Novel mutations in HINT1 gene cause autosomal recessive axonal neuropathy with neuromyotonia in two cases of sensorimotor neuropathy and one case of motor neuropathy

Accepted Manuscript

Novel mutations in HINT1 gene cause autosomal recessive axonal neuropathy with neuromyotonia in Chinese patients. Linchao Meng , Jun Fu , He Lv , Wei Zhang , Zhaoxia Wang , Yun Yuan PII: DOI: Reference:

S0960-8966(17)31219-1 10.1016/j.nmd.2018.05.003 NMD 3549

To appear in:

Neuromuscular Disorders

Received date: Revised date: Accepted date:

8 August 2017 3 May 2018 9 May 2018

Please cite this article as: Linchao Meng , Jun Fu , He Lv , Wei Zhang , Zhaoxia Wang , Yun Yuan , Novel mutations in HINT1 gene cause autosomal recessive axonal neuropathy with neuromyotonia in Chinese patients., Neuromuscular Disorders (2018), doi: 10.1016/j.nmd.2018.05.003

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Highlights We describe novel mutations of HINT1 in 3 Chinese patients with ARAN-NM.

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This is the first report of HINT1 related ARAN-NM in Chinese population.

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We expand the genotypic spectrum of HINT1 mutations.

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1

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Department of Neurology, First Hospital, Peking University, Beijing 100034, China

Novel mutations in HINT1 gene cause autosomal recessive axonal neuropathy with

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neuromyotonia in Chinese patients.

1

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Linchao Meng1, Jun Fu1, He Lv1, Wei Zhang1, Zhaoxia Wang1, Yun Yuan1

Department of Neurology, Peking University First Hospital, 8 Xishiku Street,

Xicheng District, Beijing 100034, China.

E-mail adress of each author: Linchao Meng ([email protected]), Jun

([email protected]),

He

Lv

Zhaoxia

Wang

([email protected])

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Yuan([email protected])

([email protected]),

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Fu([email protected]),

Corresponding author: Yun Yuan

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E-mail: [email protected] Fax: +86-10-66176450

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Tel: +86-10-83572110

This research was supported by a grant from the Twelfth Five-year Plan of China (No. 2011ZX09307-001-07).

Conflict of Interest The authors declare that they have no conflicts of interest. 2

Wei

Zhang

，

Yun

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Abstract Autosomal recessive axonal neuropathy with neuromyotonia (ARAN-NM) is a rare disease caused by mutations of histidine triad nucleotide binding protein 1 (HINT1) gene. ARAN-NM has been reported mainly in European countries and is little

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reported so far in China. Here, we described novel mutations of HINT1 in three Chinese patients with ARAN-NM from unrelated families. Patient 1 was a 14-year-old girl who had presented with progressive distal weakness since two years

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old. After that, she reported weakness of both feet, and difficulty in muscle relaxation after making a fist. Patient 2 was a 18-year-old boy, who had presented with progressive distal weakness of all limbs with feet drop since the age of ten and loss of

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ambulation at age 15. Patient 3 was a 26-year-old man who had been afflicted with

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weakness and atrophy of distal lower limbs since sixteen years old, complaining about muscle stiffness of lower limbs when standing and walking and contraction of finger

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flexion muscles when releasing a forced grip. Electrodiagnostic testing revealed an

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axonal motor or motor-sensory neuropathy with or without myokymic discharges. Sural biopsy showed no pathological changes in patient 1 and mild axonal

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neuropathies with demyelination in patient 2 and 3. Genetic analysis proved HINT1 with novel compound heterozygous c.112T>C (p.C38R) and c.171G>C (p.K57N) mutations in patient 1, homozygous c.112T>C (p.C38R) mutation in patient 2, as well as compound heterozygous c.112T>C (p.C38R) and c.98T>C (p.F33S) mutations in patient 3. Our study, for the first time, confirmed ARAN-NM in Chinese population. The geneitc findings can help expand the genotypic spectrum of HINT1 mutations. 3

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Key words: HINT1, hereditary neuropathy, neuromyotonia Introduction Hereditary peripheral neuropathies comprise a heterogeneous spectrum of disorders, for which more than 100 different subtypes have been identified, with each

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having its own specific clinical characteristics, pathophysiology and prognosis [1,2]. In addition to length dependent neuropathies, there were several types of hereditary peripheral neuropathies with unusual symptoms, such as the central nerve system

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involvement in X-linked Charcot-Marie-Tooth disease [3,4], focal segmental glomerulosclerosis in INF2 mutations associated with dominant inherited intermediate Charcot-Marie-Tooth neuropathy [5], cardiomyopathy in hereditary transthyretin

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amyloidosis [6], C12orf65 gene related distal motor neuropathy with optic atrophy[7]. A distal motor neuropathy with neuromyotonia had been described by Hahn et al in

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1991[8]. In 1998 Maurelli M, et al reported two siblings affected by hereditary motor

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and sensory neuropathy type II with neuromyotonia[9]. In 2012, the recessive mutations in the gene encoding the histidine triad nucleotide binding protein 1

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(HINT1) were identified by Zimon et al in axonal neuropathy with neuromyotonia

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(ARAN-NM) with an autosomal recessive pattern[10]. Since then, several families of European origin with HINT1 mutations have been described [11-15]. Here, we described three Chinese patients with ARAN-NM caused by mutations in HINT1. This is the first report for HINT1 related ARAN-NM in Chinese populations. Patients and Methods Patient 1 was a 14-year-old girl, the only daughter of unrelated parents (Figure1). 4

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There were no other family members with the history of neuromuscular disorders. She developed weakness of both hands, especially finger extension at the age 2. The disease developed slowly to her feet with weakness of bilateral toe dorsiflexion at age 8. Starting from age 11, she reported difficulty in muscle relaxation after making a fist.

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At the time of diagnosis, her hands were more severely affected than her feet, but only with very mildly affected motor function. Her sensory was intact. Muscle strength was 3/5 for finger extension, 4/5 for toe dorsiflexion and normal for proximal muscles

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in each limb (grades 0–5 on the Medical Research Council scale). Muscle atrophy occurred in intrinsic hand muscles. A decrease of the biceps brachii, triceps, patellar, and Achilles reflexes was observed. She had marked difficulty releasing a forced grip.

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The level of serum creatine kinase (CK) was 1122 U/L (0-190 U/L). Sensory conduction velocity (SCV) was within normal limits in all examined nerves. Motor

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conduction velocity (MCV) and amplitude of compound muscle action potentials

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(CMAPs) were unremarkable in the right median nerve, but reduced CMAPs (1.67mv) were observed in the left ulnar nerve. The distal motor latency (DML) was prolonged

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in the right median nerve (4.73ms) and left ulnar nerve (4.48ms). MCV of the

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common peroneal nerve was mildly slow (39.4m/s), with prolonged DML (6.55ms) and reduced CMAPs (0.37mv). Electromyography (EMG) revealed complex repetitive discharges (CRD) on the bilateral extensor digitorum communis, right caput mediale musculi gastrocnemii and left vastus medialis obliquus, and myokymic discharges on the right vastus medialis obliquus (Figure2). Except the left vastus medialis obliquus, EMG showed a neurogenic pattern in all the examined muscles. 5

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Therapeutic trial with carbamazepine was partially effective for her difficulty of releasing a forced grip.

Patient 2 was an 18-year-old boy, the only son of unrelated parents (Figure 1). There were no family members with similar symptoms and no parental consanguinity.

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He had suffered from progressive gait impairment, with feet drop and bilateral strephenopodia since the age of 10. From the age of 11, he had weakness of both hands with contracture of finger joints. At the age of 15, he lost ambulation and was

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wheelchair-bound due to severe strephenopodia, pes cavus and contracture of Achilles tendon. Muscle strength was 2/5 for finger extension and toe dorsiflexion, while proximal muscle strength was 4/5 for upper limbs and 3/5 for lower limbs. Mild

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atrophy of intrinsic hand muscles and calf muscles was seen. Deep tendon reflexes

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were absent in any of the extremities. CK level was not tested. SCV was within normal limits in the left median nerve, but slow (40.1m/s) with reduced sensory nerve

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action potential (SNAP) (4.2μv) in the left ulnar nerve. SNAP was not evocable in

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the left superficial peroneal nerve. MCV was within normal limits in the right median nerve (54.1m/s) with prolonged distal motor latency DML (4.94ms) and reduced

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amplitude of CMAP (3.0mv). MCV was slightly slow (48.2m/s) in the left ulnar nerve with prolonged DML (4.44ms) and reduced CMAPs (3.0mv). CMAP of the common peroneal nerve was not recordable. EMG showed a neurogenic pattern with CRD. Patient 3 was a 26-year-old male, the only son of unrelated parents. No family history of neuromuscular disorders was found in his family. There were no abnormalities for his developmental milestones. He presented with progressive 6

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numbness, weakness with atrophy of distal lower limbs at age of 16 years. At the same time he noticed muscle stiffness of lower limbs during standing and walking. There was no obvious weakness of upper limbs, but he felt contraction of finger flexion muscles when releasing a forced grip. Neurological examination revealed loss

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of sensation in the distal lower limbs. Muscle strength was 4/5 in distal muscles of the lower limbs, compared to 5/5 in proximal muscles of all limbs. He had atrophy of bilateral calf. Bilateral Achilles reflexes were absent. After he made a fist, the finger

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flexion muscles still shrank. The CK level was 576 U/L. SCV was within normal limits in the left median nerve, but slow in the left ulnar nerve (41.7m/s) and left superficial peroneal nerve (28.8m/s).MCV and amplitude of CMAP were within

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normal limits in the right median nerve and left ulnar nerve, but prolonged DML was found in both nerves (4.23ms and 3.25ms). MCV of the common peroneal nerve was

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slow (34.7m/s) with reduced amplitude of CMAP (0.92mv). EMG displayed a

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neurogenic pattern with CRD, myokymic discharges. Because of very mild symptom, he refused the treatment of myorelaxants.

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The clinical and electrophysiological features of the 3 patients were summarized

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in Table 1. Family pedigrees for 3 families were shown in Figure 3. The study was approved by the Ethics Committee of Peking University First

Hospital. Written informed consent was signed by each participant before initiation of the investigation in compliance with the Declaration of Helsinki. Sural nerve biopsies were taken in the calf for the three patients. Some of the samples were fixed in 4% formaldehyde, paraffin-embedded, 8-µm sections, and 7

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stained with hematoxylin and eosin (HE), congo-red, as well as luxol fast blue (LFB). The HINT1 mutations from the three patients were analyzed. Five ml of peripheral blood and extracted genomic DNA were obtained by the method of salt precipitation. Next generation sequencing (NGS) was used to identify HINT1

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mutations and sanger sequencing with specific primers to confirm the mutations. NGS was conducted using a NGS panel covering all exons and their flanking sequences of genes known to cause hereditary motor and sensory neuropathy (HMSN). Segregation

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analysis of the mutations was performed in the parents of these patients. When novel mutations were found, 100 healthy control participants of Chinese origin were screened. Allele frequencies were analyzed with the Genome Aggregation Database HomoloGene

Web

server

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(http://gnomad.broadinstitute.org).

(http://www.ncbi.nlm.nih.gov/sites/entrez?db=homologene) was used to compare

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HINT1 sequences between different species. Predictions of the functional effect of

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HINT1 mutations were completed with Mutation Taster (mutationtaster.org), PolyPhen 2 (genetics.bwh.harvard.edu/pph2), and SIFT (sift.jcvi.org) prediction

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software in silico.

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We performed Single Nucleotide Polymorphisms (SNP) array genotyping on the

Illumina Global Screening Array platform to clarify whether it was a founder mutation, if all the patients carried the same mutation.

Results Nerve Biopsy 8

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Sural nerve biopsy of patient 1 showed no pathological changes (Figure 4). Patient 2 and 3 had a normal density of myelinated fibers with a few regenerating clusters and thin myelinated fibers. Genetic Analysis

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It was found that patient 1 had compound heterozygous c.112T>C and c.171G>C mutations in HINT1, leading to amino acid substitutions of Cystine to Arginine (p.C38R) and Lysine to Asparagine (p.K57N). Her parents were heterozygous carriers.

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Her father carried the c.112T>C mutation, whereas her mother carried the c.171G>C mutation. Homozygous c.112T>C (p.C38R) mutation in HINT1 was found for patient 2, each parent of whom carried the same heterozygous mutation, respectively. Patient

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3 had compound heterozygous c.112T>C and c.98T>C mutations in HINT1, causing amino acid substitutions of Cystine to Arginine (p.C38R) and phenylalanine to Serine

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(p.F33S). His parents were heterozygous carriers. His father carried the c.112T>C

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mutation, whereas his mother carried the c.98T>C mutation.

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None of the above mutations (p.C38R, p.K57N, p.F33S) has ever been reported in the literature. According to the Genome Aggregation Database, p.K57N was not

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detected, and population allele frequencies of p.C38R (0.00001250) and p.F33S (0.000004071) were very low. P.C38R, p.K57N, p.F33S were predicted to be disease causing by Mutation Taster, probably damaging (score of 1.000 for p.C38R and 0.984 for p.K57N) and possibly damaging (score of 0.520 for p.F33S) by PolyPhen 2, and tolerated by SIFT (score of 0.00 for all three mutations). Among vertebrates, the amino acid residues C38, K57 and F33 were relatively evolutionarily conserved 9

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(Figure 5). The three families came from different provinces in China, northeast, east and south. Interestingly, all the patients carried c.112T>C (p.C38R) mutation in HINT1. It was important to charity whether it was a founder mutation. A SNP array identified a

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5.3-Mb homozygous region on chromosome 5q23.3q31.1 (genomic coordinates 127,330,108–132,641,670 GRCh37) carried by patient 2, in whom the SNP array was performed (Figure 6). This region encompassed the HINT1 gene. This variant

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occurred in the homozygous state in the context of no evidence for parental consanguinity, suggesting that the variant should arise as a founder mutation in Chinese population. The other two patients shared a smaller haplotype of 2.1-Mb

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encompassing the HINT1, as a result of meiotic recombination.

Discussion

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HINT1 related ARAN-NM has been identified since 2012[10]. Clinically, as in

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the case of our patients, the onset age is reported to be between 3 and 28 years of age, mostly in the first decade of life. Actually, the progression of the disease is very slow,

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and none of the reported patients lost ambulation until the sixth decade of life, but patient 2 in our study needed help of a wheelchair, indicating the relatively severe phenotype with early motor dysfunction [10, 16, 17, 18]. Our patients had gait impairment, distal weakness, leg stiffness, difficulties in releasing grip after a strong voluntary hand contraction, atrophy of intrinsic hand and calf muscles, foot deformities, finger contractures and mild distal sensory impairment, which indicated 10

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motor-greater-than-sensory polyneuropathy. Neuromyotonia, characterized clinically by muscle twitching at rest (visible myokymia), cramps, which can be triggered by voluntary or induced muscle contraction, and impaired muscle relaxation, or pseudomyotonia, which is present in 70-80% patients with the disease, was present in

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two of our three patients[12,19]. Hyperckemia is also a common feature for ARAN-NM, probably as a result of neuromyonia, highlighting the need to differentiate this hereditary neuropathy from muscle diseases, particularly when

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sensory element is less prominent[15].

Electrophysiological studies of peripheral nerves for the three patients were compatible with motor and sensory polyneuropathy, or pure motor polyneuropathy.

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CMAP and SNAP were decreased or absent, though conduction velocities of motor and sensory fibres were normal or mildly slow, indicating axonal impairment. EMG

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displayed CRD for all the patients, myokymic discharges for patient 1 and 3, but no

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neuromyotonic discharges in any of them. Neuromyotonic or myokymic discharges are absent in around 20-30% of the patients, making accurate diagnosis difficult for

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some cases if clinical and electrophysiological characteristics alone are taken into

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consideration[10,13,20]. Sural nerve biopsies of our three patients showed no or only mild pathological

changes, indicating motor-predominant characteristics for HINT1 related ARAN-NM. As hyperCKemia is common in the disease probably because of neuromyotonia, muscle biopsies are sometimes conducted to show the neuropathic process and type 2 fiber atrophy in order to rule out the possibility of myopathies[14,17]. 11

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In the study, we identified novel mutations (p.C38R, p.K57N, p.F33S) of HINT1 as the genotypes for the three Chinese patients with ARAN-NM. So far, less than 100 patients have been diagnosed with HINT1 related ARAN-NM, mainly in Europe and slightly in North America (Table 2) [12, 14, 15, 17]. Previous reports have identified

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p.R37P as the most common mutation for ARAN-NM, especially in the Czech populations [10, 16], where mutations in the HINT1 gene are the fifth most frequent

known type of hereditary neuropathy after CMT1A/HNPP and mutations in the GJB1

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gene and the MPZ gene [16]. Other variants, including p.C84R, p.H112N et al, have also been reported since 2012. Our study has expanded the genotypic spectrum of HINT1 mutations. We have proved the same novel p.C38R mutation in the three

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patients may result from a founder mutation in Chinese population. More case reports and studies are needed. Interestingly, an Europe research showed absence of HINT1

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mutations in a UK and Spanish cohort of patients with inherited neuropathies,

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supporting that the disease is not homogeneously distributed among European population[21].

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The exact pathogenesis about HINT1 mutations causing ARAN-NM is unknown.

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It has been proved that HINT1 mutations are loss-of-function mutations from functional studies [10]. Studies from Seburn KL showed that HINT1 knockout mice moved more slowly and for a smaller fraction of time than wild-type mice, had normal behavior and motor test results, and lacked significant electrophysiological or functional alterations [22]. Conclusions 12

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In conclusion, HINT1 mutations can cause ARAN-NM. In Chinese population, genotypes of HINT1 mutations may be different from European populations, indicating the genetic heterogeneity in different regions of the world. So far as we know, our study is the first report of HINT1 related ARAN-NM in Chinese population,

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expanding the genotypic spectrum of HINT1 mutations References 1.

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Loss-of-function mutations in HINT1 cause axonal neuropathy with neuromyotonia. Nat Genet 2012; 44: 1080-3

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Absence of HINT1 mutations in a UK and Spanish cohort of patients with inherited neuropathies. J Neurol 2015; 262: 1984-6.

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12. Peeters K, Chamova T, Tournev I, Jordanova A. Axonal neuropathy with neuromyotonia:

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there is a HINT. Brain 2017; 140: 868-77. 13. Zhao H, Race V, Matthijs G, De Jonghe P, Robberecht W, Lambrechts D, et al. Exome

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Eur J Hum Genet 2014; 22: 847–50.

14. Jerath NU, Shy ME, Grider T, Gutmann L. A case of neuromyotonia and axonal motor neuropathy: A report of a HINT1 mutation in the United States. Muscle Nerve 2015; 52: 1110-3. 15. Veltsista D, Chroni E. A first case report of HINT1-related axonal neuropathy with neuromyotonia in a Greek family. Clin Neurol Neurosurg 2016; 148: 85-7. 14

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16. Laššuthová P, Brožková DŠ, Krůtová M, Neupauerová J, Haberlová J, Mazanec R, et al. Mutations in HINT1 are one of the most frequent causes of hereditary neuropathy among Czech patients and neuromyotonia is rather an underdiagnosed symptom. Neurogenetics 2015; 16: 43-54.

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18. Rauchenzauner M, Fruhwirth M, Hecht M, Kofler M, Witsch-Baumgartner M, Fauth C. A Novel variant in the HINT1 gene in a girl with autosomal recessive axonal neuropathy with neuromyotonia: thorough neurological examination gives the clue. Neuropediatrics

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20. Boaretto F, Cacciavillani M, Mostacciuolo ML, Spalletta A, Piscosquito G, Pareyson D,

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et al. Novel loss-of-function mutation of the HINT1 gene in a patient with distal motor axonal neuropathy without neuromyotonia. Muscle Nerve 2015; 52: 688–9.

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21. Horga A, Cottenie E, Tomaselli PJ, Rojas-García R, Salvado M, Villarreal-Pérez L, et al.

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Absence of HINT1 mutations in a UK and Spanish cohort of patients with inherited neuropathies. J Neurol 2015; 262: 1984-6.

22. Seburn KL, Morelli KH, Jordanova A, Burgess RW. Lack of neuropathy-related phenotypes in hint1 knockout mice. J Neuropathol Exp Neurol 2014; 73: 693–701.

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Figure legends

Figure 1. Clinical findings for patient 1 (A, B) and patient 2 (C, D). It was difficulty

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in muscle relaxing after making a fist for patient 1 (A: at rest, B: after making a fist).

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in patient 2 (C, D).

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Contracture of finger joints, atrophy of intrinsic hand, and strephenopodia were seen

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Myokymic discharges for patient 1.

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Figure 2.

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Figure 3. Family pedigrees for the three families. Arrows indicate the family

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probands.(A. patient 1 family; B. patient 2 family; C. patient 3 family)

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Figure 4. Sural nerve pathology for patient 1, with nearly normal features (A. HE; B.

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Toluidine blue-stained)

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Figure 5. Chromatograms for HINT1 mutations for three patients and their parents (A,B and C); sequence conservation analysis for amino acid residues C38, K57 and F33 (D) 20

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Figure 6. View of complete chromosome 5 on the SNP array for paitent 2 who

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carried HINT1 c.112T>C (p.C38R) homozygous mutation.

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Table 1． Clinical and electrophysiological features of the 3 patients Author Patient 1 Patient 2 Age at onset in 2 10 years Onset symptoms Weakness of both Gait impairment hands All proximal and distal limbs

Yes

No

No No

Yes No

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Distal upper and lower limbs

Numbness, weakness with atrophy of distal lower limbs Distal lower limbs

Yes No Yes

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Distribution of muscle weakness Symptoms of neuromyotonia Foot deformity Sensory symptoms NCS EMG

Patient 3 16

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Motor neuropathy Motor and sensory Motor and sensory Neurogenic pattern neuropathy neuropathy with CRD, Neurogenic pattern Neurogenic pattern Serum creatine myokymic with CRD with CRD, myokymic kinase discharges discharges 1122 U/L Not tested 576 U/L NCS, nerve conduction studies; EMG, electromyography; CRD, complex repetitive discharges.

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Genotype

p.R37P+ p.R37P p.R37P+ p.C84R p.R37P+ p.G89V p.R37P+ p.H112N p.H51R+ p.C84R p.Q62X+ p.G93D p.H112N+ p.H112N p.W123 X + p.W123 X p.R37P+ p.R37P p.R37P+ p.Q106 X p.H112N+ p.H112N p.E34K+ p.E34K p.H51Ffs*18

Laššuthová et al.[16]

19(21)

Caetano et al.[17] Rauchenzauner et al.[18] Boaretto et al.[20] Jerath et al.[14] Zhao et al.[13]

1(1)

HMSN, HMN

10/12**

6

HMSN

1/1

1(1)

3

HMSN

1/1

1(1)

Early HMN childhood 13 HMN

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Table 2． Publication review of cases with HINT1 related ARAN-NM Author Families Age at Neuropathy Cases with (Cases) onset in type neuromyotonia years (Clinical and/or EMG) Zimoń et 33(50) 3-25 HMSN, 39/50 al.[10] HMN

p.R37P+ p.R37P 2(2) 17, 28 HMN 0/2 p.C84R+ p.C84R p.C84R+ p.H114R Veltsista et 1(2) 4, 17 HMSN 2/2 p.R37P+ al.[15] p.R37P **Not available for some cases; EMG, electromyography; HMSN, hereditary motor and sensory neuropathy; HMN, hereditary motor neuropathy

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1(1)

0/1 1/1