The hereditary macular dystrophies are progressive degenerations of the central retina and contribute significantly to irreversible visual loss in developed countries. Among these disorders, Sorsby's fundus dystrophy (SFD), an autosomal dominant condition, provides an excellent mendelian model for the study of the genetically complex age-related macular degeneration (AMD), the most common maculopathy in the elderly. Recently, we mapped the SFD locus to 22q13-qter. This same region contains the gene for tissue inhibitor of metalloproteinases-3 (TIMP3), which is known to play a pivotal role in extracellular matrix remodeling. We have now identified point mutations in the TIMP3 gene in affected members of two SFD pedigrees. These mutations are predicted to disrupt the tertiary structure and thus the functional properties of the mature protein.

Stargardt disease (STGD) is a common autosomal recessive maculopathy of early and young-adult onset and is caused by alterations in the gene encoding the photoreceptor-specific ATP-binding cassette (ABC) transporter (ABCA4). We have studied 144 patients with STGD and 220 unaffected individuals ascertained from the German population, to complete a comprehensive, population-specific survey of the sequence variation in the ABCA4 gene. In addition, we have assessed the proposed role for ABCA4 in age-related macular degeneration (AMD), a common cause of late-onset blindness, by studying 200 affected individuals with late-stage disease. Using a screening strategy based primarily on denaturing gradient gel electrophoresis, we have identified in the three study groups a total of 127 unique alterations, of which 90 have not been previously reported, and have classified 72 as probable pathogenic mutations. Of the 288 STGD chromosomes studied, mutations were identified in 166, resulting in a detection rate of approximately 58%. Eight different alleles account for 61% of the identified disease alleles, and at least one of these, the L541P-A1038V complex allele, appears to be a founder mutation in the German population. When the group with AMD and the control group were analyzed with the same methodology, 18 patients with AMD and 12 controls were found to harbor possible disease-associated alterations. This represents no significant difference between the two groups; however, for detection of modest effects of rare alleles in complex diseases, the analysis of larger cohorts of patients may be required.

PurposeUsing exome sequencing, the underlying variants in many persons with autosomal recessive diseases remain undetected. We explored autosomal recessive Stargardt disease (STGD1) as a model to identify the missing heritability.MethodsSequencing of ABCA4 was performed in 8 STGD1 cases with one variant and p.Asn1868Ile in trans, 25 cases with one variant, and 3 cases with no ABCA4 variant. The effect of intronic variants was analyzed using in vitro splice assays in HEK293T cells and patient-derived fibroblasts. Antisense oligonucleotides were used to correct splice defects.ResultsIn 24 of the probands (67%), one known and five novel deep-intronic variants were found. The five novel variants resulted in messenger RNA pseudoexon inclusions, due to strengthening of cryptic splice sites or by disrupting a splicing silencer motif. Variant c.769-784C>T showed partial insertion of a pseudoexon and was found in cis with c.5603A>T (p.Asn1868Ile), so its causal role could not be fully established. Variant c.4253+43G>A resulted in partial skipping of exon 28. Remarkably, antisense oligonucleotides targeting the aberrant splice processes resulted in (partial) correction of all splicing defects.ConclusionOur data demonstrate the importance of assessing noncoding variants in genetic diseases, and show the great potential of splice modulation therapy for deep-intronic variants.

Photoreceptor ribbon synapses release glutamate in response to graded changes in membrane potential evoked by vast, logarithmically scalable light intensities. Neurotransmitter release is modulated by intracellular calcium levels. Large Ca 2ϩ -dependent chloride currents are important regulators of synaptic transmission from photoreceptors to second-order neurons; the molecular basis underlying these currents is unclear. We cloned human and mouse TMEM16B, a member of the TMEM16 family of transmembrane proteins, and show that it is abundantly present in the photoreceptor synaptic terminals in mouse retina. TMEM16B colocalizes with adaptor proteins PSD95, VELI3, and MPP4 at the ribbon synapses and contains a consensus PDZ class I binding motif capable of interacting with PDZ domains of PSD95. Furthermore, TMEM16B is lost from photoreceptor membranes of MPP4-deficient mice. This suggests that TMEM16B is a novel component of a presynaptic protein complex recruited to specialized plasma membrane domains of photoreceptors. TMEM16B confers Ca 2ϩ -dependent chloride currents when overexpressed in mammalian cells as measured by halide sensitive fluorescent protein assays and whole-cell patch-clamp recordings. The compartmentalized localization and the electrophysiological properties suggest TMEM16B to be a strong candidate for the long sought-after Ca 2ϩ -dependent chloride channel in the photoreceptor synapse.

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Retinitis pigmentosa (RP) is a degenerative disease of the retina leading to progressive loss of vision and, in many instances, to legal blindness at the end stage. The RP28 locus was assigned in 1999 to the short arm of chromosome 2 by homozygosity mapping in a large Indian family segregating autosomal-recessive RP (arRP). Following a combined approach of chromatin immunoprecipitation and parallel sequencing of genomic DNA, we identified a gene, FAM161A, which was shown to carry a homozygous nonsense mutation (p.Arg229X) in patients from the original RP28 pedigree. Another homozygous FAM161A stop mutation (p.Arg437X) was detected in three subjects from a cohort of 118 apparently unrelated German RP patients. Age at disease onset in these patients was in the second to third decade, with severe visual handicap in the fifth decade and legal blindness in the sixth to seventh decades. FAM161A is a phylogenetically conserved gene, expressed in the retina at relatively high levels and encoding a putative 76 kDa protein of unknown function. In the mouse retina, Fam161a mRNA is developmentally regulated and controlled by the transcription factor Crx, as demonstrated by chromatin immunoprecipitation and organotypic reporter assays on explanted retinas. Fam161a protein localizes to photoreceptor cells during development, and in adult animals it is present in the inner segment as well as the outer plexiform layer of the retina, the synaptic interface between photoreceptors and their efferent neurons. Taken together, our data indicate that null mutations in FAM161A are responsible for the RP28-associated arRP.

PurposeStargardt disease (STGD1) is an autosomal recessive retinopathy, caused by mutations in the retina-specific ATP-binding cassette transporter (ABCA4) gene. To establish the mutational spectrum and to assess effects of selected deep intronic and common genetic variants on disease, we performed a comprehensive sequence analysis in a large cohort of German STGD1 patients.MethodsDNA samples of 335 STGD1 patients were analyzed for ABCA4 mutations in its 50 coding exons and adjacent intronic sequences by resequencing array technology or next generation sequencing (NGS). Parts of intron 30 and 36 were screened by Sanger chain-terminating dideoxynucleotide sequencing. An in vitro splicing assay was used to test selected variants for their splicing behavior. By logistic regression analysis we assessed the association of common ABCA4 alleles while a multivariate logistic regression model calculated a genetic risk score (GRS).ResultsOur analysis identified 148 pathogenic or likely pathogenic mutations, of which 48 constitute so far unpublished ABCA4-associated disease alleles. Four rare deep intronic variants were found once in 472 alleles analyzed. In addition, we identified six risk-modulating common variants. Genetic risk score estimates suggest that defined common ABCA4 variants influence disease risk in carriers of a single pathogenic ABCA4 allele.ConclusionsOur study adds to the mutational spectrum of the ABCA4 gene. Moreover, in our cohort, deep intronic variants in intron 30 and 36 likely play no or only a minor role in disease pathology. Of note, our findings demonstrate a possible modifying effect of common sequence variants on ABCA4-associated disease.