Major histocompatibility complex (MHC) class II deficiency is a rare form of primary immunodeficiency disorder (PID) that follows an autosomal recessive pattern of inheritance. Worldwide, over 150 patients have been reported with this deficiency (1). About two-third of these patients have a North African descent (Algeria, Morocco, and Tunisia) (1) and belong to complementation group B. A high prevalence of group A deficiency is reported in the European countries (2). A defect in the factors that regulate the expression of MHC class II genes resulting in the absence or reduced expression of MHC class II molecules on the immune cells is thought to be the underlying cause of MHC class II deficiency (3). The MHC class II molecules also referred to as human leukocyte antigens (HLAs) are polygenic and highly polymorphic cell-surface glycoproteins that assemble as α and β chain heterodimers (3). Humans have three isotypes of HLA-class II molecules: HLA-DR, -DP, and -DQ, which are present on resting B cells, monocytes, dendritic cells, activated T lymphocytes, Langerhans cells, and epithelial cells in the thymus and intestine (4). Somatic fusion experiments supported the description of four complementation groups (A, B, C, and D) (1). Subsequently, four regulatory proteins were identified, deficiencies of which are associated with MHC class II deficiency: class II transactivator (CIITA) under group A (1), RFX containing ankyrin repeats (RFXANK) under group B (5), the fifth member of the RFX (RFX5) under group C (6), and RFX-associated protein (RFXAP) under group D (7). The RFXANK, RFX5, and RFXAP proteins are three subunits of the ubiquitously expressed RFX complex, which bind directly to the promoters of all MHC class II genes and associate with other pleiotropic factors to form the MHC class II enhanceosome (1).

The development and thymic shaping of CD4⁺ T helper (Th) cells require MHC class II-mediated antigen presentation to the T cell receptor (TCR) of CD4 cells. Hence, MHC class II deficiency affects both the cell-mediated and humoral immunity due to a defect in CD4⁺ Th cell development and lack of helper T cell-dependent antibody production by B cells. Affected children typically present with severe respiratory and gastrointestinal tract infections and are diagnosed within their first year of life. However, there are some reports with a milder clinical presentation and diagnosis later up to 15 years of age (3). Diagnosis involves an immunophenotypic finding of a reduced absolute CD4⁺ Th count, inverse CD4:CD8 ratio, an absent or reduced expression of HLA-DR on lymphocytes and monocytes, hypogammaglobulinemia, and a genetic defect in any of the four genes producing the regulatory proteins.

Allogeneic hematopoietic stem cell transplantation (HSCT), preferably from an HLA-identical sibling, is currently the only available curative treatment for this disorder (3).

We describe here the clinical, immunological, and molecular findings of MHC class II deficiency in five patients with novel mutations for the first time from India.

Major histocompatibility complex class II deficiency is reported majorly in the Mediterranean region and is prevalent in areas with high consanguinity. To the best of our knowledge, this is the first report of MHC class II deficiency from India. Our patients presented clinical symptoms within their first year of life indicating the severity of this form of PID. Recurrent lower respiratory tract infection was the predominant clinical presentation.

In this study, patients displayed a reduced absolute CD4⁺ Th cell count and absent HLA-DR expression on B lymphocytes and monocytes. Notably, these patients presented with normal or elevated absolute B lymphocyte count that could potentially lead to misdiagnosis of MHC class II deficiency as T⁻B⁺ form of severe combined immunodeficiency (SCID). Thus, diagnosis of MHC class II deficiency may be confounded unless HLA-DR expression is evaluated on the immune cells.

Major histocompatibility complex class II deficiency is caused by mutation in genes (CIITA, RFXANK, RFX5, and RFXAP) that encode any one of the four regulatory factors that control the transcription of MHC class II genes. RFXANK gene mutation that accounts for more than two-thirds of all reported cases (1) was not identified in our series. Three of our patients had RFXAP gene defect. The RFXAP protein consists of three regions, which are rich in acidic amino acids (DE), glutamine (Q), or basic amino acids (RK) reminiscent of a nuclear localization signal (12). Till date, only five different RFXAP gene mutations have been reported (2) with most of the mutations affecting the nuclear localization signal or DE regions. The RFXAP gene defect in exon 1 of our patients P1 and P2 also affects the nuclear localization signal and the mutation in intron 2 of patient P5 is expected to affect the formation of C-terminal domain, which is required for binding to the RFX complex (13).

Fifth member of the RFX has a DNA-binding domain (DBD) (7) located in residues 90–166 and 407–614, which binds to the ssDNA of the X-boxes before transcription (2) and is believed to interact with CIITA (14). Of the six RFX5 gene mutations reported in the literature, five mutations result in a premature stop codon before the second DBD sequence (2). In line with this finding, the mutation in Exon 11 of RFX5 of our patient (P3) also leads to the premature truncation of the protein by the creation of a premature stop codon just before the second DBD and could potentially be the underlying cause of MHC class II deficiency.

The expression of CIITA is essential for the transcription of all MHC class II genes (15). Till date, 12 different mutations have been reported in the CIITA gene, which are either missense or nonsense mutations (2). Patient P4 described in this study harbored a novel nonsense mutation in the CIITA gene, which affects the transcription of MHC class II genes.

Despite such genetic heterogeneity, these patients had a similar clinical presentation. Traditional Sanger sequencing of individual genes is time consuming in such cases and delays the diagnosis. The strategies that are usually employed for genetic diagnosis of MHC class II deficiency are founder mutation screening and study of polymorphic markers that flank the MHC class II genes in case of consanguinity. Another approach involves a functional test to identify the gene defect, which is based on the direct correction of the genetic defect by transduction of cells from affected individuals with lentiviral vectors encoding CIITA, RFXANK, RFX5, or RFXAP to diagnose and classify MHC class II-deficient patients (16). Currently, with the application of next-generation sequencing (NGS) technology, screening of all the four causative factors can be performed at the same time, thereby reducing the cost and time for getting a diagnosis. Thus, we opted for NGS for genetic confirmation of MHC class II deficiency in our patients.

Hematopoietic stem cell transplantation is the only known curative treatment for MHC class II expression deficiency. However, the success rate is reported to be poor in MHC class II-deficient patients compared to other forms of SCID (17). This is attributed to severe infections, multiorgan failure, and acute graft-versus-host disease observed in relation to pre-existing (pre-HSCT) viral infections (18). As stated in a recent report, the reason for low survival rate in these patients may be due to the presentation of donor antigens by donor antigen-presenting cells to recipient T cells leading to graft rejection (19). Even with immunosuppression, residual host immunity in these patients is reported to be adequate to cause graft rejection. A need for a second transplant was observed in multiple reports of these patients (19). Our data support this trait, where a poor survival outcome was observed in our patients (P2 and P3). Although patient P2 had a presymptomatic diagnosis and received an early transplant, the patient expired within 8 days of transplant due to severe sepsis. For this reason, PND is highly useful in families affected with MHC class II deficiency. As the decision for termination of pregnancy is based on the test results, the preferred procedure for PND is genetic confirmation in the index case and parents and then performing PND by chorionic villus sampling or amniocentesis. However, in cases where a molecular diagnosis is not available in the index case at the time of PND, families can opt for phenotypic PND on cordocentesis sample at 18 weeks of gestation by flow cytometry. Although cordocentesis is done at a later stage of pregnancy (16–18 weeks), the results are usually available within 24 h of the procedure thus providing sufficient time to the family for safe termination of pregnancy (i.e., up to 20th week of gestation). One of our affected families (Patient P1) approached us for PND at a late gestational age of pregnancy. As genetic diagnosis was not available in the index case at the time of PND and we were limited by time for performing the molecular study, phenotypic PND on cordocentesis sample was provided to the family. The fetus was found to be affected with MHC class II deficiency and was eventually terminated (8).

Detectable TREC levels (range, 65–154 copies/reaction) were measured in all the patients. TREC-based newborn screening (NBS) has proven to be extremely beneficial in picking up classical forms of SCID (20); however, TREC levels in MHC class II patients have been reported to be higher than the NBS TREC cut-off for SCID and other T cell lymphopenia conditions (21, 22). Our study also supports this finding and highlights that MHC class II deficiency may be missed by TREC-based NBS programs.

This study was carried out in accordance with the recommendations of Institutional Ethics Committee of National institute of Immunohaematology for research on human Subjects in accordance with the Declaration of Helsinki. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Institutional Ethics Committee of National institute of Immunohaematology.

JA analyzed the data and wrote the manuscript. MG, AD, SM, MK, and GH were involved in performing laboratory investigations of the different cases. MD and NS supervised the clinical care of the patients. PT helped in the collection of samples and the clinical details. RV provided the genetic data. MM supervised the study and reviewed the manuscript.

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.