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Approach to Molecular Diagnosis of Chronic Granulomatous Disease (CGD): an Experience from a Large Cohort of 90 Indian Patients

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Abstract

Background

Chronic granulomatous disease (CGD) is characterized by mutation in any one of the five genes coding NADPH oxidase components that leads to functional abnormality preventing the killing of phagocytosed microbes by affecting the progression of a respiratory burst. CGD patients have an increased susceptibility to infections by opportunistic and pathogenic organisms. Though initial diagnosis of CGD using a nitroblue tetrazolium (NBT) test or dihydrorhodamine (DHR) test is relatively easy, molecular diagnosis is challenging due to involvement of multiple genes, presence of pseudogenes, large deletions, and GC-rich regions, among other factors. The strategies for molecular diagnosis vary depending on the affected gene and the mutation pattern prevalent in the target population. There is a paucity of molecular data related to CGD for Indian population.

Method

This report includes data for a large cohort of CGD patients (n = 90) from India, describing the diagnostic approach, mutation spectrum, and novel mutations identified. We have used mosaicism in mothers and the expression pattern of different NADPH components by flow cytometry as a screening tool to identify the underlying affected gene. The techniques like Sanger sequencing, next-generation sequencing (NGS), and Genescan analysis were used for further molecular analysis.

Result

Of the total molecularly characterized patients (n = 90), 56% of the patients had a mutation in the NCF1 gene, 30% had mutation in the CYBB gene, and 7% each had mutation in the CYBA and NCF2 genes. Among the patients with NCF1 gene mutation, 82% of the patients had 2-bp deletion (DelGT) mutations in the NCF1 gene. In our cohort, 41 different mutations including 9 novel mutations in the CYBB gene and 2 novel mutations each in the NCF2, CYBA, and NCF1 genes were identified.

Conclusion

Substantial number of the patients lack NCF1 gene on both the alleles. This is often missed by advanced molecular techniques like Sanger sequencing and NGS due to the presence of pseudogenes and requires a simple Genescan method for confirmation. Thus, the diagnostic approach may depend on the prevalence of affected genes in respective population. This study identifies potential gene targets with the help of flow cytometric analysis of NADPH oxidase components to design an algorithm for diagnosis of CGD in India. In Indian population, the Genescan method should be preferred as the primary molecular test to rule out NCF1 gene mutations prior to Sanger sequencing and NGS.

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Funding

This work was funded by the Indian Council of Medical Research (ICMR). It is also supported by the Foundation of Primary Immunodeficiency (FPID). Manasi Kulkarni is the recipient of Senior Research Fellowship from ICMR (ICMR SRF F. no. 45/8/2014-HAE-BMS).

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Authors and Affiliations

  1. National Institute of Immunohaematology-ICMR, 13th floor, KEM Hospital Campus, Mumbai, Parel, 400012, India

    Manasi Kulkarni, Gouri Hule, Priyanka Kambli, Jahnavi Aluri, Maya Gupta, Aparna Dalvi, Snehal Mhatre & Manisha Madkaikar

  2. Department of Blood Cell Research, Sanquin Blood Supply Organization, Amsterdam, The Netherlands

    Martin de Boer & Karin van Leeuwen

  3. Bai Jerbai Wadia Children Hospital, Mumbai, Parel, India

    Prasad Taur & Mukesh Desai

Authors
  1. Manasi Kulkarni
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  2. Gouri Hule
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  12. Manisha Madkaikar
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Contributions

MK performed the experiments, analyzed the data, and wrote the manuscript. GH, JA, MG, AD, and SM were involved in performing laboratory investigations of the different cases. MD supervised the clinical details of the patients. PT helped in the collecting of samples and the details of the patients. MB and KL provided the NGS data of the patients. MK and PK are involved in molecular analysis of patients. MM supervised the study and reviewed the manuscript.

Corresponding author

Correspondence to Manisha Madkaikar.

Ethics declarations

Pateint’s sample and additional samples (parents and siblings) were collected in the case of a strong family history after obtaining a proper informed consent. A clinical protocol has been followed per the guidelines of the Institutional Ethics Committee and Declaration of Helsinki.

Conflict of Interest

The authors declare that they have no conflict of interest.

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Kulkarni, M., Hule, G., de Boer, M. et al. Approach to Molecular Diagnosis of Chronic Granulomatous Disease (CGD): an Experience from a Large Cohort of 90 Indian Patients. J Clin Immunol 38, 898–916 (2018). https://doi.org/10.1007/s10875-018-0567-y

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  • DOI: https://doi.org/10.1007/s10875-018-0567-y

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