JM2, encoding a fork head-related protein, is mutated in X-linked autoimmunity-allergic disregulation syndrome

Abstract

X-linked autoimmunity-allergic disregulation syndrome (XLAAD) is an X-linked recessive immunological disorder characterized by multisystem autoimmunity, particularly early-onset type 1 diabetes mellitus, associated with manifestations of severe atopy including eczema, food allergy, and eosinophilic inflammation. Consistent with the allergic phenotype, analysis of two kindreds with XLAAD revealed marked skewing of patient T lymphocytes toward the Th2 phenotype. Using a positional-candidate approach, we have identified in both kindreds mutations in JM2, a gene on Xp11.23 that encodes a fork head domain-containing protein. One point mutation at a splice junction site results in transcripts that encode a truncated protein lacking the fork head homology domain. The other mutation involves an in-frame, 3-bp deletion that is predicted to impair the function of a leucine zipper dimerization domain. Our results point to a critical role for JM2 in self tolerance and Th cell differentiation.

Figure 1

Haplotype analysis of pedigrees XLAAD-100 and XLAAD-200. Circles, females; squares, males. Filled symbols, affected individuals; open symbols, unaffected individuals; forward slash through symbol, deceased individual. X chromosome haplotypes are schematically represented by side bars, while numbers next to the side bars represent the sizes (in bp) of alleles of markers listed in the inset. XLAAD-200-16, -17, -18, and -29 died of fetal hydrops. A question mark refers to deceased males whose genotype could not be determined. Haplotypes for X chromosome markers are shown. A crossover proximal to marker DXS1068 in individual XLAAD-200-4 defines DXS1223 as the proximal flanking marker for XLAAD, and a second recombination in XLAAD-200-27 distal to DXS6800 defines DXS6789 as the distal flanking marker for the XLAAD locus.

Figure 2

Enhanced Th2 cytokine expression in T lymphocytes of XLAAD patients. RNase protection assay analysis of cytokine gene expression in control and patient PHA-derived T-cell lymphoblasts is shown. Peripheral blood mononuclear cells of XLAAD-100 index case (patient 1) and XLAAD-200-27 (patient 2) and their respectively related healthy male controls were stimulated with PHA for 5 days. The resultant lymphoblasts were either left untreated (Medium) or activated with phorbol ester (PMA) and calcium ionophore (Iono) for 1 hour, as indicated. Total RNA was isolated and examined for cytokine expression by RNase protection assay using a commercially available kit (Pharmingen). L32 and GAPDH are transcripts of household genes that are used as internal controls. Results are representative of three different experiments.

Figure 3

Structural features of JM2 protein. (a) Domain organization. A leucine zipper (Zip) domain is present halfway through the protein at amino acids 189–210, while the fork head homology domain (FKH) extends from amino acids 287–373. (b) Homology of JM2 fork head domain sequence with fork head domains of other proteins including the Drosophila transcription factor Genesis and the human fork head related–proteins FKLH13, 7, 11, and 12. Red color indicates residue identity, while green indicates homology. Homology was established by searching the Conserved Domain Database (www.ncbi.nlm.nih.gov/Structure/), and alignment was accomplished using the Clustalw program (www.ebi.ac.uk/clustalw/). The organization of a canonical fork head domain is schematically illustrated. It includes three α helices (H1, 2, and 3), and a connecting loop (T′). Three β strands (S1, 2, and 3), and two wings (W1 and 2) arranged in order (H1-S1-H2-T′-H3-S2-W1-S3-W2) (15, 16).

Figure 4

Identification of a 5′ splice junction mutation in JM2 IVS9 of XLAAD-100 index case. (a) Analysis of JM2 IVS9 5′ splice junction site sequence in the index case and his sibling brother control. An Α→G transition at position +4 of the 5′ splice junction site is noted in the patient sequence (indicated by an arrowhead). (b and c) Exon 9 skipping in XLAAD-100 index case. (b) RT-PCR analysis of JM2 open reading frame 3′ end (bp 639–1036) in patient and control mRNA transcripts, revealing faster migration of patient RT-PCR product on agarose gel electrophoresis as compared with control product. MWM, molecular weight markers. (c) Sequence analysis of RT-PCR products shown in b, confirming the presence of exon 9 deletion and frame shift alteration in patient sequence. (d) Schematic representation of predicted mutant JM2 protein product in XLAAD-100 index case, showing truncation of the protein just before the fork head homology domain.

Figure 5

Identification of a 3-bp deletion in exon 7 of affected XLAAD-200 individuals. (a) Analysis of amplified genomic exon 7 sequence of XLAAD-200-28 and his sibling control XLAAD-200-29 showing deletion of bp 15–17 in the patient’s exon 7 sequence. The same mutation was confirmed by cDNA sequencing (data not shown). (b) Schematic representation of JM2 Zip domain and its homology to the Zip domain of human N-myc. The deleted glutamic residue (E), located in the second heptad, is boldface. A plus sign indicates homologous amino acid residues.