Larsen Syndrome and Allelic Filamin B Skeletal Disorders

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1 Larsen Syndrome and Allelic Filamin B Skeletal Disorders John M. Graham, Jr. and Deborah Krakow Medical Genetics Institute Cedars-Sinai Medical Center David Geffen School of Medicine at UCLA

2 Outline Spondylocarpotarsal syndrome Filamin B Larsen syndrome Atelosteogenesis I, III and Boomerang dysplasia Role of Filamin B in the skeleton

3 Spondylocarpotarsal synostosis syndrome Rare autosomal recessive Clinical findings Scoliosis Short stature Craniofacial abnormalities Cleft palate Clubfoot Hearing loss Joint laxity Renal abnormalities occur R00-325

4 SCT Radiographic Findings Scoliosis Vertebral fusion in the thoracic region

5 CT Analysis in SCT Anterior & posterior fusions Narrow disc space Bilateral and unilateral fusions = wedged vertebrae

6 SCT Radiological Findings - Carpal Fusions Age-matched Normal Control R00-325

7 Spondylocarpotarsal Syndrome R R R R00-084

8 The Filamin Gene Family Intracellular actin-binding filamentous proteins Three paralogues in humans FLNA, FLNB and FLNC Cell migration and scaffolding functions Normal human fibroblasts Green : filamin A Red: filamin B

9 Filamin Protein Domain Structure Fln A ABD H1 CH1 CH H aa Fln B ABD H1 H2 CH1 CH aa Fln C ABD H1 H2 CH1 CH aa ABD - Actin Binding Domain CH - Calponin Homology domains - Filamin Repeat Structure (resembles C1 immunoglobulin structure) H1 - Hinge Region - Alternative Splicing

10 Cellular Roles for Filamin filamin

11 Binding Partners of Filamin Protein SITE FUNCTION Protein SITE FUNCTION Coagulation-associated Tissue factor Glycoprotein 1bα Ion channels HCN1 Kir 3.13 Kv4.2 potassium channel Integrins β1a, β1d, β2, β3, β7 Immune regulation FcγR1 SEK TRAF2 FAP52 Signal transduction RalA, RhoA, Rac1, Cdc42 Caveolin SHIP SMADs μ opioid receptor Insulin receptor β arrestins Calmodulin Thyrotropin receptor Ca ++ sensing receptor ?? C-term 20-24? ??? ABD? Haemostasis Binds vwf pacemaker K + flux K + flux Mechanoprotection Binds IgG Inflammation Inflammation Focal adhesions G-protein signalling Endocytosis RegulatesPIP 3 TGFβ signalling Endorphin receptor Insulin signalling Actin binding TSH signalling Monitors [Ca 2+ ] Proteases Epithin Furin Granzyme B Presenilin 1, 2 Cell adhesion CEACAM Migfilin Sarcomeric proteins FATZ1 myotilin Neurotransmitter receptors Dopamine D2, D3 receptors Glutamate receptor ECM molecules Decorin Nuclear functions Androgen receptor BRCA2 FOXC1 PEBP2beta/CBFbeta? C- term???? C- t e r m C term?? Protease Proteolysis proteolysis Cell adhesion Cell ECM binding Z disc protein Anchoring protein Neurotransmission Neurotransmission Proteoglycan Androgen signaling DNA damage repair Transcription factor Transcriptional regulation

12 Nonsense Mutations in Repeat Regions of FLNB Produce SCT ABD CH1 CH H1 H2 24 SCT is a Filamin B-less Condition Normal SCT Carriers

13 Joint Dislocation Disorders - Disordered Skeletogenesis

14 Larsen Syndrome In 1950 Larsen described 6 sporadic cases with multiple joint dislocations and unusual facies (subsequent AD transmission): Anterior dislocation of the knees Dislocated hips and elbows Equinovarus or valgus feet Cylindrical fingers with spatulate thumbs Flat face, hypertelorism, frontal bossing, depressed nasal bridge AD Larsen syndrome mapped to chromosome 3p14-21 Genetic heterogeneity with some cases of proposed autosomal recessive inheritance Larsen et al., Journal of Pediatrics 37: , 1950 Vujec et al., Amer J Hum Genet. 57: , 1995

15 Larsen Syndrome - Clinical Features CRANIOFACIAL Prominent forehead Hypertelorism Mid-face flattening Depressed nasal bridge Cleft palate Mild micrognathia

16 Larsen Syndrome Bicknell et al., J Med Genet (2006)

17 Larsen Syndrome - Clinical Features SKELETAL Dislocated hips, knees, and elbows Flexion contractures Joint laxity Equinovarus or valgus feet Cylindrical fingers Spatulate thumbs Syndactyly Kyphoscoliosis Cervical kyphosis

18 Larsen Syndrome Radiography Large joint dislocations Short humeri, distal tapering Short fibulae Adult Newborn

19 HANDS AND FEET Metacarpophalangeal dislocations Interphalangeal dislocations Short metacarpals, metatarsals and distal phalanges Carpal fusions Accessory ossicles of hands, wrists, elbows, feet, knees Delayed coalescence of calcaneal ossification centers 4 years Control 11 years

20 Larsen Syndrome Cervical kyphosis instability Vertebral dislocation Scoliosis Vertebral fusions Segmentation defects Vertebral hypoplasia Platyspondyly Spondylolysis Dysraphism Adult 13 months Newborn 6 years

21 Mutations in FLNB Cause Larsen Syndrome G1834R** ABD H1 H2 CH1 CH F161C D190Y L234V E227K*** G361S G363E L delN G1586R* V1592D I1599F P1603L G1691S****** * Recurrent mutations

22 Lessons from Studies of FLNB The molecular pathology of FLNB Clinical markers for the diagnosis of Larsen syndrome. Frequency of complications in Larsen syndrome Farrington-Rock et al., (2006); Bicknell et al., (2006)

23 Larsen Syndrome Hands Bicknell et al., J Med Genet (2006)

24 Larsen syndrome Spinal Anomalies Bicknell et al., J Med Genet (2006)

25 Common features of Larsen Syndrome and Atelosteogenesis I, III, & BD Clinical Features: Facial dysmorphism - Prominent forehead - Hypertelorism - Depressed nasal bridge - Mid-face hypoplasia - Micrognathia Cleft palate Airway laxity Congenital joint dislocations Foot deformities Omphalocele (BD) Radiographic Features: Vertebral anomalies - Segmentation defects - Long Bone Hypoplasia - Fusions Clubbed humerus Short metacarpals

26 Atelosteogenesis I, III and Boomerang Dysplasia Autosomal Dominant Disorders Lethal Skeletal Dysplasia (Respiratory Distress) Clinical findings Craniofacial abnormalities Cleft palate Short limbs Abnormal hands Multiple dislocations Small chest Clubfoot

27 Joint Dislocation & Disordered Skeletogenesis in Atelosteogenesis AOI/Boomerang Atelosteogenesis I Boomerang Dysplasia Atelosteogenesis III AOIII

28 Mutations in FLNB Cause AOI/AOIII, & BD ABD CH1 CH H1 H2 24 W148R G168S*** L171Q L171R A173V G181V C183W S188P A201V M202V* Q203P G210V S229P S235P G751R delinslh;dg* 1583delD S1602P G1612D A1643S G1691S * Recurrent mutation

29 Germline and Somatic Mosaicism for FLNB Mutations Recurrence of characteristic Larsen syndrome in siblings of apparently unaffected parents Asymmetrical Larsen syndrome has been reported in two cases suggesting somatic mosaicism Somatic mosaicism suggested by the presence of more severe findings in the offspring of a LS syndrome parent, whose child had clinical features of Atelosteogenesis

30 Germline Mosaicism Initially reported as recessive inheritance, but germline mosaicism suggested due to recurrence of Larsen syndrome in the offspring of an affected individual

31 Somatic Mosaicism Somatic mosaicism was postulated due to the presence of more severe findings in the offspring of a mildly affected parent. I 1 2 II 1

32 Summary of FLNB Mutations Mutations in FLNB cause a spectrum of skeletal dysplasias AR SCT is produced by the nonsense mutations in FLNB - filamin B-less disorder LS, AOI, AOIII & BD are caused by missense mutations in FLNB predominantly clustered in CHD2 and surrounding the first hinge region (H1) Larsen syndrome Atelosteogenesis 3 Atelosteogenesis 1 Boomerang Increasing Phenotypic Severity

33 Filamin B Expression (E14)

34 Red -FLNB Green- FLNA Filamin B Localizes to the Cleavage Furrow of Proliferating Chondrocytes

35 Filamin B expression at E10.5 Flnb+/+ Flnb+/-

36 FlnB +/- mouse at E12.5 Control ß-gal stained embryo

37 E16 filamin B +/-

38 E12.5 filamin B +/-

39 E14.5 flnb -/- axial skeleton Alcian blue flnb-lacz

40 Filamin b Expression Alcian blue flnb-lacz 2 3 c c 4 5 E14.5

41 Newborn Flnb-/- mice smaller than their littermates Flnb +/+ Flnb+/- Flnb-/- Flnb -/-

42 Birth Weights of RRF239 Mice Weight (g) Fln B +/+ Fln B +/- Fln B -/- Genotype

43 Flnb-/- mice smaller than their littermates Weight (g) Flnb+/+ n=9 Flnb+/- n=12 Flnb-/- n= Postnatal Day

44 pines from skeletal preparations of RRF239 P1 Mice FlnB +/- FlnB -/- Cervical Thoracic

45 P60 Flnb-/- mice; Sternal Fusions Flnb+/+ Flnb-/-

46 P60 Flnb-/- mice Vertebral Fusions; cervical, thoracic and lumbar Flnb+/+ Flnb-/- Flnb-/- Flnb+/+

47 Micro-CT Analysis of P1 Flnb-/- mice SCANCO Medical AG Flnb-/- Flnb+/-

48 Flnb -/- p60 Micro-CT 5mm

49 Flnb -/- Carpal Fusions WT flnb -/- 2 3 c 4/5 4/5 E15.5

50 P60 Flnb-/- mice - Carpal Fusions Flnb+/+ 1mm Flnb-/-

51 E15.5 Etiology of the Vertebral Fusions Alcian blue E14.5 flnb-lacz WT flnb -/-

52 Flnb -/- : Altered BMP signaling BMP Chondrogenesis? Loss of boundary limits? Change in cell fate TGFb Joint fusions

53 Summary Filamin B -/- mouse phenocopies the AR human disorder SCT While previously considered to be a ubiquitously expressed protein, its expression pattern is highly restricted to the developing skeleton The absence of filamin B leads to a progressive vertebral and carpal fusion disorder One mechanism may be due to enhanced intrinsic ability to respond to BMP signaling and a diminished ability to respond to TGFb signaling Potential further mechanism of disease maybe that filamin B is expressed in cells destined to represent the vertebral joint structures and there has been a change in cell fate (or boundary limits) due to changes in signaling pathways

54 Dan Cohn Amy Merrill-Brugger Claire Farrington-Rock Natalia Camacho Vince Funari Matt Rock Stuart Tompson Marc Firestein Ralph Lachman David Rimoin BayGenomics Skeletal Dysplasia Research Group Clinical Collaborators Supported by NIH HD2267