Identification 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 classified as pathogenic and the other two variants c.970A>T and c.422_423del are classified 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 classified as pathogenic, while c.3817C>T and c.3467T>A are classified as likely pathogenic (Table 1).
Clinical features of five genetically diagnosed patients
The detailed clinical features of five 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 five 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 first. Reflexes of five 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, finger-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 3 (II-1 in Family 3, Fig. 1C) was a 57-year-old male with involuntary limb movements for 4 years. At age 53, he noted episodic involuntary movements of lower limbs. The frequency and region developed progressively. At age 56, the involuntary movements affected the upper limbs and neck, and gait disturbance was significant with frequent falls. Neurological examinations showed general choreiform movements and gait unsteadiness.
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 paddyfield one year after diagnosis. Case 1, Case 4 and Case 5 progressed to bed after 2-3 years after diagnosis, and Case 5 finally died from pneumonia. Case 2 had mildest symptoms and was able to take care of himself 4 years after diagnosis.