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Various approaches have been employed to identify the genes involved in BMD variation: association studies, genome scans, and candidate gene linkage analyses.
Several association studies have been performed so far, focusing on functional candidate genes coding for structural bone proteins (COL1A1, osteocalcin), hormones (PTH), hormone receptors (ESR1, CALCR, VDR, PTHR1), and cytokines (IL-6, TGFb-1) involved in bone metabolism, as reviewed in Ralston SH, 2002 [36]. This approach often produced conflicting results in different populations (e.g. Caucasoid, Asiatic) and the results were unable to confirm common genetic factors.
Genomewide linkage approaches identified several chromosomal regions linked to BMD. A few of them have been confirmed also in independent studies [9, 12, 20, 22, 38, 42].
To date there are some large linkage or linkage and TDT studies on candidate genes and bone traits (1, 8, 11, 26, 35, 45, 46]. These studies investigated the role of numerous candidate genes (hormone receptors, collagen, cytokynes) in BMD variation. Suggestive linkage was described between PTHR1 (Parathyroid Hormone Receptor 1) and BMD [11]. Linkage and association were found with the osteocalcin gene [1, 8] and the VDR gene [8, 35] detected a marginally significant result for a within-family association between BMD and the ESR1 gene.
In the present study we performed a candidate gene linkage study and a family-based association study (TDT) investigating the involvement of Estrogen Receptor a (ESR1 located on chromosome 6q25.1), Collagen type I a1 (COL1A1 chr.17q21.31-q22), Calcitonin Receptor (CALCR chr. 7q21.3) and Vitamin D Receptor (VDR, chr.12q12-q14), genes as putative loci contributing to BMD variation at different sites (femur and lumbar spine) in a sample of 118 Italian families.
METHODS
Subjects
A total of 567 subjects (118 families) were recruited through an osteopenic/osteoporotic proband at the Valeggio s/Mincio Center for Bone Disorders. The sample consisted of 60 nuclear and 58 multiplex families, including 465 sib pairs. The average family size was 6.35 (distribution: 25.4% families of 4 individuals, 15.3% of 5 individuals and 13.6% of 8 individuals, the other families were smaller). All subjects gave informed consent for the study. A structured, self-managed questionnaire was utilized to obtain data on age, menopause and number of pregnancies, breast-feeding, fractures, exercise, smoking, alcohol intake, estrogens, and calcium supplement.
Height and weight were measured using a stadi-ometer and a calibrated balance-beam scale, respectively. The exclusion criteria were: present or past endocrine or metabolic bone diseases (primary hyperparathyroidism, hyperthyroidism, diabetes, anorexia, rheumatoid arthritis); treatment with thyroid hormones, cytostatic drugs, bisphosphonates, estrogens, calcitonin or corticosteroids; dietary calcium intake lower than 500 mg/day.
Bone densitometry
BMD was measured at the lumbar spine (L2-L4) and proximal femur (neck, Ward's triangle trochanteric and intertrochanteric region, total hip) by dualenergy X-ray absorptiometry (DEXA). The measurements were made using a Hologic QDR 2000 (4500) instrument (Hologic Inc. Waltham, MA U.S.A.).
Genotyping
For 562 individuals DNA was extracted from whole blood according to standard procedures (“salting out” method), and amplified by PCR with Euro-biotaq (Eurobio) using specific oligonucleotide primers for ESR1 PvuII-XbaI [21], COL1A1 Sp1 [31], CALCR C1377T [33], and VDR-FokI [13] polymorphisms. The amplified fragments were subsequently digested with the appropriate restriction enzyme (purchased by New England Biolabs): XbaI and PvuII for ESRI, ApaI for COL1A1 Sp1, AluI for CALCR, FokI for VDR, respectively. The digestion products were resolved on 3% agarose gels (ESR1 XbaI and PvuII, VDR FokI, and CALCR AluI) or 6% acrylamide gels (COL1A1 Sp1), and stained with ethidium bromide.
Statistical analyses
Hardy-Weinberg equilibrium was performed on family founders for each polymorphism by the X2 test. Mendelian inheritance of all the marker alleles within each family was checked by the computer program MERLIN available at
http: //www .sph.umich.edu/csg/abecasis/Merlin/. The most likely haplotype in each individual was constructed and selected by MERLIN. Linkage Disequilibrium between ESR1 PvuII and XbaI alleles was assessed evaluating the expected compared to the estimated genotype distributions by the X2 test. Quantitative linkage was computed by variance component analysis implemented in MERLIN. Both quantitative and qualitative analyses were performed. For the qualitative analyses all the subjects were arranged into two classes, affected and non-affected, according to the BMD value. The following two cut off BMD values were chosen: Z<-1 for cut off 1, Z<-
1.5 for cut off 2. Family-based association between the genetic markers and the bone phenotype was tested with the UNPHASED package (http: //www .hgmp.mrc.ac.uk/~fdudbrid/ software/unp hased/).
RESULTS
Descriptive statistics for BMD at hip and spine, and covariates as age and BMI are reported in Tab. 1.
Allele frequencies were estimated for the five polymorphisms (ESR1 PvuII, ESR1 XbaI, COL1A1 Sp1, CALCR C1377T, VDR FokI) on the 116 family founder members (Tab. 2). The genotype frequencies
of each polymorphism were in Hardy-Weinberg equilibrium.
ESR1 PvuII and XbaI polymorphisms showed a strong linkage disequilibrium (Tab. 3), as previously described [2]. The PvuII/XbaI T/G haplotype (expected frequency 0.2) was not detected in the estimated haplotypes.
Table 1
Descriptive statistic for the 567 subjects in 118 families
Phenotype Probands non Probands
Age (yrs) 57 ±14 50 ± 17
BMI (Kg/m2) 24 ± 3.6 25 ± 4
BMD Ward’s triangle (g/cm2) 0.43 ± 0.12 0.59 ± 0.16
BMD trochanter (g/cm2) 0.56 ± 0.37 0.7 ± 0.32
BMD total hip (g/cm2) 0.7 ± 0.12 0.86 ± 0.15
BMD femoral neck (g/cm2) 0.6 ± 0.97 0.75 ± 0.14
BMD intertrochanter (g/cm2) 0.82 ± 0.14 0.99 ± 0.17
BMD lumbar spine (g/cm2) 0.74 ± 0.122 0.9 ± 0.16
Ward’s triangle Z score -1 ± 0.97 -0.18 ± 1
trochanter Z score -1.3 ± 1 -0.36 ±1.1
total hip Z score -1.4 ± 0.9 -0.52 ± 1
femoral neck Z score -1.4 ± 0.9 -0.54 ± 1.1
intertrochanter Z score -1.5 ± 0.9 -0.62 ± 1
lumbar spine Z score -1.8 ± 1.2 -0.96 ± 1.2
Anthropometric characteristics of the subjects enrolled in the study.
BMD is reported both as g/cm2 and as Z score.
Table 2
Allele frequencies calculated on 116 founders
Polymorphism polymorphic nucleotide Conventional annotation Observed frequency
CALCR C1377T C T 0.26 0.74
G S 0 8
COL1A1 Sp1 T s 0.2
ESR1 PvuII C P 0.36
T p 0.64
ESR1 XbaI G X 0.31
A x 0.69
VDR FokI C F 0.6
T f 0.4
Allele frequencies were calculated on unrelated subjects for CALCR, COL1A1, ESR1, and VDR polymorphisms.
The annotation used in the literature to indicate the allele variant is reported together with the dimorphic nucleotide.
Table 3
Linkage Disequilibrium of ESR1 PvuII and XbaI
Haplotype (PvuII/XbaI) expected frequency estimated frequency
T/A p/x 0.45 0.65
C/G P/X 0.11 0.31
C/A P/x 0.24 0.034
T/G p/X 0.2 0
DI=1 according to Lewontin 1964 R2=0.78 according to Devlin and Risch 1995
Linkage analysis
A variance component analysis was performed. Both single and multipoint linkage analyses were carried out. A potential linkage of ESR1 PvuII/XbaI haplotype with various femoral sites was observed (intertrochanter and femoral neck p=0.05, Ward’s triangle p=0.03) (Tab. 4). ESR1 showed the highest LOD values with multipoint analysis. No significant results were found for the COL1A1, CALCR or VDR polymorphisms tested.
Transmission Disequilibrium Test
ESR1 polymorphisms were analyzed separately or in haplotype combination.
At first a quantitative TDT was performed, assuming BMD as a continuous phenotype. No significant results were observed for any of the examined polymorphisms.
Subsequently a qualitative TDT was performed according to a BMD cut off value (Z score<-1 or Z score<-1.5). Under these settings, a distortion was observed for ESR1 PvuII/XbaI haplotype transmission (Tab. 5). ESR1 PvuII/XbaI T/A haplotype was consistently underepresented in affected sibs at lumbar spine and several femoral sites for both cut off values of BMD. C/G and/or C/A haplotypes showed an association with low BMD at lumbar spine and femur.
TDT also showed preferential transmission in affected subjects of COL1A1 Sp1 T allele at Ward’s triangle (Tab. 5).
DISCUSSION
The strong, polygenic component of a complex trait such as osteoporosis is quite evident. Twin and family studies [14, 18, 37] have shown that genetic factors play an important role in regulating Bone Mineral Density (BMD), the best known predictor of osteoporosis, with an estimated heritability of up to 84%. Different approaches have been employed in order to identify the genes involved in BMD variation. Complex segregation analyses and genome screens suggested that several genetic factors are associated to BMD or related phenotypes.
Loci
We report a linkage and association study performed with polymorphic markers of four candidate genes for bone mineral density: ESR1 (LocusLink ID: 2099), COL1A1 (LocusLink ID: 1277), CALCR
(LocusLink ID: 799) and VDR (LocusLink ID: 7421). These genes have been extensively investigated by population association studies for their role in bone metabolism. Estrogen Receptor a is predominantly expressed in cortical bone osteoblasts, osteo-cytes and osteoclasts [3] and estrogen deficiency in aging women results in postmenopausal acceleration of bone loss.
COL1A1 codes for a1 chains of type I collagen, the most abundant component of bone organic matrix and mutations in this gene cause a monogenic disorder, Osteogenesis Imperfecta, characterized by osteopenia and bone fragility [7]. A G/T variant at Sp1 binding site in intron 1 has been associated to poor bone quality and reduced bone strength [27].
Calcitonin Receptor once bound to its ligand inhibits osteoclastic bone resorption [34].
Vitamin D by interacting with its receptor induces calcemic and phosphatemic effects leading to normal bone mineralization and remodeling [16].
Study approach
Association studies are very popular in the osteoporosis field but they often lead to conflicting results, presumably because of exposure to different environmental factors, false positive outcomes, population admixture and/or linkage disequilibrium of the polymorphic markers with different causative alleles in various populations. Transmission Disequilibrium Test (TDT) circumvents population stratification/admixture by testing association within families.
To date there are a few linkage studies of the candidate genes mentioned above to bone traits [6, 8,
11, 26, 35, 45]. Linkage analysis is a useful approach if there are no candidate genes, in order to clarify the contribution of a chromosomal region to the phenotype. Linkage analysis may suffer from a limited statistical power when compared to association studies. TDT may suggest if a particular allele is associated to the phenotype variation. We decided to employ both approaches, linkage and TDT, initially to investigate the chromosomal regions for causative genes, and then for putative associated alleles. In our study the polymorphisms were at first utilized as markers for linkage analysis and subsequently a within-family association analysis (via TDT) was performed in order to investigate the hypothetical preferential allele transmission to affected sibs.
Table 4
Quantitative non parametric linkage of ESR1 haplotype and BMD
marker densitometric site p value LOD
ESR1 PvuII/XbaI intertrochanter 0.05 0.56
femoral neck 0.05 0.62
Total hip 0.09 0.4
Ward’s triangle 0.03 0.75
Table 5
Transmission Disequilibrium Test of ESR1 haplotype and BMD
marker densitometric site cut off Global p value Transmitted allele or haplotype Non transmitted allele or haplotype
ESR1 (PvuII/XbaI) Lumbar spine 1 0.01 C/G T/A
2 0.03 T/A
Total hip 1 0.003 C/G T/A
2 0.02 C/A T/A
Trochanter 1 0.006 C/G T/A
2 0.004 C/G T/A
Ward’s triangle 1 0.03 T/A
2 0.04 T/A
Intertrochanter 2 0.01 C/A T/A
COL1A1 Sp1 Ward’s triangle 1 0.05 T G
2 0.03 T G
BMD was treated as a qualitative trait. Transmission Disequilibrium Test was performed by setting as affected the individuals with Z score<-1 (cut off 1), or Z score<-1.5 (cut off 2).
ESR1
To date ESR1 contribution to bone phenotype has been mainly investigated by population association studies both in Caucasian and Asian populations. The results were controversial even when studies of homogeneous ethnicity were accounted for [2, 15, 23, 41, 44]. In a recent large meta-analysis by Ioannidis [19] including 18917 individuals, neither XbaI nor PvuII polymorphisms/haplotypes were significantly associated with BMD.
In our study ESR1PvuII/XbaI multipoint analysis with variance component showed a moderate linkage with intertrochanter, femoral neck and Ward’s triangle.
Only three large linkage studies between ESR1 and bone traits have been published so far. Duncan [11] found a moderate evidence of linkage between BMD at lumbar spine and a microsatellite DNA marker mapping 3,3 Mb from the ESR1 gene according to ensembl (http://www.ensembl.org). A paper by Qin et al. [35] reports linkage data (p<0.05) of PvuII and XbaI polymorphisms with different femoral sites. Recently a study by Zhao [46] did not find significant linkage of the PvuII polymorphism with BMD. Although the positive results did not reach high significance, they still could suggest some involvement of the chromosomal region encompassing the ESR1 gene in BMD variation.
In our study TDT analysis revealed that XbaI and PvuII polymorphisms alone did not reach a significant association at any site, while the T/A haplotype was preferentially non-transmitted to affected individuals. An association was observed between C/G or C/A haplotypes with low BMD of spine or femur.
Among the TDT studies published so far on different candidate genes [1, 17, 25, 35, 46, 47], two
studies have been reported investigating the role of ESR1 polymorphisms in osteoporosis. Qin described a marginally significant evidence for within-family association between XbaI and lumbar spine BMD in osteoporotic young females. They found no association when performing a haplotype analysis. The study by Zhao did not provide any association between either the PvuII polymorphism or six other intragenic SNPs and BMD. The different results may be due to different haplotype distribution or to linkage disequilibrium of the polymorphic markers with different causative alleles in the different populations.
COL1A1
In this study we could not confirm a linkage of COL1A1 with lumbar spine or femoral BMD. Only two other linkage studies on COL1A1 locus have been published to date, none of which reached a strong significance level. Duncan [11] found a moderate evidence of linkage between femoral neck BMD and a microsatellite DNA marker mapping close to COL1A1. Brown detected a weak linkage between femoral neck bone density and Sp1 and a microsatellite polymorphism in a twin and family study [6].
In our study TDT showed a preferential, although not highly significant, transmission of the T allele to affected sibs at Ward’s triangle BMD in our family set. A recent paper by Long could not demonstrate a significant association between COL1A1 Sp1 and BMD at spine and hip [26]. The different results between the two studies may be due to the different mean age of the subjects. A number of association studies have been published to date investigating COL1A1 Sp1A role in BMD variation. Recently a meta-analysis [28], including 26 studies, confirmed
the association of the T allele with a modest BMD reduction.
Additional functional assays confirmed a role of the Sp1 transcription factor binding site polymorphism in collagen gene expression. The presence of the T allele leads to an imbalance in type 1 versus type 2 collagen chain production, with a consequent hypothetical reduction in bone strength [27].
All these data seem to confirm some involvement of COL1A1 in bone mineral density variation.
CALCR
This is the first linkage and TDT study published so far, investigating the contribution of Calcitonin Receptor on BMD variation. We did not find any significant result and thus no clear involvement of this gene could be demonstrated. Furthermore the numerous genome wide linkage studies published to date [9, 12, 20, 22, 38, 42] failed to demonstrate any involvement of the 7q21.3 region, where the CALCR gene maps, with bone traits. Only a few association studies were published on CALCR [4, 5, 30, 32, 39] with controversial outcomes. All these data seem to exclude the CALCR gene as a QTL for BMD.
VDR
In our study linkage analysis did not show any significant result between VDR FokI polymorphism and BMD at any site. Literature data on VDR linkage are a little more abundant compared to the other loci cited above. Among the papers published so far, Zee [45], Brown [6] and Wynne [43] could not support any evidence for linkage between bone density and microsatellites flanking the VDR locus. Duncan et al. (1999) on the other hand indicated some linkage between lumbar spine and femoral neck BMD with microsatellite markers close to VDR. Recently a large study by Deng [8], including 630 subjects from 53 families, showed that VDR ApaI and FokI intragenic polymorphisms are linked to spine BMD variation.
TDT analysis did not show any allele transmission disequilibrium in our families. Deng [8] described an association between VDR ApaI and FokI polymorphisms and lumbar spine BMD.
In conclusion, we observed that among the five gene polymorphisms examined, the ESR1 PvuII/ XbaI haplotypes showed linkage and preferential transmission. These findings enforce the idea of ESR1 gene variants as valuable BMD markers, despite some conflicting results in population-based association studies [2, 15, 23, 41, 44].
The COL1A1 Sp1 polymorphism showed barely significant preferential transmission. It would be helpful to replicate the study in order to confirm these findings.
AcKnowledgements
This work was supported by MIUR (Italian Ministry of Education and Research) grants (to M
Mottes, PF Pignatti) and by the Consorzio per gli
studi universitari-Universita di Verona.
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УДК 612.741.16:612.65+612.648
АКТИВНОСТЬ ДВИГАТЕЛЬНЫХ ЕДИНИЦ ЗДОРОВЫХ ДЕТЕЙ И ДЕТЕЙ С СИНДРОМОМ ДВИГАТЕЛЬНЫХ НАРУШЕНИЙ НА ПЕРВОМ ГОДУ ЖИЗНИ
© Соколов А.Л., Зарипова Ю.Р., Мейгал А.Ю.
Кафедра физиологии человека и животных Петрозаводского государственного университета, Петрозаводск, Карелия
E-mail: meigal@petrsu. ru
В статье представлены сравнительные данные об активности двигательных единиц (ДЕ) у детей первого года жизни, начиная с раннего неонатального возраста, в группах здоровых детей и детей с синдромом двигательных нарушений (СДН). Установлено наличие двух паттернов импульсации ДЕ - «периодического» (~25% всех ДЕ), характеризующегося группами импульсов с высокой частотой импульсации, и «стационарного» паттерна (~75%) в виде длительной низкочастотной импульсации. Показано, что для здоровых детей на протяжении первого года жизни характерно постепенное уменьшение пропорции «периодического» паттерна импульсации ДЕ, а также снижение частоты импульсации ДЕ. Для ДЕ детей с СДН была характерна более высокая вариабельность межимпульсных интервалов, а также неизменность пропорции «периодического» паттерна ДЕ и частоты импульсации ДЕ в течение первого года. Сделан вывод о задержке созревания двигательного аппарата у детей с СДН на уровне мотонейронного пула.
Ключевые слова: двигательные единицы, электромиография, дети, двигательные нарушения.
MOTOR UNIT ACTIVITY IN HEALTHY INFANTS AND IN INFANTS WITH MOTOR
DYSFUNCTION SYNDROME Sokolov A.L., Zaripova Yu.R., MeigalA.Yu.
Human & Animal’s Physiology Department of the Petrozavodsk State University, Petrozavodsk, Karelia
The objective of the study was to compare parameters of the motor unit (MU) activity between the group of healthy infants and the group of infants suffering from motor dysfunction syndrome (MDS). The healthy children were characterized by two clearly different patterns of the MU firing - the “periodic” pattern with high frequency of discharging and the “stationary” pattern with lower discharge frequency. Within the first year of life the —periodic” pattern has diminished and the MU firing rate has decreased. In contrast, in the MDS group had the specific age dynamics of the MU impulsing patterns and firing rate. In addition, the MUs in the MDS group were characterized by greater variability of interspike intervals. In conclusion, development of the motor system of infants with MDS during the first year of life is retarded on the level of motoneuronal pool.
Keywords: motor units, electromyography, child, motor dysfunction syndrome.
Согласно данным ВОЗ, у более чем 10% родившихся детей имеются различные по степени тяжести неврологические расстройства, которые проявляются в виде синдрома двигательных нарушений (СДН). СДН включает в себя изменения мышечного тонуса, его топографии, а также изменение характера спонтанной моторной активности, нарушение краниальной иннервации и угнетение рефлексов. Отдаленные последствия СДН варьируют от минимальных повреждений центральной нервной системы до тяжелых органических поражений. В структуре детской инвалидности поражения ЦНС составляют около 50% [8]. Сравнительные исследования на уровне интерференционной электромиограммы (иЭМГ) здоровых детей и детей с СДН уже выполнены. Установлено, что двигательная система ребенка первого года жизни с СДН, по данным амплитудно-частного анализа иЭМГ, обладает выраженной способностью «догонять» имеющееся отставание в развитии [3, 5].
Двигательная единица (ДЕ) является элементарным функционально-анатомическим звеном
двигательной системы, и по ее характеристикам можно судить о двигательных стратегиях в целом [12]. У детей ДЕ в раннем периоде новорож-денности практически не исследованы, хотя имеются гистохимические данные об онтогенезе мышечных волокон ребенка на протяжении всего раннего возраста [9]. Сравнение характеристик импульсации ДЕ здоровых детей и детей с СДН в течение первого года жизни позволило бы выявить отличия в созревании их двигательного аппарата, могло бы указать на потенциальные маркеры патологии уже в раннем детском возрасте, однако по электромиографии ДЕ у детей имеются единичные работы [7].
В этой связи нам представлялось принципиально важным сравнить импульсную активность ДЕ у здоровых детей первого года жизни и детей с СДН, с особым вниманием к наиболее ранним периодам созревания, например раннему неонатальному возрасту, и провести их сравнительный анализ для выявления возможных маркеров патологии двигательной системы на уровне ДЕ.
