P4HA1 mutations cause a unique congenital disorder of connective tissue involving tendon, bone, muscle and the eye.

Collagen prolyl 4-hydroxylases (C-P4Hs) play a central role in the formation and stabilization of the triple helical domain of collagens. P4HA1 encodes the catalytic α(I) subunit of the main C-P4H isoenzyme (C-P4H-I). We now report human bi-allelic P4HA1 mutations in a family with a congenital-onset disorder of connective tissue, manifesting as early-onset joint hypermobility, joint contractures, muscle weakness and bone dysplasia as well as high myopia, with evidence of clinical improvement of motor function over time in the surviving patient. Similar to P4ha1 null mice, which die prenatally, the muscle tissue from P1 and P2 was found to have reduced collagen IV immunoreactivity at the muscle basement membrane. Patients were compound heterozygous for frameshift and splice site mutations leading to reduced, but not absent, P4HA1 protein level and C-P4H activity in dermal fibroblasts compared to age-matched control samples. Differential scanning calorimetry revealed reduced thermal stability of collagen in patient-derived dermal fibroblasts versus age-matched control samples. Mutations affecting the family of C-P4Hs, and in particular C-P4H-I, should be considered in patients presenting with congenital connective tissue/myopathy overlap disorders with joint hypermobility, contractures, mild skeletal dysplasia and high myopia.

Absence of the ER Cation Channel TMEM38B/TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta.

Recessive osteogenesis imperfecta (OI) is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP3R-mediated Ca2+ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca2+, associated with deficient store-operated calcium entry, although SERCA and IP3R have normal stability. Notably, steady state ER Ca2+ is unchanged in TRIC-B deficiency, supporting a role for TRIC-B in the kinetics of ER calcium depletion and recovery. The disturbed Ca2+ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca2+-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50-70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca2+ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes.

Genetic Defects in TAPT1 Disrupt Ciliogenesis and Cause a Complex Lethal Osteochondrodysplasia.

The evolutionarily conserved transmembrane anterior posterior transformation 1 protein, encoded by TAPT1, is involved in murine axial skeletal patterning, but its cellular function remains unknown. Our study demonstrates that TAPT1 mutations underlie a complex congenital syndrome, showing clinical overlap between lethal skeletal dysplasias and ciliopathies. This syndrome is characterized by fetal lethality, severe hypomineralization of the entire skeleton and intra-uterine fractures, and multiple congenital developmental anomalies affecting the brain, lungs, and kidneys. We establish that wild-type TAPT1 localizes to the centrosome and/or ciliary basal body, whereas defective TAPT1 mislocalizes to the cytoplasm and disrupts Golgi morphology and trafficking and normal primary cilium formation. Knockdown of tapt1b in zebrafish induces severe craniofacial cartilage malformations and delayed ossification, which is shown to be associated with aberrant differentiation of cranial neural crest cells.

Mutations in WNT1 cause different forms of bone fragility.

We report that hypofunctional alleles of WNT1 cause autosomal-recessive osteogenesis imperfecta, a congenital disorder characterized by reduced bone mass and recurrent fractures. In consanguineous families, we identified five homozygous mutations in WNT1: one frameshift mutation, two missense mutations, one splice-site mutation, and one nonsense mutation. In addition, in a family affected by dominantly inherited early-onset osteoporosis, a heterozygous WNT1 missense mutation was identified in affected individuals. Initial functional analysis revealed that altered WNT1 proteins fail to activate canonical LRP5-mediated WNT-regulated ß-catenin signaling. Furthermore, osteoblasts cultured in vitro showed enhanced Wnt1 expression with advancing differentiation, indicating a role of WNT1 in osteoblast function and bone development. Our finding that homozygous and heterozygous variants in WNT1 predispose to low-bone-mass phenotypes might advance the development of more effective therapeutic strategies for congenital forms of bone fragility, as well as for common forms of age-related osteoporosis.

Prolyl 3-hydroxylase 1 deficiency causes a recessive metabolic bone disorder resembling lethal/severe osteogenesis imperfecta.

A recessive form of severe osteogenesis imperfecta that is not caused by mutations in type I collagen has long been suspected. Mutations in human CRTAP (cartilage-associated protein) causing recessive bone disease have been reported. CRTAP forms a complex with cyclophilin B and prolyl 3-hydroxylase 1, which is encoded by LEPRE1 and hydroxylates one residue in type I collagen, alpha1(I)Pro986. We present the first five cases of a new recessive bone disorder resulting from null LEPRE1 alleles; its phenotype overlaps with lethal/severe osteogenesis imperfecta but has distinctive features. Furthermore, a mutant allele from West Africa, also found in African Americans, occurs in four of five cases. All proband LEPRE1 mutations led to premature termination codons and minimal mRNA and protein. Proband collagen had minimal 3-hydroxylation of alpha1(I)Pro986 but excess lysyl hydroxylation and glycosylation along the collagen helix. Proband collagen secretion was moderately delayed, but total collagen secretion was increased. Prolyl 3-hydroxylase 1 is therefore crucial for bone development and collagen helix formation.

Kuskokwim syndrome, a recessive congenital contracture disorder, extends the phenotype of FKBP10 mutations.

Recessive mutations in FKBP10 at 17q21.2, encoding FKBP65, cause both osteogenesis imperfecta (OI) and Bruck syndrome (OI plus congenital contractures). Contractures are a variable manifestation of null/missense FKBP10 mutations. Kuskokwim syndrome (KS) is an autosomal recessive congenital contracture disorder found among Yup'ik Eskimos. Linkage mapping of KS to chromosome 17q21, together with contractures as a feature of FKBP10 mutations, made FKBP10 a candidate gene. We identified a homozygous three-nucleotide deletion in FKBP10 (c.877_879delTAC) in multiple Kuskokwim pedigrees; 3% of regional controls are carriers. The mutation deletes the highly conserved p.Tyr293 residue in FKBP65's third peptidyl-prolyl cis-trans isomerase domain. FKBP10 transcripts are normal, but mutant FKBP65 is destabilized to a residual 5%. Collagen synthesized by KS fibroblasts has substantially decreased hydroxylation of the telopeptide lysine crucial for collagen cross-linking, with 2%-10% hydroxylation in probands versus 60% in controls. Matrix deposited by KS fibroblasts has marked reduction in maturely cross-linked collagen. KS collagen is disorganized in matrix, and fibrils formed in vitro had subtle loosening of monomer packing. Our results imply that FKBP10 mutations affect collagen indirectly, by ablating FKBP65 support for collagen telopeptide hydroxylation by lysyl hydroxylase 2, thus decreasing collagen cross-links in tendon and bone matrix. FKBP10 mutations may also underlie other arthrogryposis syndromes.

Lack of cyclophilin B in osteogenesis imperfecta with normal collagen folding.

Osteogenesis imperfecta is a heritable disorder that causes bone fragility. Mutations in type I collagen result in autosomal dominant osteogenesis imperfecta, whereas mutations in either of two components of the collagen prolyl 3-hydroxylation complex (cartilage-associated protein [CRTAP] and prolyl 3-hydroxylase 1 [P3H1]) cause autosomal recessive osteogenesis imperfecta with rhizomelia (shortening of proximal segments of upper and lower limbs) and delayed collagen folding. We identified two siblings who had recessive osteogenesis imperfecta without rhizomelia. They had a homozygous start-codon mutation in the peptidyl-prolyl isomerase B gene (PPIB), which results in a lack of cyclophilin B (CyPB), the third component of the complex. The proband's collagen had normal collagen folding and normal prolyl 3-hydroxylation, suggesting that CyPB is not the exclusive peptidyl-prolyl cis-trans isomerase that catalyzes the rate-limiting step in collagen folding, as is currently thought.

Absence of FKBP10 in recessive type XI osteogenesis imperfecta leads to diminished collagen cross-linking and reduced collagen deposition in extracellular matrix.

Recessive osteogenesis imperfecta (OI) is caused by defects in genes whose products interact with type I collagen for modification and/or folding. We identified a Palestinian pedigree with moderate and lethal forms of recessive OI caused by mutations in FKBP10 or PPIB, which encode endoplasmic reticulum resident chaperone/isomerases FKBP65 and CyPB, respectively. In one pedigree branch, both parents carry a deletion in PPIB (c.563_566delACAG), causing lethal type IX OI in their two children. In another branch, a child with moderate type XI OI has a homozygous FKBP10 mutation (c.1271_1272delCCinsA). Proband FKBP10 transcripts are 4% of control and FKBP65 protein is absent from proband cells. Proband collagen electrophoresis reveals slight band broadening, compatible with ≈10% over-modification. Normal chain incorporation, helix folding, and collagen T(m) support a minimal general collagen chaperone role for FKBP65. However, there is a dramatic decrease in collagen deposited in culture despite normal collagen secretion. Mass spectrometry reveals absence of hydroxylation of the collagen telopeptide lysine involved in cross-linking, suggesting that FKBP65 is required for lysyl hydroxylase activity or access to type I collagen telopeptide lysines, perhaps through its function as a peptidylprolyl isomerase. Proband collagen to organics ratio in matrix is approximately 30% of normal in Raman spectra. Immunofluorescence shows sparse, disorganized collagen fibrils in proband matrix.

Prolyl 3-hydroxylase 1 and CRTAP are mutually stabilizing in the endoplasmic reticulum collagen prolyl 3-hydroxylation complex.

Null mutations in cartilage-associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1/LEPRE1) cause types VII and VIII OI, respectively, two novel recessive forms of osteogenesis imperfecta (OI) with severe to lethal bone dysplasia and overmodification of the type I collagen helical region. CRTAP and P3H1 form a complex with cyclophilin B (CyPB) in the endoplasmic reticulum (ER) which 3-hydroxylates the Pro986 residue of alpha1(I) and alpha1(II) collagen chains. We investigated the interaction of complex components in fibroblasts from types VII and VIII OI patients. Both CRTAP and P3H1 are absent or reduced on western blots and by immunofluorescence microscopy in cells containing null mutations in either gene. Levels of LEPRE1 or CRTAP transcripts, however, are normal in CRTAP- or LEPRE1-null cells, respectively. Stable transfection of a CRTAP or LEPRE1 expression construct into cells with null mutations for the transfected cDNA restored both CRTAP and P3H1 protein levels. Normalization of collagen helical modification in transfected CRTAP-null cells demonstrated that the restored proteins functioned effectively as a complex. These data indicate that CRTAP and P3H1 are mutually stabilized in the collagen prolyl 3-hydroxylation complex. CyPB levels were unaffected by mutations in either CRTAP or LEPRE1. Proteasomal inhibitors partially rescue P3H1 protein in CRTAP-null cells. In LEPRE1-null cells, secretion of CRTAP is increased compared with control cells and accounts for 15-20% of the decreased CRTAP detected in cells. Thus, mutual stabilization of P3H1 and CRTAP in the ER collagen modification complex is an underlying mechanism for the overlapping phenotype of types VII and VIII OI.

A founder mutation in LEPRE1 carried by 1.5% of West Africans and 0.4% of African Americans causes lethal recessive osteogenesis imperfecta.

PURPOSE: Deficiency of prolyl 3-hydroxylase 1, encoded by LEPRE1, causes recessive osteogenesis imperfecta (OI). We previously identified a LEPRE1 mutation exclusively in African Americans and contemporary West Africans. We hypothesized that this allele originated in West Africa and was introduced to the Americas with the Atlantic slave trade. We aimed to determine the frequency of carriers for this mutation among African Americans and West Africans, and the mutation origin and age. METHODS: Genomic DNA was screened for the mutation using PCR and restriction digestion, and a custom TaqMan genomic single-nucleotide polymorphism assay. The mutation age was estimated using microsatellites and short tandem repeats spanning 4.2 Mb surrounding LEPRE1 in probands and carriers. RESULTS: Approximately 0.4% (95% confidence interval: 0.22-0.68%) of Mid-Atlantic African Americans carry this mutation, estimating recessive OI in 1/260,000 births in this population. In Nigeria and Ghana, 1.48% (95% confidence interval: 0.95-2.30%) of unrelated individuals are heterozygous carriers, predicting that 1/18,260 births will be affected with recessive OI, equal to the incidence of de novo dominant OI. The mutation was not detected in Africans from surrounding countries. All carriers shared a haplotype of 63-770 Kb, consistent with a single founder for this mutation. Using linkage disequilibrium analysis, the mutation was estimated to have originated between 650 and 900 years before present (1100-1350 CE). CONCLUSION: We identified a West African founder mutation for recessive OI in LEPRE1. Nearly 1.5% of Ghanians and Nigerians are carriers. The estimated age of this allele is consistent with introduction to North America via the Atlantic slave trade (1501-1867 CE).

Dissecting the phenotypic variability of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heterogeneous family of collagen type I-related diseases characterized by bone fragility. OI is most commonly caused by single-nucleotide substitutions that replace glycine residues or exon splicing defects in the COL1A1 and COL1A2 genes that encode the α1(I) and α2(I) collagen chains. Mutant collagen is partially retained intracellularly, impairing cell homeostasis. Upon secretion, it assembles in disorganized fibrils, altering mineralization. OI is characterized by a wide range of clinical outcomes, even in the presence of identical sequence variants. Given the heterotrimeric nature of collagen I, its amino acid composition and the peculiarity of its folding, several causes may underlie the phenotypic variability of OI. A deep analysis of entries regarding glycine and splice site collagen substitution of the largest publicly available patient database reveals a higher risk of lethal phenotype for carriers of variants in α1(I) than in α2(I) chain. However, splice site variants are predominantly associated with lethal phenotype when they occur in COL1A2. In addition, lethality is increased when mutations occur in regions of importance for extracellular matrix interactions. Both extracellular and intracellular determinants of OI clinical severity are discussed in light of the findings from in vitro and in vivo OI models. Combined with meticulous tracking of clinical cases via a publicly available database, the available OI animal models have proven to be a unique tool to shed light on new modulators of phenotype determination for this rare heterogeneous disease.

Antagonism Between PEDF and TGF-ß Contributes to Type VI Osteogenesis Imperfecta Bone and Vascular Pathogenesis.

Osteogenesis imperfecta (OI) is a heterogeneous genetic disorder of bone and connective tissue, also known as brittle bone disease. Null mutations in SERPINF1, which encodes pigment epithelium-derived factor (PEDF), cause severe type VI OI, characterized by accumulation of unmineralized osteoid and a fish-scale pattern of bone lamellae. Although the potent anti-angiogenic activity of PEDF has been extensively studied, the disease mechanism of type VI OI is not well understood. Using Serpinf1(-/-) mice and primary osteoblasts, we demonstrate that loss of PEDF delays osteoblast maturation as well as extracellular matrix (ECM) mineralization. Barium sulfate perfusion reveals significantly increased vessel density in the tibial periosteum of Serpinf1(-/-) mouse compared with wild-type littermates. The increased bone vascularization in Serpinf1(-/-) mice correlated with increased number of CD31(+)/Endomucin(+) endothelial cells, which are involved in the coupling angiogenesis and osteogenesis. Global transcriptome analysis by RNA-Seq of Serpinf1(-/-) mouse osteoblasts reveals osteogenesis and angiogenesis as the biological processes most impacted by loss of PEDF. Intriguingly, TGF-ß signaling is activated in type VI OI cells, and Serpinf1(-/-) osteoblasts are more sensitive to TGF-ß stimulation than wild-type osteoblasts. TGF-ß stimulation and PEDF deficiency showed additive effects on transcription suppression of osteogenic markers and stimulation of pro-angiogenic factors. Furthermore, PEDF attenuated TGF-ß-induced expression of pro-angiogenic factors. These data suggest that functional antagonism between PEDF and TGF-ß pathways controls osteogenesis and bone vascularization and is implicated in type VI OI pathogenesis. This antagonism may be exploited in developing therapeutics for type VI OI utilizing PEDF and TGF-ß antibody. © 2022 American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

SMAD3 mutation in LDS3 causes bone fragility by impairing the TGF-ß pathway and enhancing osteoclastogenesis.

Loss-of-function mutations in SMAD3 cause Loeys-Dietz syndrome type 3 (LDS3), a rare autosomal-dominant connective tissue disorder characterized by vascular pathology and skeletal abnormalities. Dysregulation of TGF-ß/SMAD signaling is associated with abnormal skeletal features and bone fragility. To date, histomorphometric and ultrastructural characteristics of bone with SMAD3 mutations have not been reported in humans and the exact mechanism by which SMAD3 mutations cause the LDS3 phenotype is poorly understood. Here, we investigated bone histomorphometry and matrix mineralization in human bone with a SMAD3 mutation and explored the associated cellular defect in the TGF-ß/SMAD pathway in vitro. The index patient had recurrent fractures, mild facial dysmorphism, arachnodactyly, pectus excavatum, chest asymmetry and kyphoscoliosis. Bone histomorphometry revealed markedly reduced cortical thickness (-68 %), trabecular thickness (-32 %), bone formation rate (-50 %) and delayed mineralization. Quantitative backscattered electron imaging demonstrated undermineralized bone matrix with increased heterogeneity in mineralization. The patient's SMAD3 mutation (c.200 T > G; p.I67S), when expressed from plasmid vectors in HEK293 cells, showed reduced phosphorylation and transcription factor activity compared to normal control and SMAD3 (p.S264Y), a gain-of-function mutation, somatic mosaicism of which causes melorheostosis. Transfection study of the patients' SMAD3 (p.I67S) mutation displayed lower luciferase reporter activity than normal SMAD3 and reduced expression of TGF-ß signaling target genes. Patient fibroblasts also demonstrated impaired SMAD3 protein stability. Osteoclastogenic differentiation significantly increased and osteoclast-associated genes, including ACP5 (encoding TRAP), ATP6V0D2, and DCSTAMP, were up-regulated in CD14 (+) peripheral blood mononuclear cells (PBMCs) with the SMAD3 (p.I67S) mutation. Upregulation of osteoclastogenic genes was associated with decreased expression of TGF-ß signaling target genes. We conclude that bone with the SMAD3 (p.I67S) mutation features reduced bone formation, and our functional studies revealed decreased SMAD3 activation and protein stability as well as increased osteoclastogenesis. These findings enhance our understanding of the pathophysiology of LDS3 caused by SMAD3 mutations. Emerging therapies targeting in the TGF-ß/SMAD pathway also raise hope for treatment of LDS3.

COL1 C-propeptide cleavage site mutations cause high bone mass osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is most often caused by mutations in the type I procollagen genes (COL1A1/COL1A2). We identified two children with substitutions in the type I procollagen C-propeptide cleavage site, which disrupt a unique processing step in collagen maturation and define a novel phenotype within OI. The patients have mild OI caused by mutations in COL1A1 (Patient 1: p.Asp1219Asn) or COL1A2 (Patient 2: p.Ala1119Thr), respectively. Patient 1 L1-L4 DXA Z-score was +3.9 and pQCT vBMD was+3.1; Patient 2 had L1-L4 DXA Z-score of 0.0 and pQCT vBMD of -1.8. Patient BMD contrasts with radiographic osteopenia and histomorphometry without osteosclerosis. Mutant procollagen processing is impaired in pericellular and in vitro assays. Patient dermal collagen fibrils have irregular borders. Incorporation of pC-collagen into matrix leads to increased bone mineralization. FTIR imaging confirms elevated mineral/matrix ratios in both patients, along with increased collagen maturation in trabecular bone, compared to normal or OI controls. Bone mineralization density distribution revealed a marked shift toward increased mineralization density for both patients. Patient 1 has areas of higher and lower bone mineralization than controls; Patient 2's bone matrix has a mineral content exceeding even classical OI bone. These patients define a new phenotype of high BMD OI and demonstrate that procollagen C-propeptide cleavage is crucial to normal bone mineralization.

Null mutations in LEPRE1 and CRTAP cause severe recessive osteogenesis imperfecta.

Classical osteogenesis imperfecta (OI) is a dominant genetic disorder of connective tissue caused by mutations in either of the two genes encoding type I collagen, COL1A1 and COL1A2. Recent investigations, however, have generated a new paradigm for OI incorporating many of the prototypical features that distinguish dominant and recessive conditions, within a type I collagen framework. We and others have shown that the long-sought cause of the recessive form of OI, first postulated in the Sillence classification, lies in defects in the genes encoding cartilage-associated protein (CRTAP) or prolyl 3-hydroxylase 1 (P3H1/LEPRE1). Together with cyclophilin B (PPIB), CRTAP and P3H1 comprise the collagen prolyl 3-hydroxylation complex, which catalyzes a specific posttranslational modification of types I, II, and V collagen, and may act as a general chaperone. Patients with mutations in CRTAP or LEPRE1 have a lethal to severe osteochondrodystrophy that overlaps with Sillence types II and III OI but has distinctive features. Infants with recessive OI have white sclerae, undertubulation of the long bones, gracile ribs without beading, and a small to normal head circumference. Those who survive to childhood or the teen years have severe growth deficiency and extreme bone fragility. Most causative mutations result in null alleles, with the absence or severe reduction of gene transcripts and proteins. As expected, 3-hydroxylation of the Pro986 residue is absent or severly reduced, but bone severity and survival length do not correlate with the extent of residual hydroxylation. Surprisingly, the collagen produced by cells with an absence of Pro986 hydroxylation has helical overmodification by lysyl hydroxylase and prolyl 4-hydroxylase, indicating that the folding of the collagen helix has been substantially delayed.

Substitution of murine type I collagen A1 3-hydroxylation site alters matrix structure but does not recapitulate osteogenesis imperfecta bone dysplasia.

Null mutations in CRTAP or P3H1, encoding cartilage-associated protein and prolyl 3-hydroxylase 1, cause the severe bone dysplasias, types VII and VIII osteogenesis imperfecta. Lack of either protein prevents formation of the ER prolyl 3-hydroxylation complex, which catalyzes 3Hyp modification of types I and II collagen and also acts as a collagen chaperone. To clarify the role of the A1 3Hyp substrate site in recessive bone dysplasia, we generated knock-in mice with an α1(I)P986A substitution that cannot be 3-hydroxylated. Mutant mice have normal survival, growth, femoral breaking strength and mean bone mineralization. However, the bone collagen HP/LP crosslink ratio is nearly doubled in mutant mice, while collagen fibril diameter and bone yield energy are decreased. Thus, 3-hydroxylation of the A1 site α1(I)P986 affects collagen crosslinking and structural organization, but its absence does not directly cause recessive bone dysplasia. Our study suggests that the functions of the modification complex as a collagen chaperone are thus distinct from its role as prolyl 3-hydroxylase.

Bruck syndrome 2 variant lacking congenital contractures and involving a novel compound heterozygous PLOD2 mutation.

Bruck syndrome (BRKS) is the rare disorder that features congenital joint contractures often with pterygia and subsequent fractures, also known as osteogenesis imperfecta (OI) type XI (OMIM # 610968). Its two forms, BRKS1 (OMIM # 259450) and BRKS2 (OMIM # 609220), reflect autosomal recessive (AR) inheritance of FKBP10 and PLOD2 loss-of-function mutations, respectively. A 10-year-old girl was referred with blue sclera, osteopenia, poorly-healing fragility fractures, Wormian skull bones, cleft soft palate, congenital fusion of cervical vertebrae, progressive scoliosis, bell-shaped thorax, restrictive and reactive pulmonary disease, protrusio acetabuli, short stature, and additional dysmorphic features without joint contractures. Iliac crest biopsy after alendronate treatment that improved her bone density revealed low trabecular connectivity, abundant patchy osteoid, and active bone formation with widely-spaced tetracycline labels. Chromosome 22q11 deletion analysis for velocardiofacial syndrome, COL1A1 and COL1A2 sequencing for prevalent types of OI, and Sanger sequencing of LRP5, PPIB, FKBP10, and IFITM5 for rare pediatric osteoporoses were negative. Copy number microarray excluded a contiguous gene syndrome. Instead, exome sequencing revealed two missense variants in PLOD2 which encodes procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (lysyl hydroxylase 2, LH2); exon 8, c.797G>T, p.Gly266Val (paternal), and exon 12, c.1280A>G, p.Asn427Ser (maternal). In the Exome Aggregation Consortium (ExAC) database, low frequency (Gly266Val, 0.0000419) and absence (Asn427Ser) implicated both variants as mutations of PLOD2. The father, mother, and sister (who carried the exon 12 defect) were reportedly well with normal parental DXA findings. BRKS2, characterized by under-hydroxylation of type I collagen telopeptides compromising their crosslinking, has been reported in at least 16 probands/families. Most PLOD2 mutations involve exons 17-19 (of 20 total) encoding the C-terminal domain with LH activity. However, truncating defects (nonsense, frameshift, splice site mutations) are also found throughout PLOD2. In three reports, AR PLOD2 mutations are not associated with congenital contractures. Our patient's missense defects lie within the central domain of unknown function of PLOD2. In our patient, compound heterozygosity with PLOD2 mutations is associated with a clinical phenotype distinctive from classic BRKS2 indicating that when COL1A1 and COL1A2 mutation testing is negative for OI without congenital contractures or pterygia, atypical BRKS should be considered.

Deficiency of cartilage-associated protein in recessive lethal osteogenesis imperfecta.

Classic osteogenesis imperfecta, an autosomal dominant disorder associated with osteoporosis and bone fragility, is caused by mutations in the genes for type I collagen. A recessive form of the disorder has long been suspected. Since the loss of cartilage-associated protein (CRTAP), which is required for post-translational prolyl 3-hydroxylation of collagen, causes severe osteoporosis in mice, we investigated whether CRTAP deficiency is associated with recessive osteogenesis imperfecta. Three of 10 children with lethal or severe osteogenesis imperfecta, who did not have a primary collagen defect yet had excess post-translational modification of collagen, were found to have a recessive condition resulting in CRTAP deficiency, suggesting that prolyl 3-hydroxylation of type I collagen is important for bone formation.

COL1A1 C-propeptide mutations cause ER mislocalization of procollagen and impair C-terminal procollagen processing.

Mutations in the type I procollagen C-propeptide occur in ~6.5% of Osteogenesis Imperfecta (OI) patients. They are of special interest because this region of procollagen is involved in α chain selection and folding, but is processed prior to fibril assembly and is absent in mature collagen fibrils in tissue. We investigated the consequences of seven COL1A1 C-propeptide mutations for collagen biochemistry in comparison to three probands with classical glycine substitutions in the collagen helix near the C-propeptide and a normal control. Procollagens with C-propeptide defects showed the expected delayed chain incorporation, slow folding and overmodification. Immunofluorescence microscopy indicated that procollagen with C-propeptide defects was mislocalized to the ER lumen, in contrast to the ER membrane localization of normal procollagen and procollagen with helical substitutions. Notably, pericellular processing of procollagen with C-propeptide mutations was defective, with accumulation of pC-collagen and/or reduced production of mature collagen. In vitro cleavage assays with BMP-1 ±â€¯PCPE-1 confirmed impaired C-propeptide processing of procollagens containing mutant proα1(I) chains. Overmodified collagens were incorporated into the matrix in culture. Dermal fibrils showed alterations in average diameter and diameter variability and bone fibrils were disorganized. Altered ER-localization and reduced pericellular processing of defective C-propeptides are expected to contribute to abnormal osteoblast differentiation and matrix function, respectively.

Consortium for osteogenesis imperfecta mutations in the helical domain of type I collagen: regions rich in lethal mutations align with collagen binding sites for integrins and proteoglycans.

Osteogenesis imperfecta (OI) is a generalized disorder of connective tissue characterized by fragile bones and easy susceptibility to fracture. Most cases of OI are caused by mutations in type I collagen. We have identified and assembled structural mutations in type I collagen genes (COL1A1 and COL1A2, encoding the proalpha1(I) and proalpha2(I) chains, respectively) that result in OI. Quantitative defects causing type I OI were not included. Of these 832 independent mutations, 682 result in substitution for glycine residues in the triple helical domain of the encoded protein and 150 alter splice sites. Distinct genotype-phenotype relationships emerge for each chain. One-third of the mutations that result in glycine substitutions in alpha1(I) are lethal, especially when the substituting residues are charged or have a branched side chain. Substitutions in the first 200 residues are nonlethal and have variable outcome thereafter, unrelated to folding or helix stability domains. Two exclusively lethal regions (helix positions 691-823 and 910-964) align with major ligand binding regions (MLBRs), suggesting crucial interactions of collagen monomers or fibrils with integrins, matrix metalloproteinases (MMPs), fibronectin, and cartilage oligomeric matrix protein (COMP). Mutations in COL1A2 are predominantly nonlethal (80%). Lethal substitutions are located in eight regularly spaced clusters along the chain, supporting a regional model. The lethal regions align with proteoglycan binding sites along the fibril, suggesting a role in fibril-matrix interactions. Recurrences at the same site in alpha2(I) are generally concordant for outcome, unlike alpha1(I). Splice site mutations comprise 20% of helical mutations identified in OI patients, and may lead to exon skipping, intron inclusion, or the activation of cryptic splice sites. Splice site mutations in COL1A1 are rarely lethal; they often lead to frameshifts and the mild type I phenotype. In alpha2(I), lethal exon skipping events are located in the carboxyl half of the chain. Our data on genotype-phenotype relationships indicate that the two collagen chains play very different roles in matrix integrity and that phenotype depends on intracellular and extracellular events.