We have confirmed that mutations in DNAJC5
cause autosomal dominant ANCL 
. However, ANCLs are disorders clinically and genetically very heterogeneous 
. The subsequent difficulty in performing an accurate diagnosis had contributed as a limiting factor in the identification of its genetic cause 
. However, to date, two different groups have been able to concurrently and independently identify the same DNAJC5
gene and same mutations using different and complementary approaches, which consolidate and validate the results 
(shown here). However, mutations in DNAJC5
are currently explaining approximately 25% of the autosomal dominant ANCLs 
Therefore, It is possible that other forms of ANCLs may have another genetic cause.
This is the first replication study of the identification of mutations in the DNAJC5 gene in ANCLs. By performing whole-exome sequencing in a multigenerational family with autosomal dominant ANCL (), we have identified a novel single-nucleotide variation (c.344T>G) in DNAJC5. In addition, using Sanger sequencing we found an in-frame single codon deletion (c.346_348 delCTC) in an independent family (). Thus, these variants fit genetic criteria for disease-causing mutations: they are present in unrelated families (); they exhibit perfect segregation with disease status (), they are not present in any healthy controls tested, they are located in evolutionarily highly conserved residues (), and they are predicted to functionally affect the encoded protein (CSPα) ().
Whole exome sequencing has proved useful to identify the pathogenic variant in monogenic disease 
. It is a rapid and cost-effective method that only requires sequencing in a small number of individuals. To date, different approaches to whole exome sequencing have been used including various designs, filtering methodologies and analytical strategies 
. Some examples are sequencing several affected individuals from different families, sequencing two affected individuals from the same family, and combining whole-exome sequencing data with linkage data, among others 
. Our design included two distantly-related affected individuals and a healthy sibling of one of the affected (and first cousin of the other affected individual) (). By selecting the two most distantly-related affected individuals available, the number of non pathogenic variants due to relatedness is dramatically reduced ().
In fact, 674±38 novel non-synonymous variants were found in each sample, but only 95 were shared by the two affected individuals. Subtracting the variants in common between the control and affected individuals, 25 SNSs were remaining (). Taking into account that on average 50% of genetic variants are shared between siblings and 12.5% between first cousins, we expected approximately 40 SNSs to remain after filtering against the control. To control for the false positive variants due to technical errors associated with Next-Generation Sequencing Platforms, all the three samples were run in the same flow cell. Thus, it is likely that the number of shared variants by both affected individuals was initially overestimated, and these artifacts were then filtered out by comparing with the control.
As done in other studies 
, additional affected family members were genotyped to identify the variants that are present in all of the affected individuals. A major challenge of whole-exome sequencing is to uniquely identify the causative variant. Most of the current approaches are based on first, removing candidate variants (non-synonymous, non-sense and splice-site variants) that are present in public databases (1000 genomes project and dbSNP), and second, on selecting only the variants present in the affected individuals. As a result, we found three unique variants located in the PDCD6IP
genes, which were present in all the affected family members but not in public databases. Interestingly, these three genes also exhibited the highest values based upon their GERP scores (). In order to elucidate the real cause of ANCL in this family, a large series of population controls were screened to verify if these variants in the PDCD6IP and LIPJ genes were present in healthy individuals. Only 16 and 8 heterozygous carriers for the variants on the PDCD6IP
genes were found, respectively. It is important to note that these variants were not present in the 1000 Genomes Project or in the dbSNP Database at the time of the analysis. Similar to others, 
this study clearly shows that pathogenic or causative variants can be identified by performing exome-sequencing in a small number of family members. It is now clear that by selecting two affected family members distant in the pedigree and one unaffected sibling of one of the affected individuals, and by running the three samples simultaneously, the number of potential candidate variants is significantly decreased. Our study also shows that although the public databases are very useful in removing commonly found variants (), they are still not comprehensive enough to eliminate all possible rare variants and unequivocally identify the causative mutation. Most importantly, screening a large number of non-affected individuals is still necessary.
gene encodes CSPα, which is a key element of the synaptic molecular machinery and accounts for 1% of all vesicle proteins 
, as well as part of the general exocytotic machinery 
. The synaptic vesicle localization and chaperone activity of CSPα suggests that it may function in rescuing synaptic proteins that have been unfolded by activity-dependent stress 
. Deletion of CSPα in flies and mice results in neurodegeneration and impairs synaptic function 
. CSPα has mostly been found associated with vesicles; however, it has a weak membrane affinity. Furthermore, there is an inverse correlation between membrane targeting of CSPα palmitoylation and adequate intracellular trafficking 
. Site-directed mutations that enhance membrane association such as p.C121-124L prevent adequate palmitoylation and lead to accumulation of CSPα in the ER and Golgi apparatus 
. In contrast, residue changes such as p.C113-119S (p.L115R, p.L116del, shown here in ) reduce binding to the membrane resulting in inadequate palmitoylation. They exhibit a localized punctuated pattern throughout the cytoplasm 
, co-localize with markers of ER–Golgi intermediate complex (Golgi SNARE proteins), and show a significant reduction in synaptic regions 
A potential neuronal-specific effect of this disproportionate and persistent CSPα missorting is a depletion of CSPα 
and possibly some of its SNARE partners the synapse, leading to a disruption in neurotransmission and synaptic dysfunction as displayed by the CSPα null animal models (mimicking loss of function) 
CSPα self-associates, forming oligomers 
. The pG83–C136 residues constitute the core region for CSPα oligomerization 
. Our analysis revealed that a more localized region between residues pF110-P138 has a tendency to form antiparallel ß-sheets species (See: Figure S1
), consistent with reports of the CSPα-CSPα dimers that are stable to temperature- and SDS-resistant particle 
. We did not find any significant increase in the intrinsic tendency of the mutations to aggregate (). However, the unattached form of the protein can induce conformational changes that facilitate a more reactive oligomeric state (). The effective increase in concentration of soluble CSPα produced by these mutations can also increase the level of macromolecular crowding, which in turn may dramatically enhance the own propensity of CSPα to aggregate, as has been shown for α-synuclein 
In a macromolecular crowded environment, the equilibrium between protein folding and protein-protein interactions is driven towards the lower volume (folded to unfolded) species 
. CSPα has been shown to have a high affinity for unfolded proteins 
. In a crowded environment, this can increase the likelihood of CSPα to interact with itself (more stable CSPα-CSPα dimers can be generated) and with other amyloidogenic partners such as α-synuclein 
. Recently, it has been shown that CSPα-CSPα dimers appear to be the main form of CSPα found in the brains of carriers of the ANCLs mutation 
Another potential target of abnormal interactions of CSPα are the synaptic proteins, especially those which are central to synaptic vesicles exocytosis, including proteins from the v- and t-SNAREs complex, and the putative Ca2+ sensor synaptotagmin 1, which undergoes palmitoylation in the golgi 
. Abnormal CSPα-CSPα dimers may impede appropriate synaptic vesicle targeting and subsequently, disrupt neurotrasmission. Indeed, recently it was shown that increasing the CSPα dimerization inhibits synaptic transmission 
Synaptic dysfunction has been consistently reported in several human and animal models of NCLs 
. Several NCL-encoded proteins have been found in synaptic compartments 
. Furthermore, signs of synaptic dysfunction (reduction in synaptic vesicle number) and degeneration have been demonstrated in PPT1 deficient neurons in vitro 
, and synaptic pathology (redistribution of SNARE complex and aggregates of Syp/SNAP25) occurs early on in disease progression in the congenital form of NCL 
. Synaptic involvement in two different mouse models of INCL was also recently demonstrated 
. The PPT1 null animal model displays alterations in the endocyclic\recycling pathway of synaptic vesicles associated with the impairment of depalmitoylation of the SNARE proteins 
, unlike CSPα null models that do not exhibit any primary defects in the endocytosis process or vesicle recycling 
. To date, the mechanisms causing synaptic vulnerability in NCLs remains poorly understood.
Our genetic results confirm DNAJC5
is the disease-causing gene of some ANCLs with autosomal dominant inheritance 
. The in silico
analysis suggests reduced the membrane binding and subsequent missorting of CSPα may play a crucial role in the pathogenicity of these mutations. This mislocalization can by itself affect the palmitoylation status and the propensity to aggregate. Thus, a dominant-negative mechanism resulting from CSPα propensity to self aggregate may be involved in the pathogenicity. The mutated CSPα may aggregate with the wild type, induces mislocalization and subsequent reduction of CSPα levels in the synapse
Since CSPα is a synaptic protein and the null animal models show a progressive neurodegenerative phenotype, a better understanding of the cellular and molecular characteristics of synaptic vulnerability will be important for our understanding of NCLs pathogenesis and for the effective development of therapeutic approaches.