Second Report of Chronic Granulomatous Disease in Jordan: Clinical and Genetic Description of 31 Patients From 21 Different Families, Including Families From Lybia and Iraq

Abstract

Chronic granulomatous Disease (CGD) is a rare innate immunodeficiency disorder caused by mutations in one of the six genes (CYBA, CYBB, NCF1, NCF2, NCF4, and CYBC1/EROS) encoding the superoxide-producing nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase complex in phagocytes. In the Western population, the most prevalent form of CGD (about two-thirds of all cases) is the X-linked form (X-CGD) caused by mutations in CYBB. The autosomal recessive forms (AR-CGD), due to mutations in the other genes, collectively account for the remaining one-third of CGD cases. We investigated the clinical and molecular features of 22 Jordanian, 7 Libyan, and 2 Iraqi CGD patients from 21 different families. In addition, 11 sibling patients from these families were suspected to have been died from CGD as suggested by their familial and clinical history. All patients except 9 were children of consanguineous parents. Most of the patients suffered from AR-CGD, with mutations in CYBA, NCF1, and NCF2, encoding p22 ^phox , p47 ^phox , and p67 ^phox proteins, respectively. AR-CGD was the most frequent form, in Jordan probably because consanguineous marriages are common in this country. Only one patient from non-consanguineous parents suffered from an X91⁰ CGD subtype (0 indicates no protein expression). AR67⁰ CGD and AR22⁰ CGD appeared to be the most frequently found sub-types but also the most severe clinical forms compared to AR47⁰ CGD. As a geographical clustering of 11 patients from eight Jordanian families exhibited the c.1171_1175delAAGCT mutation in NCF2, segregation analysis with nine polymorphic markers overlapping NCF2 indicates that a common ancestor has arisen ~1,075 years ago.

Keywords: Jordan; NADPH oxidase; autosomal recessive; chronic granulomatous disease; founder mutation; innate immunodeficiency.

Figure 1

Pedigree of Jordanian, Lybian, and Iraqi families. CGD was diagnosed by measurement of NADPH oxidase activity in neutrophils as described in Patients and Methods. All families contained consanguineous marriages, except families R, Z, AB, AH, and AN. Diagonal shaded box: highly suspicious but functionally and genetically not tested (H1, R5, S8, Z6, Z4, AD1, AD3, AD4 and AD5, AI3 and AJ1). Hlty, healthy; NGT, not genetically tested; GT, genetically tested; Open symbols: no mutation in CGD genes among the tested members; filled symbols: homozygous for the CGD mutations; half-filled symbols: heterozygous for the mutation; cross-out symbols: deceased patients. (A) CGD patients from families H, M, N, O, P, Q, AK, AM, AN, and AD suffering from AR67⁰ CGD. (B) CGD patients from families S, W, Z, AH, AI, and AJ suffering from AR22⁰ CGD. (C) CGD patients from families T, Y, X, and AB suffering from AR47⁰ CGD. T and X are related families. These families were also related to family B (21). (D) CGD patients from family R suffering from X⁰ CGD.

Figure 2

Age at diagnosis of the 31 CGD patients from Jordan, Iraq, and Lybian according to the CGD types. Twenty-two Jordanian, seven Lybian, and two Iraqi CGD patients from 21 different families were investigated. Sixteen patients suffered from AR67⁰ CGD, eight patients from AR22⁰ CGD, six from AR47⁰CGD, and only one patient had X-CGD.

Figure 3

Phenotypic and genotypic characterization of the missense mutation c.252 G>T at the 3' end of exon 3 of CYBB in the X91⁰ CGD patient from family R. (A) NADPH oxidase activity measured by SOD sensitive-cytochrome c reduction with 5 × 10⁵ purified neutrophils stimulated by PMA (80 ng/mL). RB = mother, R9 = CGD patient. (B) Western-blot of p22^phox and gp91^phox analysis of soluble extracts (50 μg of protein in each lane) from purified neutrophils of Family R's members (RB mother, R9 CGD patient). The p67^phox expression was used to control the protein load. (C) Analysis of mutated p67^phox mRNA. (D) Analysis of mutated NCF2 gene. (E) Consequence on the sequence of gp91^phox protein. The absence of exon 3 corresponds to the amino acid deletion p.Ser48_Ala84del in the second transmembrane domain of gp91^phox with a probable structural disorganization of the protein.

Figure 4

Phenotypic and genotypic characterization of the new mutation of the AR67⁰CGD patient from family M. (A) NADPH oxidase activity measured by SOD sensitive-cytochrome c reduction with 5 × 10⁵ purified neutrophils stimulated by PMA (80 ng/mL). (B) Western blot of cytosolic factors p47^phox and p67^phox analysis of soluble extracts (50 μg of protein in each lane) from purified neutrophils of family M (MA father, MB mother, M2 CGD patient, M1 sister). The p47^phox expression was used to control the protein load. (C) Analysis of mutated p67^phox mRNA. (D) Analysis of mutated NCF2 gene. (E) Consequence on the sequence of p67^phox protein. The molecular consequence is an amino acid change p.ValValAla204-206AlaAlaGlu and p.Ser207-Glu226del in the activation domain of p67^phox protein that could be a potential interacting domain with NOX2.

Figure 5

Geographical clustering of the Jordanian AR67 families sharing the c.1171_1175delAAGCT mutation in NCF2. The ancestor origin of each family is marked on the Jordan and Palestine map by a red dot.