Potential of using CRISPR-CAS9 to treat BRCA1 and BRCA2 associated hereditary breast cancer

Breast cancer is one of the major causes of death. About 22.9% of all cancer in women is breast cancer (Pohlreich et al., 2005). Although there are many known risk factors, genetics play a major role in breast cancer. Of which, germ-line mutations in BRCA1(BReast-CAncer susceptibility gene 1) and BRCA2 (BReast-CAncer susceptibility gene 2) genes are responsible for about 90% of all hereditary breast cancer cases, as well as most of hereditary ovarian cancer cases (Calderón-Garcidueñas et al., 2005). The BRCA1 and BRCA2 genes code for Tumor Suppressor Gene proteins which act as cell growth suppressors. BRCA1 and BRCA2 also help repair damaged DNA (Mehrgou & Akouchekian, 2016). Damage to one of these two genes could result in impaired repair of damaged DNA, which can lead to the accumulation of unrepaired DNA mutations and eventually lead to cancer. (Mehrgou & Akouchekian, 2016). The most common mutation forms for BRCA1 and BRCA2 are small insertions, deletions, nonsense mutations, missense mutations that cause premature transcription terminations and altered splicing (Pylkäs et al., 2008). Breast Information Core reports that the majority of cancerous mutations in BRCA1 and BRCA2 genes result in the generation of a truncated protein through nonsense, frameshift, and splicing mutations (Pohlreich et al., 2005). There is significant morbidity and mortality associated with BRCA1 and BRCA2-associated hereditary breast cancer. Clustered regularly interspersed short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas), combined as CRISPR/Cas9 has garnered tremendous interest in the scientific community for its myriad applications from diagnostics, imaging, and most importantly in genome editing (Zhan et al., 2019). Two RNAs activate and guide Cas9, a DNA endonuclease, to bind specific DNA sequences. Cas9, then, subsequently cleaves the DNA at that site (Zhan et al., 2019). When a double-strand break is introduced in the target sequence by the Cas9 endonuclease, there are two different mechanisms of repair that are used by the host cell to respond. (1) The host cell may respond with a non-homologous end joining (NHEJ), a repair mechanism that often leads to insertions or deletions. This may be beneficial if the study, in question, aims to render a target gene functionless. (2) The second method is via homology-directed repair which recombines DNA donor templates to reconstruct damaged DNA (Zhan et al., 2019). This feature is helpful as it allows for site-specific editing, if the target gene in question, contains a mutation that leads to reduced expression, no expression, or results in cancer. In this thesis, I wish to explore the use of CRISPR-Cas9 as a possible tool for preventative therapy in BRCA1 and BRCA2-associated hereditary breast cancer. The specific aims of the following thesis include: 1. A comprehensive review of literature on CRISPR-Cas9 and breast cancer, specifically due to mutations in BRCA1 and BRCA2 genes. 2. Investigation into current evidence for CRISPR-Cas9 use in diseases. 3. Conclusion on the challenges and limitations of CRISPR-Cas9 as a potential tool for preventative therapy for BRCA1 and BRCA2- associated hereditary breast cancer

Defining the clinical, molecular and imaging spectrum of adaptor protein complex 4-associated hereditary spastic paraplegia

Abstract Bi-allelic loss-of-function variants in genes that encode subunits of the adaptor protein complex 4 (AP-4) lead to prototypical yet poorly understood forms of childhood-onset and complex hereditary spastic paraplegia: SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1) and SPG52 (AP4S1). Here, we report a detailed cross-sectional analysis of clinical, imaging and molecular data of 156 patients from 101 families. Enrolled patients were of diverse ethnic backgrounds and covered a wide age range (1.0–49.3 years). While the mean age at symptom onset was 0.8 ± 0.6 years [standard deviation (SD), range 0.2–5.0], the mean age at diagnosis was 10.2 ± 8.5 years (SD, range 0.1–46.3). We define a set of core features: early-onset developmental delay with delayed motor milestones and significant speech delay (50% non-verbal); intellectual disability in the moderate to severe range; mild hypotonia in infancy followed by spastic diplegia (mean age: 8.4 ± 5.1 years, SD) and later tetraplegia (mean age: 16.1 ± 9.8 years, SD); postnatal microcephaly (83%); foot deformities (69%); and epilepsy (66%) that is intractable in a subset. At last follow-up, 36% ambulated with assistance (mean age: 8.9 ± 6.4 years, SD) and 54% were wheelchair-dependent (mean age: 13.4 ± 9.8 years, SD). Episodes of stereotypic laughing, possibly consistent with a pseudobulbar affect, were found in 56% of patients. Key features on neuroimaging include a thin corpus callosum (90%), ventriculomegaly (65%) often with colpocephaly, and periventricular white-matter signal abnormalities (68%). Iron deposition and polymicrogyria were found in a subset of patients. AP4B1-associated SPG47 and AP4M1-associated SPG50 accounted for the majority of cases. About two-thirds of patients were born to consanguineous parents, and 82% carried homozygous variants. Over 70 unique variants were present, the majority of which are frameshift or nonsense mutations. To track disease progression across the age spectrum, we defined the relationship between disease severity as measured by several rating scales and disease duration. We found that the presence of epilepsy, which manifested before the age of 3 years in the majority of patients, was associated with worse motor outcomes. Exploring genotype-phenotype correlations, we found that disease severity and major phenotypes were equally distributed among the four subtypes, establishing that SPG47, SPG50, SPG51 and SPG52 share a common phenotype, an ‘AP-4 deficiency syndrome’. By delineating the core clinical, imaging, and molecular features of AP-4-associated hereditary spastic paraplegia across the age spectrum our results will facilitate early diagnosis, enable counselling and anticipatory guidance of affected families and help define endpoints for future interventional trials.</jats:p