X‐linked recessive Menkes disease: identification of partial gene deletions in affected males

Abstract

Menkes disease is an X-linked recessive lethal disorder of copper metabolism, caused by defects in the ATP7A gene. Partial gene deletions comprise about 15% of the mutations causing Menkes disease. We have previously demonstrated identification of partial ATP7A deletions in patients by Southern blot analysis. In the present study, we report the use of three fast and reliable polymerase chain reaction (PCR)-based methods for the identification of partial ATP7A deletions in Menkes disease patients. First we demonstrate the use of multiplex PCR, a fast method for identification and rough localization of partial gene deletions, in which two exons of ATP7A are coamplified. Second, we present PCR amplification of genomic DNA across the deletion junctions, a method enabling identification of the deletion breakpoints and hence the exact size of the deletion. Finally, application of reverse transcription PCR (RT-PCR) for identification and localization of gene deletions at the cDNA level is demonstrated. By studying the mutation at the cDNA level the predicted effect of the mutation on the amino acid sequence and consequently the protein structure and function can be inferred. We demonstrate characterization of partial gene deletions in five patients, and in three of these we were able to determine the breakpoint sequences.

Screening of 383 unrelated patients affected with Menkes disease and finding of 57 gross deletions inATP7A

“…In our hands ddF turned out to be more efficient compared to SSCP [Tümer et al, 1997] in finding ATP7A mutations (unpublished observation). Multiplex PCR is an alternative and more relevant method for deletion screening compared to Southern blot analyses or single exon-PCR [Poulsen et al, 2002a]. Recently Liu et al [2002] also published a genomic DNA-based technique using multiplex PCR, heteroduplex analysis, and direct sequencing, in a serial fashion to screen for mutations in MD patients.…”

“…The 369-bp fragment including the translated sequence was analyzed as described previously [Tümer et al, 1997] 0 -GGCAGAAATATGAT-CAATTAC-3 0 ). Multiplex-PCR was carried out by using different primer sets in the same PCR reaction as described previously [Poulsen et al, 2002a]. When used as a hybridization probe, PCR product was extracted with an equal volume of chloroform and approximately 2.5-3 ml was used for a single hybridization.…”

“…PCR amplifications were performed as described previously [Poulsen et al, 2002a] using primers spanning the deletions. The primer sequences are available upon request.…”

A comparison of the mutation spectra of Menkes disease and Wilson disease

“…We included only those mutations within the coding region, omitting the variants identified within regulatory regions. The large genomic deletions identified in MNK patients, which account for 15%-20% of all MNK mutations (Chelly et al 1993), were also excluded (Poulsen et al 2002;Tümer et al 1994aTümer et al , 1994b. There is an apparent lack of large genomic deletions among WND patients, which may be attributable to a difference in the chromosomal localization of ATP7A (X chromosome) and ATP7B (chromosome 13) and the general genomic stability and chromosomal contexts of each (Tümer et al 1999).…”

Neonatal Erythroderma as a First Manifestation of Menkes Disease

“…This was confirmed by multiplex polymerase chain reaction, using primer sets flanking selected exons in the same polymerase chain reaction as described previously. 1 The same mutation was present in his mother. The resulting transcript is expected to be out of reading frame.…”