4613347637

868 Lctrcrs

868 Lctrcrs

Figurę 1 Detcction of tht tzx> somatic mutarions by hasrodupley analysis. (A) DGGE results jar case 1. DNA exiracicJ /roni a lymphoblastoid celi linę Ćant 1) and a fresk blood sample (lane 2). The homoduplex band corresponding to the deleted allelc ii missir.g w. DNA cxtracudfirom fymphoeyies. Hi, heterodupUx; Ha, homoduplex; 4Ó%-90% formamidc gradient. (B) CSGE results for case J (lanes 1-3) and case 2 (lam 5). Case 1: the somatic nturatian is shcrton on DNA extractcd front a fresh blood sample (lane 1)•> the fajfer (lane 2) and the mother (lane 3) do not carry the 26 bp deletion. Case 2: a somatic deleiion zvas detected on DNA extracted from lymphocytcs as indicaicd by the absence of the deleted homoduplex band (lane 5). Lane 4 deptets a CSGE paltem of a 31 bp deletion localised in the deletion pront region of the MECP2 gene: the Tioo homodup!cx bands ort of equal intensity.

binding protein 2 (MeCP2) invoIved in iran-scriptional silencing. This disorder most fre-quentlv occurs sporadicaily and resuJrs from a de novo mutation, aJrhough a few familia] cases have bcen reponed. Many studies' " have shown that the MECP2 gene is mutated in approximately 80% of patienrs with dassicaJ RTT and the MECP2 mutation spectrum

includes missense, nonsense, and frameshift mutarions, as well as iarger rearrangemems like deletions encompassing a few hundred bp.'^u The failure to detect MECP2 murations in the remaining 20% may indicate the presence of mutarions in unexplored regions of the MECP2 gene, such as regulatory elemems or non-coding regions, notably in the new first exon’ or in an additional RTT locus.

Here, we report for the first rime mosaicism for a somatic MECP2 mutation found in rwo unreiated females affeered with RTT. These two girls were diagnosed according to the International criteria of the Rett Syndrome Diagnostic Criteria Work Group.

Case reports

The first patiem (case 1) is 13 years old. She suffers from classical Retr syndrome with 7/9 of the necessary criteria, 4/8 of the supportive criteria, and nonę of the exdusion criteria.' Morę specificaily, she had a normal neonatal period and head circumference at birth and a phase of sociai withdrawaf at the age of 12 months when she iost purposefuj hand skills and deve!oped stereorypic hand movements, ataxia, and apraxia. She suffereć from breathing dysfunc-tion and peripheral vasomotor disrurbances. She had severelv irnpaired developmem bur acquirea independent walking ar the age of 24 months. However, she did not acouire micro^ cephaly or deve!op epilepsy.

The secona parient (case 2) was reported as an arypical case of RTT without any period of regression. Both menral and motor develcp-ment were verv slow. At the age of 4 years, she had acąuired microcephaly (-2 SD) and had very limited ambulation, but her hand use was correct without hand wringing movemenrs. She developed epilepsy and progressive scolio-sis. She is a placid girl without usefui speech bur she communicates well by eye movements.

Methods and results

For case 1, an initial study on DNA extracted from a lymphoblastoid celi linę by denaturing gradient gel eiectrophoresis (DGGE) and sequencing showed that she carried a 26 bp deletion starting at position 1165. To confirm this mutation, DNA was exrracted from a fresh blood sample and the deletion was assessed by airect sequenc:ng. Surprisingly and despite a careful examination of the sequence, we did not find the 26 bp deletion with DNA extractcd from leucocytes. This sample was reanalysed by DGGE and heterodup!exes were detected while the homoduplex corresponding to the deleted band was absent (fig 1A). Wc confirmed this result by conformarion sensitive gel eiectrophoresis (CSGE) analysis, which showed the hererodup!exes but not the mutant homoduplex (fig IB). The results obtained from peripheral blood lymphocytes suggested mosaicism for a somatic mutation.

Jn order ro determine the level of mosaicism, we used a semiquantitative approach based on fiuorescent PCR. The MECP2 gene exon 3 portion contaimng the deletion was PCR amplified, the reverse primer being conjugated

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