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. 2022 Jul 8;13:883446. doi: 10.3389/fimmu.2022.883446

Targeted Gene Sanger Sequencing Should Remain the First-Tier Genetic Test for Children Suspected to Have the Five Common X-Linked Inborn Errors of Immunity

Koon-Wing Chan 1, Chung-Yin Wong 1, Daniel Leung 1, Xingtian Yang 1, Susanna F S Fok 1, Priscilla H S Mak 1, Lei Yao 1, Wen Ma 1, Huawei Mao 2, Xiaodong Zhao 3, Weiling Liang 4, Surjit Singh 5, Mohamed-Ridha Barbouche 6, Jian-Xin He 7, Li-Ping Jiang 3, Woei-Kang Liew 8, Minh Huong Thi Le 9, Dina Muktiarti 10, Fatima Johanna Santos-Ocampo 11, Reda Djidjik 12, Brahim Belaid 12, Intan Hakimah Ismail 13, Amir Hamzah Abdul Latiff 14, Way Seah Lee 15, Tong-Xin Chen 16, Jinrong Liu 7, Runming Jin 17, Xiaochuan Wang 18, Yin Hsiu Chien 19, Hsin-Hui Yu 20, Dinesh Raj 21, Revathi Raj 22, Jenifer Vaughan 23, Michael Urban 24, Sylvia van den Berg 25, Brian Eley 26, Anselm Chi-Wai Lee 27, Mas Suhaila Isa 28, Elizabeth Y Ang 28, Bee Wah Lee 28,29, Allen Eng Juh Yeoh 28,29, Lynette P Shek 29,30, Nguyen Ngoc Quynh Le 31, Van Anh Thi Nguyen 32, Anh Phan Nguyen Lien 33, Regina D Capulong 34, Joanne Michelle Mallillin 35, Jose Carlo Miguel M Villanueva 36, Karol Anne B Camonayan 37, Michelle De Vera 38, Roxanne J Casis-Hao 39, Rommel Crisenio M Lobo 40, Ruby Foronda 41, Vicky Wee Eng Binas 42, Soraya Boushaki 12,43, Nadia Kechout 44, Gun Phongsamart 45, Siriporn Wongwaree 45, Chamnanrua Jiratchaya 45, Mongkol Lao-Araya 46, Muthita Trakultivakorn 46, Narissara Suratannon 47, Orathai Jirapongsananuruk 48, Teerapol Chantveerawong 49, Wasu Kamchaisatian 50, Lee Lee Chan 51, Mia Tuang Koh 15, Ke Juin Wong 52, Siew Moy Fong 52, Meow-Keong Thong 53, Zarina Abdul Latiff 54, Lokman Mohd Noh 54,55, Rajiva de Silva 56, Zineb Jouhadi 57, Khulood Al-Saad 58, Pandiarajan Vignesh 5, Ankur Kumar Jindal 5, Amit Rawat 5, Anju Gupta 5, Deepti Suri 5, Jing Yang 1, Elaine Yuen-Ling Au 59, Janette Siu-Yin Kwok 60, Siu-Yuen Chan 1, Wayland Yuk-Fun Hui 1, Gilbert T Chua 1, Jaime Rosa Duque 1, Kai-Ning Cheong 61, Patrick Chun Yin Chong 62, Marco Hok Kung Ho 62, Tsz-Leung Lee 61, Wilfred Hing-Sang Wong 1, Wanling Yang 1, Pamela P Lee 1, Wenwei Tu 1, Xi-Qiang Yang 3, Yu Lung Lau 1,*
PMCID: PMC9304939  PMID: 35874699

Abstract

To address inborn errors of immunity (IEI) which were underdiagnosed in resource-limited regions, our centre developed and offered free genetic testing for the most common IEI by Sanger sequencing (SS) since 2001. With the establishment of The Asian Primary Immunodeficiency (APID) Network in 2009, the awareness and definitive diagnosis of IEI were further improved with collaboration among centres caring for IEI patients from East and Southeast Asia. We also started to use whole exome sequencing (WES) for undiagnosed cases and further extended our collaboration with centres from South Asia and Africa. With the increased use of Next Generation Sequencing (NGS), we have shifted our diagnostic practice from SS to WES. However, SS was still one of the key diagnostic tools for IEI for the past two decades. Our centre has performed 2,024 IEI SS genetic tests, with in-house protocol designed specifically for 84 genes, in 1,376 patients with 744 identified to have disease-causing mutations (54.1%). The high diagnostic rate after just one round of targeted gene SS for each of the 5 common IEI (X-linked agammaglobulinemia (XLA) 77.4%, Wiskott–Aldrich syndrome (WAS) 69.2%, X-linked chronic granulomatous disease (XCGD) 59.5%, X-linked severe combined immunodeficiency (XSCID) 51.1%, and X-linked hyper-IgM syndrome (HIGM1) 58.1%) demonstrated targeted gene SS should remain the first-tier genetic test for the 5 common X-linked IEI.

Keywords: inborn errors of immunity, primary immunodeficiency diseases, targeted gene, Sanger sequencing, whole exome sequencing, next generation sequencing

Introduction

Inborn errors of immunity (IEI), previously known as primary immunodeficiency diseases (PIDD), arise from intrinsic defects in immunity, with most due to genetic mutations, and comprise over 400 diseases that could present with a diverse range of disorders including infection, autoimmunity, inflammation, malignancy, and allergy (1, 2). These multitudes of disorders could present with a wide spectrum of phenotypes of varying severities, resulting in difficulty recognising and diagnosing IEI promptly and accurately, especially in resource-limited countries and regions (3).

With rapid advance in both immunological and genetic studies in IEI including newborn screening for severe combined immunodeficiency (SCID) over the last 20 years, the prognosis of patients with IEI living in resource-rich countries and regions have improved enormously due to rapid and accurate genetic diagnosis with treatment tailored to specific IEI, together with family counseling regarding recurrence risk and reproductive choices (3–5). However, for most countries and regions of Asia and Africa, many patients with suspected IEI now still do not have ready access to these diagnostic and therapeutic approaches, let alone 20 years ago, resulting in underdiagnosis of IEI and a protracted diagnostic odyssey for many families (6).

To improve awareness and recognition of IEI in our region, we started to offer e-consultation and genetic investigations free of charge for patients suspected to have IEI referred to us by our collaborators since 2001. This was built on our paediatric immunology service started in 1988, with us having rapidly acquired the in-house capacity to diagnose IEI genetically and treat the more common IEI effectively (7–17). With more experience, we started to offer the research based targeted gene Sanger sequencing (SS) for the 5 common X-linked IEI, namely X-linked agammaglobulinemia (XLA), Wiskott-Aldrich syndrome (WAS), X-linked chronic granulomatous disease (XCGD), X-linked hyper-IgM (HIGM1) and X-linked severe combined immunodeficiency (XSCID), to our collaborators in South-East Asia and mainland China initially, followed by those in South Asia and Africa. The collaboration has resulted in providing accurate genetic diagnosis leading to appropriate management of these patients as well as increasing awareness of IEI in these countries and regions (18–31).

Over the years, we have increased the number of targeted genes subjected to SS to more than 80, as well as helped our collaborators in setting up their local genetic diagnostic service through sharing of protocols and primers, resulting in local centres with expertise and diagnostics for IEI without the need to refer patients with suspected IEI to us for genetic diagnosis (32–42).

Since 2009, we started to use next generation sequencing (NGS) to investigate patients with suspected IEI whose genetic mutations could not be identified by targeted gene SS. In the same year, we established the Asian Primary Immunodeficiency (APID) Network to provide an electronic platform for both data management and better consultative service for our collaborators (43, 44).

In this study, we aimed to review the role of targeted gene SS in the diagnostic pathway for patients with suspected IEI referred to us from 2001 to 2021, to define which suspected IEI should be subjected to targeted gene SS before offering NGS, with criteria that the gene is the most commonly found to be causal among all the genes that are associated with that clinical phenotype, and with at least a 50% diagnostic rate using one round of SS.

Materials and Methods

Patients

Patients with suspected IEI referred to us from different centres over a 20-year period (2001–2021) were included. Various diagnostic work up including laboratory tests and immunological assays were done in the referring centres. Referring clinicians would send us the clinical details and laboratory findings, which would be deposited in our APID network database. Only those patients with clinical presentation indicative of IEI would be followed up (currently can refer to the IUIS phenotypic classification) (2). Cases with HIV infection or other known causes of immune compromise would be excluded. One or several rounds of e-consultation would be conducted between the referring clinicians and the corresponding author who ultimately decided on which targeted gene SS would be done, with clinical and laboratory criteria specific to each top X-linked gene applied listed here below. X-linked genes would be normally sequenced in boys born of non-consanguineous marriages with a non-conflicting family history only, e.g., without affected sisters. Onset of recurrent bacterial infections or enteroviral infections approximately after 6 months of age, and if available, very low IgG level and B cell count would prompt the immediate sequencing of the BTK gene. The WAS gene was sequenced in boys with recurrent bacterial, viral, and fungal infections, eczema, and importantly, thrombocytopenia. The CYBB gene would be sequenced in boys with recurrent bacterial and fungal infections, BCGitis or BCGosis, and if available, a positive nitroblue tetrazolium test (NBT) or dihydrorhodamine (DHR) 123 test. The IL2RG gene was sequenced in boys presenting in first few months of life with recurrent severe infections, low absolute lymphocyte count, and if available, a very low T or NK cell count. The CD40LG gene was sequenced in boys with recurrent sinopulmonary infections, liver and biliary tract disease, and if available, a high IgM level accompanied by low IgG and IgA levels. Additional or more advanced laboratory investigations were normally not requested before proceeding to genetic testing as most patients were referred from resource-limited settings. Less than 5% of referral cases were not offered genetic testing due to insufficient clinical details. Once genomic DNA were received, genetic diagnosis by research-based targeted gene SS was then performed by our centre free of charge. The study was approved by the Clinical Research Ethics Review Board of The University of Hong Kong and Queen Mary Hospital (Ref. no. UW 08-301).

Targeted Gene SS

Genomic DNA was isolated from peripheral blood of patients by different centres, with consent obtained from parents or guardians before blood collection. Polymerase chain reaction (PCR) primer pairs covering entire coding region and flanking splice sites were designed for individual IEI genes. Research-based targeted gene SS was performed by PCR or long PCR direct SS of both sense and antisense strands of DNA as described in our previous studies (19, 20, 22–25). Homology analyses with reference sequences were performed by Basic Local Alignment Search Tool (BLAST). Mutations, identified by bioinformatics analysis, were described with reference to Human Genome Variation Society (HGVS) nomenclature (45). For those patients with typical phenotypes including the 5 common IEI, relevant single targeted gene SS has been offered in the first round of screening, e.g., BTK(Bruton tyrosine kinase) gene for XLA, WAS (WASP actin nucleation promoting factor) gene for WAS, CYBB (cytochrome b-245 beta chain) gene for XCGD, IL2RG (interleukin 2 receptor subunit gamma) gene for XSCID and CD40LG (CD40 ligand) gene for XHIM. For the other IEI, targeted gene or gene panel SS were offered at the same time. Further targeted gene tests were performed if no causal mutation identified in the previous round of SS.

Results

From 2001 to 2021, 1,376 patients with suspected IEI have been referred from different centres as shown in Figure 1 . We have developed 84 different IEI targeted gene tests according to the diversity of IEI cases referred. Totally, we have performed 2,024 targeted gene SS for all these IEI patients referred, with 744 patients identified to have disease-causing mutations. The positive diagnostic rates among patients and tests are 54.1% (744 out of 1,376 patients) and 36.8% (744 out of 2,024 SS) respectively, with 1.47 SS performed per patient on average. The details of the mutations were described in the Tables 1 – 4 , and Supplementary Tables 1, 2 . Tables 1 – 4 , and Supplementary Table 1 show all causal mutations found in the corresponding genes of the 5 common IEI while Supplementary Table 2 for all other IEI genes.

Figure 1.

Figure 1

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Map showing 72 referring centres in 17 countries. (Created with Datawrapper).

Table 1.

Causal mutations identified in WAS gene (Reference Sequence LRG_125) of the WAS patients.

Patient ID Gene Mutant allele cDNA/nucleotide change Protein change Mutant type
WAS-016A WAS X-linked LRG_125t1:c.35G>C
LRG_125t1:c.62del		 G12A
N21Tfs*24		 Missense
Frameshift
WAS-051A WAS X-linked LRG_125t1:c.58C>T Q20X Missense
WAS-149A WAS X-linked LRG_125t1:c.91G>A E31K Missense
WAS-039A WAS X-linked LRG_125t1:c.116T>G L39R Missense
WAS-083A WAS X-linked LRG_125t1:c.134C>T T45M Missense
WAS-102A WAS X-linked LRG_125t1:c.134C>T T45M Missense
WAS-088A WAS X-linked LRG_125t1:c.167C>T A56V Missense
WAS-056A WAS X-linked LRG_125t1:c.190T>A W64R Missense
WAS-025A WAS X-linked LRG_125t1:c.217T>C C73R Missense
WAS-045A WAS X-linked LRG_125t1:c.218G>A C73Y Missense
WAS-055A WAS X-linked LRG_125t1:c.223G>A V75M Missense
WAS-048A WAS X-linked LRG_125t1:c.245C>A S82Y Missense
WAS-121A WAS X-linked LRG_125t1:c.256C>T R86C Missense
WAS-030A WAS X-linked LRG_125t1:c.257G>A R86H Missense
WAS-082A WAS X-linked LRG_125t1:c.257G>A R86H Missense
WAS-101A WAS X-linked LRG_125t1:c.257G>A R86H Missense
WAS-137A WAS X-linked LRG_125t1:c.257G>A R86H Missense
WAS-148A WAS X-linked LRG_125t1:c.257G>A R86H Missense
WAS-044A WAS X-linked LRG_125t1:c.257G>T R86L Missense
WAS-097A WAS X-linked LRG_125t1:c.300G>C E100D Missense
WAS-070A WAS X-linked LRG_125t1:c.397G>A E133K Missense
WAS-131A WAS X-linked LRG_125t1:c.397G>A E133K Missense
WAS-136A WAS X-linked LRG_125t1:c.397G>A E133K Missense
WAS-151A WAS X-linked LRG_125t1:c.397G>A E133K Missense
WAS-001A WAS X-linked LRG_125t1:c.1354G>T E452X Missense
WAS-049A WAS X-linked LRG_125t1:c.1376C>T
LRG_125t1:c.1421T>A		 P459L
M474K		 Missense
Missense
WAS-071A WAS X-linked LRG_125t1:c.1378C>T P460S Missense
WAS-154A WAS X-linked LRG_125t1:c.97C>T Q33* Nonsense
WAS-110A WAS X-linked LRG_125t1:c.100C>T R34* Nonsense
WAS-152A WAS X-linked LRG_125t1:c.100C>T R34* Nonsense
WAS-160A WAS X-linked LRG_125t1:c.100C>T R34* Nonsense
WAS-123A WAS X-linked LRG_125t1:c.107_108del F36* Nonsense
WAS-029A WAS X-linked LRG_125t1:c.121C>T R41* Nonsense
WAS-078A WAS X-linked LRG_125t1:c.121C>T R41* Nonsense
WAS-112A WAS X-linked LRG_125t1:c.121C>T R41* Nonsense
WAS-128A WAS X-linked LRG_125t1:c.184G>T E62* Nonsense
WAS-050A WAS X-linked LRG_125t1:c.290G>A W97* Nonsense
WAS-100A WAS X-linked LRG_125t1:c.100C>T R34* Nonsense
WAS-119A WAS X-linked LRG_125t1:c.306C>G Y102* Nonsense
WAS-158A WAS X-linked LRG_125t1:c.403C>T Q135* Nonsense
WAS-106A WAS X-linked LRG_125t1:c.454C>T Q152* Nonsense
WAS-006A WAS X-linked LRG_125t1:c.472C>T Q158* Nonsense
WAS-023A WAS X-linked LRG_125t1:c.631C>T R211* Nonsense
WAS-028A WAS X-linked LRG_125t1:c.631C>T R211* Nonsense
WAS-033A WAS X-linked LRG_125t1:c.631C>T R211* Nonsense
WAS-087A WAS X-linked LRG_125t1:c.631C>T R211* Nonsense
WAS-107A WAS X-linked LRG_125t1:c.631C>T R211* Nonsense
WAS-124A WAS X-linked LRG_125t1:c.631C>T R211* Nonsense
WAS-126A WAS X-linked LRG_125t1:c.631C>T R211* Nonsense
WAS-127A WAS X-linked LRG_125t1:c.631C>T R211* Nonsense
WAS-018A WAS X-linked LRG_125t1:c.995dup N335* Nonsense
WAS-117A WAS X-linked LRG_125t1:c.1317_1318delinsTT Q440* Nonsense
WAS-138A WAS X-linked LRG_125t1:c.1336A>T K446* Nonsense
WAS-125A WAS X-linked LRG_125t1:c.330dup T111Hfs*11 Frameshift
WAS-004A WAS X-linked LRG_125t1:c.350del F117Sfs*10 Frameshift
WAS-034A WAS X-linked LRG_125t1:c.410_419del F137Sfs*121 Frameshift
WAS-155A WAS X-linked LRG_125t1:c.431_432insT K144Nfs*25 Frameshift
WAS-032A WAS X-linked LRG_125t1:c.436del Q146Kfs*115 Frameshift
WAS-072A WAS X-linked LRG_125t1:c.442dup R148Kfs*21 Frameshift
WAS-094A WAS X-linked LRG_125t1:c.472_473dup Q158Hfs*104 Frameshift
WAS-019A WAS X-linked LRG_125t1:c.566del P189Qfs*72 Frameshift
WAS-015A WAS X-linked LRG_125t1:c.587_588del G196Afs*10 Frameshift
WAS-002A WAS X-linked LRG_125t1:c.649_652dup P218fs*5 Frameshift
WAS-003A WAS X-linked LRG_125t1:c.649_652dup P218fs*5 Frameshift
WAS-021A WAS X-linked LRG_125t1:c.647_659dup P222Tfs*4 Frameshift
WAS-113A WAS X-linked LRG_125t1:c.665dup A223Sfs*2 Frameshift
WAS-027A WAS X-linked LRG_125t1:c.735del K245Nfs*16 Frameshift
WAS-008A WAS X-linked LRG_125t1:c.950del P317Hfs*128 Frameshift
WAS-059A WAS X-linked LRG_125t1:c.1001del G334Vfs*111 Frameshift
WAS-010A WAS X-linked LRG_125t1:c.1006_1007del K336Gfs*158 Frameshift
WAS-058A WAS X-linked LRG_125t1:c.1023_1024del L342Afs*152 Frameshift
WAS-156A WAS X-linked LRG_125t1:c.1052dup P352Tfs*143 Frameshift
WAS-012A WAS X-linked LRG_125t1:c.1092del G366Afs*79 Frameshift
WAS-084A WAS X-linked LRG_125t1:c.1143del P383Lfs*62 Frameshift
WAS-141A WAS X-linked LRG_125t1:c.1190del
LRG_125t1:c.1188_1199del		 P397Rfs*48
P401_P404del		 Frameshift
In-frame Deletion/Insertion
WAS-057A WAS X-linked LRG_125t1:c.1219_1235dup P413Gfs*38 Frameshift
WAS-007A WAS X-linked LRG_125t1:c.1265_1275del A422Gfs*69 Frameshift
WAS-118A WAS X-linked LRG_125t1:c.1271dup L425Pfs70 Frameshift
WAS-099A WAS X-linked LRG_125t1:c.1295del G432Efs*13 Frameshift
WAS-011A WAS X-linked LRG_125t1:c.120_132+1dup Splicing
WAS-009A WAS X-linked LRG_125t1:c.132+1G>T Splicing
WAS-075A WAS X-linked LRG_125t1:c.133-1G>A Splicing
WAS-047A WAS X-linked LRG_125t1:c.687G>T G229= Splicing
WAS-120A WAS X-linked LRG_125t1:c.274-2A>C Splicing
WAS-031A WAS X-linked LRG_125t1:c.360+1G>A Splicing
WAS-129A WAS X-linked LRG_125t1:c.360+5G>C Splicing
WAS-040A WAS X-linked LRG_125t1:c.361-7T>G Splicing
WAS-109A WAS X-linked LRG_125t1:c.361-1G>A Splicing
WAS-096A WAS X-linked LRG_125t1:c.559+1G>A Splicing
WAS-115A WAS X-linked LRG_125t1:c.559+2T>C Splicing
WAS-063A WAS X-linked LRG_125t1:c.734+2T>C Splicing
WAS-020A WAS X-linked LRG_125t1:c.735-1G>A Splicing
WAS-024A WAS X-linked LRG_125t1:c.735-1G>A Splicing
WAS-150A WAS X-linked LRG_125t1:c.735-1G>A Splicing
WAS-054A WAS X-linked LRG_125t1:c.777+1G>A Splicing
WAS-114A WAS X-linked LRG_125t1:c.777+1G>A Splicing
WAS-134A WAS X-linked LRG_125t1:c.777+1G>A Splicing
WAS-133A WAS X-linked LRG_125t1:c.777+2dup Splicing
WAS-061A WAS X-linked LRG_125t1:c.777+3G>C Splicing
WAS-014A WAS X-linked LRG_125t1:c.777+3_777+6del Splicing
WAS-130A WAS X-linked LRG_125t1:c.777+3_777+6del Splicing
WAS-013A WAS X-linked LRG_125t1:c.931+2T>C Splicing
WAS-104A WAS X-linked LRG_125t1:c.1338+1G>A Splicing
WAS-139A WAS X-linked LRG_125t1:c.1338+2T>G Splicing
WAS-022A WAS X-linked LRG_125t1:c.1453+1G>C Splicing
WAS-111A WAS X-linked LRG_125t1:c.1453+2T>A Splicing
WAS-103A WAS X-linked EX1-EX2del
LRG_125t1:c.1378C>T		 P460S Gross Deletion
Missense
WAS-089A WAS X-linked EX1-EX12del Gross Deletion
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Repeated mutations are in bold. WAS, WASP actin nucleation promoting factor; WAS, Wiskott–Aldrich Syndrome. *translation termination (stop) codon.

Table 4.

Causal mutations identified in CD40LG gene (Reference Sequence LRG_141) of the HIGM1 patients.

Patient ID Gene Mutant allele cDNA/nucleotide change Protein Change Mutant Type
XHIM-061A CD40LG X-linked LRG_141t1:c.346G>T G116C Missense
XHIM-020A CD40LG X-linked LRG_141t1:c.418T>G W140G Missense
XHIM-030A CD40LG X-linked LRG_141t1:c.430G>A G144R Missense
XHIM-025A CD40LG X-linked LRG_141t1:c.482T>A L161Q Missense
XHIM-050A CD40LG X-linked LRG_141t1:c.676G>A G226R Missense
XHIM-029A CD40LG X-linked LRG_141t1:c.680G>A G227E Missense
XHIM-049A CD40LG X-linked LRG_141t1:c.692T>G L231W Missense
XHIM-037A CD40LG X-linked LRG_141t1:c.761C>T T254M Missense
XHIM-058A CD40LG X-linked LRG_141t1:c.761C>T T254M Missense
XHIM-047A CD40LG X-linked LRG_141t1:c.415C>T Q139* Nonsense
XHIM-011A CD40LG X-linked LRG_141t1:c.419G>A W140* Nonsense
XHIM-014A CD40LG X-linked LRG_141t1:c.420G>A W140* Nonsense
XHIM-001A CD40LG X-linked LRG_141t1:c.654C>A C218* Nonsense
XHIM-022A CD40LG X-linked LRG_141t1:c.654C>A C218* Nonsense
XHIM-010A CD40LG X-linked LRG_141t1:c.103del Q35Rfs*2 Frameshift
XHIM-004A CD40LG X-linked LRG_141t1:c.291_299delinsG D97Efs*13 Frameshift
XHIM-024A CD40LG X-linked LRG_141t1:c.511_512del I171Lfs*29 Frameshift
XHIM-017A CD40LG X-linked LRG_141t1:c.158_161del I53Kfs*13 Frameshift
XHIM-054A CD40LG X-linked LRG_141t1:c.158_161del I53Kfs*13 Frameshift
XHIM-052A CD40LG X-linked LRG_141t1:c.489del R165Dfs*26 Frameshift
XHIM-016A CD40LG X-linked LRG_141t1:c.599del R200Nfs*42 Frameshift
XHIM-002A CD40LG X-linked LRG_141t1:c.616_619del L206Efs*35 Frameshift
XHIM-003A CD40LG X-linked LRG_141t1:c.719_720del N240Sfs*3 Frameshift
XHIM-019A CD40LG X-linked LRG_141t1:c.157-2A>G Splicing
XHIM-021A CD40LG X-linked LRG_141t1:c.410-2A>G Splicing
XHIM-036A CD40LG X-linked LRG_141t1:c.289-28_302del Splicing
XHIM-051A CD40LG X-linked LRG_141t1:c.156+1G>A Splicing
XHIM-053A CD40LG X-linked LRG_141t1:c.346+2T>A Splicing
XHIM-056A CD40LG X-linked LRG_141t1:c.289-1G>C Splicing
XHIM-057A CD40LG X-linked LRG_141t1:c.347-1G>C Splicing
XHIM-007A CD40LG X-linked LRG_141t1:c.289-2A>G Splicing
XHIM-009A CD40LG X-linked LRG_141t1:c.289-2A>G Splicing
XHIM-055A CD40LG X-linked EX1_EX2del Gross Deletion
XHIM-005A CD40LG X-linked EX1_EX5del Gross Deletion
XHIM-008A CD40LG X-linked EX1_EX5del Gross Deletion
XHIM-018A CD40LG X-linked LRG_141t1:c.288+259_409+652delinsTCGT Gross Deletion
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Repeated mutations are in bold. CD40LG, CD40 ligand; HIGM1, X-linked immunodeficiency with hyper-IgM type 1. *translation termination (stop) codon.

Table 2.

Causal mutations identified in CYBB gene (Reference Sequence LRG_53) of the XCGD patients.

Patient ID Gene Mutant allele cDNA/nucleotide change Protein change Mutant type
XCGD-110A CYBB X-linked LRG_53t1:c.-65C>T Regulatory
XCGD-072A CYBB X-linked LRG_53t1:c.376T>C C126R Missense
XCGD-018A CYBB X-linked LRG_53t1:c.577T>C S193P Missense
XCGD-004A CYBB X-linked LRG_53t1:c.613T>A F205I Missense
XCGD-044A CYBB X-linked LRG_53t1:c.626A>G H209R Missense
XCGD-077A CYBB X-linked LRG_53t1:c.665A>G H222R Missense
XCGD-062A CYBB X-linked LRG_53t1:c.911C>G
EX11-EX13del		 P304R Missense
Gross Deletion
XCGD-067A CYBB X-linked LRG_53t1:c.925G>A E309K Missense
XCGD-013A CYBB X-linked LRG_53t1:c.935T>A M312K Missense
XCGD-145A CYBB X-linked LRG_53t1:c.985T>C C329R Missense
XCGD-058A CYBB X-linked LRG_53t1:c.1014C>A H338Q Missense
XCGD-060A CYBB X-linked LRG_53t1:c.1016C>A P339H Missense
XCGD-111A CYBB X-linked LRG_53t1:c.1022C>T T341I Missense
XCGD-008A CYBB X-linked LRG_53t1:c.1025T>A L342Q Missense
XCGD-125A CYBB X-linked LRG_53t1:c.1075G>A G359R Missense
XCGD-121A CYBB X-linked LRG_53t1:c.1154T>G I385R Missense
XCGD-038A CYBB X-linked LRG_53t1:c.1234G>A G412R Missense
XCGD-078A CYBB X-linked LRG_53t1:c.1244C>T P415L Missense
XCGD-005A CYBB X-linked LRG_53t1:c.1498G>C D500H Missense
XCGD-136A CYBB X-linked LRG_53t1:c.1546T>C W516R Missense
XCGD-103A CYBB X-linked LRG_53t1:c.1548G>C W516C Missense
XCGD-043A CYBB X-linked LRG_53t1:c.1583C>G P528R Missense
XCGD-120A CYBB X-linked LRG_53t1:c.84G>A W28* Nonsense
XCGD-106A CYBB X-linked LRG_53t1:c.123C>G Y41* Nonsense
XCGD-128A CYBB X-linked LRG_53t1:c.217C>T R73* Nonsense
XCGD-095A CYBB X-linked LRG_53t1:c.271C>T R91* Nonsense
XCGD-142A CYBB X-linked LRG_53t1:c.388C>T R130* Nonsense
XCGD-029A CYBB X-linked LRG_53t1:c.469C>T R157* Nonsense
XCGD-074A CYBB X-linked LRG_53t1:c.469C>T R157* Nonsense
XCGD-101A CYBB X-linked LRG_53t1:c.469C>T R157* Nonsense
XCGD-032A CYBB X-linked LRG_53t1:c.676C>T R226* Nonsense
XCGD-076A CYBB X-linked LRG_53t1:c.676C>T R226* Nonsense
XCGD-107A CYBB X-linked LRG_53t1:c.676C>T R226* Nonsense
XCGD-137A CYBB X-linked LRG_53t1:c.676C>T R226* Nonsense
XCGD-138A CYBB X-linked LRG_53t1:c.676C>T R226* Nonsense
XCGD-019A CYBB X-linked LRG_53t1:c.868C>T R290* Nonsense
XCGD-084A CYBB X-linked LRG_53t1:c.868C>T R290* Nonsense
XCGD-108A CYBB X-linked LRG_53t1:c.868C>T R290* Nonsense
XCGD-147A CYBB X-linked LRG_53t1:c.868C>T R290* Nonsense
XCGD-080A CYBB X-linked LRG_53t1:c.1328G>A W443* Nonsense
XCGD-059A CYBB X-linked LRG_53t1:c.1399G>T E467* Nonsense
XCGD-014A CYBB X-linked LRG_53t1:c.1437C>A Y479* Nonsense
XCGD-006A CYBB X-linked LRG_53t1:c.1555G>T E519* Nonsense
XCGD-028A CYBB X-linked LRG_53t1:c.77_78del F26Cfs*8 Frameshift
XCGD-083A CYBB X-linked LRG_53t1:c.126_130delinsTTTC R43Ffs*18 Frameshift
XCGD-009A CYBB X-linked LRG_53t1:c.713del V238Gfs*4 Frameshift
XCGD-118A CYBB X-linked LRG_53t1:c.714_715insTA H239Yfs*4 Frameshift
XCGD-139A CYBB X-linked LRG_53t1:c.722_726delTAACA I241fs*243 Frameshift
XCGD-115A CYBB X-linked LRG_53t1:c.725_726del T242Sfs*3 Frameshift
XCGD-037A CYBB X-linked LRG_53t1:c.742del I248Sfs*7 Frameshift
XCGD-003A CYBB X-linked LRG_53t1:c.742dup I248Nfs*36 Frameshift
XCGD-102A CYBB X-linked LRG_53t1:c.742dup I248Nfs*36 Frameshift
XCGD-113A CYBB X-linked LRG_53t1:c.742dup I248Nfs*36 Frameshift
XCGD-030A CYBB X-linked LRG_53t1:c.857_867del V286Afs*58 Frameshift
XCGD-092A CYBB X-linked LRG_53t1:c.1038del E347Rfs*39 Frameshift
XCGD-079A CYBB X-linked LRG_53t1:c.1313del K438Rfs*64 Frameshift
XCGD-010A CYBB X-linked LRG_53t1:c.1327del W443Gfs*59 Frameshift
XCGD-073A CYBB X-linked LRG_53t1:c.1332del C445Afs*57 Frameshift
XCGD-126A CYBB X-linked LRG_53t1:c.1565del T522Kfs*11 Frameshift
XCGD-134A CYBB X-linked LRG_53t1:c.1599_1602del V534Sfs*12 Frameshift
XCGD-090A CYBB X-linked LRG_53t1:c.1619_1626dup A543Kfs*7 Frameshift
XCGD-075A CYBB X-linked LRG_53t1:c.70_72del F24del In-frame Deletion/Insertion
XCGD-007A CYBB X-linked LRG_53t1:c.646_648del F216del In-frame Deletion/Insertion
XCGD-048A CYBB X-linked LRG_53t1:c.1164_1166delinsATC 388_389delinsES In-frame Deletion/Insertion
XCGD-129A CYBB X-linked LRG_53t1:c.1322_1324del F441del In-frame Deletion/Insertion
XCGD-045A CYBB X-linked LRG_53t1:c.45+1G>A Splicing
XCGD-100A CYBB X-linked LRG_53t1:c.45+1G>A Splicing
XCGD-119A CYBB X-linked LRG_53t1:c.45+1G>C Splicing
XCGD-143A CYBB X-linked LRG_53t1:c.45+2delT Splicing
XCGD-017A CYBB X-linked LRG_53t1:c.46-1G>C Splicing
XCGD-132A CYBB X-linked LRG_53t1:c.141+1_141+2del Splicing
XCGD-093A CYBB X-linked LRG_53t1:c.141+3A>T Splicing
XCGD-001A CYBB X-linked LRG_53t1:c.252G>A A84= Splicing
XCGD-002A CYBB X-linked LRG_53t1:c.252G>A A84= Splicing
XCGD-104A CYBB X-linked LRG_53t1:c.252G>A A84= Splicing
XCGD-114A CYBB X-linked LRG_53t1:c.252G>A A84= Splicing
XCGD-015A CYBB X-linked LRG_53t1:c.253-1G>A Splicing
XCGD-089A CYBB X-linked LRG_53t1:c.674+6T>C Splicing
XCGD-109A CYBB X-linked LRG_53t1:c.675-1G>T Splicing
XCGD-042A CYBB X-linked LRG_53t1:c.804+2T>C Splicing
XCGD-071A CYBB X-linked LRG_53t1:c.1150_1151+2delAAGT Splicing
XCGD-098A CYBB X-linked LRG_53t1:c.1151+1G>A Splicing
XCGD-099A CYBB X-linked LRG_53t1:c.1314+2T>G Splicing
XCGD-023A CYBB X-linked LRG_53t1:c.1315-2A>C Splicing
XCGD-061A CYBB X-linked EX1-EX13del Gross Deletion
XCGD-041A CYBB X-linked EX7-EX11del Gross Deletion
XCGD-116A CYBB X-linked EX8-EX13del Gross Deletion
XCGD-026A CYBB X-linked LRG_53t1:c.1713A>T *571Yext*8 Extension
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Repeated mutations are in bold. CYBB, cytochrome b-245 beta chain; XCGD, X-linked chronic granulomatous disease. *translation termination (stop) codon.

Table 3.

Causal mutations identified in IL2RG gene (Reference Sequence LRG_150) of the XSCID patients.

Patient ID Gene Mutant allele cDNA/nucleotide change Protein change Mutant type
IL2RG-062A IL2RG X-linked LRG_150t1:c.3G>T M1I Start Lost
IL2RG-043A IL2RG X-linked LRG_150t1:c.202G>A E68K Missense
IL2RG-089A IL2RG X-linked LRG_150t1:c.202G>A E68K Missense
IL2RG-080A IL2RG X-linked LRG_150t1:c.252C>A N84K Missense
IL2RG-142A IL2RG X-linked LRG_150t1:c.272A>G Y91C Missense
IL2RG-063A IL2RG X-linked LRG_150t1:c.304T>C C102R Missense
IL2RG-048A IL2RG X-linked LRG_150t1:c.340G>T G114C Missense
IL2RG-027A IL2RG X-linked LRG_150t1:c.365T>C I122T Missense
IL2RG-005A IL2RG X-linked LRG_150t1:c.371T>C L124P Missense
IL2RG-064A IL2RG X-linked LRG_150t1:c.383T>C F128S Missense
IL2RG-111A IL2RG X-linked LRG_150t1:c.386T>A V129D Missense
IL2RG-049A IL2RG X-linked LRG_150t1:c.618T>A H206Q Missense
IL2RG-008A IL2RG X-linked LRG_150t1:c.670C>T R224W Missense
IL2RG-047A IL2RG X-linked LRG_150t1:c.670C>T R224W Missense
IL2RG-112A IL2RG X-linked LRG_150t1:c.675C>A S225R Missense
IL2RG-041A IL2RG X-linked LRG_150t1:c.676C>T R226C Missense
IL2RG-123A IL2RG X-linked LRG_150t1:c.676C>T R226C Missense
IL2RG-004A IL2RG X-linked LRG_150t1:c.677G>A R226H Missense
IL2RG-115A IL2RG X-linked LRG_150t1:c.694G>C G232R Missense
IL2RG-079A IL2RG X-linked LRG_150t1:c.709T>C W237R Missense
IL2RG-015A IL2RG X-linked LRG_150t1:c.722G>T S241I Missense
IL2RG-009A IL2RG X-linked LRG_150t1:c.854G>A R285Q Missense
IL2RG-014A IL2RG X-linked LRG_150t1:c.854G>A R285Q Missense
IL2RG-020A IL2RG X-linked LRG_150t1:c.854G>A R285Q Missense
IL2RG-022A IL2RG X-linked LRG_150t1:c.854G>A R285Q Missense
IL2RG-025A IL2RG X-linked LRG_150t1:c.854G>A R285Q Missense
IL2RG-061A IL2RG X-linked LRG_150t1:c.854G>A R285Q Missense
IL2RG-083A IL2RG X-linked LRG_150t1:c.854G>T R285L Missense
IL2RG-076A IL2RG X-linked LRG_150t1:c.979_980delinsTT E327L Missense
IL2RG-122A IL2RG X-linked LRG_150t1:c.979G>A E327K Missense
IL2RG-132A IL2RG X-linked LRG_150t1:c.184T>A
LRG_150t1:c.204G>C		 C62S
E68D		 Missense
Missense
IL2RG-147A IL2RG X-linked LRG_150t1:c.181C>T Q61* Nonsense
IL2RG-067A IL2RG X-linked LRG_150t1:c.202G>T E68* Nonsense
IL2RG-075A IL2RG X-linked LRG_150t1:c.306C>A C102* Nonsense
IL2RG-012A IL2RG X-linked LRG_150t1:c.376C>T Q126* Nonsense
IL2RG-103A IL2RG X-linked LRG_150t1:c.376C>T Q126* Nonsense
IL2RG-007A IL2RG X-linked LRG_150t1:c.562C>T Q188* Nonsense
IL2RG-033A IL2RG X-linked LRG_150t1:c.562C>T Q188* Nonsense
IL2RG-023A IL2RG X-linked LRG_150t1:c.711G>A W237* Nonsense
IL2RG-096A IL2RG X-linked LRG_150t1:c.811G>T G271* Nonsense
IL2RG-098A IL2RG X-linked LRG_150t1:c.865C>T R289* Nonsense
IL2RG-141A IL2RG X-linked LRG_150t1:c.865C>T R289* Nonsense
IL2RG-146A IL2RG X-linked LRG_150t1:c.865C>T R289* Nonsense
IL2RG-104A IL2RG X-linked LRG_150t1:c.929G>A W310* Nonsense
IL2RG-032A IL2RG X-linked LRG_150t1:c.982C>T R328* Nonsense
IL2RG-028A IL2RG X-linked LRG_150t1:c.127del T43Pfs*28 Frameshift
IL2RG-003A IL2RG X-linked LRG_150t1:c.310_311delinsG H104Afs*43 Frameshift
IL2RG-016A IL2RG X-linked LRG_150t1:c.359dup E121Gfs*47 Frameshift
IL2RG-055A IL2RG X-linked LRG_150t1:c.362del E121Gfs*26 Frameshift
IL2RG-088A IL2RG X-linked LRG_150t1:c.362del E121Gfs*26 Frameshift
IL2RG-074A IL2RG X-linked LRG_150t1:c.406_415del R136Gfs*8 Frameshift
IL2RG-018A IL2RG X-linked LRG_150t1:c.421del Q141Rfs*6 Frameshift
IL2RG-017A IL2RG X-linked LRG_150t1:c.507del Q169Hfs*2 Frameshift
IL2RG-058A IL2RG X-linked LRG_150t1:c.507del Q169Hfs*2 Frameshift
IL2RG-120A IL2RG X-linked LRG_150t1:c.658_659del T220Vfs*8 Frameshift
IL2RG-040A IL2RG X-linked LRG_150t1:c.741dup S248Efs*55 Frameshift
IL2RG-097A IL2RG X-linked LRG_150t1:c.741del S248Afs*25 Frameshift
IL2RG-001A IL2RG X-linked LRG_150t1:c.835del V279Cfs*15 Frameshift
IL2RG-002A IL2RG X-linked LRG_150t1:c.855-72_925-11del T286Pfs*57 Frameshift
IL2RG-145A IL2RG X-linked LRG_150t1:c.115+1G>A Splicing
IL2RG-118A IL2RG X-linked LRG_150t1:c.115+2T>C Splicing
IL2RG-143A IL2RG X-linked LRG_150t1:c.270-2A>G Splicing
IL2RG-035A IL2RG X-linked LRG_150t1:c.270-15A>G Splicing
IL2RG-059A IL2RG X-linked LRG_150t1:c.270-15A>G Splicing
IL2RG-129A IL2RG X-linked LRG_150t1:c.455-2A>T Splicing
IL2RG-144A IL2RG X-linked LRG_150t1:c.757_757+1delinsTC Splicing
IL2RG-113A IL2RG X-linked LRG_150t1:c.854+3G>T Splicing
IL2RG-006A IL2RG X-linked LRG_150t1:c.854+5G>A Splicing
IL2RG-011A IL2RG X-linked LRG_150t1:c.854+5G>A Splicing
IL2RG-042A IL2RG X-linked LRG_150t1:c.855-2A>C Splicing
IL2RG-121A IL2RG X-linked LRG_150t1:c.855-2A>T Splicing
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Repeated mutations are in bold. IL2RG, interleukin 2 receptor subunit gamma; XSCID, X-linked severe combined immunodeficiency. *translation termination (stop) codon.

Among the patients with the 5 common IEI referred, 903 single targeted gene SS were performed in the first round of screening with 611 causal mutations identified (67.7%), with the positive diagnostic rate ranging from 51.1% (IL2RG gene mutations for XSCID) to 77.4% (BTK gene mutations for XLA) ( Figure 2 ). XLA is the most common referred IEI with the highest positive diagnostic rate. For the other typical and atypical IEI patients (including those with negative finding after screening for the 5 common IEI), a total of 1,121 targeted gene SS (single or multiple rounds of SS may have been done for each patient) were performed with causal mutations identified in 133 (11.9%; Table 5 and Figure 3 ). Among the 5 common IEI, the locations of causal mutations were shown in Figures 4 – 8 . The mutations identified include missense, nonsense, frameshift, and splicing variants. In addition, uncommon mutations such as gross deletion, in-frame deletion/insertion, start loss, stop loss and regulatory variants were identified.

Figure 2.

Figure 2

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Number of patients with first round of targeted gene SS (Sanger Sequencing) performed, and number of patients with mutations identified. IEI, inborn errors of immunity; SS, Sanger sequencing; BTK, Bruton tyrosine kinase; WAS, WASP actin nucleation promoting factor; CYBB, cytochrome b-245 beta chain; IL2RG, interleukin 2 receptor subunit gamma; CD40LG; CD40 ligand.

Table 5.

Number of patients with targeted gene SS performed, and number of patients with mutations identified.

IEI genes Patients with targeted gene SS Patients with mutations identified %
NCF2 10 7 70.0
ITGB2 13 9 69.2
NOD2 4 2 50.0
RFXANK 2 1 50.0
TTC7A 2 1 50.0
FOXP3 6 2 33.3
ADA 3 1 33.3
AK2 3 1 33.3
PIK3CD 7 2 28.6
DOCK8 8 2 25.0
IKBKG 4 1 25.0
STAT3 62 15 24.2
JAK3 22 5 22.7
IL10RA 14 3 21.4
IL12RB1 64 13 20.3
AIRE 10 2 20.0
NLRP3 16 3 18.8
IL7R 22 4 18.2
CYBA 56 10 17.9
ELANE 40 6 15.0
RAG2 78 10 12.8
RAG1 82 10 12.2
STAT1 53 6 11.3
SH2D1A 46 5 10.9
TNFRSF13B 13 1 7.7
DCLRE1C 55 4 7.3
IFNGR1 51 3 5.9
XIAP 21 1 4.8
FASLG 21 1 4.8
PRF1 32 1 3.1
IL12B 55 1 1.8
FAS 20 0 0.0
UNC13D 16 0 0.0
ICOS 16 0 0.0
AICDA 13 0 0.0
CASP10 12 0 0.0
MVK 10 0 0.0
CD40 10 0 0.0
UNG 10 0 0.0
IL10RB 9 0 0.0
RAB27A 9 0 0.0
NLRP12 7 0 0.0
CD79A 7 0 0.0
HAX1 7 0 0.0
TNFRSF1A 7 0 0.0
TYK2 7 0 0.0
LIG4 6 0 0.0
CARD9 6 0 0.0
RASGRP1 6 0 0.0
ZAP70 6 0 0.0
IL10 5 0 0.0
IL24 5 0 0.0
IRAK4 5 0 0.0
CD19 4 0 0.0
NCF4 4 0 0.0
PNP 3 0 0.0
IFNGR2 3 0 0.0
CLEC7A 3 0 0.0
MYD88 3 0 0.0
PRKCD 3 0 0.0
MAGT1 2 0 0.0
IL12A 2 0 0.0
ITK 2 0 0.0
STAT5B 2 0 0.0
STK4 2 0 0.0
TCF3 1 0 0.0
IL2RA 1 0 0.0
CXCR4 1 0 0.0
LRBA 1 0 0.0
TCIRG1 1 0 0.0
CLCN7 1 0 0.0
FERMT3 1 0 0.0
GATA2 1 0 0.0
IL1RN 1 0 0.0
IL36RN 1 0 0.0
IRF8 1 0 0.0
LAT 1 0 0.0
PGM3 1 0 0.0
PSMB8 1 0 0.0
Total 1121 133 11.9
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Official gene symbols approved by HGNC were used. Approved full gene names are available in HGNC. IEI, inborn errors of immunity; SS, Sanger sequencing; HGNC, HUGO Gene Nomenclature Committee. Sum of patients are in bold.

Figure 3.

Figure 3

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Number of patients with targeted gene SS performed, and number of patients with mutations identified. Official gene symbols approved by HGNC were used. Approved full gene names are available in HGNC. IEI, inborn errors of immunity; SS, Sanger sequencing; HGNC; HUGO Gene Nomenclature Committee.

Figure 4.

Figure 4

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Distribution of casual mutations in various exons, exon-intron junctions and corresponding domains of BTK gene. The upper diagram shows the distribution and frequency of amino acid mutations in various protein domains; while the lower diagram shows the locations of splice site mutations and large deletions of the gene. BTK, Bruton tyrosine kinase; XLA, X-linked agammaglobulinemia; PH, Pleckstrin homology; SH2, Src homology 2; SH3. Src homology 3.

Figure 8.

Figure 8

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Distribution of casual mutations in various exons, exon-intron junctions and corresponding domains of IL2RG gene. The upper diagram shows the distribution and frequency of amino acid mutations in various protein domains; while the lower diagram shows the locations of splice site mutations and large deletions of the gene. CD40LG; CD40 ligand; HIGM1, X-linked immunodeficiency with hyper-IgM type 1.

Figure 5.

Figure 5

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Distribution of casual mutations in various exons, exon-intron junctions and corresponding domains of WAS gene. The upper diagram shows the distribution and frequency of amino acid mutations in various protein domains; while the lower diagram shows the locations of splice site mutations and large deletions of the gene. WAS, WASP actin nucleation promoting factor; WAS, Wiskott Aldrich Syndrome; PBD, P21-Rho-binding domain; WH1, WASP homology 1 domain; WH2, WASP homology 2 domain.

Figure 6.

Figure 6

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Distribution of casual mutations in various exons, exon-intron junctions and corresponding domains of CYBB gene. The upper diagram shows the distribution and frequency of amino acid mutations in various protein domains; while the lower diagram shows the locations of splice site mutations and large deletions of the gene. CYBB, cytochrome b-245 beta chain; XCGD, X-linked chronic granulomatous disease.

Figure 7.

Figure 7

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Distribution of casual mutations in various exons, exon-intron junctions and corresponding domains of IL2RG gene. The upper diagram shows the distribution and frequency of amino acid mutations in various protein domains; while the lower diagram shows the locations of splice site mutations and large deletions of the gene. IL2RG, interleukin 2 receptor subunit gamma; XSCID, X-linked severe combined immunodeficiency.

Discussions

Using one single round of targeted gene SS in our study was successful in diagnosing 611 of the 903 patients (67.7%) suspected to have one of the 5 common IEI, i.e., XLA (77.4%), WAS (69.2%), XCGD (59.5%), XHIM (58.1%), and XSCID (51.1%), definitively. These 5 IEI are X-linked which renders the genetic diagnosis more readily and accurately achieved. At the clinical level, a positive family history of maternal uncles or male cousins affected with similar clinical and immunological phenotypes, suggestive of X-linked pattern of inheritance, will be the first clue. Moreover, the clinical and immunological phenotypes of these 5 IEI are relatively uniform, except for XSCID, which could have multiple phenotypes due to hypomorphic mutations of IL2RG gene as well as presence of multiple genes giving rise to similar immunological phenotypes. The immunophenotype of these 5 IEI is more easily defined by laboratory tests which are less technically demanding and more available, such as complete blood count, lymphocyte subsets, immunoglobulin profile, and the nitroblue tetrazolium test (6). Though the diagnostic resources and experience of referring clinicians could differ among different centres, affecting the accuracy of the diagnosis for these 5 IEI, our findings demonstrated that the individual positive diagnostic rate is much higher than that for the other IEI (11.9%), see Supplementary Figure 1 . In addition, referring clinicians can learn from our e-consultation and diagnostic algorithm to further improve the diagnostic rate. More importantly, these 5 IEI occur at much higher rates than the rest of the 400 IEI, resulting in a higher level of awareness among paediatricians, hence earlier recognition, and referral for definitive genetic diagnosis than the less common IEI.

At the genetic diagnostic level, X-linked IEI is easier to diagnose than autosomal recessive IEI in non-consanguineous population, because identification of causal mutation in a single allele is sufficient. Moreover, there is no pitfall of missing the identification of heterozygous gross deletion by Sanger sequencing as in autosomal IEI with PCR still positive in such cases. For the X-linked genes, gross deletion will be picked up by negative PCR, and then one can confirm the deletion in each exon by multiplex PCR, co-amplification of both target and reference gene, with normal control. Due to limitation of our primers design, causal mutations within those intronic and regulatory regions may not be included in the PCR regions, and hence cannot be identified. Nevertheless, the strengths of targeted gene SS include >99% high accuracy, fast turnaround time, low cost, with fewer variants of uncertain significance and no secondary findings (3, 4). Therefore, doing one round of single specific targeted gene SS remains the first-tier genetic test for patients suspected to have one of these 5 common IEI in our laboratory.

Apart from these 5 common IEI, there were 2 more IEI with over 50% genetic diagnostic success rates in our study using targeted gene SS, i.e., leucocyte adhesion deficiency type 1 (LAD1) and autosomal recessive chronic granulomatous disease (AR-CGD) due to neutrophil cytosolic factor2 (NCF2) gene mutations. For LAD1, the clinical and immunological phenotype is uniform with little variation, and LAD1 occurs at a much higher frequency than the other two types of LAD. With flow cytometric analysis of CD18, followed by integrin subunit beta 2 (ITGB2) gene SS, LAD1 can be diagnosed easily (46). Our one round of single targeted gene SS was successful in diagnosing 9 of the 13 patients (69.2%) suspected to have LAD1. As for AR-CGD due to NCF2gene mutations, the success rate of targeted gene SS in making the genetic diagnosis was 70% in our study (7 out of 10 patients), but this was achieved by doing multiple AR-CGD genes at the same time, after failing to identify the genetic mutation for CYBB gene in male patients suspected to have CGD. Therefore, the 70% success rate was not after doing just one round of single targeted gene SS, but after multiple rounds of targeted gene SS of genes responsible for AR-CGD.

For the rest of the IEI, the success rates of achieving genetic diagnosis for each of these IEI after targeted gene SS were mostly under one-third, and in most cases, we had to do multiple rounds of targeted gene SS, with an overall success rate of only 10.9%. Therefore, whole exome sequencing (WES) is now our preferred first-tier genomic test for all the IEI except the 5 most common X-linked IEI and LAD1. However, exceptions do occur, such as AR-CGD due to NCF1 gene, which has pseudogenes, rendering both SS and WES not able to identify the causal mutations due to poor and limited coverage of sequences shared with pseudogenes. Fortunately, 97% of affected alleles in patients previously reported with p47-phox deficiency carry a hot spot mutation of “GT”deletion (ΔGT) in exon 2 of neutrophil cytosolic factor 1 (NCF1) gene (47). One can therefore simply identify the hot spot mutation by GeneScan® analysis as shown in Supplementary Figure 2 before proceeding to sequencing of the coding region. This approach was adopted by us to save time and cost

All in all, we were able to diagnose 744 of the 1376 patients (54.1%) referred to us suspected to have IEI, using targeted genes SS, with an average of 1.47 such tests per patient (ranging from 1 to 10). However, 632 of these 1376 patients (45.9%) of the referred patients remained genetically undiagnosed after single or multiple rounds of targeted gene SS.

With the availability of WES in 2009, we deployed this technology for selected undiagnosed IEI patients. Our first WES case for a male infant with early-onset inflammatory bowel disease (IBD) in 2009 resulted in the discovery of interleukin 10 receptor subunit alpha (IL10RA) gene mutations as the underlying cause of early-onset IBD (27), at about the same time when aberrant interleukin 10 (IL10) pathway was implicated as the underlying cause for early-onset IBD by another group using linkage analysis (48). Since then, we have incorporated WES more readily into our diagnostic algorithm, because of the cost coming down as well as developing our own in-house bioinformatic tools and analysis, resulting in discovery of novel IEI (49, 50). We shall review in future our experience in using WES for patients with suspected IEI who remain undiagnosed genetically after targeted gene SS. Comparison between targeted gene SS and NGS (whole exome sequencing WES) in our institutional service has been shown in Supplementary Figure 3 . In general, WES will have wider applications, but longer turnover time compared with SS under the service provided by our centre. However, if both the financial and human resource (laboratory staffs and bioinformaticians) is not a limiting factor, rapid WES may be considered to set up for those urgent cases with immediate clinical management decision (51).

In conclusion, single targeted gene SS should remain the first-tier genetic test for patients suspected to have one of the 5 common X-linked IEI before offering genomic tests such as WES or targeted gene panel (52). Flow chart of our current diagnostic algorithm, with the description of progressive changes in our bioinformatic analysis, has been provided as reference ( Supplementary Figure 4 ). We propose IEI centres in less resourced Asian and African countries and regions could consider setting up targeted gene SS for these 6 IEI which would yield a high enough success rate of genetic diagnosis in a significant number of IEI patients to become cost-effective (6, 53).

Data Availability Statement

The original contributions presented in the study are included in the article/ Supplementary Material Further inquiries can be directed to the corresponding author.

Ethics Statement

The studies involving human participants were reviewed and approved by Clinical Research Ethics Review Board of The University of Hong Kong and Queen Mary Hospital (Ref. no. UW 08-301). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

Author Contributions

Y-LL conceptualized the study. YL, XY, WT, PL, WY, and DL designed the study. K-WC, C-YW, SF, and PM performed genetic study. K-WC, and DL curated mutations. PL and DL phenotyped the patients. K-WC, C-YW, XY, and DL analyzed data. K-WC and C-YW drafted the manuscript. Other authors referred patients and provided clinical care and clinical data. All authors critically reviewed the manuscript. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by the Hong Kong Society for Relief of Disabled Children and Jeffrey Modell Foundation.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2022.883446/full#supplementary-material

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Data Availability Statement

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