The mode of Scheuermann’s disease inheritance and its phenotypic traits in probands and their relatives were studied in 90 pedigrees (90 probands and 385 relatives). The disorder was identified as a genetically related pathology inherited by autosomal dominant type, controlled by a mutant major gene, as a kyphotic deformity without signs of vertebral bodies’ anomaly and torsion. Morphological and biochemical studies showed disturbance in the structure of vertebral growth plate anterior aspects at the level of deformity, defects in proliferation and differentiation of chondrocytes, and change in proteoglycan spectrum in cells and matrix. Twelve candidate genes were studied in chondrocytes isolated from vertebral growth plates of patients with Scheuermann’s disease. The study results included disorder in the IHH gene expression and preservation of the expression of PAX1, two aggrecan isoforms, link protein, types I and II collagen, lumican, versican, growth hormone and growth factor receptor genes, and proliferation gene. Preservation of the SOX9 gene (transcription gene) probably indicates posttranscriptional genetic disorders. The study is under way.
The juvenile kyphotic deformity of the spine has been known since antiquity but was identified by Scheuermann as a nosological entity only in 1921. Scheuermann argued that this pathology was largely associated with aseptic necrosis of the ossification centers of the ring apophyses. This theory was further supported by Jones and Wise [
There are reports of aggregations of Scheuermann’s disease in families and a number of reports suggesting a hereditary character of this disorder [
However, Scheuermann’s disease is still generally considered as a hereditary disorder of unknown etiology [
Our objective was to study, in correctly ascertained pedigrees, the mode of inheritance and to identify hereditary phenotypic traits of the disorder and candidate genes.
Ninety probands age 9–18 years with Scheuermann’s disease and their 385 relatives underwent a clinical genetic examination. They were classified into three groups. Children and adolescents with a verified diagnosis of Scheuermann’s disease: 110 individuals (probands and sibs), age 9–18 years, 62 were boys (56.4%) and 48 were girls (43.6%), the male and female ratio was 1.3 : 1, Grade I 12.7%, Grade II—in 66.4%, Grade III—in 20.9%. Adults with a verified diagnosis of Scheuermann’s disease: 295 individuals, their age ranged from 21 to 69 years, male and female ratio was 1.36 : 1. Pedigrees of families with Scheuermann’s disease. Each pedigree included only one proband: 90 families, thirty-four pedigrees included 1st degree relatives (parents and sibs of the proband), fifty-six pedigrees had a more complex structure—they included 2nd and 3rd degrees relatives (grandmothers, grandfathers, aunts, uncles, and cousins).
In all 385 members of pedigrees have been examined.
All the probands and their relatives with Scheuermann’s disease admitted to specialized clinic underwent complete clinical examination including X-ray and MRI and survey by geneticist. The following characteristics were described: sex, age, kyphotic deformation degree and rigidity, and changes of vertebral body structure and shape. The diagnosis of Scheuermann’s disease was based on both clinical and radiographic signs. All cases with kyphotic deformity of 25–44° of Cobb angle were classified as Scheuermann’s disease Grade I, with 45–65° as Grade II and with 65° and more as Grade III. This classification was made with a regard for progressive structural changes of the spine column tissues.
a hypothesis of genetic control absence which assumes the equal transmission probabilities (environmental hypothesis) will be rejected; hypothesis with Mendelian probabilities and that with transmission probabilities will not differ significantly. Hypotheses were compared using the likelihood ratio test [
To enable an ultrastructural analysis, the material was fixed in 4% buffered paraformaldehyde, postfixed in 1% OsO4, dehydrated with alcohol in increasing concentrations, and embedded in Epon-Araldite. Ultrathin sections were prepared using an LKB Ultratome, counterstained, and examined under a Hitachi-600 electron microscope.
The GAG counts in vertebral body growth plates were derived from hexuronic acid content [
The GAGs’ composition in vertebral body GP was resolved by separation using gel electrophoresis in 1% agarose gel [
The level of
Primers and conditions for cartilage-related mesoderm marker RT-PCR.
Gene name | Primers name | Primer sequence | MgCl2 concentration, mM | Annealing temperature, °C | Target fragment size, bp |
---|---|---|---|---|---|
AGGRE | ACAN f |
5′-ATCGTCACCCCCGAGGAGCAG-3′ |
3 | 60 | 379 |
| |||||
LUM | LUM2 f |
5′-GTGACTGGGCTGGGTCTCCCC-3′ |
4 | 58 | 329 |
| |||||
HAPLN1 | HAPLN1 f |
5′-GGCAGAACACAGTGCCCGGAGTC-3′ |
4 | 58 | 317 |
| |||||
GHR | GHR f |
5′-GGCGAAGCTCGGAGGTCCTACA-3′ |
4 | 58 | 568 |
| |||||
TGFBR | TGFBR f |
5′-GGCGAGCGGTCTTGCCCATC-3′ |
4 | 58 | 468 |
| |||||
COL1A1 | COL1A1 f |
5′-ATCCAGCTGACCTTCCTGCG-3′ |
4 | 58 | 301 |
| |||||
COL2A1 | COL2A1 f |
5′-GAAACCATCAATGGTGGCTTCC-3′ |
4 | 58 | 322 |
| |||||
IHH3 | IHH3 f |
5′-ACCACATCAGACCGCGACCGCAAT-3′ |
4 | 58 | 441 |
| |||||
SOX9 | SOX9 f |
5′-GCGTCAACGGCTCCAGCAAGA-3′ |
4 | 58 | 369 |
| |||||
PAX1 | PAX1 f |
5′-ATCAGCCGCATCCTGCGCAACAA-3′ |
4 | 65 | 376 |
Grade I Scheuermann’s disease developed most often in children 9–11 years of age. In general, by the age 12–15 most patients had Grade II; by the age 16–18 they had Grade III Scheuermann’s disease. The incidence was 1.3 times more frequent in boys than in girls, which is in good agreement with the literature [
Scheuermann’s disease clinically manifests as a kyphotic deformity assessed radiographically by the Cobb method. Patients with Scheuermann’s disease Grade I had 25–44° of kyphosis with a curve apex at T7-T8. A kyphosis in Grade II patients amounted 45–65° with an apex also at T7-T8. Grade III curve exceeded 65 degrees, being markedly rigid. All grades showed various radiographic spine abnormalities with various incidences. There was no vertebral body torsion. Table
Radiographic characteristics of Scheuermann’s disease grades in probands.
Radiographic signs | Scheuermann’s disease grade | ||
---|---|---|---|
I ( |
II ( |
III ( |
|
Kyphosis (Cobb angle degrees) | 25–45 | 45–65 | >65 |
Number of segments involved | 3-4 | 4-5 | 5–7 |
| |||
Percentage of patients with a sign presence | |||
| |||
Endplate irregularities | 65 | 100 | 68 |
Wedge-shaped vertebral body, >5 degrees | 62 | 96 | 100 |
Schmorl’s nodules | 38 | 93 | 100 |
Vertebral body osteoporosis | 31 | 64 | 68 |
Narrowing of intervertebral space | 26 | 92 | 93 |
Apophysis fragmentation | 19 | 69 | 52 |
The mode of Scheuermann’s disease phenotypic trait inheritance was explored in detail in the 78 relatives of probands (45 males and 33 females), age 21–69, with a documented diagnosis (Table
Distribution of relatives of probands with Scheuermann’s disease depending on a kyphosis degree.
Relation degree | Total | Relatives with kyphosis (degrees) | |||||
---|---|---|---|---|---|---|---|
25–45 | 45–65 | >65 | |||||
|
% |
|
% |
|
% | ||
I–III | 78 (100%) | 41 | 52 | 31 | 40 | 6 | 8 |
I | 41 (153%) | 21 | 51 | 18 | 44 | 2 | 5 |
II | 23 (29%) | 14 | 61 | 6 | 26 | 3 | 13 |
III | 14 (18%) | 6 | 43 | 7 | 50 | 1 | 7 |
All members of this group had a marked rigid kyphotic deformity. The curve apex, as in probands, was at the T7-T8 in 80% of cases. Vertebral body changes had an equal incidence in relatives of all three degrees of relation (Table
Radiographic characteristics of relatives of probands with Scheuermann’s disease.
Radiographic signs | Relation degree | |||||||
---|---|---|---|---|---|---|---|---|
I–III ( |
I ( |
II ( |
III ( |
|||||
|
% |
|
% |
|
% |
|
% | |
Wedge-shaped vertebral body | 69 | 89 | 38 | 93 | 15 | 65 | 13 | 97 |
Platyspondylia | 9 | 12 | 2 | 56 | 8 | 35 | — | — |
Narrowing of intervertebral space | 72 | 92 | 38 | 93 | 23 | 100 | 13 | 92 |
Vertebral body osteoporosis | 63 | 81 | 33 | 80 | 23 | 100 | 9 | 66 |
Schmorl’s nodules | 33 | 42 | 15 | 37 | — | 12 | 13 | 93 |
Endplate irregulations | 2 | 3 | — | — | — | — | 2 | 14 |
Apophysis fragmentation | — | — | — | — | — | — | — | — |
Osteochondrosis signs | 61 | 78 | 33 | 80 | 20 | 87 | 8 | 55 |
Spondylosis | 44 | 56 | 25 | 61 | 7 | 30 | 9 | 66 |
Arthrosis | 26 | 33 | 12 | 29 | 19 | 83 | — | — |
As in the first group (children and adolescents with Scheuermann’s disease), this was associated with narrowing of intervertebral space (92%), but vertebral body osteoporosis was more frequent and prominent (81%), while Schmorl’s nodes manifested only in 42% of cases. Endplate irregularities were fewer (3%). There was no evidence of apophysis fragmentation. Such a distribution of radiographic signs is caused by the age structure of the group. It is remarkable that the examined relatives aged 40 and more had shown osteochondrosis (78%), spondylosis (56%), and arthrosis (33%) considered as secondary delayed changes.
The study of disorder inheritance mode was based on the 88 pedigrees ascertained through a proband with Scheuermann’s disease. The analysis has shown that Scheuermann’s disease frequency among the closest relatives of the probands was 0.143 (
Coexistence of idiopathic scoliosis and Scheuermann’s disease in families raises an issue of a single genetic nature of these pathologies: red: idiopathic scoliosis; blue: Scheuermann’s disease.
The test of possibility of monogene diallel control of Scheuermann’s disease was analysed by segregation analysis. Table
Segregation analysis of Scheuermann’s disease.
Genetic parameter | Classical Mendelian model | Dominant model | ||
---|---|---|---|---|
Obtained model of Mendelian transmission probabilities | Model of random transmission probabilities | Model of equal transmission probabilities | ||
|
0.136 | 0.136 |
0.134 | 0.421 |
|
0.0 | 0.0 | 0.0 | 0.008 |
|
1.0 | 1.0 | 1.0 | 0.981 |
|
1.0 | 1.0 | 1.0 | 0.981 |
|
0.0 | 0.0 | 0.0 | 0.073 |
|
0.434 | 0.432 |
0.433 | 0.321 |
|
0.419 | 0.432 | 0.433 | 0.321 |
|
1.0* | 1.0* | 1.0* | 0.699 |
|
0.5* | 0.5* | 0.496 | 0.699 |
|
0.0* | 0.0* | 0 | 0.699 |
LH | 160.285 | LH1 = 160.286 | LH2 = 160.285 | LH3 = 170.834 |
AIC | 334.570 | 330.572 | 336.570 | 353.668 |
The first column contains genetic parameters values for the model assuming that penetrances of all genotypes can take arbitrary values. The second column contains the parameters of dominant model assuming that
Thus, the results of clinical and genetic investigation suggest that Scheuermann’s disease is a genetically dependent pathology inherited by autosomal dominant type and controlled by a mutant major gene. Deformation manifests during the adolescent growth spurt and progresses from Grade I to Grade III. To ascertain a precise mode of inheritance we made attempts to identify gene candidates having been chosen on the basis of morphological, biochemical, and molecular genetic studies of spine structural components affected by Scheuermann’s disease.
The vertebral body growth plate (at the level of deformity) in the ventral aspects is significantly narrowed and is presented by a degraded, fragmented matrix which reveals disordered arrangement of chondrocytes (Figure
Vertebral growth plate at the level of deformity: (a) cell and matrix disorganization (GP ventral aspects); (b) structural organization of GP is preserved (GP dorsal aspects). Hematoxylin-eosin staining, ×200.
Histochemical study revealed trace reaction to low-polymeric chondroitin sulfate (Figure
Proteoglycans in cells and matrix of vertebral growth plate at the level of deformity: (a) ventral aspects; (b) dorsal aspects. Hale’s reaction, ×200.
This is in agreement with biochemical findings (Figure
Foregram of ventral and dorsal aspects of GP of patient with III Grade Scheuermann’s disease 1, 9: standard; 2: native GAG sample from the growth plate; 3, 4, and 5: GAG samples from the growth plate, annulus fibrosus, and nucleus pulposus, respectively, treated with keratanase; 6, 7, and 8: GAG samples from the growth plate, annulus fibrosus, and nucleus pulposus, respectively, treated with chondroitinase.
Ultrastructure of columnar chondrocytes in vertebral growth plate at the level of deformity: (a) ventral aspects; (b) dorsal aspects, ×5000.
Dorsal aspects are presented by cells of the columnar layer (Figure
Barrier-trophic function of collagen-bound proteoglycans: (a) chondron’s architectonics, columnar area of dorsal aspects. Semithin section. (b) Chondroblast is located in the center and surrounded by “free” and collagen-bound proteoglycans. Electron microscopy, ×5000.
In the vertebral body growth plates, GAG counts lowered from 20.00 in ventral aspects to 3.00
Pathogenetic mechanism of spinal deformity development is a growth asymmetry underlaid by disorders in chondroblast proliferation and differentiation and in structural organization of the growth plate, as well as changes in the proteoglycan spectrum.
Morphohistochemical and biochemical findings on proteoglycan spectrum alteration in ventral aspects of the growth plate—increase in keratin sulfate and decrease in chondroitin sulfate amount—gave a ground for investigation of the most representative proteoglycan gene in the growth plate—the aggrecan gene. It is known that aggrecan fulfills informational, barrier, and receptor functions and regulation of chondroblast differentiation and proliferation in the growth process [
It was emerged that aggrecan gene, isoform 1 and isoform 2, and lumican are gene expressed both in ventral and dorsal aspects of the growth plate (morphological and biochemical findings (Figure
RT-PCR analysis of gene expression: 1: specimen from the growth plate of a healthy child; 2–5: specimens from growth plates of children with Scheuermann’s disease; RT: negative control.
A probability of disorder in chondroblast receptor function was not excluded. This supposition was based on the absence of monolayer in cultured chondroblasts from growth plates of Scheuermann’s patients. We did not reveal any disorder in expression of gene receptor both to growth hormone and to transforming growth factor. Types 1 and 2 collagen genes were also expressed well.
Expression of the PAX1 gene typical only for fetal life or period of somite formation turned to be unusual. Expression of the IHH gene only in one studied case also remained unexplained, as well as proliferation gene expression in the absence of growth plate zonality. Proliferative activity is morphologically evident, though the process of columnar structure formation does not go on, but irregular proliferation is observed. Since transcription gene SOX9 is expressed in all cases, one can suppose that changes are caused by posttranslational disorders.
The paper summarizes longitudinal investigations of spinal deformities associated with Scheuermann’s disease. Morphological and biochemical findings are presented selectively as possible markers of genetic pathology. Major gene dependency of the Scheuermann’s disease was proved, and phenotypic criteria of inheritance were presented. Pathogenetic mechanisms of the disease development were identified: disorder in structural organization of ventral aspects of vertebral growth plates at the level of deformity, change in proteoglycan spectrum (keratin sulfate increase and chondroitin sulfate decrease). This study was followed up by identification of supposed candidate genes responsible for main structural transformations in a provisional cartilage.
The study showed disorder in expression of IHH and PAX1 genes the latter is normally expressed only during embryogenesis. Expression of two aggrecan gene isoforms, link protein, receptors to growth hormone and growth factor, and types I and II collagen was preserved. High proliferation gene expression against the background of disordered proliferative activity of chondrocytes in the ventral aspects of the growth plate remains unexplained. Expression of the SOX9 gene or transcription gene may be the evidence of disorder in regulation at a posttranslational level. These findings are now verified. The paper objective was to share the obtained results with colleagues and propose joint investigations.
The authors render heartful thanks to Professor P. M. Borodin, Professor O. L. Serov, and Professor S. M. Zakian for the afforded opportunity to work in the laboratory of the Institute of Cytology and Genetics (Siberian Branch of the Russian Academy of Sciences) and to Dr J. Fairbanks for his assistance in editing the English version of the paper.