R3HDM1 haploinsufficiency is associated with mild intellectual disability.

R3HDM1 (R3H domain containing 1) is an uncharacterized RNA-binding protein that is highly expressed in the human cerebral cortex. We report the first case of a 12-year-old Japanese male with haploinsufficiency of R3HDM1. He presented with mild intellectual disability (ID) and developmental delay. He had a pericentric inversion of 46,XY,inv(2)(p16.1q21.3)dn with breakpoints in intron 19 of R3HDM1 (2q21.3) and the intergenic region (2p16.1). The R3HDM1 levels in his lymphoblastoid cells were reduced to approximately half that of the healthy controls. However, the expression of MIR128-1, in intron 18 of R3HDM1, was not affected via the pericentric inversion. Knockdown of R3HDM1 in mouse embryonic hippocampal neurons suppressed dendritic growth and branching. Notably, the Database of Genomic Variants reported the case of a healthy control with a 488-kb deletion that included both R3HDM1 and MIR128-1. miR-128 has been reported to inhibit dendritic growth and branching in mouse brain neurons, which directly opposes the novel functions of R3HDM1. These findings suggest that deleting both R3HDM1 and MIR128-1 alleviates the symptoms of the disease caused by loss-of-function mutations in R3HDM1 only. Thus, haploinsufficiency of R3HDM1 in the patient may be the cause of the mild ID due to the genetic imbalance between R3HDM1 and MIR128-1.

Clinical and molecular genetic characterization of two female patients harboring the Xq27.3q28 deletion with different ratios of X chromosome inactivation.

A heterozygous deletion at Xq27.3q28 including FMR1, AFF2, and IDS causing intellectual disability and characteristic facial features is very rare in females, with only 10 patients having been reported. Here, we examined two female patients with different clinical features harboring the Xq27.3q28 deletion and determined the chromosomal breakpoints. Moreover, we assessed the X chromosome inactivation (XCI) in peripheral blood from both patients. Both patients had an almost overlapping deletion at Xq27.3q28, however, the more severe patient (Patient 1) showed skewed XCI of the normal X chromosome (79:21) whereas the milder patient (Patient 2) showed random XCI. Therefore, deletion at Xq27.3q28 critically affected brain development, and the ratio of XCI of the normal X chromosome greatly affected the clinical characteristics of patients with deletion at Xq27.3q28. As the chromosomal breakpoints were determined, we analyzed a change in chromatin domains termed topologically associated domains (TADs) using published Hi-C data on the Xq27.3q28 region, and found that only patient 1 had a possibility of a drastic change in TADs. The altered chromatin topologies on the Xq27.3q28 region might affect the clinical features of patient 1 by changing the expression of genes just outside the deletion and/or the XCI establishment during embryogenesis resulting in skewed XCI.

Relation between cognitive and cerebello-thalamo-cortical functions in healthy elderly people: Evidence from the Yakumo Study.

Relations between cognitive and cerebello-thalamo-cortical functions in healthy elderly people (65-75 years old) were examined by longitudinal behavioral data. Based on the individually calculated cognitive decline ratio in D-CAT (digit cancelation test) and in LMT (Logical Memory Test) during the period of 11 years, participants were classified into the Decline and the Maintain groups and group differences in the postural tremor measures (Quotient of Romberg) were compared. Significant group differences were shown in the postural tremor measure in D-CAT that reflects prefrontal function, but it was not the case in LMT. These results strengthened our previous findings that suggest a strong relation between the cerebello-thalamo-cortical function and the prefrontal cortex function using behavioral measures. Findings provide evidence that to strengthen postural function such as physical exercise is effective for slowing cognitive decline with age.

Clinical and genetic characterization of a patient with SOX5 haploinsufficiency caused by a de novo balanced reciprocal translocation.

Lamb-Shaffer syndrome (OMIM: 616803) is a neurodevelopmental disorder characterized by developmental delay, mild to moderate intellectual disability, speech delay, and mild characteristic facial appearance caused by SOX5 haploinsufficiency on chromosome 12p12.1. There are clinical variabilities among the patients with genomic alterations, such as intragenic deletions, a point mutation, and a chromosomal translocation of t(11;12)(p13;p12.1), in SOX5. We report herein a 5-year-old Japanese male with a de novo balanced reciprocal translocation t(12;20)(p12.1;p12.3) presenting a mild intellectual disability, speech delay, characteristic facial appearance, and autistic features. We determined the translocation breakpoints of the patient to be in intron 4 of SOX5 and the intergenic region in 20p12.3 via FISH and nucleotide sequence analyses. Thus, the present patient has SOX5 haploinsufficiency affecting 2 long forms of SOX5 and is the second reported case of Lamb-Shaffer syndrome caused by a de novo balanced reciprocal translocation. This report confirmed that haploinsufficiency of the 2 long forms of SOX5 presents common clinical features, including mild intellectual disability and autistic features, which could be useful for the clinical diagnosis of Lamb-Shaffer syndrome.

Random monoallelic expression of genes on autosomes: Parallels with X-chromosome inactivation.

Genes are generally expressed from their two alleles, except in some particular cases such as random inactivation of one of the two X chromosomes in female mammals or imprinted genes which are expressed only from the maternal or the paternal allele. A lesser-known phenomenon is random monoallelic expression (RME) of autosomal genes, where genes can be stably expressed in a monoallelic manner, from either one of the parental alleles. Studies on autosomal RME face several challenges. First, RME that is based on epigenetic mechanisms has to be distinguished from biased expression of one allele caused by a DNA sequence polymorphism in a regulatory element. Second, RME should not be confused with transient monoallelic expression often observed in single cell analyses, and that often corresponds to dynamic bursting of expression. Thanks to analyses on clonal cell populations, the existence of RME in cultured cells is now well established. Future studies of RME in vivo will have to overcome tissue heterogeneity and certain technical limitations. Here, we discuss current knowledge on autosomal RME, as well as possible mechanisms controlling these expression patterns and potential implications for development and disease, drawing parallels with what is known for X-chromosome inactivation, a paradigm of random monoallelic expression.

G9a histone methyltransferase activity in retinal progenitors is essential for proper differentiation and survival of mouse retinal cells.

In vertebrate retinal development, various transcription factors are known to execute essential activities in gene regulation. Although epigenetic modification is considered to play a pivotal role in retinal development, the exact in vivo role of epigenetic regulation is still poorly understood. We observed that G9a histone methyltransferase, which methylates histone H3 at lysine 9 (H3K9), is substantially expressed in the mouse retina throughout development. To address in vivo G9a function in the mouse retina, we ablated G9a in retinal progenitor cells by conditional gene knock-out (G9a Dkk3 CKO). The G9a Dkk3 CKO retina exhibited severe morphological defects, including photoreceptor rosette formation, a partial loss of the outer nuclear layer, elevated cell death, and persistent cell proliferation. Progenitor cell-related genes, including several cyclins, Hes1, Chx10, and Lhx2, are methylated on histone H3K9 in the wild-type retina, but they were defective in H3K9 methylation and improperly upregulated at late developmental stages in the G9a Dkk3 CKO retina. Notably, conditional depletion of G9a in postmitotic photoreceptor precursors (G9a Crx CKO) led to the development of an almost normal retina, indicating that G9a activity mainly in retinal progenitor cells, but not in photoreceptor precursors, is essential for normal terminal differentiation of and survival of the retina. Our results suggest that proper epigenetic marks in progenitor cells are important for subsequent appropriate terminal differentiation and survival of retinal cells by repressing progenitor cell-related genes in differentiating retinal cells.

An essential role for RAX homeoprotein and NOTCH-HES signaling in Otx2 expression in embryonic retinal photoreceptor cell fate determination.

The molecular mechanisms underlying cell fate determination from common progenitors in the vertebrate CNS remain elusive. We previously reported that the OTX2 homeoprotein regulates retinal photoreceptor cell fate determination. While Otx2 transactivation is a pivotal process for photoreceptor cell fate determination, its transactivation mechanism in the retina is unknown. Here, we identified an evolutionarily conserved Otx2 enhancer of ∼500 bp, named embryonic enhancer locus for photoreceptor Otx2 transcription (EELPOT), which can recapitulate initial Otx2 expression in the embryonic mouse retina. We found that the RAX homeoprotein interacts with EELPOT to transactivate Otx2, mainly in the final cell cycle of retinal progenitors. Conditional inactivation of Rax results in downregulation of Otx2 expression in vivo. We also showed that NOTCH-HES signaling negatively regulates EELPOT to suppress Otx2 expression. These results suggest that the integrated activity of cell-intrinsic and -extrinsic factors on EELPOT underlies the molecular basis of photoreceptor cell fate determination in the embryonic retina.

Analysis of transcriptional regulatory pathways of photoreceptor genes by expression profiling of the Otx2-deficient retina.

In the vertebrate retina, the Otx2 transcription factor plays a crucial role in the cell fate determination of both rod and cone photoreceptors. We previously reported that Otx2 conditional knockout (CKO) mice exhibited a total absence of rods and cones in the retina due to their cell fate conversion to amacrine-like cells. In order to investigate the entire transcriptome of the Otx2 CKO retina, we compared expression profile of Otx2 CKO and wild-type retinas at P1 and P12 using microarray. We observed that expression of 101- and 1049-probe sets significantly decreased in the Otx2 CKO retina at P1 and P12, respectively, whereas, expression of 3- and 4149-probe sets increased at P1 and P12, respectively. We found that expression of genes encoding transcription factors involved in photoreceptor development, including Crx, Nrl, Nr2e3, Esrrb, and NeuroD, was markedly down-regulated in the Otx2 CKO at both P1 and P12. Furthermore, we identified three human retinal disease loci mapped in close proximity to certain down-regulated genes in the Otx2 CKO retina including Ccdc126, Tnfsf13 and Pitpnm1, suggesting that these genes are possibly responsible for these diseases. These transcriptome data sets of the Otx2 CKO retina provide a resource on developing rods and cones to further understand the molecular mechanisms underlying photoreceptor development, function and disease.

Negative regulation of ciliary length by ciliary male germ cell-associated kinase (Mak) is required for retinal photoreceptor survival.

Cilia function as cell sensors in many organs, and their disorders are referred to as "ciliopathies." Although ciliary components and transport machinery have been well studied, regulatory mechanisms of ciliary formation and maintenance are poorly understood. Here we show that male germ cell-associated kinase (Mak) regulates retinal photoreceptor ciliary length and subcompartmentalization. Mak was localized both in the connecting cilia and outer-segment axonemes of photoreceptor cells. In the Mak-null retina, photoreceptors exhibit elongated cilia and progressive degeneration. We observed accumulation of intraflagellar transport 88 (IFT88) and IFT57, expansion of kinesin family member 3A (Kif3a), and acetylated α-tubulin signals in the Mak-null photoreceptor cilia. We found abnormal rhodopsin accumulation in the Mak-null photoreceptor cell bodies at postnatal day 14. In addition, overexpression of retinitis pigmentosa 1 (RP1), a microtubule-associated protein localized in outer-segment axonemes, induced ciliary elongation, and Mak coexpression rescued excessive ciliary elongation by RP1. The RP1 N-terminal portion induces ciliary elongation and increased intensity of acetylated α-tubulin labeling in the cells and is phosphorylated by Mak. These results suggest that Mak is essential for the regulation of ciliary length and is required for the long-term survival of photoreceptors.

Blimp1 suppresses Chx10 expression in differentiating retinal photoreceptor precursors to ensure proper photoreceptor development.

The zinc finger transcription factor Blimp1 plays fundamentally important roles in many cell lineages and in the early development of several cell types, including B and T lymphocytes and germ cells. Although Blimp1 expression in developing retinal photoreceptor cells has been reported, its function remains unclear. We identified Blimp1 as a downstream factor of Otx2, which plays an essential role in photoreceptor cell fate determination. To investigate Blimp1 function in the mouse retina, we ablated Blimp1 in the developing retina by conditional gene targeting. In the Blimp1 conditional knockout (CKO) retina, the number of photoreceptor cells was markedly reduced in the differentiated retina. We found that the numbers of both bipolar-like cells and proliferating retinal cells increased noticeably, with ectopic localizations in the postnatal developing retina. In contrast, a reduction of the number of photoreceptor precursors was observed during development. Forced expression of Blimp1 by in vivo electroporation suppressed bipolar cell genesis in the developing retina. Multiple genes involved in bipolar development, including Chx10, were upregulated in the Blimp1 CKO retina. Furthermore, we showed that Blimp1 can bind to the Chx10 enhancer and repress Chx10 enhancer activity. These results suggest that Blimp1 plays a crucial role in photoreceptor development by repressing genes involved in bipolar cell fate specification and retinal cell proliferation in differentiating photoreceptor precursors.

Pikachurin, a dystroglycan ligand, is essential for photoreceptor ribbon synapse formation.

Exquisitely precise synapse formation is crucial for the mammalian CNS to function correctly. Retinal photoreceptors transfer information to bipolar and horizontal cells at a specialized synapse, the ribbon synapse. We identified pikachurin, an extracellular matrix-like retinal protein, and observed that it localized to the synaptic cleft in the photoreceptor ribbon synapse. Pikachurin null-mutant mice showed improper apposition of the bipolar cell dendritic tips to the photoreceptor ribbon synapses, resulting in alterations in synaptic signal transmission and visual function. Pikachurin colocalized with both dystrophin and dystroglycan at the ribbon synapses. Furthermore, we observed direct biochemical interactions between pikachurin and dystroglycan. Together, our results identify pikachurin as a dystroglycan-interacting protein and demonstrate that it has an essential role in the precise interactions between the photoreceptor ribbon synapse and the bipolar dendrites. This may also advance our understanding of the molecular mechanisms underlying the retinal electrophysiological abnormalities observed in muscular dystrophy patients.