Robert D. Blank

Collagen and Bone Strength

BONE STRENGTH IS a complex property which we understand in only a limited and incomplete fashion. Bone density and bone strength are highly but imperfectly correlated. The relative amounts and properties of the mineral (apatite) and the organic matrix (mainly type I collagen) in bone, as well as the organization of the bone at both the microscopic and macroscopic scales (microarchitecture and anatomy, respectively), determine its mechanical strength. That there is a strong genetic component to bone density and, more generally, to bone strength is well established, but we still know relatively little about the contributions of specific genes to bone mineral content and bone strength. As we discuss below, the evidence for the importance of various candidate genes is uneven in its quality but is compelling in the case of the type I collagen genes. Readers of this journal know that bone is a metabolically active tissue, with bone resorption and deposition proceeding as ongoing processes. Bone modeling in response to load, as described by Wolff, enables bones to adapt their material structures to optimize both their mechanical properties and their mass. Osteoporosis in many but not all settings, including those associated with estrogen insufficiency, steroid use, nutritional abnormalities, or excessive exercise, develops when these opposing processes are unbalanced. Whether the cause is osteoblastic insufficiency or osteoclastic overactivity, the result at the tissue level is always a loss of bone mass/density and deleterious changes in bone architecture. At the inorganic molecular level, the smaller mineral crystals disappear, and the larger more brittle crystals persist. There is some suggestion that collagen structure and properties may also be altered at the organic level. In an article by Puustjärvi et al. on page 321 of this Journal issue, the authors probe the mechanism of bone loss associated with heavy exercise. They report that longterm running significantly reduced bone density in beagle dog vertebrae without changing the bone’s mechanical (indentation) properties. The authors suggest that changes in the orientation of the collagen network, without changes in collagen maturation (cross-linking) and content (hydroxyproline per tissue), contributed to the maintenance of strength in the face of decreased mineral density. The purpose of this commentary is to review additional documentation for that view while indicating the limitations of the present study. First, we briefly review genotypephenotype associations of collagen. Next, the impact of collagen mutations on the microscopic structure of the connective tissue are considered. Then, we summarize what is known relating these microarchitectural changes to biomechanical tests. Finally, we consider how Puustjärvi et al.’s paper fits into this background.

Mouse chromosome 4.

This year’s report incorporates 78 new genetic markers into the consensus linkage map. Of these markers, ten have a known, mapped human homolog. The murine gene, followed by the human homolog and the human chromosomal location in parenthesis are as follows: Cpt2 4 CPT2 (1p32); Cyp2j5 and Cyp2j6 4 CYP2J2 (1p31.3-p31.2); Guca1b 4 GUCA2B (1p34-p33); Htr6 4 HTR6 (1p36-1p35); Hub 4 ELAVL2 (9p21); Hud 4 ELAVL4 (1p34); Matn1 4 MATN1 (1p35); Mmp16 4 MMP16 (8q21.3-q22.1); Tgfbr1 4 TGFBR1 (9q33-q34). In the case of the Cyp genes, two genes have been identified in mouse while only a single gene has been identified in humans. Mouse Chromosome (Chr) 4 shares significant stretches of linkage homology with human Chr 1, 6, 8, 9 and 21 (34245; 23572). The entire distal half of mouse Chr 4 is homologous with human Chr 1p. There have been four changes in nomenclature of genetic loci on Chr 4. Gene names and/or symbols that have been changed by the Nomenclature Committee include: Cerr1 changed to Cer1; Dana changed to D4H1s1733E; Zie changed to Gklf; and Etl2 changed to Il11ra2.

A novel locus for adolescent idiopathic scoliosis on chromosome 12p.

Adolescent idiopathic scoliosis (AIS) is a common disorder with strong evidence for genetic predisposition. Quantitative trait loci (QTLs) for AIS susceptibility have been identified on chromosomes. We performed a genome‐wide genetic linkage scan in seven multiplex families using 400 marker loci with a mean spacing of 8.6 cM. We used Genehunter Plus to generate linkage statistics, expressed as homogeneity (HLOD) scores, under dominant and recessive genetic models. We found a significant linkage signal on chromosome 12p, whose support interval extends from near 12pter, spanning approximately 10 million bases or 31 cM. Fine mapping within the region using 20 additional markers reveals maximum HLOD = 3.7 at 5 cM under a dominant inheritance model, and a split peak maximum HLOD = 3.2 at 8 and 18 cM under a recessive inheritance model. The linkage support interval contains 95 known genes. We found evidence suggestive of linkage on chromosomes 1, 6, 7, 8, and 14. This study is the first to find evidence of an AIS susceptibility locus on chromosome 12. Detection of AIS susceptibility QTLs on multiple chromosomes in this and other studies demonstrate that the condition is genetically heterogeneous.

Clinical Value of Monitoring BMD in Patients Treated With Bisphosphonates for Osteoporosis

Osteoporosis is a common disease with serious medical and economic consequences. Despite great efforts to educate healthcare professionals and the public, it remains underdiagnosed and undertreated. BMD testing by DXA is an extraordinarily useful clinical tool for assessment of fracture risk and to diagnose osteoporosis before the first fracture occurs. DXA is the only technology for measuring BMD that can be used with FRAX, the World Health Organization fracture risk assessment algorithm that is becoming widely used throughout the world. Several organizations recommend serial DXA testing for monitoring pharmacologic therapy of osteoporosis. The utility of BMD testing to monitor therapy was questioned almost a decade ago when the concept of ‘‘regression to the mean’’ was raised. Although this concept has relevance at a population level, it was subsequently refuted as misleading and irrelevant to the clinical management of individual patients. A study published recently in BMJ by Bell et al. raises the question anew. Despite the title, ‘‘Value of Routine Monitoring of Bone Mineral Density after Starting Bisphosphonate Treatment: Secondary Analysis of Trial Data,’’ the authors conclude that monitoring BMD ‘‘in postmenopausal women in the first three years after starting treatment with a potent bisphosphonate is unnecessary and may be misleading.’’ The authors go on to state that ‘‘routine monitoring should be avoided in this early period.’’ These conclusions are based on a secondary analysis of pooled data from the two arms of the Fracture Intervention Trial (FIT) in which postmenopausal women with low BMD were randomized to alendronate or placebo. They conclude that using BMD to monitor response to treatment with alendronate was of no value because (1) >97% of the patients on treatment ultimately showed an increase in BMD and (2) the withinsubject variability was considerable. Several of the same authors used a similar approach in a post hoc analysis of the Perindopril Protection Against Recurrent Stroke Study (PROGRESS) to conclude that monitoring the initial blood pressure response after perindopril (an angiotensinconverting enzyme inhibitor) therapy was unnecessary. The purpose of this commentary is to address issues raised by Bell et al. and to place the need for BMD monitoring into an appropriate clinical context. Whereas we applaud all efforts to apply the best available medical evidence to clinical decision-making, the validity and applicability of evidence should be closely scrutinized before making recommendations. The conclusion of the recent BMJ article, that monitoring therapy with BMD testing is unnecessary, rests on four assumptions: (1) the goal of monitoring is to document effectiveness by showing an increase in BMD, (2) the increase in BMD in virtually all treated subjects in FIT can be expected to occur in patients in clinical practice, (3) the response to one bisphosphonate in a clinical trial is indicative of the response to all bisphosphonates in clinical practice, and (4) within-person

Synteny‐defined candidate genes for congenital and idiopathic scoliosis

Idiopathic scoliosis (IS) is a common but poorly understood syndrome. Congenital scoliosis (CS) is less common but comparably unexplored. Previous studies suggest that each has a significant genetic component. However, the occurrence of scoliosis in the presence of other hereditary connective tissue syndromes raises the possibility that IS and CS are in fact a heterogeneous group of disorders with varied pathogenetic mechanisms. Mouse mutations have proven informative in identifying genes that are important in the development of the musculoskeletal system and provided important mechanistic insights regarding their roles in human disease. We sought to identify candidate genes for human IS and CS by reviewing mouse mutations with phenotypes affecting the axial skeleton. We performed a systematic review using the Mouse Genome Database (MGD), the Genome Database (GDB), and the Online Mendelian Inheritance in Man (OMIM) world-wide-web sites with additional searches performed based on the results of this initial search. We identified approximately 400 mouse mutations, reviewed approximately 250 of these for vertebral phenotypes, assessed 45 of these for synteny conservation between mouse and man, and identified 28 mouse mutations for which 29 credible candidates for human scoliosis could be identified based on mouse phenotypic and mapping data. For each of these, we have synthesized information about the mouse mutant phenotype, mapping data, information regarding molecular pathogenesis when a specific causative gene has been identified, and information regarding plausible candidates based on map position when the causative gene has not been identified. Among these were three loci for which the mutant gene had been identified and the human homologue was known. Some of the mouse mutants have phenotypes similar to human syndromes.

Bone Strength and Related Traits in HcB/Dem Recombinant Congenic Mice

Fracture susceptibility depends jointly on bone mineral content (BMC), gross bone anatomy, and bone microarchitecture and quality. Overall, it has been estimated that 50‐70% of bone strength is determined genetically. Because of the difficulty of performing studies of the genetics of bone strength in humans, we have used the HcB/Dem series of recombinant congenic (RC) mice to investigate this phenotype. We performed a comprehensive phenotypic analysis of the HcB/Dem strains including morphological analysis of long bones, measurement of ash percentage, and biomechanical testing. Body mass, ash percentage, and moment of inertia each correlated moderately but imperfectly with biomechanical performance. Several chromosome regions, on chromosomes 1, 2, 8, 10, 11, and 12, show sufficient evidence of linkage to warrant closer examination in further crosses. These studies support the view that mineral content, diaphyseal diameter, and additional nonmineral material properties contributing to overall bone strength are controlled by distinct sets of genes. Moreover, the mapping data are consistent with the existence of pleiotropic loci for bone strength‐related phenotypes. These findings show the importance of factors other than mineral content in determining skeletal performance and that these factors can be dissected genetically.

Interobserver Reproducibility of Criteria for Vertebral Body Exclusion

We studied reproducibility of the ISCD vertebral exclusion criteria among four interpreters. Surprisingly, agreement among interpreters was only moderate, because of differences in threshold for diagnosing focal structural defects and choice of which vertebra among a pair discordant for T‐score, area, or BMC to exclude. Our results suggest that reproducibility may be improved by specifically addressing the sources of interobserver disagreement.

An analysis of PAX1 in the development of vertebral malformations.

Due to the sporadic occurrence of congenital vertebral malformations, traditional linkage approaches to identify genes associated with human vertebral development are not possible. We therefore identified PAX1 as a candidate gene in vertebral malformations and congenital scoliosis due to its mutation in the undulated mouse. We performed DNA sequence analysis of the PAX1 gene in a series of 48 patients with congenital vertebral malformations, collectively spanning the entire vertebral column length. DNA sequence coding variants were identified in the heterozygous state in exon 4 in two male patients with thoracic vertebral malformations. One patient had T9 hypoplasia, T12 hemivertebrae and absent T10 pedicle, incomplete fusion of T7 posterior elements, ventricular septal defect, and polydactyly. This patient had a CCC (Pro)→CTC (Leu) change at amino acid 410. This variant was not observed in 180 chromosomes tested in the National Institute of Environmental Health Sciences (NIEHS) single nucleotide polymorphism (SNP) database and occurred at a frequency of 0.3% in a diversity panel of 1066 human samples. The second patient had a T11 wedge vertebra and a missense mutation at amino acid 413 corresponding to CCA (Pro)→CTA (Leu). This particular variant has been reported to occur in one of 164 chromosomes in the NIEHS SNP database and was found to occur with a similar frequency of 0.8% in a diversity panel of 1066 human samples. Although each patients mother was clinically asymptomatic and heterozygous for the respective variant allele, the possibility that these sequence variants have clinical significance is not excluded.

A Missense T(Brachyury) Mutation Contributes to Vertebral Malformations

No major susceptibility genes for sporadically occurring congenital vertebral malformations (CVM) in humans have been identified to date. Body patterning genes whose mutants cause axial skeletal anomalies in mice are candidates for human CVM susceptibility. T (also known as Brachyury) and TBX6 are critical genes needed to establish mesodermal identity. We hypothesized that mutations in T and/or TBX6 contribute to the pathogenesis of human CVMs. The complete T and TBX6 coding regions, splice junctions, and proximal 500 bp of the promoters were sequenced in 50 phenotyped patients with CVM. Three unrelated patients with sacral agenesis, Klippel‐Feil syndrome, and multiple cervical and thoracic vertebral malformations were heterozygous for a c.1013C>T substitution, resulting in a predicted Ala338Val missense alteration in exon 8. A clinically unaffected parent of each patient also harbored the substitution, but the variant did not occur in an ethnically diverse, 443‐person reference population. The c.1013C>T variant is significantly associated with CVM (p < 0.001). Alanine 338 shows moderate conservation across species, and valine at this position has not been reported in any species. A fourth patient harbored a c.908–8C>T variant in intron 7. This previously unreported variant was tested in 347 normal control subjects, and 11 heterozygotes and 2 T/T individuals were found. No TBX6 variants were identified. We infer that the c.1013C>T substitution is pathogenic and represents the first report of an association between a missense mutation in the T gene and the occurrence of sporadic CVMs in humans. It is uncertain whether the splice junction variant increases CVM risk. TBX6 mutations do not seem to be associated with CVM. We hypothesize that epistatic interactions between T and other developmental genes and the environment modulate the phenotypic consequences of T variants.

Clinical Presentations and RET Protooncogene Mutations in Seven Multiple Endocrine Neoplasia Type 2 Kindreds

Multiple endocrine neoplasia type 2 (MEN 2) is a group of related autosomal dominant cancer syndromes caused by mutations in the RET protooncogene. A subset of familial Hirschsprungs disease, aganglionic megacolon, is also caused by mutations in this gene.