Mutation Analysis of GARS and Genotype-Phenotype Correlation in CMT2D/HMN5A in Chinese Patients

Background CMT2D is a rare subtype of axonal CMT, caused by a variant of the glycyl-tRNA synthetase (GARS) gene which is also a disease-causing gene of distal spinal muscular atrophy type V (dSMA-V) or hereditary motor neuropathy 5A (HMN5A). There were only several cases reported in China, all lacking an epidemiological study of CMT2D/ HMN5A. Methods 206 patients of Chinese Han descent, clinically diagnosed with inherited peripheral neuropathy (IPN), were recruited in this study from December 20, 2012 to July 31, 2019. All patients underwent a detailed medical history screening, a neurological examination, a laboratory examination, several electrophysiological studies, and genetic testing. Results A total of 206 unrelated patients underwent genetic analysis. Four variants of GARS from four different families were found, including c.794C > T (p.S265F), c.374A > G (p.E125G), c.1000A > T (p.I334F), and c.781T > G (p.Y261D), with the rst three being considered pathogenic. For the three pathogenic variant carriers, one was diagnosed with CMT2D, while the two others were diagnosed with HMN5A.


Introduction
Inherited peripheral neuropathies (IPN) include a large heterogenous group of hereditary diseases with more than 100 causative genes reported to date. The main categories of IPN include hereditary motor and sensory neuropathy (HMSN), also called Charcot-Marie-Tooth disease (CMT), hereditary sensory and autonomic neuropathy (HSAN), and distal hereditary motor neuropathy (dHMN) [1].
Charcot-Marie-Tooth disease (CMT), the most common IPN with a worldwide incidence of 1 in 2500, comprises a group of clinically and genetically heterogeneous peripheral neuropathies [2,3]. It is roughly classi ed into Type 1 (CMT1; demyelinating) and Type 2 (CMT2; axonal) according to median nerve motor conduction velocity. More than 80 genes mutations associated with CMT have been reported [4]. Owing to the development of molecular genetics, the classi cation of CMT has been re ned.
The variant of glycyl-tRNA synthetase (GARS) gene causes Charcot-Marie-Tooth disease type 2D (CMT2D) and distal spinal muscular atrophy type V (dSMA-V), also called hereditary motor neuropathy 5A (HMN5A) [4]. In China, one family with CMT2D associated with variant of c.999G>T (p.E333D) and the other family with HMN5A associated with variant of c.383T>G (p.L128R) have been reported [5,6], and no epidemiological study of CMT2D/HMN5A has been reported yet.
In this study, we reported four variants of GARS, including c. 794C>T  This study was approved by the Chinese PLA General Hospital Ethics Committee, in accordance with the principles stated in the Declaration of Helsinki. Informed 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 beyond the normal reference values [7,8].

Sural nerve biopsy
Sural nerve biopsy was performed on Patient 1 with GARS variant with informed consent. A segment of nerve was xed in 3% glutaraldehyde in 0.1 M phosphate buffer with pH 7.4. Cross-sections of 1 mm thickness were post-xed 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 by using next generation sequencing (NGS). IPN-associated genes, especially CMT-associated genes were examined. Brie y, genomic DNA was extracted from the leukocytes of fresh whole blood samples obtained from the patients. Target genes were captured by GenCap target region probe (MyGenostics Inc, Medford, MA, USA) and ampli ed by polymerase chain reaction. The eluted DNA was nally ampli ed 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].
Depth reading of NGS identi ed PMP22 duplications/deletions, and multiplex ligation-dependent probe analysis (MLPA) was applied to con rm the results. Sanger direct sequencing was used to con rm and detect variants in the patients and their family members [7,8].

Results
Four patients, two males and two females, were identi ed with genetic analysis, with GARS variants  [7] reported to cause HMN5A [10]. His bilateral distal lower limbs weakness and atrophy started at the age of 13 and subsequently weakness of bilateral distal upper limbs developed. No subjective sensory abnormality endorsed. Physical examination showed muscle atrophy in bilateral thenar eminence, interosseous muscle, tibialis anterior muscle and calf muscle, with weakness of extremities more severe in distal lower limbs. Deep tendon re exes were decreased, and bilateral Babinski signs were absent. Super cial sensation was lost in distal extremities while deep sensation was intact.
NCS showed axonal motor neuropathy (Table 1). EMG showed active and chronic denervation potentials in both upper and lower limbs. Sural nerve biopsy showed uncompaction of myelin sheath, axonal degeneration and necrosis of peripheral nerve myelin sheath and axon ( Figure 1). Patient 1 had family history with some of the family members having similar presentations (Table 2, Figure 2a). III 5, 27-yearold male, son of the proband, presented with distal lower limbs weakness at the age of 12, and then upper limbs weakness at the age of 14. Physical examination showed weakness of distal extremities and super cial sensory loss in gloving-sock pattern ( Table 2). NCS of III 5 showed axonal motor neuropathy ( Table 1) and EMG showed active and chronic denervation potentials in both upper and lower limbs.
Sanger test con rmed that affected family members (II1, II3, II4, II6, III1 and III3) carried c.794C>T variant and unaffected members (III2 and III4) did not. According to the standards and guidelines of ACMG, the variant was considered to be pathogenic.
Patient 2 was a 34-year-old male who carried c.374A>G variant reported to cause both CMT2D and HMN5A [4,7]. He presented with bilateral upper limbs weakness at the age of 12 and bilateral lower limbs weakness later on. No subjective sensory abnormality reported. Some family members had similar presentations, indicating autosomal dominant inheritance ( gure 2b). His mother (II 8) and son (IV 2) shared c.374A>G variant con rmed by using Sanger test. Physical examination showed prominent weakness and muscle atrophy of bilateral hands and mild weakness of bilateral distal lower limbs, with decreased deep tendon re exes and negative bilateral Babinski signs. Super cial and deep sensation were intact. NCS showed axonal motor neuropathy (Table 1). EMG showed active and chronic denervation potentials in upper and lower limbs. According to the standards and guidelines of ACMG, the variant was considered to be pathogenic. Patient 3 was a 21-year-old female with c.1000A>T variant reported to cause HMN5A [13]. Weakness of bilateral distal lower limbs appeared about 1 year prior to evaluation. No subjective sensory abnormality was noticed. Her father and brother had similar presentations and her brother (II 2) carried the same variant ( gure 2c). Physiological examination showed mild weakness and atrophy of bilateral distal upper and lower limbs. Deep tendon re exes were decreased, and bilateral Babinski signs were absent.
Super cial and deep sensation were intact. NCS showed axonal motor neuropathy (Table 1). EMG showed active and chronic denervation potentials in upper and lower limbs. According to the standards and guidelines of ACMG, the variant was considered to be pathogenic.  Table 1). The variant was predicted to be damaging/probably damaging. It was also not present in the 1000 Genomes Project database. The variant was considered to be of uncertain signi cance according to the ACMG guideline. Other ancillary examinations including complete blood count, basic metabolic panel, vitamin B12, homocysteine, cerebral spinal uid (CSF) test, lung CT, PET-CT etc, were within normal limits, except for positive anti-Hu antibody in serum and CSF. With 7 years' follow-up, symptoms persisted and no malignancy was detected. Reexamination of NCS showed an improvement of sensory nerve conduction (Table 1).

Discussion
In 2003, Antonellis et al. con rmed that GARS gene was the pathogenic gene of CMT2D/HMN5A for the rst time [11]. Up to now, only 20 variants of GARS gene have been reported to be associated with CMT2D/HMN5A [5,12,13]. Classic phenotype of CMT2D/HMN5A is weakness and atrophy of upper extremities, especially the thenar eminence and the rst dorsal interosseous muscle groups. The main distinguishing characteristic of the two disorders is sensory de cits in a gloving-stock pattern in patients with CMT2D. Sensory loss may vary between family members and may be normal in some. Patient 2 and Patient 3 in our study presented with classic phenotype of HMN5A, while the Patient 1 who presented with prominent super cial sensory loss in distal extremities was diagnosed as CMT2D. Upper limbs were the most frequent onset sites, and lower limbs onset (in Patient 1) could also be seen [9]. Compared with HMN5A, weakness of lower limbs in patients with CMT2D tended to be more prominent [9]. Most patients were of adolescent onset, while infant onset was also reported and tended to be more severe [14][15][16]. Severity of phenotype could be varying even in one family [14,16].
Motor peripheral neuropathy in the rst three patients were con rmed with electrophysiological studies.
NCS of the Patient 1 and Patient 2 showed distinctively lower CMAP in median nerve than in ulnar nerve, and this imbalance involvement argued against a primary length-dependent distal axonopathy and was more in favor of a motor neuronopathy [9]. Sensory loss was the characteristic of CMT2D, and in other studies, sensory nerve action potential amplitude was decreased or diminished in CMT2D patients [6,12]. In our study, normal sensory nerve conduction was revealed in the Patient 1, however, sural nerve biopsy showed uncompaction of the myelin sheath, axonal degeneration and necrosis of peripheral nerve myelin sheath and axon, which indicated the diagnosis of CMT2D. Not only large myelinated bers, but also small-to middle-size bers may occur in CMT2D [9,16].
A Korean patient with c.794C>T (p.S265F) was diagnosed with HMN5A [17], however our patient with c.794C>T (p.S265F) was diagnosed with CMT2D, because sensory nerve damage was revealed in sural nerve biopsy [10]. Previous studies showed that the mutation c.374A>C (p.E125G) was associated with both CMT2D and HMN5A [9,11]. Coexistence of CMT2D and HMN5A phenotypes caused by same variant remained unknown etiology. Recently, the GARS c.794C>A (p.S265Y) variant was reported in a Malian family with CMT2D [12], and c.373G>A (p.E125K) was associated to an infant patient with failure to thrive and severe muscle weakness [18]. These two amino acid sites might be critical to GARS.
Previously, c.1000A>T (p.I334F) variant was reported in a patient with HMN5A [16], and in our study, we found Patient 3, a 21-year-old female also carried c.1000A>T variant. Unlike c.794C>T (p.S265F) and c.374A>G (p.E125G) which occupied in the core catalytic domain of GARS, c.1000A>T (p.I334F) was located in the anticodon binding domain.
An unreported GARS gene variant c.781T>G (p.Y261D) was found in Patient 4. This variant was predicted as pathogenic by using different prediction tools and located in the core catalytic domain of GARS, the same as c.794C>T (p.S265F) and c.374A>G (p.E125G). However, no genetic cosegregation was found in this family and no pure sensory neuropathy associated with GARS gene variant was reported yet. According to the guidelines of ACMG, the pathogenicity of this mutation was considered indeterminate.
With subacute onset of sensory neuropathy, long-term positive anti-Hu antibody, and no any other possible reason of sensory neuropathy, the patient was considered anti-Hu antibody neuropathy [19][20][21][22]. However, malignancy was not con rmed in seven-year of follow-up. Asymptomatic carriers with pathogenic variant were found in different studies [12,23], so we cannot exclude the possibility that descendants of Patient 4 may show symptoms later. Continuous follow-up should be conducted on Patient 4 and her family members.
An American study that screened 100 patients diagnosed with dSMA, HMN, or motor axonal CMT for variants in GARS found 3 variants [16], while a Taiwan study reported two heterozygous variants found from 340 CMT patients (0.6%) [24]. Another American study showed very low frequency of GARS gene variant, only 0.4% of 3216 CMT patients [25]. In Asia, one Japanese study reported that one diseasecausing mutation in one patient was found from 89 patients with axonal CMT (1.1%).
[26] In this study,  (Table 3). So far, as GARS variants are rare, few studies focus on the mutation frequency of GARS. The issue of the above studies on the mutation frequency of GARS is that the inclusion criteria of patients were inconsistent, which makes horizontal comparison di cult among people from different countries and regions. Studies with larger sample size, especially multicenter studies with uni ed inclusion criteria of patients are required to assess the mutation frequency of GARS among different groups of people.
In general, GARS variant is a rare cause of CMT and the phenotype tends to be CMT2D or HMN5A. Clinical characteristics, NCS, and even sural nerve biopsy and skin biopsy are essential to distinguish them. As the advance of next-generation sequencing technologies including disease-speci c gene panels, whole-exome sequencing and whole-genome sequencing etc, novel likely pathogenic genes and variants would be found increasingly [7]. In this study, the results suggest the importance of comprehensive assessment by using clinical phenotype, ancillary tests and genetic evidence to evaluate the pathogenicity of genetic variants in patients suspected as IPN. Declarations