Identification of novel pathogenic F13A1 mutation and novel NBEAL2 gene missense mutation in a pedigree with hereditary congenital factor XIII deficiency

Accepted Manuscript Identification of novel pathogenic F13A1 mutation and novel NBEAL2 gene missense mutation in a pedigree with hereditary congenital factor XIII deficiency

Siyuan Jia, Yunyan He, Meirong Lu, Ning Liao, Yonghong Lei, Nikuze Lauriane, Kairong Liang, Hongying Wei PII: DOI: Reference:

S0378-1119(19)30334-8 https://doi.org/10.1016/j.gene.2019.03.067 GENE 43754

To appear in:

Gene

Received date: Revised date: Accepted date:

22 February 2019 26 March 2019 28 March 2019

Please cite this article as: S. Jia, Y. He, M. Lu, et al., Identification of novel pathogenic F13A1 mutation and novel NBEAL2 gene missense mutation in a pedigree with hereditary congenital factor XIII deficiency, Gene, https://doi.org/10.1016/j.gene.2019.03.067

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ACCEPTED MANUSCRIPT Identification of novel pathogenic F13A1 mutation and novel NBEAL2 gene missense mutation in a pedigree with hereditary congenital factor XIII deficiency Siyuan Jia1*, Yunyan He2*, Meirong Lu1, Ning Liao2, Yonghong Lei2 , Nikuze Lauriane2 , Kairong Liang2 & Hongying Wei2#

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Affiliations: 1Guangxi Medical University; and 2Department of Pediatrics, The First

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Affiliated Hospital of Guangxi Medical University, Guangxi Key Laboratory of Thalassemia Research, No.6, Shuangyong Road, Qingxiu District, Nanning, Guangxi

#

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province, 530021, P.R.China

Address correspondence to: Hongying Wei, MD, PhD, Department of Pediatrics,

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The First Affiliated Hospital of Guangxi Medical University, Guangxi Key Laboratory of Thalassemia Research, No.6, Shuangyong Road, Qingxiu District, Nanning, province,

530021,

[email protected] *

P.R.China,

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Guangxi

Fax:

+8607715356780,

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Siyuan Jia and Yunyan He contribute equally to this manuscript.

1

E-mail:

ACCEPTED MANUSCRIPT Abstract The genetic defects of a 12-year-old patient with factor XIII deficiency (FXIIID) and eight pedigree members suspected with FXIIID were studied. Clinical diagnosis, pedigree investigation, phenotypic study and genetic analysis were performed. DNA sequence analysis revealed that the proband had a novel deletion mutation of F13A1

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gene (NM_000129: exon 12: c.1652delC: p.Thr551LysfsTer26) which he inherited from both the parents who were heterozygous for the same 1652delC deletion. This

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frameshift (p.Thr551LysfsTer26) led in homozygous form to severe FXIIID. Additionally, a homozygous missense mutation of NBEAL2 gene (NM_015175: exon

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13: c.1367C>T: p.Ala456Val) was identified in the proband. Again, the mutation was inherited from both the parents who were heterozygous for the same c.1367C>T novel

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mutation. Other members of the pedigree were also revealed to be heterozygous for the same proband’s F13A1 and NBEAL2 genes mutations. We first report a pedigree

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with pathogenic F13A1 gene mutation and a novel mutant NBEAL2 gene.

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Keywords: F13A1, NBEAL2, FXIIID, GPS

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ACCEPTED MANUSCRIPT Introduction Hereditary factor XIII deficiency (FXIIID) is a rare inherited blood disease characterized by lifelong bleeding tendency with an incidence of about 1 per 2 million in the general population. Consanguineous marriage can increase the risk (Dorgalaleh and Rashidpanah, 2016). Mutations of the F13A1 gene or F13B gene are causative,

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F13A1 gene mutations are the main cause (Ivaskevicius et al., 2007). The F13A1 gene is located on chromosome 6p24-25 and comprises 15 exons and 4 introns. In The

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Human Gene Mutation Database (HGMD), only 183 F13A1 gene mutations and 20 F13B genetic variations have been reported (Sun et al., 2018). In affected patients,

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clinical manifestations can range from micro skin bleeding to fatal intracranial hemorrhage resulting from FXIII variations. Due to scarcity and heterogeneity,

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FXIIID is easily misdiagnosed or underdiagnosed. The rarity and normal standard coagulation screening tests add to the diagnosis difficulty. Rich clinical experience,

misdiagnosis (Karimi et al., 2018).

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further confirmed test and genetic analysis can decrease missed diagnosis or

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Today, identification of causative NBEAL2 mutations facilitates the genetic diagnosis

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of most Grey platelet syndrome (GPS) patients. GPS is an uncommon bleeding disorder characterized by platelet defects. A reduction or absence of α-granules in platelets lead to platelets enlarged, with a grey appearance on light microscopy of

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Wright-stained peripheral blood smears (Nurden and Nurden, 2007). With the help of next-generation sequencing, GPS-associated NBEAL2 mutations are coming to

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accumulate (Pluthero et al., 2018). Quite recently, Orna Steinberg-Shemer and Hannah Tamary found a variation (c.7225-1G.C) in NBEAL2 gene and concluded that a blood smear is the key to identify GPS (Steinberg-Shemer and Tamary, 2018). The genetic mechanism of molecular defects in GPS remains unclear. Methods A 12-year-old boy was born from parents with a history of consanguineous marriage. He had a history of left iliac fossa hematoma and left axillary hematoma when he was 11 years old. The left iliac fossa hematoma was dissected and followed by postoperative bleeding. He was admitted to our pediatric inpatient department 3

ACCEPTED MANUSCRIPT presenting with fever, back pain for 3 days and lower limb weakness for 4 days. The pain in the lower back and cervical spine was worsen with activity. Four days before being referred to our department, he was diagnosed at a local hospital with “subarachnoid

hemorrhage

with

hydrocephalus”.

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treatment

included

anti-inflammatory therapy and symptomatic treatment, but the patient condition did

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not improve. The following day the patient developed sudden numbness of the lower extremities, accompanied by difficulty in urinating. He was taken to our hospital

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where brain CT demonstrated hydrocephalus and lumbar CT didn’t show any abnormality. Lumbar puncture revealed a bloody cerebrospinal fluid. He was admitted

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to the Department of Neurosurgery for signs of neurological dysfunction; “spinal cord lesions to be investigated” was the initial diagnosis. He was suspected of bladder

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retention and got a Foley catheter inserting and other symptomatic treatment in the Department of Neurosurgery. He was later referred to our department for further

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treatment. The investigations revealed normal platelet count and the mean platelet volume, prothrombin time (PT), activated partial thromboplastin time (aPTT),

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thrombin time (TT) were within normal range (Table 1). Morphological evaluation of

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peripheral blood smear by optical microscope showed normal platelets. Platelets were scattered and large platelets could be found occasionally. Transmission electron microscope showed no significant reduction in the platelet α-granules (Figure 3).

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FXIII activity was measured by FXIII semiquantitative test and fibrinogen was completely dissolved within 2 hours. Factor XIII antigen assay showed the content

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was less than 1% (normal range 65-135%). A total of nine individuals of three generations of the pedigree participated in our research (Figure 1 and 2). All subjects and their legal guardians gave written informed consent to the investigation according to the Declaration of the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University and the 1975 Declaration of Helsinki. The parents of the patient are first cousins. His mother is his father’s cousin’s daughter. DNA samples were extracted from peripheral blood cells using the Qiagen FlexiGene DNA Kit (#512206, Qiagen, Germany) and were stored at -80℃. A panel of sequencing primers was designed for the sequencing of the exons and ±10 bp 4

ACCEPTED MANUSCRIPT flanking introns of blood and immune-related genes using the Agilent SureDesign online design tool. Ten nanograms of a DNA sample were used for targeted gene capture and library construction. Next-generation sequencing (NGS) samples were sequenced on the NEXTSEQ 500 sequencer (Illumina) according to the manufacturer's protocol. Primer design, synthesis, and the NGS test were conducted

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by Kangso Medical Inspection (Beijing, China) according to standard laboratory procedures. Sequences were aligned to human genome version 19 (HG19) using BWA

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(v0.7.15). ANNOVAR(v2016-02-01), RefSeq Ensembl and UCSC were used to locate the mutations. The potential functional impacts of the identified variants were

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assessed using SIFT (http://sift.jcvi.org/) (Kumar et al., 2009) and PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) (Adzhubei et al., 2010) and MutationTaster

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(http://www.mutationtaster.org/) (Schwarz et al., 2010). Results

missense

mutation

in

exon

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The results analysis of the patient’s F13A1 gene showed a novel homozygous 12

(NM_000129:

exon

12:

c.1652delC:

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p.Thr551LysfsTer26). The C deletion at nucleotide 1652 caused Thr551LysfsTer26;

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the numbering of the amino acids is according to the Swiss-Prot P02671. The 1652delC mutation was not found in his maternal grandmother and paternal grandfather. The remaining analyzed pedigree members carried the 1652delC in

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heterozygous form (Figure 4). This variant was confirmed to be a novel mutation associated with patient’s phenotype. The mutation was considered as a likely

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pathogenic mutation by American College of Medical Genetics and Genomics (ACMG) (Richards et al., 2015). Additionally, a novel homozygous missence mutation in exon 13 of NBEAL2 gene (NM_015175: exon 13: c.1367C>T: p.Ala456Val) was revealed in the patient. This missense mutation (C to T transition at nucleotide 1367) led to amino acid substitute at 456th locus, namely, alanine was substituted by valine, and the numbering of the amino acids is according to the Swiss-Prot P02671. The c.1367C>T mutation was not found in the proband’s paternal grandfather, paternal uncle and maternal grandmother. The remaining analyzed pedigree members carried the c.1367C>T mutation in heterozygous form (Figure 5). 5

ACCEPTED MANUSCRIPT No literature has been reported on the pathogenicity of this mutation. ACMG predicted it as a mutation of uncertain significance. More genetic data are needed. This patient received treatment with fresh frozen plasma and cryoprecipitate. After 4 weeks of treatment, the patient improved and was discharged. Defecating and urinating were recovered. Muscle strength increased to level IV.

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Discussion Clinically, FXIIID is often overlooked for a rare occurrence and unawareness. A

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delayed diagnosis and treatment is common. Since 1960, Duckert et al reported the first case of FXIIID cases in the world, more than 500 cases of inherited FXIIID have

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been identified (Schroeder and Kohler, 2013). Limited medical resources and tradition for consanguineous marriages contribute to the high incidence in some developing

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countries. Common bleeding symptoms including the umbilical cord bleeding, ear bleeding, epistaxis and joint bleeding have been reported in the literature (Naderi et

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al., 2016; Li et al., 2018). A cerebral hemorrhage, which was the initial symptom of the patient in our study, is not common as the first presentation. The patient had

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hematomyelia and hematuria. F13A1 is a non-glycosylated single polypeptide chain

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molecule with a molecular mass of 83 kDa. It consists of 731 amino acids omitting the initiator methionine, contains an activation peptide, a beta-sandwich, a catalytic and two beta-barrel domains. The structural domains of F13A1 include the following

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amino acid residues, AP-FXIII: 1–37, beta-sandwich: 38-184, catalytic core domain: 185-515, beta-barrel 1: 516-628 and beta-barrel 2: 629-731(Komaromi et al., 2011).

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Our study for the first time describes a pedigree with FXIIID caused by novel F13A1 mutation, combined with novel NBEAL2 mutation. To clarify the diagnosis, a high-throughput genetic analysis was conducted. Previous studies showed that FXIIID can be caused by missense mutations in F13A1, such as Pro166Leu and Gln415Arg (Biswas et al., 2014). To the best of our knowledge, the F13A1 mutation (NM_000129: exon 12: c.1652delC: p.Thr551LysfsTer26) detected in the current pedigree has not been reported. In this pedigree, the novel mutation occurred in the beta-barrel 1 domain of F13A1, c.1652delC in exon 12 resulted in p.Thr551Lys mutation, followed by 26 altered amino acids and termination codon downstream, leading to the 6

ACCEPTED MANUSCRIPT production of pathological protein, which ultimately affected the activity of F13A1. The pedigree study and ACMG evaluation revealed that the mutation was linked to FXIIID. We considered the novel mutation as a pathogenic mutation for FXIIID. Further clarification of the pathogenic role of the missense variants is needed. Interestingly, sequencing of genomic NBEAL2 detected a missense mutation, known

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to have deleterious effects on gene expression, missense-mediated mRNA disrupted and nonfunctional protein included. NBEAL2 encodes a protein containing a BEACH

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domain, crucial for dense tubular system of platelets and vesicular trafficking (Cullinane et al., 2013). NBEAL2 mutation can generate nonsense codons, frameshifts,

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mRNA splicing in most reported GPS cases (Pluthero et al., 2018). However, morphological evaluation of the blood smear by light microscopy and electron

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microscope did not reveal grey platelets or decreased α-granules. The novel homozygous missence mutation in NBEAL2 was considered as variants of uncertain

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significance. The finding cannot be diagnostic criteria of GPS. Our study identified new missense mutations in F13A1 and NBEAL2 gene and it is the

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first report of mutation of NBEAL2 in a pedigree with FXIIID. The diagnosis and

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differential diagnosis of the two diseases are important. There are significant similarities between FXIIID, GPS and myelodysplastic syndrome, immune thrombocytopenia, etc. After the diagnosis, we provided the corresponding treatment

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with fresh frozen plasma and cryoprecipitate in case of fatal bleeding complications that are spontaneous or caused by minor trauma. The patient improved.

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Conclusions

Novel mutations of F13A1 and NBEAL2 genes were identified, enriching gene mutations’ spectrum. The experience we shared can provide some help for FXIIID and GPS diagnosis and treatment research, preventing missed or delayed similar cases in the future. Genetic analysis is an effective method to confirm the molecular diagnosis of intractable unexplained bleeding. Statement of Contribution Siyuan Jia, Yunyan He and Hongying Wei conceptualized and designed the study, 7

ACCEPTED MANUSCRIPT drafted the initial manuscript, and reviewed and revised the manuscript. Meirong Lu， Nikuze Lauriane, Kairong Liang and Yong Hong Lei coordinated and supervised data collection, and critically reviewed the manuscript for important intellectual content. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.

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Acknowledgements This study was supported by grants from the National Natural Science Foundation of

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China (No.81360093) and Guangxi Key Laboratory of Thalassemia Research (16-380-34).

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Conflicts of Interest

The authors have no financial relationships relevant to this article to disclose. The

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authors have no conflicts of interest relevant to this article to disclose. References

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Adzhubei, I.A., Schmidt, S., Peshkin, L., Ramensky, V.E., Gerasimova, A., Bork, P., Kondrashov, A.S. and Sunyaev, S.R., 2010. A method and server for predicting damaging missense mutations. Nat Methods 7, 248-9.

Biswas, A., Ivaskevicius, V., Thomas, A., Varvenne, M., Brand, B., Rott, H., Haussels, I., Ruehl, H.,

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Scholz, U., Klamroth, R. and Oldenburg, J., 2014. Eight novel F13A1 gene missense

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mutations in patients with mild FXIII deficiency: in silico analysis suggests changes in FXIII-A subunit structure/function. Ann Hematol 93, 1665-76. Cullinane, A.R., Schaffer, A.A. and Huizing, M., 2013. The BEACH is hot: a LYST of emerging roles for BEACH-domain containing proteins in human disease. Traffic 14, 749-66.

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Dorgalaleh, A. and Rashidpanah, J., 2016. Blood coagulation factor XIII and factor XIII deficiency. Blood Rev 30, 461-475. Ivaskevicius, V., Seitz, R., Kohler, H.R., Schroeder, V., Muszbek, L., Ariens, R.A.S., Seifried, E.,

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Oldenburg, J. and Grp, S., 2007. International Registry on Factor XIII Deficiency: A basis formed mostly on European data. Thrombosis and Haemostasis 97, 914-921. Karimi, M., Peyvandi, F., Naderi, M. and Shapiro, A., 2018. Factor XIII deficiency diagnosis: Challenges and tools. International Journal of Laboratory Hematology 40, 3-11. Komaromi, I., Bagoly, Z. and Muszbek, L., 2011. Factor XIII: novel structural and functional aspects. J Thromb Haemost 9, 9-20. Kumar, P., Henikoff, S. and Ng, P.C., 2009. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc 4, 1073-81. Li, B., Borhany, M., Abid, M., Kohler, H.P. and Schroeder, V., 2018. Identification of a novel nonsense mutation leading to congenital factor XIII deficiency. Thromb Res 165, 83-85. Naderi, M., Tabibian, S., Menegatti, M., Kalantar, E., Kazemi, A., Zaker, F. and Dorgalaleh, A., 2016. Disseminated intravascular coagulation with positive D-dimer: a controversial clinical feature in severe congenital factor XIII deficiency in southeast Iran. Blood Coagul Fibrinolysis 27, 8

ACCEPTED MANUSCRIPT 933-935. Nurden, A.T. and Nurden, P., 2007. The gray platelet syndrome: clinical spectrum of the disease. Blood Rev 21, 21-36. Pluthero, F.G., Di Paola, J., Carcao, M.D. and Kahr, W.H.A., 2018. NBEAL2 mutations and bleeding in patients with gray platelet syndrome. Platelets 29, 632-635. Richards, S., Aziz, N., Bale, S., Bick, D., Das, S., Gastier-Foster, J., Grody, W.W., Hegde, M., Lyon, E., Spector, E., Voelkerding, K., Rehm, H.L. and Committee, A.L.Q.A., 2015. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular

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Pathology. Genet Med 17, 405-24.

Schroeder, V. and Kohler, H.P., 2013. Factor XIII deficiency: an update. Semin Thromb Hemost 39,

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632-41.

Schwarz, J.M., Rodelsperger, C., Schuelke, M. and Seelow, D., 2010. MutationTaster evaluates

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disease-causing potential of sequence alterations. Nat Methods 7, 575-6. Steinberg-Shemer, O. and Tamary, H., 2018. Gray platelet syndrome mimicking atypical autoimmune lymphoproliferative syndrome: the key is in the blood smear. Blood 131, 2737.

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Sun, L., Yan, Q., Wang, Y., Luo, H., Du, P., Hassan, R., Liu, L. and Jiang, W., 2018. Pathogenicity analysis of variations and prenatal diagnosis in a hereditary coagulation factor XIII deficiency

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family. Hematology, 1-9.

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Table 1 Clinical and laboratory characteristics of family members Family member

Age

Bleeding

(years)/Sex

history

Platelets

MPV

PT

APTT

(×10 /L)

(fl)

(s)

(s)

303.30

7.69

12.00

29.30

9

Myelapoplexy

IV10

12/M

III9

32/F

Negative

135.80

11.47

11.70

33.20

II6

41/M

Negative

155.90

8.35

10.00

28.60

IV11

10/F

Negative

258.00

7.30

11.00

I2

70/F

Negative

240.00

7.20

10.8

II5

45/M

Negative

153.40

8.25

II7

58/M

Negative

230.00

11.00

Negative

250-350

M

Normal value

hematuria

D E 9-12

TT

Fib

(s)

(g/L)

T P

FXIII

I R

C S U

antigen (%)

FXIII urea solubility Clot lysis

PM

8.9

4.52

<1.00

10.60

3.52

72.00

N

Normal

11.40

3.00

80.00

N

Normal

12.50

2.35

82.00

N

Normal

24.60

11.00

3.87

79.00

N

Normal

at 30 minutes

Normal

11.00

N A 27.50

10.30

3.49

81.00

N

Normal

9.50

23.40

13.50

3.50

78.00

N

Normal

9-15

23-40

9-15

2-5

65-135

N

Normal

T P E

28.10

MPV: Mean Platelet Volume; PT: Prothrombin Time; APTT: Activated Partial Thromboplastin Time; TT: Thrombin Time; Fib: Fibrinogen N: No clot lysis at 24 hours; PM: Platelet Morphology; IV10: proband; Ⅲ9: proband’s mother; Ⅱ6: proband’s father; Ⅳ11: proband’s sister; I2:

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proband’s paternal grandmother; II5: proband’s paternal uncle; II7: proband’s maternal grandfather

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Figure Legends

Figure 1. The family tree of the hereditary FXIII deficiency. The proband (IV10)

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is indicated by a black-filled square (male) and an arrow. The proband’s sister (IV11) is heterozygous, as indicated by a half-black-filled circle (female). The proband’s

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father (II6) is heterozygous, as indicated by a half-black-filled square (male). The proband’s maternal grandmother (II8) is wild-type and is indicated by a circle. The

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proband’s paternal grandfather (I1) is wild-type and is indicated by a square

Figure 2. Pedigree chart of the family with NBEAL2 missense mutation p.Ala456Val. The proband is indicated by a black-filled square (male) and an arrow. The proband’s sister (IV11) is homozygous, as indicated by a black-filled circle (female). The proband’s father (II6) is heterozygous, as indicated by a half-black-filled square (male). The proband’s paternal grandmother (I2) is 11

ACCEPTED MANUSCRIPT heterozygous, as indicated by a half-black-filled circle (female). The proband’s maternal grandmother (II8) is wild-type and is indicated by a circle. The proband’s

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paternal grandfather (I1) is wild-type and is indicated by a square

Figure 3. Findings of transmission electron microscopy of platelets in the

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pedigree. (A) ~ (C): Findings of transmission electron microscopy of platelets in proband (IV10), sibling(IV11) and paternal grandmother (I2). No significant

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reduction in α-granules were observed in platelets for NBEAL2 p.Ala456Val

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homozygote or heterozygote. α-granules in platelets are indicated with arrows

Figure 4. Representative chromatograms from Sanger sequencing of the F13A1 gene in the pedigree. DNA sequencing of FXIII gene in our study revealed homozygous c.1652delC (p.Thr551LysfsTer26) mutation in the proband and 12

ACCEPTED MANUSCRIPT heterozygous

c.1652delC (p.Thr551LysfsTer26) mutation in six of the studied

pedigree members. A, proband (IV10). B, proband’s father (Ⅱ6) C, proband’s mother (Ⅲ9)D, proband’s paternal grandfather (Ⅰ1), no mutation E, proband’s paternal grandmother (Ⅰ2) F, proband’s maternal grandfather (Ⅱ7) G, proband’s maternal grandmother (Ⅱ8), no mutation H, proband’s paternal uncle (Ⅱ5) I, proband’s sister

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(Ⅳ11)

Figure 5. Representative chromatograms from Sanger sequencing of the NBEAL2 gene in the pedigree. Sequencin.g of c.1367C>T (p.Ala456Val) in the NBEAL2 gene, A, proband (IV10), homozygote. B, proband’s father (Ⅱ6), heterozygote. C, proband’s mother (Ⅲ 9), heterozygote. D, proband’s paternal grandfather (Ⅰ1), no mutation. E, proband’s paternal grandmother (Ⅰ2), heterozygote. F, proband’s

maternal

grandfather(Ⅱ8),

heterozygote.

G,

proband’s

maternal

grandmother(Ⅱ7), no mutation H, proband’s paternal uncle (Ⅱ5), no mutation. I, proband’s sister (Ⅱ5), homozygote 13

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Abbreviations: FXIIID: factor XIII deficiency GPS: Grey platelet syndrome F13A1: Coagulation Factor XIII A Chain NBEAL2: Neurobeachin Like 2

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HGMD: The Human Gene Mutation Database PT: prothrombin time

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APTT: activated partial thromboplastin time

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TT: thrombin time NGS: next-generation sequencing

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ACMG: American College of Medical Genetics and Genomics

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ACCEPTED MANUSCRIPT

Highlights Novel mutations of F13A1 and NBEAL2 genes were identified, enriching gene mutations’ spectrum. The experience we shared can provide some help for FXIIID and GPS diagnosis and

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treatment research, preventing missed or delayed similar cases in the future. Genetic analysis is an effective method to confirm the molecular diagnosis of

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intractable unexplained bleeding.

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