LAUSR

Pathogenic variants in the MME gene cause dominant and recessive lateonset axonal hereditary neuropathy, that is, axonal CharcotMarieTooth syndrome (LOCMT2). Here, we report nextgeneration sequencing (NGS) and Sanger sequencing (SS) results of 28 LOCMT2 patients carrying either the repeatedly reported c.467del p.(Pro156Leufs*14) or the c.440–2A>C variants. We demonstrate that an intronic ATrepeat in close proximity to these two mutations is frequently causing an allele dropout during SS that result in false genotyping in a considerable proportion of patients. This may result in an incorrect diagnosis, which has a considerable clinical impact for genetic counselling and prognosis. Recent studies have demonstrated that both heterozygous and biallelic variants in MME (encoding the metalloprotease neprilysin) are a frequent cause of LOCMT2 (MIM: 617017). The heterozygotes variants cause a milder phenotype with reduced penetrance. Besides the large spectrum of rare or even single pathogenic MME variants, the frameshift deletion c.467del p.(Pro156Leufs*14) and the splice site mutation c.440–2A>C have been recurrently reported in patients with autosomal dominant and autosomal recessive LOCMT2. Although PCR is considered to be a robust technology and a reliable tool to be used for routine diagnosis, allelespecific sequence variations occasionally may provoke amplification failure of one of the two alleles at a given locus. Such an allele dropout has also been shown for the c.467del mutation in MME in one consanguineous family. In this study, registries at the Medical University of Vienna and Telemark Hospital Trust were searched for LOCMT2 individuals carrying the MME variants NM_007289.3:c.467del p.(Pro156Leufs*14) and NM_007289.3:c.440–2A>C. We ascertained 28 individuals from 16 families (MH1MH16) afflicted with LOCMT2. For segregation analysis three healthy family members were also included. The families originated from Austria, Germany, Norway and Sweden. Whole exome sequencing, NGSbased multigene panel sequencing or SS of the MME gene was performed and analysed as reported previously. SS was used to confirm MME variants detected by NGS and for segregation analysis in families. Due to conflicting results between NGS and SS at both laboratories, sequencing was repeatedly carried out using primers either including or excluding the adjacent ATrepeat of variable size, c.439+33_439+48AT[815], located 57 bp 5’ of exon 6 (online supplemental material 1). Subsequently, six additional enzymes and two additional conditions for the original AccuPrime Taq DNA Polymerase System were tested to unravel the PCR enzymes’ ability to amplify both the short and the long ATrepeat. A full list of the primers, enzymes and conditions used is described in the online supplemental material 1. Moreover, the length of the intronic ATrepeats was assessed on 179 selected DNA samples by using NGS data, fragment length analysis (FLA) and/or multiplex ligationdependent probe amplification (MLPA). FLA details are described in the online supplemental material 1, MLPA and NGS followed procedures as described. 3 Tracking of the c.467del p.(Pro156Leufs*14) and the c.440–2A>C MME mutations previously detected by NGS or SS revealed conflicting results in 7/28 (25%) of the patients when using the original SS primers including the ATrepeat (table 1). In three families, MH1, MH2 and MH6, the c.467del variant was first detected as heterozygous by NGS, but turned out to be homozygous by SS in several family members. On the other hand, in family MH14, the index patient was tested heterozygous for the c.440–2A>C variant by NGS, whereas the same mutation was absent by SS. To unravel these discrepancies, an alternative primerset Diagnostics