Isolation of Chromosome 21 Specific DNA Probes and Their Use in the Study of Down's Syndrome

Galt, James (1988) Isolation of Chromosome 21 Specific DNA Probes and Their Use in the Study of Down's Syndrome. PhD thesis, University of Glasgow.

Abstract

Down's syndrome is caused by the presence in an individual of three copies of gene loci in a critical region in the long arm of chromosome 21. Usually a complete extra copy of chromosome 21 is present (trisomy) and at birth this has an overall frequency of 1 in 700. The most important clinical effect is severe mental retardation, and Down's syndrome represents the most commonly identified cause of human mental handicap. The phenotype also includes dysmorphic features, malformations of major organs, an increased risk of leukaemia and presenile Alzheimer-type dementia. Older cytogenetic studies of chromosomal heteromorphisms have shown that in 80% of cases the additional chromosome in trisomy 21 is of maternal origin. Surprisingly this proportion did not seem to be increased in older mothers (over 35 years) who are known to have an increased frequency of Down's syndrome offspring. The cytogenetic studies were, however, uninformative in many cases. The main aim of the present project was to isolate polymorphic DNA probes from chromosome 21 which could be used to study nondisjunction in Down's syndrome families and to construct a genetic linkage map of chromosome 21. Cloned sequences derived from the critical region were to be analysed for expression in an attempt to identify expressed genes of pathogenic importance. Two chromosome 21-specific recombinant libraries were screened and of 486 bacteriophage clones initially selected, 29 recombinants were further analysed. Fourteen of these were shown to contain highly repetitive DNA sequences and were not studied further. The remaining 15 clones contained single- or low-copy number sequences and these were regionally mapped on chromosome 21 using a panel of somatic cell hybrids. Five of the single-copy sequences (JG21/D21S86, JG62/P21S90, JG72/P21S92, JG90/D21S95, JG99/D21S97) mapped in the 21q21-q22.1 region. A further six (JG12/D21S85, JG22/P21S87, JG24/P21S88, JG63/D21S91, JG81/D21S94, JG108/D21S99) were localised in the 21q22.1-qter region and one (JG77/D21S93) in the 21q22.1-q22.2 region. Probe JG373 (D21S101) was mapped in the distal part of band 21q22.3. The remaining two DNA sequences (JG51 and JG73) are mildly repetitive and were mapped to the pericentromeric region of chromosome 21. Although these clones were isolated separately from the same recombinant library and have different human insert sizes, they appear to detect homology with the same series of sequences in the human genome, which are present on chromosome 21 and on other chromosomes. The results obtained with these two probes suggest that they are part of the alphoid repeat family, members of which are present at the centromere of every human chromosome. Eight of the probes isolated in the present project were tested with a total of 25 restriction endonucleases and five restriction fragment length polymorphisms (RFLPs) were discovered : JG77/D21S93 detects an Msp I polymorphism, with alleles of 6kb and 3kb, of frequencies 0.67 and 0.33, respectively; JG81/D21S94 detects two RFLPs, with the enzymes Pvu II and Eco RV - the allele sizes and frequencies (in parenthesis) for these two RFLPs are 8.5kb (0.83)/8kb(0.17) for Pvu II and 5kb(0. 88)/4. 5kb(0.12) for Eco RV; JG90/D21S95 detects an RFLP with the enzyme Nde II, with allele sizes/frequencies of 2.2kb(0.7) and 1.8kb(0.3); the other RFLP is detected by JG99/D21S97 with the enzyme Pst I and has 7kb(0.16) and 6.6kb(0.84) alleles. The Msp I RFLP detected by JG77/D21S93 was used in conjunction with 4 other previously described polymorphic chromosome 21 probes to analyse the origin of nondisjunction in a total of 33 Down's syndrome families. Cytogenetic analysis by Q-banding was also used in some of these families. The parental origin of the additional chromosome was determined in 12 cases (36%) - in 9 of these (75%) the additional chromosome was of maternal origin, while in the other 3 (25%) it was derived from the father. These proportions are in agreement with those obtained previously from studies of cytogenetic heteromorphisms. (Abstract shortened by ProQuest.).

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Genetics
• Date of Award: 1988
• Depositing User: Enlighten Team
• Unique ID: glathesis:1988-77658
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 14 Jan 2020 11:53
• Last Modified: 14 Jan 2020 11:53
• URI: https://theses.gla.ac.uk/id/eprint/77658