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Summer Research Fellowship Programme of India's Science Academies

Molecular characterisation of inherited phagocytic defect

Syed Mohammad Hammad

Aligarh Muslim University (AMU), Aligarh 202002

Dr. Manisha Madkaikar

Director, National Institute of Immunohaematology, ICMR, 13th floor, K E M Hospital, Parel, Mumbai 400012

Abstract

Phagocytic defects account for predisposition of patients to bacterial and fungal infections. The defect can be at any level of phagocyte cell function i.e., tethering, rolling, activation, adhesion, diapedesis or killing. Chronic Granulomatous Disease (CGD) is an inheritable heterogenous disease resulting from an inability of phagocytes to kill microbes that they have ingested. It leads to recurrent life-threatening infections with bacteria and fungi and dysregulated granuloma formation. CGD is attributed to defects in the NADPH complex responsible for generating superoxides and killing ingested microorganisms. It could be due to mutations in genes coding any of the five members -gp91phox, p47phox, p22phox, p67phox, and p40phox. The gene for gp91phox-CYBB causes X-linked CGD, whereas CYBA, NCF1, NCF2, and NCF4 genes (for p22phox, p47phox, p67phox and p40phox respectively) causes autosomal recessive (AR)-CGD. NBT and flow-cytometry based DHR assay is used for initial confirmation of CGD. Abnormalities in DHR assay in male patients along with mosaic pattern in carrier mother suggest XL-CGD, and direct way for molecular analysis. The rest are analysed for NADPH component expression by flow cytometry using mAbs selective for proteins. Abnormal 7D5 mAb expression could be due to a defect in any one of the membrane-bound components (gp91phox or p22phox), whereas reduced expression of specific mAbs for other intracellular genes paves way for sequencing of the respective genes. Genomic DNA are selectively amplified by PCR and sequenced by Sanger sequencing to ultimately analyse them for variants. In-silico analysis of variants by various prediction tools is employed to predict the functional impact of the variants found. Novel variants, if any, demand for functional analysis in the lab to substantiate the prediction done by the tools. Effects of variation found can also be analysed at RNA level by sequencing cDNA for any alteration in length.

Keywords: Chronic Granulomatous Disease (CGD), CYBB gene, PCR, NADPH complex, prediction tools, splice-site mutations

Abbreviations

CGD Chronic Granulomatous Disease 
 NADPHNicotinamide adenine dinucleotide phosphate 
 NBT nitroblue tetrazolium
 DHR dihydrorohodamine
 PMAphorbol myristate acetate 
XL-CGD X linked-CGD 
 AR-CGDAutosomal Recessive CGD 
 HSFHuman Splicing Finder 
 BLAST

Basic Local Alignment Search Tool

 CYBB Cytochrome B-245 Beta Chain

INTRODUCTION

Background

Phagocytic defects predispose the patient to recurrent bacterial and fungal infection. Chronic Granulomatous Disease is an inheritable phagocytic defect caused due to the failure of neutrophils to produce microbicidal reactive oxygen species. It is caused due to the mutation in any of the five components of NADPH oxidase responsible in superoxide production. The most common form of the disease is X-linked CGD caused due to mutation in CYBB gene located at Xp21.1-p11.4. The CYBB gene codes for the large subunit of flavocytochrome b558, gp9lphox, the heme and flavin-containing core of NADPH oxidase. It has 13 exons and spans approximately 33kbp while its cDNA is approximately 1700 bp long.

An intronic mutation was found in -5 position of exon 5 of CYBB gene and analysed in various prediction tools. The predictions are then substantaited with cDNA analysis of the patient to detect any alteration in the length of cDNA. It was assumed that the mutation lies in the polypyrimidine tract of the intron and can lead to partial loss of exon 5 sequence.

Objectives of the Research

The research aims to compare the prediction of the effect of a novel mutation found in CYBB gene obtained by In-Silico analysis tools like Mutation Taster and Human Splicing Finder with that of cDNA analysis in laboratory. cDNA of CYBB gene from exon 1 to 5 are to be analysed to detect any alteration due to the mutation.

Scope

The research focuses on In-Silico and cDNA analysis of a patient with Chronic Granulomatous Disease. Only two prediction tools- Mutation Taster and Human Splicing Finder are employed for predicting the effect of the novel mutation in genomic DNA of the patient. cDNA of the patient, his father and mother are amplified along with that of the normal control and sequenced to detect the alteration in the cDNA and mRNA sequence.

LITERATURE REVIEW

Phagocytosis is the cornerstone to innate immunity system and involves the cellular uptake of particulate ligand (size~1µm) by blood leukocytes and other cells [ ​Greenberg S and Grinstein S, 2002​ ]. It is carried out by the interaction of receptors (Fc, C3, pattern-recognition, etc), lectins and other molecules of the phagocytes with the particulate (antibody, microbes or apoptotic cells) [ ​Sharon N, 1984​]. It is followed by the wrapping up of the plasma membrane around the ingested pathogen into a phagosome. Phagosome detaches from the plasma membrane and fuses with hydrolytic enzymes containing lysosomes to form phagolysosome, which ultimately destroys the pathogen.

Phagocytosis in mammals is mainly the work of PMNs (Neutrophils), monocytes and macrophages. These cell types have often been referred to as ‘professional phagocytes’ [ ​Michel Rabinovitch, 1995​ ]. They are specially equipped to find and destroy pathogens. Neutrophils however are qualitatively and quantitatively the most important phagocytic cell against acute bacterial infections. They are formed in the marrow and chemotactic substances guide them to accumulate at sites of tissue damage, infection or inflammation using the bloodstream as a transport system [ ​Rabinovitch M, 1995​].

Neutrophils have a definite pathway for ingesting microbes or any foreign particle and contributing to immunity. It involves tethering, rolling, activation, adhesion, diapedesis and killing [ ​Andrews T and Sullivan KE, 2003​ ]. Generally, microbes are coated with antibody (opsonization) or complement which facilitates the engulfment of the particles. Neutrophils are capable of recognizing bacteria directly, often via TLRs (toll-like receptors). In addition, the neutrophil integrins, CD11b/CD18 (Mac-1) and CD11c/CD18 (p150), recognize and bind some bacterial species directly [ ​Bullock WE, 1993​ ]. Cross-linking of these receptors with pathogens activates the neutrophils for digestion. PMNs then degranulate, releasing their toxic products into phagosomes [ ​J. G. Hirsch, 1960​ ]. Ingestion and cross-linking stimulate the respiratory burst [ ​Wade BH and Mandell GL, 1983​ ]. In it, there occurs a rapid influx of oxygen into the neutrophils through activation of a NADPH-cytochrome b-dependent oxidase that reduces oxygen to a superoxide. Superoxide is itself weakly antimicrobial and the follow up products hydrogen peroxide and HOCl by superoxide dismutase and myeloperoxidase respectively have strong antimicrobial property. These oxygen species along with the lysosomal enzymes serve to kill the ingested microbes.

NADPH oxidase plays an important role in the generation of hydrogen peroxide and other reactive oxygen species (ROS) [ ​B J McMurrich, 1976​ ]. It has five structural components: gp91phox, p47phox, p22phox, p67phox, and p40phox (encoded by genes CYBB, NCF1, CYBA, NCF2 and NCF4 respectively) and one regulatory subunit Rac2. gp91phox and p22phox (cytochrome b558) are embedded primarily in the membrane of specific granules and plasma membrane [ ​Yuk-fun HuiYuk-fun Hui, Molecular studies of X-linked chronic granulomatous disease​ ] and are constitutively associated [ ​A J Jesaitis, 1987​ ]. Generation of the superoxide in NADPH occurs by a one-electron reduction of oxygen via the gp91phox subunit, using reduced NADPH as the electron donor. The other subunits are vital to the activation of NADPH.

Abnormality in any of the five components of NADPH complex leads to a rare functional neutrophil disorder known as Chronic Granulomatous Disease. However, recently mutation in regulatory subunit Rac2 has been also identified as the cause of CGD-like syndrome [ ​David A. Williams, 2013​]. CGD follows both autosomal recessive (AR) and X-linked pattern of inheritance [omim.org]. X-linked CGD is caused due to the mutation in CYBB gene located at Xp21.1-p11.4, whereas defects in CYBA, NCF1, NCF2, and NCF4 (located at 16q24.2, 7q11.23, 1q25.3 and 22q12.3 resp) [omim.org] cause autosomal recessive-CGD. XL-CGD accounts for >65% cases of CGD in majority of developing countries, however AL-CGD is more frequent in countries like Turkey, Egypt, Oman and Iran where consanguineous marriage is common [​​Manisha Madkaikar, 2018​​]. Defect in any of the components lead to predisposition of patients to bacterial and fungal infection. Reports show that XL-CGD is more severe than AR-CGD. However, within AL-CGD there is no difference between AR-p22phox, AR- p67phox, and AR-p47phox gene defects [ ​J WINKELSTEIN, 1998​ ].

XL-CGD is most common form of CGD predisposing the patient to recurrent bacterial and fungal infections. The mutations causing XL-CGD can be point mutations or deletion or insertion in CYBB gene sequence. In majority of cases it is caused due to mutation in exonic region, [ ​​Andrew R. Cross, 2001​​] however intronic mutation leading to it are no less common. [​​de Boer M and Bolscher BG and Dinauer MC and Orkin SH and Smith CI and Ahlin A and Weening RS and Roos D, 1992​​]. Intronic mutations affecting branch-site, 5' and 3' recognition site and polypyridimine tract can lead to aberrant splicing. [​Monika Gos, 2019​]. Deep intronic mutations can also create a new splice-site and form abnormal mRNA transcript.[​Andrew R. Cross, 2001​] These mutations can lead to exon skipping, shortening, inclusion of an intron and thus functionally affect the translation process..

A novel mutation can be assessed for its probable effect using various prediction tools. These prediction tools provide the results in the form of numerical scores based on certain algorithms [​Dominik Seelow, 2014​] ​and each tools have different threshold value for evaluating pathogenetic effects. It is due to this reason the tools are not consistent with each other and require functional analysis to substantiate the findings [​Toshiyuki Fukao, 2017​]. ​Any changes in mRNA transcript and hence the polypeptide it is coding for can be assessed by analyzing the cDNA sequence. The cDNA sequence of the patient when compared with normal sequence clearly gives the alteration in the sequence if any. Real-time PCR instead of general PCR can also be employed to evaluate mRNA transcripts quantitatively [​Stephan E. Philipp, 2016​]. ​Further a more reliable technique i.e. minigene assay can also be performed to detect aberrant splicing. These techniques thereby confirm or contradict the statements of the various prediction tools.

METHODOLOGY

Patient

The patient is a 1 year old male with a history of recurrent infections in lung, liver and skin since the age of 4 months. He had intermittent high grade fever, bronchopneumonia with nutritional anemia at the age of 8 months followed by liver abscess, perianal abscess with severe malnutrition at the age of 9 months. Peripheral blood sample of the patient and his mother were collected in EDTA anticoagulated vacutainer and processed at the National Institute of Immunohaematology (NIIH), Mumbai, India. Clinical protocol as per the guidelines of the Institutional Ethics Committee and Declaration of Helsinki was followed.

NBT and DHR Assay

Neutrophil function test was performed using nitroblue tetrazolium (NBT), dihydrorohodamine-123 (DHR), and phorbol myristate acetate (PMA) (a protein-kinase activator) in the laboratory (Sigma-Aldrich) [NIIH, Mumbai]. Functional neutrophils readily reduce NBT to blue formazan after oxidative stimulation with PMA whereas CGD cells remain colorless. Flow cytometry based DHR assay detects reduced level of ROS, especially hydrogen peroxide and abnormal levels hint at defective NADPH complex activity. A stimulation index (SI) was calculated for gated neutrophils by estimating the ratio of median fluorescent intensity (MFI) of phorbol myristate acetate (PMA)-stimulated and PMA-unstimulated cells in DHR assay. The SI value of the patient as well as that of mother was always paired and simultaneously processed with a healthy control sample at the time of diagnosis [​​Manisha Madkaikar, 2018​​]. The results were obtained by the lab professionals and thereafter observed by me.

NADPH Component Expression by Flow Cytometry

NADPH oxidase component expression for gp91phox or p22phox was evaluated for the patient with carrier mother deduced through DHR test. Anti-Flavocytochrome b558 (Human) mAb or 7D5mAb expression was checked by flow cytometry. Gating of neutrophils, T cells, monocytes, and B cells was done using FSC and SSC. Abnormal 7D5 expression hinted at defect in either gp91phox or p22phox which are functionally associated. It directs the way for molecular analysis of CYBB gene first in case of male child with carrier mother and CYBA gene first in case of female child. In this assay too, I only observed the reported results.

Molecular Diagnosis

Genomic DNA and RNA were isolated from peripheral blood using Qiagen extraction kit. The concentration and purity of DNA was determined by “Invitrogen Qubit 4 Flurometer” (QUBIT 1X dsDNA HS AssayKit) and “NANODROP 1000” Spectrophotometer respectively. cDNA was obtained from the extracted RNA using Random Primer, and its concentration was subsequently determined. cDNAs of the patient’s father and mother were also obtained.

Molecular analysis of genomic DNA

Primer sets for CYBB and CYBA genes were designed using NCBI Primer BLAST and tested for selectivity by “In-Silico PCR- BIOTEC”. All the exons along with >20bps of introns around exon-intron boundaries were amplified and run on agarose 1.2% agarose gel. Amplicons were then purified and sequenced by Sanger sequencing (ABI 3130Xl genetic analyzer, Applied Biosystems, USA). Results were then analyzed in NCBI BLASTn program with reported gene database. Variants found were checked for pathogenocity in In-Silico analysis tools: “Mutation Taster” and “Human Splicing Finder”. Novel variants required functional analysis in laboratory to substantiate the results obtained by prediction tools.

Molecular analysis of cDNA

Result of the In-silico analysis of the variant found at -5 position of exon 5 of CYBB gene demanded for functional analysis. It was hypothesized that any deleterious effect on the coding exon and thereafter proteins can be detected by analyzing the cDNA sequence. The concentrations of cDNA of the patient, normal control, father and mother of patient were determined by “Invitrogen Qubit 4 Flurometer” (QUBIT 1X dsDNA HS AssayKit). Specific primer pair for cDNA of CYBB gene covering exon 1 to exon 5 was obtained from SIGMA-ALDRICH.

Standardisation of PCR for cDNA amplification

Primers 0.8µl each of CYBB exon1-5F and CYBBexon1-5R were added in the reaction mixture of 25µl as given below in the protocol. cDNA of the normal person used (conc. 220ng) was taken in the reaction mixture such that 25µl mixture has 10-20ng of cDNA. Gradient PCR was then set up for the temperature ranges 54-66°C and 3 tubes with no enhancers, with MgCl2 alone, and along with DMSO were kept for temperatures 59.8°C, 64.2°C and 66°C each in TAKARA PCR Thermal Cycler. The reaction condition involved initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30sec, annealing at respective temp for 45 sec, and extension at 72°C for 45sec, and further final extension at 72°C for 7 min. It was then analyzed in 1.2% agarose gel with 1X TAE buffer. Distinct bands were obtained at 60°C with MgCl2 as enhancer.

DEPC-water (µl) Complete 10X buffer (µl) dNTP 25mM (µl) Taq polymerase (µl) MgCl2 (µl) 25mM DMSO (µl) CYBB1-5F 10pM(µl) CYBB1-5R 10pM(µl) cDNA (µl)
18.65 2.5 0.5 0.25 - - 0.8 0.8 1.5
16.65 2.5 0.5 0.25 2 - 0.8 0.8 1.5
15.65 2.5 0.5 0.25 2 1 0.8 0.8 1.5

The concentration of patient's cDNA as well as that of his father and mother was low and therefore higher number of cycles need to be taken. Normal cDNA was diluted to get the concentration corresponding to that of patient (~2ng/µl), and PCR was set up for 40 cycles at 60°C with MgCl2 and thereafter analyzed. Concentration of ~6ng and ~14ng were taken in 25µl reaction mixture. The one which gives promising result can then be done with the patient's sample.

DEPC-water (µl) Complete 10X buffer (µl) dNTP 25mM (µl) Taq polymerase (µl) MgCl2 (µl) 25mM CYBB1-5F 10pM(µl) CYBB1-5R 10pM(µl) cDNA (µl)
15.15 2.5 0.5 0.25 2 0.8 0.8 3
11.15 2.5 0.5 0.25 2 0.8 0.8 7

Polymerase chain reaction for patient, and his father and mother

cDNA samples were in limited amount and therefore optimum concentration for amplification was determined by re-standardization. Polymerase Chain reaction was set up in “GeneAmp PCR System” taking concentration of cDNA in 25µl reaction mixture for normal control, patient, patient’s father and mother as ~6ng, ~6ng, ~3.6ng, and ~3.7ng respectively. The reaction condition involved initial denaturation at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 30sec, annealing at 60°C for 45 sec, and extension at 72°C for 45sec, and further final extension at 72°C for 7 min. It was then analyzed in 1.2% agarose gel prepared from 1.5X TAE and 1µl/10ml of ETBR. The samples were run along with “100bp DNA Ladder RTU (Ready-to-use)” [GeneDireX, Inc.] for 1hr 10min at 120V and then analysed by UV rays in BIO-RAD instrument. The amplified product were then purified using “GeneAll cleaning kit” that removes extra components like dNTPs, polymerase, misprimed sequences, remaining primers, etc.

sample DEPC-water (µl) Complete 10X buffer (µl) dNTP 25mM (µl) Taq polymerase (µl) MgCl2 (µl) 25mM CYBB1-5F 10pM(µl) CYBB1-5R 10pM(µl) cDNA (µl)
Normalcontrol 15.15 2.5 0.5 0.25 2 0.8 0.8 3
patient 15.15 2.5 0.5 0.25 2 0.8 0.8 3
F/0 patient 12.15 2.5 0.5 0.25 2 0.8 0.8 6
M/0 patient 11.15 2.5 0.5 0.25 2 0.8 0.8 7

Mother’s sample (sample 0) did not show any band during UV analysis. Due to the limited cDNA quantity and necessity to evaluate mother’s sample in case of X-linked disease, PCR product was reused as template after cleanup. Along with it, mother’s cDNA of concentration ~4.2ng was also taken in 25µl reaction mixture. PCR was set up in ‘TAKARA thermo cycler” with the reaction condition similar as above except for 45 cycles instead of 40 and different composition of the reaction mixture as:

Mother’s sample DEPC-water (µl) Complete 10X buffer (µl) dNTP 25mM (µl) Taq polymerase (µl) MgCl2 (µl) 25mM CYBB1-5F 10pM(µl) CYBB1-5R 10pM(µl) cDNA (µl)
Sample 1 9.85 2.5 075 0.30 2 0.8 0.8 8
Sample 2* 6.85 2.5 0.75 0.30 2 0.8 0.8 11

*Sample 2 used amplicons as the template.

The product were analysed in 1.2% agarose gel along with 100bp DNA Ladder. Sample 2 showed three bands and required isolation of each band separately. For isolation, both sample 1 and 2 were cleaned by GeneAll Cleanup kit and again run on 3% agarose gel with 1X TAE buffer, along with “1kb DNA ladder RTU” (GeneDireX) for approximately 1hr 10 min till the bands become sharp. No bands were seen for Sample 1, while sample 2 had two sharp bands and one light band in the middle. All 3 bands were cut along with the gel and put into separate eppendorf tube. They were then purified from the gel using kit.

Sequence PCR for the amplified products

The amplified products of the normal control, the patient, his father and bands obtained from sample 2 were taken for sequence PCR. Primers CYBB exon1-5F and CYBB exon1-5R are diluted to 3.5pM and PCR is set up for 25 cycles of denaturation at 96°C for 10 sec and annealing at 60°C for 4 min. The reaction mixture has components as given.

Reaction DEPC Water BigDye Buffer RR Buffer Primer 3.5pM Purified Product
1X 5µl 2µl 1µl 1µl 1µl

The sequence PCR product is then cleaned by GeneAll Cleaning kit using NR buffer for binding, NW buffer for washing and DEPC water for elution.

Sequencing

The sequencing PCR product were fed into the sequencer (3130xl Genetic Analyzer, Applied Biosystems) after a short denaturation step at 95˚C for 5 min. Automated DNA Sequencers generate a four-color chromatogram showing the results of the sequencing run in chromatogram file (Software- Chromas).

In-Silico analysis

On sequencing, the sequences obtained were compared with the reported gene database of cDNA of CYBB gene at NCBI using the BLASTN program.

RESULTS AND DISCUSSION

Neutrophil Function Test

Reports of NBT slide test and flow-cytomtery based DHR assay suggest abnormal neutrophil function. Zero percent (0%) positivity for NBT and DHR assay in patient while 25% and 21.2% positivity with mosaic pattern were observed in NBT and DHR assay respectively of mother. SI value of 1.125 was observed in case of patient, while the normal control has almost 14 SI value. The result suggests defective neutrophil function due to X-linked CGD. The mother is showing a clear mosaic pattern suggestive of carrier status.

20190802_162343.jpg
    Flow cytometry based DHR assay showing 0% positivity in case of patient while 21% positivity for mother's sample with mosaic pattern showing carier status.

    NADPH Oxidase Component Expression

    Gp91phox and p22phox are functionally associated membrane bound components. The functionality of p22phox was determined by evaluating 7D5mAb expression. The result shows reduced expression with SI value of ~1.5 in patient, while the mother exhibited mosaic pattern suggesting abnormality in membrane-bound component particularly in gp91phox with X-linked inheritance pattern. It paves way for molecular analysis of CYBB gene and CYBA gene.

    20190802_162402.jpg
      row1: patient's expression, row2: normal person's expression, row 3: patient's mother expression. 

      Results for Genomic DNA Analysis

      No disease-causing variants were found in 13 exons of CYBB gene or 16 exons of CYBA gene. A novel intronic variant in the acceptor region of intron 4 of CYBB gene leading to substitution of nucleotide T at -5 position with A (g.13650T>A) was predicted to be disease-causing in “Mutation-Taster” tool. Chromograph of mother had heteropeak at the variant site which was obvious for X-linked CGD carrier. The mutation was not reported in CYBB gene by either Human Gene Mutation Database or Single Nucleotide Polymorphism Database.

      Screenshot (87).png
        Patient's genomic DNA shows mutation (T>A) in acceptor site of intron 4 of CYBB gene
        Screenshot (88).png
          Genomic DNA of mother shows heterpeak at the same site 

          Screenshot (89).png
            Father's DNA normal
            Gene CYBB
            Phy.location Chr23: 37652913T>A
            Transcript id ENST00000378588
            NM id NM_00397
            Alteration region intron
            DNA changes g.13650T>A
            cDNA changes c.337-5T>A
            Variation id N/A
            Prediction (Mutation Taster) ·         Protein features (might be) affected ·         Splice site changes

            The “Human Splicing Finder” (HSF) which combines 12 different algorithms to identify and predict the effects of mutations on splicing signals suggested no alteration in splicing due to it. There was an inconsistency in the prediction of both tools. In-Silico analysis tools work on scoring on the basis of certain algorithm and do not guarantee accuracy and hence functional analysis was required.

             Results for cDNA Analysis

            Primers CYBB exon1-5F (5’-ATGGGGAACTGGGCTGTGAA-3’) and CYBB exon1-5R (5’-CTTTATTCTCTTTCGAGCAA-3’) were standardized for targeted amplification of the cDNA. Distinct bands were observed at 60°C with MgCl2 (35 cycles). However, patient’s cDNA was not amplified. It could have been due to either skipping of exon 5 where primers would bind or lower concentration of the cDNA. Therefore, normal cDNA was diluted to the concentration of the cDNA of patient and the reaction was set up for 40 cycles. It was observed that PCR tube with ~6ng of cDNA of normal showed promising band in 1.2% gel. However, the one with ~14ng did not show band due to unexplainable reason.

            cDNA of normal control, patient, and his father and mother were amplified again with the primers at the standardised condition (40cycles) and bands were analysed along with 100bp DNA ladder. It was seen that the patient's cDNA was shortened and corresponded to ~400bp of ladder. Father’s cDNA corresponded with the normal cDNA at length ~550bp. cDNA of mother did not show any amplification. The truncated amplicon of the patient suggested possible skipping. However had it been total exon 5 skipping the primer would not have attached and amplified the region. This suggested that a part of exon was skipped and a new 3'-splice site acceptor has been created within exon. For confirmation, complete sequence was required.

            It was necessary to examine mother’s amplified products for deciphering the inheritance of the mutation. Due to limited amount of sample, the amplified product was again taken as template and in another PCR tube, a little higher amount of original cDNA was taken than what was earlier. The reaction was set up again with higher amount of dNTPs and Taq and 45 cycles of denaturation, annealing and extension. It was again run on 3% agarose gel and analysed. 3 bands corresponding to ~550bp, ~450bp and ~500bp (faint) were seen in the sample amplified by taken amplicon as template. No bands were seen for the other sample. The three bands were separately extracted from the gel, cleaned and then proceeded for sequencing.

            Screenshot (79).png
              Standardisation of cDNA primers CYBB exon1-5F and CYBB exon1-5R. Distinct band was visible at 60 degree celsius with MgCl2 (well 1).
              Screenshot (81)_1.png
                Analysis of amplification of normal control, patient, father and mother cooresponding to 100bp DNA ladder. Well 1: ladder, well 2: normal control, well 3: patient cDNA 

                Screenshot (83).png
                  Analysis of mother's cDNA taking higher concentration (well 3) than earlier and using amplicon as template (well 4) with normal control (well 2) along with 100 bp ladder 

                  Screenshot (82).png
                    Analysis of mother's cDNA in 3% agarose gel and 1X TAE buffer run along with 1kp DNA ladder. Wells 4 and 6 show no band and contained origibal cDNA as template in higher concentration. Wells 8 and 10 have bands and are obtained using amplicon as template. 3 bands are seen in wells 8 and 10.

                    Sequences for the patient, normal control and father were obtained. Only the lowest band from the sample 2 of mother that was amplified by taking amplicon as template could be sequenced by Sanger sequencing. These sequences were then compared with reported sequence "Homo sapiens cytochrome b-245 beta polypeptide isoform 1 (CYBB) mRNA, partial cds" through NCBI BLAST program. For normal control as well as father of the patient, complete cDNA sequences from exon 1 to exon 5 of CYBB gene were seen. It was obvious since the disease is X-linked and father has no complications. However, a contradiction to our hypothesis was observed in case of sequence of patient's cDNA and that of the lower band of mother from sample 2. It was hypothesised that the mutation would lead to skipping of a part of exon 5 and creation of new 3' splice site acceptor since the primer used binded to exon 5. But analysis of sequences using BLAST program showed that exon 4 and exon 5 are intact as such in patient's cDNA and in lower band of Sample 2. Further to our surprise sequences of exon 2 and 3 were missing. Exon 1 would obviously remain intact as it is where primer was attached. It must be due to a possible deep inronic mutation possibly in intron 2 that led to loss of both exon 2 and 3 presumably from the amplified product. It was hence due to these missing exons 2 and 3 a truncated band was seen in the electrophoresis gel. It need to be clearly understood that truncated band corresponded to roughly 400 bp (normal corresponds to 550 bp) while exon 2 and 3 in total have length of approximately 210bp. Therefore further analysis is a prerequisite before stating which exon or a part of it is actually lost. However it was thereby confirmed that the novel mutation g.13650T>A in CYBB gene in intron 4 did not alter the nucleotide sequence of exon 5 or exon 4. Hence it can be suggested that this mutation do not produce any disease causing effect contradictory to the prediction by Mutation Taster tool.

                    kaam2.png
                      Sequences obtained by SANGER SEQUENCER compared with reference database showing the loss of sequences in the case of patients and the lower band of his mother. Result shows no loss of sequences of exon 4 and 5 while the sequences before that were missing. 

                      CONCLUSION AND RECOMMENDATIONS

                      NBT and flow cytomtery based DHR assay along with NADPH component expression test results suggested that the patient has Chronic Granulomatous Disease with X-linked inheritance pattern. These tests in turn directs the way for molecular analysis of CYBB gene first and CYBA gene later coding for gp91phox and p22phox respectively. Genomic DNA analysis of all exons along with >20bps of introns from exon-intron boundaries showed that there is g.13650T>A (c.337-5T>A) novel mutation in the intron 4 of CYBB gene. In-silico analysis using Mutation Taster and Human Splicing Finder gave results that were inconsistent with each other. Mutation Taster tools predicted the mutation to be disease-causing due to possible splice-site changes, while HSF suggested no possible alteration in splicing. cDNAs of CYBB gene from exon 1 to exon 5 of patient, control, father and mother of the patient were amplified and run in agarose gel. A truncated band of the patient suggested some part of exon was missing from the cDNA sequence. It was hypothesised that a new 3'-splice-site acceptor must have been created in exon 5 region that led to a part of it missing. However sequences obtained when compared with reference database using NCBI BLAST program showed a contradictory result. It was observed that complete sequence of exon 4 and 5 remained intact. So the truncation observation must have been due to a possible loss of some parts of exon 2 and 3 which were not found in the final sequence of patient and the lower band of mother. These two also corresponded to each other in the agarose gel. Thus it was concluded that the novel mutation in intron 4 do not produce any truncation neither in exon 4 nor in exon 5 of CYBB gene.

                      Mother's cDNA from original sample could not be amplified and amplicons had to used again as a template which needs to be considered before stating about the cDNA sequence of mother. Of the three bands obtained using amplicon as template only the one that corresponded to patient's amplicon was properly sequenced and analysed. Thus, a more proper approach for analysis of mother's cDNA need to be taken. Further an analysis taking other sets of primer to clearly pin-point the lossed exon should also be done.

                      The research has certain limitations and shortcomings. Firstly, primer sets covering exon 1 to 6 should have been taken instead of primers encompassing 1 to 5 if a possible loss of exon 5 had to be investigated. Less cDNA sample of the mother was one of the major shortcomings and due to it amplicons were used as template. Further a more reliable method like Minigene assay or real-time PCR was not used.

                      The research concluded that the novel mutation g.13650T>A in intron 4 of CYBB gene do not change or affect the sequence of exon 4 and exon 5. Further it also suggests that prediction of In-silico analysis tools can be inconsistent with each other and to the result observed. It was also seen that sequences of exon 2 and 3 were missing and thus re-evaluation of genomic DNA need to be done to detect any disease-causing deep intronic mutation in intron 2 or 3 of CYBB gene.

                      ACKNOWLEDGEMENTS

                      The two months of immense learning and experience at National Institute of Immunohaematology, Mumbai has helped me develop greater interest in learning new methods and looking at science particularly immunology in a different way. .

                      I take this opportunity to express my profound gratitude and deep regards to my guide Dr. Manisha Madkaikar for her exemplary guidance, monitoring and constant encouragement throughout the course of this project.

                      I express my deepest thanks to Dr. Sneha, Dr. Priyanka Kambli, and all lab mates for the endless support and constant guidance throughout these 58 days. They were always eager to help and encouraged learning by doing. They were calm enough when I committed mistakes and suggested me methods to tackle every odd situations.

                      I would also like to thanks my father, mother, grandmother and brothers for believing in me and giving me moral as well as financial support. Further I would like to express my deepest gratitude to my maternal aunt and uncle for the wonderful stay in Mumbai.

                      Last but not the least, i would thank IAS-NASI-INSA for their support throughout the programme and for giving me this great opportunity.

                      Bibliography

                      https://www.omim.org

                      https://asia.ensembl.org/index.html

                      https://blast.ncbi.nlm.nih.gov/Blast.cgi

                      http://www.mutationtaster.org

                      http://www.umd.be/HSF/

                      http://www4a.biotec.or.th/cgi-bin/webPcr

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