<p>Fuchs endothelial corneal dystrophy (FECD) is a complex, late-onset disorder that is a frequent indication for corneal transplantation and affects women more frequently than men. Although linkage and association studies in patients of European and Asian ancestry have started to explain the genetic basis of this disorder, the mechanism by which FECD develops is still unclear. Three projects were undertaken to help elucidate the genetic basis of FECD. The first project examined a large, multigenerational family that exhibited strong familial clustering of FECD and identified evidence of linkage to chromosome 18. This locus that has also been implicated through work on large and small FECD families as well as unrelated patients. The second project examined African-Americans with FECD and is the first work to examine this population of patients with respect to the FECD phenotype. Novel variants in three FECD candidate genes, <italic>COL8A2</italic>, <italic>SLC4A11</italic>, and <italic>ZEB1</italic> were identified at approximately the same rate as observed in patients of other ancestries, reinforcing the notion that these genes only contribute to a small fraction of FECD genetic susceptibility. Finally, the influence of environmental factors on FECD susceptibility was examined through the use of a risk factor questionnaire given to cases and controls at the time of study enrollment. Several factors, including gender, age, and cataracts, were found to significantly affect FECD risk. Gender and the number of cataract surgeries were found to significantly interact with a genetic variant, rs613872 in the <italic>TCF4</italic> locus on chromosome 18 that has been consistently and reproducibly associated with FECD, to influence FECD susceptibility. Together, these findings indicate that the genetic basis of FECD is complex, and recent advances in the field show promise in accelerating the pace of discovery that will hopefully develop better FECD treatments in the near future.</p> / Dissertation

The expansion of repetitive sequences leads to more than 40 neurological, neurodegenerative and neuromuscular diseases. These diseases are frequently characterized by ongoing DNA repeat instability upon transmission, worsening of disease severity and decreasing age of onset with each successive generation. The mechanism of repeat instability and contribution of repeat instability to disease pathogenesis are unknown. My thesis examines the contribution of cis-elements – sequences around and within repeats as well as surrounding epigenetic environments – to repeat instability, and discusses their possible contribution to repeat diseases. Here I identify the first cis-element regulating repeat instability, a DNA binding site for a trans factor protein, CTCF. Loss of CTCF binding at the spinocerebellar ataxia type 7 disease locus induces somatic and germline instability in an age- and tissue-specific manner in mice. CTCF protects against instability in an epigenetic manner, and may function at other disease loci also possessing CTCF binding sites near the repeat. Given that CTCF flanks many repeat loci and is often situated between a replication origin and disease-associated repeat, I assess the role of CTCF on replication and instability at the myotonic dystrophy repeat locus. Templates with CTCF binding sites reduce overall replication efficiency in primate cells that may be partly due to replication fork stalling. Mutating CTCF binding sites can alter the stability of the repeat depending on the distance from the origin of replication to the repeat. Finally I examine chromatinization of (ATTCT)n repeats from the spinocerebellar ataxia type 10 locus. These repeats induce very strong nucleosome formation, and at physiological conditions form even more strongly on (ATTCT)n repeats with interruptions that are also found in some patients. These data contribute to the understanding of repeat instability in the causation of many diseases, and suggest that the presence of cis-elements at repeat-associated disease loci alter the behaviour of repeats.

No description available.

The expansion of repetitive sequences leads to more than 40 neurological, neurodegenerative and neuromuscular diseases. These diseases are frequently characterized by ongoing DNA repeat instability upon transmission, worsening of disease severity and decreasing age of onset with each successive generation. The mechanism of repeat instability and contribution of repeat instability to disease pathogenesis are unknown. My thesis examines the contribution of cis-elements – sequences around and within repeats as well as surrounding epigenetic environments – to repeat instability, and discusses their possible contribution to repeat diseases. Here I identify the first cis-element regulating repeat instability, a DNA binding site for a trans factor protein, CTCF. Loss of CTCF binding at the spinocerebellar ataxia type 7 disease locus induces somatic and germline instability in an age- and tissue-specific manner in mice. CTCF protects against instability in an epigenetic manner, and may function at other disease loci also possessing CTCF binding sites near the repeat. Given that CTCF flanks many repeat loci and is often situated between a replication origin and disease-associated repeat, I assess the role of CTCF on replication and instability at the myotonic dystrophy repeat locus. Templates with CTCF binding sites reduce overall replication efficiency in primate cells that may be partly due to replication fork stalling. Mutating CTCF binding sites can alter the stability of the repeat depending on the distance from the origin of replication to the repeat. Finally I examine chromatinization of (ATTCT)n repeats from the spinocerebellar ataxia type 10 locus. These repeats induce very strong nucleosome formation, and at physiological conditions form even more strongly on (ATTCT)n repeats with interruptions that are also found in some patients. These data contribute to the understanding of repeat instability in the causation of many diseases, and suggest that the presence of cis-elements at repeat-associated disease loci alter the behaviour of repeats.

Limb girdle muscular dystrophies (LGMDs) are a clinically and genetically heterogeneous group of myopathies characterized by weakness and wasting of the proximal musculature. There are currently seventeen loci associated with different LGMDs, seven with an autosomal dominant mode of inheritance (LGMD1A–1G) and 10 with an autosomal recessive mode of inheritance (LGMD2A– 2J). The cumulative worldwide prevalence of LGMD is thought to be ~1/15,000. In the Hutterite population of North America there is an over-representation of autosomal recessive LGMD with a prevalence estimated to be >1/400. The objective of this work was to delineate the genetic basis of LGMD in this large genetically isolated population. A genome-wide scan was performed on Hutterite LGMD patients and their families in order to locate the mutant gene. This allowed us to identify a novel locus at chromosome region 9q31-33 that was named LGMD2H. Extensive haplotyping and mutation screening led to the discovery of c.1459G>A in TRIM32 as the causative mutation of LGMD2H. We then found that this same mutation was the cause of another previously described myopathy in the Hutterites, sarcotubular myopathy (STM)[reference awaiting publishers decision]. Analysis of the TRIM32 gene product revealed that it is a potential E3-ubiquitin ligase, is expressed in many human tissues including muscle and brain, and has a punctate cytoplasmic distribution. During the analysis of the LGMD2H region, it became apparent that there were Hutterite LGMD patients not linked to the LGMD2H locus. In order to identify the causative gene(s) in the remaining families, we performed a genome-wide scan. A locus at chromosome 19q13 was found to correspond to disease inheritance, the site of a previously described LGMD locus, LGMD2I. No causative gene had yet been identified at this locus so haplotyping and mutation screening was performed. We were able to identify c.826C>A in FKRP as the causative mutation in our remaining cohort of LGMD patients. The same mutation has since been found in many other populations, and is apparently a relatively common cause of LGMD. We obtained DNA from 19 non-Hutterite LGMD2I patients of diverse origins with c.826C>A and determined that it is an old founder mutation. There is no further evidence of any other loci causing autosomal recessive myopathy in the Hutterites. With the identification of c.1459G>A in TRIM32 and c.826C>A in FKRP we appear to have delineated the genetic cause of all myopathies of increased prevalence in the Hutterite population. To date, we have been able to provide accurate, non-invasive, diagnosis to over 70 patients and have provided carrier testing to approximately 120 at-risk family members. This kind DNA-based approach is not feasible in the general population due the enormous amount of locus, allelic, and clinical heterogeneity among myopathy patients. / May 2005

Myotilin is a muscle-specific Z-disc protein with putative roles in myofibril assembly and structural upkeep of the sarcomere. Several myotilin point mutations have been described in patients with Limb-Girdle Muscular Dystrophy Type 1A (LGMD1A), myofibrillar myopathy (MFM), spheroid body myopathy (SBM), and distal myopathy, four similar adult-onset, progressive, and autosomal dominant muscular dystrophies--collectively called the myotilinopathies. It is not yet known how myotilin mutations cause muscle disease. To investigate myotilin's role in the pathogenesis of muscle disease, I have created and characterized transgenic mice expressing mutant (Thr57Ile) myotilin under the control of the human skeletal alpha-actin promoter. Like LGMD1A and MFM patients, these mice develop progressive myofibrillar pathology that includes Z-disc streaming, excess myofibrillar vacuolization, and plaque-like myofibrillar aggregation. These aggregates become progressively larger and more numerous with age. I show that the mutant myotilin protein properly localizes to the Z-disc, and also heavily populates the aggregates, along with several other Z-disc associated proteins. Whole muscle physiological analysis reveals that the extensor digitorum longus (EDL) muscle of transgenic mice exhibits significantly reduced maximum specific isometric force compared to littermate controls. Intriguingly, the soleus and diaphragm muscles are spared of any abnormal myopathology and show no reductions in maximum specific force. These data provide evidence that myotilin mutations promote aggregate-dependent contractile dysfunction. To better understand myotilin function, I also created two separate lines of myotilin domain deletion transgenic mice: one expresses a deletion of the N-terminal domain and the second expresses a deletion of the minimal alpha-actinin binding site. Studies in these mice show that 1) the N-terminal domain of myotilin may be required for normal localization to the Z-disc; 2) interaction with alpha-actinin is not required for localization of myotilin to the Z-disc; and 3) deletion of the alpha-actinin binding site causes an aggregation phenotype similar to that of the TgT57I mouse and myotilinopathy patients. In sum, I have established a promising patho-physiological mouse model that unifies the diverse clinical phenotypes of the myotilinopathies. This mouse model promises to be a key resource for understanding myotilin function, unraveling LGMD1A pathogenesis, and investigating therapeutics. / Dissertation

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2010.

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / "December 2002." Typescript. Vita. Includes bibliographical references (leaves 141-160). Also issued on the Internet.

Attaining an understanding of the mechanisms underpinning development has been amongst the cardinal scientific challenges of our age. The transition from a single cell organism to the level of complexity evidenced in higher eukaryotes has been facilitated by the advent of intricate developmental networks involving a plethora of factors that synergise to allow for precise spatio-temporal expression of the proteins present in higher organisms. Development is often portrayed as a domino like cascade of events stemming from relatively uncomplicated origins that go on to branch out and form associations and interactions amongst multitudinous actors that will inexorably lead towards a higher state of order. Transcription factors occupy a central position within this tapestry of interactions. They regulate expression of the various required proteins and they provide the cues for the developmental events that will eventually shape an organism. These factors frequently remain unknown until some occurrence causes developmental processes to fail and inadvertently focus attention on the factors that facilitate development. Myotonic dystrophy is a useful paradigm of such a developmental dysfunction that has led to the discovery of a transcription factor integral to both muscle development and gonadogenesis in both Drosophila and higher eukaryotes.

<p>Muscular dystrophy is a devastating disease for which no cures or preventative treatments are currently available. There has been great progress in the identification of genetic mutations that cause some forms of muscle disease; however, genetic heterogeneity is the rule rather than the exception. Molecular diagnosis of these disorders is challenging because the large number of known causative genes makes exhaustive clinical testing very expensive and the similarity of clinical presentation makes selection of likely candidate genes difficult. The Duke Limb-Girdle Muscular Dystrophy (LGMD) group strives to identify the mutations causing disease in affected members of families with molecularly undiagnosed, dominantly inherited forms of muscular dystrophy while potentially identifying and characterizing genes and mutations that have not been previously shown to be involved in muscle disease pathogenesis.</p><p>One strategy to identify disease-causing mutations in families with many affected individuals is to perform linkage analysis to identify a region of the genome that is likely to contain the disease-causing variant followed by candidate gene sequencing within the peak to isolate the mutation. Linkage analysis was performed for a family with a molecularly undiagnosed form of scapuloperoneal muscular dystrophy. Three suggestive linkage regions were identified on chromosomes 3, 4, and 14 respectively. Each affected individual in the family carried a heterozygous deletion of a lysine residue at position 1784 in exon 37 of MYH7, which maps to one of the linked loci. Other groups have also identified this variant in individuals with similar symptoms. The deletion of the lysine residue is likely the causative mutation in this family.</p><p>An alternate strategy for mutation identification is exome capture and sequencing. This approach may be used to screen genes that are known to be involved in muscle disease pathogenesis while potentially identifying candidate disease-causing variants in genes that have not previously been shown to be involved in the disease. This strategy was evaluated through the analysis of exome sequencing data obtained from multiple affected family members in two families with different muscle disease symptoms. Variant filtration and Sanger sequencing follow-up were performed to identify those variants located in genes known to be involved in skeletal and/or cardiac muscle disease that fit the expected inheritance patterns in each family and are rarely identified in the general population, and likely functional. The mutation, desmin IVS3+3 A>G, was identified in the first family, and the mutation, filamin C p.W2710X, was identified in the second family. These mutations segregated with affection status in the complete families. They have been identified in other individuals with similar phenotypes and were found to affect the proteins by functional analyses. Therefore, the mutations likely caused disease in affected members of these two families as well.</p><p>For families in which initial analysis of exome sequencing data does not reveal a likely disease-causing variant in genes in which mutations are known to cause skeletal muscle disease, it is necessary to determine whether the exome sequencing data provided deep, high-quality coverage at each base in their coding and splicing regions. Coverage variability in genes known to be involved in skeletal and/or cardiac muscle disease was examined for eleven example exomes. After duplicate removal, the mean coverage per exome ranged from 42X to 84X, and 85.7%-92.8% of the expected capture region was covered at ¡Ý10X as calculated with the command GATK ¨CDepthOfCoverage. Depth and quality of coverage were examined in the coding and splicing regions of 102 genes that are known to be involved in skeletal and/or cardiac muscle disease; they were found to vary across different exome captures. Some regions were not well covered in any of the exomes sequenced. The results indicate that while many causative genes are well-covered, gaps exist which may interfere with the identification of some disease-causing mutations. In some cases, these gaps may be filled by increasing overall coverage.</p><p>The initial screen indicated that mutations in known disease genes may be frequent in the Duke LGMD families, so the use of a single exome in a family to screen genes that are known to be involved in muscle disease was attempted. Exome sequence analysis was performed for single affected individuals from seven families with multiple affected family members. For each exome, the candidate set was restricted to variants found within the coding and splicing regions of 102 skeletal and/or cardiac muscle disease genes. Strict filters were applied to identify extremely rare, high quality variants located within those genes, and Sanger sequencing follow-up was performed to determine which variants segregated with affection status in the complete family. In five of the seven families, potential disease-causing variants were found in a heterozygous state in all affected individuals. When possible, functional testing of these alleles, preferably in an in vivo model, would be beneficial to assist in determining whether each allele is likely to be pathogenic.</p><p>The described work utilized linkage analysis followed by candidate gene sequencing as well as exome capture and sequencing to attempt to isolate the mutations responsible for muscular dystrophy in affected family members. While linkage analysis may continue to be important to identify regions of the genome that are identical by descent in extended families, the use of next generation sequencing technologies to isolate mutations that cause rare, highly penetrant disorders in smaller families can be effective. However, it is necessary to examine the depth and quality of coverage within the consensus coding and splicing regions of genes in which mutations are known to cause a similar phenotype to that found in a family of interest. In the future, functional follow-up will be important to assist in the interpretation of variants of unknown significance.</p> / Dissertation