Multisystem Anomalies in Severe Combined Immunodeficiency with Mutant BCL11B

Abstract

Background: Severe combined immunodeficiency (SCID) is characterized by arrested T-lymphocyte production and by B-lymphocyte dysfunction, which result in life-threatening infections. Early diagnosis of SCID through population-based screening of newborns can aid clinical management and help improve outcomes; it also permits the identification of previously unknown factors that are essential for lymphocyte development in humans.

Methods: SCID was detected in a newborn before the onset of infections by means of screening of T-cell-receptor excision circles, a biomarker for thymic output. On confirmation of the condition, the affected infant was treated with allogeneic hematopoietic stem-cell transplantation. Exome sequencing in the patient and parents was followed by functional analysis of a prioritized candidate gene with the use of human hematopoietic stem cells and zebrafish embryos.

Results: The infant had "leaky" SCID (i.e., a form of SCID in which a minimal degree of immune function is preserved), as well as craniofacial and dermal abnormalities and the absence of a corpus callosum; his immune deficit was fully corrected by hematopoietic stem-cell transplantation. Exome sequencing revealed a heterozygous de novo missense mutation, p.N441K, in BCL11B. The resulting BCL11B protein had dominant negative activity, which abrogated the ability of wild-type BCL11B to bind DNA, thereby arresting development of the T-cell lineage and disrupting hematopoietic stem-cell migration; this revealed a previously unknown function of BCL11B. The patient's abnormalities, when recapitulated in bcl11ba-deficient zebrafish, were reversed by ectopic expression of functionally intact human BCL11B but not mutant human BCL11B.

Conclusions: Newborn screening facilitated the identification and treatment of a previously unknown cause of human SCID. Coupling exome sequencing with an evaluation of candidate genes in human hematopoietic stem cells and in zebrafish revealed that a constitutional BCL11B mutation caused human multisystem anomalies with SCID and also revealed a prethymic role for BCL11B in hematopoietic progenitors. (Funded by the National Institutes of Health and others.).

Figure 1. Clinical Findings

Panel A shows the features of the patient (shown with informed consent). The image on the left shows increased intraorbital distance, short palpebral fissures, abnormal nasal creases, and micrognathia; the image on the right shows ear tag, loose skin folds, and hirsutism. Panel B shows the percentage of peripheral-blood chimerism after infusions of hematopoietic stem cells (red arrows) from a matched unrelated donor. Chimerism has been stable for more than 2 years. Panel C shows T-cell–receptor diversity measured by spectratyping before and 6 months after hematopoietic stem-cell transplantation. Numbers in the upper left of each panel indicate the specific T-cell–receptor Vβ gene family analyzed.

Figure 2 (facing page). Role of BCL11B in Human Hematopoietic Stem Cells and Zebrafish Development

Panels A and B show lymphocyte frequency (left axis, solid lines) and BCL11B messenger RNA (mRNA) expression relative to that of housekeeping gene GAPDH (right axis, dashed lines) during in vitro differentiation of CD34+ human cord-blood cells cultured on the indicated OP9 monolayers. I bars indicate standard deviations. Panels C and D show differentiation of CD34+ human cord-blood cells transduced with green fluorescent protein (GFP) alone, mutant BCL11B–GFP, or BCL11B small interfering RNA (siRNA)–GFP lentivirus. Panel C shows the percentage of CD19+ B cells developing on OP9 monolayers, and Panel D shows the percentage of CD3+ T cells on OP9-DL1 monolayers. An asterisk indicates P<0.05. Panel E shows the effect of morpholino (bcl11ba-ATG-MO; see Fig. S4 in the Supplementary Appendix) knockdown of bcl11ba on T cell development at 5 days after fertilization in transgenic lck:GFP zebrafish embryos. Numbers in the lateral view are the fractions of embryos that have the depicted phenotype. Panel F shows the craniofacial cartilage of control versus bcl11ba morphant zebrafish embryos that were assessed at 5 days after fertilization by Alcian blue staining of craniofacial cartilage structures: AC denotes auditory capsular, CB ceratobranchial, CH ceratohyal, EP ethmoid plate, ME Meckel’s cartilage, and PQ palatoquadrate. Dorsal and lateral views are depicted. Panel G shows measurements of the distance between the eyes, and Panel H shows the distances depicted graphically as the mean for five embryos of each type; T bars indicate standard deviations.

Figure 3 (facing page). Dominant Negative Effect of Mutant BCL11B in Zebrafish and Jurkat T Cells

Panel A shows T-cell development in the zebrafish thymus at 5 days after fertilization after either human mutant BCL11B or mCherry as a control was overexpressed by injection of mRNA (25 pg) into transgenic lck:GFP embryos. Panels B and C show increased intraocular distance (arrows) and disruption of cartilaginous cranial structures 5 days after fertilization in zebrafish overexpressing human p.N441K BCL11B (as in Panel A). The intraocular distance increased from a mean of 1.0 mm to 2.5 mm (five embryos, P<0.001). Panel D shows the ability of intact but not mutant human BCL11B to rescue the arrest in T-cell development and correct the craniofacial abnormalities caused by bcl11ba knockdown. Rescue of T-cell development was evaluated after heat-inducible reexpression (30 hours after fertilization) and whole-mount in situ hybridization (WISH) analysis with an lck probe at 5 days after fertilization to identify thymocytes (within dashed red outlines in the lateral view). The restoration of craniofacial cartilage structure (red arrows) was assessed at 5 days after fertilization with the use of Alcian blue staining. Numbers indicate the proportions of embryos that had the depicted phenotypes. Panel E shows the production of interleukin-2 by Jurkat cells that were stimulated with phorbol myristate acetate (PMA) and ionomycin. The cells were transfected with a constant amount of vector DNA, with increasing proportions of p.N441K BCL11B, GFP vector, or BCL11B siRNA. I bars indicate standard deviations. Panel F shows chromatin immunoprecipitation (ChIP) after stimulation, with PMA and ionomycin, of Jurkat cells (left panel) transfected with FLAG-tagged wild-type or p.N441K BCL11B, and untransfected Jurkat cells (right panel). The fold change in DNA was calculated as the ratio of output DNA to input DNA in cells stimulated with PMA and ionomycin relative to the ratio in unstimulated cells. ChIP in untransfected cells was performed with anti-BCL11B antibody, as a control. Panel G shows the ability of BCL11B to heterodimerize, assessed by transfection of distinctly tagged wild-type (WT) and mutant (mut) BCL11B into Jurkat cells and coprecipitation with the indicated anti-tag antibodies. Extracts were produced from unstimulated Jurkat cells and from cells stimulated with PMA and ionomycin (PMA+Io). Panel H shows verification, by quantitative polymerase chain reaction (PCR), of BCL11B binding sites in genomic loci detected by ChIP-seq analysis of human cord-blood progenitors that were expanded in vitro. DNA bound by FLAG-tagged wild-type or mutant BCL11B was isolated by coprecipitation with anti-FLAG antibody. Fold changes were calculated as the relative quantity of the DNA binding site of interest (BCL11B_1, BCL11B_2, or TACC1), determined by quantitative PCR in ChIP-seq samples from human cord-blood progenitors transduced with FLAG-tagged wild-type BCL11B relative to that in samples transduced with FLAG-tagged mutant BCL11B. Two canonical BCL11B binding sites within the BCL11B locus were bound more extensively (one site 5 times more extensively and one site 10 times more extensively) than were the sites in the p.N441K mutant (left panel). Conversely, a novel TACC1 site (isoform 1, NM_006283.2) was found to have greater binding by the p.N441K mutant than by wild-type BCL11B.

Figure 4. Role of BCL11B in Migration of Hematopoietic Progenitor Cells in Zebrafish and Humans

Panel A shows c-myb WISH staining at 36 hours (1.5 days) after fertilization to examine the localization of hematopoietic stem and progenitor cells. The fractions indicate the numbers of zebrafish that had the depicted phenotype out of the total number examined. Displaced hematopoietic stem and progenitor cells are marked by black (control morpholino oligonucleotide [MO] injected) and red (bcl11ba-MO injected) arrows. Panel B shows the relative ccr9b expression, measured by quantitative PCR in control embryos and bcl11ba morphants 1.5 and 5 days after fertilization. Expression in triplicate samples was quantified, normalized to that of β-actin and depicted as mean fold change (the expression in experimental embryos relative to the expression in control-injected embryos, which was defined as 1); I bars indicate standard deviations. Panel C shows the effect of morpholino (ccr9b-ATG-MO) knockdown of ccr9b, either alone or in combination with bcl11ba-ATG-MO, on thymic seeding at 3.5 days after fertilization in transgenic cd41:GFP zebrafish embryos (red circles). Numbers are the fraction of embryos that had the depicted phenotype. Panel D shows the expression, normalized to that of housekeeping gene GAPDH, of BCL11B, CCR7, and CCR9 mRNA in human cord-blood CD34+ cells transduced with lentivirus encoding GFP alone or BCL11B siRNA–GFP. Panel E shows the in vitro transwell migration of human CD34+ cells, transduced as in Panel C, in response to CCL19 or CCL25 (100 ng per milliliter) or no chemokine.