Clinical heterogeneity and novel pathogenic variants of Chinese patients with neuroacanthocytosis

Background: Neuroacanthocytosis (NA) syndromes are a group of exceedingly rare diseases, including McLeod syndrome (MLS) and chorea-acanthocytosis (ChAc). The characteristic phenotypes comprise a variety of movement disorders, including chorea, dystonia, and parkinsonism. We aimed to investigate the clinical heterogeneity and novel pathogenic variants of Chinese patients with neuroacanthocytosis. Results: Three novel XK variants (c.942G>A, c.970A>T, c.422_423del) were identied in three index MLS patients and three novel VPS13A variants (c.3817C>T, c.9219C>A, c.3467T>A) in two index ChAc patients. In addition, we summarized the genotypes and phenotypes of reported MLA patients and ChAc patients in the Chinese population. Conclusion: Our study expands the genetic spectrum of XK and VPS13A and helps the clinical diagnosis of neuroacanthocytosis. chorea-acanthocytosis NA families and identied three novel XK variants (c.942G > A, c.970A > T, c.422_423del) in 3 index MLS patients and three novel VPS13A variants (c.3817C > T, c.9219C > A, c.3467T > A) in two index ChAc patients. In addition, we summarized the genotypes and phenotypes of reported MLA patients and ChAc patients in the Chinese population. Our study expands the genetic spectrum of XK and VPS13A and helps the clinical diagnosis of neuroacanthocytosis.


Introduction
Neuroacanthocytosis (NA) is a group of syndromes, including McLeod syndrome (MLS), choreaacanthocytosis (ChAc), Huntington's disease-like 2 (HDL2), and pantothenate kinase-associated neurodegeneration (PKAN). The acanthocytosis is a frequent nding in MLS and ChAc, but only occurs in approximately 10% of HDL2 and PKAN patients [1,2]. The "core" neuropathologic nding in NA syndromes is the degeneration affecting the basal ganglia, thus their clinical presentations can be remarkably similar [3]. The characteristic phenotypes comprise psychiatric and cognitive symptoms, and a variety of movement disorders including chorea, dystonia, and parkinsonism. The age of onset, inheritance pattern and ethnic background provide diagnostic clues in each condition, but clinical diagnosis remains challenging. Genetic diagnosis provides a de nitive diagnosis and is important for genetic counseling.
In the two core NA syndromes, MLS is inherited in an X-linked way with pathogenic variants of XK gene, while ChAc is an autosomal recessive disorder caused by pathogenic variants in VPS13A. It is estimated that there are likely a few hundred of MLS patients and around one thousand ChAc patients in the world [3]. Although more than 50 NA patients have so far been reported in China [4][5][6][7][8][9][10][11][12][13], most of which were not con rmed by speci c molecular tests [4]. The distribution of pathogenic variants within XK and VPS13A has not been conclusively determined in China.
Targeted next-generation sequencing (NGS) has been successfully utilized to diagnose neurological diseases which may be di cult to differentiate from clinical symptoms. In this study, we collected 14 unrelated Chinese patients who exhibited involuntary movements and had been ruled out the Huntington's disease (HD) by the HTT gene testing. We performed targeted NGS in these patients and 5 index patients were diagnosed with MLS or ChAc by Sanger sequencing veri cation. Three novel XK variants and three novel VPS13A variants were identi ed.

Subjects
Fourteen unrelated patients who presented suspected chorea with negative HTT genetic testing and their familial members were recruited between June 2015 and March 2019. The neurological examinations and clinical evaluations were performed by at least two senior neurologists. Routine blood tests and radiological examinations were performed. The study was approved by the Ethics Committee of Second A liated Hospital, Zhejiang University School of Medicine. Written informed consent was obtained from each participant.

Acanthocyte examination
Blood was collected into the tube containing ethylene-diamine-tetracetic acid (EDTA) and examined by scanning electron microscopy (SEM). Red cells were xed in a 1% solution of glutaraldehyde and stored at 4°C until the time of examination. The red cells were absorbed onto tissue paper and gold-coated with an Emscope Sputter Coater. They were then examined in a Nova nano 450 electron microscope operating at 5 kV. The acanthocyte rate was counted as the method described previously [14].

Genetic analysis
Genomic DNA was extracted from peripheral EDTA-treated blood by Blood Genomic Extraction Kit (Qiagen, Hilden, Germany). A customized panel was designed to cover 54 genes for chorea and dystonia, including VPS13A, XK, PRNP, 10 genes of neurodegeneration with brain iron accumulation (NBIA), 4 genes of primary familial brain calci cation (PFBC), and other 37 genes of hereditary dystonia (Supplemental Table 1). A detailed protocol of NGS was reported in our previous study [15]. Sanger sequencing was performed on an ABI 3500xl Dx Genetic Analyzer (Applied Biosystems, Foster City, USA) as our previous report to verify the ltered potential variants [16]. Co-segregation analysis was carried out in families with genetic variants. SIFT and PolyPhen-2 were used to predict the pathogenicity of the identi ed variants.

Results
Identi cation of novel XK and VPS13A variants After targeted NGS and Sanger sequencing, three novel hemizygous variants within XK gene were found in three index patients ( Fig. 1A-C). Among these three variants, two are nonsense (c.942G>A and c.970A>T) and one is frameshift (c.422_423del). They are all absent in 1000G, gnomAD and our NGS database containing 500 Chinese matched controls. According to American College of Medical Genetics and Genomics (ACMG) standards [17], c.942G>A is classi ed as pathogenic and the other two variants c.970A>T and c.422_423del are classi ed as likely pathogenic (Table 1).
In addition, three novel variants within VPS13A gene were detected in another two index patients, one of whom harbored a homozygous variant (c.3817C>T) and the other harbored compound heterozygous variants (c.3467T>A and c.9219C>A) ( Fig. 1D-E). Among these variants, two are nonsense (c.3817C>T and c.9219C>A) and one is missense (c.3467T>A). The three variants are at extremely low frequency in gnomAD (<=2) and absent in 1000G and our NGS database. The missense variant c.3467T>A was predicted to be deleterious by SIFT and PolyPhen-2. According to ACMG standards, c.9219C>A is classi ed as pathogenic, while c.3817C>T and c.3467T>A are classi ed as likely pathogenic (Table 1).

Clinical features of ve genetically diagnosed patients
The detailed clinical features of ve NA patients are summarized in Table 2. Three MLS index patients were all males with no family history of NA ( Fig. 1A-C). Two ChAc index patients were females, one of which was from consanguineous marriage ( Fig.1D) and the other had a positive family history (Fig.1E).
All ve patients had mild to moderate elevated creatine kinase (CK). Four patients had generalized chorea-like symptoms, and the brain magnetic resonance imaging (MRI) of them revealed atrophy of either caudate or putamina ( Fig. 2A-D). One MLS case (Case 2) showed gait disturbance, mild involuntary movements and markedly elevated CK, which made it confused with myopathy at rst. Re exes of ve NA patients were all reduced. Two of them received electromyogram (EMG) and showed axonal neuropathy. Apart from one case undetected, the acanthocyte rate of peripheral blood in four patients was between 18-32%, which was easily recognized in electron microscopy ( Fig. 2E-F). Only Case 2 received the Kell blood group phenotype in blood centre. The Kell antigen was absent in his erythrocytes, as well as the K, Kpa and Kpb antigens. The k antigen was weakly positive.
Case 1 (II-2 in Family 1, Fig. 1A) was a 47-year-old male with involuntary tongue and limb movements for over 20 years. At age 43, he noted progressive gait disturbance and tendency to fall. He went to the hospital and was diagnosed with "chorea". Then he was treated with risperidone and discontinued after several months. At age 47, he had a general tonic-clonic seizure and was treated with valproate.
Neurological examinations showed dysarthria and 4/5 weakness of left proximal lower limb.
Case 2 (II-4 in in Family 2, Fig. 1B) was a 41-year-old male with gait disturbance for 4 years. At age 37, he noted progressive gait disturbance and tendency to fall. His serum CK was markedly elevated, which could be over 4000U. Therefore, he was previously diagnosed with "metabolic myopathy". Neurological examinations showed mild postural instability and slight wadding gait. When he was sitting, mild involuntary movements of his toes and trunk were noted, just like akathisia. The muscle force, muscle tone, nger-to-nose test and heel-to-knee test were normal, which suggested that his gait instability was due to mild involuntary movements in trunk and lower limbs. Case 4 (II-2 in Family 4, Fig. 1D) was a 35-year-old female with involuntary limb movements for over 10 years. Her parents had a consanguineous marriage. At early of 20s, she presented mild involuntary movements which did not affect daily life. The symptoms worsen gradually and she developed dysarthria, drooling, orofacial dyskinesias with tongue and lip biting in the following years. At age 35, she had two generalized tonic-clonic seizures and was treated with levetiracetam. Neurological examinations showed general choreiform movements with dysarthria and orofacial dyskinesias.
Case 5 (II-2 in Family 5, Fig. 1E) was a 61-year-old female with involuntary limb movements for over 10 years. At age of about 50, she began to present with progressive general involuntary movements and then gradually developed dysarthria, orofacial dyskinesias and shoulder shrugging. Her neurological examinations showed general choreiform movements with dysarthria and orofacial dyskinesias. She had a 50-year-old brother who presented with similar symptoms at age of 40s. The brother could not walk and stayed in bed in a rehabilitation hospital for many years. He did not undergo the genetic testing.
The prognosis of most cases was poor. Case 3 died from falling in the paddy eld one year after diagnosis. Case 1, Case 4 and Case 5 progressed to bed after 2-3 years after diagnosis, and Case 5 nally died from pneumonia. Case 2 had mildest symptoms and was able to take care of himself 4 years after diagnosis.

Discussion
Neuroacanthocytosis syndromes are all exceedingly rare with an estimate prevalence of less than 1 to 5 per 1,000,000 for each disorder worldwide [3]. Currently, 43 pathogenic variants of XK gene have been included in the Human Gene Mutation Database (HGMD, v2019.4), including 13 missense/ nonsense variants, 6 splicing variants, 12 small deletion/ insertion/ indels and 12 gross deletions. In the Chinese population, six pathogenic variants of XK gene have been reported (Table 3), two of which were included in HGMD. Combined with three novel variants in the current study, there are 9 pathogenic XK variants including ve nonsense variants, two small deletions and two gross deletions. ChAc are relatively more common than MLS. There are 126 pathogenic variants of VPS13A in HGMD, including 41 missense/ nonsense variants, 25 splicing variants, 48 small deletion/ insertion/ indels and 12 gross deletion/ insertion/ complex rearrangements. Combined with three novel variants in our study, there are 10 pathogenic VPS13A variants in the Chinese population (Table 1 and Table 3), including 7 missense/ nonsense variants, one splicing variant and two small insertion/ deletions. These 19 pathogenic variants were all from single families and we did not nd any hotspot variant in the Chinese population.
MLS and ChAc share a number of similar features [18] and there is a striking phenotypic overlap in these two disorders, including age at onset, chorea, dysarthria, orofacial dyskinesia, seizures, psychiatric symptoms and brain MRI, but some clues may help to differentiate them. First, in genetics, MLS is inherited in an X-link way and ChAc in an autosomal recessive manner. Therefore, the family history and the gender can give important clues to help diagnosis. Besides, the large deletion in XK gene often involves multiple adjacent genes such as DMD, CYBB, RPGR and OTC, and leads to concomitant disorders such as dystrophinopathy, chronic granulomatous disease, retinitis pigmentosa and ornithine transcarbamylase de ciency [19]. Although our patients did not have large deletions, two Chinese patients with chronic granulomatous had been reported that their XK genes were involved in multigenic deletions [7]. It is important to screen MLS phenotype in males with chronic granulomatous disease for complication monitoring and management. In clinical manifestations, the orolingual dystonia is more frequently presented in ChAc. In our study and previous reports in the Chinese population, all ChAc patients had orolingual dystonia, but less than half MLS had the condition. In addition, the severity often disproportionate to other involuntary movements in ChAc. For example, self-mutilating tongue and lip biting, which present in 40% of Caucasians [20] and 85% (6/7) of reported Chinese ChAc patients, are very rare in MLS patients [18].
In the other hand, MLS patients seemed to have higher CK levels than ChAc in the Chinese population. About 78% of MLS patients (7/9) had CK above 1000 U/L, whereas only one of six ChAc patient reached this level. Moreover, MLS patients can have muscle complaints with mild involuntary movements, which often lead to a confusion with myopathy [5,6]. However, the EMG usually show axonal neuropathy, so sometimes these patients can be misdiagnosed with Kennedy's disease, which is also an X-link neurological disease manifests with proximal weakness, hyperCKemia, and axonal neuropathy-mimic EMG [21]. Therefore, it is important to pay attention to mild involuntary movement and differentiate MLS in males with both hyperCKemia and a neuropathic EMG. The acanthocyte rate in peripheral blood can usually fall 5-50% in ChAc and 8-30% in MLS [3], which is consistent with our results. However, the presence of acanthocytes may vary over time and can be absent in some cases [22][23][24][25], thus cannot be used to distinguish different NA syndromes.

Conclusion
In conclusion, we reported ve Chinese NA families and identi ed three novel XK variants (c.942G > A, c.970A > T, c.422_423del) in 3 index MLS patients and three novel VPS13A variants (c.3817C > T, c.9219C > A, c.3467T > A) in two index ChAc patients. In addition, we summarized the genotypes and phenotypes of reported MLA patients and ChAc patients in the Chinese population. Our study expands the genetic spectrum of XK and VPS13A and helps the clinical diagnosis of neuroacanthocytosis.

Ethics approval and consent to participate
The study protocol was in accordance with the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of Second A liated Hospital, Zhejiang University School of Medicine. Written informed consents were obtained from all the participants.

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Written informed consents for publication were obtained from all the participants.

Availability of data and material
The data that support the ndings of this study are available from the corresponding author upon reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This work was supported by the research foundation for distinguished scholar of Zhejiang University to Zhi-Ying Wu (188020-1938 10101/089).

Authors' contributions
Hao Yu and Liang Zhang: data acquisition, analysis and interpretation, and manuscript preparation; Xiao-Yan Li: data acquisition, analysis and interpretation; Zhi-Ying Wu: study design and conception, data acquisition, analysis and interpretation, critical revision of the manuscript.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. TableS1.docx