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.     

An ABCA4 genomic deletion in patients with Stargardt disease

Stargardt disease (STGD1) segregates with mutations in the ABCA4 (ABCR) locus. However, mutations of the ABCA4 coding region detected by sequencing account for only 66-80% of disease chromosomes. We hypothesized a potential contribution of otherwise undetected genomic rearrangements of the ABCA4 region. To investigate this hypothesis, we performed genomic Southern analysis on samples from 96 STGD families in which we had identified either one or no ABCA4 mutations by conventional methods. Among 192 chromosomes evaluated, we found one deletion (0.52%), IVS17-905_IVS18+35del, that spans 1,030 bp and eliminates exon 18 of ABCA4. By conceptual translation, this alteration creates an in-frame deletion of 30 amino acids, G885_H915del, and cosegregates with the disease in this family, implying a disease-associated allele. STGD subjects with this deletion were found to have a second mutant ABCA4 allele, 2588G>C. DNA sequence analysis of the deletion junction revealed consensus DNA topoisomerase I sites at both breakpoints that may predispose to nonhomologous recombination. Using deletion-specific PCR, we found the same allele in 2 of 308 STGD subjects (0.32%), in 1 of 96 age-related macular degeneration (AMD) subjects (0.52%), and in 2 of 480 (0.2%) individuals with no known eye diseases, but it was absent in a control group consisting of 96 individuals over age 60 and with normal eye examinations. In vitro biochemical studies of the cloned G885_H915del mutation revealed diminished expression, suggesting that partial deletion of the putative nucleotide-binding domain I leads to either misfolding or defective membrane interactions and eventually reduces the protein function in the retinopathy-affected subjects. Our experiments suggest that genomic alterations contribute to only a small fraction of retinopathy-associated alleles.     

Evaluation of the common variants of the ABCA4 gene in families with Stargardt disease and autosomal recessive retinitis pigmentosa

Stargardt disease (STGD) is one of the most common autosomal recessive retinal dystrophies with an estimated incidence of one in 10,000. It affects the central retina (macula). Retinitis pigmentosa (RP) comprises a large and exceptionally heterogeneous group of hereditary disorders of the retina. It is caused by the loss of photoreceptors. The condition is a degenerative disorder characterized by retinal pigment deposits and has an estimated incidence of one in 4,000. Although, to date, 45 known loci have been identified, none of them independently account for a substantial portion of RP. Recently, the photoreceptor cell-specific ATP-binding cassette transporter (ABCA4) gene was found to be mutated in patients with STGD as well as autosomal recessive RP. In order to further understand the contribution of this gene to the susceptibility to STGD and RP, we analyzed three unrelated STGD families and one autosomal recessive RP family specifically for the more common variants (A1038V, G1961E, 2588G-->C, R943Q or 2828G-->A) in the ABCA4 gene. Our analyses employing standard techniques such as polymerase chain reaction, restriction fragment length polymorphism, and direct DNA sequencing of amplified products were able to identify one common variant (R943Q) in all three STGD families but not in the RP family. All three affected STGD individuals, however, were heterozygous for this variation, and this alteration did not segregate with the disease and was also present in the normal controls. Similar analysis of other common variants revealed no pathogenic mutations in the STGD and RP families. It is likely that the variant identified in this study represents a rare polymorphism (non-pathogenic). Although, at present we cannot eliminate the possibility of this gene as a candidate gene, future extensive studies on this as well as other candidate genes may uncover the susceptibility gene for these recessive forms of the disorders in these families.     

Molecular analysis of the ABCA4 gene in Turkish patients with Stargardt disease and retinitis pigmentosa

The clinical importance of sequence variations in the ABCA4 gene has been extensively discussed during the last decade. Mutations in the ABCA4 gene are involved in several forms of inherited retinal degenerations. We screened all 50 exons of the ABCA4 gene in a cohort of 5 Stargardt Disease (STGD) and 35 autosomal recessive retinitis pigmentosa (arRP) patients of Turkish descent to assess the nature of ABCA4 mutant alleles in this population. Our results revealed the presence of three novel mutations: c.160T>G (p.C54G), c.2486C>T (p.T829M), and c.973-6C>A; two mutations previously reported, c.634C>T (p.R212C) and c.4253+4C>T, and several polymorphic changes in the ABCA4 gene among Turkish patients affected with Stargardt and arRP. To our knowledge this report represents the first published study of ABCA4 mutations in the Turkish population resulting in STGD.     

Novel mutations in CRB1 and ABCA4 genes cause Leber congenital amaurosis and Stargardt disease in a Swedish family

This study aimed to identify genetic mechanisms underlying severe retinal degeneration in one large family from northern Sweden, members of which presented with early-onset autosomal recessive retinitis pigmentosa and juvenile macular dystrophy. The clinical records of affected family members were analysed retrospectively and ophthalmological and electrophysiological examinations were performed in selected cases. Mutation screening was initially performed with microarrays, interrogating known mutations in the genes associated with recessive retinitis pigmentosa, Leber congenital amaurosis and Stargardt disease. Searching for homozygous regions with putative causative disease genes was done by high-density SNP-array genotyping, followed by segregation analysis of the family members. Two distinct phenotypes of retinal dystrophy, Leber congenital amaurosis and Stargardt disease were present in the family. In the family, four patients with Leber congenital amaurosis were homozygous for a novel c.2557C>T (p.Q853X) mutation in the CRB1 gene, while of two cases with Stargardt disease, one was homozygous for c.5461-10T>C in the ABCA4 gene and another was carrier of the same mutation and a novel ABCA4 mutation c.4773+3A>G. Sequence analysis of the entire ABCA4 gene in patients with Stargardt disease revealed complex alleles with additional sequence variants, which were evaluated by bioinformatics tools. In conclusion, presence of different genetic mechanisms resulting in variable phenotype within the family is not rare and can challenge molecular geneticists, ophthalmologists and genetic counsellors.     

Cosegregation and functional analysis of mutant ABCR (ABCA4) alleles in families that manifest both Stargardt disease and age-related macular degeneration.

Mutations in ABCR (ABCA4) have been reported to cause a spectrum of autosomal recessively inherited retinopathies, including Stargardt disease (STGD), cone-rod dystrophy and retinitis pigmentosa. Individuals heterozygous for ABCR mutations may be predisposed to develop the multifactorial disorder age-related macular degeneration (AMD). We hypothesized that some carriers of STGD alleles have an increased risk to develop AMD. We tested this hypothesis in a cohort of families that manifest both STGD and AMD. With a direct-sequencing mutation detection strategy, we found that AMD-affected relatives of STGD patients are more likely to be carriers of pathogenic STGD alleles than predicted based on chance alone. We further investigated the role of AMD-associated ABCR mutations by testing for expression and ATP-binding defects in an in vitro biochemical assay. We found that mutations associated with AMD have a range of assayable defects ranging from no detectable defect to apparent null alleles. Of the 21 missense ABCR mutations reported in patients with AMD, 16 (76%) show abnormalities in protein expression, ATP-binding or ATPase activity. We infer that carrier relatives of STGD patients are predisposed to develop AMD.     

筛选反义寡核苷酸抑制哮喘大鼠CD4+T细胞IL-4表达的研究

目的探讨不同序列的反义寡核苷酸对哮喘大鼠CD4+T细胞IL-4表达的抑制作用.方法采用脂质体转染技术,将不同的反义寡核苷酸(AS-1反外显子-1;AS-2反外显子-2;AS-3反翻译终止部位)和空白对照分别转入经免疫磁珠阴性分离的哮喘大鼠CD4+T淋巴细胞,细胞培养28小时后,用ELISA和半定量RT-PCR分别检测细胞培养上清IL-4和细胞内IL-4 mRNA的水平.结果不同组RT-PCR结果(IL-4/β-actin相对吸光度)AS-1、AS-2、 AS-3及空白组分别为0.261 5±0.147 6、0.288 5±0.141 1、1.101 2±0.364 1及1.206 8±0.383 6(F=22.597,P＜0.01).ELISA检测培养上清液IL-4结果AS-1、AS-2、 AS-3及空白组分别为13.800±7.233、15.329±7.358、52.643±12.075及58.286±14.100(F=34.976,P＜0.01).经AS1、2干预后细胞内IL-4mRNA和细胞培养上清IL-4的水平均较AS-3和空白组干预后 IL-4的水平低(P＜0.01).结论IL-4反义寡核苷酸能够抑制哮喘大鼠CD4+T淋巴细胞IL-4和IL-4mRNA表达;不同序列IL-4反义寡核苷酸抑制作用存在差异.     

Genetic variants of ABCA1 modify Alzheimer disease risk and quantitative traits related to beta-amyloid metabolism

Linkage studies have provided evidence that one or more loci on chromosome 9q influence Alzheimer disease (AD). The gene encoding the ATP-binding cassette A1 transporter (ABCA1) resides within proximity of previously identified linkage peaks and represents a plausible biological candidate for AD due to its central role in cellular lipid homeostasis. Several single nucleotide polymorphisms (SNPs) spanning ABCA1 have been genotyped and haplotype-based association analyses performed in four independent case-control samples, consisting of over 1,750 individuals from three European populations representing both early and late-onset AD. Prominent effects were observed for a common (H2) and rarer haplotype (H5) that were enriched in AD cases across studied populations (odds ratio [OR] 1.59, 95% confidence interval [CI] 1.36-1.82; P<0.00001 and OR 2.90; 95% CI 2.54-3.27; P<0.00001, respectively). Two other common haplotypes in the studied region (H1 and H3) were significantly under-represented in AD cases, suggesting that they may harbor alleles that decrease disease risk (OR 0.79, 95% CI 0.64-0.94; P=0.0065 and OR 0.70, 95% CI 0.46-0.93; P=0.011, respectively). While findings were significant in both early and late-onset samples, haplotype effects were more distinct in early-onset materials. For late-onset samples, ancillary evidence was obtained that both single marker alleles and haplotypes of ABCA1 contribute to variable cerebrospinal fluid tau and beta amyloid (Abeta42) protein levels, and brain Abeta load. Results indicate that variants of ABCA1 may affect the risk of AD, providing further support for a genetic link between AD and cholesterol metabolism.     

Transcriptional expression of survivin and its splice variants in endometriosis

Survivin is a novel inhibitor of apoptosis (IAP), and the two splice variants of survivin (survivin-2B and survivin-EX3) have been identified. Gene expression levels of survivin, survivin-2B and survivin-EX3 in 56 ectopic (16 peritoneal red and 16 peritoneal black lesions and 24 ovarian endometriomata) and 13 eutopic endometrial tissues surgically obtained from 42 women with endometriosis (group A) were compared with those in 16 control eutopic endometrium from 16 women without endometriosis (group B) by quantitative RT-PCR analysis. Survivin mRNA expression levels in ectopic endometriotic tissues were significantly higher than those in eutopic endometrium of groups A and B over the whole cycle. Red peritoneal lesions had higher gene expression levels of survivin than black lesions. In contrast, all tissue samples examined showed relatively lower gene expression levels of survivin-2B and survivin-EX3. No cyclic variation was found in survivin and the two splice variants, both in ectopic and in eutopic endometrium. Although there was no significant difference in the ratio of survivin-2B/survivin between ectopic and eutopic endometrium, the ratio of survivin-EX3/survivin in peritoneal endometriotic lesions was significantly higher than that of eutopic endometrium of groups A and B. These results suggest that survivin and survivin-EX3 may be closely linked to escape from apoptosis and the development of endometriosis.     

Distribution of NBCn2 (SLC4A10) splice variants in mouse brain

The five known Na-coupled HCO₃^– transporters (NCBTs) of the solute carrier 4 (SLC4) family play important roles in pH regulation and transepithelial HCO₃^– transport. Nearly all of the NCBTs have multiple splice variants. One particular NCBT, the electroneutral Na/HCO₃^– cotransporter NBCn2 (SLC4A10), which is predominantly expressed in brain, has three known splice variants—NBCn2-A, -B, and -C—as well as a potential variant-D. It is important to know the tissue-specific expression of the splice variants for understanding the physiological roles of NBCn2 in central nervous system. In the present study, we developed three novel rabbit polyclonal antibodies against NBCn2: (1) anti-ABCD, which recognizes all four variants; (2) anti-BD, which recognizes NBCn2-B and -D; (3) anti-CD, which recognizes NBCn2-C and -D. By western blotting, we examined the expression and distribution of NBCn2 splice variants in five brain regions: cerebral cortex, subcortex, cerebellum, hippocampus, and medulla. The expression pattern revealed with anti-ABCD is distinct from those revealed with anti-BD and anti-CD. Moreover, by using immunoprecipitation in combination with western blotting, we demonstrate that NBCn2-D does indeed exist and that it is predominantly expressed in subcortex, to a lesser extent in medulla, but at very low levels in cortex, cerebellum, and hippocampus. NBCn2-A may be the dominant variant in mouse brain as a whole, and may also dominate in cerebral cortex, cerebellum, and hippocampus. Immunohistochemistry with anti-ABCD shows that NBCn2 is highly expressed in choroid plexus, cortex, molecular layer of cerebellum, hippocampus, and some specific regions of the brainstem.