Expanding the clinical and immunological phenotypes of PAX1-deficient SCID and CID patients

Abstract

Paired box 1 (PAX1) deficiency has been reported in a small number of patients diagnosed with otofaciocervical syndrome type 2 (OFCS2). We described six new patients who demonstrated variable clinical penetrance. Reduced transcriptional activity of pathogenic variants confirmed partial or complete PAX1 deficiency. Thymic aplasia and hypoplasia were associated with impaired T cell immunity. Corrective treatment was required in 4/6 patients. Hematopoietic stem cell transplantation resulted in poor immune reconstitution with absent naïve T cells, contrasting with the superior recovery of T cell immunity after thymus transplantation. Normal ex vivo differentiation of PAX1-deficient CD34⁺ cells into mature T cells demonstrated the absence of a hematopoietic cell-intrinsic defect. New overlapping features with DiGeorge syndrome included primary hypoparathyroidism (n = 5) and congenital heart defects (n = 2), in line with PAX1 expression during early embryogenesis. Our results highlight new features of PAX1 deficiency, which are relevant to improving early diagnosis and identifying patients requiring corrective treatment.

Keywords: Hypoparathyroidism; Inborn errors of immunity; Otofaciocervical syndrome; PAX1; SCID; thymus.

Fig. 1

Family pedigrees of patients. Pedigrees and results of Sanger sequencing in patients with PAX1 variants. Double lines indicate consanguinity; filled black circles or squares depict the patients; half-filled black circles or squares depict the carrier state; diagonal lines indicate deceased individuals. Gray dashed circles or squares indicate individuals with ear abnormalities and hearing loss. Triangles indicate miscarriages. Males and females are distinguished by squares and circles, respectively.

Fig. 2

Variants in PAX1 deficiency are distributed throughout the gene. (A) (A) Schematic diagram of PAX1 protein domains with variants. Asterisk (*) indicates a premature stop codon. Previously reported mutations are shown by red color. (B) The PAX1 variants with allele frequencies and CADD scores (NR: Not-reported). (C) Analysis of the P162L variant located in the paired box domain of PAX1. a) Structural model of the paired box domain, highlighting the two helical bundles (II and III). The structure was colored according to the model quality score TopScore (scores close to 0 indicate a high-quality model and scores close to 1 a low-quality model). The position of the variant is highlighted with a gray sphere and a red arrowhead. b) Co-evolutionary network of P162 (shown in red). Co-evolving residues are shown in blue. The pair-wise co-evolutionary coupling scores are shown over yellow dashed lines (score of 1.0 means perfectly coupled, and 0 means not coupled). c) Residue distribution found at position 162 on a multiple sequence alignment of PAX1 homologs. The residues are grouped according to their physicochemical properties (P: proline (WT residue). R; K; H: positively charged. D, E: negatively charged. N, Q: long polar. S, T: short polar. A, V, G: small aliphatic. L, I, M: large aliphatic. Y, F: aromatic). (D) Luciferase assay showing reduced transcriptional activity of mutant PAX1 proteins, corresponding to the PAX1 variants detected in patients. The promoter region of Nkx3–2 was used to drive luciferase expression. Results of 3 independent experiments are presented (means ± SEM). P value was calculated with one-way ANOVA and adjusted by Bonferroni-Dunn multiple comparisons test. ***P < 0.001; ****P < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Clinical phenotypes of PAX1-deficient patients showing specific features. (A) The main manifestations of PAX1 deficiency. (B) Representative pictures of patients' phenotypes: (P1) Facial dysmorphisms (bilateral microtia, nasal root flattening, hypertelorism, epicanthus, and micrognathia), alopecia, and Omenn-like generalized rash (resolved with HSCT). (P2) Dysmorphisms characterized by low-set ears, short nose with low nasal bridge, hypertelorism, epicanthus, micrognathia, long philtrum, and thin upper lip. (P3) Micro- and retrognathia, maxillary hypoplasia, telecanthus, bilateral epicanthus, small and anteverted nostrils, microtia, low set ears, down slanting palpebral fissures, and thin upper lip. (P4) Facial dysmorphism including hypertelorism, flattened nasal root, epicanthus, flat philtrum, long eyelashes, right facial paralysis, bilateral preauricular cupping, microtia, short neck, and microdontia. (P5) Low-set ears with pre-auricular pits, overfolded ears, and a hypoplastic mandible with retrognathia. Flat nasal bridge with nevus flammeus (arrow) / asymmetric face. (C) Radiological findings of the patients. P1–1: Chest X-ray with no thymus shadow, butterfly (C4-T1) and split (T2-T4) vertebral bodies (black arrows), and hooked distal clavicles (white arrows). P1–2 and P1–3: Increased lumber dextroscoliosis centralized at the level of L1 over time (Cobb angles: 5.2^° (black) vs. 33.3^° (red)). P1–4: Thoracolumbar kyphosis. P2–1: A rudimentary thymus shadow on chest X-ray. P2–2: Hypoplastic vertebrae (L4-L5) on X-ray spin (white arrows). P2–2 and P2–3: Increased lumber levoscoliosis centralized at the level of L2 over time (Cobb angles (red lines): 1.8^°vs. 18.8^°). P3–1: Chest X-ray shows hooked distal clavicles (arrows) and multiple nodular opacities with pneumonic infiltration (arrow head). P3–2: Temporal bone computed tomography demonstrates bilateral external auditory canal atresia (white arrows). P3–3: C6 and C7 butterfly vertebrae on X-ray graph (white square). P4–1: Hooked distal clavicles (arrows). P4–2: Gibbus on chest X-ray graph (white arrows). P5–1: Aplastic or fully obstructed external auditory meatus, with no eardrum visible. Hypoplastic ossicles and increased sclerosis in the cochlear and semicircular canal region. P5–2 and P5–3: Hyperkyphosis and lumbar levoscoliosis. P6–1: Prominent mid-face with beak-like appearance. P6–2 and P6–3: Scoliosis on X-ray of the whole spine. HSCT: Hematopietic stem cell transplantation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Diminished T cells and defective proliferation in PAX1 deficiency. (A) A dimensional reduction analysis showing the comparison of T, B, and NK cells between patients (P) and healthy controls (HC). (B) A dimensional reduction analysis showing the comparison of T cell subpopulations between patients (P) and healthy controls (HC). (C) Flow cytometric analysis of percentages of CD69, CD25, and proliferation in CD4⁺ and CD8⁺ T cells of patients and healthy controls in unstimulated and stimulated (anti-CD3 and anti-CD28) conditions. CD4⁺ naive T cells (Tn, CD4⁺CD45RA⁺CCR7⁺), central memory CD4⁺ T cells (Tcm, CD4⁺CD45RA⁻CCR7⁺), effector memory CD4⁺ T cells (Tem, CD4⁺CD45RA⁻CCR7⁻), terminally differentiated effector memory CD4⁺ T cells (Tex, CD4⁺CD45RA⁺CCR7⁻), CD8⁺ naive T cells (Tn, CD8⁺CD45RA⁺CCR7⁺), central memory CD8⁺ T cells (Tcm, CD8⁺CD45RA⁻CCR7⁺), effector memory CD8⁺ T cells (Tem, CD8⁺CD45RA⁻CCR7⁻), terminally differentiated effector memory CD8⁺ T cells (Tex, CD8⁺CD45RA⁺CCR7⁻). CTV: Cell Trace Violet. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Skewed T cell receptor beta repertoire in PAX1 deficiency. (A) Individual V gene usage in patients compared to controls. Red bars represent the TRBV genes of the patients. Blue bars represent the TRBV genes of the healthy controls. Blue error bars indicate a significant difference in gene usage frequency compared to healthy controls (HCs). (B) Principle component analysis-based K-means clustering analysis of the V gene usage in the patients and healthy controls. V gene usage in CD4⁺ T cells of the patients and healthy controls. (C) Repertoire overlap analysis of clonotypes shared between patients and healthy controls. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)