Chronic necrotizing granulomatous skin lesions and MHC class I deciency syndrome due to TAP2 deciency

Major histocompatibility complexes class I (MHC- I) deciency, also known as bare lymphocyte syndrome type 1 (BLS-1), is a rare autosomal recessively inherited immunodeciency disorder with remarkable clinical and biological heterogeneity. Transporter associated with antigen processing (TAP) is a member of the ATP-binding cassette superfamily of transporters and consists of two subunits, TAP1 or TAP2. Any defect resulting from a mutation or deletion of these two subunits, adversely affects the peptide translocation in the endoplasmic reticulum, which is an important process for the proper assembly of MHC- I molecules. To date, few patients with reduced cell surface expression of MHC-I molecules have been reported. Herein; we described two Iranian cases with 2 and 3 decades delayed diagnosis of chronic necrotizing granulomatous skin lesions due to TAP2 deciency without pulmonary involvement; and then segregation analysis in family members was performed by PCR and Sanger sequencing among 10 members of their family and found three homozygous mutation in three asymptomatic patients. I deciency. indicated the same homozygous mutation in the TAP2 gene.


Introduction
Human lymphocyte antigen (HLA) class I de ciency, also known as bare lymphocyte syndrome type 1 (BLS-1), was rst identi ed in 1978 and is a rare autosomal recessively inherited immunode ciency disorder with remarkable clinical and biological heterogeneity. BLS type 1 is characterized by severely de cient expression of HLA class I, whereas the defective expression of HLA class II is observed in BLS type 2, which the latter causes life threating infections in early childhood [1].
HLA class I molecules, also called major histocompatibility complexes class I (MHC-I), appear on the surface of all nucleated human cells. They are heterodimer molecules that consist of a heavy chain or α subunit and a light chain called β2-microglobulin. The α subunit contains the α1, α2, and α3 domains.
The peptide-binding site is formed by the α1 and α2 domains and binds to antigenic peptides with 8-10 amino acids that are derived from the proteasome by degradation of cytosolic proteins.
Endogenous proteins are processed proteolytically by the proteasome. Subsequently, the generated small peptides are transferred to the endoplasmic reticulum (ER) lumen by a transporter associated with antigen processing (TAP). TAP is a member of the ATP-binding cassette (ABC) superfamily of transporters and consists of two subunits, including TAP1 or TAP2. Any defect resulting from a mutation or deletion of TAP1 or TAP2 hampers the peptide translocation in the ER, which is an important process for the proper assembly of HLA class I molecules in the ER. Therefore, TAP defects result in attenuated expression of HLA class 1 molecules on the cell surface and impair the intracellular antigen presentation pathway [2][3][4].
In patients with TAP de ciency, the expression levels of MHC-I molecules on the cell surface are signi cantly lower (30-100-fold) than in normal hosts. As a result, these patients suffer from bacterial infections, including sinusitis, chronic respiratory tract in ammation, and granulomatous skin lesions.
The latter occurs in about 50% of patients. It is well understood that mutations in the TAP2 subunit cause more serious problems than mutations in the TAP1 subunit [4][5][6]. The disease is extremely rare and usually appears late in childhood, usually when they become adults. Their HLA genotype is homozygous, and their parents are usually rst cousins [4]. Herein; we evaluated a genetic study of an Iranian family with MHC class I de ciency syndrome due to TAP2 de ciency. We rst described the index case, and then we analyzed the sequencing of the patient, her parents, and also other family members.

Study Participants
Ten members of an Iranian family were included in the study. Blood samples were taken for DNA analysis and the participants' genomes were sequenced.

Genotyping and Analysis
Samples were collected from the subjects after obtaining written informed consent. Genomic DNA was isolated from peripheral blood samples using the Exgene™ Blood SV kit (GeneAll Cat. No. 105-101 / 105-152), according to the kit's protocol.
Paired-end sequencing (2 × 101bp) was performed on the NextSeq2000 sequencer (Illumina, San Diego, CA). WES libraries were prepared and captured according to the Agilent Technologies Sure-Select protocol. The sequence data were mapped to the human reference genome (hg-19, NCBI37) using the Burrows-Wheeler Alignment (BWA) method. About 99% of the mapped reads were covered and the mean coverage of the target regions was ∼75 times. Variants were identi ed with the Genome Analysis Toolkit, SAMtools, and the Picard Tools.

Variant validation: PCR and Sanger sequencing
Validation of TAP2 gene, single nucleotide deletion variant identi ed by whole-exome sequencing (WES) in the index patient, and segregation analysis in family members was done by PCR and Sanger sequencing. This speci c primers pair designed for the test: F: 5′-CTGTGGCTCCTTGGGAAAAC-3′, R: 5′-CAGCACCTGGAAGAGGAGAA -3′.

Results
Medical history of the index case Patient II.2, the index patient, was a 46-year-old woman who was referred to our hospital, in February 2021, with a 20-year history of skin lesions involving the lower limb. Her medical history was unremarkable except for these chronic skin lesions on her right leg and foot. The lesions started on the right foot, which gradually grew larger and also increased in number [ Figure 1]. The skin lesions were itchy papules and nodules on the right leg and foot, accompanied by right leg edema and foot dis gurement. The patient was hemodynamically stable and complete blood counts, liver function tests, renal function tests, and comprehensive metabolic panels were unremarkable. Before the admission, skin lesion biopsies had been performed several times during the last years. The histopathologic examination showed hyperkeratosis, parakeratosis, acanthosis, and mild spongiosis of the epidermis. Dermis revealed a large area of necrosis centered on the deep dermis including thickened and hyalinized collagen bers and some vessels. The necrotic area was surrounded by a granulomatous in ammation containing aggregates of histiocytes, Langhans type multinucleated giant cells, numerous lymphocytes, and plasma cells. Periodic acid Schiff (PAS), Ziehl Neelsen and modi ed acid fast staining were all negative. These ndings were consistent with necrotizing granulomatous in ammation [ Figure 2]. No microorganisms were detected and the patient had no documented evidence of tuberculosis or sarcoidosis. Differential diagnoses included deep mycosis, mycobacterial infections, and also mycetoma. The result of the polymerase chain reaction (PCR) of the MTB complex was negative. In addition, mycological and mycobacterial cultures were negative.
Six months, and then eight months of empirical anti-TB treatment were ineffective. After being diagnosed with sarcoidosis, she was treated with corticosteroids and methotrexate, but the lesions did not improve. Two years ago, the patient was diagnosed with probable deep mycosis and was given itraconazole and also a course of voriconazole which were not bene cial. The skin lesions continued to exacerbate periodically without responding to other treatments.
Based on history exacerbated numerous skin lesions on the right leg and foot accompanied by dis gurement, actinomycetoma, or eumycetoma was probably diagnosed for the patient. The empirical treatment consisted of amphotericin B deoxycholate, clindamycin, and co-trimoxazole were initiated. The patient had no respiratory signs and symptoms. Computed tomography of the chest revealed normal ndings. Magnetic resonance imaging of the right foot was also normal with no evidence for osteomyelitis. After a few days, the exacerbating episode was diminished modestly with antibacterial therapy [ Figure 3].
In connection with the long-lasting and non-healing chronic necrotizing granulomatous skin lesions over several years, possible underlying immunode ciency was considered. Therefore, the immunological workups including ow-cytometry and also WES were performed. The patient was discharged 16 days after admission, taking co-trimoxazole double-strength tablets every 8 hours, clindamycin 600 mg every 8 hours, penicillin v every 6 hours, and terbina ne 500 mg every 12 hours. Laboratory data are shown (Table 1-4).      Figure 4, 5].
Parents and the patient family members Patient I.4, index patient's second cousin, was a 61-year-old patient who has been developing hypopigmented ulcerative patches and plaques with itching, erythema, and severe edema involving lower limbs for 33 years ago with nodules around the nose [ Figure 6]. Microscopic examination of the skin biopsies revealed hyperkeratosis, parakeratosis, spongiosis, and acanthosis of the epidermis. The dermis showed a dense in ltration of mixed in ammatory cells, including some multinucleated giant cells surrounding a large central area of necrosis. These ndings were compatible with the necrotizing granulomatous reaction. Ziehl-Nielsen staining for acid-fast bacilli and PAS staining were negative. MTB PCR results were also found negative. Six months of empirical anti-tuberculosis treatment did not show positive results. Since TAP2 de ciency was proven for the index patient, this patient was also suspected of having MHC class I de ciency. WES test indicated the same homozygous mutation in the TAP2 gene. Hence, the patient had the same disorder as the index patient.
Patient II.2 had three brothers and three sisters. Sequence analyzes of brothers, including case II.4 (40 years old) and II.8 (28 years old) revealed a homogeneous mutation of TAP2 de ciency. Both cases were asymptomatic, except for a history of recurrent minor aphthous oral ulcers. Case II.6 (33 years old), one of the sisters of the index patient, was also found to have homozygous TAP2 de ciency but was completely asymptomatic. Case II.7 (31 years old), another sister of the index patient, was heterozygous for this mutation with no symptom. Case II.3 and II.5, sister and brother of patient II.2, represented the wild type of TAP2. Case I.2 (68 years old) and I.3 (60 years old), the father and mother of patient II.2, were heterozygous for this mutation and were also asymptomatic.

Discussion
Bare lymphocyte syndrome is an entity due to mutations in genes of human leukocyte antigen. To date, few patients with reduced cell surface expression of MHC-I molecules have been reported. On the other hand, MHC-II de ciency is also a rare autosomal recessive combined immunode ciency which affects thymic epithelium and also both marrow-derived cells, leading to impaired antigen presentation, defective maturation and activation of CD4+ lymphocyte. Patients are susceptible to multiple severe infections including bacteria, fungi and also viruses and death in early childhood is common [7]. In contrast to severe MHC-II de ciency, these patients have characteristic clinical manifestations that are not usually life-threatening [8]. MHC-I de ciency is not often diagnosed because it is scarce and can remain asymptomatic for decades [9]. MHC-I molecules appear at variable levels on the surface of all nucleated cells in the body. Besides the role of presenting intracellular antigenic peptides to cytotoxic T lymphocytes, these molecules also have a signi cant role in modulating the activity of cells carrying MHC-I binding receptors, including natural killer (NK) cells and T cells [10,11]. Hence, malignant or virusinfected cells that cannot express HLA class I molecules are killed by NK and T cells. It has also been suggested that diminished levels of HLA class I molecules in TAP-de cient patients lead to the attacking of self-cells by NK cells, a phenomenon called missing self-recognition [12]. Indeed, it seems that neutrophils are the main culprit in the pathogenesis of TAP de ciency due to chemo-attraction or activation resulting from ine cient pathogen clearance. In some cases, chronic cutaneous granulomatous lesions involving the skin have been described. No speci c pathogens have been identi ed in these lesions. Although histopathology of sinuses and skin lesions may reveal necrotizing granulomatous in ammation, but this nding had not been identi ed in the lungs of TAP de cient patients [4].
Among patients with MHC type 1 de ciency, recurrent bacterial infections of the upper respiratory tract including sinusitis or otitis media, also nasal polyps have been reported. The infection may extend to the lower respiratory tract and cause bronchitis, pneumonia, and bronchiectasis. Haemophilus in uenza, Streptococcus pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa are the most common organisms developing the infection [13,14].
Cutaneous manifestations are necrotizing granulomatous lesions from a pustule or subcutaneous nodule, with progressive extension and also ulceration in an asymmetrical distribution over the legs, or hands, the former being more common. Mycobacteria, fungi, or other pathogens have never been identi ed in these lesions [4].
Infectious complications in the central nervous system can be misdiagnosed with granulomatosis with polyangiitis (formerly Wegener's granulomatosis) and make a challenging issue. Discrimination of this entity from other disease conditions including chronic granulomatous disease, common variable immunode ciency, granulomatosis with polyangiitis, sarcoidosis, mycobacterial infections, and cystic brosis is necessary [4].
Mutations in the TAP-2 subunit are associated with failure of MHC-I expression on cells leading to the abnormal selection of CD8+ T cells in the thymus. Decreased number of alpha/beta TCR-positive CD8 T cells were observed in patients with TAP de ciency. On the ip side, the patients represent a higher proportion of gamma/ delta-positive CD8+ T cells. Despite normal levels of NK cells, including CD3-CD56/CD16+ and also CD3-CD8+ NK cells in the patients, their function is abnormal [17]. However, ow cytometry is an important diagnostic tool for HLA class II de ciency. The absence of cellular and humoral immune responses to foreign antigens and inactivation of T cells are the most signi cant immunological feature of BLS type 2. In this disorder, although the total number of circulating T and B lymphocytes is normal, a decrease in CD4+ T lymphocytes is noted, which varies from patient to patient [18]. The complete absence of HLA-DR expression on B cells and monocytes is diagnostic [19].
Due to the low number of patients, there is no strong evidence of treatment. Bone marrow transplantation and also gene therapy have been treatment options for HLA II de ciency, but therapeutic options for HLA I de ciency are principally based on the prevention of infections [20]. The treatment includes antibiotic therapy for respiratory infections with or without chest physiotherapy. However, in addition to antibiotics, intravenous immunoglobulin may be useful in some patients [4]. For chronic sinusitis, sinus surgery is not recommended as it can exacerbate the condition and in cases of cutaneous lesions, local therapy can be helpful [4]. In a study by Law-Ping-Man et al. treatment with long-term chloroquine was used as an anti-in ammatory and also immunomodulatory agent (3.5 mg/kg/d), leading to persistence of the plaques without progression. Immunomodulatory treatment with interferon and immunosuppressive therapy, consisting of corticosteroids in combination with other agents such as azathioprine, cyclophosphamide, methotrexate, or cyclosporine is not recommended due to exacerbation of lesions and this type of therapy is contraindicated in TAP-de cient patients [4,7,21].
We presented two patients who developed chronic, slowly progressive granulomatous lesions one of them on the right lower limb and the other on the face and also lower limbs. The skin lesions of our patients were considered severe and not spontaneously healed. These two patients lack the other characteristic feature of this disorder, including recurrent bacterial infections of the respiratory tract with subsequent bronchiectasis. Our ndings showed that TAP-de cient patients represented immunity against infections. It has been shown that MHC-I expression mainly occurs using TAP-dependent pathways, but there are also TAP-independent pathways that enable patients to have su cient immunity against infections [22]. Figure 1 Papules and nodules on the right leg and foot with leg edema and also foot dis gurement.

Figure 2
Histopathologic examination revealed ndings consistent with necrotizing granulomatous in ammation.

Figure 3
Improvement of exacerbating episode after antimicrobial therapy.  Pedigree of an Iranian family with TAP 2 de ciency.