Abstract Breast cancer is a common malignancy affecting women around the world. Approximately 10 percent of breast cancer patients have a hereditary form of the disease. Women with an inherited alteration in one of the BRCA1 and BRCA2 genes have an increased risk of developing these cancers at a young age (before menopause), and often have multiple family members with the disease. A total of 6 families with multiple cases of breast cancer were identified from southern Taiwan, and five of these families were found to have missense mutations in the BRCA1 or BRCA2 genes. One novel missense mutation of A5885C (Gln1886Pro), as well as new silent mutation of A4806G (Thr1526), in the exon 11 of the BRCA2 gene was found in one(A) family. The second(F) family was found to have three missense mutations of C2731T (Pro871Leu), A3232G (Glu1038Gly) and A3667G (Lys1183Arg) in the exon 11 of the BRCA1 gene. It is very unusual to have three previously reported BRCA1 mutations in the same family and these three mutations are located on the same chromosome. Two missense mutations of A3232G (Glu1038Gly) in exon 11 and A4956G (Ser1613Gly) in exon 16, as well as silent mutations of T2430C (Leu771) and T4427C (Ser1436), of the BRCA1 gene were found in the third(E) family. The missense mutation of A4956G (Ser1613Gly) in exon 16, as well as silent mutation of T4427C (Ser1436), of the BRCA1 are found in the fourth(C) and fifth(D) family. The sixth(B) families were found to possess only one silent mutation of T4035C (Val1269) in the BRCA2 gene. The amino acid changes might cause the protein structure unstable and these could explain the moderate role of BRCA mutations in the pathogenesis of breast cancer.

Missense mutations account for more than 50% of the mutations known to be involved in human inherited diseases. Missense classification is a challenging task that involves sequencing of the genome, identifying the variations, and assessing their deleteriousness. This is a very laborious, time and cost intensive task to be carried out in the laboratory. Advancements in bioinformatics have led to several large-scale next-generation genome sequencing projects, and subsequently the identification of genome variations. Several studies have combined this data with information on established deleterious and neutral variants to develop machine learning based classifiers. There are significant issues with the missense classifiers due to which missense classification is still an open area of research. These issues can be classified under two broad categories: (a) Dataset overlap issue - where the performance estimates reported by the state-of-the-art classifiers are overly optimistic as they have often been evaluated on datasets that have significant overlaps with their training datasets. Also, there is no comparative analysis of these tools using a common benchmark dataset that contains no overlap with the training datasets, therefore making it impossible to identify the best classifier among them. Also, such a common benchmark dataset is not available. (b) Inadequate capture of vital biological information of the protein and mutations - such as conservation of long-range amino acid dependencies, changes in certain physico-chemical properties of the wild-type and mutant amino acids, due to the mutation. It is also not clear how to extract and use this information. Also, some classifiers use structural information that is not available for all proteins. In this study, we compiled a new dataset, containing around 2 - 15% overlap with the popularly used training datasets, with 18,036 mutations in 5,642 proteins. We reviewed and evaluated 15 state-of-the-art missense classifiers - SIFT, PANTHER, PROVEAN, PhD-SNP, Mutation Assessor, FATHMM, SNPs&GO, SNPs&GO3D, nsSNPAnalyzer, PolyPhen-2, SNAP, MutPred, PON-P2, CONDEL and MetaSNP, using the six metrics - accuracy, sensitivity, specificity, precision, NPV and MCC. When evaluated on our dataset, we observe huge performance drops from what has been claimed. Average drop in the performance for these 13 classifiers are around 15% in accuracy, 17% in sensitivity, 14% in specificity, 7% in NPV, 24% in precision and 30% in MCC. With this we show that the performance of these tools is not consistent on different datasets, and thus not reliable for practical use in a clinical setting. As we observed that the performance of the existing classifiers is poor in general, we tried to develop a new classifier that is robust and performs consistently across datasets, and better than the state-of-the-art classifiers. We developed a novel method of capturing long-range amino acid dependency conservation by boosting the conservation frequencies of substrings of amino acids of various lengths around the mutation position using AdaBoost learning algorithm. This score alone performed equivalently to the sequence conservation based tools in classifying missense mutations. Popularly used sequence conservation properties was combined with this boosted long-range dependency conservation scores using AdaBoost algorithm. This reduced the class bias, and improved the overall accuracy of the classifier. We trained a third classifier by incorporating changes in 21 important physico-chemical properties, due to the mutation. In this case, we observed that the overall performance further improved and the class bias further reduced. The performance of our final classifier is comparable with the state-of-the-art classifiers. We did not find any significant improvement, but the class-specific accuracies and precisions are marginally better by around 1-2% than those of the existing classifiers. In order to understand our classifier better, we dissected our benchmark dataset into: (a) seen and unseen proteins, and (b) pure and mixed proteins, and analysed the performance in detail. Finally we concluded that our classifier performs consistently across each of these categories of seen, unseen, pure and mixed protein.

Breast Cancer susceptibility gene 1 (BRCA1) & Breast Cancer Breast cancer is one of the most common malignancies affecting women worldwide. About 5-10% of all cases are estimated to be familial. Mutations in the BRCA1 (Breast Cancer susceptibility gene 1) gene account for about 15-20% of inherited breast cancer cases and 60-80% of families predisposed to both breast and ovarian cancer. BRCA1 mutations also result in susceptibility to early-onset breast and ovarian cancer. The human BRCA1 gene encodes a multi-domain 1,863 amino acid nuclear protein that is expressed in a wide variety of adult human tissues. The N-terminal end of BRCA1 contains a RING-finger domain. Exon 11 of BRCA1 contains two nuclear localization signals towards its N-terminal for targeting BRCA1 to the nucleus. The carboxyl terminus contains two BRCT (BRCA1 C-terminal) domains and a transcriptional activation domain. This study was carried out to functionally characterize BRCA1 and to find out the percentage in which BRCA1 gene is mutated in Indian familial breast and/or ovarian cancer families. The work has been divided into three sections: 1. Identification & characterization of a BRCA1 Associated Protein 2 (BAP2). 2. Germ-line BRCA1 mutation Analysis in Indian Breast and/or Ovarian Cancer Families. 3. Characterization of a novel missense mutation (E116K) in BRCA1. BRCA1 is known to interact with large number of proteins and is involved in various cellular functions like tumorigenesis, transcription, DNA damage repair, cell-cycle control, ubiquitinylation, genetic stability, cell growth and apoptosis. The interacting partners of BRCA1 have given a lot of clue about the functions of this complex protein. In the first project, we used the yeast two-hybrid system to identify novel interacting proteins of BRCA1. We used the 1-500 amino acid region of BRCA1 as bait in library screen and picked up a novel clone (clone 89) showing interaction with BRCA1. Clone 89 contains approximately 2.3 Kb long cDNA sequence. Using the nucleotide blast search, we obtained a full-length cDNA of approximately 5.4 Kb (KIAA0657) that is located on chromosome 2, 2q36.1 region. We have named this new protein BRCA1 Associated Protein 2 (BAP2). Translation of this coding sequence gave a protein that has homology to Titin protein. This protein, which has 1,236 amino acids, contains 9 Immunoglobulin like domains. The homologues of this protein exists in many other organisms but the function is not known. We have confirmed the interaction between BRCA1 and c89 using in vitro GST pull-down assay. We have studied the influence of BAP2 on various functions of BRCA1 like transcription, colony suppression and cell cycle. In the transcription assays, BAP2 activated p21 promoter activity perhaps by using endogenous BRCA1 as simultaneous ectopic expression of truncated BRCA1 (containing aa 1-500) abolished this activity. Further, BAP2 also increased the ability of BRCA1 to activate p21 promoter suggesting that BAP2 may act as a co-activator of BRCA1 functions. Surprisingly, we observed that BAP2 inhibited p53-mediated transcription both in the absence and presence of BRCA1. BAP2 failed to inhibit colony growth by itself as well as in combination with BRCA1. In the cell-cycle study, we found that BAP2 did not have any significant effect on cell cycle profile by itself. However, it drastically augmented the G2/M arrest mediated by BRCA1. Thus we conclude that we have identified a novel interacting protein of BRCA1 that regulates certain functions of BRCA1. Detection of mutations is of central importance in the study of genetic and malignant diseases. Mutation detection helps us in understanding the protein structure, function and expression. More than that, it is also important for pre-symptomatic/antenatal diagnosis, confirmation of the genetic cause of the disease and the mode of inheritance of a disease in a particular family, the prediction of clinical phenotype and the potentiation of diagnostic analysis in the case of families with incomplete pedigrees or with new mutations. Therefore, the importance of direct mutation analysis cannot be understated. The second project deals with screening of mutations in BRCA1 gene in 50 familial breast and/or ovarian cancer families using the technique of Conformation Sensitive Gel Electrophoresis (CSGE). CSGE can be used to detect mismatches in DNA heteroduplexes that contain one strand of wild type and one strand of mutated DNA. In a collaborative study with Kidwai Memorial Hospital for Oncology, Bangalore, we screened 50 families suffering from breast and/or ovarian cancer. We detected 13 mutations in this study out of which 3 are novel and 10 have already been reported earlier (Breast Information Core). All the mutations obtained in our study result in truncation of the BRCA1 protein either because of non-sense mutation or frame-shift mutation. Interestingly, 8 of the mutations detected are 185delAG mutations – the most commonly occurring mutation in Ashkenazi Jewish population. From this study, we conclude that BRCA1 is mutated in 26% of familial breast and/or ovarian cancer cases in India. Genetic testing in individuals with family history of breast, ovarian or both has become very common. It is difficult to interpret the result of genetic screen if a DNA change in the gene does not result in truncation of the protein. Rare missense changes of unknown functional and pathogenic significance are called unclassified variants. It is important to study the functional implications of these unclassified variants in order to determine the risk associated with the presence of such variations. The third project deals with characterization of one such missense variation. In an earlier mutation analysis study for BRCA1 gene in breast cancer samples, we found a novel missense variation resulting in Glu116Lys (E116K) change. In order to determine if this variant is a disease associated missense mutation or a benign sequence alteration; we introduced this variation into full length BRCA1 cDNA and studied its effect on the known functions of BRCA1, namely, transcription, colony suppression and cell cycle. We found that E116K is defective for activating transcription. However, it continued to inhibit growth in colony formation assay and arrest cells in G2/M phase of cell cycle. We conclude that E116K mutation results in loss of transactivation function of BRCA1 but has no effect on colony formation and cell cycle regulation; thus it can be categorized as a novel missense mutation.

The peroxisome is a subcellular organelle that carries out a diverse range of metabolic functions, including the b-oxidation of very long chain fatty acids, the breakdown of peroxide and the a-oxidation of fatty acids. Disruption of peroxisome metabolic functions leads to severe disease in humans. These diseases can be broadly grouped into two categories: those in which a single enzyme is defective, and those known as the peroxisome biogenesis disorders (PBDs), which result from a generalised failure to import peroxisomal matrix proteins (and consequently result in disruption of multiple metabolic pathways). The PBDs result from mutations in PEX genes, which encode protein products called peroxins, required for the normal biogenesis of the peroxisome. PEX1 encodes an AAA ATPase that is essential for peroxisome biogenesis, and mutations in PEX1 are the most common cause of PBDs worldwide. This study focused on the identification of mutations in PEX1 in an Australasian cohort of PBD patients, and the impact of these mutations on PEX1 function. As a result of the studies presented in this thesis, twelve mutations in PEX1 were identified in the Australasian cohort of patients. The identified mutations can be broadly grouped into three categories: missense mutations, mutations directly introducing a premature termination codon (PTC) and mutations that interrupt the reading frame of PEX1. The missense mutations that were identified were R798G, G843D, I989T and R998Q; all of these mutations affect amino acid residues located in the AAA domains of the PEX1 protein. Two mutations that directly introduce PTCs into the PEX1 transcript (R790X and R998X), and four frameshift mutations (A302fs, I370fs, I700fs and S797fs) were identified. There was also one mutation found in an intronic region (IVS22-19A>G) that is presumed to affect splicing of the PEX1 mRNA. Three of these mutations, G843D, I700fs and G973fs, were found at high frequency in this patient cohort. At the commencement of these studies, it was hypothesised that missense mutations would result in attenuation of PEX1 function, but mutations that introduced PTCs, either directly or indirectly, would have a deleterious effect on PEX1 function. Mutations introducing PTCs are thought to cause mRNA to be degraded by the nonsense-mediated decay of mRNA (NMD) pathway, and thus result in a decrease in PEX1 protein levels. The studies on the cellular impact of the identified PEX1 mutations were consistent with these hypotheses. Missense mutations were found to reduce peroxisomal protein import and PEX1 protein levels, but a residual level of function remained. PTC-generating mutations were found to have a major impact on PEX1 function, with PEX1 mRNA and protein levels being drastically reduced, and peroxisomal protein import capability abolished. Patients with two missense mutations showed the least impact on PEX1 function, patients with two PTC-generating mutations had a severe defect in PEX1 function, and patients carrying a combination of a missense mutation and a PTC-generating mutation showed levels of PEX1 function that were intermediate between these extremes. Thus, a correlation between PEX1 genotype and phenotype was defined for the Australasian cohort of patients investigated in these studies. For a number of patients, mutations in the coding sequence of one PEX1 allele could not be identified. Analysis of the 5' UTR of this gene was therefore pursued for potential novel mutations. The initial analyses demonstrated that the 5' end of PEX1 extended further than previously reported. Two co-segregating polymorphisms were also identified, termed –137 T>C and –53C>G. The -137T>C polymorphism resided in an upstream, in-frame ATG (termed ATG1), and the possibility that the additional sequence represented PEX1 coding sequence was examined. While both ATGs were found to be functional by virtue of in vitro and in vivo expression investigations, Western blot analysis of the PEX1 protein in patient and control cell extracts indicated that physiological translation of PEX1 was from the second ATG only. Using a luciferase reporter approach, the additional sequence was found to exhibit promoter activity. When examined alone the -137T>C polymorphism exerted a detrimental effect on PEX1 promoter activity, reducing activity to half that of wild-type levels, and the -53C>G polymorphism increased PEX1 promoter activity by 25%. When co-expressed (mimicking the physiological condition) these polymorphisms compensated for each other to bring PEX1 promoter activity to near wild-type levels. The PEX1 mutations identified in this study have been utilised by collaborators at the National Referral Laboratory for Lysosomal, Peroxisomal and Related Genetic Disorders (based at the Women's and Children's Hospital, Adelaide), in prenatal diagnosis of the PBDs. In addition, the identification of three common mutations in Australasian PBD patients has led to the implementation of screening for these mutations in newly referred patients, often enabling a precise diagnosis of a PBD to be made. Finally, the strong correlation between genotype and phenotype for the patient cohort investigated as part of these studies has generated a basis for the assessment of newly identified mutations in PEX1.

Melanopsin (Opn4) is the opsin photopigment of intrinsically photosensitive retinal ganglion cells (ipRGCs). It has a conserved opsin structure and activation mechanism, yet demonstrates unusual functional properties that suggest it will possess unique structure-function relationships. The aim of this thesis was to characterise key OPN4 residues by examining the impact of non-synonymous mutations on melanopsin function. A genotype-driven screen of a chemically-mutagenized mouse archive led to the identification of a novel Opn4 mutant, S310A, located at a known opsin spectral tuning site. Action spectra from ipRGC and pupil light responses (PLR) of Opn4^S310A mice revealed no change in wavelength of peak sensitivity. However, Opn4^S310A PLR was significantly less sensitive at longer wavelengths, consistent with a short-wavelength shift in spectral sensitivity. This suggests S310A acts as a spectral tuning site in melanopsin. Next, the impact of naturally-occurring missense variants in human melanopsin (hOPN4) was examined in vitro. Fluorescent calcium imaging of 16 hOPN4 variants expressed in HEK293 cells revealed four hOPN4 variants abolished or attenuated responses to light (Y146C, R168C, G208S and S308F). These variants were located in conserved opsin motifs for chromophore binding or hydrogen-bond networks, functional roles apparently shared by melanopsin. Finally, two hOPN4 single nucleotide polymorphisms (SNPs) P10L and T394I, associated with abnormal non-image forming behaviour in humans, were explored in vivo. Using targeted viral-delivery of hOPN4 SNPs to mouse ipRGCs, a range of OPN4-driven behaviours, such as circadian photoentrainment and pupil light responses, were found to be comparable with hOPN4 WT control. Multi-electrode array recordings of ipRGCs transduced with hOPN4 T394I virus had significantly attenuated sensitivity and faster response offset, indicating this site may be functionally important for melanopsin activity but compensatory rod and cone input limits changes to non-image forming behaviour.

<p>  </p> <p>With the discovery and treatment of any disease comes the important question of its genetic prevalence. This is especially important for animals under strict breeding control, such as dogs, because this can provide essential information regarding breeding pair decisions. Thus, the focus of this thesis is to investigate the genetic prevalence of three different diseases: 1) Factor VII Deficiency (FVIID), 2) Collie Eye Anomaly (CEA), and 3) Progressive Rod-Cone Degeneration-Progressive Retinal Atrophy (prcd-PRA). Factor VII Deficiency (FVIID) is a clotting disorder observed in both humans and dogs, characterized by impeded function of the Factor VII protein. In dogs, FVIID is caused by a single nucleotide substitution (c.407G>A) in the <em>F7 </em>gene. This mutation, identified in a colony of research Beagles, is also present in dogs with a wide variety of distantly-related breed backgrounds and in mixed-breed dogs, suggesting an ancient, ancestral origin. Given the relatively common presence of this variant, it was hypothesized that this genetic mutation could be contributing to excessive bleeding in canine autopsy cases that could not be attributed to typical causes. DNA from formalin-fixed paraffin-embedded tissues (n = 67 cases) were Sanger sequenced for the FVIID c.407G>A mutation, and all were determined to be homozygous wild-type. Therefore, the tested variant is not associated with the unexplained bleeding in these cases, and it is not a logical diagnostic test to apply to similar cases in the future.</p> <p><br></p> <p>CEA and prcd-PRA are ophthalmic genetic diseases of concern often included in commercial genetic testing panels. A large dataset spanning 15+ years provided by a commercial partner company (OptiGen/Wisdom Panel, Kinship) encompassed dogs tested for the CEA-associated <em>NHEJ1</em> deletion (n = 33,834 dogs) and the prcd-PRA causal mutation in <em>PRCD</em> (n = 86,667 dogs). Disease trends were observed graphically and analyzed with Chi-square goodness-of-fit testing and regression modeling for disease status and genotype classification. Both diseases had a statistically significant change in genotype frequencies from the first year of data to the last; both diseases also had a negative association between progression of time and overall probability of a dog being disease-positive or a carrier/heterozygous. This suggests that genetic testing results are being incorporated into breeding decisions, although affected dogs were still being identified by the end of this study. Different breeds, AKC groups, FCI groups, genetic clades, and geography were also investigated to determine impact on overall disease trend. </p>

Ahnak ist ein ubiquitäres Protein, das an einer Vielzahl biologischer Prozesse beteiligt ist. In der Herzmuskelzelle ist Ahnak überwiegend am Sarkolemma lokalisiert und bindet an Aktin und an die regulatorischen Beta2-Untereinheit des L-Typ-Kalzium-Kanals. Das Ziel dieser Arbeit war die Funktion von Ahnak im Herzen mit Hilfe eines Knock-out-Maus-Modells und in Bindungsstudien zu untersuchen. Morphologische Untersuchungen zeigten, dass das Längenwachstum adulter Kardiomyozyten bei Ahnakdefizienz signifikant reduziert war. Die Kontraktionseigenschaften adulter isolierter Ahnak-defizienter Kardio-myozyten (im Alter von 6 Monaten) waren ebenfalls verändert. Die Kontraktions- und Relaxaktionsgeschwindigkeiten waren erhöht. Eine Erhöhung des diastolischen Kalzium-Spiegels zeigten die Kardiomyozyten schon im Alter von 3 Monaten. Diese beobachteten phänotypischen Veränderungen lassen vermuten, dass die Aktivität des L-Typ-Kalzium-Kanals erhöht ist. In dieser Arbeit konnte das PXXP-Motiv, in der C-terminalen Ahnak-Domäne, als die hochaffine Beta2-Bindungsstelle (KD ~ 60 nM) identifiziert werden. Substitution von Prolin gegen Alanin verringerte zwar die Bindung zur Beta2-Untereinheit dramatisch (KD ~ 1 µM), hob sie aber nicht auf. In weiteren Bindungsstudien zeigte sich, dass die natürlich vorkommende Missensmutation I5236T die Bindung zur regulatorischen Beta2-Untereinheit verstärkte, dagegen verminderte die PKA-abhängige Phosphorylierung der beiden Proteinpartner die Bindung. Experimente am ganzen isoliert perfundierten Herzen zeigten, dass Ahnak-Knock-Out-Herzen geringer Beta-adrenerg stimulierbar waren. Ahnak scheint wie eine physiologische Bremse des kardialen Kalzium-Kanals zu wirken. / Ahnak is an ubiquitous protein with in unique structure, which has been implicated in cell type specific functions. In cardiomyocytes, ahnak is predominantly localized at the sarcolemma and is associated with actin and with the regulatory beta2 subunit of the L-type calcium-channel. The aim of this work was to unravel the function of ahnak in the heart, using a knock-out-mouse model and binding studies. Morphological studies showed a significant decrease in the cell-length of ahnak deficient cardiomyocytes. The contractile parameters of isolated adult ahnak deficient cardiomyocytes (in the age of 6 month) were altered. The development of tension and relaxation were increased. An increase of diastolic calcium was already observed at the age of 3 month. In general the observed phenotypic changes suggested an increased activity of the L-type calcium-channel. In this study, a PXXP-motif, which locates in ahnaks C-terminus, was identified as the high affinity beta2 subunit binding site (KD ~ 60 nM). Substitution of both proline residues by alanine reduced, but did not abolish the binding (KD ~ 1 µM). Further binding studies revealed that the natural occurring ahnak missense mutation I5236T increases the binding affinity to the regulatory beta2 subunit. By contrast PKA dependant phosphorylation of both protein partners decreases the interaction. In studies with isolated perfused working heart preparations, the ahnak deficient hearts were less beta-adrenergic stimulated than hearts from wild type. Taken together ahnak seems to be a physiological brake of the cardiac calcium-channel.