Identification of three novel mutations in the FANCA, FANCC, and ,ITGA2B genes by whole exome sequencing

Samira Negahdari; Mina Zamani; Tahereh Seifi; Sahar Sedighzadeh; Neda Mazaheri; Jawaher Zeighami; Alireza Sedaghat; Alihossein Saberi; Mohammad Hamid; Bijan keikhaei; Ramin Radpour; Gholamreza Shariati; Hamid Galehdari

doi:10.4103/ijpvm.IJPVM_462_19

ORIGINAL ARTICLE

Year : 2020 | Volume : 11 | Issue : 1 | Page : 117

Identification of three novel mutations in the FANCA, FANCC, and ,ITGA2B genes by whole exome sequencing

Samira Negahdari¹, Mina Zamani², Tahereh Seifi², Sahar Sedighzadeh², Neda Mazaheri¹, Jawaher Zeighami¹, Alireza Sedaghat³, Alihossein Saberi⁴, Mohammad Hamid⁵, Bijan keikhaei⁶, Ramin Radpour⁷, Gholamreza Shariati⁴, Hamid Galehdari⁶
¹ Narges Genetics Diagnostic Laboratory, Ahvaz, Iran
² Narges Genetics Diagnostic Laboratory; Department of Genetics, Faculty of Sciences, Shahid Chamran University of Ahvaz, Ahvaz, Iran
³ Narges Genetics Diagnostic Laboratory; Health Research Institute, Diabetes Research Center, Ahvaz Jundishapur Universityof medical Sciences, Ahvaz, Iran
⁴ Narges Genetics Diagnostic Laboratory; Department of Genetics, Ahvaz Jundishapur University of medical Sciences, Ahvaz, Iran
⁵ Narges Genetics Diagnostic Laboratory, Ahvaz; Department of Molecular Medicine, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
⁶ Health Research Institute, Research Centre of Thalassemia and Hemoglobinopathies, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
⁷ Tumor Immunology, Department for BioMedical Research (DBMR), University of Bern, Bern; Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland

Date of Submission	14-Dec-2019
Date of Acceptance	27-Mar-2020
Date of Web Publication	06-Aug-2020

Correspondence Address:
Gholamreza Shariati
Narges Genetics Diagnostic Laboratory, Ahvaz, Iran; Department of Genetics, Ahvaz Jundishapur University of medical Sciences, Ahvaz
Iran
Hamid Galehdari
Health Research Institute, Research Centre of Thalassemia and Hemoglobinopathies, Ahvaz Jundishapur University of Medical Sciences, Ahvaz
Iran

Source of Support: None, Conflict of Interest: None

Check

DOI: 10.4103/ijpvm.IJPVM_462_19

Abstract

Background: Various blood diseases are caused by mutations in the FANCA, FANCC, and ITGA2B genes. Exome sequencing is a suitable method for identifying single-gene disease and genetic heterogeneity complaints. Methods: Among families who were referred to Narges Genetic and PND Laboratory in 2015-2017, five families with a history of blood diseases were analyzed using the whole exome sequencing (WES) method. Results: We detected two novel mutations (c.190-2A>G and c.2840C>G) in the FANCA gene, c. 1429dupA mutation in the FANCC gene, and c.1392A>G mutation in the ITGA2B gene. The prediction of variant pathogenicity has been done using bioinformatics tools such as Mutation taster PhD-SNP and polyphen2 and were confirmed by Sanger sequencing. Conclusions: WES could be as a precise tool for identifying the pathologic variants in affected patient and heterozygous carriers among families. This highly successful technique will remain at the forefront of platelet and blood genomic research.

Keywords: Blood platelets, congenital abnormalities, DNA, Fanconi anemia, sequence analysis

How to cite this article:
Negahdari S, Zamani M, Seifi T, Sedighzadeh S, Mazaheri N, Zeighami J, Sedaghat A, Saberi A, Hamid M, keikhaei B, Radpour R, Shariati G, Galehdari H. Identification of three novel mutations in the FANCA, FANCC, and ,ITGA2B genes by whole exome sequencing. Int J Prev Med 2020;11:117

How to cite this URL:
Negahdari S, Zamani M, Seifi T, Sedighzadeh S, Mazaheri N, Zeighami J, Sedaghat A, Saberi A, Hamid M, keikhaei B, Radpour R, Shariati G, Galehdari H. Identification of three novel mutations in the FANCA, FANCC, and ,ITGA2B genes by whole exome sequencing. Int J Prev Med [serial online] 2020 [cited 2021 Jul 24];11:117. Available from: https://www.ijpvmjournal.net/text.asp?2020/11/1/117/291427

Introduction

Fanconi anemia (FA; MIM no. 227650) is an autosomal or X-linked recessive genetic disorder that causes bone marrow failure.^[1] The mutations in several FANCC (A, B, C, D1, D2, E, F, G, I, J, L, M, N, O, P and Q) genes are associated with this disorder. It is estimated that one of every 300 people carry the mutation in FANCC genes [Figure 1].^[2] The FANC proteins FANCA, B, C, E, F, G, and L constructed the complex known as the FA central complex. The FA complex triggers FANCD2 and FANCI proteins (ID complex).^[3] Subsequently, these two proteins bring DNA repair proteins to the place of interstrand cross-linkers (ICLs), so the cross-link will be detached and replication of DNA can come to an end.^[4],[5] The majority of (about 85%) FA patients have mutations in the FANCA, FANCC, and FANCG genes but are altered in various populations.^[6]FANCA gene is located on chromosem16q24.3 that is about 80 kb length with 43 exons and shows a much higher mutational frequency than other FA genes, which accounts for 60-65%.^[7],[8] FA complementation group A is an autosomal recessive disease with various clinical symptoms, such as small stature and low birth weight, renal abnormalities, skeletal malformations, hyperpigmentation of skin (Cafe-au-lait spots), hypogonadism and infertility, mental retardation, and hematological problems. These patients have an increased risk of certain cancers.^[9],[10] The FA complementation group C is prompted by mutations in the FANCC gene located on chromosome 9q22.32. FA complementation group C associated with anemia, leukopenia, thrombopenia, cardiac, renal and limb malformations, dermal pigmentary changes, and susceptibility to the development of malignancies.^[11],[12] Glanzmann's syndrome is a hereditary genetic disorder with severe platelet failure, which has a long bleeding time and a normal platelet count. Mutations in ITGA2B (αIIb) or ITGB3 (β3) genes that are located on chromosome 17 (q21-22) may lead to Glanzmann's syndrome type I or type II with an autosomal recessive inheritance.^[13],[14] In this disorder, the platelet contains defective or small amounts of integrin αIIbβ3 at the platelet surface, which is a glycoprotein receptor for fibrinogen.^[15] The platelets of patients with this condition due to the lack of fibrinogen-binding sites do not have the ability to bind to fibrinogen. As a result, the bleeding time will be significantly longer.^[16],[17]

Figure 1: Human FANC genes^[18]

Click here to view

In this study, we used the whole exome sequencing (WES) method in five patients with a history of clinically different anemia to identify the disease-causing mutations. Thus, we consider WES as a well-organized method to compete with a traditional molecular diagnosis of blood and other genetic disorders.

Methods

Samples and study design

Among families who were referred to Narges Genetic and PND Laboratory in 2015-2017, four families from Khuzestan with a history of blood diseases were analyzed [Figure 1], [Figure 2], [Figure 3], [Figure 4]. This study was confirmed by the Medical Ethics Committee of Ahvaz University of Medical Sciences. The informed consent form was completed and signed by all members of the family who entered the study [Table 1].

Table 1: Characteristics of patients in the current study

Click here to view

Figure 2: Pedigree and chromatograms of the affected dead child and his parents (FANCA/c.2840C>G)

Click here to view

Figure 3:Pedigree and chromatograms of the affected child and her parents (FANCC/c.1429_1430 insA)

Click here to view

Figure 4: Pedigree and chromatograms of the affected child and his parents (FANCA/g.2043A>G)

Click here to view

DNA sample isolation

To obtain genomic DNA, 10 mL of peripheral blood (PB) was collected in ethylenediamine tetraacetic acid (EDTA)-containing tubes from the patients. Deoxyribonucleic acid (DNA) was extracted from PB-derived leukocytes of the family members using the standard salting out protocol. The quality of the DNA was evaluated using the NanoDrop spectrophotometer (ASP-2680, ACTGene Inc., NJ, USA).

Whole exome sequencing and data analysis

The WES was performed by the Illumina-HiSeq 2000 genome analyzer platform by × 100 depth of sequencing and 5Gb output (CNAG; Macrogen Company). Among the variants detected, all previously reported mutations and probable pathogenic variants including novel non-synonymous ones were confirmed by Sanger sequencing. The variants were queried against the public version of the Human Gene Mutation Database (HGMD, http://www.hgmd.cf.ac.uk/). The prediction of variant pathogenicity has been done using Mutation taster and Human Splicing Finder (HSF). We used PredictSNP and PolyPhen-2 for the prediction of protein functions in mutated forms. The allele frequency of each variation was detected using the Genome Aggregation Database (gnomAD), the Exome Aggregation Consortium (ExAC) and 1000 Genome project (http://www. 1000genomes.org/), and the exome database located at our laboratory (including the results of 800 Iranian WES).

Sanger sequencing

After confirming the PCR results using electrophoresis on 1% agarose gel, polymerase chain reaction (PCR) products were sent for Sanger sequencing. Primers were designed by Oligo software (Oligo, USA) according to the sequences of each gene obtained from gene bank. Primer sequences were available upon request. All the identified mutations were confirmed via Sanger sequencing with an ABI Prism 3700 instrument (Applied Biosystems, CA, USA). The outcomes were evaluated using the Chromas Lite 2.1.1 software and then compared with the reported gene sequence using the BLASTN program.

Results

Using the WES method, coding regions, splice site, frameshift variants, indels, and a part of the intronic region were analyzed. We removed all the common variants (minor allele frequency, MAF >1%) and reported homozygote individuals for c.2840C>G and c.1392A>G variants in ITGA2B were zero (n = 0). We did not find g.2043A>G variants in FANCA gene, c. 1429_1430 ins A frameshift variant in FANCC gene in gnomAD, ExAC, and 1000G databases. Finally, after the filtering steps, the genes were compared with those that were identified by examining the texts as candidate genes in blood diseases [Table 2]. Sanger sequencing confirmed the existence of the detected mutations in patients and family members. Other family members were also screened for the mutation [Figure 2], [Figure 3], [Figure 4], [Figure 5]. We used HSF software to predict the potential splice sites c. 1392A > G variant in ITGA2B gene [Figure 6].

Table 2: Location, predicted pathogenicity of the variation in the patients and HGMD report for each change

Click here to view

Figure 5: Pedigree and chromatograms of the affected child and his parents (ITGA2B/ c. 1392A > G)

Click here to view

Figure 6: Human Splicing Finder (HSF) prediction result for 1392A>G variant identified in ITGA2B gene

Click here to view

Discussion

WES is a suitable method for identifying single-gene disease and genetic heterogeneity complaints. This technology has created genetic studies that allow the sequencing of human pathogenic variants in a relatively short time and low cost. In the present study, we identified a previously reported frameshift homozygous single nucleotide variation c.2840C>G (HGMD ID: CM970493) in the dead child of a couple who was subjected for the prenatal diagnosis (PND) test. In addition, a novel nonsense homozygous single nucleotide variation g.2043A>G was also identified in the splice acceptor of the FANCA gene and novel disease-causing and pathogenic homozygous c.1429_1430 ins a frameshift mutation in the FANCC gene. The result of in silico analysis using mutation taster, polyPhen-2, and PredictSNP2 show that these are disease-causing and pathogenic variants. In addition, these variants are not present in our exome database or in the Population frequency databases including 1000G, ExAC, and GenomAD.

FANCA protein plays an important role in binding to the chromatin in the DNA repair process.^[19] Various studies have reported that there is a FANCA mutation in about 60% of patients with Fanconi disease.^{[1],[19],[20]} Several types of indels and point mutations have been reported in patients with Fanconi disease.^{[2],[7],[21],[22]} Knies et al. used WES to screen four FA patients to evaluate the efficiency of exome sequencing for FA genotyping and described WES as an appreciated and adequately safe method for the molecular analysis of FA gene's variants in competition with the traditional genetic testing/screening strategies.^[20] Ameziane et al. showed that the WES method was able to recognize different mutations of BRCA2, FANCD2, FANCI, and FANCL genes in novel unclassified FA patients indicating that WES could be the first test to diagnose FA.^[4]

After FANCA, the mutation in FANCC is the most commonly diagnosed mutation in patients with FA.^[23],[24] Fadaee et al. used NGS and multiplex ligation-dependent probe amplification for 48 Iranian families with FA patients and reported that NGS is the precise method for identification of a pathogenic mutation in FA patients.^[25]

FANCC gene mutations are abundant in Ashkenazi Jews and the Japanese people.^[26],[27] Considering the severe sensitivity of FANCC-deficient cells, various studies suggested that FANCC protein has an important role in chromosome stability during the DNA repair process.^[28] There are also several reports showing the function of FANCC as an intracellular antioxidant to reduce the activity of reductase.^[29],[30]

We could also identify a novel c.1392A>G mutation in the splice region of the ITGA2B gene from a patient presenting blood disorders, using the WES method. Frequencies of reported homozygote variants in the gnomAD browser and in our Exome database are counted as zero (n = 0). This variant was predicted as pathogenic using mutation taster and PredictSNP2 (93% disease). Since the c.1392A>G mutation makes no change in the amino acid sequence, the disease phenotype seems to be the result of a change and loss of mRNA splicing pattern. Analysis of c.1392A>G mutation in the splice region of ITGA2B gene showed that this mutation breaks the splice site and therefore is regarded as pathogenic [Figure 6]. Functional studies are necessary to assess the mechanism of disease development due to this splice region mutation more accurately. Glanzmann's disease has a high frequency in cultures with more rates of consanguineous marriage such as Indians, Iranians, and Iraqi Jews.^[31],[32] As clinical manifestations of blood disorders vary from person to person, many studies using NGS technology have been conducted to identify different causative mutations involved in inherited platelet disorders (IPDs).^{[33],[34],[35]} Buitrago et al. identified 114 novel missense variants in ITGA2B and 68 novel missense variants in ITGB3 genes by WES and reported that WES had 69-98% sensitivity in identifying Glanzmann mutations.^[36]

Conclusions

The WES method has a significant benefit such as high coverage and requires a low amount of genomic samples, which improves the sensitivity of mutation detection compared with traditional methods. Given that, most of our patients were reported in families with a history of consanguinity, WES could serve as a precise method for identifying the heterozygous carriers among the screened families for further investigations. This highlights the major impact of the WES method for screening and identification of platelet and blood-related genomic variants.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Acknowledgments

The authors are indebted to the patients and family members for their cooperation.

Financial support and sponsorship

This study has been funded by Narges Genetic and PND Laboratory, Ahwaz, Iran.

Conflicts of interest

There are no conflicts of interest.

References

1.	Moghrabi NN, Johnson MA, Yoshitomi MJ, Zhu X, Al-Dhalimy MJ, Olson SB, et al. Validation of Fanconi anemia complementation Group A assignment using molecular analysis. Genet Med 2009;11:183-92.
2.	Kee Y, D'Andrea AD. Expanded roles of the Fanconi anemia pathway in preserving genomic stability. Genes Dev 2010;24:1680-94.
3.	Kottemann MC, Smogorzewska A. Fanconi anaemia and the repair of Watson and Crick DNA crosslinks. Nature 2013;493:356-63.
4.	Ameziane N, Sie D, Dentro S, Ariyurek Y, Kerkhoven L, Joenje H, et al. Diagnosis of fanconi anemia: Mutation analysis by next-generation sequencing. Anemia 2012;2012:132856.
5.	Bogliolo M, Schuster B, Stoepker C, Derkunt B, Su Y, Raams A, Trujillo JP, et al. Mutations in ERCC4, encoding the DNA-repair endonuclease XPF, cause Fanconi anemia. Am J Hum Genet 2013;92:800-6.
6.	Bagby G. Recent advances in understanding hematopoiesis in Fanconi Anemia. F1000Res 2018;7:105.
7.	Solomon PJ, Margaret P, Rajendran R, Ramalingam R, Menezes GA, Shirley AS, et al. A case report and literature review of Fanconi Anemia (FA) diagnosed by genetic testing. Ital J Pediatr 2015;41:38.
8.	Hess J, Unger K, Orth M, Schötz U, Schüttrumpf L, Zangen V, Gimenez-Aznar I, et al. Genomic amplification of Fanconi anemia complementation group A (FancA) in head and neck squamous cell carcinoma (HNSCC): Cellular mechanisms of radioresistance and clinical relevance. Cancer Lett 2016;386:87-99.
9.	Bogliolo M, Surralles J. Fanconi anemia: A model disease for studies on human genetics and advanced therapeutics. Curr Opin Genet Dev 2015;33:32-40.
10.	Solanki A, Mohanty P, Shukla P, Rao A, Ghosh K, Vundinti BR. FANCA gene mutations with 8 novel molecular changes in Indian Fanconi anemia patients. PLoS One 2016;11:e0147016.
11.	Cerabona D, Sun Z, Nalepa G. Leukemia and chromosomal instability in aged Fancc-/- mice. Exp Hematol 2016;44:352-7.
12.	Sertorio M, Amarachintha S, Wilson A, Pang Q. Loss of fancc impairs antibody-secreting cell differentiation in mice through deregulating the wnt signaling pathway. JImmunol 2016;196:2986-94.
13.	Kulkarni B, Ghosh K, Shetty S. Second trimester prenatal diagnosis in Glanzmann's Thrombasthenia. Haemophilia 2016;22:e99-100.
14.	Ghosh A, Kumar S, Chacko R, Charlu AP. Total extraction as a treatment for anaemia in a patient of Glanzmann's thrombasthenia with chronic gingival bleed: Case report. JClin Diagn Res 2016;10:ZD11-2.
15.	Nurden AT, Pillois X, Wilcox DA. Glanzmann thrombasthenia: State of the art and future directions. Semin Thromb Hemost 2013;39:642-55.
16.	Maclachlan A, Watson SP, Morgan NV. Inherited platelet disorders: Insight from platelet genomics using next-generation sequencing. Platelets 2017;28:14-9.
17.	Coller BS, Shattil SJ. The GPIIb/IIIa (integrin alphaIIbbeta3) odyssey: a technology-driven saga of a receptor with twists, turns, and even a bend. Blood 2008;112:3011-25.
18.	Dong H, Nebert DW, Bruford EA, Thompson DC, Joenje H, et al. Update of the human and mouse Fanconi anemia genes. Hum Genomics 2015;9:32.
19.	Moghadam AA, Mahjoubi F, Reisi N, Vosough P. Investigation of FANCA gene in Fanconi anaemia patients in Iran. Indian J Med Res 2016;143:184-96. [PUBMED] [Full text]
20.	Knies K, Schuster B, Ameziane N, Rooimans M, Bettecken T, de Winter J, et al. Genotyping of fanconi anemia patients by whole exome sequencing: advantages and challenges. PLoS One 2012;7:e52648.
21.	Auerbach AD. Fanconi anemia and its diagnosis. Mutat Res 2009;668:4-10.
22.	Centra M, Memeo E, d'Apolito M, Savino M, Ianzano L, Notarangelo A, et al. Fine exon-intron structure of the Fanconi anemia group A (FAA) gene and characterization of two genomic deletions. Genomics 1998;51:463-7.
23.	Aftab I, Iram S, Khaliq S, Israr M, Ali N, Jahan S, et al. Analysis of FANCC gene mutations (IVS4+4A>T, del322G, and R548X) in patients with Fanconi anemia in Pakistan. Turk J Med Sci 2017;47:391-8.
24.	Niedzwiedz W, Mosedale G, Johnson M, Ong CY, Pace P, Patel KJ, et al. The Fanconi anaemia gene FANCC promotes homologous recombination and error-prone DNA repair. Mol Cell 2004;15:607-20.
25.	Esmail Nia G, Fadaee M, Royer R, Najmabadi H, Akbari MR. Profiling Fanconi anemia gene mutations among Iranian patients. Arch Iran Med 2016;19:236-40.
26.	Gillio AP, Verlander PC, Batish SD, Giampietro PF, Auerbach AD, et al. Phenotypic consequences of mutations in the Fanconi anemia FAC gene: An International Fanconi Anemia Registry study. Blood 1997;90:105-10.
27.	de Vries Y, Lwiwski N, Levitus M, Kuyt B, Israels SJ, Arwert F, Zwaan M, et al. A Dutch Fanconi anemia FANCC founder mutation in Canadian manitoba mennonites. Anemia 2012;2012:865170.
28.	Pace P, Johnson M, Tan WM, Mosedale G, Sng C, Hoatlin M, et al. FANCE: The link between Fanconi anaemia complex assembly and activity. EMBO J 2002;21:3414-23.
29.	Pang Q, Keeble W, Diaz J, Christianson TA, Fagerlie S, Rathbun K, et al. Role of double-stranded RNA-dependent protein kinase in mediating hypersensitivity of Fanconi anemia complementation group C cells to interferon gamma, tumor necrosis factor-alpha, and double-stranded RNA. Blood 2001;97:1644-52.
30.	Joenje H, Patel KJ. The emerging genetic and molecular basis of Fanconi anaemia. Nat Rev Genet 2001;2:446-57.
31.	Sandrock-Lang K, Oldenburg J, Wiegering V, Halimeh S, Santoso S, Kurnik K, et al. Characterisation of patients with Glanzmann thrombasthenia and identification of 17 novel mutations. Thromb Haemost 2015;113:782-91.
32.	Poon MC, d'Oiron R, Zotz RB, Bindslev N, Di Minno MN, Di Minno G, et al. The international, prospective Glanzmann Thrombasthenia Registry: Treatment and outcomes in surgical intervention. Haematologica 2015;100:1038-44.
33.	Jones ML, Murden SL, Bem D, Mundell SJ, Gissen P, Daly ME, et al. Rapid genetic diagnosis of heritable platelet function disorders with next-generation sequencing: Proof-of-principle with Hermansky-Pudlak syndrome. J Thromb Haemost 2012;10:306-9.
34.	Westbury SK, Turro E, Greene D, Lentaigne C, Kelly AM, Bariana TK, et al. Human phenotype ontology annotation and cluster analysis to unravel genetic defects in 707 cases with unexplained bleeding and platelet disorders. Genome Med 2015;7:36.
35.	Simeoni I, Stephens JC, Hu F, Deevi SV, Megy K, Bariana TK, et al. A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders. Blood 2016;127:2791-803.
36.	Buitrago L, Rendon A, Liang Y, Simeoni I, Negri A, ThromboGenomics Consortium, et al. AlphaIIbbeta3 variants defined by next-generation sequencing: predicting variants likely to cause Glanzmann thrombasthenia. Proc Natl Acad Sci U S A 2015;112:E1898-907.