Clinical overview of patient Second child of healthy, non-consanguineous parents Well in intermediate neonatal period, but collapsed at 17 hours with hypoglycaemia Cardiopulmonary arrest Resuscitation with several doses of IV hydrocortisone (hormone produced by adrenal gland) Artificially ventilated for 4 days Hyponatraemic (↓ blood [Na]) No adrenal glands on USS
Clinical overview of patient Glycerol kinase deficiency Undetectable random cortisol level Reduced oxygen supply to tissues Raised serum CKs Blind at 10 months Significant motor delay Feeding well Diagnosed with a contiguous gene syndrome causing both DMD and glycerol kinase deficiency (GKD)
Genetic investigations Normal 46,XY male karyotype. QF-PCR of selected exons of dystrophin gene showed loss of exon 52, (confirmed using alt primers); all other exons present incl. exs 50 & 51. Ex 52 del is out of frame – severe DMD phenotype, but no explanation for the contiguous gene deletion syndrome. primers designed at each end of GK gene – entirely deleted.
QF-PCR of selected 3’ dystrophin exons Ex 52 del
One or two events? Two apparent non-contiguous deletions of ex52 dystrophin and the entire GK gene locus: Two separate mutational events OR A single more complex rearrangement RT-PCR of dystrophin transcript in overlapping sections yielded products from reactions both 5’ and 3’ of exon 52, but no reaction which transversed exons 51-53.
Determination of breakpoints Breakpoint analysis of ex 52 deletion: Introns 51 & 52 each re-iteratively divided up for long- range PCRs. Breakpoints (3 & 4) pinpointed within 1 or 2kb Large-scale deletion screen of the ~2Mb region telomeric of dystrophin showed: FTHL17 to be deleted. Exons 6-11 of IL1RAPL1 deleted (exons 1-5 present) Breakpoints in intron 5 IL1RAPL1 (bkpt 1) and FTHL17- DMD region (bkpt 2) roughly determined by deletion analysis
Xp21 chromosomal region Region of deletion Ex52 deleted Breakpoint 1 Breakpoint 2 Breakpoint 3 Breakpoint 4 Dystrophin ex 78 present FTHL17 & GK genes telomeric to dystrophin deleted IL1RAPL1 exs 1-5 present, but exs 6-11 deleted Deletion of ~1.6Mb 600kb present
Characterisation of rearrangement Breakpoints roughly determined, but nature of rearrangement unknown, so: Amplify patient DNA using different combinations of primers from region closest to each of the 4 breakpoints Amplification achieved from breakpoints 1 & 3 and breakpoints 2 & 4 Products sequenced
The rearrangement Junction between breakpoints 1 & 3 is clean, occurs within Alu repeat sequences (~80% identical between two regions involved). Junction between breakpoints 2 & 4 shows little similiarity between two regions, and shows 15 novel bp insertion. 600 kb region between two deletions correlates to exs 53-79 of dystrophin which are inverted.
IL1RAPL1 E1-E11 3 511 Dys E1-E79 50607079 IL1RAPL1 E1-E5 3 5 Dys E53-E79 53607079 Dys E1-E51 50 Deleted Inverted FTHL17 1 2 3 4 PRU124953 1-32-4 Normal b) 2000 kb050010001500 MAGE-B1,2,3DAX-1 IL1RAPL1 Dystrophin FTHL17 GK a) Deleted region Del
Characterisation of chimeric ILRAPL1/dystrophin mRNA product (bkpts 1& 3) Theory: Chimeric mRNA formed from fusion of IL1RAPL1 and dystrophin genes Ex5 of IL1RAPL1 spliced to ex 53 of the inverted 3’ of the dystrophin gene Transcription of IL1RAPL1 gene passes from intron 5 into sense strand of intron 52 of dystrophin gene Transcription of dystrophin gene should traverse intron 51 & enter the antisense strand of the 3’ end of the same gene – premature termination likely. RT-PCR on RNA from muscle biopsy to confirm Forward primer in ex1 IL1RAPL1 Reverse primer in ex58 dystrophin Product of 1086 bp (expected size)
N-terminal 234 residues of IL1RAPL1 C-terminal 1132 residues of dystrophin Ig1Ig2 Spec21Spec22Spec23Spec24 Cys-RichC-terminal Signal peptide Exon 5Exon 53 LTVTVERIQETTNQWD a) b) ILRAPL1/dystrophin RNA sequence
Characterisation of partially inverted dystrophin mRNA (bkpts 2 &4) RT-PCR using F primer in exon 49 of dystrophin showed: 658 bp product comprising 3’ end of ex 49 spliced to exs 50 & 51, followed by 215 bp of int 51 and then consensus AUUAAA polyadenylation signal
Phenotype Muscular dystrophy, high CKs - single exon deletion & partial inversion of dystrophin gene Glycerol kinase deficiency – deletion of GK gene Congenital adrenal hypoplasia – NROB1 (DAX1) gene deleted Learning difficulties associated with mutations in IL1RAPL1 gene
Origin of mutation - linkage studies QF-PCR showed RD’s mother, CMD, to be a carrier of the ex52 del (and therefore of the Xp21 rearrangement). CMD has inherited the high risk X from her father, PD. PD not affected, therefore de novo mutation in CMD or PD is germline mosaic for inversion.
Detection of inversions At the time (2001), this inversion would not have been detected using our regular DMD testing strategy (karyotype & QF-PCR) – only investigated further as doctors noted DMD, GKD, CAH etc and patient diagnosed with contiguous gene disorder. Much of this work wouldn’t be necessary if referred now – this rearrangement detectable by arrays. BUT only detectable as is complex with deletion as well as inversion:
IL1RAPL1 High density aCGH Dystrophin Deletion of dystrophin exon 52 -2 -3 +6 +5 +4 +3 +2 +1 0 -4 -5 -6 Inversion of exs 53 to 79 of dystrophin Deletion including 11 genes +7 -7
Reference & acknowledgements A complex deletion-inversion-deletion event results in a chimeric IL1RAPL1-dystrophin transcript and a contiguous gene deletion syndrome. Wheway et al, JMG (2003); 40: 127-131 Lab work carried out by: Jo McCauley (Wheway) Michael Yau Vandana Nihalani David Ellis Vicky Cloke