Identification of Cytochrome b‐245, beta‐chain gene mutations, and clinical presentations in Iranian patients with X‐linked chronic granulomatous disease

Abstract Background X‐linked chronic granulomatous disease (X‐CGD) is an immunodeficiency disorder caused by defects in the gp91phox subunit that leads to life‐threatening infections. We aimed to identify CYBB gene mutations and study clinical phenotypes in Iranian patients with probable X‐CGD. Methods We studied four unrelated Iranian patients with probable X‐CGD and their families recruited in several years. We isolated genomic DNA from whole blood and performed Sanger sequencing in the CYBB gene's coding and flanking regions. We also performed pathogenicity predictions using in silico tools. Results We detected four different mutations, including a novel insertion mutation in exon 5 (p.Ile117AsnfsX6), in the patient. Bioinformatics analysis confirmed the pathogenic effect of this mutation. We predicted protein modeling and demonstrated lost functional domains. The patient with the insertion mutation presented pneumonia and acute sinusitis during his life. We also detected three other known nonsense mutations (p.Arg157Ter, p.Arg226Ter, and p.Trp424Ter) in the CYBB gene. The patient with p.Arg157Ter developed lymphadenitis and pneumonia. Moreover, the patient with inflammatory bowel disease showed p.Arg226Ter and the patient with tuberculosis presented p.Trp424Ter. We detected different clinical features in the patients compared to other Iranian patients with the same mutations. Conclusion Our results expand the genetic database of patients with X‐CGD from Iran and make it much easier and faster to identify patients with X‐CGD. Our results also help to detect carriers and enable prenatal diagnosis in high‐risk families as a cost‐effective strategy.


| INTRODUC TI ON
Chronic granulomatous disease (CGD) (OMIM #306400) is a primary immunodeficiency described by the total absence or low degrees of reactive oxygen species (ROS) generation. ROS is a result of a defect in the nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) complex in phagocytes. Chronic granulomatous disease is a rare disorder with repeated and life-threatening bacterial and fungal infections that involves different organs such as lymph nodes, gastrointestinal tract, and lungs. 1 It is accompanied with pneumonia, abscess, and granuloma formation in response to chronic inflammation. 2,3 Mutations in five different genes can cause CGD with autosomal recessive or X-linked recessive inheritance patterns. CYBA, NCF1, NCF2, and NCF4 genes situated on an autosomal chromosome encode structure segments of NADPH oxidase protein (p22 phox , p47 phox , p67 phox , and p40 phox , respectively). Further, the CYBB (cytochrome b-254 beta-chain) gene (OMIM #300481) is situated on the short arm of the X chromosome at position 21.1, containing 13 exons and spanning 30 kb.
It encodes the β-chain of flavocytochrome b 245 (also called as gp91 phox or NOX2). It is a necessary component of the NADPH oxidase complex in phagocytes, such as granulocytes, monocytes, and macrophages.
The gp91 phox protein encoded by the CYBB gene comprises an N-terminal domain and a C-terminal domain. N-terminal is anticipated to form six transmembrane α-helices, and C-terminal is a cytoplasmic domain containing binding sites for flavin adenine dinucleotide (FAD) and NADPH. These domains are necessary for transferring electrons to phagosomes. 4 The CYBB gene is created by a wide variety of mutations, including deletions, splice site mutations, and missense or nonsense mutations. [5][6][7][8][9] According to the published data from the United States, European countries, and Japan, the most well-known inherited pattern was X-linked chronic granulomatous disease (X-CGD) (≥65%).
Nevertheless, among CGD patients in North Africa, the Arab world, Turkey, and Iran, the autosomal recessive inheritance pattern is more frequent. These results highlight the diverse inherited pattern and a high rate of consanguinity marriages. [10][11][12][13][14] Several studies reported mutations of the CYBB gene in CGD pa- with sepsis, pneumonia, infectious dermatitis, and recurrent or severe abscess. 9 This study aimed to identify CYBB gene mutations in Iranian patients suspected to have X-CGD and examine the relationship between these mutations and the patients' phenotype.

| Ethics statement
The ethics committee of Ahvaz Jundishapur University of Medical Sciences approved this study (IR.AJUMS.REC.1397.853). Informed consent was received from all patients and their families enrolled in the study.

| Patients
In this study, we enrolled four unrelated kindreds with patients sus-

| Family 1
In the first family with Bakhtiari ethnicity, the proband's cousin

| Family 2
In the second family with Fars ethnicity, the proband, born in 2009, was diagnosed with severe lymphadenitis and pneumonia when he was 3 months old. He was suspected to have X-CGD based on the NBT (no positive cell) and DHR (2.16 %) tests. Then, he was hospitalized several times due to pneumonia. Finally, he died at the age of eight despite taking preventative medicine. Furthermore, he had an older brother who died of severe fever when he was 40 days before the proband was born.

| Family 3
In the third family with Turkish ethnicity, the proband, born in 2017, was diagnosed with chronic gastritis and colitis when he was F I G U R E 1 A, The pedigree of the first studied family; B and C, the sequence chromatograph of the mutation c.348-349insA in the patient 1 with hemizygous state and the wild-type sequence in the CYBB gene; D, the sequence analyses of the patient's mother with heterozygous state for the mutation c.348-349insA

| Family 4
In the fourth family with Kurdish ethnicity, the proband, born in 2001, was diagnosed with the frequent abscess when he was 1 year old. He was suspected to have X-CGD based on the NBT (no positive cell) and DHR (2.6 %) tests. A few years later, he underwent surgery due to spinal tuberculosis (TB) when he was nine. He responded well to the operation and medication.

| Sequencing and mutational analysis
Genomic DNA was isolated from the EDTA whole blood of the patients and their families using the YEKTA TAJHIZ kit (cat. No: All 13 exons and exon-intron boundaries of the CYBB gene were amplified by polymerase chain reaction (PCR) with conditions and primers described previously. 18 Sanger sequencing and data analysis were performed for all PCR products in the patients using an ABI Prism 3700 apparatus (Big Dye Terminator sequencing kit, Applied Biosystems).
Meanwhile, all available mothers and siblings of the patients with the detected mutation were analyzed. We used several databases such as ENSEMBL (https://www.ensem bl.org/index.html), HGMD (http:// www.hgmd.cf.ac.uk/), and dbSNP (https://www.ncbi.nlm.nih.gov/ snp/) to investigate whether the detected variants were novel or were previously reported as a pathogenic mutation or polymorphism.
Furthermore, we used Mutation Taster (http://www.mutat ionta ster. org/) to anticipate the pathogenic role of the identified mutation.
Moreover, sequence alignment was carried out using the ENSEMBLE database and Clustal-w for a new mutation to examine the area in distinct species.
We named new variants based on guidelines of the human genome variation society using human CYBB protein sequences with the reference ENSG00000165168. The nonsense-mediated mRNA decay (NMD) ESC predictor Web site (https://nmdpr edict ion.shiny apps.io/nmdes cpred ictor/) was used to predict whether a frameshift variant would escape NMD.

| RE SULTS
We identified a novel insertion mutation (c.348-349insA) located on exon 5 of the CYBB gene with the hemizygous state for the proband of the first family ( Figure 1B). This mutation was a frameshift. The amino acid at position 122 was converted to stop codon and create premature stop codon (p.Ile117AsnfsX6). The pathological effect of the mutation was examined by in silico analysis (Mutation Taster) ( Table 1). The mutation is predicted as a disease-causing agent and is not reported in 1000 genome databases. The patient's mother and sister ( Figure 1A,I2,II2) were analyzed. The patient's healthy sister was the wild type for this region ( Figure 1C), while his mother was heterozygote for this mutation as a carrier ( Figure 1D).
According to the American College of Medical Genetics guideline, 19 the sequence variant (c.348-349insA) in the CYBB gene is classified as a pathogenic variant due to having the pathogenic very strong criterion (PVS1, a null variant in a gene where LOF is a known mechanism of disease) and the pathogenic moderate criterion (PM2, absent from controls in Exome Sequencing Project, 1000 Genomes Project, or Exome Aggregation Consortium).
Based on Mutation Taster Server, this insertion mutation is located between amino acids 103-123, which represents the transmembrane α-helices of gp91 phox . The SWISS-MODEL server was used for structural and functional analysis and revealed that this mutation caused loss of some parts of the subunit in the structure of gp91 phox (Figure 2A). Moreover, the NMD prediction Web site predicted that this premature stop codon caused NMD ( Figure 2B). This region has been conserved among species ( Figure 2C).
The I-Mutant server calculated the free energy changed (ΔΔG) value equal to −1.56 kcal/mol with a reliability index of 6 for this mutation. Therefore, the structural stability of this mutant protein largely decreased.
In the proband of the second family, a known pathogenic nonsense mutation was detected in exon 5 of the CYBB gene (c.469C>T) with hemizygous state (Figure 3A,B). The mutation caused the change in arginine to a stop codon (p.Arg157Ter).
In the third family, a known nonsense mutation was found in exon 7 of the CYBB gene (c.676C>T) with hemizygous state (Figure 3C,D).
The mutation led to the change in arginine to a stop codon (p.Ar-g226Ter), and it was located at the N-terminal of gp91 phox . The patient's mother and sister were heterozygous for this mutation as a carrier.
A known pathogenic nonsense mutation (c.1272G>A) with hemizygous state was found in exon 10 of the CYBB gene in the patient 4 ( Figure 3E,F). The mutation caused the change in tryptophan to a stop codon (p.Trp424Ter), and it was located at the cytoplasmic domain of gp91 phox . Furthermore, the patient's mother and sister were heterozygous for the mutation.

| D ISCUSS I ON
In this study, we identified four mutations in four Iranian kindreds with X-CGD patients and realized that one of the mutations was novel.
We detected a novel frameshift mutation (c.348-349insA) in obtained in their study and suggested that the methylation-induced deamination of cytosine may be a significant mechanism for nonsense X-CGD mutations in Iran. 23 Roos et al. listed 681 distinct mutations in the CYBB gene and examined their frequency. They reported that deletion (35.6%) and missense (21.3%) mutations were the most common mutations found in the CYBB gene, with 7.9% of these mutations being insertion and 14.1% of them being nonsense. 29 We reported an insertion mutation and three nonsense mutations in this article, with approximately low frequency in the CYBB gene.
In Iran and countries with consanguineous marriages, X-CGD is rare to compare to the autosomal recessive form. For this reason, few studies have been performed on the Iranian population.

| CON CLUS ION
Our data could be helpful to early diagnose patients suspected to have X-CGD, and also, to provide correct clinical diagnosis and optimize therapeutics in these children such as medication, bone marrow transplantation, and gene therapy. Furthermore, carrier screening in X-CGD patients' families could be beneficial for early diagnosis and prevention of X-CGD in children born with the disease. Meanwhile, these findings can expand the genetic database of CYBB gene mutations; however, conducting further research on X-CGD patients will be necessary.

ACK N OWLED G M ENTS
We would like to thank the patients and their families for their co-

DATA AVA I L A B I L I T Y S TAT E M E N T
The data supporting the study findings are available from the corresponding author upon reasonable request.