A cohort of 404 patients referred for hereditary neuropathy with liability to pressure palsies was tested initially for the common PMP22 whole gene deletion. 94 whole gene deletions were detected, plus three partial gene deletions, and the remaining 307 patients were screened for PMP22 point mutations. Nine point mutations were identified (8.5% of all mutations), eight of which were in exon 5, suggesting a point mutation hotspot for individuals with this condition. Sequencing analysis of PMP22 exon 5 should therefore be included as a routine diagnostic test for gene deletion-negative patients.
Hereditary neuropathy with liability to pressure palsies (HNPP) was first described in 1947 [
Autosomal dominant inheritance of HNPP was first shown in 1954 [
As PMP22 is the only gene that has been linked to HNPP, point mutation analysis of this gene in patients without a known whole or partial gene deletion is therefore likely to reveal additional mutations as mutations that reduce the functional level of PMP22 can cause a HNPP phenotype [
Between 1991 and 2014, a cohort of 404 independently-ascertained patients was collected at our laboratory following referrals from centres within the catchment area of the Wessex Regional Genetics Laboratory. As this is a laboratory-based study, we have no access to clinical data, nor do we have details of the referral criteria used by the clinicians.
This study falls in the realm of routine clinical care and consent for diagnostic testing was obtained from all patients as part of the referral process.
MLPA was carried out according to the manufacturer’s instructions with the current CMT probe mix at the time of referral (MRC-Holland, The Netherlands) either kit P033 or kit P405. Any patients referred prior to 2002 were retroactively tested with the P033 kit. MLPA PCR products (0.5 μl) were added to 0.2 μl of Genescan™-500 ROX™ Size Standard (Applied Biosystems, USA) and 9 μl of Hi-Di Formamide (Applied Biosystems, USA) and separated on an ABI 3130xl Genetic Analyzer (Applied Biosystems, USA) using a 36 cm array and 3130 POP-7™ array polymer (Applied Biosystems, USA). The run conditions were as follows: injection voltage = 1.2 kVolts, injection time = 5 seconds, oven temperature = 60˚C, run voltage = 15 kVolts, run time = 20 minutes. Subsequent data was analysed using the MLPA analysis function of the Gene Marker (version 1.85) software (SoftGenetics, USA).
Mutation analysis of the four coding exons of the PMP22 gene (exons 2-5 Ensembl transcript ENST- 00000395938, gene ENSG00000109099) was carried out by direct sequencing analysis. PCR products were first generated using a standard PCR reaction with a 25 μl volume and a 60˚C annealing temperature using the relevant exon primers; 2F-ctcctcgcaggcagaaact, 2R-ctgaaccagcaggagcacg, 3F-tccccttttccttcactcct, 3R-ccaataagcgtttccagctc, 4F-catggccagctctcctaac, 4R-actaatcattccgcagacttg, 5F-ccgctctgccatggactc and 5R-ttccctatgtacgctcagag. These products were then sequenced using the standard protocol of the Big-Dye® Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, USA ) and separated on an ABI 3130xl Genetic Analyzer (Applied Biosystems, USA ). Subsequent data was analysed using the Mutation Surveyor (version 3.1) software ( SoftGenetics , USA ).
Any missense mutations were assessed using the Alamut mutation interpretation software (version 1.5-
Location | Base change | Protein change | Mutation | Reference |
---|---|---|---|---|
Exon 2 | c.11delT | p.(Leu4fs) | Frameshift | [ |
Exon 2 | c.19_20delAG | p.(Leu7fs) | Frameshift | [ |
Exon 2 | c.31_39delCTCCACGTC | p.(Leu11fs) | Frameshift | [ |
Exon 2 | c.65C>T | p.(Ser22Phe) | Missense | [ |
Intron 2 | c.78+1G>T | Splicing | [ | |
Intron 2 | c.78+5G>A | Splicing | [ | |
Intron 2 | c.79-13T>A | ? p.(Gln27fs) | Splicing/Frameshift | This study |
Exon 3 | c.88G>A | p.(Val30Met) | Missense | [ |
Intron 3 | c.178+1G>C | Splicing | [ | |
Intron 3 | c.179-1G>C | Splicing | [ | |
Exon 4 | c.183G>A | p.(Trp61Ter) | Termination | [ |
Exon 4 | c.183G>A | p.(Trp61Ter) | Termination | [ |
Exon 4 | c.199G>A | p.(Ala67Thr) | Missense | [ |
Exon 4 | c.227delG | p.(Ser76fs) | Frameshift | [ |
Exon 4 | c.281_282insG | p.(Gly94fs) | Frameshift | [ |
Exon 4 | c.281_282insG | p.(Gly94fs) | Frameshift | [ |
Exon 4 | c.289delT | p.(Tyr97fs) | Frameshift | [ |
Exon 4 | c.297delT | p.(Gly100fs) | Frameshift | [ |
Intron 4 | c.320-1G>C | Splicing | [ | |
Intron 4/ Exon 5 | c.320-1_320delGGinsTA | Splicing | This study | |
Exon 5 | c.320G>T | p.(Gly107Val) | Missense | [ |
Exon 5 | c.328G>A | p.(Val110Met) | Missense | This study |
Exon 5 | c.353C>T | p.(Thr118Met) | Missense | [ |
Exon 5 | c.353C>T | p.(Thr118Met) | Missense | [ |
Exon 5 | c.353C>T | p.(Thr118Met) | Missense | This study |
Exon 5 | c.353C>T | p.(Thr118Met) | Missense | This study |
Exon 5 | c.364_365delCC | p.(Pro122fs) | Frameshift | [ |
Exon 5 | c.372G>A | p.(Trp124Ter) | Termination | [ |
Exon 5 | c.372G>A | p.(Trp124Ter) | Termination | [ |
Exon 5 | c.392C>G | p.(Ser131Cys) | Missense | This study |
Exon 5 | c.395A>G | p.(Tyr132Cys) | Missense | This study |
Exon 5 | c.419G>A | p.(Trp140Ter) | Termination | [ |
Exon 5 | c.433_434insC | p.(Leu145fs) | Frameshift | [ |
Exon 5 | c.434delT | p.(Leu145fs) | Frameshift | This study |
Exon 5 | c.434delT | p.(Leu145fs) | Frameshift | [ |
Exon 5 | c.469C>T | p.(Arg157Trp) | Missense | This study |
Exon 5 | c.475C>T | p.(Arg159Cys) | Missense | [ |
Interactive Biosoftware, France). Alamut uses the programs SIFT (sift.jcvi.org) and Polyphen (genetics. bwh. harvard.edu/pph2/) to predict causality. SIFT predicts whether an amino acid substitution in a protein will have a phenotypic effect, based on the degree of conservation of amino acid residues in sequence alignments derived from closely related sequences, collected through PSI-BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi). The amino acid substitution is given a score from 0 to 1 and is predicted to be damaging if the score is ≤0.05, and tolerated if the score is >0.05 [
Potential splice site mutations were analysed using the Fruitfly splice predictor program within Alamut (http://www.fruitfly.org/seq_tools/splice.html).
Dosage analysis of the PMP22 gene showed that 94 of the 404 referrals had the standard whole gene deletion, while three had a partial gene deletion—the first had a deletion of exons 1-3, the second had a deletion of exons 4 and 5 and the third had a deletion of exons 2 and 3. The remaining 307 patients were screened for point mutations within PMP22.
A single sequence change was found in nine of the 307 patients (see
This study confirms that deletions are the most common HNPP-causing mutation but also demonstrates that point mutation analysis extends the diagnostic yield for HNPP referrals. Nine sequence changes were detected in patients referred with a diagnosis of HNPP. The nine changes comprised of eight different mutations, so no point mutation founder effect was evident. Two of these mutations (c.353C>T p.(Thr118Met) and c.469C>T p.(Arg157Trp)) are listed as deleterious by Clin Var (ncbi.nlm.nih.gov/clinvar/), UniProt (uniprot.org) and dbSNP (ncbi.nlm.nih.gov/SNP/) while the other six were novel and unclassified. The p.(Thr118Met) mutation was originally published in the literature as a possible polymorphism [
Patient | Location | Base change | Protein change | Polyphen prediction | SIFT prediction |
---|---|---|---|---|---|
1 | Intron 2 | c.79-13T>A | Potential frameshift (c.78_79ins23) | Not applicable | Not applicable |
2 | Exon 5 | c.320-1_320delGGinsTA | Destruction of normal exon 4/exon 5 splicing | Not applicable | Not applicable |
3 | Exon 5 | c.328G>A | p.(Val110Met) | Probably damaging | Deleterious |
4 | Exon 5 | c.353C>T | p.(Thr118Met) | Probably damaging | Deleterious |
5 | Exon 5 | c.353C>T | p.(Thr118Met) | Probably damaging | Deleterious |
6 | Exon 5 | c.392C>G | p.(Ser131Cys) | Possibly damaging | Deleterious |
7 | Exon 5 | c.395A>G | p.(Tyr132Cys) | Possibly damaging | Deleterious |
8 | Exon 5 | c.434delT | Frameshift | Not applicable | Not applicable |
9 | Exon 5 | c.469C>T | p.(Arg157Trp) | Probably damaging | Deleterious |
causality (loss of PMP22 function) for the three unclassified amino acid substitutions (c.328G>A p.(Val110Met), c.392C>G p.(Ser131Cys) and c.395A>G p.(Tyr132Cys)), while the c.320-1_320delGGinsTA (destruction of splice site) and c.434delT (protein reading frame shift) mutations are clearly deleterious. The remaining base change, c.79-13T>A, is potentially deleterious based on the in silico data, but RNA analysis would be required to prove causality, and at present we have been unable to obtain an RNA sample from this patient.
The interesting finding from this study is the distribution of the point mutations, with eight out of nine changes clustering within a single coding exon, namely exon 5. There is a paucity of reported PMP22 mutations in the literature, but our results are supported by the Inherited Peripheral Neuropathies Mutation Database (IPNMD; molgen.vib-ua.be) and a literature search which garnered 28 other HNPP-causing PMP22 mutations (see
Mutations in PMP22 can also cause Charcot-Marie-Tooth type 1A (CMT1A) [
The majority of mutation positive cases from our cohort (n = 94) had the standard whole gene deletion, while three had a partial gene deletion. These 97 deletion cases were all detected by MLPA analysis, but the identification of nine point mutations, accounting for 8.5% of all positive cases, highlighted the importance of PMP22 sequence analysis in patients with HNPP. As eight of the nine mutations were within exon 5 of the gene (as are ~51% of all reported HNPP-causing point mutations), we would recommend that, as a minimum, sequencing of this exon is performed on all HNPP gene deletion-negative cases.