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Front. Immunol. | doi: 10.3389/fimmu.2019.02678

Opinion: All patients with Common Variable Immunodeficiency Disorders (CVID) should be routinely offered diagnostic genetic testing

 Rohan Ameratunga1*, Klaus Lehnert2 and  Seetarn Woon1
  • 1Auckland City Hospital, New Zealand
  • 2The University of Auckland, New Zealand

Common Variable Immunodeficiency Disorders (CVID) are a rare group of primary immunodeficiency disorders (PID) where late onset antibody failure leads to immune system failure. 1 Onset of symptoms can occur from early childhood to the eighth decade or later.The majority of patients with CVID present with recurrent and severe infections. Untreated, patients are predisposed to chronic suppuration of the respiratory tract, often resulting in chronic sinus disease and bronchiectasis. Approximately 25% of CVID patients suffer autoimmune or inflammatory conditions, consequent to immune dysregulation.The causes of these rare disorders are unknown in most cases. In 2003, the first genetic defect was identified in Germany. 4 Mutations of the Inducible T cell co-stimulator (ICOS), which plays a critical role in T and B cell communication, were found in patients with a CVID phenotype. There was a founder effect as all affected individuals in the Black Forest area carried the identical mutation. Different mutations of ICOS were subsequently identified in other parts of the world, confirming the existence of allelic heterogeneity.Two years later, mutations of the T cell activator, calcium modulator and cyclophilin ligand interactor (TACI) were discovered. This molecule plays an important role in B cell signalling and immunoglobulin isotype switching. Mutations of TACI were initially thought to cause CVID but subsequently it became apparent that identical mutations were also found in the general population at a frequency much greater than the incidence of CVID. It is now thought that mutations of TACI predispose to CVID or have a disease modifying effect on the disorder.We and others have suggested mutations associated with CVID should be categorised according to whether they cause the disorder or whether they modify or predispose to the condition. 6,7 Apart from TACI, mutations of other genes including BAFFR, TWEAK, MSH5 and TRAIL are also thought to predispose to, or modify the disease severity of patients with CVID.In contrast, mutations of genes such as NFKB1, NFKB2, CTLA4, TCF3 etc. are more likely to cause the condition. 8 9 Such patients are removed from the broad category of CVID and are stated to have a CVID-like disorder caused by a specific mutation. None of the current diagnostic criteria for CVID allow the diagnosis if a known disorder is identified. 6,[10][11][12] This is the basis for excluding patients with a causative mutation from the umbrella diagnosis of CVID. Since the discovery of ICOS mutations, approximately 20 genetic defects have been shown to modify or predispose to CVID or cause CVIDlike disorders. 13 CVID is genetically complex. Locus heterogeneity (genocopy) is a major feature of CVID-like disorders, making it difficult to identify the affected gene purely on clinical grounds. Mutations of several genes can result in the classical phenotype of late onset antibody failure leading to recurrent and severe infections as well as autoimmunity.Although clinical identification of individual CVID-like disorders is difficult, there may be subtle clues such as the presence of alopecia and pituitary dysfunction, which are indicative of NFKB2 defects. In other cases, a careful history may reveal severe autoimmunity, which may suggest PI3KD(activated protein kinase 3D syndrome, APDS) or CTLA4/LRBA mutations. Similarly, the presence of vasculitis in the context of hypogammaglobulinemia might indicate DADA2 deficiency. 13 In most cases however, such clues are absent.Similarly, phenotypic heterogeneity makes diagnosis difficult as the clinical manifestations can vary widely, even within the same family carrying the identical mutation. We have recently described the pleomorphic clinical presentation of a family with NFKB1 deficiency. 14 One heterozygous brother carrying the mutation was asymptomatic with normal immunoglobulins, while his heterozygous sister had severe disease with features of late onset combined immunodeficiency (LOCID). 14 We have used our CVID disease severity score (CDSS) to quantify the phenotypic severity of individual family members. 15 The phenotypic heterogeneity may be the result of variable penetrance and expressivity, epigenetic influences or epistasis caused by gene-gene interactions.As noted in the case of ICOS deficiency, CVID-like disorders also manifest allelic heterogeneity where different mutations of the same gene can result in a similar phenotype. Because of genetic and phenotypic heterogeneity, there has been understandable reluctance to routinely sequence CVID patients because of the low yield. 16 Serial Sanger sequencing of individual genes was not an efficient use of valuable resources.Given the rapid progress in the understanding of these conditions in recent years, we believe there is now a strong case for routine diagnostic genetic testing of patients with a CVID phenotype ( Table 1). This change in approach is both the result of identifying increasing numbers of genetic defects as well as advances in technology, particularly NGS. We have previously discussed diagnosing CVID in the era of genome sequencing. 13 In this current viewpoint article, we have incorporated new information from our recent studies to strengthen our arguments for routine diagnostic sequencing of patients with a CVID phenotype.NGS allows efficient sequencing of disorders with locus heterogeneity.Over the last decade, NGS has revolutionised the approach to molecular diagnosis. Multiple genes can now be sequenced simultaneously, with either gene panels or by Whole Exome Sequencing (WES) with targeted analysis. 13 In non-consanguineous populations, the causative mutation can be identified in approximately 25% of CVID patients. 19 The diagnostic yield is much higher in kindreds with more than one affected individual, founder populations or those with high rates of consanguinity. The advent of NGS is the principal reason for the feasibility of routine genetic sequencing of patients with CVID-like disorders, who have locus heterogeneity. (Table 1).Identifying the causative genetic defect is now the standard of care of PID patients. We have outlined the many overlapping advantages (and some disadvantages) in identifying the underlying genetic defect in PIDs. [21][22][23] We have listed in detail the specific advantages of genetic diagnosis in CVID-like disorders in Table 1.Identification of a causative mutation will confirm the presence of a CVID-like disorder and will enable diagnosis of patients with atypical presentations. This is particularly important given the genetic and phenotypic heterogeneity in CVID-like disorders, outlined above.Some patients with well-characterised PIDs such as X-linked lymphoproliferative (XLP) disorder or STAT3 mutations can present with predominant hypogammaglobulinemia. If other characteristic features of these disorders are not obvious, such atypical presentations may cause confusion with CVID. Given there may be specific treatments for these conditions, early identification is of paramount importance. Pre-emptive bone marrow transplantation prior to EBV infection in presymptomatic male relatives can be life-saving in XLP. 24 NGS will rapidly identify the majority of "non-CVID" patients presenting with hypogammaglobulinemia.Identification of the mutation will offer prognostic information. We have recently shown that many children with transient hypogammaglobulinemia of infancy (THI) do not recover until early adulthood. 18 CVID/ CVID-like disorders are the most important differential diagnosis until patients with THI recover. Identification of a causative mutation in a child with persistent hypogammaglobulinemia will exclude THI and will indicate the patient is likely to require long-term subcutaneous or intravenous immunoglobulin (SCIG/IVIG) therapy.Hypogammaglobulinemia can be caused by a wide range of non-immunological disorders and it can sometimes be difficult to exclude these secondary causes. If a causative genetic defect is identified, this will exclude secondary causes, such as those caused by anticonvulsant drugs, gut disease or other rare conditions. [25][26][27][28][29] As with other genetic disorders, identification of the mutation has profound implications for family members. The presence of a genetic defect may allow early diagnosis and prompt commencement of SCIG/IVIG treatment of affected family members, when they develop symptoms. These presymptomatic individuals could be made aware of possible risks and complications. This may either prevent catastrophic infections or mitigate ongoing target organ damage leading to bronchiectasis and other disabling complications.Detection of a causative mutation may allow prevention of PIDs. The specific mutation may allow prenatal diagnosis and/or preimplantation genetic diagnosis (PGD). While current technologies will not prevent disease caused by new mutations, PGD could lead to a substantial decrease in the prevalence of disease within a generation. This will result in a major reduction in the burden of suffering as well as healthcare costs. The NZ government offers free in vitro fertilization and PGD for families carrying severe genetic defects.Identification of the specific mutation may lead to new therapeutic options. Patients with mutations of CTLA 4 or LRBA may be candidates for abatacept. Those with gain of function mutations of PI3K (APDS) may improve with mTOR inhibitors such as rapamycin. Patients with a severe CVID-like disorder caused by mutations of DADA2 may benefit from early bone marrow transplantation.Discovery of the mutation may in the future lead to gene-based therapies including retroviral gene transfer or gene editing with CRISPR-Cas9. As discussed previously, off-target effects of the CRISPRCas9 system may however limit its in vivo use. 30 We are unaware of any current in vivo trials of retroviral gene therapy or CRISPR Cas9 gene editing studies in patients with CVID-like disorders.The use of NGS has resulted in new discoveries including novel mechanisms of disease. We have recently shown the existence of quantitative epistasis in a patient with a digenic inheritance leading to a CVID-like disorder. 31,32 Epistasis is the synergistic, non-linear interaction of two or more genetic loci leading either to much more severe disorder or a completely different phenotype. We have suggested the synergistic interaction of genes is termed quantitative epistasis, while those leading to a different phenotype are termed qualitative epistasis. 32 The proband had mutations of both TCF3and TACI genes, which caused a severe defect in antibody production leading to a CVID-like disorder.These two genetic loci lie in tandem, along the immunoglobulin production and isotype switching pathways. The synergistic interaction of these two mutations caused quantitative epistasis, leading to a severe CVID-like disorder. Our study confirms genes such as TACI have disease modifying effect on the severity of CVID/ CVID-like disorders and supports the separation these two groups of mutations. Such digenic patients can only be identified by NGS and are a strong argument for sequencing both groups of genes, either causing CVID-like disorders (NFKB1, NFKB2, etc.) or those modifying the severity of CVID, such as TACI. There is no single laboratory test, which identifies all patients with CVID. We have recently shown marked fluctuations in IgG levels in patients with hypogammaglobulinemia, which we have termed transient hypogammaglobuinemia of adulthood (THA). 17 Of concern was that 41% of symptomatic patients were able to normalise their IgG on at least one occasion, when measured over time. Seven of twenty-two hypogammaglobulinemic patients with bronchiectasis were also able to normalise their IgG on at least one occasion.Some of these asymptomatic patients with hypogammaglobulinemia may have a CVID-like disorder and in time will experience progressive clinical deterioration. 14 Identifying a causative genetic defect will establish the diagnosis and assist with monitoring and therapeutic decisions. Patients with profound hypogammaglobulinemia (< 3 g/l) and those who are symptomatic with persistent hypogammaglobulinemia should be considered for genetic testing. Patients with THA with subsequent sustained normal IgG levels do not need testing, with the possible exception of those with a family history of an immunological disorder. We have shown that some family members carrying mutations of CVID-like disorders can be asymptomatic with normal IgG levels. We acknowledge, THA patients will need to be carefully assessed.We have discussed the difficulties with the previous ESID/PAGID (1999) criteria for CVID. 33 They lacked precision and asymptomatic patients with trivial hypogammaglobulinemia with mildly impaired responses to the diphtheria vaccine could be designated as having CVID and offered lifelong SCIG/IVIG.We have recently compared diagnostic criteria for CVID in a long-term cohort study of patients with primary hypogammaglobulinemia. 17 We showed while there was general congruence of diagnostic criteria, there were important differences. The ICON CVID criteria require primary hypogammaglobulinemia with a single impaired vaccine response. 11 Patients are required to have a reduction in IgA and/or IgM in addition to reductions in IgG. In our study, many asymptomatic individuals with mild hypogammaglobulinemia qualified as having CVID by ICON criteria, given their impaired vaccine responses to Pneumovax® or the diphtheria vaccine. Given their excellent health over a mean duration of 106 months (to date), it is unlikely these asymptomatic patients have CVID or any other immunological disorder.In our study, both symptomatic and asymptomatic patients with hypogammaglobulinemia had excellent responses to H. influenzae type B (HIB) and tetanus vaccines. 34 Vaccine responses were thus non-discriminatory in this study. Similarly, we also recently showed some patients with THI, who subsequently recovered, had impaired vaccine responses, which could potentially lead to misdiagnosis of CVID if ICON criteria are applied. 18 IgM and particularly IgA levels can be difficult to interpret in infants and young children.Identifying the causative mutation would obviate the need to apply CVID diagnostic criteria as the patient would then be reclassified as having a CVID-like disorder. 35 We suggest patients with CVIDlike disorders are offered SCIG/IVIG on clinical grounds and vaccine challenge responses may not be necessary. Thus, the primary aim of genetic sequencing is to remove these patients from the umbrella diagnosis of CVID so they can be more accurately classified as having a specific PID.While we advocate routine diagnostic WES or WGS for all patients with a CVID phenotype, there are important caveats. We have discussed the technical limitations of NGS including lack of coverage with WES leading to errors. 13 These errors are less likely with WGS but currently this technology is more expensive than WES. NGS is not available in all parts of the world. However, at least two commercial companies are now offering these tests, one using a gene panel, while the other offers WES with targeted analysis. The company offering WES with targeted analysis releases raw data for an additional fee, which allows future analysis of gene mutations, which have yet to be discovered. With the appropriate consents and ethics approvals, this data can also be converted to parents:child trio analysis for gene-discovery research studies.Funding for these tests is a common problem. Clinical services and insurance providers have been slow to recognise the value of such technology and the far-reaching benefits of testing. 16 Prevention of a single case could lead to lifetime savings of over $2M, which would fund NGS for a large cohort of CVID patients. If funding is not immediately available from clinical services, in many cases these can be undertaken as part of research studies, with the appropriate consents. In many cases, our patients have self-funded these tests.Another important caveat is the risk of assigning disease causality to ethnic specific variants. What may seem to be a causal mutation may simply be a common benign variant (polymorphism) in an under-surveyed ethnic group. Current databases comprise mainly of individuals of Caucasian origin, while other ethnicities such as Maori are poorly represented.We have also discussed social and financial disadvantages of identifying the mutation, such as genetic discrimination in the domains of insurance or employment. 23 The Americans with Disabilities act (ADA) and the Genetic Information Non-discrimination Act of 2008 (GINA) protects Americans from such discrimination. Such enabling legislation is however not universally enacted in all jurisdictions. NZ does not currently have laws forbidding genetic discrimination.In spite of these limitations, we believe all patients with a CVID phenotype should now be routinely offered diagnostic NGS sequencing if resources permit. It is important to counsel patients before offering these studies as there is a risk of identifying variants of unknown significance (VUS). 13 If a causal mutation is not found, such patients can be enrolled in gene discovery research studies with the appropriate consents and ethics approvals.

Keywords: CVID- Common Variable Immunodeficiency Disorders, Hypogammaglobinaemia, Genetic sequence analysis, Transient hypogammaglobulinaemia, Transient Hypogammaglobulinemia of Infancy

Received: 22 Aug 2019; Accepted: 30 Oct 2019.

Copyright: © 2019 Ameratunga, Lehnert and Woon. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Prof. Rohan Ameratunga, Auckland City Hospital, Auckland, New Zealand,