A deletion-inversion-deletion event on the X

chromosome

Presented by Wendy Roworth

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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

Show QF-PCR report

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 ~1.6Mb600kb 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 5 11

Dys E1-E79

50607079

IL1RAPL1 E1-E5

3 5

Dys E53-E79

53 60 70 79

Dys E1-E51

50

Deleted DeletedInverted

FTHL17

1 23 4

PRU124953

1-3 2-4

Normal

b)

2000 kb0 500 1000 1500

MAGE-B1,2,3 DAX-1IL1RAPL1 DystrophinFTHL17GK

a)

Deleted regionDel

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

Ig1 Ig2 Spec21 Spec22 Spec23 Spec24 Cys-Rich C-terminal

Signal peptide

Exon 5 Exon 53L T V T V E R I QETT N Q W D

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.

1 3 42

1 3

4

21:32:4

4:2

3:1

1:3 2:4

3:14:2

Mutational mechanism

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

0-1

-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