Mutations in THAP11 cause an inborn error of cobalamin metabolism and developmental abnormalities

Abstract

CblX (MIM309541) is an X-linked recessive disorder characterized by defects in cobalamin (vitamin B12) metabolism and other developmental defects. Mutations in HCFC1, a transcriptional co-regulator which interacts with multiple transcription factors, have been associated with cblX. HCFC1 regulates cobalamin metabolism via the regulation of MMACHC expression through its interaction with THAP11, a THAP domain-containing transcription factor. The HCFC1/THAP11 complex potentially regulates genes involved in diverse cellular functions including cell cycle, proliferation, and transcription. Thus, it is likely that mutation of THAP11 also results in biochemical and other phenotypes similar to those observed in patients with cblX. We report a patient who presented with clinical and biochemical phenotypic features that overlap cblX, but who does not have any mutations in either MMACHC or HCFC1. We sequenced THAP11 by Sanger sequencing and discovered a potentially pathogenic, homozygous variant, c.240C > G (p.Phe80Leu). Functional analysis in the developing zebrafish embryo demonstrated that both THAP11 and HCFC1 regulate the proliferation and differentiation of neural precursors, suggesting important roles in normal brain development. The loss of THAP11 in zebrafish embryos results in craniofacial abnormalities including the complete loss of Meckel's cartilage, the ceratohyal, and all of the ceratobranchial cartilages. These data are consistent with our previous work that demonstrated a role for HCFC1 in vertebrate craniofacial development. High throughput RNA-sequencing analysis reveals several overlapping gene targets of HCFC1 and THAP11. Thus, both HCFC1 and THAP11 play important roles in the regulation of cobalamin metabolism as well as other pathways involved in early vertebrate development.

Figure 1

THAP11 mutation in an individual with a cblX-like disorder. (A) Chromatograph of Sanger sequencing shows a novel homozygous c.240C>G (p.Phe80Leu) missense mutation in the subject. (B) Protein domains of THAP11 identified using UniProt include THAP-type zinc-finger DNA-binding domain (THAP), Glutamine-rich domain (Gln) domain, Alanine-rich domain (Ala), HCFC1 binding motif (HBM), and a coiled-coil domain (CC). (C) Evolutionary conservation of Phe80 (highlighted in red) in THAP11 demonstrated using comparative analysis of orthologs from multiple species. Orthologs were identified by using BLASTP, and the alignments were performed by using ClustalW. Protein accession numbers are in parentheses.

Figure 2

Thap11 regulates craniofacial development in zebrafish. (A–D) Alcian Blue-Alizarin Red staining of 4 day post fertilization (dpf) zebrafish embryos with the following treatment; (A) non-injected, (B) thap11 morpholino (thap11 Mo)-injected, (C) injected with wild-type THAP11 mRNA or (D) co-injected with thap11 morpholino and wild-type THAP11 mRNA.

Figure 3

Thap11 regulates neural stem cell proliferation and differentiation. (A,B) 2 day post fertilization (dpf) zebrafish embryos analysed for anti-acetylated tubulin expression using antibody immunohistochemistry. Arrowhead indicates hypothalamus and asterisk labels the presumptive cerebellum. (A) Non-injected (NI), (B) thap11 morpholino (thap11 Mo). (C–H) Immunohistochemistry with anti-Sox2 (green) and anti-HuC/D (red) antibodies at 2 day post fertilization (dpf) from (C) non-injected, (D) thap11 morphants (thap11 Mo), (E) THAP11 mRNA injected (THAP11), (F) thap11 morphants co-injected with THAP11 mRNA, (G)THAP11 c. 240C>G mRNA injected, and (H) thap11 morphants co-injected with the THAP11 c240C>G. Arrowheads demonstrate regions of HuC/D expression and arrows indicate Sox2 expression. HuC/D expression is consistently observed in the optic tectum and tegmentum, regions indicated by the arrowheads in (C). A thin lining of neural precursors are present surrounding the ventricle region and indicated by the arrow. Anatomically comparable regions from serial sections are demonstrated in the treated groups. n = 9 per group. I. Bar graph showing the cell counts of Sox2+ cells in sections from C-H. NI= non-injected, MO = thap11 morpholino injected, THAP11 = wild type THAP11 mRNA injected, c240C>G = mutant THAP11 mRNA injected, MO + THAP11 = co-injected as in (F), MO + c240C>G = co-injected as in (H). The counts were carried out in 9 sections in each category (n = 9) and error bars are shown. Asterisks denote results that were statistically significant.

Figure 4

hcfc1b regulates neural stem cell differentiation. (A–F) Sox2 and HuC/D expression analysis using antibody immunohistochemistry in 2 days post fertilization (dpf) zebrafish embryos are shown. (A) non-injected embryos (NI), (B) embryos injected with HCFC1 mRNA, (C) hcfc1b morpholino-injected (hcfc1b Mo), (D) co-injected with hcfc1b Mo and wild-type HCFC1 mRNA, (E) co-injected with hcfc1b Mo and mutated HCFC1 c.217G>A (217), (F) co-injected with hcfc1b MO and mutated HCFC1 c.344C>T (344) mRNA. Arrowheads demonstrate regions of HuC/D expression and arrows indicate Sox2 expression. For A, B, C, and D, n = 12 per group. For E&F, n = 9 per group. G) Bar graph showing the cell counts of Sox2+ cells in sections from A-F. NI= non-injected, MO = hcfc1b morpholino injected, HCFC1 = wild type HCFC1 mRNA injected, MO + HCFC1 = co-injected as in (D), MO + c.217 = co-injected as in (E), MO + c.344 = co-injected as in (F). The counts were carried out in 9 sections in each category (n = 9) and error bars are shown. Asterisks denote results that were statistically significant. Cells were counted in 12 sections each of A–D, and 9 sections each of E–F. Error bars are shown * denotes significance relative non-injected control embryos and ** denotes significance relative to hcfc1b morphant embryos.

Figure 5

hcfc1b regulates neural stem cell proliferation. (A,B) Non-injected or hcfc1b morpholino (hcfc1b Mo) injected embryos were pulsed with 10mM 5-ethynyl-2’-deoxyuridine (EdU) and analysed using the EdU Click-It technology. (A’-B’). Higher magnification images of the area indicated by the arrowhead in A and B. n = 9 per group.

Figure 6

Expression of target genes in patient samples. Normalized expression of MMACHC, TMOD2, HCFC1, and THAP11 in control, cblX patients, and THAP11 patients are shown. The Reference includes average expression from 7 control individuals, cblX includes average expression from 12 patients with cblX, and Subject includes expression data from the single patient with the THAP11 c.240C > G mutation.