Genomic DNA from the proband was used to perform exome sequencing using the Agilent SureSelectXT capture kit (Agilent Technologies, Santa Clara, CA). The sample was sequenced with mean coverage of 80 to 100× on an Illumina HiSeq 2500 sequencing platform (Illumina, San Diego, CA). Sequenced data were aligned to the human reference genome (GRCh37/hg19) using the BWA program.[4, 5] Subsequent to this, the GATK-Lite pipeline was used for realignment, base recalibration, variant calling, and variant filtering.[6, 7] Filtered variants were annotated using the VariMAT pipeline (internal data analysis pipeline of MedGenome), and clinically relevant mutations were identified from published literature and various databases, including ClinVar, OMIM, SwissVar, HGMD, GWAS, ExAC, 1000-Genome, dbSNP, EVS.[8-15] Only nonsynonymous and splice-site variants detected in the targeted genes were used for clinical interpretation.
Whole-exome sequencing, performed to identify the causative gene variation, did not detect any literature-reported variations for the clinical symptoms. A homozygous nonsense variation in exon 22 of ATP13A2 gene (chr1: 17316187; G>A), which results in a stop codon and premature truncation of the protein at codon 826 (ENST00000341676:c.2476C>T, ENSP00000341115:p.Q826Ter), was detected. This variant was not found in different common variation databases, such as 1000Genome, ExAC, dbSNP, and EVS. The variant was also not detected in 100 normal controls that were screened by us. The Exome Aggregation Consortium does not report this variant in its database. After this, analysis of both the parents showed the same mutation in the heterozygous state, indicating that they are the carriers for this likely pathogenic mutation (Fig. 1).
Figure 1. (A) Integrated genomic viewer (IGV) snapshot of next-generation sequencing reads showing the nonsense variant (chr1:17316187; G>A, ENST00000341676: c.2476C>T, ENSP00000341115:p.Q826Ter) detected in ATP13A2 gene of the INDEX case is shown. The x-axis represents the reference genome (hg19/GRCh37) and the exon-22 ofATP13A2 gene. The y-axis represents the total reads detected in the sample using exome sequencing. (B) Sequence chromatogram showing the same variation in heterozygous state detected in mother, and father of index case.
Since the initial description of KRS, case reports/series have been published from various parts of the world, including Pakistan and Afghanistan. To date, no documented cases have been published from India. Initial description of KRS was a rapidly progressive disorder with early development of significant motor disabilities. However, newer publications have indicated variable phenotypes, including that of intact cognition, absence of myoclonus/tremors, and slower clinical progression. Most of these patients have good l-dopa response. In comparison to published literature, our case had a clear autosomal-recessive inheritance, with both parents being heterozygous carriers, typical onset, and early ocular signs. In contrast to classical descriptions, he did not have any cognitive deficits, has slow clinical progression, and no myoclonus or hallucinations. Our case also had a nonsense mutation, which has not been documented to date. In being the first case reported from India, and also with higher prevalence of consanguinity in Southern India, this may provide the option to screen for juvenile-onset parkinsonism and study clinical heterogeneity.