Identification of new genes implicated in centronuclear myopathy - - PowerPoint PPT Presentation

Identification of new genes implicated in centronuclear myopathy

• Dr. Johann Böhm

IGBM C, Strasbourg, France

XLCNM

CNM Severity / onset Mutated protein

Autosomal recessive (ARCNM) Amphiphysin 2 (BIN1) ++ / childhood X-linked (XLCNM) Myotubularin (MTM1) +++ / neonatal Autosomal dominant (ADCNM) Dynamin 2 (DNM2) + / adult

(Nicot et al., Nature Genet 2007) (Laporte et al., Nature Genet 1996) (Bitoun et al., Nature Genet 2005)

Patient Control

Molecular diagnosis

Patients

Functional investigations Animal model Therapeutic approaches

Molecular diagnosis

Blood sample : DNA Blood or skin : cells (DNA and protein) Muscle biopsy : histology and proteins 500 patients received over the last 15 years: 300 mutated in MTM1 - myotubularin 50 mutated in DNM2 - dynamin 5 mutated in BIN1 - amphiphysin ~150 without known cause => Other unknown genes

Molecular diagnosis

Implicated in membrane trafficking Nuclear positioning (nuclear envelope) Interaction with CNM proteins Myopathies in other organisms

120 families without mutation in MTM1/BIN1/DNM2

Linkage analysis

Positional candidates Functional candidates

Identification in 50% of patients with neuromuscular disorders Not applicable to small families or sporadic cases = no genetic counseling no therapeutic approach

Sanger sequencing (25 years): Gene by gene exploration 5 /sequencing covering 500 nucleotides Next generation sequencing (since 2008): Whole genome / exome 30 million nucleotides/hour Analysis: a few days 5000 /genome Ion Torrent (since 2011): 1 GB /2 hours Fast analysis (simple chemistry) 800-1000 /run

1573 non-synonymous variations ; 345 insertions / deletions (indels) in coding sequences

Novel disease gene discovery

Miller syndrome AR Whole-genome, one family (two affected siblings and both parents) Metachondromatosis AD Whole-genome, single proband Miller syndrome AR Exome, four cases (two siblings, two other unrelated) SchinzelGiedion syndrome AD Exome, four unrelated cases Fowler syndrome AR Exome, two unrelated cases Kabuki syndrome AD Exome, 10 unrelated cases Joubert syndrome 2 AR Exomes of 2 related individuals Non-syndromic hearing loss (DFNB82) AR exome, single case TARP syndrome X X chromosome exons, two unrelated carriers Familial exudative vitreoretinopathy AD Linkage interval + 2 candidate genes, single proband Clericuzio-type poikiloderma with neutropenia AR Linkage interval, single case Sensory/motor neuropathy with ataxia AD Linkage interval, proband and both parents Non-syndromic deafness (DFNB79) AR Linkage interval, single case

Clinical diagnosis

Congenital chloride-losing diarrhea AR Exome, single patient with suspected Bartter syndrome Primary ciliary dyskinesia AR Exome, two siblings

Molecular diagnosis

CharcotMarieTooth disease AR Whole-genome, single proband

Controls

• Confirm variant by re-sequencing
• Segregation in the family
• Sequence appropriate control populations

Ranking

• Exclude SNP (dbSNP et HapMap)
• Rank by type (stop, splice, missense)
• Rank by impact (e.g. missense variations in conserved domains?)

SNPs make up 90% of all human genetic variations, and SNPs with a minor allele frequency of at least 1% occur every 100 to 300 bases along the human genome, on average (how humans develop diseases, respond to pathogens, chemicals, drugs, etc.)

Single Nucleotide Polymorphisms

Chromosome pairs

Sexual chromosomes = determine sex

http://www.eibe.info/

http://www.simpsonspark.com/pictures_others1.php

XY XX XY XX XX XY XY XX XX

50% 50%

X-linked centronuclear myopathy (MTM)

http://www.simpsonspark.com/pictures_others1.php

19/19

Autosomal dominant CNM (DNM2)

19/19 19/19 19/19 19/19 19/19 19/19 19/19 19/19

http://www.simpsonspark.com/pictures_others1.php

66%

Autosomal recessive CNM (BIN1)

2/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2

Onset at 3.5 months: weakness, muscle atrophy, lethargy, ventroflexed neck, arched spine, short choppy gait, collapsing after short distances Routine serum biochemistry profiles unremarkable Electromyography: abnormal spontaneous activity in several muscle groups

Jake

Affected Vastus lateralis cross sections:

Hypotrophic, round shaped fibers, central nuclei Central and sub-sarcolemmal accumulations (necklace fibers) Type 1 fiber predominance ATPase NADH-TR H & E H & E NADH-TR ATPase

Control

protein-protein interaction

Phosphatase Coiled-coil PDZ-binding GRAM-PH

protein-lipid interaction

Affected male Obligate carrier

Exon 7: c.465C>A

Controls:

• worldwide panel of 237 unrelated and unaffected Labrador

retrievers

• 59 control dogs from 25 other breeds

All affected males hemizygous, all obligate carriers heterozygous

wild type N155K

20 40 60 80 100

No aggregates <50 aggregates >50 aggregates

WT WT WT N155K N155K N155K

N155K Proteasome Merge

Perspectives

• Important for the scientific understanding of the pathogenesis of CNM
• Pre-clinical therapeutic trials in rodent models cannot always be

directly extrapolated to the human condition.

• Labrador retriever represents an excellent model to establish

therapeutic approaches for centronuclear myopathies

Conclusions and Perspectives

Conclusion

• First MTM1 mutation identified in CNM in dogs
• Classical clinical and histological CNM features

Thanks!