Osteogenesis imperfecta

Genetic aspect

• 6-7/1,00,000 people world wide• 4-5 / 1,00,000 people type I – IV• The classification has been the subject of

debate• Right from Sillence1979 classification to

Shapiro 1985• Gloriuex – expanded Sillence classification

2004.

• Traditional belief – OI is Autosomal Dominant

• But recessive forms of OI have been identified.

• Mutations were initially noted only in COL1A1 and COL1A2

• Now recessive forms with CTRAP, LEPRE and PPIB identified

Pathology

• Type I collagen abnormality• Autosomal dominant except type

VII,VIII,XI• Pro alpha1 & alpha 2• Complete lack of type I collagen • Substitution of glycine residue with a bulky

side chain

Collagen synthesis

Shapiro Type Fracture RadiologyCongenita A Fracture at birth Bones radiologically

normal

Congenita B Fractures at birth Bones radiologically normal

Tarda A Fractures at or before walking age

Bone radiologically narrow and osteopenic

Tarda B Fractures at walking age Bones radiologically normal

Sillence Class Inheritance FeaturesI A AD Normal stature, blue

sclerae

I B AD above + DI

II AR Perinatally lethal, crumpled femora

III A AR Multiple fractures at birthProgressively deformingNormal sclerae

III B AR Above + DI

IV A AD Bone fragility Normal sclerae

IV B AD Above + DI

• Type I collagen – bone, skin, tendon, ligaments, blood vessels and cornea.

• Gly- X- Y • Two identical alpha 1 chains and one

alpha 2 chain• Glycine smallest amino acid

Current management

• Orthopedic managementOrthopedic management• Prevention of fractures and deformity• Correction of deformity• Rehabilitation• Improving the functional ability

Pharmacological management

• Bisphosphonate therapy• Bone mineral density• Fracture rates• Increased vertebral height

Cell based therapy

• Bone marrow transplantation• Increased body length• 45-77% increase in BMD• Decreased # rates

Horwitz EM et al. Clinical responses to bone Horwitz EM et al. Clinical responses to bone marrowmarrow

transplantation in children with severe transplantation in children with severe osteogenesisosteogenesis

imperfecta. Blood 2001; 97: 1227–1231.imperfecta. Blood 2001; 97: 1227–1231.

Gene supplementation

• mutation independent approach • targeting polymorphic sites of procollagen

genes• Supplement with collagen gene• Over express the normal collagen gene

Ex vivo gene herapy• Oim stromal cells transduced with

adenoviral vector- procollagen alpha 2(I) • Transduced cells produced 2:1 ratio alpha 1

: 2• Direct injection of vector in to skin and

femur of oim • High expression of collagen transgene in

skin and femur• Systemic transfusion of transduced human

mesenchymal stem cells with therapeutic genes

Gene targeting to bone

• Reporter gene under control of ostecalcin in MSC

• Tissue specific expression • Osteoblasts and osteocytes• Tissue specific promotors

• Dominant negative mutation – silence the abnormal gene without suppressing the normal one

Collagen biochemical testing

• Skin biopsy• Dermal fibroblasts – collagen from skin

cells• Sensitivity 90% in non lethal forms• Sensitivity 98% - lethal forms

Collagen molecular testing

• DNA based Gene sequence analysis • Blood sample• COL1A1• COL1A2• Sensitivity 95%

Recessive mutation analysis

• CRTAP• LEPRE 1(P3H1) – 1p34.1• Skin biopsy

LEPRE-1(1p34.1)

Type VIII osteogenisis imperfecta leprecanFailure of prolene 3- hydroxylation (prolyl 3-hydroxylase 1)Abnormal collagen folding

CTRAP ( cartilage associated protein) 3p22.3

• Works with leprecan and cyclophilin B• Modifies proline in collagen molecule• Proline 3- hydroxylation needed for normal

folding of the collagen• Type VII OI

Molecular Location on chromosome 3: base pairs 33,155,449 to 33,189,264

CTRAP –related OItype II/III/VII

• Analysis of entire coding region – sequence analysis or mutation analysis

• Prenatal diagnosis • Carrier testing

LEPRE1- OItype II/III/VIII

• Analysis of entire coding region – sequence analysis or mutation analysis

• Prenatal diagnosis • Carrier testing

COL1A1

• Pro aplha 1• Ehlers Danlos syndrome- arginine -

>cystine• Interferes with collagen building proteins • Type I OI – reduced production• Type II,III,IV- abnormally shortened non

functional collagen• Altered C terminus

COL1A1- 17q21.33

Increased risk of Osteoporosis – polymorphism

Infantile cortical hyperostosis

Arginine to cystine

•Pro alpha 2 chain•Segment deletion of alpha 2 cahin•Mutation in both copies – typical EDS•Severe forms of OI type II/III/IV

COL1A2 (7q 22.1)

OI COL1A2

• Deletions• Altered sequence ( change of Gly-)• Substitution – c terminus• EDS + OI duplicationDeletionsAbnormally short version pro alpha 2

COL1A1/2- related OI type I/II/III/IV

• Analysis of the entire coding region• Sequence analysis of select exons• Analysis of the entire coding region-

mutation scanning• Linkage analysis• Deletion‘/ duplication analysis• Prenatal diagnosis

OIType I – V ADType VII,III ( rarely) – AR 60 % mild OI - denovo mutation 100 % lethal OI – denovo mutationAD cases – 50% chance inheritanceProband cultured cells expresses – abnormal

collagen Chorionic villus sampling – 10-12 wks – at risk

pregnancy

High risk pregnancy

• Molecular genetic testing of COL1A1, COL1A2

• Prenatal USG – highly specialized centers• Lethal forms identified < 20 wks

Type VI – mode of inheritance not known Defective mineralization, Fish scale appearance of iliac crest under

polarized light Type V hypertrophic callus Type VI/ VII – rhizomelic shortening Type V & Vi has not been mapped.

Clinical testing

• Sequence analysis – c DNA - coding sequence mutations

• Genomic DNA – m RNA sequence alteration/ stability.

• RNA for cDNA – dermal fibroblasts• DNA isolated from any tissue

Genetic testing

Mutations detected

Mutation detection rate

cDNA sequence analysis

Missense mutation, small deletions/ insertiions,exon skipping mutations

OI type I 98%OI type II 98%OI type III 60-70%Oi type IV 70%

Genomic DNA analysis

Non sense mutations Missense mutationsSplice site mutations

Interpretation of results

• Analysis of genomic DNA may miss instances of whole gene deletion unless attention is paid to the state of common polymorphic nucleotides

• Sequence variants of primers – monoallelic amplification( small deletions missed)

• Failure to identify mutation,collagen quantity,structure- R/o OI

• Exon skipping/ small deletions- characterize at genomic level to be precise of mutation

• Characterize genomic mutation to account for protein level abnormality

Types of sequence alterations that may be detected

Pathogenic sequence alteration reported in the literature

Sequence alteration predicted to be pathogenic but not reported in the literature

Unknown sequence alteration of unpredictable clinical significance

Sequence alteration predicted to be benign but not reported in the literature

Benign sequence alteration reported in the literature

Possibilities if a sequence alteration is not detected

Patient does not have a mutation in the tested gene (e.g., a sequence alteration exists in another gene at another locus)

Patient has a sequence alteration that cannot be detected by sequence analysis (e.g., a large deletion, a splice site deletion)

Patient has a sequence alteration in a region of the gene (e.g., an intron or regulatory region) not covered by the laboratory's test

Testing strategy for proband

• The sensitivity of molecular genetic testing = sensitivity of structural/quantitative analysis

OI type I

• Premature termination of codon• Frame shift mutations• Splice mutations• Decreased production• Type Ib – altered sequence of type I

collagen

OI type II,III,IV• Pro aplha 1 or 2 mutations• Substitution of glycine in the triple helical

domain with serine , arginine, typtophan, cysteine – first portion of glycine codon

• Exon skipping beyond exon 14 in pro alpha 1 and beyond exon 25 in pro alpha 2 – lethal

• Upstream mutation – laxity • Mutation in carboxy terminal affects chain

association

• Pro alpha substitution - arginine, valine, glutamic acid, aspartic acid, and tryptophan at carboxy terminal – 70 % lethal

• Much more variability with glycine residue mutations in pro alpha 2 chain

Mosaicism

• AD mutations noted in OI II/III/IV• Mosaicism for non lethal mutations – no

phenotypic feature of OI• Mosaicism for lethal forms - mild OI –

even if majority of the somatic cells carry mutation

Penetrance

• AD OI – heterozygous individuals• 100% penetrance• Variable expressivity

Risk to family members AD

• Parents of proband• Milder forms of OI – affected parent +• Denovo mutations severity• Recommendations

• Proband with apparent denovo mutation – clinical examination of parents

• Mutation in proband identified =Mol. Gen . Test

Sibs of proband

• 50 % if parents of proband +• 5% if parents of proband – because of

mosaicism• Offspring of proband – 50%• Other members- if parents affected then at

risk

Risk to family members

• Parents of proband obligate heterozygotes• Heterozygotes ( carriers) asymptomatic• Sibs of proband – 25% affected , 50 %

asymptomatic carrier, 25% unaffected• Risk of unaffectd sib being a carrier is 2/3• Offspring – obligate carriers• Sibs of probands parents have 50%

chance of beign carriers

Carrier detection

• Not available for clinical testing

Genetic counselling

• Both parents have no e/o disease- denovo mutation

• One parent has germ line mosaicism• Undisclosed adoption/ alternate paternity

Prenatal test

• Amniocentesis – 15-18 wks of gestation (MGT)

• CVS – 12 wks ( MBT, BIOchem)• Done if disease causing allele of affected

family member has been done.• CVS – produce less type I procollagen –

biochem false +

• Inability to identify a mutation, however, does not eliminate the diagnosis of OI in the fetus.

Phenotype vs genotype

• Similar phenotype – deletions/duplications/of single aminoacids/ gly-x-y triplets or exon skipping events

• Mutations affectinf 5’ end affecting aminoterminal end – mild clinical phenotype.