Hairless Streaks in Cattle Implicate TSR2 in Early Hair Follicle Formation

Four related cows showed hairless streaks on various parts of the body with no correlation to the pigmentation pattern. The stripes occurred in a consistent pattern resembling the lines of Blaschko. The non-syndromic hairlessness phenotype observed occurred across three generations of a single family and was compatible with an X-linked mode of inheritance. Linkage analysis and subsequent whole genome sequencing of one affected female identified two perfectly associated non-synonymous sequence variants in the critical interval on bovine chromosome X. Both variants occurred in complete linkage disequilibrium and were absent in more than 3900 controls. An ERCC6L missense mutation was predicted to cause an amino acid substitution of a non-conserved residue. Analysis in mice showed no specific Ercc6l expression pattern related to hair follicle development and therefore ERCC6L was not considered as causative gene. A point mutation at the 5'-splice junction of exon 5 of the TSR2, 20S rRNA accumulation, homolog (S. cerevisiae), gene led to the production of two mutant transcripts, both of which contain a frameshift and generate a premature stop codon predicted to truncate approximately 25% of the protein. Interestingly, in addition to the presence of both physiological TSR2 transcripts, the two mutant transcripts were predominantly detected in the hairless skin of the affected cows. Immunohistochemistry, using an antibody against the N-terminal part of the bovine protein demonstrated the specific expression of the TSR2 protein in the skin and the hair of the affected and the control cows as well as in bovine fetal skin and hair. The RNA hybridization in situ showed that Tsr2 was expressed in pre- and post-natal phases of hair follicle development in mice. Mammalian TSR2 proteins are highly conserved and are known to be broadly expressed, but their precise in vivo functions are poorly understood. Thus, by dissecting a naturally occurring mutation in a domestic animal species, we identified TSR2 as a regulator of hair follicle development.

Mechanisms of rapid sympatric speciation by sex reversal and sexual selection in cichlid fish

Mechanisms of speciation in cichlid fish were investigated by analyzing population genetic models of sexual selection on sex-determining genes associated with color polymorphisms. The models are based on a combination of laboratory experiments and field observations on the ecology, male and female mating behavior, and inheritance of sex-determination and color polymorphisms. The models explain why sex-reversal genes that change males into females tend to be X-linked and associated with novel colors, using the hypothesis of restricted recombination on the sex chromosomes, as suggested by previous theory on the evolution of recombination. The models reveal multiple pathways for rapid sympatric speciation through the origin of novel color morphs with strong assortative mating that incorporate both sex-reversal and suppressor genes. Despite the lack of geographic isolation or ecological differentiation, the new species coexists with the ancestral species either temporarily or indefinitely. These results may help to explain different patterns and rates of speciation among groups of cichlids, in particular the explosive diversification of rock-dwelling haplochromine cichlids.

Mutation Update of the CLCN5 Gene Responsible for Dent Disease 1

Dent disease is a rare X-linked tubulopathy characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressive renal failure, and variable manifestations of other proximal tubule dysfunctions. It often progresses over a few decades to chronic renal insufficiency, and therefore molecular characterization is important to allow appropriate genetic counseling. Two genetic subtypes have been described to date: Dent disease 1 is caused by mutations of the CLCN5 gene, coding for the chloride/proton exchanger ClC-5; and Dent disease 2 by mutations of the OCRL gene, coding for the inositol polyphosphate 5-phosphatase OCRL-1. Herein, we review previously reported mutations (n = 192) and their associated phenotype in 377 male patients with Dent disease 1 and describe phenotype and novel (n = 42) and recurrent mutations (n = 24) in a large cohort of 117 Dent disease 1 patients belonging to 90 families. The novel missense and in-frame mutations described were mapped onto a three-dimensional homology model of the ClC-5 protein. This analysis suggests that these mutations affect the dimerization process, helix stability, or transport. The phenotype of our cohort patients supports and extends the phenotype that has been reported in smaller studies.

Global patterns of DNA polymorphism in noncoding regions in human populations

DNA sequence variation is currently a major source of data for studying human origins, evolution, and demographic history, and for detecting linkage association of complex diseases. In this dissertation, I investigated DNA variation in worldwide populations from two ∼10 kb autosomal regions on 22q11.2 (noncoding) and 1q24 (introns). A total of 75 variant sites were found among 128 human sequences in the 22q11.2 region, yielding an estimate of 0.088% for nucleotide diversity (π), and a total of 52 variant sites were found among 122 human sequences in the 1q24 region with an estimated π value of 0.057%. The data from these two regions and a 10 kb noncoding region on Xq13.3 all show a strong excess of low-frequency variants in comparison to that expected from an equilibrium population, indicating a relatively recent population expansion. The effective population sizes estimated from the three regions were 11,000, 12,700, and 8,600, respectively, which are close to the commonly used value of 10,000. In each of the two autosomal regions, the age of the most recent common ancestor (MRCA) was estimated to be older than 1 million years among all the sequences and ∼600,000 years among non-African sequences, providing first evidence from autosomal noncoding or intronic regions for a genetic history of humans much more ancient than the emergence of modern humans. The ancient genetic history of humans indicates no severe bottleneck during the evolution of humans in the last half million years; otherwise, much of the ancient genetic history would have been lost during a severe bottleneck. This study strongly suggests that both the “out of Africa” and the multiregional models are too simple for explaining the evolution of modern humans. A compilation of genome-wide data revealed that nucleotide diversity is highest in autosomal regions, intermediate in X-linked regions, and lowest in Y-linked regions. The data suggest the existence of background selection or selective sweep on Y-linked loci. In general, the nucleotide diversity in humans is low compared to that in chimpanzee and Drosophila populations. ^

Positional candidate cloning of the RP10 form of retinitis pigmentosa

Retinitis pigmentosa (RP) is a genetically heterogeneous group of retinal degenerations that affects over one million people worldwide. To date, 11 autosomal dominant, 13 autosomal recessive, and 5 X-linked forms of retinitis pigmentosa have been identified through linkage analysis, but the disease-causing genes and mutations have been found for only half of these loci. My research uses a positional candidate cloning approach to identify the gene and mutations responsible for one type of autosomal dominant retinitis pigmentosa, RP10. The premise is that identifying the genes and mutations responsible for disease will provide insight into disease mechanisms and provide treatment options. Previous research mapped the RP10 locus to a 5cM region on chromosome 7q31 between markers D7S686 and D7S530. Linkage and fine-point haplotype analysis was used to reduce and refine the RP10 disease interval to a 4cM region located between D7S2471 and a new marker located 45,000bp telomeric of D7S461. In order to identify genes located in the RP10 interval, an extensive EST map was created of this region. Five EST clusters from this map were analyzed to determine if mutations in these genes cause the RP10 form of retinitis pigmentosa. The genomic structure of a known metabotrophic glutamate receptor, GRMS8, was determined first. DNA sequencing of GRM8 in RP10 family members did not identify any disease-causing mutations. Four other EST clusters (A170, A173, A189, and A258) were characterized and determined to be part of the same gene, UBNL1 (ubinuclein-like 1). The full-length mRNA sequence and genomic structure of UBNL1 was determined and then screened in patients. No disease-causing mutations were identified in any of the RP10 family members tested. Recent data made available with the release of the public and Celera genome assemblies indicates that UBNL1 is outside of the RP10 disease region. Despite this complication, characterization of UBNL1 is still important in the understanding of normal visual processes and it is possible that mutations in UBNL1 could cause other forms of retinopathy. The EST map and list of RP10 candidates will continue to aid others in the search for the RP10 gene and mutations. ^

From gene to protein: Investigating the disease etiology of retinitis pigmentosa

Retinitis pigmentosa (RP) is a name given to a group of inherited retinal dystrophies that lead to progressive photoreceptor degeneration, and thus, visual impairment. It is evident at both the clinical and the molecular level that these are heterogeneous disorders, with wide variation in severity, mode of inheritance, and phenotype. The genetics of RP are not simple; the disease can be inherited in dominant, recessive, X-linked, and digenic modes. Autosomal dominant RP (adRP) results from mutations in at least ten mapped loci, but there may be dozens of genetic loci where mutations can cause RP. To date, there are over a hundred genes known to cause retinal degenerative diseases, and less than half of these have been cloned (RetNet). Among the dozens of retinitis pigmentosa loci known to exist, only a few have been identified and the remainders are inferred from linkage studies. Today, the genes for seven of the twelve-adRP loci have been identified, and these are rhodopsin, peripherin/RDS, NRL, ROM1, CRX, RP13 and RP1. My research projects involved a combination of the continued search for genes involved in retinal dystrophies, as well the investigation into the role of peripherin/RDS and RP1 in the disease etiology of autosomal dominant RP. ^ Most of the mutations leading to inherited retinal disorders have been identified in predominately retina expressed genes like rhodopsin, peripherin/RDS, and RP1. Expressed sequence tags (ESTs) that were retina-specific were culled from sequence databases and, together with laboratory analysis, were analyzed as potential candidate genes for retinal dystrophies. Thirteen of the fifty-five identified retina-specific ESTs mapped to within candidate regions for inherited retinopathies. One of these is RP1L1, a homologue of RP1 and a potential cause of adRP. ^ Once a disease-associated gene has been identified, elucidating the role of that gene in the visual process is essential for understanding what happens when the process is defective as it is in adRP. My next projects involved investigating the role of a novel 5′ donor +3 splice site mutation on the mRNA of peripherin/RDS in adRP affected individuals, and comparative sequencing in RP1 to define conserved regions of the protein. Comparative sequencing is a powerful way to delineate critical regions of a sequence because different regions of a gene have different functions, and each region is subject to different levels of functional or structural constraints. Establishing a framework of conserved domains is beneficial not only for structural or functional studies, but can also aid in determining the potential effects of mutations. With the completion of sequencing of human genome, and other organisms such as Saccharomyces cerevisiae, Caenorhabditis elegans , and Drosophila, the facility of comparative sequencing will only increase in the future. Comparative sequencing has already become an established procedure for pinpointing conserved regions of a protein, and is an efficient way to target regions of a protein for experimental and/or evolutionary analysis. ^

Downstream targets of the Rhox5 homeobox gene

The X-linked mouse Rhox gene cluster contains over 30 homeobox genes that are candidates to regulate multiple steps in male and female gametogenesis. The founding member of the Rhox gene cluster, Rhox5, is an androgen-dependent gene expressed in Sertoli cells that promotes the survival and differentiation of the adjacent male germ cells. To decipher downstream signaling pathways of Rhox5, I used in vivo and in vitro microarray profiling to identify and characterize downstream targets of Rhox5 in the testis. This led to the identification of many Rhox5 -regulated genes, two of which I focused on in more detail. One of them, Unc5c, encodes a pro-apoptotic receptor with tumor suppressor activity that I found is negatively regulated by Rhox5 through a Rhox5-response element in the Unc5c 5' untranslated region (5' UTR). Examination of other mouse Rhox family members revealed that Rhox2 and Rhox3 also have the ability to downregulate Unc5c expression. The human RHOX protein RHOXF2 also had this ability, indicating that Unc5c repression is a conserved Rhox-dependent response. The repression of Unc5c expression by Rhox5 may, in part, mediate Rhox5's pro-survival function in the testis, as I found that Unc5c mutant mice have decreased germ cell apoptosis in the testis. This along with my other data leads me to propose a model in which Rhox5 is a negative regulator upstream of Unc5c in a Sertoli-cell pathway that promotes germ-cell survival. The other Rhox5-regulated gene that I studied in detail is insulin II (Ins2). Several lines of evidence, including electrophoretic mobility shift anaylsis, promoter mutagenesis, and chromatin immuoprecipitation analysis indicated that Ins2 is a direct target of Rhox5. Structure-function analysis identified homeodomain residues and the RHOX5 amino-terminal domain crucial for conferring Ins2 inducibility. Rhox5 regulates not only the Ins2 gene but also genes encoding other secreted proteins regulating metabolism (adiponectin and resistin), the rate-liming enzyme for monosaturated fatty acid biosynthesis (SCD-1), and transcription factors crucial for regulating metabolism (the nuclear hormone receptor PPARγ). I propose that the regulation of some or all of these molecules in Sertoli cells is responsible for the Rhox5-dependent survival of the adjacent germ cells. ^

A cross-sectional study of disparities in suicide ideation between Hispanic and non-Hispanic white female adolescents

Suicide is recognized as a major public health and clinical problem in the United States. One fifth of adolescents in the United States seriously consider suicide each year, and about 8% of high school students attempt suicide at least once. Hispanic ethnicity constitutes a risk factor for suicidal ideation and suicide attempts, with Hispanic females at highest risk. Nevertheless, published studies on suicidal behavior in Hispanic female adolescents are extremely limited and focus on suicidal ideation in school samples. Given the severity of the problem and the paucity of information on this topic, more research on ethnic differences in suicidal ideation in community samples of high-risk children is urgently needed. This cross-sectional study delineated differences in suicide ideation between Hispanic female adolescents and non-Hispanic white female adolescents attending a mental health clinic and examined the association of ethnicity with suicide ideation independent of other known risk factors. Data were accrued between June 2004 and December 2008 in a Harris County Mental Health and Mental Retardation Association (MHMRA) clinic. Data were limited to adolescents who were Harris County Residents between the ages of 10 to 17 years when they were admitted to the clinic. The objective of this study was to determine whether differences in socio-demographic and clinical variables play a significant role in ethnic disparities in suicide ideation. A series of logistic regressions were performed to estimate the association between ethnicity and suicide ideation after controlling for potentially confounding factors. ^ Results showed an interaction between Hispanic ethnicity and having a history of treatment: Hispanic girls having history of treatment had lower odds of having suicide ideation than Hispanic girls without such a history. After adjusting for treatment history, family problems, substance use, juvenile justice involvement, current treatment, and age, Hispanic girls had 1.86 times the odds of having suicide ideation than non Hispanic girls (OR=1.86, 95% CI=0.88-1.46). Although additional studies on community samples of high risk adolescents are needed to verify these findings, our study highlights the fact that Hispanic girls are at significantly higher risk and need to be targeted for prevention and treatment efforts. ^

Transcriptional and translational regulators of gene expression in germ cell development

Germ cell development is a highly coordinated process driven, in part, by regulatory mechanisms that control gene expression. Not only transcription, but also translation, is under regulatory control to direct proper germ cell development. In this dissertation, I have focused on two regulators of germ cell development. One is the homeobox protein RHOX10, which has the potential to be both a transcriptional and translational regulator in mouse male germ cell development. The other is the RNA-binding protein, Hermes, which functions as a translational regulator in Xenopus laevis female germ cell development. ^ Rhox10 is a member of reproductive homeobox gene X-(linked (Rhox) gene cluster, of which expression is developmentally regulated in developing mouse testes. To identify the cell types and developmental stages in which Rhox10 might function, I characterized its temporal and spatial expression pattern in mouse embryonic, neonatal, and adult tissues. Among other things, this analysis revealed that both the level and the subcellular localization of RHOX10 are regulated during germ cell development. To understand the role of Rhox10 in germ cell development, I generated transgenic mice expressing an artificial microRNA (miRNA) targeting Rhox10. While this artificial miRNA robustly downregulated RHOX10 protein expression in vitro, it did not significantly reduce RHOX10 expression in vivo. So I next elected to knockdown RHOX10 levels in spermatogonial stem cells (SSCs), which I found highly express both Rhox10 mRNA and RHOX10 protein. Using a recently developed in vitro culture system for SSCs combined with a short-hairpin RNA (shRNA) approach, I strongly depleted RHOX10 expression in SSCs. These RHOX10-depleted cells exhibited a defect in the ability to form stem cell clusters in vitro. Expression profiling analysis revealed many genes regulated by Rhox10, including many meiotic genes, which could be downstream of Rhox10 in a molecular pathway that controls SSC differentiation. ^ RNA recognition motif (RRM) containing protein, Hermes is localized in germ plasm, where dormant mRNAs are also located, of Xenopus oocytes, which implicates its role in translational regulator. To understand the function of Hermes in oocyte meiosis, I used a morpholino oligonucleotide (MO) based knockdown approach. Microinjection of Hermes MO into fully grown oocytes, which are arrested in meiotic prophase, caused acceleration of oocytes reentry into meiosis (i.e., maturation) upon progesterone induction. Using a candidate approach, I identified at least three targets of Hermes: Ringo/Spy, Xcat2, and Mos. Ringo/Spy and Mos are known to have functions in oocyte maturation, while Ringo/Spy, Xcat2 mRNA are localized in the germ plasm of oocytes, which drives germ cell specification after fertilization. This led me to propose that Hermes functions in both oocyte maturation and germ cell development through its ability to regulate 3 crucial target mRNAs. ^

THE DISCHARGED PSYCHIATRIC PATIENT: POST-HOSPITAL ADJUSTMENT AND FACTORS AFFECTING REHOSPITALIZATION

Discharged psychiatric patients were studied six months post-discharge to determine those demographic, social and clinical characteristics affecting positive or negative adjustment and the degree to which the use of mental health services and medication compliance mediated the effects. With the exception of those with primary or secondary diagnoses of OBS, substance abuse or mental retardation, sixty-three psychiatric subjects between the ages of eighteen and sixty-four were chosen from all admissions into the hospital and interviewed six months after discharge using a specially designed questionnaire.^ The subjects' adjustment to community living was found to be marginal. Although not engaged in destructive activities, over half were living with their family members who supported them financially and emotionally. Most were unemployed and had been so for a long time. Others worked sporadically and frequently changed residences. Most did have substantial social ties with extended family and with friends with whom they interacted regularly, but one-fourth were socially isolated. Almost three-quarters continued to obtain regular mental health services after discharge and followed medication instructions under the supervision of their physician. The use of mental health services after discharge and the use of medication did not appear to affect the subjects' community adaption or their rate of rehospitalization.^ Forty percent of those discharged were rehospitalized by the end of the follow-up period. Four levels of risk of rehospitalization emerged. The highest risk was associated with a history of five or more prior hospitalizations, living alone, and social isolation. One third or more of the subjects expressed a need for more counseling, leisure time activities, case-manager assistance, vocational guidance, supervised housing, and placement into a transitional residential treatment program.^ Recommendations were made to enhance the ability to predict recidivism, to develop interorganizational casework management programs linking the patient and family to the community mental health system and to create computerized tracking and monitoring programs that systematically report patient treatment regimen and progress cross-sectionally and longitudinally. ^

Dyrk1A Influences Neuronal Morphogenesis Through Regulation of Cytoskeletal Dynamics in Mammalian Cortical Neurons

Down syndrome (DS) is the most frequent genetic cause of mental retardation. Cognitive dysfunction in these patients is correlated with reduced dendritic branching and complexity, along with fewer spines of abnormal shape that characterize the cortical neuronal profile of DS. DS phenotypes are caused by the disruptive effect of specific trisomic genes. Here, we report that overexpression of dual-specificity tyrosine phosphorylation-regulated kinase 1A, DYRK1A, is sufficient to produce the dendritic alterations observed in DS patients. Engineered changes in Dyrk1A gene dosage in vivo strongly alter the postnatal dendritic arborization processes with a similar progression than in humans. In cultured mammalian cortical neurons, we determined a reduction of neurite outgrowth and synaptogenesis. The mechanism underlying neurite dysgenesia involves changes in the dynamic reorganization of the cytoskeleton.

Resistance to apoptosis caused by PIG-A gene mutations in paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder resulting from mutations in an X-linked gene, PIG-A, that encodes an enzyme required for the first step in the biosynthesis of glycosylphosphatidylinositol (GPI) anchors. PIG-A mutations result in absent or decreased cell surface expression of all GPI-anchored proteins. Although many of the clinical manifestations (e.g., hemolytic anemia) of the disease can be explained by a deficiency of GPI-anchored complement regulatory proteins such as CD59 and CD55, it is unclear why the PNH clone dominates hematopoiesis and why it is prone to evolve into acute leukemia. We found that PIG-A mutations confer a survival advantage by making cells relatively resistant to apoptotic death. When placed in serum-free medium, granulocytes and affected CD34+ (CD59−) cells from PNH patients survived longer than their normal counterparts. PNH cells were also relatively resistant to apoptosis induced by ionizing irradiation. Replacement of the normal PIG-A gene in PNH cell lines reversed the cellular resistance to apoptosis. Inhibited apoptosis resulting from PIG-A mutations appears to be the principle mechanism by which PNH cells maintain a growth advantage over normal progenitors and could play a role in the propensity of this disease to transform into more aggressive hematologic disorders. These data also suggest that GPI anchors are important in regulating apoptosis.

Genetic basis of tolerance to O2 deprivation in Drosophila melanogaster

The ability to tolerate a low-O2 environment varies widely among species in the animal kingdom. Some animals, such as Drosophila melanogaster, can tolerate anoxia for prolonged periods without apparent tissue injury. To determine the genetic basis of the cellular responses to low O2, we performed a genetic screen in Drosophila to identify loci that are responsible for anoxia resistance. Four X-linked, anoxia-sensitive mutants belonging to three complementation groups were isolated after screening more than 10,000 mutagenized flies. The identified recessive and dominant mutations showed marked delay in recovery from O2 deprivation. In addition, electrophysiologic studies demonstrated that polysynaptic transmission in the central nervous system of the mutant flies was abnormally long during recovery from anoxia. These studies show that anoxic tolerance can be genetically dissected.

Phosphorylation of two regulatory tyrosine residues in the activation of Bruton’s tyrosine kinase via alternative receptors

Mutation of Bruton’s tyrosine kinase (Btk) impairs B cell maturation and function and results in a clinical phenotype of X-linked agammaglobulinemia. Activation of Btk correlates with an increase in the phosphorylation of two regulatory Btk tyrosine residues. Y551 (site 1) within the Src homology type 1 (SH1) domain is transphosphorylated by the Src family tyrosine kinases. Y223 (site 2) is an autophosphorylation site within the Btk SH3 domain. Polyclonal, phosphopeptide-specific antibodies were developed to evaluate the phosphorylation of Btk sites 1 and 2. Crosslinking of the B cell antigen receptor (BCR) or the mast cell Fcɛ receptor, or interleukin 5 receptor stimulation each induced rapid phosphorylation at Btk sites 1 and 2 in a tightly coupled manner. Btk molecules were singly and doubly tyrosine-phosphorylated. Phosphorylated Btk comprised only a small fraction (≤5%) of the total pool of Btk molecules in the BCR-activated B cells. Increased dosage of Lyn in B cells augmented BCR-induced phosphorylation at both sites. Kinetic analysis supports a sequential activation mechanism in which individual Btk molecules undergo serial transphosphorylation (site 1) then autophosphorylation (site 2), followed by successive dephosphorylation of site 1 then site 2. The phosphorylation of conserved tyrosine residues within structurally related Tec family kinases is likely to regulate their activation.

Tissue-specific knockout of the mouse Pig-a gene reveals important roles for GPI-anchored proteins in skin development

Glycosylphosphatidylinositol (GPI)-anchored proteins are widely distributed on plasma membranes of eukaryotes. More than 50 GPI-anchored proteins have been shown to be spatiotemporally expressed in mice with a deficiency of GPI-anchor biosynthesis that causes embryonic lethality. Here, we examine the functional roles of GPI-anchored proteins in mouse skin using the Cre-loxP recombination system. We disrupted the Pig-a gene, an X-linked gene essential for GPI-anchor biosynthesis, in skin. The Cre-mediated Pig-a disruption occurred in skin at almost 100% efficiency in male mice bearing two identically orientated loxP sites within the Pig-a gene. Expression of GPI-anchored proteins was completely absent in the skin of these mice. The skin of such mutants looked wrinkled and more scaly than that of wild-type mice. Furthermore, histological examination of mutant mice showed that the epidermal horny layer was tightly packed and thickened. Electron microscopy showed that the intercellular space was narrow and there were many small vesicles embedded in the intercellular space that were not observed in equivalent wild-type mouse skin preparations. Mutant mice died within a few days after birth, suggesting that Pig-a function is essential for proper skin differentiation and maintenance.