EMQN best practice guidelines for the laboratory diagnosis of osteogenesis imperfecta

Abstract

Osteogenesis imperfecta (OI) comprises a group of inherited disorders characterized by bone fragility and increased susceptibility to fractures. Historically, the laboratory confirmation of the diagnosis OI rested on cultured dermal fibroblasts to identify decreased or abnormal production of abnormal type I (pro)collagen molecules, measured by gel electrophoresis. With the discovery of COL1A1 and COL1A2 gene variants as a cause of OI, sequence analysis of these genes was added to the diagnostic process. Nowadays, OI is known to be genetically heterogeneous. About 90% of individuals with OI are heterozygous for causative variants in the COL1A1 and COL1A2 genes. The majority of remaining affected individuals have recessively inherited forms of OI with the causative variants in the more recently discovered genes CRTAP, FKBP10, LEPRE1,PLOD2, PPIB, SERPINF1, SERPINH1 and SP7, or in other yet undiscovered genes. These advances in the molecular genetic diagnosis of OI prompted us to develop new guidelines for molecular testing and reporting of results in which we take into account that testing is also used to 'exclude' OI when there is suspicion of non-accidental injury. Diagnostic flow, methods and reporting scenarios were discussed during an international workshop with 17 clinicians and scientists from 11 countries and converged in these best practice guidelines for the laboratory diagnosis of OI.

Figure 1

Critical steps in collagen type I biosynthesis and indication of genes known to be involved in OI. Type I collagen is the major structural protein in bone, tendon and ligament. It is first synthesized in the rough endoplasmic reticulum (rER) as type I procollagen, containing C- and N-terminal propeptides. In the rER, the two α1(I)-collagen chains encoded by COL1A1 and the one α2(I)-collagen chain encoded by COL1A2 comprising predominantly Gly-X-Y triplets, align and assemble in the C- to-N direction to form a triple helix. During folding, collagen is modified by, among others, specific enzymes that hydroxylate lysine and proline residues and glycosylate hydroxylysyl residues. This process is called post-translational modification and it stops when triple helix assembly is complete. The CRTAP/P3H1/CyPB complex encoded by the CRTAP, LEPRE1 and PPIB genes, is responsible for the 3-hydroxylation of P986 (p.P1164 counting from the methionine that initiates translation) but will most likely also act as a cis-trans isomerase and a molecular chaperone. FKBP65 encoded by FKBP10 also acts as a molecular chaperone for type I procollagen. The protein product of PLOD2 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2) hydroxylates telopeptide lysines in the rER. HSP47 encoded by SERPINH1 is thought to maintain the stability of the triple helix. After folding, the procollagen molecules are transported through the Golgi apparatus and plasma membrane (PM) into the extracellular matrix (ECM) where cleavage of the N-and C-terminal propeptides occurs and collagen molecules aggregate to form fibrils.

Figure 2

Preferred diagnostic flow in OI. The approach to diagnosis is designed to maximize the likelihood that causative variants will be identified in all affected individuals or assign those without causative variants to research pools. This flow assumes that the clinical diagnosis of OI is well established according to the traditional diagnostic criteria. With clear evidence of OI from radiological and clinical examination, further analysis should proceed according to the proposed strategy. Functional analysis consists of analysis of proteins and mRNA/cDNA from cultured fibroblasts and also includes COL1A1 null allele testing in certain selected cases.