Eve L. Bingham

NOVEL MUTATIONS IN XLRS1 CAUSING RETINOSCHISIS, INCLUDING FIRST EVIDENCE OF PUTATIVE LEADER SEQUENCE CHANGE

Juvenile retinoschisis is an X‐linked recessive disease caused by mutations in the XLRS1 gene. We screened 31 new unrelated patients and families for XLRS1 mutations in addition to previously reported mutations for 60 of our families (Retinoschisis Consortium, Hum Mol Genet 1998;7:1185–1192). Twenty‐three different mutations including 12 novel ones were identified in 28 patients. Mutations identified in this study include 19 missense mutations, two nonsense mutations, one intragenic deletion, four microdeletions, one insertion, and one intronic sequence substitution that is likely to result in a splice site defect. Two novel mutations, c.38T→C (L13P) and c.667T→C (C223R), respectively, present the first genetic evidence for the functional significance of the putative leader peptide sequence and for the functional significance at the carboxyl terminal of the XLRS1 protein beyond the discoidin domain. Mutations in 25 of the families were localized to exons 4–6, emphasizing the critical functional significance of the discoidin domain of the XLRS1 protein. Hum Mutat 14:423–427, 1999.

Differentiation of human embryonic stem cells to a parathyroid-like phenotype.

Iatrogenic hypoparathyroidism is the most common complication of cervical endocrine surgery. Current management is limited and palliative. As the molecular steps in parathyroid development have been defined, they may be replicable in vitro, with a goal of cellular replacement therapy. Human embryonic stem cell (hESC) lines were investigated as a model for parathyroid regeneration in vitro. BG01 was selected as a model based on expression of genes of interest in embryoid bodies (EBs). Established strategies for mouse embryonic stem cell differentiation into definitive endoderm were modified and extended to maximize the expression of definitive markers of parathyroid development. The optimal approach included the use of Activin A at 100 ng/mL with BG01 cells grown on murine embryonic fibroblasts for 5 days under conditions of increasing serum concentration. After 5 days, the cells were allowed to mature further in tissue culture without murine fibroblasts but with continuous Activin A. Our strategy produced differentiated cell cultures that expressed intermediate markers of endoderm and parathyroid development (CXCR4, EYA1, Six1, and Pax1), as well as markers of committed parathyroid precursors or developed parathyroid glands (glial cell missing-2 [Gcm2], CCL21, calcium sensing receptor [CaSR], and parathyroid hormone [PTH]). We further characterized the cells by testing conditioned medium from various time points in our differentiation scheme for the presence of PTH. We found that by keeping the cells in culture 2 weeks after the withdrawal of Activin A, the cells were able to produce PTH. Further in vivo work will be needed to demonstrate proper functionality of the cells developed in this way.

Linkage study of best's vitelliform macular dystrophy (VMD2) in a large North American family

Bests vitelliform macular dystrophy (VMD2) is an autosomal dominant retinal dystrophy for which the underlying biochemical cause is unknown. We used 11 genetic markers in the vicinity of the VMD2 gene in our study of a large North American family in which macular dystrophy characteristics overlap the broad definition of Bests disease. Significant evidence for linkage was found for markers D11S956 (Z = 5.88, theta = 0.04) and FCER1B (Z = 4.31, theta = 0.00). Recombination events localized the disease gene to the 5-cM interval D11S956-UGB, a genetic inclusion interval that substantially overlaps the VMD2 inclusion interval defined by recombinants at FCER1B and UGB observed by other research groups. The resulting exclusion of ROM1 from the genetic inclusion interval eliminates ROM1 defects as a possible cause of the disease in this family. Linkage studies of many families, including those that share most but not all features with classical Bests disease, will be needed to properly evaluate genetic heterogeneity and the range of phenotypic variation that can result from VMD2 defects.

X-linked juvenile retinoschisis: Localization between (DXS1195, DXS418) and AFM291wf5 on a single YAC

We studied 17 pedigrees with 108 affected males with X-linked juvenile retinoschisis (RS; McKusick No. 31270) and have analyzed all of the known polymorphic markers in the RS region of Xp22.1-p22.2 between DXS987 and DXS41. By haplotype analyses we found 7 individuals who showed crossovers in this interval surrounding RS. We previously reported AFM291wf5 as the centromeric boundary, and this remains unchanged in the present study. A new recombination was identified on the telomeric side at (DXS1195, DXS418). Our data support the locus order Xpter--(DXS987, DXS207, DXS1053, DXS43)--(DXS1195, DXS418)--(RS, DXS257, DXS999)--(AFM291wf5, DXS443)--DXS1052--(DXS1226, DXS274, DXS41)--Xcen; loci grouped in parentheses could not be mutually ordered by our genetic data. Physical mapping has indicated a distance of at most 900-1,000 kb between (DXS1195, DXS418) and AFM291wf5. No recombination was observed between RS and DXS257 which lies in our new interval of interest, but one critical individual was not informative with this marker. Our data now define the smallest RS inclusion interval. This interval is contained on a single YAC from which we have identified expressed sequences as candidate genes for RS.

Differentiation of precursors into parathyroid-like cells for treatment of hypoparathyroidism

BACKGROUND Hypoparathyroidism is the most frequent permanent complication of thyroid surgery. Our hypothesis is that human precursor cells in culture can be differentiated into parathyroid cells and used to reconstitute function. Human embryonic stem cells (hESCs) are a stable model to study differentiation into parathyroid-like cells. In prior work, the BG01-hESC line was stimulated to form parathyroid-like cells. This cell line is no longer available, however, and additional studies were needed to confirm and extend prior observations. METHODS Increasing concentrations of fetal bovine serum and timed exposure to Activin A were used to differentiate H1-hESC into parathyroid-like cells. The potential benefit of Sonic hedgehog exposure on parathyroid-like cell development also was evaluated by serial alterations of culture conditions. Calcium-sensing receptor (CaSR), GCM2, and PTH expression (RT-PCR) and PTH protein secretion (ELISA) were used as markers of differentiated cells. RESULTS We successfully modified our prior protocol to generate cells that express CaSR, GCM2, and PTH RNA from undifferentiated H1-hESC. The cells also secreted PTH. CONCLUSION We replicated parathyroid differentiation using H1-hESC cells. Our data advance the project toward in vitro differentiation of precursor cells isolated from individual patients for autotransplantation.

Searching for Genotype-Phenotype Correlations in X-Linked Juvenile Retinoschisis

We have analyzed 145 RS families for mutations in the XLRS1 gene and have sought clinical correlation of phenotype severity with the type and location of mutation in a subset of these families. Some of the RS families exhibited consistency of either severe or mild clinical phenotypes in multiple affected male members. Intrafamilial variability of clinical phenotypes was encountered in affected males of several other RS families.

Refined genetic mapping of juvenile X-linked retinoschisis

Juvenile X-linked retinoschisis (RS) is an eye disease that causes acuity reduction and peripheral visual field loss typically beginning early in life. In further work towards positional cloning of the RS gene, we restudied our previously reported seven large American families and one additional new family, with a total of 63 affected males. RS linkage analysis using microsatellite repeat markers gave the following results: DXS207 (Z = 24.89, theta = 0.01), DXS987 (Z = 24.04, theta 0.01) and DXS999 (Z = 14.70, theta = 0.00). Recombination events in four individuals were studied further with additional markers (AFM291wf5, DXS443, DXS1052, DXS274 and DXS1226), and a flanking interval was obtained (DXS43, DXS207, DXS987)-RS-(AFM291wf5, DXS443). This study moves the RS centromeric boundary to (AFM291wf5, DXS443), about 5.5 cM closer than the previously reported boundary at DXS274 and narrows the RS inclusion interval to about 3.7 cM (using distances from CEPH family data).

Comparison of two decellularized dermal equivalents

Immunologically inert allogeneic acellular dermal scaffolds provide a matrix with molecular architecture close to native tissues, which synthetic scaffolds cannot. Not all nature‐derived scaffolds possess the same biological and physical properties. The different properties of scaffolds supporting cellular growth used for manufacturing tissue engineered grafts could lead to different implantation results. The scaffold properties should be carefully considered in order to meet the expected outcomes of tissue engineered grafts. In this report, we evaluated the cellular growth on AlloDerm® and Allopatch, 2 acellular scaffolds derived from human cadaver skin, using a fabricated 3D organotypic culture with primary human oral keratinocytes to produce an ex vivo produced oral mucosa equivalent (EVPOME). A well stratified epithelium could be constructed on both scaffolds. AlloDerm® and Allopatch EVPOMEs were also implanted into severe combined immunodeficiency mice to compare the ingrowth of blood vessels into the dermal component of the two EVPOMEs. Blood vessel counts were 3.3 times higher (p = .01) within Allopatch EVPOMEs than within AlloDerm® EVPOMEs. An oral and skin keratinocyte co‐culture, separated by a physical barrier to create a cell‐free zone, was used to evaluate cell migration on AlloDerm® and Allopatch. Slower cell migration was observed on Allopatch than on AlloDerm®.

Blue Cone Monochromacy

Blue cone monochromacy (BCM) is an X-linked ocular disorder in which affected males have normal short-wavelength-sensitive (blue) cone and rod function but lack medium-(green) and long-wavelength-sensitive (red) cone function. Affected males characteristically have visual acuities of 20/100 to 20/200, myopia, nystagmus, and minimal foveal granularity with otherwise normal fundus findings.1

The MHC Restriction of a Monoclonal Antigen-Specific I-J+ TsF2 Maps to a Location Outside the I-J Subregion

Negative regulation of the immune response is mediated by distinct subpopulations of T lymphocytes (1–6). Current information suggests that at least three separate suppressor Tcells (Ts) and their respective mediators (TsF) are involved in a defined sequence of events. While the mechanisms by which these TsF effect suppression remain unclear, it is important to note that many of the described TsF not only contain an I-J major histocompatibility complex (MHC) determinant but must react with an MHC-controlled gene product on an acceptor cell.