Molecular cytogenetics - Its use in human gene mapping and the detection and definition of subtle chromosomal aberrations

Morrison, Norma (2004) Molecular cytogenetics - Its use in human gene mapping and the detection and definition of subtle chromosomal aberrations. PhD thesis, University of Glasgow.

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This work involved the investigation of three different molecular cytogenetic approaches, Fluorescence In Situ Hybridisation (FISH), Comparative Genomic Hybridisation (CGH) and Primed In Situ (PRINS) labelling, and their utilisation in chromosomal localisation of DNA sequences, breakpoint definition and the detection of cryptic abnormalities. As a development of earlier mapping in this department using non-fluorescent ISH, simple and reliable localisation, by FISH, of unique cloned sequences in a range of vectors was optimised and applied. Among the sequences localised were myotonic dystrophy protein kinase-related Cdc42-binding kinase beta (MRCKbeta) and telomerase reverse transcriptase (hTERT), to 14q32.31~q32.32 and 5p15.33 respectively. Probes were sourced, and their map localisations confirmed or refined, for application to two cases of chomosomal abnormality which required breakpoint definition. The first was a patient with der(12)(12qter→12p13.3::21q22.3→21q11.2::21q11.2→21qter). Breakpoint mapping with 21q-specific clones showed that the 21 q region monosomic in this individual was proximal to BAG sequence 268F23, which lies at 16.03Mb from 21pter, and included marker D21S1911 at 15.06Mb from 21pter. The breakpoint of at least one of the two chromosome 21 components of the derivative chromosome 12 lies between these two loci. This has provided a focussed region for further biochemical and molecular study. The second case was a rare proximal duplication of 8p with a mild phenotype. The duplication was confirmed to be direct, rather than inverted, and the breakpoints defined as dup(8)(p11.1p21.1~p21.2). This abnormality had not previously been reported. Concurrently, FISH probes were sourced, extracted and optimised for use in diagnostic investigation of cryptic segmental aneusomy in DiGeorge/velocardiofacial (DGA/CF), Rubinstein-Taybi and Wolf-Hirschhorn syndrome patients, and in potential carriers of Duchenne and Becker muscular dystrophy. Chromosome 22q11.2-specific cosmids were used to investigate 161 patients with features of DiGeorge/velocardiofacial syndrome, detecting 34 deletions. Following reports of hemizygosity at 22q11.2 in patients with non-syndromic conotruncal defects, a series of 24 patients with tetralogy of Fallot were examined for microdeletion at this locus and 4 deletions were disclosed. Plasmid probes for4p16.3 and 11 pi 5.5 were used to determine a cryptic t(4;11) rearrangement in a child for whom there was clinical suspicion of Wolf-Hirschhorn syndrome. This provided a means to carrier determination and prenatal diagnosis for her large, extended family. Cosmids specific for 16p13 were used for FISH assessment of a group of 15 patients with suspected Rubinstein-Taybi syndrome. No microdeletions were found with the original RT1 cosmid probe, but work now continues with 4 additional cosmids which span the Rubinstein-Taybi syndrome critical region. Prior to and during the introduction of a quantitative PCR method in this department a range of 20 exon-specific dystrophin probes was used to provide an unequivocal, simple method of carrier determination in families with inherited microdeletion at Xp21. Ninety seven female carriers were diagnosed in a total of 83 families using this FISH approach. Following the report that ~6% of cases of idiopathic mental retardation might be associated with a cryptic subtelomeric rearrangement (Flint et al 1995), the novel commercial Multiprobe-T device was initially appraised then used to screen a series of 100 patients. This disclosed three cryptic chromosomal abnormalities, namely a der(9)t(3;9), der(9)t(9;16) and der(18)t(10;18). Successful clinical application by Bryndorf et al (1995) prompted optimisation and assessment of CGH in the investigation of a selection of patients with apparent or suspected constitutional chromosomal aberrations. Abnormalities detected Included a subtle deletion at 17p11.2, and a deletion involving 1q25-q31 in an individual with an apparently balanced translocation of chromosomes 5 and 6 but abnormal phenotype. In combination with FISH, CGH refined characterisation of an apparent but unresolvable abnormality of chromosome 3, demonstrating duplication of 3p24.2-p25 and deletion of the 3p subtelomeric region. This study also involved the introduction of PRINS technology to this department and evaluation of its potential for robust detection of unique target sequences. Technical modifications assessed included, proteinase K and ligase pretreatments and use of Tyramide Signal Amplification (TSA). Slide pretreatment in Carnoy's fixative then 2xSSC, and use of an alternative, relatively inexpensive, enzyme not previously reported in PRINS (Dynazyme), were identified as beneficial. PRINS with commercial repetitive PRINS kits and 'in house' repetitive sequence primers was successful, but no satisfactory results were obtained with primers for unique or very low copy sequences. The future role of these methods in mapping and investigation of segmental aneusomy is discussed and array CGH, a high resolution refinement of CGH, reviewed.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Genetics, bioinformatics.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Boyd, Dr. Elizabeth
Date of Award: 2004
Depositing User: Enlighten Team
Unique ID: glathesis:2004-71176
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 17 Aug 2021 09:02
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