EFMR (MIM 300088) is a condition characterized by seizure onset in infancy or early childhood (6-36 months) and cognitive impairment. The disorder is sex-limited, with the phenotype being restricted to females. Males are apparently spared, with normal cognitive function and absence of seizures. Evidence for the trait being due to a gene on the X chromosome derives from the pattern of inheritance and from genetic linkage to Xq22 (ref. 1
). EFMR is recognizable in multi-generational pedigrees by the unique sex-limited expression pattern, wherein affected females are connected through unaffected transmitting males1
We ascertained five new families carrying EFMR on the basis of their inheritance pattern, electroclinical features and gene localization to Xq22 (ref. 4
and S.M., unpublished data). To identify the underlying genetic defect, we resequenced 737 genes from the Vertebrate Genome Annotation (VEGA) database in probands from three families. We identified a single nonsense nucleotide change 2012C>G (S671X) in the PCDH19
gene in family 3 (). Families 1 and 2 initially did not show any changes.
Figure 1 Mutations in PCDH19 cause EFMR. Pedigrees of the seven EFMR families showing the characteristic inheritance pattern of affected females and transmitting males. Each of the X chromosome-encoded PCDH19 mutations segregated with the EFMR clinical phenotype. (more ...)
Subsequent comparative sequence analysis of the annotated PCDH19
ORF revealed that it was incomplete, consistent with a previous report5
. We annotated the complete 3,447-bp ORF of PCDH19
, which consists of six exons. The full-length, processed PCDH19
mRNA is 9,765 nt long, with exon 2 alternatively spliced (Supplementary Fig. 1
online). Sequencing of the entire PCDH19
ORF identified a missense change 1322T>A (V441E) in family 1, a nonsense change 253C>T (Q85X) in family 2, a frameshift change 2030_2031 insT (L677fsX717) in family 4 and a frameshift change 357delC (I119fsX122) in family 5. Finally, we identified a frameshift change 1091_1092insC (P364fsX375) in the original EFMR-bearing family (family 6, ref. 1
) (). In summary, we identified PCDH19
nucleotide changes in all six families bearing EFMR that we studied. Another unique nucleotide change (1671C>G, N557K) was identified by screening a cohort of 87 females that included both isolated and familial cases with epilepsy and varying degrees of mental impairment. We found that the nucleotide change in this small family, with two affected girls with epilepsy and autism spectrum disorder, arose de novo
in their carrier father on the grandmaternal X chromosome (, family 7).
All seven nucleotide changes () segregated with the clinical phenotype in the respective families () and were not present in 250 male probands from families with putative X-linked mental retardation or in 750 control (350 male and 400 female) X chromosomes. Further, we did not detect any other potentially deleterious nucleotide changes in probands from the three families in any of the other 736 X-chromosome genes sequenced (data not shown). Taken together, the predicted loss of function of all seven PCDH19
changes (see below), the high degree of conservation of the residues affected by the two missense changes () and our PCDH19
mRNA studies (see below) strongly support PCDH19
as the gene whose mutation causes EFMR. Moreover, PCDH19
is located at Xq22 (Ensembl database) within the original linkage region1
. EFMR locus homogeneity in these families has now been molecularly defined, and the combined lod score with markers in the Xq22 region reached 11.9 (refs. 1
and S.M., unpublished data). Among the seven families bearing EFMR studied, there were two (of 68) carrier females who had been classified (at the time of the original assessment) as unaffected. Therefore, the estimated penetrance of the known EFMR mutations in females was >90%.
Figure 2 Structure and expression analysis of PCDH19. (a) Schematic diagram of the PCDH19 protein showing the signal peptide, extracellular cadherin (EC), transmembrane (TM) and cytoplasmic (CM) domains. The positions of the mutations found in the families bearing (more ...) PCDH19
was expressed predominantly in neural tissues and at different developmental stages. By RNA blot analysis using a PCDH19
-specific probe, we detected a transcript size of approximately 9.8 kb from various areas of the male and female adult human brain ( and below). We also identified PCDH19
mRNA expression in primary skin fibroblasts. This allowed us to examine the consequences of the PCDH19
mutations 253C>T, Q85X (family 2) and 2012C>G, S671X (family 3) on the stability of their respective mRNAs. Both mutations introduce a premature termination codon (PTC) into the PCDH19
mRNA. Such PTC-containing mRNAs are usually recognized by the nonsense-mediated decay (NMD) surveillance complexes and efficiently degraded6
. We found that the PTC mutations in family 2 () and family 3 (data not shown) led to PCDH19
mRNA degradation by NMD. Inhibition of translation by cycloheximide treatment of skin fibroblast cells preserved the PTC mutation-containing mRNA (). The absence of mutant PCDH19
mRNAs could not be attributed to skewing of X-inactivation. We confirmed random X-inactivation in DNA isolated from blood and skin fibroblast cultures of each affected female available (data not shown), in agreement with previous data1
encodes a 1,148-amino acid protein belonging to the protocadherin δ2 subclass of the cadherin superfamily of cell-cell adhesion molecules. PCDH19 contains a signal sequence, six extracellular cadherin (EC) repeats, a transmembrane domain and a cytoplasmic region with conserved CM1 and CM2 domains (). The biological role of the PCDH19 protein is not known. However, members of the protocadherin family are predominantly expressed in the nervous system7
and are postulated to be involved in the establishment of neuronal connections and in signal transduction at the synaptic membrane9
, also a δ2 protocadherin, is required for growth of striatal axons and thalamocortical projections11
. Usher syndrome type 1, involving combined hearing loss and blindness, was the first human disorder to be associated with mutations in a PCDH
. Mutations in PCDH19
have now been associated with epilepsy and mental retardation.
All mutations identified in this study were located in the large PCDH19 extracellular domain () containing the cadherin repeats, which facilitate cell-cell interactions. We predict five of these mutations to be complete loss-of-function mutations as a consequence of NMD degradation of their respective PTC-containing mRNAs ( and data not shown). Given the similarity among the clinical phenotypes associated with all seven PCDH19
mutations, it is reasonable to suggest that the remaining two mutations, missense mutations leading to V441E and N557K substitutions, also lead to loss of PCDH19
function. The N557K mutation affects an invariant asparagine residue within the EC5 domain (). The equivalent asparagine residue of EC1 of classical cadherins (for example, Asn100 of N-cadherin13
) and protocadherins (for example, Asn101 of Pcdhα14
) is essential for calcium binding and thus for the adhesive function of the EC1 domain13
. The valine residue at position 441 (in EC4, or the equivalent of Val96 in EC1 of N-cadherin13
or Val97 in EC1 of Pcdhα14
) is also highly conserved () and in close proximity to the calcium-binding site13
. We speculate that both PCDH19
missense variants adversely affect PCDH19 adhesive function through impaired calcium binding.
To investigate the expression of PCDH19 in the developing mouse and human central nervous system (CNS), we performed in situ hybridization analysis. In mice, Pcdh19 had widespread expression in both the embryonic and adult brain, including the developing cortex and hippocampus ( and Supplementary Note online), which is consistent with our finding that mutations of this gene in humans are associated with cognitive impairment. In human tissue, in addition to PCDH19, we also investigated the expression pattern of PCDH11X and PCDH11Y, two paralogous protocadherin genes, whose expression and function we speculate to be relevant to EFMR (see and below). Each of the three PCDH genes was expressed in developing cortical plate, amygdala and subcortical regions and in the ganglionic eminence (). However, PCDH11X/Y expression showed a marked sexual dimorphism in the caudate nucleus: virtually absent expression in the male and high expression in the female (). Additionally, closer inspection of the amygdala showed that PCDH19 was expressed in lateral nuclei, whereas PCDH11X/Y was more medial.
Figure 3 Expression of Pcdh19 in the developing mouse CNS. (a-l) Expression at embryonic day 15.5 (a-f) and postnatal day 2 (g-l) (representative of two males and two females studied). (a,b) Adjacent coronal sections through the hippocampal region stained with (more ...)
Figure 4 Expression of PCDH11X/Y and PCDH19 in midgestation developing human CNS. (a,b) Autoradiography in situ hybridization of female (f; panel a) and male (m; panel b) coronal sections through the basal regions of the brain using probes for PCDH11X/Y and PCDH19 (more ...)
Typically, in most X-linked dominant conditions, males are more severely affected and often die prenatally. In EFMR, the transmitting males are apparently normal (that is, without epilepsy or intellectual disability), but with some obsessive traits reported anecdotally4
. Previous hypotheses to explain the EFMR expression pattern1
considered either a dominant negative effect of the mutant protein in carrier females (for example, similar to that of the C-terminally truncated P-cadherin in malignant melanoma15
) or the presence of a compensatory or rescue factor in males. The dominant negative effect of mutant PCDH19
is unlikely, based on our NMD results (see above), and PCDH19
does not have a Y-chromosome paralog to provide male rescue. Sexually dimorphic expression and function16
and/or relevant compensatory genes is also a possibility.
We propose an alternative hypothesis, based on the identity of the EFMR gene, that invokes functional rescue by a related, but nonparalogous, protocadherin gene in males. The PCDH19
gene is subject to X inactivation (Supplementary Figs. 2
online). Therefore, hemizygous transmitting males will have a homogeneous population of PCDH19-negative cells, whereas affected females are likely to be mosaics comprising PCDH19-negative and PCDH19-wild type cells. Such tissue mosaicism may scramble cell-cell communication, which manifests clinically as EFMR. A similar mechanism was proposed for the craniofrontonasal syndrome CFNS (MIM 304110)18
. We hypothesize that the absence of PCDH19
function in males may potentially be compensated for by the related but nonparalogous protocadherin gene PCDH11Y
, a Y-chromosome gene that is expressed in human brain19
has an X chromosome paralog, PCDH11X
, that has strong sequence similarity19
. However, the differences in brain expression patterns between these two genes may account for differential ability to compensate for absence of PCDH19
. The potential role of PCDH11Y
(and that of its paralog PCDH11X
) in the EFMR phenotype will need to be addressed in the future by engineering the appropriate animal models.
Cadherins are a large family of genes with crucial functions in human brain21
. Our data directly implicate the protocadherin gene family in epilepsy and intellectual disability. On the basis of our observations, molecular diagnosis will result in wider recognition of EFMR, especially in smaller families and single cases, with important consequences for counseling.