ABCA4-associated disease as a model for missing heritability in autosomal recessive disorders: novel noncoding splice, cis-regulatory, structural, and recurrent hypomorphic variants

Bauwens, Miriam; Garanto, Alejandro; Sangermano, Riccardo; Naessens, Sarah; Weisschuh, Nicole; Zaeytijd, Julie De; Khan, Mubeen; Sadler, Françoise; Balikova, Irina; Cauwenbergh, Caroline Van; Rosseel, Toon; Bauwens, Jim; Leeneer, Kim De; Jaegere, Sarah De; Laethem, Thalia Van; Vries, Meindert De; Carss, Keren; Arno, Gavin; Fakin, Ana; Webster, Andrew R.; l'Argentière, Thomy de Ravel de; Sznajer, Yves; Vuylsteke, Marnik; Kohl, Susanne; Wissinger, Bernd; Cherry, Timothy J.; Collin, Rob W.J.; Cremers, Frans P.M.; Leroy, Bart P.; Baere, Elfride De

doi:10.1038/s41436-018-0420-y

Cited by 130 publications

(178 citation statements)

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“…In addition, eight recently discovered causal deep‐intronic variants (c.769–784C>T, c.859–506G>C, c.859–540C>G, c.1937+435C>G, c.4539+1100A>G, c.4539+1106C>T, c.4539+2064C>T and c.5197–557G>T) (Bauwens et al, ; Sangermano et al, ) as well as 300 bp upstream and 300 bp downstream sequences were captured using 174 smMIPs (Table S1 and Figure S1). Larger segments were sequenced as they may carry novel causal deep‐intronic variants that may affect the recognition of the same pseudoexons by the splicing machinery.…”

Section: Methodsmentioning

confidence: 99%

“…Previously reported pathogenic variants with high AFs such as c.2588G>C and c.5603A>T (AFs in non‐Finnish European [nFE] in gnomAD 0.00784 and 0.06647, respectively) (Cornelis et al, ; F. P. Cremers et al, ; Schulz et al, ; Zernant et al, , ), were selected separately. Known deep‐intronic variants were selected based on prior knowledge from literature (Albert et al, ; Bauwens et al, , ; Braun et al, ; Sangermano et al, ; Schulz et al, ; Zernant et al, ).…”

Section: Methodsmentioning

confidence: 99%

“…Copy‐number variations (CNVs), deep‐intronic variants (Bauwens et al, ; Bax et al, ; Braun et al, ; Zernant et al, ), and a low‐penetrant frequent coding variant (p.Asn1868Ile) (Runhart et al, ; Zernant et al, ) explained about 10% of this missing heritability. Recently, upon sequence analysis of the entire ABCA4 gene, eight novel and one known deep‐intronic variant explained approximately 65% of the remaining unsolved cases (Bauwens et al, ; Sangermano et al, ; Zernant et al, ). As the phenotype is specific and no other gene has been described to be mutated in typical STGD1 cases since ABCA4 was identified 22 years ago (Allikmets et al, ), hundreds of cases have remained genetically unsolved due to the low‐sensitive mutation scanning techniques that were used such as single strand conformation polymorphism (Klevering et al, ; Maugeri et al, ), denaturing high performance liquid chromatography (Maia‐Lopes et al, ), and arrayed primer extension (Jaakson et al, ).…”

Section: Introductionmentioning

confidence: 99%

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Cost‐effective molecular inversion probe‐based ABCA4 sequencing reveals deep‐intronic variants in Stargardt disease

et al. 2019

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Purpose Stargardt disease (STGD1) is caused by biallelic mutations in ABCA4, but many patients are genetically unsolved due to insensitive mutation‐scanning methods. We aimed to develop a cost‐effective sequencing method for ABCA4 exons and regions carrying known causal deep‐intronic variants. Methods Fifty exons and 12 regions containing 14 deep‐intronic variants of ABCA4 were sequenced using double‐tiled single molecule Molecular Inversion Probe (smMIP)‐based next‐generation sequencing. DNAs of 16 STGD1 cases carrying 29 ABCA4 alleles and of four healthy persons were sequenced using 483 smMIPs. Thereafter, DNAs of 411 STGD1 cases with one or no ABCA4 variant were sequenced. The effect of novel noncoding variants on splicing was analyzed using in vitro splice assays. Results Thirty‐four ABCA4 variants previously identified in 16 STGD1 cases were reliably identified. In 155/411 probands (38%), two causal variants were identified. We identified 11 deep‐intronic variants present in 62 alleles. Two known and two new noncanonical splice site variants showed splice defects, and one novel deep‐intronic variant (c.4539+2065C>G) resulted in a 170‐nt mRNA pseudoexon insertion (p.[Arg1514Lysfs*35,=]). Conclusions smMIPs‐based sequence analysis of coding and selected noncoding regions of ABCA4 enabled cost‐effective mutation detection in STGD1 cases in previously unsolved cases.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cost‐effective molecular inversion probe‐based ABCA4 sequencing reveals deep‐intronic variants in Stargardt disease

et al. 2019

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“…Possible reasons for these results are as follows: (1) targeted exome sequencing and WES cannot detect gross deletions, gross insertions, or complex rearrangement variants (Broadgate, Yu, Downes, & Halford, 2017), which might have been present in these families; (2) the sequence depth and coverage in this study was insufficient to call all variants accurately; (3) novel STGD-associated genes may have been filtered out during our raw data analysis; (4) deep-intronic variants in ABCA4 that are potentially associated with autosomal recessive STGD could not be captured through targeted exome sequencing and WES (Albert et al, 2018;Bauwens et al, 2015Bauwens et al, , 2019Bax et al, 2015;Braun et al, 2013;Sangermano et al, 2014;Zernant et al, 2014); and (5) diseasecausing variants with high minor allele frequency, which had not been reported previously may have been filtered out, for example, c.5603A > T, p.(Asn1868Ile) (Cremers, Cornelis, Runhart, & Astuti, 2018;Runhart et al, 2018;Zernant et al, 2017). Possible reasons for these results are as follows: (1) targeted exome sequencing and WES cannot detect gross deletions, gross insertions, or complex rearrangement variants (Broadgate, Yu, Downes, & Halford, 2017), which might have been present in these families; (2) the sequence depth and coverage in this study was insufficient to call all variants accurately; (3) novel STGD-associated genes may have been filtered out during our raw data analysis; (4) deep-intronic variants in ABCA4 that are potentially associated with autosomal recessive STGD could not be captured through targeted exome sequencing and WES (Albert et al, 2018;Bauwens et al, 2015Bauwens et al, , 2019Bax et al, 2015;Braun et al, 2013;Sangermano et al, 2014;Zernant et al, 2014); and (5) diseasecausing variants with high minor allele frequency, which had not been reported previously may have been filtered out, for example, c.5603A > T, p.(Asn1868Ile) (Cremers, Cornelis, Runhart, & Astuti, 2018;Runhart et al, 2018;Zernant et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

Application of targeted exome and whole‐exome sequencing for Chinese families with Stargardt disease

Dan

Huang

Xing

et al. 2019

Annals of Human Genetics

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Objective: The aim of this study was to investigate pathogenic variants and molecular etiologies of Stargardt disease (STGD) in a cohort of Chinese families. Materials and Methods:A cohort of 12 unrelated STGD families diagnosed on the basis of clinical manifestations underwent analysis by targeted exome or whole-exome sequencing. Bioinformatics analysis, Sanger sequencing, and cosegregation analysis of available family members were used to validate sequencing data and confirm the presence of disease-causing genes.Results: Using targeted exome and whole-exome sequencing, we found that eight families had disease-causing variants in the ABCA4 gene, one family had only one heterozygous variant in the ABCA4 gene, and the remaining three families have not been identified with any disease-causing variants for STGD. We identified 15 variants in the ABCA4 gene; of these, five variants have not been previously described for STGD. Conclusion:The findings in this study expand the data regarding the frequency and spectrum of variants in the ABCA4 gene, thus potentially enriching our understanding of the molecular basis of STGD. Moreover, they constitute clues for future STGD diagnosis and therapy.

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“…A region encompassing the entire ABCA4 gene (chr1:94337885‐94703604, hg19) was enriched using a custom HaloPlex Target enrichment kit (Agilent Technologies, Belgium), followed by NGS (MiSeq, Illumina, San Diego, California). Data were analyzed as described previously …”

Section: Methodsmentioning

confidence: 99%

Functional characterization of novel MFSD8 pathogenic variants anticipates neurological involvement in juvenile isolated maculopathy

et al. 2019

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Biallelic MFSD8 variants are an established cause of severe late‐infantile subtype of neuronal ceroid lipofuscinosis (v‐LINCL), a severe lysosomal storage disorder, but have also been associated with nonsyndromic adult‐onset maculopathy. Here, we functionally characterized two novel MFSD8 variants found in a child with juvenile isolated maculopathy, in order to establish a refined prognosis. ABCA4 locus resequencing was followed by the analysis of other inherited retinal disease genes by whole exome sequencing (WES). Minigene assays and cDNA sequencing were used to assess the effect of a novel MFSD8 splice variant. MFSD8 expression was quantified with qPCR and overexpression studies were analyzed by immunoblotting. Transmission electron microscopy (TEM) was performed on a skin biopsy and ophthalmological and neurological re‐examinations were conducted. WES revealed two novel MFSD8 variants: c.[590del];[439+3A>C] p.[Gly197Valfs*2];[Ile67Glufs*3]. Characterization of the c.439+3A>C variant via splice assays showed exon‐skipping (p.Ile67Glufs*3), while overexpression studies of the corresponding protein indicated expression of a truncated polypeptide. In addition, a significantly reduced MFSD8 RNA expression was noted in patient's lymphocytes. TEM of a skin biopsy revealed typical v‐LINCL lipopigment inclusions while neurological imaging of the proband displayed subtle cerebellar atrophy. Functional characterization demonstrated the pathogenicity of two novel MFSD8 variants, found in a child with an initial diagnosis of juvenile isolated maculopathy but likely evolving to v‐LINCL with a protracted disease course. Our study allowed a refined neurological prognosis in the proband and expands the natural history of MFSD8‐associated disease.

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ABCA4-associated disease as a model for missing heritability in autosomal recessive disorders: novel noncoding splice, cis-regulatory, structural, and recurrent hypomorphic variants

Cited by 130 publications

References 39 publications

Cost‐effective molecular inversion probe‐based ABCA4 sequencing reveals deep‐intronic variants in Stargardt disease

Cost‐effective molecular inversion probe‐based ABCA4 sequencing reveals deep‐intronic variants in Stargardt disease

Application of targeted exome and whole‐exome sequencing for Chinese families with Stargardt disease

Functional characterization of novel MFSD8 pathogenic variants anticipates neurological involvement in juvenile isolated maculopathy

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