Cultured Thymus Tissue Implantation for Congenital Athymia Post FDA Approval: Auto-Immunity and Novel Genetic Mutations

Introduction

Congenital athymia is an ultra-rare cellular immune deficiency characterized by absent T cell thymic emigrants, significant T cell dysfunction, subsequent risk for infection, autoimmunity and malignancy often resulting in early mortality. Cultured thymus tissue implantation (CTTI) is a curative option for athymic infants, a technique pioneered at Duke University, currently only available at Duke or Great Ormond Street Hospital. Etiologies of congenital athymia include DiGeorge syndrome, CHARGE syndrome, FOXN1 deficiency and diabetic embryopathy. Affected infants display absent T cell receptor excision circles (TREC) on newborn screen with persistent T cell lymphopenia specifically with low/absent naïve T cells. Presentations include a typical phenotype (TP) defined as low naïve T cells with absent T cell function based on mitogen proliferation or an atypical phenotype (AP) with low naïve T cells, expanded memory T cells and normal proliferation to mitogens. An AP may have signs of autologous GVHD (auGVHD), such as rash, lymphadenopathy, eosinophilia. Aim of this study was to depict patients treated at Duke since the US Food and Drug Administration approval in 2021.

Methods

This retrospective study was approved by IRB and includes all patients who underwent CTTI from March 2022 to July 2024. All patients were identified with abnormal TRECs on newborn screening and underwent evaluation with complete and differential blood counts, extended lymphocyte phenotyping by flow cytometry and genetic studies to confirm absence of SCID gene mutations. Physical exam with or without imaging established associated syndromic features. T cell function using ex vivo mitogen stimulation of blood mononuclear cells with phytohemagglutinin (PHA) was performed. TP was defined as circulating naïve T cells (CD3+CD45RA+CD62L) <50/mm³ or <5% of the total T cell count within 3 months of CTTI, with a PHA response < 30% of the lower limit of normal for the laboratory and no autoimmunity history. AP was defined as >100/mm³ total T cells but naïve T cells (CD45RA+CD62L+CD3+) <50/mm³ or <5% of the total T cell count with a PHA response >30% of the lower limit of normal for the laboratory, with or without autoimmunity. Patients received immunosuppression based on disease phenotype and PHA response. Descriptive statistics were used.

Results

In this cohort 34 patients (67.5% male) received CTTI at a median age of 31.5 (range 8-66) months.

Common syndromic conditions included: Complete DiGeorge syndrome (47%), CHARGE syndrome (26.5%), diabetic embryopathy (11.7%). Identified genetic mutations included: 22q11.2 deletion (47%), CHD7 mutation (14.7%), FOXI3 mutation (2.9%), TBX1 mutation (2.9%), and PAX2 mutation (2.9%). No genetic mutation was identified in 4 patients with CHARGE syndrome.

Novel mutations putatively associated with athymia were observed in 2 patients with AP:

• EXTL3 compound heterozygous mutation (c.1537 C>G p.R513G) and (c2305 G>C p.V769L) with facial dysmorphism, global hypotonia, cleft palate, atrial septal defect, and dilated aortic root
• TP63 de novo mutation (c.1040G>A, p.Cys347Tyr) with bilateral corneal clouding, facial dysmorphism, cleft palate, chronic otorrhea, hearing loss, and anorectal malformation

AP was present in 82.4% of our cohort, the majority (64.2%) with >1 symptom of auGVHD, such as rash (60.7%), eosinophilia (35.7%), lymphadenopathy (17.8%), diarrhea (7.1%), hemolytic anemia (3.5%), transaminitis (3.5%) and alopecia (3.5%). Oligoclonal T cell expansion was identified in 42.9% of AP patients. TP was present in 17.6% of patients. Maternal GVHD without symptoms was observed in 1 patient with TP based on chimerism. Eosinophilia rash, and oligoclonal T cell expansion was observed in 1 patient with TP (classified as TP due to low PHA response). Median age at CTTI of AP patients was 35.5 (range 8-66) months versus 17.5 (range 8-24) months for TP patients (p-value < 0.01).

Conclusion:

Two novel putative mutations of EXTL3 compound heterozygous (c.1537 C>G p.R513G) and (c2305 G>C p.V769L) and TP63 (c.1040G>A, p.Cys347Tyr) associated with athymia were identified in the cohort. Infants with AP were older, more likely to have auGVHD, with a relatively high incidence of oligoclonal T cell expansion. Early identification of athymic infants based on initial newborn screening is necessary to prevent complications and optimize outcomes.

SA & FC, JS & KM have equal contributions.