Mendelian Susceptibility to Mycobacterial Disease (MSMD): Clinical, Immunological, and Genetic Features of 22 Patients from 15 Moroccan Kindreds

The first molecular evidence of a monogenic predisposition to mycobacteria came from the study of Mendelian susceptibility to mycobacterial disease (MSMD). We aimed to study this Mendelian susceptibility to mycobacterial diseases in Moroccan kindreds through clinical, immunological, and genetic analysis. Patients presented with clinical features of MSMD were recruited into this study. We used whole blood samples from patients and age-matched healthy controls. To measure IL-12 and IFN-γ production, samples were activated by BCG plus recombinant human IFN-γ or recombinant human IL-12. Immunological assessments and genetic analysis were also done for patients and their relatives. Our study involved 22 cases from 15 unrelated Moroccan kindreds. The average age at diagnosis is 4 years. Fourteen patients (64%) were born to consanguineous parents. All patients were vaccinated with the BCG vaccine, and twelve of them (55%) developed locoregional or disseminated BCG infections. The other symptomatic patients had severe tuberculosis and/or recurrent salmonellosis. Genetic mutations were identified on the following genes: IL12RB1 in 8 patients, STAT1 in 7 patients; SPPL2A, IFNGR1, and TYK2 in two patients each; and TBX21 in one patient, with different modes of inheritance. All identified mutations/variants altered production or response to IFN-γ or both. Severe forms of tuberculosis and complications of BCG vaccination may imply a genetic predisposition present in the Moroccan population. In the presence of these infections, systematic genetic studies became necessary. BCG vaccination is contraindicated in MSMD patients and should be delayed in newborn siblings until the exclusion of a genetic predisposition to mycobacteria.


Introduction
Mendelian susceptibility to mycobacterial diseases (MSMD) is a rare group of inborn errors of immunity (IEI) characterized by selective susceptibility to infections caused by weakly virulent mycobacteria, such as Bacille Calmette-Guérin (BCG) vaccine and various environmental mycobacteria (EM), in otherwise healthy individuals [1]. This condition affects about 1/10,000 individuals worldwide [2][3][4]. Mycobacterial infectious diseases generally begin in childhood and have a wide range of clinical manifestations, from localized to disseminated, and acute to chronic infections, with one or more mycobacterial species that may or may not recur [3]. MSMD patients are also susceptible to the more virulent Mycobacterium tuberculosis [5], and about half of them develop non-typhoidal salmonellosis of varying severity [3]. In specific genetic disorders, patients also suffer from chronic mucocutaneous candidiasis (CMC), while in others, patients develop viral infections and, more rarely, parasitic infections [3,4,6].
To date, 35 genetic disorders caused by mutation of 19 genes (IFNGR1, IFNGR2, IFNG, IL12RB1, IL12RB2, IL23R, IL12B,  ISG15, USP18, ZNFX1, TBX21, STAT1, TYK2, IRF8, CYBB,  JAK1, RORC, NEMO, and SPPL2A) have been shown to cause isolated or syndromic MSMD [4,[7][8][9][10][11][12]. Patients with isolated MSMD are sensitive only to mycobacterial infections, whereas patients with syndromic MSMD suffer from a mycobacterial infection in the context of one or a few other diseases [4,13]. These disorders are defined based on the impact of the mutation (null or hypomorphic), the mode of inheritance (dominant or recessive, autosomal or X-linked), the protein expression (normal, low, or absent), and the function affected (e.g., phosphorylation, binding to DNA or both) [3,4,6]. Despite this clinical and genetic heterogeneity, almost all genetic etiologies of MSMD alter the interferon-gamma (IFN-γ)-mediated immunity, by impairing or abolishing IFN-γ production or the response to this cytokine or both [3,14]. It was proven that the human IFN-γ level is a quantitative trait that defines the outcome of mycobacterial infection [14]. Defects associated with a residual production or response to IFN-γ show incomplete penetrance [3][4][5][6]14]. The only known disorders to be fully penetrants in isolated MSMD patients are autosomal recessive (AR) complete IFN-γR1, IFN-γR2, and IFN-γ deficiencies [4,15]. AR complete IL-12Rβ1 deficiency is found in about 60% of diagnosed patients as the most common genetic cause of MSMD; it results in lack of expression or expression of non-functional IL-12Rβ1 [4,16]. Despite this progress, no genetic disorder has yet been identified for about half of all MSMD patients [4].
The study of patients with MSMD has greatly improved our understanding of the pathogenesis of mycobacterial diseases, allowing better therapeutic approaches and genetic counseling to the affected families. Patients with defects in IFN-γ production may benefit from treatment with recombinant human IFNγ, in addition to antibiotics. In contrast, hematopoietic stem cell transplantation (HSCT) is the only medical option to date for patients with completely defective responses to IFN-γ [3]. Besides, the BCG vaccine is contraindicated in patients with MSMD, as most patients are diagnosed after being vaccinated and developing BCG complications. In Morocco, the mandatory use of the BCG vaccine, the endemicity of tuberculosis (TB), and the high rate of consanguinity could be the triggering factors for MSMD in this population. The objective of this work is to investigate the clinical, immunological, and genetic features of Moroccan patients with MSMD.

Materials and Methods
Patients presented with clinical features of MSMD, including complicated local/regional (BCG-itis) or systemic, disseminated reactions (BCG-osis) to BCG vaccination, unusually severe, persistent, and/or recurrent infections with mycobacteria, and/or TB and/or salmonella and/or CMC, as well as genetically confirmed patients, were recruited into this study. Patients were vaccinated with BCG during the first month of their life according to the Moroccan national immunization plan [17]. Diagnosis of BCG-osis was done according to the European Society for Immunodeficiency (ESID) diagnostic criteria [18]. BCG-osis is confirmed in more than one remote site beyond the vaccination site and/or at least in one blood or bone marrow culture [19]. Local BCG-itis is defined as a local abscess at the injection site (≥ 10 mm × 10 mm) and/or severe BCG scar ulceration. Regional BCG-itis is the involvement of any regional lymph nodes or other regional lesions beyond the vaccination site, including axillary, supraclavicular, cervical, and ipsilateral lymph nodes [19,20]. Clinical CMC was followed according to the questionnaire used in the past [21]. Thus, we included the date and patient age at the time of the episode of CMC, sites involved (nails, skin, oral mucosa, genital mucosa, others), clinical signs and duration before treatment, use of antibiotics during or before candidiasis, clinical features for each episode, antifungal treatment and duration, and time to clinical remission. Patients were diagnosed and followed up in the department of pediatric infectious and immunological diseases at Casablanca Children's Hospital. A patient was referred to the Neurosurgery Department of Mohammed V Hospital in Rabat. HIV-positive patients were excluded from the study. Written informed consent was obtained from the parents. The favorable opinion of the Ethics Committee of the Faculty of Medicine and Pharmacy of Casablanca is granted for this study. A questionnaire was conducted summarizing demographic information including age, sex, age at onset and diagnosis, treatments, consanguinity, and family history of similar diseases in relatives or ancestors.

Genomic DNA Extraction and Whole-Exome Sequencing (WES)
DNA was extracted from peripheral whole blood by the phenol-chloroform method or with the iPrep Pure Link gDNA Blood Kit and the iPrep Instruments (Life Technologies, Thermo Fisher Scientific). Genetic analyses were done for all patients and their family members when available using the WES for all index cases and Sanger sequencing for relatives or to confirm mutations. The WES method used has been described elsewhere [22,23]. The genetic mutations were confirmed by PCR amplification followed by Sanger sequencing. All calls with ≤ 4 × read coverage and SNP quality expressed in ≤ 30 scales have been filtered. The frequencies of the variants were filtered according to data from the variant exome server (http:// evs. gs. washi ngton. edu/ EVS/) and 1000 Genomes (http:// brows er. 1000g enomes. org/ index. html). All variants have been annotated with ANNOVAR21. All mutations found in MSMD patients were confirmed by Sanger sequencing on genomic DNA. Familial segregation was made if DNA from relatives was available.

Immunological Screening, Exploration of the IL-12/ IFN-γ Axis on Whole Blood
Immunological assays, including lymphocyte subsets (CD3+, CD4+, CD8+, CD16+, CD19+ cells), seric immunoglobulin (IgA, IgM, IgG, and IgE) levels, nitro blue tetrazolium chloride (NBT), and dihydrorhodamine (DHR) tests, were performed. Whole blood activation test was performed to assess both the production of IL-12 and IFN-γ and the response to these two cytokines [24]. Blood samples taken on heparin were placed in cell culture plates with the medium alone (RPMI 1640, PAA) or stimulated by BCG alone (M. bovis-BCG), BCG plus recombinant human IL-12 (20 ng/ml; R&D Systems), or BCG plus recombinant IFN-γ (5,000 IU/ml; Imukin, Boehringer Ingelheim). Stimulated and unstimulated blood samples were incubated for 48 h at 37 °C, in an incubator with an atmosphere containing 5% CO 2 . The blood samples were then collected and centrifuged at 1700 rpm for 10 min at 4 °C. The cell-culture supernatants were stored at − 80 °C.
The IFN-γ and IL-12p40 present in the supernatants were quantified by ELISA using the Human Quantikine IFN-γ and IL-12p40 kits, according to the manufacturer's recommendations (R&D Systems). The IL-12 response was considered normal if the amount of IFN-γ produced in response to stimulation with BCG + IL-12 was at least 10 times that produced in response to stimulation with BCG alone. The IFN-γ response was considered normal if the amount of IL12-p40 produced in response to stimulation with BCG + IFN-γ was at least twice that produced in response to stimulation with BCG alone [24]. IL-12p40 has been measured as a proxy for both IL-12 and IL-23 production. As positive controls for these assays, we used 29 unrelated healthy adult subjects (age range: 26-54 years) who had been vaccinated with BCG. A T-cell line of P8 was generated by infecting frozen peripheral blood mononuclear cells with Herpesvirus saimiri (T-saimiri cell line), and a secretion of IFN-γ after IL-12 stimulation was evaluated by ELISA as previously described [25,26]. All the experiments were performed in the same laboratory, and at least one healthy control was tested at the same time as the patient. Written informed consent was obtained from all participating healthy controls.

Demographic and Genetic Characteristics of 22 Patients from 15 Moroccan Kindreds
Our study involved 22 patients (11 females and 11 males) from 15 unrelated families from different regions of Morocco. The mean age of patients is 4 years (1-17 years). The median age at onset is 6 months, while the average age at diagnosis is 4 years. Fourteen patients (64%) were born to consanguineous parents. WES was performed for all patients, and Sanger sequencing was used to confirm the mutations in the patients and their relatives [27][28][29][30][31]. Eleven mutations have been identified in six genes: IL12RB1 in 8 patients (P1 -P8), STAT1 in 7 patients (P13-P19), SPPL2A in two patients (P11 and P12), IFNGR1 in two patients (P9, P10), TYK2 in two patients (P20, P21), and TBX21 in one patient (P15). Except for four heterozygous AD mutations identified in the STAT1 gene, all identified mutations (7/11) were homozygous with AR inheritance (Tables 1 and 2). Six patients from four unrelated families (P1-P6) had the same homozygous nonsense mutation c.913A>T (p.K305*), in the exon 9 of the IL12RB1 gene. Another homozygous nonsense mutation c.631C>T (p.R211*) in the exon 7 and a frameshift deletion mutation c.315del in the exon 4 of the IL12RB1 gene were found in two patients (P7 and P8, respectively). This frameshift mutation has not been previously reported. One missense mutation c.295T>C (p.W99R) in the exons 4 and one frameshift deletion c.131delC (p.P44fs*) in the exon 2 of IFNGR1 gene were identified in two patients (P9 and P10, respectively). Two patients (P11 and P12) are monozygotic twin sisters; they had the same essential splicing-site mutation in the intron 6 of SPPL2A. Four different monoallelic mutations of STAT1, c.1492C>G (p.L498V), c.2102A>G (p.Y701C), c.469G>A (p.E157K), and c.2120T>A (p.I707T) were identified in seven patients (P13-P19) from four unrelated families. These mutations are responsible for AD partial STAT1 deficiency. The p.Y701C mutation was not identified in the parents of patient P14, indicating that it is probably a de novo mutation. Two other brothers (P20 and P21) had a homozygous frameshift insertion c.3315_3316insC in the exon 23 of TYK2. Finally, a patient (P22) has a homozygous indel (insertion and/or deletion) mutation in exon 1 of the TBX21 gene (Table 1). Overall, 15 individuals with AR deficiency, including one asymptomatic homozygous sibling, and 7 individuals with AD deficiency, including two asymptomatic heterozygous members, were identified ( Fig. 1).

Immunological Data in Symptomatic MSMD Patients
NBT and/or DHR tests were performed for 12 symptomatic patients and showed normal phagocytic activity of neutrophils in all of them. Serum levels of immunoglobulins (IgA, IgM, IgG, and IgE) and lymphocyte subsets count (CD3+, CD4+, CD8+, CD16+, CD19+ cells) in samples from 14 symptomatic patients were without abnormalities and within the reference ranges for the majority of patients. Two patients (P7 and P20) had slightly low CD4+ cell counts. One patient (P17) had increased lymphocyte subsets count and elevated serum levels of all immunoglobulins. Nine other patients had elevated serum levels of IgG and IgM. Results were compared with the reference values using the PID Phenotypical Diagnosis app [32] and are presented in Table 3. As there were no obvious immunological abnormalities in the tests performed, we evaluated the functionality of the IL-12/IFN-γ axis by examining the levels of IL-12p40 and IFN-γ in the supernatant of whole blood samples after stimulation with BCG, with or without IFN-γ and IL-12. Cytokine evaluation was performed on samples from 12 patients and 29 healthy controls. Patients with AR complete IL-12Rβ1 deficiency (P1-P5, P8) showed no response to stimulation with IL-12, while patients with AR complete SPPL2A deficiency (P12, P13) and AD partial STAT1 defect (P14, P15) had normal responses to IL-12 in terms of IFN-γ production. The T-saimiri cell line of P8 does not secrete IFN-γ as described previously in other IL-12Rβ1 deficient patients [15,25,26]. Patients with AR complete TYK2 deficiency (P20, P21) showed a subnormal response to IL-12 in vitro ( Fig. 2A). As for the response to IFN-γ, patients with AD partial STAT1 deficiency showed an impaired but not abolished response to IFN-γ. The other patients showed a normal response to IFN-γ in terms of IL-12p40 production (Fig. 2B). In two patients with AR complete IFN-γR1 deficiency, a significant increase of IFN-γ in plasma (1123 pg/ml in P9 and 500 pg/ml in P10) was observed with no response to it in vitro.

Treatment and Survival of Patients
The management of mycobacterial infections in these patients consisted of antitubercular medications based on rifampin, isoniazid, pyrazinamide, and ethambutol combined with other antibiotics, including ciprofloxacin and clarithromycin, or antiviral, mainly ganciclovir and aciclovir. One patient (P2) also received recombinant IFN-γ treatment (50 µg/m 2 three times a week) after the failure of the initial treatment, with good clinical improvement. One patient (P10) with AR complete IFN-γR1 deficiency received immunoglobulin substitution in a short period that was prescribed during the episode of macrophage activation syndrome due to an uncontrolled acute mycobacterial disease. No patient underwent hematopoietic stem cell transplantation (HSCT). Five patients underwent different surgeries including intestinal segmental resection (P2), abscess drainage (P1, P13, P20), and tracheostomy (P7). The evolution of clinical symptoms was marked by the reduction in the frequency of repeated infections in ten cases of our cohort. Four patients died, P2 due to acute intestinal intussusception, P6 from severe pulmonary tuberculosis, and P13 and P20 from multifocal tuberculosis ( Table 2).  [4,10,11]. It has been noted that AR primary immunodeficiencies (PIDs) or IEIs are more common in populations with high rates of consanguinity, such as East Asian and North African, including the Moroccan population [33,34]. In our cohort, 14 patients (64%) were born to consanguineous parents. Biallelic mutations of IL12RB1 are the most common genetic cause of MSMD and are found in about 45-60% of diagnosed patients worldwide [3,4,35]. Likewise, AR complete IL-12Rβ1 deficiency was identified in 42% of symptomatic patients of this cohort. Six of them had the same homozygous mutation p.K305*, as the commonest mutation in MSMD patients from Morocco. The p.K305* mutation has been reported previously in two patients with BCG-osis and Salmonella infections. Both patients were born to consanguineous parents from Morocco [16,36]. The R211* mutation was reported in a Turkish patient with no complications from the BCG vaccine, but with recurrent salmonellosis, mycobacterial, and fungal infections, he also suffered from Henoch-Schonlein purpura in lower extremities and other autoimmune manifestations [37]. Similarly, P7, carrying the same mutation, had recurrent salmonella infections and autoimmune manifestations, including Henoch-Schonlein purpura and arthralgia of the lower extremities and hepatosplenomegaly. However, inflammatory and autoimmune manifestations have been reported in some MSMD cases, particularly in patients with AR complete IL-12Rß1 deficiency [38,39]. P8 carries a novel frameshift mutation c.315_316del in the IL12RB1 gene that has not been previously reported in any genetic database. All these three mutations lead to the appearance of a stop codon, resulting in a nonfunctional truncated protein that is not expressed on the cell's surface. This explains the inability to produce IFN-γ in response to stimulation with IL-12 in these patients. Although the IL-12Rβ1 chain is part of the IL-12 receptor as well as the IL-23 receptor, which also impairs IL-23-dependent IL-17 production resulting in CMC in some patients with complete IL-12Rβ1 deficiency, none of our patients was reported to have this infection. TYK2 is a Janus kinase (JAK) involved in various signaling pathways, including responses to IL-12, IL-23, IFNα/β, and IL-10. Once activated, TYK2 phosphorylates the intracellular part of the receptor and the recruited STATs [40]. Patients with TYK2 deficiency have mycobacterial and/ or viral diseases. Interestingly, only 30% of AR complete TYK2 deficient patients developed BCG disease after vaccination [12]. P20 did not develop any complications from the BCG vaccine or other infections until the age of 13 when she contracted M. tb and suffered from TB. Cells from TYK2deficient patients have impaired, but not abolished, responses to IL-12, IL-23, and IFN-α/IFN-β due to residual TYK2independent responses implying other molecules, such as other JAK kinases [31]. As expected, whole blood from P20 and P21 showed an impaired (subnormal) response to IL-12 in terms of IFN-γ production, probably accounting for the incomplete clinical penetrance to low virulent mycobacteria in these patients [31]. The impaired response to IFN-α/IFN-β underlies the viral infections in TYK2-deficient patients [31].

Discussion
STAT1 is a transcription factor involved in the response to type I (IFN-α/β), type II (IFN-γ), and type III (IFN-λ) IFNs [41,42]. AD STAT1 deficiency underlies relatively mild BCG and mycobacterial infections compared to AR STAT1 deficiencies, which lead to severe and potentially fatal mycobacterial and viral infections [42][43][44][45]. Multifocal osteomyelitis occurs frequently in patients with AD STAT1 deficiency [29,46]. We have identified four monoallelic mutations in five patients suffering from multifocal mycobacterial osteomyelitis and granulomatous tuberculoid dermatitis with osteoma, local BCG-itis associated with granulomatous tuberculoid dermatitis without caseous necrosis, and HSV skin infections, and from fatal multifocal TB (M. tb+). All mutants STAT1 were normally expressed but showed impaired phosphorylation (E157K) or DNAbinding (E157K and L498V) ability [29,47]. Y701C mutant abolished STAT1 phosphorylation and DNA-binding after IFN-γ stimulation in vitro [48]. I707T is a new mutation that is currently under functional characterization. These mutations resulted in impaired production of IL-12 upon stimulation with IFN-γ. The mutants exert a dominant negative effect over GAF (STAT1 homodimers) activity and a recessive effect on the activity of ISGF3 (heterodimer of STAT1/ STAT2/IRF9), explaining the occurrence of mycobacterial infections and the absence of viral diseases in these patients.
AR complete IFN-γR1 and IFN-γR2 deficiencies are responsible for the most severe forms of MSMD. Over 50% of patients die before the age of 10 years from severe and disseminated mycobacterial infection and/or life-threatening BCG infections [15,46,49]. Patients also suffer from EM, salmonellosis, and viral infections, associated with signs of macrophage activation in some patients [15]. Two rare homozygous mutations p.W99R and p.P44fs* of the IFNGR1 gene were detected in two patients (P9, P10) with severe adverse reactions to the BCG vaccine, CMV infection, and macrophage activation in the presence of splenomegaly and pancytopenia [30]. These mutations lead to the expression of a nonfunctional receptor, explaining the absence of IL-12p40 production in response to stimulation with IFN-γ in IFN-γR1 deficiency [24] and the high plasma levels of IFN-γ in these patients. Measurement of the IL-12p40 subunit does not allow a distinction between IL-12 and IL-23 production in response to IFN-γ, since they both share this subunit. Given the importance of IL-23 in antimycobacterial immunity as well as its involvement in inflammatory and autoimmune manifestations, measuring the production of each separately should be considered in future studies.
Complete AR SPPL2A deficiency was found in two monozygotic twin sisters from Morocco (P13, P12) and a patient from Turkey [28]. All three children had BCGosis and a decreased number of conventional dendritic cells type 2 (cDC2). The identified splice-site mutations lead to a loss of SPPL2a protein production (c.733+1G>A) or the production of a truncated protein (c.1328-1G>A) in an overexpression system [28]. SPPL2A is a transmembrane protease with multiple substrates, including the N-terminal fragment (NTF) of CD74 which is expressed by HLA class II+ antigen-presenting cells (APC) [50]. The selective depletion of cDC2 is probably due to the accumulation of the non-cleaved toxic CD74 N-terminal fragment in these cells [50]. Moreover, SPPL2a-deficient memory T H 1 cells showed impaired IFN-γ production in response to mycobacterial antigens in vitro [50].
T-bet or T-box protein 21 (TBX21) is a transcription factor that governs the development or function of several IFN-γ-producing lymphocytes. This new genetic etiology of MSMD was described for the first time in P22, who suffered from BCG-osis and persistent upper respiratory hyperresponsiveness [8]. T-bet-deficient mice are highly vulnerable to mycobacteria. Thus, mycobacterial disease and T-bet deficiency in this patient are consistent with the data in mice. The patient expressed normal amounts of TBX21 RNA, but protein expression was diminished [8]. Cellular immunophenotyping showed a strong diminution of circulating NK, invariant NKT, and Th1 cells in vivo, which are potent IFN-γ producing cells [8]. The discovery of inherited T-bet deficiency provides a unique opportunity to analyze the role of this transcription factor in various human leukocyte subsets, especially in Th cells.
These results confirm that the integrity of IFN-γmediated immunity is required for host defense against mycobacterial infection. In response to activation signals induced by pattern recognition receptors (PRRs), IL-12, IL-23, and ISG15 are secreted by dendritic cells, macrophages, and neutrophils. These cytokines bind to their receptors (IL-12Rβ1/2, IL-23R, and ISG15R (LFA-1)) on T-helper and NK cells, inducing the production of IFN-γ, IL-17, and TNF and promoting proliferation and differentiation of naïve T-helper cells into antigen-specific Th1 cells. As well, secreted IFN-γ binds to its receptor (IFN-γR) on the surface of macrophages and dendritic cells enhancing the production of IL-12 and their ability to eliminate intracellular microorganisms, such as Mycobacteria and Salmonella sp. (Fig. 2C).

Conclusion
This work shows that severe forms of TB and complications related to BCG vaccines may be related to a genetic predisposition present in the Moroccan population. Therefore, early diagnosis and genotyping of suspected patients and relatives are crucial. In our cohort, early-diagnosed patients had good recovery with no subsequent unusual infections, regardless of the genetic defect. Biallelic mutations of IL12RB1 were the most prevalent among Moroccan MSMD patients. If it is therefore necessary to enlarge the cohort, a systematic search for a genetic predisposition to mycobacteria is essential in presence of any severe or multifocal form of M. tb infection or complications of the BCG vaccine. We also propose to delay BCG vaccination from the neonatal period in siblings of affected families until the assertion of a genetic predisposition to Mycobacteria. Because genetic studies require more time and cost, functional tests, such as IFN-γ release and cytokine assays, can be targeted tools for early diagnosis of inborn errors of immunity, as despite their genetic and clinical heterogeneity, a large proportion of IEIs involves common and/or overlapping physiological phenotypes. and the French National Research Agency (ANR) under the "Investments for the future" program (grant number ANR-10-IAHU-01), ANR-GENMSMD/ANR-16-CE17-0005-01 (for JB), ANRS project ECTZ170784-ANRS0073 to SBD, and the SCOR Corporate Foundation for Science.
Data Availability All data are included in the manuscript.

Declarations
Ethics Approval The study was approved by the ethics committee of the Faculty of Medicine and Pharmacy Casablanca of Hassan II University.

Consent to Participate
Written informed consent was obtained from the guardians of the pediatric patients or directly from adult relatives and controls.

Consent for Publication
The subject has given consent for findings based on his samples and history to be published.

Competing Interests
The authors declare no competing interests.