Identification of the variant Ala335Val of MED25 as responsible for CMT2B2: molecular data, functional studies of the SH3 recognition motif and correlation between wild-type MED25 and PMP22 RNA levels in CMT1A animal models

Alejandro Leal 0 1 2 3 4 5 6 Kathrin Huehne 0 1 2 3 4 5 6 Finn Bauer 0 1 2 3 4 5 6 Heinrich Sticht 0 1 2 3 4 5 6 Philipp Berger 0 1 2 3 4 5 6 Ueli Suter 0 1 2 3 4 5 6 Bernal Morera 0 1 2 3 4 5 6 Gerardo Del Valle 0 1 2 3 4 5 6 James R. Lupski 0 1 2 3 4 5 6 Arif Ekici 0 1 2 3 4 5 6 Francesca Pasutto 0 1 2 3 4 5 6 Sabine Endele 0 1 2 3 4 5 6 Ramiro Barrantes 0 1 2 3 4 5 6 Corinna Berghoff 0 1 2 3 4 5 6 Martin Berghoff 0 1 2 3 4 5 6 Bernhard Neundrfer 0 1 2 3 4 5 6 Dieter Heuss 0 1 2 3 4 5 6 Thomas Dorn 0 1 2 3 4 5 6 Peter Young 0 1 2 3 4 5 6 Lisa Santolin 0 1 2 3 4 5 6 Thomas Uhlmann 0 1 2 3 4 5 6 Michael Meisterernst 0 1 2 3 4 5 6 Michael Sereda 0 1 2 3 4 5 6 Gerd Meyer zu Horste 0 1 2 3 4 5 6 Klaus-Armin Nave 0 1 2 3 4 5 6 Andr Reis 0 1 2 3 4 5 6 Bernd Rautenstrauss 0 1 2 3 4 5 6 0 A. Leal School of Biology and Neuroscience Research Program, University of Costa Rica , San Jose, Costa Rica 1 A. Leal School of Biology and Institute for Health Research (INISA), University of Costa Rica , San Jos, Costa Rica 2 J. R. Lupski Department of Molecular and Human Genetics, Baylor College of Medicine , One Baylor Plaza, Room 601B, Houston, TX 77030, USA 3 G. Del Valle Laboratory of Neurology , Neurolab, San Jos, Costa Rica 4 B. Morera School of Biological Sciences, Universidad Nacional , Heredia, Costa Rica 5 M. Berghoff Klinikum der Justus-Liebig-Universitt , 35385 Gieen, Germany 6 R. Barrantes School of Biology, University of Costa Rica , San Jose, Costa Rica Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous disorder. All mendelian patterns of inheritance have been described. We identified a homozygous p.A335V mutation in the MED25 gene in an extended Costa Rican family with autosomal recessively inherited Charcot-Marie-Tooth neuropathy linked to the CMT2B2 locus in chromosome 19q13.3. MED25, also known as ARC92 and ACID1, is a subunit of the human activator-recruited cofactor (ARC), a family of large transcriptional coactivator complexes related to the yeast Mediator. MED25 was identified by virtue of functional association with the activator domains of multiple cellular and viral transcriptional activators. Its exact physiological function in transcriptional regulation remains obscure. The - CMT2B2-associated missense amino acid substitution p. A335V is located in a proline-rich region with high affinity for SH3 domains of the Abelson type. The mutation causes a decrease in binding specificity leading to the recognition of a broader range of SH3 domain proteins. Furthermore, Med25 is coordinately expressed with Pmp22 gene dosage and expression in transgenic mice and rats. These results suggest a potential role of this protein in the molecular etiology of CMT2B2 and suggest a potential, more general role of MED25 in gene dosage sensitive peripheral neuropathy pathogenesis. Charcot-Marie-Tooth disease (CMT) or hereditary motor and sensory neuropathy (HMSN) represents a group of frequent, clinically and genetically heterogenous disorders of peripheral nerves [13]. Two major CMT forms are distinguished by electrophysiological criteria: the demyelinating CMT (HMSN) type 1 and the axonal CMT type 2 [3]. Autosomal dominant CMT type 1 is most frequently caused by a 1.4-Mb tandem duplication including the PMP22 gene (CMT1A [MIM #118220]) [4, 5]. For the dominantly inherited axonal CMT type 2A mutations in the Mitofusin 2 gene (MFN2 [MIM *608507]) [6] are the most frequently observed variations. Intermediate types of CMT have also been described [7, 8]. To create animal models of the most frequently observed form of CMT, CMT1A, transgenic mice and rats that carry altered copy numbers of the PMP22 gene have been generated [911]. These animals recapitulate many pathological hallmarks of the human disease [12]. Both the autosomal recessive demyelinating (CMT4 [MIM #214400]) and the autosomal recessive axonal forms (ARCMT2 [MIM #605588, %605589]) are less frequently observed. For the autosomal recessive axonal ARCMT2, the first locus was mapped on chromosome 1q21.2q21.3 [13]. Subsequently, De Sandre-Giovannoli et al. identified a homozygous R298C mutation in the LMNA gene in three consanguineous Algerian families with severe and early onset [14]. Furthermore, mutations in the gangliosideinduced differentiation-associated protein 1 (GDAP1) gene on chromosome 8q21.1 have been identified in three families of Spanish ancestry with ARCMT2 [15]. These patients were characterized by childhood onset, distal muscle weakness, sensory deficits, and vocal chord paresis in some of the patients [16]. We investigated an extended Costa Rican family (CR-P) with Spanish and Amerindian ancestors that segregated an autosomal recessive CMT type 2 neuropathy that mapped to chromosome 19q13.3 (ARCMT2 [MIM %605589]) [17, 18]. Here we provide genetic and functional data to support the contention that the homozygous p.A335V mutation in the gene for Mediator 25 (MED25) is responsible for the neuropathy segregating in the family CR-P from Costa Rica. Materials and methods The clinical and electrophysiological investigations took place in the city hospital of Palmares in Costa Rica, 31 family members underwent genetic analysis; clinical and electrophysiological evaluations were performed on 11 subjects. Eight of these studied subjectsthree males and five femaleswere clinically diagnosed with motor and sensory neuropathy. Informed consent was obtained from all study participants. The age at onset of the chronic symmetric sensory-motor neuropathy was 28 to 42 years (mean 33.8 5.3), the electrophysiologic data reflected a primary axonal degenerative process with mild myelin impairment in the course of the disease [18]. The electrophysiological investigations were described in detail elsewhere [18]. Genomic DNA was extracted from peripheral blood samples by the salting-out method. DNA was diluted in HPLCH20 to a concentration of approximately 50 ng/ml and stored at 4C. The critical interval in chromosome 19q13.3 could be refined to 1 Mb between marker D19S604 and a SNP (c.901 T>C) identified in the nuclear receptor subfamily 1, group H, member 2 (NR1H2) gene by homozygosity mapping. Primers flanking all exons of 53 genes corresponding to 487 amplicons in the critical interval were designed using the Primer3 program. All 19 exons of MED25 have also been sequenced. Oligonucleotide sequences and PCR conditions are available on request. Bidirectional direct sequencing was implemented using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) on an ABI 3730 capillary sequencer (Applied Biosystems) for all variations found with unidirectional sequencing. Sequence traces were evaluated using the DNAStar software package. A BAC-contig on the critical interval was constructed using public databases and confirmed with Celera scaffolds. This interval, included on the public contig NT_011109 and the Celera-Scaffold GA_x2KMHMQE7RG (Celera Publication Site) of chromosome 19q, was analysed (...truncated)