MOLECULAR CHARACTERIZATION OF HEMOPHILIA A IN THE REPUBLIC OF MACEDONIA
Sukarova-Stefanovska E1, Zisovski N2, Muratovska O2, Kostova S3, Efremov GD1,*
*Corresponding Author: Professor Dr. Georgi D. Efremov, Research Centre for Genetic Engineering and Biotechnology, Macedonian Academy of Sciences and Arts, Bul. Krste Misirkov 2, 1000, Skopje, Republic of Macedonia; Tel: +389 2 3120 253; Fax: +389 2 3115 434; e-mail: gde@manu.edu.mk
page: 27

RESULTS

The results of Southern blot analysis for detection of an inversion in intron 22, were obtained after radiography. Hybridization signals of 21.5, 16.0 and 14.0 kb fragments were displayed when normal DNA was analyzed; 20.0, 17.5 and 14.0 kb fragments when DNA with an inversion type 1 was analyzed, and 20.0, 16.0 and 15.5 kb fragments when DNA with the inversion type 2 was analyzed (Fig. 1). The inversion in the fVIII gene was identified in 19 out of 50 patients, all with a severe form of the disease. The incidence of the intron 22 inversion in hemophilia A patients from the Republic of Macedonia was 38% (19/50) of all screened patients, or 67% (19/29) of patients with a severe form of hemophilia A. Among 19 inversions found in hemophilia A patients from the Republic of Macedonia, 18 (94.7%) had the distal inversion type 1, while one patient had the rare form of inversion, type 3A. None of the hemophilia A patients from the Republic of Macedonia had the proximal inversion type 2. The incidence and type of inversion among our patients of different ethnic groups is shown in Table 1. The mothers of 15 cases with inver­sions were also studied, and all were found to be heterozy­gotes for the inversion.

The patients who were excluded for the presence of an inversion in intron 22, were further analyzed for the pres­ence of single base substitutions and small deletions/ insertions in the coding regions of the fVIII gene. A total of 11 different point mutations were found in 15 unrelated hemophilia A patients (Table 2). Four of these sequence changes were novel single base substitutions [ATGAGG (MetArg), exon 1, codon 19; GCTCCT (AlaPro), exon 3, codon 78; TGCGGC (CysGly), exon 24, codon 2174, and TATGAT (TyrAsp), exon 25, codon 2256]; while seven identified point mutations [GAA TAA (GluTerm), exon 7, codon 272; TATTGT (Tyr Cys), exon 10, codon 473; CGGTGG (ArgTrp), codon 527, exon 11; GCCACC (AlaTyr), exon 14, codon 704; CGCTGC (ArgCys), exon 14, codon 1689; CGATGA (ArgTerm), exon 14, codon 1696; CGC TGC (ArgCys), exon 23, codon 2159] had already been reported in the hemophilia A database. The mutation Arg2159 Cys appears to be frequent among hemophilia A patients from the Republic of Macedonia, since the same mutation was found in four unrelated patients with a mild form of the disease.

 

Figure 1. Southern blot analysis for detection of the inversion in intron 22 of the fVIII gene. A) Lane 1: patient with inversion type 1; lane 2: carrier of inversion type 1; lane 3: patient with inversion type 2; lane 4: carrier of inversion type 2; lane 5: normal control. B) Lane 1: patient with inversion type 3A; lane 2: patient with inversion type 1; lane 3: carrier of inversion type 1; lane 4: normal control.

 

 

 

Table 1. Incidence and type of inversion in intron 22 of the factor VIII gene among 50 studied patients of different ethnic origins from the Republic of Macedonia

 

 

Ethnic origin

 

n

 

Inversion in int22h

 

%

Type of Inversion

Type 1

Type 2

Type 3A

 

Macedonian

 

29

 

7

 

24.1

 

7

 

 

 

Albanian

 

15

 

9

 

60.0

 

8

 

 

1

 

Serb

 

4

 

2

 

50.0

 

2

 

 

 

Gypsy

 

2

 

1

 

50.0

 

1

 

 

 

Total

 

50

 

19

 

38.0

 

18

 

 

1

 

Table 2. Mutations and sequence changes identified in hemophilia A patients from the Republic of Macedonia (novel mutations are shown in bold)

 

 

Patient

 

Nationality

 

Severity

 

fVIII:C

 

Exon

 

Codon

 

Nucleotide
Change

 

Amino Acid
Change

 

Domain

 

Ref.

 

   H-45

 

Albanian

 

severe

 

1

 

1

 

19

 

ATG>AGG

 

Met>Arg

 

signal peptide

 

24

 

   H-1

 

Albanian

 

severe

 

1.2

 

3

 

78

 

GCT>CCT

 

Ala>Pro

 

A1

 

24

 

   H-32

 

Albanian

 

severe

 

2

 

7

 

272

 

GAA>TAA

 

Glu>Stop

 

A1

 

18

 

   H-123

 

Macedonian

 

mild

 

20

 

10

 

473

 

TAT>TGT

 

Tyr>Cys

 

A2

 

20

 

   H-128

 

Macedonian

 

moderate

 

8

 

11

 

527

 

CGG>TGG

 

Arg>Trp

 

A2

 

20

 

   H-54

 

Macedonian

 

mild

 

13

 

14

 

704

 

GCC>ACC

 

Ala>Tyr

 

A2

 

21

 

   H-97

 

Macedonian

 

moderate

 

6

 

14

 

1689

 

CGC>TGC

 

Arg>Cys

 

A2

 

22

 

   H-52

 

Macedonian

 

severe

 

1

 

14

 

1696

 

CGA>TGA

 

Arg>Stop

 

A3

 

19

 

   H-53

 

Serbian

 

mild

 

18

 

23

 

2159

 

CGC>TGC

 

Arg>Cys

 

C1

 

19

 

   H-33

 

Serbian

 

mild

 

16

 

23

 

2159

 

CGC>TGC

 

Arg>Cys

 

C1

 

19

 

   H-125

 

Macedonian

 

mild

 

18

 

23

 

2159

 

CGC>TGC

 

Arg>Cys

 

C1

 

19

 

   H-129

 

Macedonian

 

mild

 

10

 

23

 

2159

 

CGC>TGC

 

Arg>Cys

 

C1

 

19

 

   H-14

 

Macedonian

 

moderate

 

6

 

24

 

2174

 

TGC>GGC

 

Cys>Gly

 

C2

 

24

 

   H-36

 

Macedonian

 

moderate

 

3.5

 

24

 

2174

 

TCG>GGC

 

Cys>Gly

 

C2

 

24

 

   H-25

 

Macedonian

 

mild

 

30

 

25

 

1689

 

TAT>GAT

 

Tyr>Asp

 

C2

 

Unpublished data




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