Xq22 deletions and correlation with distinct neurological disease traits in females: Further evidence for a contiguous gene syndrome.

Xq22 deletions that encompass PLP1 (Xq22-PLP1-DEL) are notable for variable expressivity of neurological disease traits in females ranging from a mild late-onset form of spastic paraplegia type 2 (MIM# 312920), sometimes associated with skewed X-inactivation, to an early-onset neurological disease trait (EONDT) of severe developmental delay, intellectual disability, and behavioral abnormalities. Size and gene content of Xq22-PLP1-DEL vary and were proposed as potential molecular etiologies underlying variable expressivity in carrier females where two smallest regions of overlap (SROs) were suggested to influence disease. We ascertained a cohort of eight unrelated patients harboring Xq22-PLP1-DEL and performed high-density array comparative genomic hybridization and breakpoint-junction sequencing. Molecular characterization of Xq22-PLP1-DEL from 17 cases (eight herein and nine published) revealed an overrepresentation of breakpoints that reside within repeats (11/17, ~65%) and the clustering of ~47% of proximal breakpoints in a genomic instability hotspot with characteristic non-B DNA density. These findings implicate a potential role for genomic architecture in stimulating the formation of Xq22-PLP1-DEL. The correlation of Xq22-PLP1-DEL gene content with neurological disease trait in female cases enabled refinement of the associated SROs to a single genomic interval containing six genes. Our data support the hypothesis that genes contiguous to PLP1 contribute to EONDT.

PMD patient mutations reveal a long-distance intronic interaction that regulates PLP1/DM20 alternative splicing.

Alternative splicing of the proteolipid protein 1 gene (PLP1) produces two forms, PLP1 and DM20, due to alternative use of 5' splice sites with the same acceptor site in intron 3. The PLP1 form predominates in central nervous system RNA. Mutations that reduce the ratio of PLP1 to DM20, whether mutant or normal protein is formed, result in the X-linked leukodystrophy Pelizaeus-Merzbacher disease (PMD). We investigated the ability of sequences throughout PLP1 intron 3 to regulate alternative splicing using a splicing minigene construct transfected into the oligodendrocyte cell line, Oli-neu. Our data reveal that the alternative splice of PLP1 is regulated by a long-distance interaction between two highly conserved elements that are separated by 581 bases within the 1071-base intron 3. Further, our data suggest that a base-pairing secondary structure forms between these two elements, and we demonstrate that mutations of either element designed to destabilize the secondary structure decreased the PLP1/DM20 ratio, while swap mutations designed to restore the structure brought the PLP1/DM20 ratio to near normal levels. Sequence analysis of intron 3 in families with clinical symptoms of PMD who did not have coding-region mutations revealed mutations that segregated with disease in three families. We showed that these patient mutations, which potentially destabilize the secondary structure, also reduced the PLP1/DM20 ratio. This is the first report of patient mutations causing disease by disruption of a long-distance intronic interaction controlling alternative splicing. This finding has important implications for molecular diagnostics of PMD.

Dual roles for Prox1 in the regulation of the chicken betaB1-crystallin promoter.

PURPOSE: Lens fiber cell differentiation is marked by the onset of betaB1-crystallin expression and is controlled by the cooperative action of a set of transcription factors including Prox1, an atypical homeodomain protein. Previously, the authors reported that Prox1 directly interacts with the OL2 element found in the chicken betaB1-crystallin basal promoter to activate the expression of this gene. Here they mapped the location of activating and repressing sequences of the full-length chicken betaB1-crystallin promoter (-432/+30) in lens epithelial cells, annular pad cells, and intact lens and characterized Prox1-binding sites found in this region. METHODS: Transfection analysis and transgenic mice were used to characterize upstream regions of the chicken betaB1-crystallin gene. DNaseI footprinting and chromatin immunoprecipitation was performed to identify Prox1-binding sites, and transfection analyses were used to characterize these sites functionally. RESULTS: Sequences between -152 and -432 of the chicken betaB1-crystallin promoter mediated either promoter activation or repression, depending on the stage of lens differentiation tested. Two new Prox1-binding sites were found in this region that bound Prox1 more avidly than the OL2 element. However, neither binding site conferred Prox1-mediated activation on a heterologous promoter; instead, each allowed Prox1 to repress promoter function. CONCLUSIONS: The function of the upstream region of the chicken betaB1-crystallin promoter changes depending on cellular context. These data suggest that Prox1 function as a transcriptional activator could be regulated at the DNA level based on the characteristics of the responsive elements.

Morpholino Antisense Oligomers as a Potential Therapeutic Option for the Correction of Alternative Splicing in PMD, SPG2, and HEMS.

DNA variants of the proteolipid protein 1 gene (PLP1) that shift PLP1/DM20 alternative splicing away from the PLP1 form toward DM20 cause the allelic X-linked leukodystrophies Pelizaeus-Merzbacher disease (PMD), spastic paraplegia 2 (SPG2), and hypomyelination of early myelinating structures (HEMS). We designed a morpholino oligomer (MO-PLP) to block use of the DM20 5' splice donor site, thereby shifting alternative splicing toward the PLP1 5' splice site. Treatment of an immature oligodendrocyte cell line with MO-PLP significantly shifted alternative splicing toward PLP1 expression from the endogenous gene and from transfected human minigene splicing constructs harboring patient variants known to reduce the amount of the PLP1 spliced product. Additionally, a single intracerebroventricular injection of MO-PLP into the brains of neonatal mice, carrying a deletion of an intronic splicing enhancer identified in a PMD patient that reduces the Plp1 spliced form, corrected alternative splicing at both RNA and protein levels in the CNS. The effect lasted to post-natal day 90, well beyond the early post-natal spike in myelination and PLP production. Further, the single injection produced a sustained reduction of inflammatory markers in the brains of the mice. Our results suggest that morpholino oligomers have therapeutic potential for the treatment of PMD, SPG2, and HEMS.

Subfertility in mice harboring a mutation in betaB2-crystallin.

PURPOSE: betaB2-crystallin is one of the most abundant proteins of the adult ocular lens of mammals although it is expressed at lower levels in several extralenticular locations. While mutations in betaB2-crystallin are known to result in lens opacities, alterations in tissues besides the lens have not been previously investigated in these mutants. Since we found mice harboring the Crybb2Phil mutation bred poorly, here we assess the contribution of betaB2-crystallin to mouse fertility and determine the expression pattern of betaB2-crystallin in the testis. METHODS: The expression pattern of betaB2-crystallin in the testis was analyzed by rt-PCR, western blotting, and immunohistochemistry. The fecundity of wildtype and Crybb2Phil mice was analyzed by quantitative fertility testing. The morphology of testes and ovaries was assessed by hematoxylin and eosin staining. RESULTS: In the mouse testis, betaB2-crystallin mRNA is found at low levels at birth, but its expression upregulates in this tissue as the testis is primed to initiate spermatogenesis. Western blotting detected betaB2-crystallin protein in sperm obtained from mice, cattle, and humans while immunolocalization detected this protein in developing sperm from the spermatocyte stage onward. Male and female mice homozygous for a 12 nucleotide inframe deletion mutation in betaB2-crystallin are subfertile when analyzed on a Swiss Webster derived background due to defects in egg and sperm production. However, mice harboring the same mutation on the C57Bl/6 genetic background did not exhibit any defects in reproductive function. CONCLUSIONS: betaB2-crystallin is expressed in developing and mature sperm and mice of both sexes harboring the Philly mutation in the betaB2-crystallin gene are subfertile when analyzed on a Swiss Webster genetic background. While these data are suggestive of a role for betaB2-crystallin in fertility, definitive determination of this will await the creation of a betaB2-crystallin null mouse.

Altered PLP1 splicing causes hypomyelination of early myelinating structures.

OBJECTIVE: The objective of this study was to investigate the genetic etiology of the X-linked disorder "Hypomyelination of Early Myelinating Structures" (HEMS). METHODS: We included 16 patients from 10 families diagnosed with HEMS by brain MRI criteria. Exome sequencing was used to search for causal mutations. In silico analysis of effects of the mutations on splicing and RNA folding was performed. In vitro gene splicing was examined in RNA from patients' fibroblasts and an immortalized immature oligodendrocyte cell line after transfection with mutant minigene splicing constructs. RESULTS: All patients had unusual hemizygous mutations of PLP1 located in exon 3B (one deletion, one missense and two silent), which is spliced out in isoform DM20, or in intron 3 (five mutations). The deletion led to truncation of PLP1, but not DM20. Four mutations were predicted to affect PLP1/DM20 alternative splicing by creating exonic splicing silencer motifs or new splice donor sites or by affecting the local RNA structure of the PLP1 splice donor site. Four deep intronic mutations were predicted to destabilize a long-distance interaction structure in the secondary PLP1 RNA fragment involved in regulating PLP1/DM20 alternative splicing. Splicing studies in fibroblasts and transfected cells confirmed a decreased PLP1/DM20 ratio. INTERPRETATION: Brain structures that normally myelinate early are poorly myelinated in HEMS, while they are the best myelinated structures in Pelizaeus-Merzbacher disease, also caused by PLP1 alterations. Our data extend the phenotypic spectrum of PLP1-related disorders indicating that normal PLP1/DM20 alternative splicing is essential for early myelination and support the need to include intron 3 in diagnostic sequencing.

Proteomic and sequence analysis of chicken lens crystallins reveals alternate splicing and translational forms of beta B2 and beta A2 crystallins.

PURPOSE: To characterize the adult chicken lens proteome using mass spectrometry and two-dimensional gel electrophoresis (2-DE). METHODS: Lens proteins from 10-week old chickens were separated by gel filtration and reversed-phase chromatography, and whole protein masses were measured with electrospray mass spectrometry. Water-soluble lens proteins were separated by 2-DE and identified by tandem mass spectrometry of in-gel digests. RESULTS: Whole protein masses were consistent with all major chicken lens crystallin sequences, except for beta B2 and beta B3. Subsequent cDNA sequencing revealed errors in published sequences translating into 2- and 7-amino-acid differences, respectively, for beta B2 and beta B3, which were in better agreement with the measured masses. Previously uncharacterized forms of beta A2 and beta B2 were observed. The novel form of beta A2 had four fewer amino acids, was more abundant, and resulted from translation at a second start codon. The novel form of beta B2 contained 14 additional amino acids in the interdomain linker and resulted from alternate splicing within intron 4 of the transcript. All examined crystallins, except beta A3, for which data could not be obtained, were N-terminally acetylated, and all beta-crystallins lacked an initial methionine, except for the smaller beta A2 form. In-gel digests identified 29 proteins on the 2-DE map and indicated that truncation occurs within N-terminal extensions of beta-crystallins during lens maturation. CONCLUSIONS: The complementary techniques 2-DE, mass spectrometry, and DNA sequencing were used to provide the most complete description of the adult chicken lens proteome to date and identified alternate forms of beta A2 and beta B2.

Ectopic Pax6 expression disturbs lens fiber cell differentiation.

PURPOSE: Pax6 is a transcription factor necessary for the specification and subsequent formation of the ocular lens. It is expressed in all lens cells at early stages of development. After lens formation, Pax6 expression is maintained in the lens epithelium, whereas its level abruptly decreases in differentiated fiber cells. This study is to test the hypothesis that normal fiber cell differentiation would be perturbed by sustained Pax6 expression. METHODS: Transgenic mice expressing the canonical form of mouse Pax6 were created under the control of a modified mouse alphaA-crystallin promoter. The phenotypic changes in the transgenic lens were analyzed by light and electron microscopy. The effect of ectopic Pax6 expression on the lens fiber cells was investigated by in situ hybridization, immunohistochemical staining, real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and two-dimensional (2-D) gel electrophoresis. RESULTS: Transgenic mice from seven different lines all had cataracts with severity that correlated with the transgene expression level in lens fiber cells. In severely affected lines, a lumen was present between the apical surfaces of the epithelial and fiber cells, suggesting that secondary fiber cell elongation is incomplete. Electron microscopy analysis showed that the ball-and-socket interdigitations between neighboring fiber cells were underdeveloped or attenuated in the transgenic lens. Most interesting, elevated levels of Pax6 in fiber cells reduced the protein levels of transcription factor cMaf, which is known to be essential in fiber cell differentiation. Furthermore, the total amount of lens proteins was 60% less than normal in the Pax6 transgenic lens. Among the crystallins examined, the relative ratio of intact betaB1-crystallin protein to total lens protein was significantly reduced. Real-time reverse transcriptase PCR showed that the ratio of betaB1-crystallin transcript levels to total mRNA levels were reduced by 87%. CONCLUSIONS: The data demonstrate that high levels of Pax6 expression disrupt normal fiber cell differentiation and maturation.

The Zeb proteins δEF1 and Sip1 may have distinct functions in lens cells following cataract surgery.

PURPOSE: Posterior capsular opacification (PCO), the most prevalent side effect of cataract surgery, occurs when residual lens epithelial cells (LECs) undergo fiber cell differentiation or epithelial-to-mesenchymal transition (EMT). Here, we used a murine cataract surgery model to investigate the role of the Zeb proteins, Smad interacting protein 1 (Sip1) and δ-crystallin enhancer-binding factor 1 (δEF1), during PCO. METHODS: Extracapsular extraction of lens fiber cells was performed on wild-type and Sip1 knockout mice. Protein expression patterns were assessed at multiple time points after surgery using confocal immunofluorescence. ßB1-Crystallin mRNA levels were measured using quantitative RT-PCR. We used Transfac searches to identify δEF1 binding sites in the ßB1-crystallin promoter and transfection analysis to test the ability of δEF1 to regulate ßB1-crystallin expression. RESULTS: δEF1, which, in other systems, can activate fibrotic genes (e.g., α-smooth muscle actin) and repress epithelial genes, upregulates by 48 hours after fiber cell removal. In culture, δEF1 repressed ßB1-crystallin promoter activity, suggesting that it may also turn off lens gene expression following surgery, contributing to "fibrotic PCO" development. Sip1 also upregulates in LECs by 48 hours, but analysis of Sip1 knockout lenses demonstrated that Sip1 does not play a major role in EMT or fiber cell differentiation after surgery. However, Sip1 knockout LECs do express the ectodermal marker keratin 8, suggesting that Sip1 may limit the reprogramming of residual LECs to an embryonic state. CONCLUSIONS: Zeb transcription factors likely play important, but distinct roles in PCO development after cataract surgery.

General utility of the chicken betaB1-crystallin promoter to drive protein expression in lens fiber cells of transgenic mice.

Transgenic mouse technology has been very valuable for the study of lens fiber cells since they can not be propagated in cell culture. The targeting of transgenes to the lens has traditionally been done with the alphaA-crystallin promoter. However, while lens-specific, transgenic lines made with the alphaA-crystallin promoter express the transgene at levels 100-300-fold lower than endogenous alphaA-crystallin. Here we propose an alternative, the chicken betaB1-crystallin promoter (-432/+30). Transgenic mice made with this promoter have successfully expressed CAT, d/n m-calpain, Weel, and betaB2-crystallin mRNA at levels comparable to the endogenous betaB1-crystallin gene and no eye abnormalities such as cataracts, have resulted. All of the transgenic lines made with the chicken betaB1-crystallin promoter have expressed the transgene in the lens fiber cells, and the best lines express at levels close to endogenous betaB1-crystallin. While RNA expression is very high, only moderate protein expression has been achieved, implying that the high protein expression of the crystallins is partially controlled at the level of translation. Thus, the chicken betaB1-crystallin promoter directs high level RNA expression to lens fiber cells, which may be especially useful for the expression of ribozyme and anti-sense RNAs in addition to ectopic proteins.