Novel Resequencing Chip Customized to Diagnose Mutations in Patients With Inherited Syndromes of Intrahepatic Cholestasis

Liu, Cong; Aronow, Bruce J.; Jegga, Anil G.; Wang, Ning; Miethke, Alex; Mourya, Reena; Bezerra, Jorge A.

doi:10.1053/j.gastro.2006.10.034

Cited by 95 publications

(81 citation statements)

References 50 publications

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“…7,8,12,[28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][57][58][59] This study showed that some ABCB11 missense mutations were associated with splicing defects, most caused protein processing defects, and the two functionally analyzed showed a reduction in taurocholate transport. Additionally, the ability to modulate splicing and protein processing defects was examined; some mutations were amenable to such treatments.…”

Section: Discussionmentioning

confidence: 75%

Missense mutations and single nucleotide polymorphisms in ABCB11 impair bile salt export pump processing and function or disrupt pre-messenger RNA splicing #

et al. 2009

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The gene encoding the human bile salt export pump (BSEP), ABCB11, is mutated in several forms of intrahepatic cholestasis. Here we classified the majority (63) of known ABCB11 missense mutations and 21 single-nucleotide polymorphisms (SNPs) to determine whether they caused abnormal ABCB11 pre-messenger RNA splicing, abnormal processing of BSEP protein, or alterations in BSEP protein function. Using an in vitro minigene system to analyze splicing events, we found reduced wild-type splicing for 20 mutations/SNPs, with normal mRNA levels reduced to 5% or less in eight cases. The common ABCB11 missense mutation encoding D482G enhanced aberrant splicing, whereas the common SNP A1028A promoted exon skipping. Addition of exogenous splicing factors modulated several splicing defects. Of the mutants expressed in vitro in CHO-K1 cells, most appeared to be retained in the endoplasmic reticulum and degraded. A minority had BSEP levels similar to wild-type. The SNP variant A444 had reduced levels of protein compared with V444. Treatment with glycerol and incubation at reduced temperature overcame processing defects for several mutants, including E297G. Taurocholate transport by two assessed mutants, N490D and A570T, was reduced compared with wild-type. Conclusion: This work is a comprehensive analysis of 80% of ABCB11 missense mutations and singlenucleotide polymorphisms at pre-mRNA splicing and protein processing/functional levels. We show that aberrant pre-mRNA splicing occurs in a considerable number of cases, leading to reduced levels of normal mRNA. Thus, primary defects at either the protein or the mRNA level (or both) contribute significantly to BSEP deficiency. These results will help to develop mutation-specific therapies for children and adults suffering from intrahepatic cholestasis due to BSEP deficiency. (HEPATOLOGY 2009;49:553-567.)

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Section: Discussionmentioning

confidence: 75%

Missense mutations and single nucleotide polymorphisms in ABCB11 impair bile salt export pump processing and function or disrupt pre-messenger RNA splicing #

et al. 2009

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“…High-density oligonucleotide microarrays offer the opportunity to conduct both tests on a single analytical platform. Although single-nucleotide polymorphism arrays have been used extensively, little is known about the sensitivity of mutation detection with ABR (4,6 ). Our data suggest that the HCM1 array is an effective alternative to conventional capillary sequencing.…”

Section: Discussionmentioning

confidence: 80%

Array-Based Resequencing Assay for Mutations Causing Hypertrophic Cardiomyopathy

et al. 2008

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Background: Dissecting the complex genetic basis of hypertrophic cardiomyopathy (HCM) may be key to both better understanding and optimally managing this most prevalent genetic cardiovascular disease. An array-based resequencing (ABR) assay was developed to facilitate genetic testing in HCM. Methods: An Affymetrix resequencing array and a single long-range PCR protocol were developed to cover the 3 most commonly affected genes in HCM, MYH7 (myosin, heavy chain 7, cardiac muscle, beta), MYBPC3 (myosin binding protein C, cardiac), and TNNT2 [troponin T type 2 (cardiac)]. Results: The assay detected the underlying point mutation in 23 of 24 reference samples and provided pointers toward identifying a G insertion and a 3-bp deletion. The comparability of array-based assay results to conventional capillary sequencing was ≥99.9%. Both techniques detected 1 heterozygous variant that was missed by the other method. Conclusions: The data provide evidence that ABR can substantially reduce the high workload previously associated with a genetic test for HCM. Therefore, the HCM array could facilitate large-scale studies aimed at broadening the understanding of the genetic and phenotypic diversity of HCM and related cardiomyopathies.

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“…Although its first application was in SNP discovery and genotyping, recent reports have evaluated resequencing arrays as a means of finding human disease-causing mutations in hypogonadotrophic hypogonadism, 22 congenital myasthenia, 22 retinitis pigmentosum, 27 lung cancer, 28 and intrahepatic cholestasis. 29 All of these studies achieved overall call rates exceeding 90% using methods based on adaptive background genotype calling scheme algorithms of Cutler et al, 20 which …”

Section: Genementioning

confidence: 99%

Mutations causing severe combined immunodeficiency: detection with a custom resequencing microarray

Lebet

Chiles²,

Hsu

et al. 2008

Genetics in Medicine

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Purpose: Mutation diagnosis of severe combined immunodeficiency is challenging because of the multiplicity of disease genes and large number of disease-causing mutations, including unique ones that continue to be found.A resequencing microarray could facilitate mutation detection, increasing the chance of diagnosing infants early for optimal rescue by hematopoietic stem cell transplantation. Methods: After analyzing cumulative mutations, we developed a custom Affymetrix GeneChip® microarray including probes representing exons and flanking regions of severe combined immunodeficiency disease genes. DNA samples were analyzed by array versus standard dideoxy genomic sequencing. We tested males and their mothers with X-linked IL2RG variants and patients and carriers with autosomal variants in IL7R, JAK3, and DCLRE1C. Results: New, unique severe combined immunodeficiency mutations are frequent. Resequencing array call rates of 95-98% exceeded GeneChip product specifications, and all of 47 point mutations in known samples were detected, as were the sites of 12 of 22 disease-causing insertions and deletions. Each gene had particular nucleotides that were often not called correctly and had to be excluded from analysis; exclusion rates ranged from 0.4% (hemizygous IL2RG) to 9.2% (heterozygous JAK3). Conclusion:Microarray resequencing is a promising technology for severe combined immunodeficiency mutation diagnosis that

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Novel Resequencing Chip Customized to Diagnose Mutations in Patients With Inherited Syndromes of Intrahepatic Cholestasis

Cited by 95 publications

References 50 publications

Missense mutations and single nucleotide polymorphisms in ABCB11 impair bile salt export pump processing and function or disrupt pre-messenger RNA splicing #

Missense mutations and single nucleotide polymorphisms in ABCB11 impair bile salt export pump processing and function or disrupt pre-messenger RNA splicing #

Array-Based Resequencing Assay for Mutations Causing Hypertrophic Cardiomyopathy

Mutations causing severe combined immunodeficiency: detection with a custom resequencing microarray

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