Demographic and genetic characteristic 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.
Sanger sequencing or WES were used in all patients to detect genetic etiologies [23-27]. 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 (Table 1).
Six patients from four unrelated families (P1–P6) had the same homozygous nonsense mutation c.913A>T (p.K305*), in the exon 9 of 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. Two homozygous missense mutations c.295T>C (p.W99R) and c.131delC (p.Y269fs*) in the exons 4 and 2 of IFNGR1 gene were identified in two patients (P9 and P10). 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. The p.Y701C mutation was not identified in the parents of patient P14, indicating that it is a de novo mutation. Another dizygotic twin patients (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 the exon 1 of 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).
Clinical diseases in 19 symptomatic patients
Axillary, cervical, supraclavicular and/or deep abdominal lymphadenopathy with or without fistula has been observed in all symptomatic patients except P6, P14, P15 and P16. All patients were vaccinated with BCG vaccine according to the Moroccan national immunization plan. Twelve of them (55%) developed different infections with M. bovis BCG vaccine strain. Loco-regional BCG infections or BCG-itis were diagnosed in eight patients (P1, P2, P3, P4, P8, P11, P12, and P17). Disseminated BCG infection or BCG-osis was diagnosed in three cases (P8, P15 and P22) (Table 2). Local BCG-itis was observed in one patient (P14), who presented with tuberculoid granulomatous dermatitis without necrosis, eczematiform lesions and herpes simplex virus (HSV) infection. The other six symptomatic patients (P6, P7, P13, P16, P20 and P21) did not develop BCG infections. P6 had severe pulmonary tuberculosis at the age of 12 years. P13 presented with pulmonary and cerebral tuberculosis at the age of 13 years. P16 had cutaneous tuberculosis. P20 presented with pulmonary tuberculosis and meningitis with deep abdominal lymphadenopathy and psoas abscess (M. tb+) and her brother P21 suffered from meningitis of unknown origin, recurrent otitis and urinary tract infections (Table 2). Three individuals, including two with AD STAT1 deficiency (P18 and P19) and one with IL-12Rβ1 deficiency (P5), were asymptomatic for infectious disease including mycobacterial infections.
Three patients developed disseminated salmonella infections (P2, P3, P7), which was the only infectious disease in P7. This patient had Henoch-Schonlein purpura, arthralgia, laryngeal granuloma, hepato-splenomegaly and inflammatory chest swelling in the context of recurrent infections with Salmonella enteritidis. Three cases (P2, P7, P8) had dysenteric syndrome caused by Salmonella enteritidis.
Viral infections were observed in four patients, including cytomegalovirus (CMV) infection in three patients with mild (P9, P22) and severe (P10) CMV viremia (PCR CMV at 6760 IU/ml 15,829 IU/ml and 468,848 IU/ml, respectively). Patient P14 had herpes simplex virus (HSV) infection of the skin. Three patients (P1, P2 and P10) had signs of sepsis, with positive blood culture for Salmonella enteritidis (P2) and Enterobacter cloacae (P10) (Table 2).
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. One patient (P17) had increased lymphocyte subsets count and elevated serum level 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 [28] and are presented in Table 3.
As there were no obvious immunological abnormalities in the tests performed, we evaluated the functionality of the IL12/IFNγ axis by examining the levels of IL-12p70 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 (Figure 2). 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. Patients with AR complete TYK2 deficiency (P20, P21) showed a subnormal response to IL-12 in vitro. 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 this cytokine (Figure 2). In two patients with AR complete IFN-γR1 deficiency (P9 and P10) a significant increase of IFN-γ on plasma was observed with no response to it in vitro (data not shown).
Treatment and survival of patients
The management of mycobacterial infections in these patients consisted of anti-tubercular medications based on rifampin, isoniazid, pyrazinamide, and ethambutol combined with other antibiotic, including ciprofloxacin and clarithromycin, or antiviral, mainly ganciclovir and aciclovir. One patient (P2) also received recombinant IFNγ treatment (50 mg/kg three times a week) after failure of initial treatment, with good clinical improvement. One patient (P10) with complete IFNGR1 deficiency received immunoglobulin substitution. No patient underwent hematopoietic stem cell transplantation (HSCT). Five patients underwent different surgeries including intestinal segmental resection (P2), abscess drainage (P1, P13, P20), 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 several salmonella enteritidis infections, P6 from severe pulmonary tuberculosis, P13 and P20 from multifocal tuberculosis (Table 2).