In sexually reproducing individuals, intraorganismal genetic heterogeneity (IGH) or mosaicism is thought to occur infrequently while genetic homogeneity is presumed the norm. In organisms that undergo modular development, such as long-lived plants, IGH has been substantially documented. In Arabidopsis thaliana we have shown that non-parental DNA that is inherited at low but detectable rates can also manifest on single plants as genotypically distinct somatic sectors suggesting that even short-lived annual plants show IGH. The underlying mechanism responsible for generating this type of IGH remains unknown. In order to better understand this phenomenon I have tested the hypothesis that among genome changes that occur in response to stress, these putative triggers also up-regulate IGH. Metabolic stress, cold stress, mechanical damage and ROS exposure were examined. To test for IGH, transgene markers and polymorphic molecular markers were used. Also, presented in this thesis is work investigating the effect of in vitro propagation through tissue culture on IGH frequencies. Regenerated plants as well as undifferentiated callus tissue were genotyped and assayed for sequence reversions. Molecular genotyping revealed an outcome contrary to that predicted by the initial hypothesis showing instead that a high frequency of restoration occurred in the progeny of un-treated control plants. With the exception of samples passed through tissue culture, molecular marker changes, including single and double reversions of alleles, were detected in every line at some low level Furthermore, many of the revertants were found to be genetic mosaics. DNA sequence analyses revealed that sequences flanking three molecular markers that had undergone reversion were near identical to the great-grandparent of the sequenced individual. These results suggest that stress is perhaps an inhibitor of restoration. Although there may be other explanations for the results described in this thesis, the evidence implicates genome restoration as a mechanism for generating IGH.

Thesis (M.S.)--The University of North Carolina at Greensboro, 2009. / Directed by Vincent Henrich; submitted to the Dept. of Biology. Title from PDF t.p. (viewed May 27, 2010). Includes bibliographical references (p. 43-45).

No description available.

Down Syndrome is a human condition caused by an extra copy of a #21 chromosome. At least one to two percent of free (not translocated) trisomy 21 cases are mosaics, i.e., they have two or more distinct cell lines. Usually, one cell line is 47 ,XX or XY ,+21 while the other cell line is normal 46,XX or 46,XY. The purpose of the study was to establish the etiologies of the separate cell lines by determining whether the zygote was trisomic or normal. Meiotic non-disjunction in the formation of a gamete could lead to a trisomic zygote; loss of a #21 chromosome during a later mitotic division could then lead to a chromosomally normal cell line. Alternatively, a mitotic error in a normal embryo can produce a trisomy 21 cell line.

It is known that age-related changes impacting multiple organ systems occur earlier in people with Down syndrome (Ds), but the biological basis underlying this trisomy 21-associated propensity for premature aging is poorly understood. Given that the trisomic/normal cells from people with mosaic Ds (mDs) are identical with regards to environmental exposures and genes (except for chromosome 21 copy number), comparisons of these isogenic trisomic/disomic cells allow one to “unmask” the cellular consequences of trisomy 21 by removing extraneous factors. The primary aim of this study was to determine if trisomy 21 results in an increase in the acquisition of age-related somatic chromosomal changes. To meet this aim, chromosome-specific telomere lengths, senescence-associated distension of satellites (SADS), and chromosomal instability frequencies were compared between the isogenic trisomic/disomic cells of people with mDs ranging from 1 to 44 years of age. Chromosome-specific telomere lengths were quantified using a Q-FISH (pantelomeric probe) method. The average trisomic cell telomere length (3.609 mean, +/- 0.082 SE) was significantly less than the average disomic cell telomere length (3.888 +/- 0.083) (n=28; p

Type 2 Diabetes Mellitus (T2DM) is a metabolic disorder of global concern that is primarily characterized by insulin resistance, relative insulin deficiency, and hyperglycemia. G- Protein Coupled Receptors (GPCRs), important mediators of cellular signaling responses, have been prime targets of drug discovery efforts in various therapeutic areas. Human G-Protein Receptor 40 (HGPR40) is highly expressed in the pancreas and has been implicated in the regulation of glucose metabolism and pathophysiology of T2DM. For effective control of diabetes, combination therapy is being considered because no single drug can completely control diabetes and its associated complications. This necessitates the identification of novel drug targets including HGPR40 which might permit development of drugs which function differently from existing drugs. This project focused on the construction of two chimeric receptor proteins named HGPR40.1-715_43.709-1013 (Chimera I) and HGPR40.1-431_43.389-1013 (Chimera II) which were made by exchanging domains within trans-membranes regions 6 and 4, of HGPR40 and HGPR43 DNA, homologous receptors which vary in amino acid sequence but which have the same three-dimensional structure. After PCR amplification of sufficient quantities of the desired gene fragments they were ligated together to form the desired recombinant chimeric proteins which were cloned into the expression vector pcDNA3.1 in two successive cloning steps. The vector contains CMV promoter, multiple cloning sites, neomycin resistance gene etc for high-level expression in a wide range of mammalian cells. The two full-length chimeras were designed to be 6452 bp and 6483 bp by exchanging either the first or the first two external domains of HGPR40 with those of HGPR43. The creation of the correct chimeras was verified by both agarose gel electrophoresis and PCR analysis. Then chimeric DNA was transformed into Escherichia coli to obtain sufficient amounts of DNA for sequencing verification of the desired construct. Upon verification, the cloned DNA was to be transfected into cultured mammalian kidney (HEK293) cells for expression of the chimeric proteins. A (FLIPR) Fluorometric Imaging Plate Reader analysis by our collaborators at Eli Lilly would have been used to measure the extent of Ca2+ efflux from the endoplasmic reticulum testing a variety of stimulatory molecules to obtain an indication of which would activate the receptor. Activation would initiate the internal (second messenger cascade) G-protein signaling pathway and result in the secretion of insulin. However, transformation of the chimeric membrane receptors into E. coli resulted in altered sequences which could not be used for the FLIPR analysis. Expression of membrane proteins in E. coli can lead to such gene rearrangements. These experiments will be repeated in the future using another type of competent cell designed to prevent genetic re-arrangements. Construction of stable clones will permit us to test numerous potential ligands to aid in development of novel therapeutic drugs targeting HGPR40 to aid in combating diabetes. / Department of Biology

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Congenital heart disease (CHD) is characterized by structural defects of the heart and great vessels. It is the most common birth defect, affecting an estimated 1% of live births, and is the leading cause of mortality among birth defects. Despite recent progress in genetic research, more than 50% of CHD cases remain unexplained. An estimated 23% are due to aneuploidies and copy number variants and up to 30% has been attributed to de novo variation, though that number ranges between 3-30% depending on CHD complexity. The contribution of somatic mosaicism, or de novo genetic mutations arising after oocyte fertilization, to congenital heart disease (CHD) is not well understood due to limitations in sample size, detection method, and validation rate. Further, the relationship between mosaicism in blood and cardiovascular tissue has not been determined. We developed a computational method, Expectation-Maximization-based detection of Mosaicism (EM-mosaic), to analyze mosaicism in exome sequences of 2530 CHD proband-parent trios. EM-mosaic accurately detected 309 mosaic mutations in blood, with 85 of 94 (90%) candidates tested independently confirmed. We found twenty-five likely damaging mosaics in plausible CHD-risk genes, affecting 1% of our cohort. Variants in these genes predicted as damaging had higher variant allele fraction than benign variants, suggesting a role in CHD. The frequency of protein-coding mosaic variants detectable in blood was 0.122 or roughly 1 in 8 individuals. Analysis of 66 individuals with matched cardiac tissue available revealed both tissue-specific and shared mosaicism, with shared mosaics generally having higher allele fraction. CHD patients often present with comorbid cardiac and extracardiac anomalies that further their impact quality of life. Neurodevelopmental disorders (NDDs) are especially prevalent in CHD cases compared to the general population, yet the underlying genetic causes remain poorly explained. Further, patients with single ventricle defects undergoing surgery often later develop arrhythmias and experience worsening ventricular function. We used a statistical approach to dissect the association between de novo variation and these clinical outcomes and found that pleiotropic mutations contribute a large fraction of the risk of acquiring NDD and abnormal ventricular function phenotypes in CHD patients. We developed a proof-of-concept rare variant risk score that combines information from de novo, rare transmitted, and copy- number variants and show that prediction of outcomes such as NDD can be improved, especially in complex CHD cases.

KIF1A is a molecular motor for membrane-bound cargo important to the development and survival of sensory neurons. KIF1A dysfunction has been associated with several Mendelian disorders with a spectrum of overlapping phenotypes, ranging from spastic paraplegia to intellectual disability. We present a novel pathogenic in-frame deletion in the KIF1A molecular motor domain inherited by two affected siblings from an unaffected mother with apparent germline mosaicism. We identified eight additional cases with heterozygous, pathogenic KIF1A variants ascertained from a local data lake. Our data provide evidence for the expansion of KIF1A-associated phenotypes to include hip subluxation and dystonia as well as phenotypes observed in only a single case: gelastic cataplexy, coxa valga, and double collecting system. We review the literature and suggest that KIF1A dysfunction is better understood as a single neuromuscular disorder with variable involvement of other organ systems than a set of discrete disorders converging at a single locus. / National Institutes of Health / Revisión por pares

A non‐mosaic trisomy 20 was discovered in all cells in two separate cultures from an age‐related genetic amniocentesis. Karyotypes of cells obtained via amniocentésis at the time of termination and of cells cultured from the placenta gave the same unambiguous results. However, the fetus, under macro‐ and microscopic analysis, showed only two minor anomalies: left simian crease and low‐set ears. These findings are more suggestive of a normal or at most mosaic trisomy 20 state. The significance of this finding for prenatal diagnosis is discussed.