Correlation between genotype and phenotype of inherited factor Ⅴ deficiency

doi:10.3969/j.issn.1674-8115.2021.08.013

Abstract

Abstract: Objective

·To perform clinical phenotype diagnosis,thrombin generation assay (TGA) and F5 analysis in FⅤD patients, evaluate clinical bleeding risk and discuss the correlation between genotype and phenotype.

Methods

·Five patients with FⅤD who visited the Department of Hematology of The Second Affiliated Hospital of Guangzhou Medical University from January to November 2020 were selected. PT and APTT were detected by routine coagulation screening. Plasma FⅤ∶C and plasma FⅧ∶C were detected by one-stage coagulation. Plasma FⅤ∶Ag, free and total TFPI was measured by ELISA. The risk of bleeding was graded according to the bleeding score recommended by ISTH.TGA was used to evaluate the risk of clinical bleeding in the patients with FⅤD. All exons and flanking sequences of F5 were analyzed by Sanger sequencing, and the mutation sites were verified by reverse sequencing. AccuCopy multiple gene copy number detection technique was used to detect F5 copy number variation (CNV) in patients with FⅤD.

Results

·All 5 probands belonged to type Ⅰ FⅤD, including 2 cases of severe FⅤD (FⅤ∶C <1%). The levels of free and total TFPI decreased in varying degrees. The lagtime and time to peak of TGA were significantly prolonged, but the levels of peak height and endogenous thrombin potential varied. Mutation analysis revealed 8 kinds of mutations, including missense, nonsense, frameshift mutations and CNV, of which missense mutations accounted for 75% of the total mutations. p.Cys603Ser, p.Leu949Trpfs*, p.Leu1262_Gln1657del were novel mutations. Large fragment deletions in exon 13?14 of proband 2 were detected by AccuCopy (c.3784_4971del, p.Leu1262_Gln1657del). Further analysis of mRNA level showed that the large fragment deletion mutation led to abnormal mRNA splicing, resulting in novel splice site with three kinds of abnormal transcripts (c.3577_4971del, c.3577_4456del, c.3331_4456del).

Conclusion

·The level of FⅤ∶C in patients with severe FⅤD is not related to the severity of bleeding, but TGA and bleeding score can well correlate the bleeding risk. The bleeding severity of FⅤD may be related to the type of F5 mutation.

Key words: inherited factor Ⅴ deficiency, bleeding score, thrombin generation assay, phenotype, genotype

CLC Number:

R554.9

Ke-ke LI, Zhao-lin CHEN, Ying FENG, Yang XIAO. Correlation between genotype and phenotype of inherited factor Ⅴ deficiency[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(8): 1074-1080.

Figures/Tables 7

TrendMD

References 32

1	Duckers C, Simioni P, Rosing J, et al. Advances in understanding the bleeding diathesis in factor Ⅴ deficiency[J]. Br J Haematol, 2009, 146(1): 17-26.
2	Lippi G, Favaloro EJ, Montagnana M, et al. Inherited and acquired factor Ⅴ deficiency[J]. Blood Coagulation Fibrinolysis, 2011, 22(3): 160-166.
3	Dahlbäck B. Pro- and anticoagulant properties of factor Ⅴ in pathogenesis of thrombosis and bleeding disorders[J]. Int J Lab Hematol, 2016, 38(): 4-11.
4	Tabibian S, Shiravand Y, Shams M, et al. A comprehensive overview of coagulation factor Ⅴ and congenital factor Ⅴ deficiency[J]. Semin Thromb Hemost, 2019, 45(5): 523-543.
5	Monković DD, Tracy PB. Functional characterization of human platelet-released factor Ⅴ and its activation by factor Xa and thrombin[J]. J Biol Chem, 1990, 265(28): 17132-17140.
6	Gremmel T, Frelinger AL, Michelson AD. Platelet physiology[J]. Semin Thromb Hemostasis, 2016, 42(3): 191-204.
7	Duckers C, Simioni P, Spiezia L, et al. Low plasma levels of tissue factor pathway inhibitor in patients with congenital factor Ⅴ deficiency[J]. Blood, 2008, 112(9): 3615-3623.
8	Rodeghiero F, Tosetto A, Abshire T, et al. ISTH/SSC bleeding assessment tool: a standardized questionnaire and a proposal for a new bleeding score for inherited bleeding disorders[J]. J Thromb Haemost, 2010, 8(9): 2063-2065.
9	Elbatarny M, Mollah S, Grabell J, et al. Normal range of bleeding scores for the ISTH-BAT: adult and pediatric data from the merging project[J]. Haemophilia, 2014, 20(6): 831-835.
10	Bridey F, Lacombe C, Sustendal L, et al. Development of a method to separate lipoprotein-bound and lipoprotein-depleted tissue factor pathway inhibitor. Measurement of free tissue factor pathway inhibitor activity[J]. Blood Coagul Fibrinolysis, 1998, 9(7): 637-643.
11	Fu QH, Zhou RF, Liu LG, et al. Identification of three F5 gene mutations associated with inherited coagulation factor Ⅴ deficiency in two Chinese pedigrees[J]. Haemophilia, 2004, 10(3): 264-270.
12	Du RQ, Lu CC, Jiang ZW, et al. Efficient typing of copy number variations in a segmental duplication-mediated rearrangement hotspot using multiplex competitive amplification[J]. J Hum Genet, 2012, 57(8): 545-551.
13	Duga S, Asselta R, Tenchini ML. Coagulation factor Ⅴ[J]. Int J Biochem Cell Biol, 2004, 36(8): 1393-1399.
14	Al-Numair NS, Ramzan K, Saleh M, et al. First description of the molecular and clinical characterization of hereditary factor V deficiency in Saudi Arabia: report of four novel mutations[J]. Blood Coagul Fibrinolysis, 2019, 30(5): 224-232.
15	黄丹丹, 陆晔玲, 戴菁, 等. 凝血酶生成试验在遗传性凝血因子缺陷症患者出血评估中的价值[J]. 中国实验诊断学, 2011, 15(3): 503-507.
16	Tripodi A. Thrombin generation assay and its application in the clinical laboratory[J]. Clin Chem, 2016, 62(5): 699-707.
17	van Doorn P, Rosing J, Duckers C, et al. Factor Ⅴ has anticoagulant activity in plasma in the presence of TFPIα: difference between FV₁ and FV₂[J]. Thromb Haemost, 2018, 118(7): 1194-1202.
18	Shao YY, Wu WM, Xu GQ, et al. Low factor V level ameliorates bleeding diathesis in patients with combined deficiency of factor Ⅴ and factor Ⅷ[J]. Blood, 2019, 134(20): 1745-1754.
19	游国岭, 陆晔玲, 戴菁, 等. F8基因大片段缺失所导致的一例重型血友病A的分子发病机制研究[J]. 中华检验医学杂志, 2013, 36(6): 534-537.
20	Park CH, Park MS, Lee KO, et al. Congenital factor Ⅴ deficiency from compound heterozygous mutations with a novel variant c.2426del (p.Pro809Hisfs*2) in the F5 gene: a case report[J]. Medicine (Baltimore), 2020, 99(5): e18947.
21	Liu HC, Shen MC, Eng HL, et al. Asp68His mutation in the A1 domain of human factor Ⅴ causes impaired secretion and ineffective translocation[J]. Haemophilia, 2014, 20(4): e318-e326.
22	梁枫萍, 程鹏. 1例遗传性凝血因子Ⅴ缺乏症家系的基因分析[J]. 重庆医学, 2018, 47(14): 1970-1974.
23	Mann KG, Kalafatis M. Factor Ⅴ: a combination of dr Jekyll and mr Hyde[J]. Blood, 2003, 101(1): 20-30.
24	Kling SJ, Griffee M, Flanders MM, et al. Factor Ⅴ deficiency caused by a novel missense mutation, Ile417Thr, in the A2 domain[J]. J Thromb Haemost, 2006, 4(2): 481-483.
25	Schrijver I, Houissa-Kastally R, Jones CD, et al. Novel factor Ⅴ C2-domain mutation (R2074H) in two families with factor V deficiency and bleeding[J]. Thromb Haemost, 2002, 87(2): 294-299.
26	傅启华, 王鸿利, 王明山, 等. 2种新的凝血因子Ⅴ基因突变导致的遗传性凝血因子Ⅴ缺乏症[J]. 中华医学杂志, 2003, 83(4): 312-315.
27	Montefusco MC, Duga S, Asselta R, et al. A novel two base pair deletion in the factor Ⅴ gene associated with severe factor Ⅴ deficiency[J]. Br J Haematol, 2000, 111(4): 1240-1246.
28	Borhany M, Ranc A, Fretigny M, et al. Molecular analysis of eight severe FⅤ-deficient patients in Pakistan: a large series of homozygous for frameshift mutations[J]. Haemophilia, 2019, 25(4): e278-e281.
29	Castoldi E, Duckers C, Radu C, et al. Homozygous F5 deep-intronic splicing mutation resulting in severe factor Ⅴ deficiency and undetectable thrombin generation in platelet-rich plasma[J]. J Thromb Haemost, 2011, 9(5): 959-968.
30	Nuzzo F, Beshlawi I, Wali Y, et al. High incidence of intracranial bleeding in factor V-deficient patients with homozygous F5 splicing mutations[J]. Br J Haematol, 2017, 179(1): 163-166.
31	Nuzzo F, Bulato C, Nielsen BI, et al. Characterization of an apparently synonymous F5 mutation causing aberrant splicing and factor Ⅴ deficiency[J]. Haemophilia, 2015, 21(2): 241-248.
32	Hunt RC, Simhadri VL, Iandoli M, et al. Exposing synonymous mutations[J]. Trends Genet, 2014, 30(7): 308-321.

Number	Forward primer	Reverse primer	Length/bp
1	GGGAGAAAGCACCCCTCTTG	TGAGTAGTAGGCCCAAGCCC	2 251
2	GAGAAGCACACACACCATGC	GGACTTTCTGGCCCTGATCG	1 060

Number	Forward primer	Reverse primer	Length/bp
1	GGGAGAAAGCACCCCTCTTG	TGAGTAGTAGGCCCAAGCCC	2 251
2	GAGAAGCACACACACCATGC	GGACTTTCTGGCCCTGATCG	1 060

Proband	PT/s	APTT/s	FⅧ∶C/%	FⅤ∶C/%	FⅤ∶Ag/%	Free TFPI/（ng·mL^-1）	Total TFPI/（ng·mL^-1）
Reference range	10.00‒16.00	22.30‒38.70	50.00‒150.00	50.00‒150.00	70.00‒140.00	5.20‒14.80	50.80‒111.60
1	29.30	65.60	90.90	2.40	3.30	4.60	68.10
2	85.10	>180.00	85.20	<1.00	<1.00	4.50	47.50
3	39.80	83.60	100.50	<1.00	<1.00	2.20	34.50
4 5	34.70 40.90	79.75 86.70	94.60 74.20	2.50 1.60	5.68 <1.00	2.50 3.90	33.80 48.00

Proband	PT/s	APTT/s	FⅧ∶C/%	FⅤ∶C/%	FⅤ∶Ag/%	Free TFPI/（ng·mL^-1）	Total TFPI/（ng·mL^-1）
Reference range	10.00‒16.00	22.30‒38.70	50.00‒150.00	50.00‒150.00	70.00‒140.00	5.20‒14.80	50.80‒111.60
1	29.30	65.60	90.90	2.40	3.30	4.60	68.10
2	85.10	>180.00	85.20	<1.00	<1.00	4.50	47.50
3	39.80	83.60	100.50	<1.00	<1.00	2.20	34.50
4 5	34.70 40.90	79.75 86.70	94.60 74.20	2.50 1.60	5.68 <1.00	2.50 3.90	33.80 48.00

Proband	LT/min	Peak height/（nmol·L^-1）	Ttpeak/min	ETP/ ［nmol·（L·min）^-1］
PPP-1	13.33	232.86	16.00	1 171.56
PPP-2	10.50	2.22	16.33	0
PPP-3	9.67	169.13	15.00	2 147.40
PPP-4	8.50	359.16	10.83	1 584.50
PPP-5	10.17	159.47	13.50	1 098.01
PPP-N	4.67	111.96	10.17	1 132.26
PRP-1	14.83	94.25	30.33	1 819.92
PRP-2	19.33	14.31	47.50	0
PRP-N	8.26	87.56	21.34	1 550.07