We studied two families (referred to as XLAAD-100 and -200) with a total of five affected males, one in XLAAD-100 and four in XLAAD-200 (Figure ). All affected children suffered from type 1 diabetes mellitus with onset in infancy (range 3 weeks to 11 months), chronic diarrhea, and allergic reactions especially to foods (Table ). Other family members, including obligate heterozygote female carriers of the XLAAD-200 kindred, were clinically unaffected. Laboratory studies revealed evidence of heightened allergic reactivity including eosinophilia and elevated IgE levels, as well as positive radioallergosorbent tests and positive immediate hypersensitivity responses by skin prick tests to food and other allergens. After in vitro stimulation of peripheral blood lymphocytes with mitogens, there was exaggerated expression of the Th2 cytokines IL-4, IL-5, IL-10, and IL-13 and decreased expression of the Th1 cytokine IFN-γ (Figure ). Both the clinical phenotype and the laboratory studies were consistent with dysregulated Th cell type 2 responses in XLAAD.
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 (more ...)
Clinical characteristics of XLAAD patients
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 (more ...)
The XLAAD susceptibility locus was mapped by screening the probands and their relatives with polymorphic markers from the X chromosomes. Two-point linkage analysis mapped the XLAAD locus to a 46 cM interval that is flanked by the polymorphic markers DXS1223 proximally and DXS6789 distally, with a lod score of 1.81 at DXS6810 (Figure ). This interval overlaps those previously defined in other XLAAD families. Importantly, the XLAAD interval overlaps with the critical interval of the Scurfy mouse gene, a murine model of dysregulated lymphocyte activation (10
exhibits several XLAAD-related features, including T-cell hyperactivation and enhanced Th2 cytokine production, cytopenia, eczema, and diarrhea (11
). This suggested that alterations in the human homologue of the mouse gene responsible for the Scurfy
mutation may also be responsible for XLAAD.
The critical region of Scurfy
has been localized to a <300 kb segment on chromosome Xp11.23–p11.22 (13
). Accordingly, coding sequences in the public domain that map to the syntenic human region were systematically screened for candidate XLAAD genes. Given evidence of enhanced Th2 lymphokine gene transcription, particular emphasis was placed on screening either established or candidate transcriptional regulators, the latter identified by BLAST search. JM2
, encoding a candidate transcription factor of previously unknown function, was identified in the syntenic human region of the Scurfy
critical interval. The JM2
open reading frame is long and is predicted to encode a 381-amino-acid-long protein that contains a fork head homology domain. This is a highly conserved DNA-binding domain that defines the HNF-3/fork head family of transcription factors and is characterized by distinct winged helix structure (15
) (Figure ). JM2 is distinguished from other fork head homology proteins by the location of the fork head homology domain at the carboxyl-terminus and by the additional presence of a leucine zipper (Zip) dimerization domain midway through the protein (Figure ). A putative nuclear localization signal is found at the carboxyl-terminus.
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 (more ...)
The critical role played by fork head homology and Zip domains in JM2 function was highlighted by the mutations found in the two XLAAD kindreds. Genomic DNA sequencing of the XLAAD-100 proband revealed the presence of an A→G substitution at position +4 of the 5′ donor splice junction of IVS9 (Figure a). This substitution was not present in the child’s two other siblings or his parents, including his mother, indicating that it arose de novo. It was also lacking in 100 X chromosomes screened for this mutation. Sequence analysis of RT-PCR–amplified JM2 mRNA transcripts revealed skipping of JM2 exon 9 in transcripts of the index case of family XLAAD-100 but not in unaffected family members or in unrelated controls (Figure , b and c). Exon 9 skipping results in a frame shift at codon 273 that gives rise to a premature stop signal at codon 286. This leads to the generation of a truncated JM2 protein that lacks the fork head homology domain (Figure d). When the RT-PCR was run at a less stringent annealing temperature (60°C instead of 65°C), a second minor RT-PCR product appeared. This product, which migrated slower than its wild-type counterpart, resulted from aberrant splicing at IVS9 5′ donor splice junction (data not shown). No wild-type product was detected under any of the RT-PCR conditions tried. These results confirmed the pathogenicity of IVS9 +4 A→G mutation due to its disruption of IVS9 5′ donor splice junction.
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 (more ...)
Analysis of the XLAAD-200 kindred revealed affected males to suffer from a 3-bp deletion in JM2 exon 7, resulting in an in-frame deletion of bp 600–602 of JM2 cDNA (Figure a). Mothers of affected males, as well as the grandmother, were heterozygous for the mutant allele, while unaffected family members and control subjects lacked this mutation. There was in XLAAD-200 an unexpectedly high incidence of fatal hydrops fetalis. In one tested baby (XLAAD-200-29), the JM2 gene was normal; the others (XLAAD-200-16, -17, -18) were not available for testing.
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 (more ...)
The mutant transcripts are predicted to encode a JM2 protein lacking glutamic acid 201 residue (ΔE201). This residue lies in the second of the three heptad repeats that constitute the JM2 Zip motif. The JM2 Zip motif is most closely related to the three-heptad Zip motif of N-myc, with virtual identity at the second heptad (17
) (Figure ). Previous studies have revealed an essential role for the N-myc Zip motif and its individual heptad repeats including the second heptad in N-myc homodimerization and in heterodimerization of N-myc with its partner protein Max (18
). Specifically, alteration of the N-myc second heptad by mutagenesis of the distal leucine residue to proline or by mutagenesis of the glutamic-lysine-glutamic (EKE) motif to alanine residues impairs homo/heterodimerization and homodimerization, respectively (19
). By analogy, it is speculated that the ΔE201 deletion may interfere with heterodimerization of JM2 with partner proteins and/or its homodimerization, resulting in failure of effector function.
Studies on the Scurfy mouse have confirmed that disease pathogenesis is mediated by CD4+
T helper cells (20
). The disease can be induced by transfer of Scurfy CD4+
T cells to normal hosts, and it is cured upon breeding of Scurfy mice into T cell–deficient mouse strains (20
). This and clinical observations in XLAAD patients of the beneficial effects of T-cell immunosuppressants point to a pivotal role for JM2
in maintaining T-cell tolerance and in regulating Th cell differentiation. The unusual association of XLAAD with type 1 diabetes mellitus and with intense allergic inflammation distinguishes JM2
from other genes whose dysfunction results in autoimmunity, including those encoding complement proteins and components of the Fas cell death pathway, and AIRE
, the defective gene in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) (23
). It will be important to determine the mechanism(s) by which JM2
regulates autoimmunity and Th cell differentiation, including its role in maintaining central versus peripheral tolerance and in regulating well-described transcriptional programs underlying Th cell lineage commitment (27
The high incidence of type 1 diabetes mellitus in XLAAD patients, which is 100% in affected males of several XLAAD families, indicates that JM2
deficiency functions as a highly penetrant single gene trigger of type 1 diabetes mellitus. Of note, there exists a male bias in the incidence of sporadic type 1 diabetes mellitus, estimated at a male-to-female ratio of 1.7, which has been linked to a susceptibility locus on Xp11 that includes JM2
). This suggests that mutations and/or polymorphisms in JM2
may more broadly contribute to the pathogenesis of sporadic type 1 diabetes mellitus. JM2
may also interact with other previously established susceptibility genes for type 1 diabetes mellitus such as HLA-DR genes, a premise suggested by the observation that male bias and linkage to Xp11 in sporadic type 1 diabetes mellitus is most prominent in HLA-DR3+
). These issues will need to be addressed in broad-based population studies on sporadic type 1 diabetes mellitus.