Patients
A total of 206 unrelated Chinese Han patients clinically diagnosed with IPN were recruited from the Neurological Department of the First Medical Center, Chinese PLA General Hospital (Beijing, China) from December 20th, 2012 to March 2nd, 2020. Patients underwent detailed history-taking, neurological examination, laboratory examination, electrophysiological studies, and genetic testing.
This study was approved by the Chinese PLA General Hospital Ethics Committee, in accordance with the principles stated in the Declaration of Helsinki. Informed written consent was obtained from each patient enrolled in this study.
Electrophysiological examination
All patients underwent nerve conduction study (NCS) in which their skin temperature was maintained at 32°C or above during the examination. NCS were performed on the median, ulnar, tibial, peroneal, and sural nerves using the Keypoint electromyography (EMG) system (Medoc Ltd, Israel). The results were measured according to the normal reference values utilized by the EMG laboratory of Chinese PLA General Hospital (median motor nerve: amplitude ≥5.0 mV, velocity ≥50.0 m/s; median sensory nerve: amplitude ≥5.0 µV, velocity ≥50.0 m/s; ulnar motor nerve: amplitude ≥5.0 mV, velocity ≥50.0 m/s; ulnar sensory nerve: amplitude ≥5.0 µV, velocity ≥50.0 m/s; tibial motor nerve: amplitude ≥5.0 mV, velocity ≥40.0 m/s; peroneal motor nerve: amplitude ≥3.0 mV, velocity ≥45.0 m/s; and sural sensory nerve: amplitude ≥6.0μV, velocity ≥50.0 m/s). NCS were considered abnormal if any of the studied parameters was found to be abnormal [7, 8].
Sural nerve biopsy
Sural nerve biopsy was performed on Patient 1 with GARS variant with informed consent. A segment of nerve was fixed in 3% glutaraldehyde buffered to pH 7.4 with 0.1 M phosphate buffer. Cross-sections of 1 mm thickness were post-fixed in 0.1 M osmic tetroxide for 2 h, dehydrated in a series of graded ethanols and propylene oxide, and embedded in epoxy resin (LX-112). Semithin sections were stained with toluidine blue or paragon. Thin sections were stained with lead citrate and uranyl acetate, and examined under an electron microscope [9].
Genetic analysis
All patients underwent genetic analysis via NGS (high throughput target sequencing). We examined IPN-associated genes, especially CMT-associated genes (Table 1). Genomic DNA was extracted from the peripheral leukocytes of fresh blood samples obtained from patients with a clinical diagnosis of IPN. Target genes were captured by GenCap target region probe (MyGenostics Inc, Medford, MA, USA) and amplified by polymerase chain reaction. The eluted DNA was finally amplified for 15 cycles according to the following procedure: 98°C for 30 s (1 cycle), 98°C for 25 s, 65°C for 30 s, 72°C for 30 s (15 cycles), and 72°C for 5 min (1 cycle) [7, 8]. The amplified product was purified using SPRI beads (Beckman Coulter, Brea, CA, USA) according to manufacturer’s protocol. Enriched libraries were sequenced using a HiSeq 2000 sequencer (Illumina, San Diego, CA, USA), which generated 100 bp paired reads [7, 8].
Depth reading of NGS identified PMP22 duplications/deletions, and multiplex ligation-dependent probe analysis (MLPA) was applied to confirm the results. Sanger direct sequencing was used to confirm and detect variants in the patients and their family members [7, 8].
The reference genome was UCSC hg19 (http://genome.ucsc.edu/). Read mapping was done using SOAP (Short Oligonucleotide Analysis Package) aligner (http://soap.genomics.org.cn/soapaligner.html) and Burrows–Wheeler Aligner (http://bio-bwa.sourceforge.net/bwa.shtml) software [7, 8]. Variant detection included the identification of single-nucleotide polymorphisms and indels using GATK and SOAPsnp (http://soap.genomics.org.cn/soapsnp.html) software [7, 8]. The genomic variants database included the 1000 Genomes Project (browser.1000genomes.org/index.html) and the single nucleotide polymorphism database (dbSNP) (http://www.ncbi.nlm.nih.gov/projects/SNP/) [7, 8].
Bioinformatics analysis
Polymorphism Phenotyping 2 (PolyPhen-2) (http://genetics.bwh.harvard.edu/pph 2/), sorting intolerant from tolerant (SIFT) (http://sift.jcvi.org/), and Mutation Taster (http://www.mutationtaster.org/) were used to predict potential functional effects of GARS mutations [7, 8]. PolyPhen-2 classified the predicted effects of amino acid substitutions on the function of human proteins as “benign,”“possibly damaging,” “probably damaging,” or “unknown.” The functional impact of the mutation was predicted as“tolerated” or “damaging” by SIFT and as “polymorphism” or “disease-causing” by Mutation Taster [7, 8]. The pathogenicity was determined by the ACMG guideline.
Table 1
List of examined Charcot–Marie–Tooth disease-associated genes
PMP22 | SBF2 | EGR2 | HSPB8 | HSPB1 | DYNC1H1 | DNAJB2 |
MPZ | SBF1 | PRX | HSPB1 | GDAP1 | MYH14 | INF2 |
LITAF | RAB7A | HK1 | HSPB3 | CCT5 | TRPV4 | GNB4 |
EGR2 | DHTKD1 | FGD4 | SETX | PRNP | AARS | YARS |
NEFL | TRIM2 | Figure 4 | DNAJB2 | NGF | SPTLC1 | KARS |
FBLN5 | PDK3 | CTDP1 | BSCL2 | HSPB8 | SPTLC2 | PLEKHG5 |
GDAP1 | AIFM1 | KIF1B | GARS | DNM2 | RAB7 | GJB1 |
MTMR2 | MARS | MFN2 | REEP1 | AARS | ATL1 | PRPS1 |
SH3TC2 | HARS | LMNA | IGHMBP2 | LRSAM1 | DNMT1 | HOXD10 |
NDRG1 | HINT1 | MED25 | SLC5A7 | KIF5A | WNK1 | IKBKAP |
KIF1A | TFG | NTRK1 | DCTN1 | DNM2 | FAM134B | SCN9A |
NEFL | BICD2 | DCTN1 | ATP7A | | | |