We identified a missense mutation p.A335V in a yet unassigned transcription activation domain of MED25 cosegregating with a peripheral neuropathy in a Costa Rican kindred. Heterozygous p.A335V was found in five out of 632 healthy Costa Rican controls and furthermore in one German healthy control supporting the recessive nature of this mutation. These observations suggest that A335V is not a private mutation, but a very rare variant, until now, only in homozygous state in CMT2B2 patients [15
]. The incidence for CMT in Costa Rica is 1:66,358 (Hardy–Weinberg, q2
), whereas that combined for the USA and Germany is 1:154,449 (Hardy–Weinberg, q2
). The difference may be explained by a potential founder effect for the p.A335V MED25 mutation due to the Spanish ancestry of immigrants in the sixteenth century of the extended Costa Rican family. Interestingly, for a recessive GDAP1 mutation (p.Q163X) a similar common haplotype shared by nonrelated patients and families with Spanish origin was described [46
There are two variants in exon 9 of MED25 reported in public databases (rs34889830 and rs4290565), nevertheless these polymorphisms were not found in additional ESTs or genomic sequences in databases and were not observed in the 377 neuropathy patients via sequence analysis. For MED25
several splice variants are known, especially for the 3′ region [35
]. One of them is the P78 cDNA expressed in prostate cancer cells [48
]. Some conserved domains, among them the von Willebrandt factor type A (vWA) domain- and Synapsin-related regions, were assigned according to homology data [35
]. An interaction with Mediator of the vWA domain was recently shown by immunoprecipitation [35
]. It was also shown that MED25 contains an activator-interacting domain (ACID) which is a functionally important target of the VP16 transcriptional activator [35
]. The MED25 VP16-binding domain was later assigned to a discrete region between amino acids 402 and 590 [35
We describe the proline-rich SH3 recognition motif comprising the p.A335V mutation. Affinity experiments using synthetic MED25 peptides showed a loss of binding specificity of the mutant as shown for the interaction with the Abl and Lck SH3 domains (Fig. ). Although both types of kinases have been associated with the nervous system [49
], their interconnection with transcription activation as suggested by the MED25
mutation in family CR-P has not yet been shown. This raises the question whether additional cofactors, mediated by their SH3 domains, might act as interaction partners of MED25
in the nervous system. At present, 839 different human SH3 domains [51
] of mostly uncharacterized binding preferences are known, which renders the existence of additional MED25
interaction partners feasible. Based on the extension of affinity to a broader range of SH3 domains detected for the MED25
mutant, however, we anticipate that the number of binding partners will be larger for the mutant than for the wildtype. Such interactions include an increased number of nonphysiological interactions and a concomitant "loss-of-specific-function". As a further consequence, the MED25 physiological function will be reduced in a dose-dependent manner by increased competitive binding to additional interaction partners. This situation could be compared to recessively inherited deletions, e. g. spinal muscular atrophy with two distinct survival motor neuron genes responsible for the resulting phenotype.
The wild-type and mutated proline-rich motif in MED25 revealed in our study an intrinsic transcription activation property in a GAL4 reporter assay indicating a novel in vivo function. The "loss-of-specific-function" induced by the A335V mutation may activate gene transcription in peripheral nerves with remarkably reduced degree and specificity for the cognate downstream target genes. The reduced, but maintained wildtype function may potentially explain a mild peripheral neuropathy with late age of onset (mean 33.8 years) in an autosomal recessive trait.
The significant correlation of Med25
dosage and disease phenotype in transgenic animals was also present in preliminary siRNA knockdown experiments (data not shown). This raises the question of how a structural membrane protein can influence the expression of a transcription activator in a yet unknown signal transduction pathway [9
, a transcription factor associated with CMT1A, DSN, and CHN, plays an essential role during peripheral nerve myelination and differentiation of Schwann cells [52
]. The recessive p.I268N mutation abrogates the binding of NAB repressors and causes, in homozygous state, a severe phenotype (CHN). This finding may reflect a threshold effect in which certain increased protein level of the downstream PNS target gene(s), e. g. PMP22 and P0, must be achieved for manifestation of phenotype [53
]. In Tr mice abnormal retention within the endoplasmic reticulum of myelinating Schwann cells was described earlier [55
]. However, the pathway from increased PMP22
dosage to myelin impairment and axonal damage remains enigmatic. Furthermore, the PNS target genes regulated by MED25
are currently completely unknown.
The p.A335V mutation was identified in patients presenting with an axonally classical peripheral neuropathy accompanied by mild myelin impairment. With regard to our expression analysis results for MED25 in sciatic nerves of transgenic animals and after injury a primary, subtle molecular lesion in the CR-P patients could be occurring in Schwann cells. The pathway from Schwann cell defect to axonal damage is unknown. MED25
is expressed in reasonable amounts in the cerebellum, cortex, DRG, and optic nerve (Fig. a). Hence, a MED25 mutation could potentially affect the central nervous system. The absence of central nervous clinical findings like ataxia or impairment of the optic nerve in the CR-P patients points to differences in molecular pathways between PNS and CNS, may be because central axons are myelinated by oligodendrocytes. In this context it is of interest that a patient with partial trisomy 19q including the MED25
gene among several others had severe central nervous impairment [56
]. Clinical and electrophysiological re-examination revealed, however, no impairment at all of the peripheral nervous system. This increased gene dosage for MED25 is in heterozygous state thus supporting the assumption of an autosomal recessive mode of inheritance for CMT2B2.
Our data demonstrate that functional effects of p.A335V include loss-of-specific-function for SH3 ligand binding that probably sequester MED25 to nonphysiological pathways. Such potential altering of the availability of MED25 may affect transcriptional activation or regulation of target genes in the peripheral nervous system. We further document that Pmp22 expression correlates with Med25 expression levels in: (1) transgenic animals, both mice and rats, wherein the Pmp22 expression is altered by effectively changing gene copy number, (2) in animals wherein the Pmp22 expression is altered by epigenetic means, and (3) in a nerve-crush/injury paradigm. These latter findings suggest that PMP22 may be at least one of the genes downstream of MED25 that is mediating the neuropathic effects of the p.A335V mutation.