The index patient (Fig. 1: IV–7) developed slowly progressive cerebellar symptoms with balance and coordination problems primarily affecting gait and manual movements at the age of 12. He rarely cried as an infant, had nystagmus and poor handwriting and was a double graduate, though needing a writer for exams. The index case’s condition was significantly exacerbated by a stroke due to thrombosis in the left transverse sinus at age 60. In this context, an MRI was acquired that showed severe cerebellar and some degree of midbrain atrophy (Fig. 2), and polycythemia vera was diagnosed and subsequently controlled with medication and phlebotomy. It was only after the stroke that vision was impaired due to the ischaemic event and subsequent falls led to parenchymal and subdural haematomas causing aphasia, disorientation, aggression, a very strong grip with stereotypy of hand and neck with the head tilting backwards. He died one year ago at the age of 70. Regarding the movement disorder, the paternal cousin of the index patient (Fig. 1: IV−3, currently at age 68) developed similar symptoms around puberty with pure cerebellar ataxia leading to frequent falls and leg fractures, for which she is now wheelchair bound. She has dysmetric slow saccades and besides poor handwriting, she does not have any further complicating symptoms being mentally alert, though speech is slow. In both affected individuals, ataxic gait and brisk lower limb reflexes suggested early onset cerebellar ataxia with retained reflexes (EOCARR). Nerve conduction studies were within normal limits. EMG recordings were suggestive of bilateral S1 segment lesion at the proximal level for both patients and additional left L5 segment lesion at proximal level for the affected cousin (IV−3). There was no hypogonadotropic hypogonadism or chorioretinal dystrophy in both of them. Clinical data was available from 22 individuals of the family (Fig. 1), whereof only the two reported cousins were found symptomatic.
Homozygosity mapping revealed only one large region of shared homozygosity by the two affected cousins on chromosome 19 (Supplementary Figure 1), spanning over 4 Mb (Chr19: 3630740–7759053). This region comprises 132 genes, from which two (ATCAY, PNPLA6) were particularly interesting candidates given the phenotype of our patients. WES revealed one synonymous change in SAFB2 (unlikely to be related with the disease) and two homozygous missense changes in PNPLA6 at c.3847G>A (p.V1283M) and c.3929A>T (p.D1310V) in exon 32, transcript ENST00000414982 (see Fig. 3a for filtering strategy). The PNPLA6 variants were confirmed by Sanger sequencing (Fig. 3b) and segregated perfectly with the disease in all 20 unaffected and two affected individuals tested (Figs. 1 and 3b): Nine heterozygous carriers were detected in four generations, and only the two affected cousins were homozygous. All exons of ATCAY and PNPLA6 were also screened by Sanger sequencing and ruled out the presence of additional variants that could have been missed by WES. Screening of 40 British patients with pure cerebellar phenotype revealed no further mutations in PNPLA6. No additional mutations in known autosomal recessive cerebellar ataxia genes were detectable on the exome.
The variants found in our consanguineous family (p.V1283M and p.D1310V, see Table 1 for summary) are not present in public databases (Exome Sequencing Project variant server (EVS), dbSNP, Exome Aggregation Consortium (ExAC), Complete Genomics 69 (cg69) and 1000 Genomes). The PNPLA6 c.3929A>T, p.D1310V was conserved across species (Table 1 and Fig. 3c), and predicted to be disease causing by three prediction tools (Table 1). Since the variant c.3847G>A, p.V1283M, with more benign prediction scores (Table 1), lies at the first nucleotide of exon 32, we investigated potential effects on splicing (e.g. exon skipping) by Sanger sequencing cDNA from the affected cousins and their carrier and wildtype siblings. No splicing changes were detected around exon 32 (see Fig. 3b).