International Journal of Laboratory Hematology The Official journal of the International Society for Laboratory Hematology
LETTER TO THE EDITOR
INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY
Letter to the Editor
Factor IX mutations in hemophilia B patients from Liaoning Province, China Dear Sir or Madam, We investigated the molecular basis of hemophilia B (HB) in 27 patients with HB from 23 families residing in Liaoning Province, China, and found a variety of mutations that cause mild-to-severe HB. The patients were recruited from No. 202 Hospital of PLA, Shenyang. The diagnosis was based on factor IX levels, as determined by a one-stage clotting assay performed on citrated, platelet-poor plasma. Eleven patients had mild HB (factor IX coagulam activity: 6~30%), eight had moderate HB (factor IX coagulam activity: 2~5%), and eight had severe HB (factor IX coagulam activity: T in family 23, c.1191 insT in family 8, and c.1331 A>G in family 19; Table 1). The basic predicted tertiary structures of sequence fragments of the normal and mutated proteins are shown in Figure 1. The change of structures may result in the change of protein function, and further cause HB. © 2013 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, e59–e61
Some report showed that HB occurs in approximately 1 in every 30 000 male live births [1], and this rate varies little within races. HB is invariably caused by the mutations in the F9 gene, and these mutations show considerable heterogeneity among different cohorts of the patients with HB [2–4]. HB is an X-linked, inherited, blood clotting disorder. It is characterized by a factor IX deficiency caused by the mutations in the F9 gene located at Xq27.1 (GenBank Accession Number: K02402.1). Hepatocytes synthesize a factor IX precursor containing an amino-terminal signal sequence and a propeptide, both of which are cleaved off in separate reactions prior to protein secretion; defects in the removal of these sequences result in a nonfunctional protein [5]. The F9 gene and factor IX protein have shown considerable sequence homology and near-identical structural organization to those of the vitamin Kdependent serine proteases coagulation factor VII, factor X, and protein C. The F9 mutations detected in the patients with HB have been associated with varying disease severity [6]. The current factor IX mutation database includes 1094 unique F9 variants, with 798 point mutations [7]. We systematically analyzed the sequence of the entire F9 gene in 23 apparently unrelated families and identified causative mutations in each family. A total of 19 different mutations were detected, including one frameshift mutation, one nonsense point mutation, two splice-site point mutations, and fifteen missense mutations. Point mutations (single-nucleotide substitutions) were the most common gene defects. This result is consistent with another study, which reported that point mutations are present in approximately 73% of patients with HB [8]. CG transitions can cause missense or nonsense mutations, depending on the codon involved and the types of transition (C-to-T or G-to-A). C-to-T transitions are particularly damaging at arginine codons of the CGA type, because they involve the translation stop codon TGA (a nonsense mutation). In this study, eight point mutations involved CG sites, indicating that the CG dinucleotide is a mutational hot spot in the F9 gene, in agreement with another study [9].
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LETTER TO THE EDITOR
Table 1. Molecular changes in the F9 gene in 23 families with hemophilia B from Liaoning Province, China
No
Location
Region
Nucleotide mutation
Amino acid mutation
11 16 12 18 2 15 14 13 1 3 22 17 23 4 5 9 6 10 8 20 7 21 19
c.127 C>T c.127 C>T c.128 G>A c.214 G>C c.214 G>C c.275 T>C c.344 A>G c.370 G>A c.370 G>A c.373 G>A c.373 G>A +1c.390 c.485 G>T c.676 C>T c.677 G>A 2c.723 c.880 C>T c.881 G>A c.1191 insT c.1226 G>A c.1237 G>A c.1241 C>T c.1331 A>G
Exon 2 Exon 2 Exon 2 Exon 2 Exon 2 Exon 3 Exon 4 Exon 4 Exon 4 Exon 4 Exon 4 Intron4 Exon 5 Exon 6 Exon 6 Intron6 Exon 8 Exon 8 Exon 8 Exon 8 Exon 8 Exon 8 Exon 8
CGG>TGG CGG>TGG CGG>CAG GAA>CAA GAA>CAA GTT>GCT TAT>TGT GAA>AAA GAA>AAA GGA>AGA GGA>AGA G>C CGA>CTA* CGG>TGG CGG>CAG A>G CGA>TGA CGA>CAA Insert T * GGA>GAA GGA>AGA CCC>CTC TAT>TGT*
Arg>Trp Arg>Trp Arg>Gln Glu>Gln Glu>Gln Val>Ala Tyr>Cys Glu>Lys Glu>Lys Gly>Arg Gly>Arg Arg>Leu Arg>Trp Arg>Gln Arg>stop Arg>Gln Gly > Trp Gly > Glu Gly>Arg Pro>Leu Tyr>Cys
Mutation type
Classification
Missense mutation Missense mutation Missense mutation Missense mutation Missense mutation Missense mutation Missense mutation Missense mutation Missense mutation Missense mutation Missense mutation Splice-site mutation Missense mutation Missense mutation Missense mutation Splice-site mutation Nonsense mutation Missense mutation Frameshift mutation Missense mutation Missense mutation Missense mutation Missense mutation
Mild Mild Mild Severe Moderate Mild Severe Moderate Moderate Mild Mild Moderate Severe Moderate Severe Mild Severe Moderate Severe Mild Moderate Mild Severe
*Novel mutation.
(a)
(d)
(b)
(e)
(c)
(f)
We identified three novel mutations, all of which caused severe HB. The first one is a G>T mutation at nucleotide position c.485 in family 23, which was a missense mutation located at codon 162. This point mutation resulted in the substitution of arginine with leucine. These two amino acids have a similar size, but quite different
Figure 1. Prediction of tertiary protein structure. We used SWISS-MODEL to predict the basic tertiary structures of sequence fragments of mutated (a, b, c) and normal (d, e, f) factor IX proteins in families 23 (a, d), 8 (b, e), and 19 (c, f). These mutations caused severe or slight changes in protein structure.
electrical properties. Arginine is an exposed amino acid, while leucine is a buried one. The different properties of arginine and leucine and the predicted secondary structure of the mutated protein suggest that the mutation may affect the tertiary protein structure and therefore protein function (Figure 1a, d). © 2013 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, e59–e61
LETTER TO THE EDITOR
The second novel point mutation observed in family 8 consisted of the insertion of a T nucleotide at position c.1191 in exon 8 of the F9 gene. This one basepair (bp) insertion occurred between codons 397 and 398 and altered the codons following codon 397. The resultant frameshift mutation led to the substitution of glycine with tryptophan at position 398 in the catalytic domain of factor IX. In addition, the fourth codon after codon 397 was changed from GAA to TGA, leading to a premature stop codon in exon 8. The truncated factor IX has different secondary and tertiary structures from the normal protein (Figure 1b, e). Moreover, the active site encoded by codon 411 was lost, which might have abolished the binding activity or other functions of factor IX. Thus, this insertion mutation was detrimental and caused severe HB. The last novel mutation detected in this study was a T>G transversion in exon 8 at nucleotide position c.1331 in the propositus of family 19. We confirmed the presence of this mutation by sequencing a second independent PCR product from the propositus. This mutation was a missense mutation that resulted in the substitution of tyrosine located at position 444 with cystine. Tyrosine is an aromatic, hydrophilic amino acid that is much larger than hydrophobic cystine. Despite these differences, this amino acid substitution did not greatly alter the tertiary protein structure (Figure 1c, f). Nevertheless, change in the secondary structure may affect protein function. Further analysis is required to determine whether this mutation alters the structure and function of the factor IX protein. No double mutations were detected in all our samples, which may due to the limited size of our samples. Our present study has confirmed the genetic causes of HB in these families and has potentiated the possibility of prenatal diagnosis for fetus. Taken together, we found three
References 1. Belvini D, Salviato R, Radossi P, Pierobon F, Mori P, Castaldo G, Tagariello G, AICH HB Study Group. Molecular genotyping of the Italian cohort of patients with hemophilia B. Haematologica 2005;90:635–42. 2. Mukherjee S, Mukhopadhyay A, Banerjee D, Chandak GR, Ray K. Molecular pathology of haemophilia B: identification of five novel mutations including a LINE 1 insertion in Indian patients. Haemophilia 2004;10:259–63. 3. Espinos C, Casana P, Haya S, Cid AR, Aznar JA. Molecular analyses in hemophilia B families: identification of six new mutations in
novel mutation types and have added them to the HB mutation database. Furthermore, via structural prediction, two novel types of the missense mutations had insignificant influence on protein structure, but the patients in these two families (families 23 and 19) had severe HB. The underlying mechanism of this phenomenon remains unclear, but may involve interindividual differences. We are intended to further investigate this finding in our future studies.
Acknowledgements We thank all the patients and their families who participated in this study. This study was supported by the National Foundation of China (No. 30973140), the Doctor starting Funding of Liaoning (No. 20111123), and the Natural Science Foundation of Liaoning (No. 201202230).
D. H. Cao*,1, X. L. Liu†,1, X. W. Ma*, J. L. Sun*, X. Z. Bai*, G. B. Qiu‡,* *Aristogenesis Center, No. 202 Hospital of PLA, Shenyang 110003, China †
Assisted reproductive technology laboratory, Shenyang Women’s and Children’s Hospital, Shenyang 110011, China ‡ Department of Laboratory Medicine, No. 202 Hospital of PLA, Shenyang 110003, China 1
These authors contributed equally to this work.
E-mail: [email protected] doi: 10.1111/ijlh.12169
the factor IX gene. Haematologica 2003;88:235–6. 4. Onay UV, Kavakli K, Kilinc Y, Gurgey A, Aktuglu G, Kemahli S, Ozbek U, Caglayan SH. Molecular pathology of haemophilia B in Turkish patients: identification of a large deletion and 33 independent point mutations. Br J Haematol 2003;120:656–9. 5. Bentley AK, Rees DJ, Rizza C, Brownlee GG. Defective propeptide processing of blood clotting factor IX caused by mutation of arginine to glutamine at position -4. Cell 1986;45:343–8. 6. Balraj P, Ahmad M, Khoo AS, Ayob Y. Factor IX mutations in haemophilia B patients
© 2013 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, e59–e61
e61
in Malaysia: a preliminary study. Malays J Pathol 2012;34:67–9. 7. Factor IX Mutation database [Internet]. http://www.biochem.ucl.ac.uk/pavithra/fix/ 8. Rallapalli PM, Kemball-Cook G, Tuddenham EG, Gomez K, Perkins SJ. An interactive mutation database for human coagulation factor IX provides novel insights into the phenotypes and genetics of hemophilia B. J Thromb Haemost 2013;11:1329–40. 9. Bowen DJ. Haemophilia A and haemophilia B; molecular insights. Mol Pathol 2002; 55:127–44.
LETTER TO THE EDITOR
INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY
Letter to the Editor
Factor IX mutations in hemophilia B patients from Liaoning Province, China Dear Sir or Madam, We investigated the molecular basis of hemophilia B (HB) in 27 patients with HB from 23 families residing in Liaoning Province, China, and found a variety of mutations that cause mild-to-severe HB. The patients were recruited from No. 202 Hospital of PLA, Shenyang. The diagnosis was based on factor IX levels, as determined by a one-stage clotting assay performed on citrated, platelet-poor plasma. Eleven patients had mild HB (factor IX coagulam activity: 6~30%), eight had moderate HB (factor IX coagulam activity: 2~5%), and eight had severe HB (factor IX coagulam activity: T in family 23, c.1191 insT in family 8, and c.1331 A>G in family 19; Table 1). The basic predicted tertiary structures of sequence fragments of the normal and mutated proteins are shown in Figure 1. The change of structures may result in the change of protein function, and further cause HB. © 2013 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, e59–e61
Some report showed that HB occurs in approximately 1 in every 30 000 male live births [1], and this rate varies little within races. HB is invariably caused by the mutations in the F9 gene, and these mutations show considerable heterogeneity among different cohorts of the patients with HB [2–4]. HB is an X-linked, inherited, blood clotting disorder. It is characterized by a factor IX deficiency caused by the mutations in the F9 gene located at Xq27.1 (GenBank Accession Number: K02402.1). Hepatocytes synthesize a factor IX precursor containing an amino-terminal signal sequence and a propeptide, both of which are cleaved off in separate reactions prior to protein secretion; defects in the removal of these sequences result in a nonfunctional protein [5]. The F9 gene and factor IX protein have shown considerable sequence homology and near-identical structural organization to those of the vitamin Kdependent serine proteases coagulation factor VII, factor X, and protein C. The F9 mutations detected in the patients with HB have been associated with varying disease severity [6]. The current factor IX mutation database includes 1094 unique F9 variants, with 798 point mutations [7]. We systematically analyzed the sequence of the entire F9 gene in 23 apparently unrelated families and identified causative mutations in each family. A total of 19 different mutations were detected, including one frameshift mutation, one nonsense point mutation, two splice-site point mutations, and fifteen missense mutations. Point mutations (single-nucleotide substitutions) were the most common gene defects. This result is consistent with another study, which reported that point mutations are present in approximately 73% of patients with HB [8]. CG transitions can cause missense or nonsense mutations, depending on the codon involved and the types of transition (C-to-T or G-to-A). C-to-T transitions are particularly damaging at arginine codons of the CGA type, because they involve the translation stop codon TGA (a nonsense mutation). In this study, eight point mutations involved CG sites, indicating that the CG dinucleotide is a mutational hot spot in the F9 gene, in agreement with another study [9].
e59
e60
LETTER TO THE EDITOR
Table 1. Molecular changes in the F9 gene in 23 families with hemophilia B from Liaoning Province, China
No
Location
Region
Nucleotide mutation
Amino acid mutation
11 16 12 18 2 15 14 13 1 3 22 17 23 4 5 9 6 10 8 20 7 21 19
c.127 C>T c.127 C>T c.128 G>A c.214 G>C c.214 G>C c.275 T>C c.344 A>G c.370 G>A c.370 G>A c.373 G>A c.373 G>A +1c.390 c.485 G>T c.676 C>T c.677 G>A 2c.723 c.880 C>T c.881 G>A c.1191 insT c.1226 G>A c.1237 G>A c.1241 C>T c.1331 A>G
Exon 2 Exon 2 Exon 2 Exon 2 Exon 2 Exon 3 Exon 4 Exon 4 Exon 4 Exon 4 Exon 4 Intron4 Exon 5 Exon 6 Exon 6 Intron6 Exon 8 Exon 8 Exon 8 Exon 8 Exon 8 Exon 8 Exon 8
CGG>TGG CGG>TGG CGG>CAG GAA>CAA GAA>CAA GTT>GCT TAT>TGT GAA>AAA GAA>AAA GGA>AGA GGA>AGA G>C CGA>CTA* CGG>TGG CGG>CAG A>G CGA>TGA CGA>CAA Insert T * GGA>GAA GGA>AGA CCC>CTC TAT>TGT*
Arg>Trp Arg>Trp Arg>Gln Glu>Gln Glu>Gln Val>Ala Tyr>Cys Glu>Lys Glu>Lys Gly>Arg Gly>Arg Arg>Leu Arg>Trp Arg>Gln Arg>stop Arg>Gln Gly > Trp Gly > Glu Gly>Arg Pro>Leu Tyr>Cys
Mutation type
Classification
Missense mutation Missense mutation Missense mutation Missense mutation Missense mutation Missense mutation Missense mutation Missense mutation Missense mutation Missense mutation Missense mutation Splice-site mutation Missense mutation Missense mutation Missense mutation Splice-site mutation Nonsense mutation Missense mutation Frameshift mutation Missense mutation Missense mutation Missense mutation Missense mutation
Mild Mild Mild Severe Moderate Mild Severe Moderate Moderate Mild Mild Moderate Severe Moderate Severe Mild Severe Moderate Severe Mild Moderate Mild Severe
*Novel mutation.
(a)
(d)
(b)
(e)
(c)
(f)
We identified three novel mutations, all of which caused severe HB. The first one is a G>T mutation at nucleotide position c.485 in family 23, which was a missense mutation located at codon 162. This point mutation resulted in the substitution of arginine with leucine. These two amino acids have a similar size, but quite different
Figure 1. Prediction of tertiary protein structure. We used SWISS-MODEL to predict the basic tertiary structures of sequence fragments of mutated (a, b, c) and normal (d, e, f) factor IX proteins in families 23 (a, d), 8 (b, e), and 19 (c, f). These mutations caused severe or slight changes in protein structure.
electrical properties. Arginine is an exposed amino acid, while leucine is a buried one. The different properties of arginine and leucine and the predicted secondary structure of the mutated protein suggest that the mutation may affect the tertiary protein structure and therefore protein function (Figure 1a, d). © 2013 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, e59–e61
LETTER TO THE EDITOR
The second novel point mutation observed in family 8 consisted of the insertion of a T nucleotide at position c.1191 in exon 8 of the F9 gene. This one basepair (bp) insertion occurred between codons 397 and 398 and altered the codons following codon 397. The resultant frameshift mutation led to the substitution of glycine with tryptophan at position 398 in the catalytic domain of factor IX. In addition, the fourth codon after codon 397 was changed from GAA to TGA, leading to a premature stop codon in exon 8. The truncated factor IX has different secondary and tertiary structures from the normal protein (Figure 1b, e). Moreover, the active site encoded by codon 411 was lost, which might have abolished the binding activity or other functions of factor IX. Thus, this insertion mutation was detrimental and caused severe HB. The last novel mutation detected in this study was a T>G transversion in exon 8 at nucleotide position c.1331 in the propositus of family 19. We confirmed the presence of this mutation by sequencing a second independent PCR product from the propositus. This mutation was a missense mutation that resulted in the substitution of tyrosine located at position 444 with cystine. Tyrosine is an aromatic, hydrophilic amino acid that is much larger than hydrophobic cystine. Despite these differences, this amino acid substitution did not greatly alter the tertiary protein structure (Figure 1c, f). Nevertheless, change in the secondary structure may affect protein function. Further analysis is required to determine whether this mutation alters the structure and function of the factor IX protein. No double mutations were detected in all our samples, which may due to the limited size of our samples. Our present study has confirmed the genetic causes of HB in these families and has potentiated the possibility of prenatal diagnosis for fetus. Taken together, we found three
References 1. Belvini D, Salviato R, Radossi P, Pierobon F, Mori P, Castaldo G, Tagariello G, AICH HB Study Group. Molecular genotyping of the Italian cohort of patients with hemophilia B. Haematologica 2005;90:635–42. 2. Mukherjee S, Mukhopadhyay A, Banerjee D, Chandak GR, Ray K. Molecular pathology of haemophilia B: identification of five novel mutations including a LINE 1 insertion in Indian patients. Haemophilia 2004;10:259–63. 3. Espinos C, Casana P, Haya S, Cid AR, Aznar JA. Molecular analyses in hemophilia B families: identification of six new mutations in
novel mutation types and have added them to the HB mutation database. Furthermore, via structural prediction, two novel types of the missense mutations had insignificant influence on protein structure, but the patients in these two families (families 23 and 19) had severe HB. The underlying mechanism of this phenomenon remains unclear, but may involve interindividual differences. We are intended to further investigate this finding in our future studies.
Acknowledgements We thank all the patients and their families who participated in this study. This study was supported by the National Foundation of China (No. 30973140), the Doctor starting Funding of Liaoning (No. 20111123), and the Natural Science Foundation of Liaoning (No. 201202230).
D. H. Cao*,1, X. L. Liu†,1, X. W. Ma*, J. L. Sun*, X. Z. Bai*, G. B. Qiu‡,* *Aristogenesis Center, No. 202 Hospital of PLA, Shenyang 110003, China †
Assisted reproductive technology laboratory, Shenyang Women’s and Children’s Hospital, Shenyang 110011, China ‡ Department of Laboratory Medicine, No. 202 Hospital of PLA, Shenyang 110003, China 1
These authors contributed equally to this work.
E-mail: [email protected] doi: 10.1111/ijlh.12169
the factor IX gene. Haematologica 2003;88:235–6. 4. Onay UV, Kavakli K, Kilinc Y, Gurgey A, Aktuglu G, Kemahli S, Ozbek U, Caglayan SH. Molecular pathology of haemophilia B in Turkish patients: identification of a large deletion and 33 independent point mutations. Br J Haematol 2003;120:656–9. 5. Bentley AK, Rees DJ, Rizza C, Brownlee GG. Defective propeptide processing of blood clotting factor IX caused by mutation of arginine to glutamine at position -4. Cell 1986;45:343–8. 6. Balraj P, Ahmad M, Khoo AS, Ayob Y. Factor IX mutations in haemophilia B patients
© 2013 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, e59–e61
e61
in Malaysia: a preliminary study. Malays J Pathol 2012;34:67–9. 7. Factor IX Mutation database [Internet]. http://www.biochem.ucl.ac.uk/pavithra/fix/ 8. Rallapalli PM, Kemball-Cook G, Tuddenham EG, Gomez K, Perkins SJ. An interactive mutation database for human coagulation factor IX provides novel insights into the phenotypes and genetics of hemophilia B. J Thromb Haemost 2013;11:1329–40. 9. Bowen DJ. Haemophilia A and haemophilia B; molecular insights. Mol Pathol 2002; 55:127–44.
