Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides

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.     

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

PurposeABCA4-associated disease, a recessive retinal dystrophy, is hallmarked by a large proportion of patients with only one pathogenic ABCA4 variant, suggestive for missing heritability.MethodsBy locus-specific analysis of ABCA4, combined with extensive functional studies, we aimed to unravel the missing alleles in a cohort of 67 patients (p), with one (p = 64) or no (p = 3) identified coding pathogenic variants of ABCA4.ResultsWe identified eight pathogenic (deep-)intronic ABCA4 splice variants, of which five are novel and six structural variants, four of which are novel, including two duplications. Together, these variants account for the missing alleles in 40.3% of patients. Furthermore, two novel variants with a putative cis-regulatory effect were identified. The common hypomorphic variant c.5603A>T p.(Asn1868Ile) was found as a candidate second allele in 43.3% of patients. Overall, we have elucidated the missing heritability in 83.6% of our cohort. In addition, we successfully rescued three deep-intronic variants using antisense oligonucleotide (AON)-mediated treatment in HEK 293-T cells and in patient-derived fibroblast cells.ConclusionNoncoding pathogenic variants, novel structural variants, and a common hypomorphic allele of the ABCA4 gene explain the majority of unsolved cases with ABCA4-associated disease, rendering this retinopathy a model for missing heritability in autosomal recessive disorders.     

Heterozygous Deep‐Intronic Variants and Deletions in ABCA4 in Persons with Retinal Dystrophies and One Exonic ABCA4 Variant

Variants in ABCA4 are responsible for autosomal‐recessive Stargardt disease and cone‐rod dystrophy. Sequence analysis of ABCA4 exons previously revealed one causative variant in each of 45 probands. To identify the “missing” variants in these cases, we performed multiplex ligation‐dependent probe amplification‐based deletion scanning of ABCA4. In addition, we sequenced the promoter region, fragments containing five deep‐intronic splice variants, and 15 deep‐intronic regions containing weak splice sites. Heterozygous deletions spanning ABCA4 exon 5 or exons 20–22 were found in two probands, heterozygous deep‐intronic variants were identified in six probands, and a deep‐intronic variant was found together with an exon 20–22 deletion in one proband. Based on ophthalmologic findings and characteristics of the identified exonic variants present in trans, the deep‐intronic variants V1 and V4 were predicted to be relatively mild and severe, respectively. These findings are important for proper genetic counseling and for the development of variant‐specific therapies.     

An Augmented ABCA4 Screen Targeting Noncoding Regions Reveals a Deep Intronic Founder Variant in Belgian Stargardt Patients

Autosomal‐recessive Stargardt disease (STGD1) is hallmarked by a large proportion of patients with a single heterozygous causative variant in the disease gene ABCA4. Braun et al. ( 2013 ) reported deep intronic variants of ABCA4 in STGD1 patients with one coding variant, prompting us to perform an augmented screen in 131 Belgian STGD1 patients with one or no ABCA4 variant to uncover deep intronic causal ABCA4 variants. This revealed a second variant in 28.6% of cases. Twenty‐six percent of these carry the same causal variant c.4539+2001G>A (V4). Haplotyping in V4 carriers showed a common region of 63 kb, suggestive of a founder mutation. Genotype–phenotype correlations suggest a moderate‐to‐severe impact of V4 on the STGD1 phenotype. In conclusion, V4 occurs in a high fraction of Belgian STGD1 patients and represents the first deep intronic founder mutation in ABCA4. This emphasizes the importance of augmented molecular genetic testing of ABCA4 in Belgian STGD1.     

RNA sequencing solved the most common but unrecognized NEB pathogenic variant in Japanese nemaline myopathy

PurposeThe diagnostic rate for Mendelian diseases by exome sequencing (ES) is typically 20–40%. The low rate is partly because ES misses deep-intronic or synonymous variants leading to aberrant splicing. In this study, we aimed to apply RNA sequencing (RNA-seq) to efficiently detect the aberrant splicings and their related variants.MethodsAberrant splicing in biopsied muscles from six nemaline myopathy (NM) cases unresolved by ES were analyzed with RNA-seq. Variants related to detected aberrant splicing events were analyzed with Sanger sequencing. Detected variants were screened in NM patients unresolved by ES.ResultsWe identified a novel deep-intronic NEB pathogenic variant, c.1569+339A>G in one case, and another novel synonymous NEB pathogenic variant, c.24684G>C (p.Ser8228Ser) in three cases. The c.24684G>C variant was observed to be the most frequent among all NEB pathogenic variants in normal Japanese populations with a frequency of 1 in 178 (20 alleles in 3552 individuals), but was previously unrecognized. Expanded screening of the variant identified it in a further four previously unsolved nemaline myopathy cases.ConclusionThese results indicated that RNA-seq may be able to solve a large proportion of previously undiagnosed muscle diseases.     

Functional analysis of two novel splice site mutations of APOB gene in familial hypobetalipoproteinemia

Familial hypobetalipoproteinemia (FHBL) is a co-dominant disorder characterized by reduced plasma levels of low density lipoprotein cholesterol (LDL-C) and its protein constituent apolipoprotein B (apoB), which may be due to mutations in APOB gene, mostly located in the coding region of this gene. We report two novel APOB gene mutations involving the acceptor splice site of intron 11 (c.1471-1G>A) and of intron 23 (c.3697-1G>C), respectively, which were identified in two patients with heterozygous FHBL associated with severe fatty liver disease. The effects of these mutations on APOB pre-mRNA splicing were assessed in COS-1 cells expressing the mutant APOB minigenes.The c.1471-1G>A APOB minigene generated two abnormal mRNAs. In one mRNA the entire intron 11 was retained; in the other mRNA exon 11 joined to exon 12, in which the first nucleotide was deleted due to the activation of a novel acceptor splice site. The predicted products of these mRNAs are truncated proteins of 546 and 474 amino acids, designated apoB-12.03 and apoB-10.45, respectively. The c.3697-1G>C APOB minigene generated a single abnormal mRNA in which exon 23 joined to exon 25, with the complete skipping of exon 24. This abnormal mRNA is predicted to encode a truncated protein of 1220 amino acids, designated apoB-26.89.These splice site mutations cause the formation of short truncated apoBs, which are not secreted into the plasma as lipoprotein constituents. This secretion defect is the major cause of severe fatty liver observed in carriers of these mutations.     

Splice variant in ARX leading to loss of C‐terminal region in a boy with intellectual disability and infantile onset developmental and epileptic encephalopathy

Pathogenic variants in the X‐chromosome Aristaless‐related homeobox (ARX) gene contribute to intellectual disability, epilepsy, and associated comorbidities in affected males. Here, we report a novel splice variant in ARX in a family with three affected individuals. The proband had early onset developmental and epileptic encephalopathy, his brother and mother had severe and mild intellectual disability, respectively. Massively parallel sequencing identified a novel c.1449‐1G>C in intron 4 of the ARX gene, predicted to abolish the splice acceptor site, retaining intron 4 and leading to a premature termination codon immediately after exon 4. As exon 5 is the last exon of the ARX gene, the premature termination codon at position p.L484* would be predicted to escape nonsense‐mediated mRNA decay, potentially producing at least some C‐terminally truncated protein. Analysis of cDNA from patient lymphoblastoid cells confirmed retention of intron 4 and loss of detectable expression of ARX mRNA across exon 4 to exon 5. We review published cases of variants that lead to altered or early termination of the ARX protein, but not complete loss of function, and are associated with phenotypes of intellectual disability and infantile onset developmental and epileptic encephalopathies, including Ohtahara and West syndromes. Taken together, this novel splice variant retaining intron 4 is likely to be the cause of the early onset developmental and epileptic encephalopathy in the proband.     

A detailed clinical and molecular survey of subjects with nonsyndromic USH2A retinopathy reveals an allelic hierarchy of disease-causing variants

Defects in USH2A cause both isolated retinal disease and Usher syndrome (ie, retinal disease and deafness). To gain insights into isolated/nonsyndromic USH2A retinopathy, we screened USH2A in 186 probands with recessive retinal disease and no hearing complaint in childhood (discovery cohort) and in 84 probands with recessive retinal disease (replication cohort). Detailed phenotyping, including retinal imaging and audiological assessment, was performed in individuals with two likely disease-causing USH2A variants. Further genetic testing, including screening for a deep-intronic disease-causing variant and large deletions/duplications, was performed in those with one likely disease-causing change. Overall, 23 of 186 probands (discovery cohort) were found to harbour two likely disease-causing variants in USH2A. Some of these variants were predominantly associated with nonsyndromic retinal degeneration (‘retinal disease-specific''); these included the common c.2276 G>T, p.(Cys759Phe) mutation and five additional variants: c.2802 T>G, p.(Cys934Trp); c.10073 G>A, p.(Cys3358Tyr); c.11156 G>A, p.(Arg3719His); c.12295-3 T>A; and c.12575 G>A, p.(Arg4192His). An allelic hierarchy was observed in the discovery cohort and confirmed in the replication cohort. In nonsyndromic USH2A disease, retinopathy was consistent with retinitis pigmentosa and the audiological phenotype was variable. USH2A retinopathy is a common cause of nonsyndromic recessive retinal degeneration and has a different mutational spectrum to that observed in Usher syndrome. The following model is proposed: the presence of at least one ‘retinal disease-specific'' USH2A allele in a patient with USH2A-related disease results in the preservation of normal hearing. Careful genotype–phenotype studies such as this will become increasingly important, especially now that high-throughput sequencing is widely used in the clinical setting.     

Cerebral hypomyelination associated with biallelic variants of FIG4

The lipid phosphatase gene FIG4 is responsible for Yunis‐Varón syndrome and Charcot‐Marie‐Tooth disease Type 4J, a peripheral neuropathy. We now describe four families with FIG4 variants and prominent abnormalities of central nervous system (CNS) white matter (leukoencephalopathy), with onset in early childhood, ranging from severe hypomyelination to mild undermyelination, in addition to peripheral neuropathy. Affected individuals inherited biallelic FIG4 variants from heterozygous parents. Cultured fibroblasts exhibit enlarged vacuoles characteristic of FIG4 dysfunction. Two unrelated families segregate the same G>A variant in the +1 position of intron 21 in the homozygous state in one family and compound heterozygous in the other. This mutation in the splice donor site of exon 21 results in read‐through from exon 20 into intron 20 and truncation of the final 115 C‐terminal amino acids of FIG4, with retention of partial function. The observed CNS white matter disorder in these families is consistent with the myelination defects in the FIG4 null mouse and the known role of FIG4 in oligodendrocyte maturation. The families described here the expanded clinical spectrum of FIG4 deficiency to include leukoencephalopathy.     

The ACMG SF v3.0 gene list increases returnable variant detection by 22% when compared with v2.0 in the ClinSeq cohort

PurposeThe American College of Medical Genetics and Genomics (ACMG) recommends the return of pathogenic and likely pathogenic (P/LP) secondary findings from exome and genome sequencing. The latest version (ACMG secondary finding [SF] v3.0) includes 14 additional genes. We interrogated the ClinSeq cohort for variants in these genes to determine the additional yield in unselected individuals.MethodsExome data from 1473 individuals (60% White, 34% African American or Black, 6% other) were analyzed. We restricted our analyses to coding variants; +1,+2,–1, and –2 splice site variants; and the pathogenic GAA variant, NM_000152.5:c.-32-13T>G. Variants were assessed with slightly modified ACMG/Association of Molecular Pathology guidelines.ResultsA total of 25 P/LP variants were identified. In total, 7 individuals had P/LP variants in genes recommended for return of heterozygous variants, namely HNF1A (1), PALB2 (3), TMEM127 (1), and TTN (2). In total, 4 individuals had a homozygous variant in a gene recommended for biallelic variant return, namely HFE, NM_000410.3(HFE):c.845G>A p.Cys282Tyr. A total of 17 P/LP variants were identified in the heterozygous state in genes recommended only for biallelic variant reporting and were not returned. The frequency of returnable P/LP variants did not significantly differ by race.ConclusionUsing the ACMG SF v3.0, the returnable P/LP variant frequency increased in the ClinSeq cohort by 22%, from 3.4% (n = 50, ACMG SF v2.0) to 4.1% (n = 61, ACMG SF v3.0).     

Autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice site mutations in the Stargardt's disease gene ABCR

Ophthalmological and molecular genetic studies were performed in a consanguineous family with individuals showing either retinitis pigmentosa (RP) or cone-rod dystrophy (CRD). Assuming pseudodominant (recessive) inheritance of allelic defects, linkage analysis positioned the causal gene at 1p21-p13 (lod score 4.22), a genomic segment known to harbor the ABCR gene involved in Stargardt's disease (STGD) and age- related macular degeneration (AMD). We completed the exon-intron structure of the ABCR gene and detected a severe homozygous 5[prime] splice site mutation, IVS30+1G->T, in the four RP patients. The five CRD patients in this family are compound heterozygotes for the IVS30+1G- >T mutation and a 5[prime] splice site mutation in intron 40 (IVS40+5G- >A). Both splice site mutations were found heterozygously in two unrelated STGD patients, but not in 100 control individuals. In these patients the second mutation was either a missense mutation or unknown. Since thus far no STGD patients have been reported to carry two ABCR null alleles and taking into account that the RP phenotype is more severe than the STGD phenotype, we hypothesize that the intron 30 splice site mutation represents a true null allele. Since the intron 30 mutation is found heterozygously in the CRD patients, the IVS40+5G->A mutation probably renders the exon 40 5[prime] splice site partially functional. These results show that mutations in the ABCR gene not only result in STGD and AMD, but can also cause autosomal recessive RP and CRD. Since the heterozygote frequency for ABCR mutations is estimated at 0.02, mutations in ABCR might be an important cause of autosomal recessive and sporadic forms of RP and CRD.      

A case–control collapsing analysis identifies retinal dystrophy genes associated with ophthalmic disease in patients with no pathogenic ABCA4 variants

PurposeVariants in the ABCA4 gene are causal for a variety of retinal dystrophy phenotypes, including Stargardt disease (STGD1). However, 15% of patients who present with symptoms compatible with STGD1/ABCA4 disease do not have identifiable causal ABCA4 variants. We hypothesized that a case–control collapsing analysis in ABCA4-negative patients with compatible symptoms would provide an objective measure to identify additional disease genes.MethodsWe performed a genome-wide enrichment analysis of “qualifying variants”—ultrarare variants predicted to impact protein function—in protein-coding genes in 79 unrelated cases and 9028 unrelated controls.ResultsDespite modest sample size, two known retinal dystrophy genes, PRPH2 and CRX, achieved study-wide significance (p < 1.33 × 10⁻⁶) under a dominant disease model, and eight additional known retinal dystrophy genes achieved nominal significance (p < 0.05). Across these ten genes, the excess of qualifying variants explained up to 36.8% oF.A.ffected individuals. Furthermore, under a recessive model, the cone–rod dystrophy gene CERKL approached study-wide significance.ConclusionOur results indicate that case–control collapsing analyses can efficiently identify pathogenic variants in genes in non-ABCA4 retinal dystrophies. The genome-wide collapsing analysis framework is an objective discovery method particularly suitable in settings with overlapping disease phenotypes.     

Functional characterization of four ATP‐binding cassette transporter A3 gene (ABCA3) variants

ABCA3 transports phospholipids across lamellar body membranes in pulmonary alveolar type II cells and is required for surfactant assembly. Rare, biallelic, pathogenic ABCA3 variants result in lethal neonatal respiratory distress syndrome and childhood interstitial lung disease. Qualitative functional characterization of ABCA3 missense variants suggests two pathogenic classes: disrupted intracellular trafficking (type I mutant) or impaired ATPase‐mediated phospholipid transport into the lamellar bodies (type II mutant). We qualitatively compared wild‐type (WT‐ABCA3) with four uncharacterized ABCA3 variants (c.418A>C;p.Asn140His, c.3609_3611delCTT;p.Phe1203del, c.3784A>G;p.Ser1262Gly, and c.4195G>A;p.Val1399Met) in A549 cells using protein processing, colocalization with intracellular organelles, lamellar body ultrastructure, and ATPase activity. We quantitatively measured lamellar body‐like vesicle diameter and intracellular ABCA3 trafficking using fluorescence‐based colocalization. Three ABCA3 variants (p.Asn140His, p.Ser1262Gly, and p.Val1399Met) were processed and trafficked normally and demonstrated well‐organized lamellar body‐like vesicles, but had reduced ATPase activity consistent with type II mutants. P.Phe1203del was processed normally, had reduced ATPase activity, and well‐organized lamellar body‐like vesicles, but quantitatively colocalized with both endoplasmic reticulum and lysosomal markers, an intermediate phenotype suggesting disruption of both intracellular trafficking and phospholipid transport. All ABCA3 mutants demonstrated mean vesicle diameters smaller than WT‐ABCA3. Qualitative and quantitative functional characterization of ABCA3 variants informs mechanisms of pathogenicity.     

Biallelic variants in OGDH encoding oxoglutarate dehydrogenase lead to a neurodevelopmental disorder characterized by global developmental delay,movement disorder,and metabolic abnormalities

PurposeThis study aimed to establish the genetic cause of a novel autosomal recessive neurodevelopmental disorder characterized by global developmental delay, movement disorder, and metabolic abnormalities.MethodsWe performed a detailed clinical characterization of 4 unrelated individuals from consanguineous families with a neurodevelopmental disorder. We used exome sequencing or targeted-exome sequencing, cosegregation, in silico protein modeling, and functional analyses of variants in HEK293 cells and Drosophila melanogaster, as well as in proband-derived fibroblast cells.ResultsIn the 4 individuals, we identified 3 novel homozygous variants in oxoglutarate dehydrogenase (OGDH) (NM_002541.3), which encodes a subunit of the tricarboxylic acid cycle enzyme α-ketoglutarate dehydrogenase. In silico homology modeling predicts that c.566C>T:p.(Pro189Leu) and c.890C>A:p.(Ser297Tyr) variants interfere with the structure and function of OGDH. Fibroblasts from individual 1 showed that the p.(Ser297Tyr) variant led to a higher degradation rate of the OGDH protein. OGDH protein with p.(Pro189Leu) or p.(Ser297Tyr) variants in HEK293 cells showed significantly lower levels than the wild-type protein. Furthermore, we showed that expression of Drosophila Ogdh (dOgdh) carrying variants homologous to p.(Pro189Leu) or p.(Ser297Tyr), failed to rescue developmental lethality caused by loss of dOgdh. SpliceAI, a variant splice predictor, predicted that the c.935G>A:p.(Arg312Lys)/p.(Phe264_Arg312del) variant impacts splicing, which was confirmed through a mini-gene assay in HEK293 cells.ConclusionWe established that biallelic variants in OGDH cause a neurodevelopmental disorder with metabolic and movement abnormalities.     

Deletions Overlapping VCAN Exon 8 Are New Molecular Defects for Wagner Disease

Wagner disease is a rare nonsyndromic autosomal‐dominant vitreoretinopathy, associated with splice mutations specifically targeting VCAN exon 8. We report the extensive genetic analysis of two Wagner probands, previously found negative for disease‐associated splice mutations. Next‐generation sequencing (NGS), quantitative real‐time PCR, and long‐range PCR identified two deletions (3.4 and 10.5 kb) removing at least one exon–intron boundary of exon 8, and both correlating with an imbalance of VCAN mRNA isoforms. We showed that the 10.5‐kb deletion occurred de novo, causing somatic mosaicism in the proband's mother who had an unusually mild asymmetrical phenotype. Therefore, exon 8 deletions are novel VCAN genetic defects responsible for Wagner disease, and VCAN mosaic mutations may be involved in the pathogenesis of Wagner disease with attenuated phenotype. NGS is then an effective screening tool for genetic diagnosis of Wagner disease, improving the chance of identifying all disease‐causative variants as well as mosaic mutations in VCAN.     

Comprehensive analysis of patients with Stargardt macular dystrophy reveals new genotype–phenotype correlations and unexpected diagnostic revisions

PurposeStargardt macular dystrophy (STGD) results in early central vision loss. We sought to explain the genetic cause of STGD in a cohort of 88 patients from three different cultural backgrounds.MethodsNext-generation sequencing using a novel capture panel was used to search for disease-causing mutations. Patients with undetermined causes were clinically reexamined and tested for copy-number variations as well as intronic mutations.ResultsWe determined the cause of disease in 67% of our patients. Our analysis identified 35 novel ABCA4 alleles. Eleven patients had mutations in genes not previously reported to cause STGD. Finally, 45% of our patients with unsolved causes had single deleterious mutations in ABCA4, a recessive disease gene. No likely pathogenic copy-number variations were identified.ConclusionThis study expands our knowledge of STGD by identifying dozens of novel alleles that cause the disease. The frequency of single mutations in ABCA4 among STGD patients is higher than that among controls, indicating that these mutations contribute to disease. Disease in 11 patients was explained by mutations outside ABCA4, underlining the need to genotype all retinal disease genes to maximize genetic diagnostic rates. Few ABCA4 mutations were observed in our French Canadian patients. This population may contain an unidentified founder mutation. Our results indicate that copy-number variations are unlikely to be a major cause of STGD.Genet Med 17 4, 262–270.