Resolving the dark matter of ABCA4 for 1054 Stargardt disease probands through integrated genomics and transcriptomics.

PURPOSE: Missing heritability in human diseases represents a major challenge, and this is particularly true for ABCA4-associated Stargardt disease (STGD1). We aimed to elucidate the genomic and transcriptomic variation in 1054 unsolved STGD and STGD-like probands. METHODS: Sequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays. RESULTS: In 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. CONCLUSION: Deep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases.

Synaptic plasticity in the substantia gelatinosa in a model of chronic neuropathic pain.

Chronic neuropathic pain (CNP) is common after peripheral nerve injuries (PNI), but is rather refractory to available anti-pain medication. Advances in neuropathic pain research have identified cellular and molecular cues triggering the onset of neuropathic pain, but the mechanisms responsible for maintenance of chronic pain states are largely unknown. Structural changes such as sprouting of injured A-fibres into the substantia gelatinosa of the dorsal horn in the spinal cord have been proposed to relate to neuropathic pain in partial PNI models. Structural changes in central pain networks may also underlie the more persistent CNP following complete sectioning of a peripheral nerve, because this type of injury results in continuous and spontaneous afferent input to the spinal cord, which can trigger central sensitization. In the present study, the left sciatic nerve was completely sectioned and a 1-cm segment was removed to maintain a chronic pathology, whereas the right sciatic nerve was left intact. Mechanical allodynia was measured up to 84 days after injury, after which synaptic changes were studied in the lumbar substantia gelatinosa. The numbers of larger sized synaptophysin-immunoreactive presynaptic boutons were found to be increased in the substantia gelatinosa ipsilateral to the nerve injury. From these data we conclude that structural synaptic changes within the substantia gelatinosa are present months after complete nerve injury and that this plasticity may be involved in maintaining neuropathic pain states.