DMD Genetics: complicated, complex and critical to understand

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1 DMD Genetics: complicated, complex and critical to understand Stanley Nelson, MD Professor of Human Genetics, Pathology and Laboratory Medicine, and Psychiatry Co Director, Center for Duchenne Muscular Dystrophy UCLA

2 Duchenne is an X linked recessive DMD: X-linked recessive, genetic lethal in males and overall frequency not changing

3 Human Genome 3 billion letters (GATC) ~24,000 genes ~ Gene for Duchenne/Becker Muscular Dystrophy identified ~5,000 Different Genetic Diseases known

4 DMD Gene Found in 1986 (Kunkel) Improved diagnosis and counseling Provided insight into Duchenne and Becker Suggested therapeutic strategies Gene therapy Exon skipping Utrophin upregulation

5 LARGEST GENE, Discovered in 1986, 79 exons

6 LARGEST GENE, Discovered in 1986, 79 exons

7 Dystrophin Protein domains

8 Normal Muscle Dystrophin

9 Muscle fibers contain myofibrils, which are bundles of myofilaments.

10 Dystrophin is an essential part of a large protein complex at the muscle membrane

11 Duchenne Muscular Dystrophy Dystrophin at the Muscle Membrane dystrophin hematoxylin and eosin staining unaffected DMD

12 Identification of DMD gene mutations proved that both Duchenne and Becker muscular dystrophy are caused by mutations in DMD Duchenne MD Becker Muscular Dystrophy

13 Western Blot in DMD and BMD Normal dystrophin MW = 427 kda DMD No dystrophin seen BMD Smaller dystrophin protein observed MWs differ in patients from different families Thompson & Thompson,

14 Histology and Dystrophin staining in DMD and BMD Muscle Normal BMD H&E Duchenne: increased connective tissue and leukocytes Dystrophin Normal: localized to myocyte membrane Becker: present but reduced Duchenne: completely absent DMD

15 DMD gene is LARGE. 79 EXONS Exon Frame Map of the Dystrophin gene Genetics of DMD and BMD

16 More Severe or typical Duchenne: OUT OF FRAME DNA MUTATION Genetics of DMD and BMD

17 SEVERE DISEASE: Because the end of the protein is not made Genetics of DMD and BMD

18 MILDER DISEASE= IN FRAME MUTATION Genetics of DMD and BMD

19 MILDER DISEASE= IN FRAME MUTATION Because the end of the protein is made Genetics of DMD and BMD

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21 DNA: CTGAGCCAACTATTGATGAA RNA: c CUGAGCCAACUAUUGAUGAA CODE: CUG AGC CAA CUA UUG AUG AA Protein LSQLLM

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23 Most mutations are in exons and are deletions Magri, et al 2010 J. Neuro

24 Single exon skipping relevance (Flanigan, et al 2009

25 Modest differences in age at loss of walking with different deletions Exon 44 skippable/others Exon 45 skippable/others R. Wang, Duchenne Connect Data, 2014

26 Small mutations

27 Dystrophin Protein domains

28 Different types of dystrophin protein are made from the DMD gene E30 e45 e51 e56 e63

29 New Diagnostic technique: Combination of RNA_SEQ and Whole Genome Sequencing for DMD mutation identification No expression after exon37

30 Assembly of RNA-Seq reads Pulled out all reads matching the end of DMD exon 37 (red) and assembled using Velvet BLAT of resulting contig showed intron 37 joined to intergenic region 5Mb upstream

31 Paired End WGS Large number of paired end reads with larger than expected insert size at two different locations

32 Assembly Assembly of whole genome reads in both regions showed a strand flip in the contig Right breakpoint strand switch Left breakpoint strand switch

33 Conclusion CDMD 1048 ~5mb region of chrx is inverted = Pathogenic mutation Allows creation of a specific clinical DNA test Mother is carrier, sister can be tested, appropriate genetic counseling now possible

34 Example 2: MUSCLE BIOPSY STILL GIVES IMPORTANT INFORMATION.

35 MUSCLE BIOPSY STILL GIVES IMPORTANT INFORMATION.?

36 RNA ANALYSIS FROM MUSCLE BIOPSY

37 Almost all families have their own distinct DMD mutation Relatively common 1 in 5,000 live male births HIGH Mutation rate Calculated at 1 in 10,000!! Often new mutation in child (Mom germline mosaic with an unknown fraction of eggs affected): mutation occurred when mom was in utero! OR Mom is a carrier (unbeknownst to her) (And her Mom or Dad may be a germline mosaic)

38 Female Carriers for DMD Majority have no overt clinical manifestations ~ 70% have slightly increased serum CK and liver enzyme tests Manifesting carriers: Non-mutated X chromosome inactivated non-randomly such that the majority of myofiber nuclei produce defective dystrophin A few percent of females have muscle weakness, some with a Duchenne-like clinical course Cardiomyopathy can develop in carrier women without muscle weakness and requires surveillance All potential carrier females should be tested

39 Summary Most Duchenne causing DMD mutations lead to complete loss of dystrophin function DMD mutations that allow some dystrophin expression usually cause a more mild phenotype (Becker) All patients should receive a specific molecular diagnosis Molecular diagnosis relevant to clinical trials DNA marker necessary for carrier testing in females A portion of patients need muscle biopsy to establish molecular diagnosis with RNA analysis or to clarify an unusual DNA mutation Whole genome sequencing/rna analysis of DMD gene can find mutations when standard clinical testing is normal. RNA sequencing in some cases clarifies exact RNA consequence of DNA mutation and is necessary.