (90.1)

211 (95.9)

2.56 (1.07 - 6.29); 0.034^§

T/G

15 (7.8)

6 (2.7)

G/D-

4 (2.1)

3 (1.4)

455A>C (DIIIa)

A/A

189 (97.9)

220 (100)

0.047^§§

A/C

3 (1.5)

C/D-

1 (0.6)

1025T>C (DAR)

T/T

178 (92.2)

211 (95.9)

1.99 (0.8 - 5.05); 0.16^§

T/C

12 (6.2)

6 (2.7)

C/D-

3 (1.6)

3 (1.4)

1136C>T (DAU)

Absence

150 (77.7)

212 (96.4)

7.65 (3.34 - 18.18); < 0.000^§

Presence

43 (22.3)

8 (3.6)

DIIIa/DAR or 1136C>T (DAU)

Absence

114 (78.6)**

203 (92.3)

3.25 (1.65 - 6.43); 0.002^§

Presence

31 (21.4)**

17 (7.7)

**Table 3.** Distributions of *DIIIa*, *DAR* and *DAU* Alleles Frequencies in the *RHD* Gene Among Individuals With HbSS and Healthy Individuals
Genotypes	HbSS (N = 137) (%)	Healthy individual group, N (%)	OR (95% CI); P
**N = 92. ^§Chi-squared Yates corrected. ^§§Fisher’s exact test. OR: odds ratio; CI: confidence interval; HbSS: sickle cell anemia.
667T>G
T/T	122 (89.1)	211 (95.9)	2.88 (1.15 - 7.38); 0.021^§
T/G	13 (9.5)	6 (2.7)
G/D-	2 (1.4)	3 ( 1.4 )
455A>C (DIIIa)
A/A	134 (97.8)	220 (100)	0.05^§§
A/C	2 (1.4)	0
C/D-	1 (0.8)	0
1025T>C (DAR)
T/T	125 (91.2)	211 (95.9)	2.25 (0.86 - 5.95); 0.11^§
T/C	11 (8.0)	6 (2.7)
C/D-	1 (0.8)	3 (1.4)
1136C>T (DAU)
Absence	109 (79.6)	212 (96.4)	6.81 (2.84 - 16.89); < 0.000^§
Presence	28 (20.4)	8 (3.6)
DIIIa/DAR or 1136C>T (DAU)
Absence	71 (77.2)**	203 (92.3)	3.53(1.67 - 7.48); < 0.000^§
Presence	21 (22.8)**	17 (7.7)

Table 3. Distributions of DIIIa, DAR and DAU Alleles Frequencies in the RHD Gene Among Individuals With HbSS and Healthy Individuals

Genotypes

HbSS (N = 137) (%)

Healthy individual group, N (%)

OR (95% CI); P

**N = 92. ^§Chi-squared Yates corrected. ^§§Fisher’s exact test. OR: odds ratio; CI: confidence interval; HbSS: sickle cell anemia.

667T>G

T/T

122 (89.1)

211 (95.9)

2.88 (1.15 - 7.38); 0.021^§

T/G

13 (9.5)

6 (2.7)

G/D-

2 (1.4)

3 ( 1.4 )

455A>C (DIIIa)

A/A

134 (97.8)

220 (100)

0.05^§§

A/C

2 (1.4)

C/D-

1 (0.8)

1025T>C (DAR)

T/T

125 (91.2)

211 (95.9)

2.25 (0.86 - 5.95); 0.11^§

T/C

11 (8.0)

6 (2.7)

C/D-

1 (0.8)

3 (1.4)

1136C>T (DAU)

Absence

109 (79.6)

212 (96.4)

6.81 (2.84 - 16.89); < 0.000^§

Presence

28 (20.4)

8 (3.6)

DIIIa/DAR or 1136C>T (DAU)

Absence

71 (77.2)**

203 (92.3)

3.53(1.67 - 7.48); < 0.000^§

Presence

21 (22.8)**

17 (7.7)

**Table 4.** Distributions of the *DIIIa*, *DAR* and *DAU* Alleles in SCD and Healthy Groups
RHD alleles	SCD patients (n = 193)	Healthy controls (n = 220)	OR (95% CI)	P
Chi-squared Yates corrected. *Fisher’s exact test. OR: odds ratio; CI: confidence interval; SCD: sickle cell disease; HbSS: sickle cell anemia; HbSC: hemoglobin SC.
667T>G	19 (9.8%)	9 (4.1%)	2.56 (1.07 - 6.29)	0.034*
DIIIa	4 (2.1%)	0	-	0.047**
DAR	15 (7.8%)	9 (4.1%)	1.99 (0.8 - 5.05)	0.16*
1136C>T (DAU)	43 (22.3%)	8 (3.6%)	7.65 (3.34 - 18.2)	< 0.000*
	HbSS (n = 137)
667T>G	15 (10.9%)	9 (4.1%)	2.88 (1.15 - 7.38)	0.021*
DIIIa	3 (2.2%)	0	-	0.05**
DAR	12 (8.8%)	9 (4.1%)	2.25 (0.86 - 5.95)	0.110*
1136C>T (DAU)	28 (22.2%) (n = 126)	8 (3.6%)	6.81 (2.84 - 16.89)	< 0.000*
	HbSC (n = 56)
667T>G	4 (7.1%)	9 (4.1%)	-	0.31**
DIIIa	1 (1.8%)	0	-	0.20**
DAR	3 (5.4%)	9 (4.1%)	-	0.71**
1136C>T (DAU)	14 (20.9%) (n = 67)	8 (3.1%)	7.00 (2.58 - 19.38)	< 0.0001*

Table 4. Distributions of the DIIIa, DAR and DAU Alleles in SCD and Healthy Groups

RHD alleles

SCD patients (n = 193)

Healthy controls (n = 220)

OR (95% CI)

*Chi-squared Yates corrected. **Fisher’s exact test. OR: odds ratio; CI: confidence interval; SCD: sickle cell disease; HbSS: sickle cell anemia; HbSC: hemoglobin SC.

667T>G

19 (9.8%)

9 (4.1%)

2.56 (1.07 - 6.29)

0.034*

DIIIa

4 (2.1%)

0.047**

DAR

15 (7.8%)

9 (4.1%)

1.99 (0.8 - 5.05)

0.16*

1136C>T (DAU)

43 (22.3%)

8 (3.6%)

7.65 (3.34 - 18.2)

< 0.000*

HbSS (n = 137)

667T>G

15 (10.9%)

9 (4.1%)

2.88 (1.15 - 7.38)

0.021*

DIIIa

3 (2.2%)

0.05**

DAR

12 (8.8%)

9 (4.1%)

2.25 (0.86 - 5.95)

0.110*

1136C>T (DAU)

28 (22.2%) (n = 126)

8 (3.6%)

6.81 (2.84 - 16.89)

< 0.000*

HbSC (n = 56)

667T>G

4 (7.1%)

9 (4.1%)

0.31**

DIIIa

1 (1.8%)

0.20**

DAR

3 (5.4%)

9 (4.1%)

0.71**

1136C>T (DAU)

14 (20.9%) (n = 67)

8 (3.1%)

7.00 (2.58 - 19.38)

< 0.0001*

**Table 5.** Distribution of Partial D Genotype Frequencies in the SCD, HbSS and Healthy Groups
RHD gene alleles	SCD (N = 193) (%)	HbSS (N = 137) (%)	Healthy individual group (N = 220) (%)
SCD: sickle cell disease; HbSS: sickle cell anemia.
DIIIa/D normal	2 (1.0 )	1 (0.7)	-
DAR/D normal	11 (5.7 )	11 (8.0)	6 (2.7)
DIIIa/D-	1 (0.5)	0	-
DAR/D-	3 (1.6)	1 (0.7)	3 (1.4)
DIIIa/DAU	1 (0.5)	1 (0.7)	-
DAR/DAU	1 (0.5)	0	-
DAU/ D-, D normal or DAU/DAU	40 (20.7)	27 (19.7)	8 (3.6)

Table 5. Distribution of Partial D Genotype Frequencies in the SCD, HbSS and Healthy Groups

RHD gene alleles

SCD (N = 193) (%)

HbSS (N = 137) (%)

Healthy individual group (N = 220) (%)

SCD: sickle cell disease; HbSS: sickle cell anemia.

DIIIa/D normal

2 (1.0 )

1 (0.7)

DAR/D normal

11 (5.7 )

11 (8.0)

6 (2.7)

DIIIa/D-

1 (0.5)

DAR/D-

3 (1.6)

1 (0.7)

3 (1.4)

DIIIa/DAU

1 (0.5)

1 (0.7)

DAR/DAU

1 (0.5)

DAU/ D-, D normal or DAU/DAU

40 (20.7)

27 (19.7)

8 (3.6)

**Table 6.** Distribution of the “C”, “c”, “E”, and “e” Antigens of the Rh System and the *DIIIa*, *DAR* and *DAU* Alleles of the Gene *RHD* Among Sickle Cell Disease Patients
RHD alleles	ccee	Ccee	ccEe	CcEe	Not conclusive
DIIIa/D normal	-	1	-	-	-
DAR/D normal	5	2	2	-	-
DIIIa/D-	-	-	-	-	1
DIIIa/DAU	1	-	-	-	-
DAR/DAU	1	-	-	-	-
DAU/D-, D normal or DAU/DAU	14	4	5	1	-
Total (%)	21 (56.8)	7 (18.9)	7 (18.9)	1 (2.7)	1 (2.7)

Table 6. Distribution of the “C”, “c”, “E”, and “e” Antigens of the Rh System and the DIIIa, DAR and DAU Alleles of the Gene RHD Among Sickle Cell Disease Patients

RHD alleles

ccee

Ccee

ccEe

CcEe

Not conclusive

DIIIa/D normal

DAR/D normal

DIIIa/D-

DIIIa/DAU

DAR/DAU

DAU/D-, D normal or DAU/DAU

Total (%)

21 (56.8)

7 (18.9)

1 (2.7)