WP07: Identification of new NCL genes
Sara Mole, PhD
University College London
To identify novel NCL genes by
- collecting new families, especially those that are consanguineous or multiply affected, focusing on those newly diagnosed from India
- excluding known gene loci from new families
- performing exome sequencing of excluded families as means of gene identification
- confirming / excluding candidate variants by segregation analysis and by screening appropriate control individual
- analysing the newly identified genes for mutations in known but genetically undefined NCL families
Collection of families with genetically undefined NCL
This task was extended to include genetically undiagnosed families received by any route, to supplement those received from India diagnosed with NCL (n=45). In addition to those families submitted by partners of DEM-CHILD, we received some Indian families through independent contact of participants by Indian clinicians. We also collected families through other European and international contacts. A total of 363 families were received requiring diagnosis from countries of the DEM-CHILD consortium plus 10 additional countries.
Excluding known gene loci from new families
Patient numbers diagnosed from DEM-CHILD countries are reflected in the epidemiology studies in WP03. In addition, 31 patients from countries outside the consortium were also found to carry mutations in known genes (CLN1, CLN2, CLN5, CLN6, CLN7, CLN8, CLN11). Mutation analysis of known genes has been performed in those families confirmed with NCL. Data on known and new mutations identified have been added into the NCL mutation database curated by partner 1. More are likely to be added beyond the end of the project.
About half the mutations were found in the gene CLN2, and 17% in both CLN6 and CLN7, suggesting that about 75% of new undiagnosed cases that have been excluded from CLN1 and CLN2 loci by rapid enzyme testing may represent CLN6 and CLN7 diseases. Interestingly no cases were found to carry mutations in CLN3, suggesting that recognition of classic juvenile CLN3 disease, is improving in these countries or that few cases are present.
Identification of new loci using exome sequencing
A total of 209 families were submitted for analysis by targeted sequencing to partner 6 with many positive hits identified, and nine families were submitted for independent analysis by partner 3.
Results of the deep NGS screens are still under inspection between partners, and there are indications for a number of yet unknown mutations. E.g., in one of the samples from India there is a deletion of 3 codons in the PCLO gene, which is one of the NCL candidate genes. The deletion could change the conformation of the encoded protein and seems to be homozygous. PCLO (homo sapiens piccolo presynaptic cytomatrix protein) is part of the presynaptic cytoskeletal matrix, which is involved in establishing active synaptic zones and in synaptic vesicle trafficking. Variations in this gene have been associated with bipolar disorder and major depressive disorder
Activities under this task on families collected from India have been negatively affected by the restriction of the Indian partner to provide samples to the project. However, some other locally held families have been subject to exome sequencing, through local collaboration. In families with childhood or teenage onset, this has resulted in the identification of one new disease gene ATP13A2/CLN12 in a Belgian family (Bras et al. 2012). All four affected siblings showed difficulties in learning from around the ages of 8 years characteristic for dementia, followed by rigidity, and akinesia. Both, vacuolated lymphocytes and ultrastructural pathology that resembled that for NCL were diagnosed but no mutation underlying the disease had been identified. Exome sequencing led to the identification of a single homozygous mutation in ATP13A2 Mutations in ATP13A2 are a known cause of Kufor-Rakeb syndrome (KRS), a rare parkinsonian phenotype with juvenile onset. These data show that NCL and KRS may share etiological features and implicate the lysosomal pathway in Parkinson's disease.
On new gene, ATP13A2 has successfully been identified in a collaborative project of partner 3.