patients for deletions of these loci
6.1 Introduction 6.2 Results
6.2.1 Long range physical mapping around the DXS442 and DXS366 loci 6.2.2 Physical linkage of the DXS442 and DXS 178 loci
6.2.3 Screening of D NA from X LA patients for deletions of the DXS442 and DXS366 loci
6.2.4 Mapping a deletion of the DXS442 locus 6.3 Discussion
6.1 Introduction
During the course of this study several new polymorphic loci were identified and probes isolated that mapped to the same interval as the X LA locus (Dietz band et a l ,
1990; Barker et al., 1991). The mapping of one of these loci, DXS265, has already been described (section 3.2.2). A genetic linkage analysis of Alport syndrome families (Barker et a l , 1991) placed two more of these loci, DXS366 and DXS442, distal to the DXS3 locus and proximal to the DXS 17 locus. The DXS3 locus was the proximal flanking marker for the X LA locus at this time. A recombination event in one of the Alport syndrome families (Barker et al., 1991) allowed DXS366 to be placed proximal to DXS442. These loci showed no recombinations with the X LA locus in a preliminary genetic linkage analysis of nine X LA families (Kwan et al., 1991). As these markers
appeared to be closely linked to the X LA locus, a long range physical map around each of these two loci was produced, and attempts were made to link them to other loci in Xq22. Probes specific for these loci were also used to screen D N A from X LA patients for possible deletions around the X LA locus.
6 .2 Results
6.2.1 Long range physical mapping around the D X S 442 and
D X S 366 ioci
D N A from normal female leucocytes was digested with a variety of rare cutting restriction enzymes singly or in combination with Eagl. The fragments produced were separated by PFGE, the gels were blotted onto nylon membranes and these were hybridised sequentially with probes specific for the DXS366 and DXS442 loci. Examples of these hybridisations are shown in Fig. 6.1 [A ]. From the results of these hybridisations a preliminary map was constructed. As a number of restriction sites could not be placed on this map, a number of additional combination digests were performed. Many o f these digests involved Pvul as the preliminary data suggested that this could be used to confirm the presence of a number of CpG islands in the vicinity of the DXS366 and DXS442 loci. Fragments were separated by PFGE, blotted and hybridised with probes specific for the DXS366 and DXS442 loci. Examples of these hybridisations are shown in Fig. 6.1 [B]. The results of all of the hybridisations are summarised in Table 6.1.
A long range physical map was constructed around the DXS366 and DXS442 loci which spans a total of 1.19Mb, this is shown in Fig. 6.2[A ]. The DXS366 and DXS442 specific probes recognise the same fragments in all digests except with ^ I . The 370kb and 280kb Sfil partial digestion products recognised by the DXS442 specific probe were also recognised by the probe specific for DXS366 but the 225kb fragment was not seen, instead fragments of 50kb and, more rarely, 150kb, were observed in hybridisations with the DXS366 specific probe. This suggests that the DXS366 and DXS442 loci lie within 280kb of each other but on opposite sides of a partially cutting Sfil site.
A number of rare cutting restriction sites are clustered on the distal side of the DXS442 locus, suggesting the presence of a well defined CpG island at this location.
Table 6.1
Long range restriction mapping around the DXS366 and DXS442 loci
Enzyme Fragment size
seen with the DXS366 specific probe
Fragment size seen with the DXS442 specific probe
Enzyme Fragment size
seen with the DXS366 specific probe
Fragment size seen with the DXS442 specific probe 550 550 Pv«I/5j jHII 360 360 Eagl 380 380 PvuHNael 280 280 Mlul >1400 >1400 PvuHNarl 280 280 Nael 280 280 PvuHNotl 750 750 Narl (280) (280) PvuHNrul 360 360 550 550 PvuHSacll 360 360 Notl >1400 >1400 PvuHSfil (50) (225) Nrul 800 800 (280) (280) Pvul 750 750 360 360 5flcII 650 650 PvuHSmal 280 280 Sfil (50) (225) (150)' BssRlHMlul 550 550 (280) (280) 370 370 NaellNarl 280 280 Smal 280 280 NaeltSfil (50) (225) 280 280 380 380 NaeHSmal 280 280 EagHMlul 380 380
Eagl! Nael 280 280 NarllSfil nd 225
EagHNarl 280 280 Narl! Smal 280 280
Eagl! Nrul 380 380
Eagl! Pvul 360 360 Notl! Nrul 800 800
EagHSacll 380 380 NotHSacll 650 650
Eagl! Sfil (50) (225)
(280) (280)
370 370
Eagl! Smal 280 280
Fragments produced as a result of incomplete digestion are shown in brackets. • denotes a fragment that was rarely seen. Fragment sizes are in kb.
The recognition sequences of the enzymes that have sites within this island indicate that there are at least 11 unmethylated CpG pairs in this island. Another possible CpG island is located proximal to the DXS366 locus. The placing of rare cutting restriction sites in the two CpG islands was carried out mostly with reference to the Pvul sites. Pvul does not cut in the CpG island distal to the DXS442 locus and cuts 80kb from the CpG island proximal to the DXS366 locus. Double digests with Pv mI were used to place
the sites for all of the enzymes that cut in the two CpG islands (Fig. 6.1 and 6.2[A ]). Digests with Notl in combination with other enzymes suggested there were no Notl sites