Chorea-acanthocytosis: Homozygous 1-kb deletion in VPS13A detected by whole-genome sequencing.

OBJECTIVE: To determine a molecular diagnosis for a large multigenerational family of South Asian ancestry with seizures, hyperactivity, and episodes of tongue biting.
METHODS: Two affected individuals from the family were analyzed by whole-genome sequencing on the Illumina HiSeq X platform, and rare variants were prioritized for interpretation with respect to the phenotype.
RESULTS: A previously undescribed, 1-kb homozygous deletion was identified in both individuals sequenced, which spanned 2 exons of the VPS13A gene, and was found to segregate in other family members.
CONCLUSIONS: VPS13A is associated with autosomal recessive chorea-acanthocytosis, a diagnosis consistent with the phenotype observed in this family. Whole-genome sequencing presents a comprehensive and agnostic approach for detecting diagnostic mutations in families with rare neurologic disorders.

Chorea-acanthocytosis (CHAC; MIM number 200150) is an autosomal recessive neurodegenerative disorder characterized by chorea and blood cells with abnormal morphology (acanthocytosis). Additional common features include dystonia, seizures, tics, and uncontrollable tongue biting. CHAC is caused by homozygous or compound heterozygous mutations in VPS13A,[1] which encodes chorein, a protein of unknown function. Here, we report a family with previously unexplained epilepsy found by whole-genome sequencing (WGS) to carry a novel mutation in VPS13A, leading to a diagnosis of CHAC.

Clinical report

A large multigeneration Pakistani family was recruited from the province of Khyber Pakhtunkhwa, Pakistan, presenting with a history of seizures, episodes of hyperactive and psychiatric problems and headache, nausea, sleep problems, and persistent tics with onset beginning late in the third decade of life. Within the family, there are 4 affected individuals across 3 generations and an additional younger participant suspected to be in the early stages of developing the same disorder.

Clinical details of index case IV:16

The index patient, a 37-year-old woman, first visited the neurologist at Shifa International Hospital, Islamabad, presenting with hyperactive behavior beginning 1 year prior, with episodes of tongue biting commencing 6 months later and 3 generalized tonic-clonic seizures during the month before the consultation. She had been diagnosed with epilepsy and migraine 3 years earlier but since developed hyperactivity and difficulty in concentrating in daily activities. She also suffered frequent tongue bites while speaking. On examination, she was found to have bilateral ptosis, but other cranial nerves were normal. Tone and power in all 4 limbs were normal, as were plantar responses. Higher mental function and speech were also typical. She was diagnosed as having a congenital syndrome with epilepsy, Tourette syndrome, and bilateral ptosis and prescribed escitalopram, procyclidine, carbamazepine, and later, haloperidol. One genetic test was sent for, namely Huntington disease, for which CAG repeats at the HD locus were normal (27 repeats). Red blood cell morphology was studied and reported normal.

Her parents were reportedly nonrelated, and the symptoms were also present in 3 other family members: participants III:1, IV:13, and V:8 in the figure. Individual V:6 was also suspected of being in the early stages of developing the same disorder. The family history and clinical features of affected individuals are described in table 1. Six additional family members (individuals III:16, III:21, V:2, V:4, V:9, and V:13 [figure]) were suspected to have a clinically separate neurologic disorder with symptoms including intellectual disability, seizures, migraine, depression, and osteoarthritis.

Figure

Pedigree of the family

“−” indicates the presence of VPS13A p.(Thr186_Leu232del). “+” indicates no deletion. Individuals without genotypes shown were not available for analysis. Black shapes indicate individuals affected with the primary disorder, and gray shapes indicate individuals affected with the second, separate phenotype. Individual V:7 died at the age of 10 years before the typical age at onset; thus, the affection status could not be determined. Numbers inside circles indicate more than 1 individual in the family. The index case is denoted by an arrow.

Table 1

Clinical features of affected individuals

Methods

DNA from 22 family members, including the 5 affected individuals, was extracted from whole blood using a standard phenol-chloroform method. Participants III-1 and IV-16 (index case) were selected for analysis by WGS. WGS was performed at The Centre for Applied Genomics (Toronto, Canada) with total genomic DNA following standard protocols. Library preparation was performed from 100 ng of DNA using the Illumina TruSeq Nano DNA Library Prep Kit following the manufacturer's recommended protocol. Libraries were pooled in equimolar quantities and sequenced on an Illumina HiSeq X platform following Illumina's recommended protocol to generate paired-end reads of 150 bases in length. Base calling and data analysis were performed using Illumina HiSeq Analysis Software version 2-2.5.55.1311. Reads were mapped to the hg19 reference sequence using Isaac alignment software (Isaac alignment software: SAAC00776.15.01.27), and SNV and small indel variants were called using the Isaac variant caller (Isaac Variant Caller [Starling]: 2.1.4.2). Rare variants were defined as those with less than 1% frequency in population databases.[2-4] CNVs were detected using the read depth methods as previously described.[5] Rare CNVs were defined as those less than 1% frequency among unrelated, unaffected individuals sequenced using the same technology.[6] Participants were screened for regions of homozygosity using PLINK (v1.90) “Runs of homozygosity” implementation.

Standard protocol approvals, registrations and patient consents

Informed consent was obtained from all participants, and this study was approved by The Hospital for Sick Children Research Ethics Board, Toronto, Canada.

Results

Through analysis of rare CNVs detected from WGS data, a 1168-bp deletion was detected, spanning exons 8 and 9 of VPS13A (chr9:79827422-79828590); c.556_696del; p.(Thr186_Leu232del). The deletion was homozygous in both individuals sequenced and would likely result in an in-frame deletion of 47 amino acids. PCR amplification and Sanger sequencing confirmed the deletion and were used to test the pattern of segregation among all family members, where DNA was available (figure). Homozygosity mapping from the WGS data for participants III-1 and IV-16 showed that the deletion resides in an extended region of homozygosity in both individuals (chr9:78,311,259-98,376,367 in III-1 and chr9:79,451,734-97,016,027 in IV-16). Individuals IV:13, V:8, and V:6 were found to be homozygous for the deletion. Family relationships were confirmed by the analysis of short tandem repeats. Analysis of single nucleotide variants and small insertion/deletions did not yield any pathogenic or likely pathogenic variants (all homozygous variants in coding regions identified in either individual are shown in table e-1, links.lww.com/NXG/A50). On peripheral blood smear, acanthocytes were detected in 3 of the 4 individuals with the primary condition (III:1, IV:13, and V:8) and in V:6, but not in the index case IV:16.

Discussion

The phenotype of the affected individuals is in keeping with the known CHAC phenotype. All 4 experienced choreoathetosis, seizures, and cognitive/psychiatric symptoms. Four members of the family were found to have acanthocytes on peripheral blood smear, supporting the diagnosis of CHAC. The molecular findings and hematology testing also confirmed the diagnosis of the youngest individual, V:6, presenting with the early stages of the condition. None of the 6 individuals with the second phenotype was homozygous for the deletion in VPS13A (3 did not carry the variant, and 3 were heterozygous), confirming that they likely have a second, distinct disorder.

There are 4 neuroacanthocytosis syndromes (CHAC, McLeod syndrome, Huntington disease-like 2, and pantothenate kinase–associated neurodegeneration) with similar but distinct phenotypic features, and the clinical presentation of CHAC also overlaps with other disorders, presenting a diagnostic challenge.[7,8] The presence of acanthocytes can be helpful to suggest a diagnosis of CHAC, but hematology results are inconsistent,[7,8] as is the case in this family. With WGS, a precise diagnosis could be rapidly achieved without the requirement of any prior awareness of the disorder by the referring clinician. Achieving a correct diagnosis now allows accurate anticipation of the course of the disorder (particularly in the case of the youngest, mildly affected individual) and facilitates appropriate treatment.[9] The diagnosis also prevents further unnecessary testing for possible nutritional or infectious etiologies and helps to remove any stigma associated with behaviors in the family.

The nature of the mutation in this family further demonstrates the benefit of using WGS compared with other nontargeted technologies. Such a small deletion would be too small to detect by microarray analysis and challenging to identify using exome sequencing. It may also not have been detected using a targeted gene or gene panel test, depending on the specific methodologies used. Small CNVs affecting VPS13A, such as the one identified in this family, have been found previously in cases with CHAC, although the deletion we describe had not been reported to date.[10]

Here, we present a large multigenerational family from Pakistan with chorea-acanthocytosis attributable to a 1168-bp, 2-exon deletion in VPS13A. This family showcases the potential for WGS as a first-tier diagnostic test in neurologic disorders.