Functional characterization of two novel splicing mutations in the OCA2 gene associated with oculocutaneous albinism type II

Rimoldi, Valeria; Straniero, Letizia; Asselta, Rosanna; Mauri, Lucia; Manfredini, Emanuela; Penco, Silvana; Gesu, Giovanni; Longo, Alessandra; Piozzi, Elena; Soldà, Giulia; Primignani, Paola

doi:10.1016/j.gene.2013.11.102

Cited by 16 publications

(8 citation statements)

References 26 publications

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“…Among ten novel indels, nine frameshift indels can be classified as pathogenic variants and a variant OCA2 _c.2373_2375delCGT (p.Val792del) can only be classified as a variant with uncertain significance (VUS). Eight variants are in or flank splicing site, including three reported previously to be related to OCA (Marti et al, ; Rimoldi et al, ), and five novel variants ( OCA2 _c.646+3A>G, OCA2 _c.2140‐2A>G, OCA2 _c.2245‐11T>G, OCA2 _c.808‐3C>G, and SLC45A2 _c.1032+1G>T), four ( OCA2 _c.646+3A>G, OCA2 _c.2140‐2A>G, OCA2 _c.808‐3C>G, and SLC45A2 _c.1032+1G>T) of which in vitro splicing assay compared with WT in HeLa and ARPE‐19 cell lines, demonstrated that brought about change in splicing (Figure ) and no change was observed between WT and MUT for analysis of variant OCA2 _c.2245‐11T>G (Data not shown). Therefore, seven splicing can be classified as pathogenic variants and OCA2 _c.2245‐11T>G can only be classified as a VUS at the current stage.…”

Section: Resultsmentioning

confidence: 96%

Comprehensive analysis of spectral distribution of a large cohort of Chinese patients with non‐syndromic oculocutaneous albinism facilitates genetic diagnosis

Zhong

Zheng

et al. 2019

Pigment Cell Melanoma Res

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Non‐syndromic oculocutaneous albinism (nsOCA) is a group of genetically heterogeneous autosomal recessive disorders with complete lack or decrease pigmentation in skin, hair, and eyes. TYR, OCA2, TYRP1, SLC45A2, SLC24A5, and LRMDA were reported to cause OCA1‐4 and OCA6‐7, respectively. By sequencing all the known nsOCA genes in 114 unrelated Chinese nsOCA patients combined with In silico analyses, splicing assay, and classification of variants according to the standards and guidelines of American College of Medical Genetics and Genomics, we detected seventy‐one different OCA‐causing variants separately in TYR, OCA2, SLC45A2, and SLC24A5, including thirty‐one novel variants (13 in TYR, 11 in OCA2, and 7 in SLC45A2). This study shows that OCA1 is the most common (75/114) and OCA2 ranks the second most common (16/114) in Chinese. 99 patients of our cohort were caused by variants of all the known nsOCA genes. Cutaneous phenotypes of OCA1, OCA2, and OCA4 patients were shown in this study. The second OCA6 case in China was identified here. These data expand the spectrum of OCA variants as well phenotype and facilitate clinical implement of Chinese OCA patients.

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Section: Resultsmentioning

confidence: 96%

Comprehensive analysis of spectral distribution of a large cohort of Chinese patients with non‐syndromic oculocutaneous albinism facilitates genetic diagnosis

Zhong

Zheng

et al. 2019

Pigment Cell Melanoma Res

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“…We analyzed mCherry-OCA2 variants containing disease-related mutations within highly conserved regions of the protein ( Figure 1—figure supplement 1A ). We chose mutations identified in patients with OCA type II through human genetic studies: V443I, a common albinism-associated mutation in a predicted luminal loop and important for melanin content in vitro ( Lee et al, 1994a , 1994b ; Sviderskaya et al, 1997 ; King et al, 2003 ; Garrison et al, 2004 ; Hongyi et al, 2007 ; Preising et al, 2007 ; Hutton and Spritz, 2008 ; Rimoldi et al, 2014 ) and K614E, in a predicted cytoplasmic loop ( Passmore et al, 1999 ). We also generated an OCA2 variant with five point mutations in the same predicted luminal loop as V443I (5mut: V443I, M446V, I473S, N476D, N489D) ( Lee et al, 1994a , 1994b ; Spritz et al, 1995 ; Hongyi et al, 2007 ; Preising et al, 2007 ) ( Figure 2A ).…”

Section: Resultsmentioning

confidence: 99%

An intracellular anion channel critical for pigmentation

Bellono

Escobar

Lefkovith

et al. 2014

eLife

110

129

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Intracellular ion channels are essential regulators of organellar and cellular function, yet the molecular identity and physiological role of many of these channels remains elusive. In particular, no ion channel has been characterized in melanosomes, organelles that produce and store the major mammalian pigment melanin. Defects in melanosome function cause albinism, characterized by vision and pigmentation deficits, impaired retinal development, and increased susceptibility to skin and eye cancers. The most common form of albinism is caused by mutations in oculocutaneous albinism II (OCA2), a melanosome-specific transmembrane protein with unknown function. Here we used direct patch-clamp of skin and eye melanosomes to identify a novel chloride-selective anion conductance mediated by OCA2 and required for melanin production. Expression of OCA2 increases organelle pH, suggesting that the chloride channel might regulate melanin synthesis by modulating melanosome pH. Thus, a melanosomal anion channel that requires OCA2 is essential for skin and eye pigmentation.DOI: http://dx.doi.org/10.7554/eLife.04543.001

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“…For the functional characterization of the candidate branch-site mutation in COL4A5 , the relevant genomic DNA region was cloned in the hybrid alpha-globin-fibronectin minigene plasmid (modified pBS-KS), as previously described [22]. In particular, a 543-bp fragment of COL4A5 (including the entire exon 29 and flanking intronic regions) was PCR amplified from the patient’s genomic DNA using the following primers: COL4A5_ex29_ Nde I_F and COL4A5_ex29_ Nde I_R (lowercase letters indicate nucleotides added to the primers to introduce the Nde I restriction site) and cloned into the modified pBS-KS vector.…”

Section: Methodsmentioning

confidence: 99%

Alport syndrome cold cases: Missing mutations identified by exome sequencing and functional analysis

et al. 2017

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Alport syndrome (AS) is an inherited progressive renal disease caused by mutations in COL4A3, COL4A4, and COL4A5 genes. Despite simultaneous screening of these genes being widely available, mutation detection still remains incomplete in a non-marginal portion of patients. Here, we applied whole-exome sequencing (WES) in 3 Italian families negative after candidate-gene analyses. In Family 1, we identified a novel heterozygous intronic variant (c.2245-40A>G) -outside the conventionally screened candidate region for diagnosis- potentially disrupting COL4A5 exon29 splicing. Using a minigene-based approach in HEK293 cells we demonstrated that this variant abolishes exon29 branch site, causing exon skipping. Moreover, skewed X-inactivation of the c.2245-40A>G allele correlated with disease severity in heterozygous females. In Family 2, WES highlighted a novel COL4A5 hemizygous missense mutation (p.Gly491Asp), which segregates with the phenotype and impacts on a highly-conserved residue. Finally, in Family 3, we detected a homozygous 24-bp in-frame deletion in COL4A3 exon1 (NM_000091.4:c.30_53del:p.Val11_Leu18del or c.40_63del24:p.Leu14_Leu21del), which is ambiguously annotated in databases, although it corresponds to a recurrent AS mutation. Functional analyses showed that this deletion disrupts COL4A3 signal peptide, possibly altering protein secretion. In conclusion, WES -together with functional studies- was fundamental for molecular diagnosis in 3 AS families, highlighting pathogenic variants that escaped previous screenings.

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Functional characterization of two novel splicing mutations in the OCA2 gene associated with oculocutaneous albinism type II

Cited by 16 publications

References 26 publications

Comprehensive analysis of spectral distribution of a large cohort of Chinese patients with non‐syndromic oculocutaneous albinism facilitates genetic diagnosis

Comprehensive analysis of spectral distribution of a large cohort of Chinese patients with non‐syndromic oculocutaneous albinism facilitates genetic diagnosis

An intracellular anion channel critical for pigmentation

Alport syndrome cold cases: Missing mutations identified by exome sequencing and functional analysis

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