PMC5389975_01
PMC5389975_02
Clinicopathological findings in the affected children. Case 2: (A) Presence of many intracellular, small, narrow base, budding, and darkly pigmented yeast-like cells (1-5 mm diameter) in the lymph node (Grocott stain, original magnification x 1,000). (B) Screenshots of video laryngoscopy showing bilateral granulomatous lesions of aryepiglottic folds. Case 3: (C) Frontal and lateral photographs of the left infracostal large abscess. Case 4: (D) Photograph of the pruritic, maculopapular erythematous rash in the back of the right hand. (E) Hyperkeratosis and regional atrophy associated with basal vacuolization and focal necrosis in addition to perivascular and interstitial inflammatory mixed infiltrate with mild nuclear fragmentation in the erythematous maculopapular rash on the left hand (hematoxylin-eosin stain, x 100). (F) Perivascular and interstitial inflammatory mixed infiltrates with neutrophils in karyorrhexis, lymphocyte, and eosinophils in the erythematous lesions of the left limb (hematoxylin-eosin stain, x 100).
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F1
He also evolved a pruritic, maculopapular erythematous rash in the neck and the back of both hands (Figure 1D), with a clinical diagnosis of pityriasis. Histopathology of an erythematous maculopapular rash on the left hand revealed hyperkeratosis and regional atrophy associated with basal vacuolization and focal necrosis in addition to perivascular and interstitial inflammatory mixed infiltrates with mild nuclear fragmentation (Figure 1E) and, thus, inconsistent with the diagnosis of pityriasis. Histopathology of the erythematous lesions of the left limb showed inflammatory mixed perivascular, and interstitial infiltrates with neutrophils in karyorrhexis, lymphocytes, and eosinophils (Figure 1F), confirming the diagnosis of leukocytoclastic vasculitis. Western European ancestry (CEU) and other persons of self-reported European ancestry from the SARP (Supplementary Figure S1). We also determined that Cases 2, 3, and 4 share the CACCAGTCCGG (forward strand) extended homozygous genotype, which spans 27.3 kb across the IL12RB1 gene (Supplementary Figure S2, Supplementary Table S1, and Supplementary data).
PMC5389975_03
Clinicopathological findings in the affected children. Case 2: (A) Presence of many intracellular, small, narrow base, budding, and darkly pigmented yeast-like cells (1-5 mm diameter) in the lymph node (Grocott stain, original magnification x 1,000). (B) Screenshots of video laryngoscopy showing bilateral granulomatous lesions of aryepiglottic folds. Case 3: (C) Frontal and lateral photographs of the left infracostal large abscess. Case 4: (D) Photograph of the pruritic, maculopapular erythematous rash in the back of the right hand. (E) Hyperkeratosis and regional atrophy associated with basal vacuolization and focal necrosis in addition to perivascular and interstitial inflammatory mixed infiltrate with mild nuclear fragmentation in the erythematous maculopapular rash on the left hand (hematoxylin-eosin stain, x 100). (F) Perivascular and interstitial inflammatory mixed infiltrates with neutrophils in karyorrhexis, lymphocyte, and eosinophils in the erythematous lesions of the left limb (hematoxylin-eosin stain, x 100).
fmicb-08-00616-g0001.jpg
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F1
He also evolved a pruritic, maculopapular erythematous rash in the neck and the back of both hands (Figure 1D), with a clinical diagnosis of pityriasis. Histopathology of an erythematous maculopapular rash on the left hand revealed hyperkeratosis and regional atrophy associated with basal vacuolization and focal necrosis in addition to perivascular and interstitial inflammatory mixed infiltrates with mild nuclear fragmentation (Figure 1E) and, thus, inconsistent with the diagnosis of pityriasis. Histopathology of the erythematous lesions of the left limb showed inflammatory mixed perivascular, and interstitial infiltrates with neutrophils in karyorrhexis, lymphocytes, and eosinophils (Figure 1F), confirming the diagnosis of leukocytoclastic vasculitis. Western European ancestry (CEU) and other persons of self-reported European ancestry from the SARP (Supplementary Figure S1). We also determined that Cases 2, 3, and 4 share the CACCAGTCCGG (forward strand) extended homozygous genotype, which spans 27.3 kb across the IL12RB1 gene (Supplementary Figure S2, Supplementary Table S1, and Supplementary data).
PMC5389975_04
Clinicopathological findings in the affected children. Case 2: (A) Presence of many intracellular, small, narrow base, budding, and darkly pigmented yeast-like cells (1-5 mm diameter) in the lymph node (Grocott stain, original magnification x 1,000). (B) Screenshots of video laryngoscopy showing bilateral granulomatous lesions of aryepiglottic folds. Case 3: (C) Frontal and lateral photographs of the left infracostal large abscess. Case 4: (D) Photograph of the pruritic, maculopapular erythematous rash in the back of the right hand. (E) Hyperkeratosis and regional atrophy associated with basal vacuolization and focal necrosis in addition to perivascular and interstitial inflammatory mixed infiltrate with mild nuclear fragmentation in the erythematous maculopapular rash on the left hand (hematoxylin-eosin stain, x 100). (F) Perivascular and interstitial inflammatory mixed infiltrates with neutrophils in karyorrhexis, lymphocyte, and eosinophils in the erythematous lesions of the left limb (hematoxylin-eosin stain, x 100).
fmicb-08-00616-g0001.jpg
PMC5389975_04_fmicb-08-00616-g0001.jpg
F1
He also evolved a pruritic, maculopapular erythematous rash in the neck and the back of both hands (Figure 1D), with a clinical diagnosis of pityriasis. Histopathology of an erythematous maculopapular rash on the left hand revealed hyperkeratosis and regional atrophy associated with basal vacuolization and focal necrosis in addition to perivascular and interstitial inflammatory mixed infiltrates with mild nuclear fragmentation (Figure 1E) and, thus, inconsistent with the diagnosis of pityriasis. Histopathology of the erythematous lesions of the left limb showed inflammatory mixed perivascular, and interstitial infiltrates with neutrophils in karyorrhexis, lymphocytes, and eosinophils (Figure 1F), confirming the diagnosis of leukocytoclastic vasculitis. Western European ancestry (CEU) and other persons of self-reported European ancestry from the SARP (Supplementary Figure S1). We also determined that Cases 2, 3, and 4 share the CACCAGTCCGG (forward strand) extended homozygous genotype, which spans 27.3 kb across the IL12RB1 gene (Supplementary Figure S2, Supplementary Table S1, and Supplementary data).
Electropherogram of the novel IL12RB1 predicted loss-of-function genotype confirmed by Sanger sequencing. The area shaded in pink indicates the homozygous Trp7Ter predicted loss-of-function genotype for the SNP variant rs150172855 (c.21G>A/p.Trp7Ter/TGG   TGA) identified in Cases 2, 3, and 4. As exemplified here with Case 1, the parents of all four cases were heterozygous carriers. The stop-gain mutation is located in exon 1 within the predicted coding region of the signal peptide. Shown are the sequencing traces for the forward strand of DNA.
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F2
We sequenced the exons of the IL12RB1 gene in Cases 2, 3, and 4 (no DNA sample was available from Case 1) and found all three to be homozygous for the autosomal recessive Trp7Ter predicted loss-of-function single nucleotide polymorphism (SNP) c.21G>A/p.Trp7Ter/TGG   TGA (Figure 2). We next sequenced the parents from the three unrelated kindreds and confirmed them all to be heterozygous for the stop-gain allele variant (Figure 2). Western European ancestry (CEU) and other persons of self-reported European ancestry from the SARP (Supplementary Figure S1). We also determined that Cases 2, 3, and 4 share the CACCAGTCCGG (forward strand) extended homozygous genotype, which spans 27.3 kb across the IL12RB1 gene (Supplementary Figure S2, Supplementary Table S1, and Supplementary data).
The Trp7Ter stop-gain mutation at the signal peptide sequence does not knock out cell-surface expression of the IL-12Rbeta1 polypeptide. Cell-surface expression of IL-12Rbeta1 protein antigen in peripheral blood mononuclear cells was ascertained by flow cytometry in a representative non-carrier control subject (A), and the heterozygous carrier mothers of case 2 (B) and case 4 (C) and the homozygous Trp7Ter predicted loss-of-function cases 2 (D), 3 (E), and 4 (F). The PHA-T-cell blasts were stained with phycoerythrin (PE)-labeled mouse 2.4E6 monoclonal antibody anti-Human CD212 (IL-12Rbeta1), or PE-labeled matched control isotype. The arrows point to the matched control isotype and CD212 specific staining intensities depicted as the shaded and open areas under the curves, respectively. In all subjects, stimulated cells exhibited increased fluorescence intensity, reflecting the presence of the CD212 specific IL-12Rbeta1 antigen at the cell surface, independently of the occurrence of the stop-gain mutation. For comparison, the expression mean percentage of positive cells in seven non-carrier controls was 88.2.
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F3
We showed ex vivo that the stop-gain mutation at the signal peptide coding sequence, paradoxically, does not knock out cell-surface expression of the IL-12Rbeta1 polypeptide (Figure 3). IL-12Rbeta1 protein antigen is present in the cell-surface of PHA-blasts from Case 2 (Figure 3D), Case 3 (Figure 3E), and Case 4 (Figure 3F). Importantly, the ex vivo and in vitro levels of the cell-surface expressed IL-12Rbeta1 polypeptide are diminished in these children, and the receptor activity is affected because the production of IFN-gamma in response to phytohemagglutinin (PHA; Figure 4A) and BCG lysate (Figure 4B) and of IL-17A in response to PHA (Figure 4C) and BCG (Figure 4D) stimuli is impaired vs. the control subjects. By imputation, the Trp7Ter stop-gain mutation creates either an exonic acceptor splice silencer (ESS) site, an acceptor splice site or a donor splice site [Supplementary Figure S3 with potentially deleterious effect(s) through RNA mis-splicing]. Whether the Trp7Ter stop-gain mutation is deleterious through exon 1 partial in-frame skipping in the IL12RB1 gene or there is transcriptional initiation from potentially alternative non-AUG start sites downstream of the annotated AUG (i.e., Val9, Leu16, or Leu17; Supplementary Figure S3) is still unclear.
The cell-surface-expressed IL-12Rbeta1 protein in the affected children is reduced and functionally impaired. Cytokine-specific Cytometric Bead Array analyses revealed reduced production of IFN-gamma in response to PHA (A) and BCG lysate (B) and of IL-17A in response to PHA (C) and BCG lysate (D) stimuli in cultured peripheral blood monocular cells from the subjects homozygous for the IL12RB1 Trp7Ter predicted loss-of-function mutation vs. the control subjects. For comparison, in five control non-carriers, the IFN-gamma mean expression values were 6,063 and 1,830 pg/mL upon stimulation with PHA or BCG lysate, respectively. For IL17-A cytokine, the mean expression values were 350.3 and 25.10 pg/mL under the same stimuli. Quantification assays were done on samples taken at least 4 months after remission (3 years for case 2, 4 months for case 3, and 2 years for case 4). Boxes, interquartile ranges; horizontal lines within boxes, median values.
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F4
IL-12Rbeta1 protein antigen is present in the cell-surface of PHA-blasts from Case 2 (Figure 3D), Case 3 (Figure 3E), and Case 4 (Figure 3F). Importantly, the ex vivo and in vitro levels of the cell-surface expressed IL-12Rbeta1 polypeptide are diminished in these children, and the receptor activity is affected because the production of IFN-gamma in response to phytohemagglutinin (PHA; Figure 4A) and BCG lysate (Figure 4B) and of IL-17A in response to PHA (Figure 4C) and BCG (Figure 4D) stimuli is impaired vs. the control subjects. Worth of note, PHA-blasts from Case 2 produced >4,000 pg/mL of IFN-gamma, which is close to the levels observed in the controls (Figure 4A), and contrasting to the situation in the younger affected brother (Case 3).