The ADAMTS18 gene is responsible for autosomal recessive early onset severe retinal dystrophy
- Ivana Peluso1,
- Ivan Conte1,
- Francesco Testa2,
- Gopuraja Dharmalingam1,
- Mariateresa Pizzo1,
- Rob WJ Collin3,
- Nicola Meola1,
- Sara Barbato1,
- Margherita Mutarelli1,
- Carmela Ziviello1,
- Anna Maria Barbarulo4,
- Vincenzo Nigro1, 5,
- Mariarosa AB Melone4, 6,
- the European Retinal Disease Consortium,
- Francesca Simonelli2 and
- Sandro Banfi1, 5Email author
© Peluso et al.; licensee BioMed Central Ltd. 2013
Received: 8 November 2012
Accepted: 24 January 2013
Published: 28 January 2013
Inherited retinal dystrophies, including Retinitis Pigmentosa and Leber Congenital Amaurosis among others, are a group of genetically heterogeneous disorders that lead to variable degrees of visual deficits. They can be caused by mutations in over 100 genes and there is evidence for the presence of as yet unidentified genes in a significant proportion of patients. We aimed at identifying a novel gene for an autosomal recessive form of early onset severe retinal dystrophy in a patient carrying no previously described mutations in known genes.
An integrated strategy including homozygosity mapping and whole exome sequencing was used to identify the responsible mutation. Functional tests were performed in the medaka fish (Oryzias latipes) model organism to gain further insight into the pathogenic role of the ADAMTS18 gene in eye and central nervous system (CNS) dysfunction.
This study identified, in the analyzed patient, a homozygous missense mutation in the ADAMTS18 gene, which was recently linked to Knobloch syndrome, a rare developmental disorder that affects the eye and the occipital skull. In vivo gene knockdown performed in medaka fish confirmed both that the mutation has a pathogenic role and that the inactivation of this gene has a deleterious effect on photoreceptor cell function.
This study reveals that mutations in the ADAMTS18 gene can cause a broad phenotypic spectrum of eye disorders and contribute to shed further light on the complexity of retinal diseases.
Inherited retinal dystrophies (IRD) are a genetically heterogeneous group of disorders that represent the most frequent causes of genetic blindness in the western world [1, 2]. They include Retinitis Pigmentosa (RP) and Leber Congenital Amaurosis (LCA), among the others. RP is the most frequent form of retinal dystrophies with an approximate incidence ranging between 1 in 3000 and 1 in 5000 individuals [1, 3, 4]. LCA is characterized by a severe visual impairment that starts in the first years of life . Both RP and LCA are generally characterized by a large extent of genetic heterogeneity. Over the last few years, about 230 genes causing inherited retinal diseases have been mapped to chromosomal locations (see RETnet web site: http://www.sph.uth.tmc.edu/RetNet/) and about 190 responsible genes have been identified. It is currently possible to determine the molecular defect underlying IRDs in up to 50% of patients , which strongly indicates the existence of additional genes responsible for these conditions.
We recently analyzed a cohort of over 400 Italian patients with autosomal recessive retinal dystrophies, including Retinitis Pigmentosa (ARRP) and LCA ( and unpublished data). Patients were screened for the presence of previously described mutations in genes with a known pathogenic role in ARRP and LCA using genotyping microchips based on the allele-specific primer extension (APEX) technique . About 70% of the analyzed patients did not harbor any previously described mutation in known LCA/ARRP genes. We reasoned that this subset of patients could be a valuable resource to identify novel retinal dystrophy genes. Therefore, we selected a subset of the latter patients, preferentially those with some evidence of belonging to consanguineous families, for homozygosity mapping genotyping followed by whole exome sequencing analysis, which has proven to be an effective strategy for the identification of novel autosomal-recessive retinal disease genes [9, 10].
We used standard methods to isolate genomic DNA from peripheral blood of the patients and their family members. Informed consent was obtained from all participating patients and families according to the Declaration of Helsinki and the studies were approved by the Research Ethics Committee of the Second University of Naples.
Ophthalmologic examination including best corrected visual acuity using Snellen charts or Teller Acuity Cards, measurement of objective refractive error after cycloplegia, slit-lamp biomicroscopy, dilated fundus examination, bilateral full-field ERGs and optical coherence tomography (OCT) recordings, was carried out as previously described .
Genotyping was performed on SNP microarray (GeneChip Genome-Wide Human SNP Array 5.0, Affymetrix, Santa Clara, CA). Array experiments were performed according to protocols provided by the manufacturer. Genotypes were called with the Genotype Console program, ver. 2.1 (Affymetrix) and regions of homozygosity were identified using PLINK , with a sliding window of 50 SNPs and allowing 2 heterozygous SNPs (miscalls) and 10 missing SNPs (no calls) per window. Regions containing more than 250 consecutive homozygous SNPs were considered to be significant homozygous regions, on average corresponding to a genomic size of 1 Megabase (Mb).
Whole exome sequencing
Whole exome enrichment was carried out using the SureSelect All Exon kit v.1 (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer instructions. Whole exome sequencing was carried out on a SOLiD 3 Plus System (Life Technologies, Carlsbad, CA, USA). Sequencing reads were mapped to the reference genome (UCSC, hg19 build) using the software BioScope v1.3 (Life Technologies, Carlsbad, CA, USA). Single nucleotide variations (SNV) and in-del mutation calling analyses were carried out using the diBayes algorithm with medium stringency settings and the SOLiD Small Indel Fragment Tool, respectively.
RNA in situ hybridization and immunohistochemistry
Medaka stocks and mRNA injections
The Cab-strain of wild type medaka fish were kept and staged as described . A morpholino (Mo; Gene Tools LLC, Oregon, USA) was designed against the ATG initiation codon and the 5′ untranslated region of the medaka ortholog of the ADAMTS18 gene (olAdamts18) whereas a control Mo carrying five mismatches (mmMo-Adamts18) was used as a control (see sequences in Additional file 1: Table S2). The specificity and inhibitory efficiencies of Mo-Adamts18 were determined as described in . Mo-Adamts18 was injected at a 90 μM concentration into one blastomere at the two-cell stage. In vitro synthesis of the human wild type ADAMTS18 mRNAs as well as of the c.T3235 > C mutated form was performed as described . Morpholino and mRNA injections were carried out as previously described [14, 17, 18].
The light-induced photoreceptor cell degeneration assay was performed as previously described . Medaka embryos were incubated with phenyl thiourea (PTU) to prevent pigmentation . Fish were sacrificed and analyzed after 5 days of constant light.
Results and discussion
Clinical features of patient A24
Identification of a mutation in the ADAMTS18 gene in patient A24
We carried out SNP genotyping analysis on a genomic DNA sample from patient A24. This analysis revealed the presence of four large homozygous regions in the genome of the patient, namely on chromosomes 8, 4, 3 and 16 for a total of 15.5 Mb (Additional file 2: Table S1).
We then performed whole exome sequencing analysis on patient A24. Over 65 million sequencing reads, corresponding to about 3.3 Gigabases (Gb) of mappable sequences were obtained using a SOLiD 3 Plus System (Life Technologies, Carlsbad, CA, USA). In total, 86.12% reads could be mapped to the reference genome (UCSC, hg19 build). About 77% of the uniquely mapped reads were aligned on the targeted exome after duplicate reads removal, with 70% of the targeted exons covered at > 20x depth. Overall, we identified 13.388 exonic variants including 6.213 non-synonymous SNVs, 152 small insertions or deletions, 71 stop-gain or stop-loss variants and 40 variants putatively affecting splice sites. Further filtering based on the exclusion of all known dbSNP variants (using dbSNP130) reduced the total number to 760 and the application of an autosomal-recessive model of inheritance for the disease left 110 sequence variants. By limiting the analysis to the regions of homozygosity and by only considering genes with reported evidence of significant expression in the eye [23, 24], we were left with only a single homozygous missense variation, namely c.T3235 > C (p.C1079R) in the ADAMTS18 gene (NM_199355) that is localized to the long arm of chromosome 16 (Figure 1B). Intriguingly, Aldahmesh et al. recently reported a homozygous missense variation  in this gene in a Saudi Arabian family with Knobloch syndrome, a rare autosomal recessive developmental disorder that affects the eye and the occipital region of the skull and the brain (On-line Mendelian Inheritance in Man (OMIM) #267750). They therefore proposed ADAMTS18 as the gene responsible for Knobloch syndrome even if they did not provide any functional evidence in support of their hypothesis . Due to the above-mentioned observations, we regarded this gene as a good candidate for a pathogenic role in the phenotype of patient A24 and therefore selected it for further analysis. ADAMTS18 is a member of the ADAMTS protein family, a family of metalloproteinases similar to the ADAM proteins (A Disintegrin-like And Metalloproteinase), but distinct by the additional presence of ThromboSpondin (TSP) motifs in the C-terminus and the lack of transmembrane domains . The c.T3235 > C mutation causes the substitution of a highly conserved cysteine residue with an arginine in one of the four C-terminal TSP type 1 motifs of the protein (Figure 1D), which are known to be important in modulating ADAMTS-mediated proteolysis  and influence protein recognition and matrix localization .
Sanger sequencing confirmed that the c.T3235 > C sequence variation was present in homozygosity in patient A24 (Figure 1C). The patient’s parents and his healthy brother were found to be heterozygous carriers of the mutation, which is in line with the autosomal recessive inheritance of the disorder in the family (Figure 1C). The variation was not present in neither the Exome Variant Server (http://evs.gs.washington.edu/EVS/) nor in over 350 Italian control DNA samples. Finally, the c.T3235 > C variation was predicted to be “disease causing” by MutationTaster (http://www.mutationtaster.org/) with high probability and “probably damaging” by Polyphen2 (http://genetics.bwh.harvard.edu/pph2/index.shtml), further supporting its putative pathogenic role.
This is the second report that describes a putative pathogenic role of the ADAMTS18 gene in human genetic diseases that affect the eye and the central nervous system (CNS). Due to the recent description of another ADAMTS18 homozygous missense mutation, i.e., the p.S179L, in a family with Knobloch syndrome , we carefully revised the clinical history and phenotype of patient A24 to detect a possible overlap with the Knobloch phenotype. However, the lack of any detectable occipital defect as well as the lack of myopia (the patient is actually hypermetropic) and of the classical signs of vitreoretinal degeneration  prompted us to exclude the diagnosis of Knobloch syndrome in patient A24 (Figure 1A and data not shown) thus suggesting that the ADAMTS18 gene is also responsible for non-Knobloch forms of retinal diseases.
To determine whether mutations in ADAMTS18 are more widely involved in retinal dystrophies, we analyzed 450 unrelated individuals who had ARRP, LCA or autosomal recessive Cone Rod dystrophy and who had significant homozygous regions (see Methods). In 9 families, ADAMTS18 was located in significantly large homozygous regions: sequence analysis of probands from these families, however, did not reveal the presence of additional mutation in ADAMTS18.
ADAMTS18 is expressed in the adult eye
ADAMTS18 was previously reported to be expressed in the developing mouse eye . We aimed at determining whether ADAMTS18 is also expressed in the adult eye, and mainly in the retina, i.e., the main target of the phenotype present in patient A24. To that purpose, we performed RT-PCR experiments on human retina cDNA (Clontech). As a result, we detected the expected size products (data not shown) using oligonucleotide primers specific for the ADAMTS18 mRNA sequence (Additional file 1: Table S2) and we confirmed their identity by Sanger sequencing. To define the sites of expression of this gene at the cellular level, we performed RNA in situ hybridization experiments on murine adult eye section collected at P60. This experiment revealed the murine Adamts18 gene to be strongly expressed in the retinal ganglion cell layer (GCL), in the inner part of the inner nuclear layer (INL) and in the retinal pigment epithelium (RPE) (Figure 1E).
In vivo analysis in medaka fish
We determined, by using a multidisciplinary strategy involving the use of advanced genomic procedures and in vivo functional analysis, that mutations in the ADAMTS18 gene, recently proposed to cause Knobloch syndrome, can also be responsible, although with a relatively low frequency, for early-onset severe retinal dystrophy possibly accompanied by other CNS features, such as autism and neurodevelopmental delay. Importantly, the results of our ADAMTS18 knockdown experiment in medaka provide for the first time in vivo functional support to the pathogenic role of this gene in Knobloch syndrome as well. Our data indicate that different mutations in the ADAMTS18 can be linked to the pathogenesis of different eye disorders and contribute to shed further light on the molecular mechanisms underlying the complexity of inherited retinal dystrophies.
We thank the participating patients and their families. We are grateful to B. Franco and G. Diez-Roux for critical reading of the manuscript. We also thank C. Gilissen and J.A. Veltman for helpful discussion; S. Crispi and the High Throughput Sequencing IGB facility for support in exome sequencing; R. Rispoli and the TIGEM Bioinformatics Core for support in exome data analysis and F. Salierno for technical support. The members of the European Retinal Disease Consortium involved in this study are Elfride De Baere, Robert K. Koenekoop, Bart P. Leroy, Frans P. Cremers, Susanne Kohl, Christian Hamel, Carmen Ayuso, Bernd Wissinger, Chris Inglehearn, Carmel Toomes and Anneke den Hollander. This work was supported by grants from the Retina Italia, RP-Liguria and the Italian Telethon Foundations (to S.B.) and by the European Union, EU FP7/2007-2013 under grant agreement no. 223143 (project acronym: TECHGENE).
Ethics approval for the work described was provided by the Second University of Naples Medical Ethical Committee.
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