Alternative titles; symbols
HGNC Approved Gene Symbol: RIPOR2
Cytogenetic location: 6p22.3 Genomic coordinates (GRCh38): 6:24,804,284-25,042,168 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 6p22.3 | ?Deafness, autosomal recessive 104 | 616515 | Autosomal recessive | 3 |
| Deafness, autosomal dominant 21 | 607017 | Autosomal dominant | 3 |
RIPOR2 plays a role in myogenic differentiation and formation of cellular filopodia (Yoon et al., 2007).
By sequencing clones obtained from a size-fractionated brain cDNA library, Nagase et al. (1997) cloned C6ORF32, which they designated KIAA0386. The deduced protein has 1,068 amino acids. RT-PCR detected moderate expression in thymus and weak expression in placenta and lung.
By subtractive hybridization between undifferentiated human term cytotrophoblasts and differentiating cytotrophoblasts to identify genes with increased expression during differentiation, Dakour et al. (1997) cloned C6ORF32, which they called PL48. The 536-amino acid protein has a calculated molecular mass of 59.8 kD and contains a transmembrane region, 2 glycosaminoglycan attachment sites, 4 N-linked glycosylation sites, and numerous potential phosphorylation sites. Northern blot analysis of spontaneously differentiating human cytotrophoblasts detected 2.8-, 3.5-, and 4.8-kb transcripts at 2 hours with increased expression over time of differentiation. PLA48 was not expressed in undifferentiated myeloid leukemia cells, but expression increased between 24 and 72 hours following DMSO-induced granulocytic lineage differentiation. Dakour et al. (1997) concluded that PL48 acted as an early response gene during spontaneous cytotrophoblast differentiation but was induced later in the DMSO-induced granulocytic differentiation pathway.
Yoon et al. (2007) cloned C6ORF32 by microarray analysis of genes with increased expression during fetal myoblast differentiation, followed by RT-PCR of human primary fetal muscle cells harvested 5 days after differentiation. C6ORF32 can encode 12 putative alternatively spliced forms. They identified 2 isoforms, which they called Iso1 and Iso2. Iso1 contains 1,018 amino acids, and Iso2 contains 591 amino acids, including an N-terminal extension of 55 amino acids compared to the PL48 species identified by Dakour et al. (1997). Immunofluorescence studies localized C6ORF32 to the cell membrane, cytoskeleton, and long cellular filopodia in primary human fetal skeletal muscle cells, and to the cytoplasm in cells undergoing mitosis. C6ORF32 was expressed in mononuclear cells, not in myotubes, upon induction of differentiation and cellular fusion, and expression was upregulated during human myoblast differentiation at both the mRNA and protein levels.
In mouse tissues, Diaz-Horta et al. (2014) found highest expression of Fam65b in the embryonic brain, followed by the inner ear and postnatal inner ear. Immunofluorescent studies showed that Fam65b was present in stereocilia of inner and outer hair cells and was targeted to the apical plasma membrane compartment. The protein was abundant at the vesicular trafficking area and showed vesicle-like structures, suggesting that it is in intracellular membrane trafficking compartments.
Yoon et al. (2007) found that RNAi knockdown of mouse C6orf32 during murine myoblast differentiation caused decreased cell fusion and severe reduction of myotube formation, characterized by decreased myogenin (MYOG; 159980) and myosin heavy chain (see 160730) expression and unaltered MyoD1 (159970) expression. The authors suggested that C6orf32 may play a role in early myogenic differentiation and that the decreased muscle cell fusion may be a secondary effect of impaired or slowed differentiation caused by C6orf32 depletion. Overexpression of mouse C6orf32 Iso2 or Iso1 in murine myoblasts and human embryonic kidney cells induced formation of long filopodia. Transfection of mutant constructs showed that N-terminal amino acids 55 to 113 of C6ORF32 are likely to mediate filopodia formation.
Using biochemistry and stochastic optical reconstruction microscopy, Zhao et al. (2016) showed that Fam65b oligomers formed a circumferential ring near the basal taper of hair cell stereocilia in mice. Taperin (TPRN; 613354), a protein near the taper, formed a dense core-like structure that was disrupted in the absence of Fam65b. Stereocilia of Fam65b-knockout mice showed disrupted mechanotransduction followed by deterioration. Yeast 2-hybrid screening revealed that Rhoc (165380) bound Fam65b. Rhoc colocalized with Fam65b in stereocilia and was required for Fam65b function.
By radiation hybrid analysis, Nagase et al. (1997) mapped the C6ORF32 gene to chromosome 6.
Gross (2022) mapped the RIPOR2 gene to chromosome 6p22.3 based on an alignment of the RIPOR2 sequence (GenBank BC001232) with the genomic sequence (GRCh38).
Autosomal Recessive Deafness 104
In 6 affected members of a consanguineous Turkish family with autosomal recessive deafness-104 (DFNB104; 616515), Diaz-Horta et al. (2014) identified a homozygous splice site mutation in the FAM65B gene (611410.0001). The mutation, which was found by whole-exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family. In vitro functional expression studies showed that the mutant protein accumulated abnormally in cytoplasmic inclusion bodies and did not reach the membrane. Mutations in the FAM65B gene were not found in 248 other families with autosomal recessive deafness or in 437 simplex cases with deafness. Diaz-Horta et al. (2014) concluded that FAM65B is a plasma membrane-associated protein of hair cell stereocilia that is essential for hearing.
Autosomal Dominant Deafness 21
In 20 affected members of a large multigenerational Dutch family (W97-056) with autosomal dominant deafness-21 (DFNA21; 607017) reported by Kunst et al. (2000), de Bruijn et al. (2021) identified a heterozygous 12-bp in-frame deletion (c.1696_1707del) in the RIPOR2 gene (611410.0002). The mutation, which was identified by exome sequencing, segregated with the disorder in the family, although there was evidence for incomplete or age-dependent penetrance. Based on these findings, analysis of existing exome datasets from large cohorts of patients with hereditary hearing loss identified 11 additional probands of Dutch origin who carried this heterozygous deletion. It segregated with the disorder in 6 families, with evidence of incomplete penetrance. Haplotype analysis indicated a founder effect. Expression of the orthologous Ripor2 mutation in the cochlear outer hair cells of wildtype mice did not visibly affect the stereocilia structure in the short term, but the mutant protein did not localize normally to the stereocilia base. Expression of the mutation into Ripor2-null mice was unable to rescue morphologic defects of outer hair cells, suggesting that the small deletion disrupts protein function. Since haploinsufficiency is an unlikely mechanism, de Bruijn et al. (2021) postulated a toxic gain-of-function effect. The deletion was present at low frequencies in the gnomAD database (0.0071-0.0077%). The heterozygous deletion was found in 0.0392% of individuals (18 of 22,952) from the southeast Netherlands with unknown hearing abilities, and the authors concluded that mutations in RIPOR2 may be a common cause of hearing loss in certain northern European populations.
Diaz-Horta et al. (2014) found that fam65b was expressed in the otic vesicle of zebrafish. Morpholino knockdown of the fam65b gene in zebrafish embryos resulted in a significant reduction in the number of saccular hair cells and neuromasts, and caused hearing loss.
In 6 affected members of a consanguineous Turkish family with autosomal recessive deafness-104 (DFNB104; 616515), Diaz-Horta et al. (2014) identified a homozygous G-to-A transition at the splice acceptor site of intron 2 of the FAM65B gene (c.102-1G-A, NM_014722.2), resulting in the in-frame skipping of exon 3, which comprises residues 34-86 (R34_D86delinsS) in the core region of the PX membrane localization domain. The mutation, which was found by whole-exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family. It was not found in the dbSNP (build 137) or Exome Variant Server databases or in 330 Turkish controls. In vitro functional expression studies in COS-7 cells showed that the mutant protein mislocalized to large puncta in the cytosol, suggesting an accumulation into dense inclusion bodies. In contrast, the wildtype protein was distributed within cell membrane trafficking compartments at the edge of the cell periphery.
In 20 affected members of a large multigenerational Dutch family (W97-056) with autosomal dominant deafness-21 (DFNA21; 607017) reported by Kunst et al. (2000), de Bruijn et al. (2021) identified a heterozygous 12-bp in-frame deletion (c.1696_1707del, NM_014722.3) in exon 14 of the RIPOR2 gene, resulting in a deletion of 4 residues (Gln566_Lys569del) from a conserved region that is present in all isoforms. The mutation, which was identified by exome sequencing, segregated with the disorder in the family, although 3 unaffected family members ranging from 23 to 51 years of age carried the mutation, indicating incomplete or age-dependent penetrance. The mutation was not present in 3 affected family members, suggesting phenocopies. Based on these findings, analysis of existing exome datasets from large cohorts of patients with hereditary hearing loss identified 11 additional probands of Dutch origin who carried this heterozygous deletion. It segregated with the disorder in 6 families, although there was evidence of incomplete penetrance. In addition, 1 affected individual in family W04-262 did not carry the mutation. Haplotype analysis indicated a founder effect. Expression of the orthologous Ripor2 mutation in the cochlear outer hair cells of wildtype mice did not visibly affect the stereocilia structure in the short term, but the mutant protein did not localize normally to the stereocilia base. Expression of the mutation into Ripor2-null mice was unable to rescue morphologic defects of outer hair cells, suggesting that the small deletion disrupts protein function. Since haploinsufficiency is an unlikely mechanism, de Bruijn et al. (2021) postulated a toxic gain-of-function effect. The deletion was present at low frequencies in the gnomAD database (0.0071-0.0077%). The heterozygous deletion was found in 0.0392% of individuals (18 of 22,952) from the southeastern Netherlands with unknown hearing abilities, and the authors concluded that mutations in RIPOR2 may be a common cause of hearing loss in certain northern European populations.
Dakour, J., Li, H., Morrish, D. W. PL48: a novel gene associated with cytotrophoblast and lineage-specific HL-60 cell differentiation. Gene 185: 153-157, 1997. [PubMed: 9055809] [Full Text: https://doi.org/10.1016/s0378-1119(96)00587-2]
de Bruijn, S. E., Smits, J. J., Liu, C., Lanting, C. P., Beynon, A. J., Blankevoort, J., Oostrik, J., Koole, W., de Vrieze, E., Cremers, C. W. R. J., Cremers, F. P. M., Roosing, S., Yntema, H. G., Kunst, H. P. M., Zhao, B., Pennings, R. J. E., Kremer, H., DOOFNL Consortium. A RIPOR2 in-frame deletion is a frequent and highly penetrant cause of adult-onset hearing loss. J. Med. Genet. 58: 96-104, 2021.
Diaz-Horta, O., Subasioglu-Uzak, A., Grati, M., DeSmidt, A., Foster, J., II, Cao, L., Bademci, G., Tokgoz-Yilmaz, S., Duman, D., Cengiz, F. B., Abad, C., Mittal, R., Blanton, S., Liu, X. Z., Farooq, A., Walz, K., Lu, Z., Tekin, M. FAM65B is a membrane-associated protein of hair cell stereocilia required for hearing. Proc. Nat. Acad. Sci. 111: 9864-9868, 2014. [PubMed: 24958875] [Full Text: https://doi.org/10.1073/pnas.1401950111]
Gross, M. B. Personal Communication. Baltimore, Md. 3/18/2022.
Kunst, H., Marres, H., Huygen, P., Van Duijnhoven, G., Krebsova, A., Van Der Velde, S., Reis, A., Cremers, F., Cremers, C. Non-syndromic autosomal dominant progressive non-specific mid-frequency sensorineural hearing impairment with childhood to late adolescence onset (DFNA21). Clin. Otolaryng. 25: 45-54, 2000. [PubMed: 10764236] [Full Text: https://doi.org/10.1046/j.1365-2273.2000.00327.x]
Nagase, T., Ishikawa, K., Nakajima, D., Ohira, M., Seki, N., Miyajima, N., Tanaka, A., Kotani, H., Nomura, N., Ohara, O. Prediction of the coding sequences of unidentified human genes. VII. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro. DNA Res. 4: 141-150, 1997. [PubMed: 9205841] [Full Text: https://doi.org/10.1093/dnares/4.2.141]
Yoon, S., Molloy, M. J., Wu, M. P., Cowan, D. B., Gussoni, E. C6ORF32 is upregulated during muscle cell differentiation and induces the formation of cellular filopodia. Dev. Biol. 301: 70-81, 2007. [PubMed: 17150207] [Full Text: https://doi.org/10.1016/j.ydbio.2006.11.002]
Zhao, B., Wu, Z., Muller, U. Murine Fam65b forms ring-like structures at the base of stereocilia critical for mechanosensory hair cell function. eLife 5: e14222, 2016. [PubMed: 27269051] [Full Text: https://doi.org/10.7554/eLife.14222]