骨骼疾病分子机制研究
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摘要
第一部分中国山东一个短指家系临床特征调查及其致病基因的定位和克隆
短指(趾)(Brachydactylies，BDs)是一组以指(趾)畸形为主要特征的手(足)遗传病。1951年，Bell根据畸形发生部位和患者受累程度的不同，将遗传性短指畸形分为A、B、C、D、E五种类型。并将A型进一步细分为A1、A2、A3三种亚型。A1型短指最早是在1903年由Farabee在他的博士论文中描述的一种常染色体显性遗传病，这也是在人类中确定的第一种常染色体显性遗传病。1979年，Fitch对短指畸形进行了重新分类。根据Fitch的重新分类，A1亚型(Brachydactyly type A1，BDA1)的特征主要包括：患者所有指(趾)中节指骨短小、缺失或与远端指骨发生融合，拇指(趾)的近端指骨一般变短；有些个体的掌骨和跖骨也变短；有些家系患者还表现为身材矮小。部分患者会伴有肌肉骨骼的异常、脊柱侧凸、眼球震颤和发育迟缓等症状。
2000年，Yang等通过对两个中国BDA1家系进行连锁分析，成功地将BDA1致病基因定位于2q35-2q36间8.1cM的区域内，对区域内的基因进行功能分析的结果提示Indian Hedgehog(IHH)基因有可能是BDA1的致病基因。2001年，Gao等在三个无血缘关系的中国BDA1大家系的患者中发现了IHH基因三个杂合错义突变，这些突变都位于IHH基因编码N-端功能区域内，从而确定IHH基因是BDA1的致病基因。但是，IHH基因错义突变并非导致BDA1的唯一致病基因。2002年，Armour发现一个加拿大A1型短指家系致病基因位于5p13.2-p13.3之间一个11cM区域内。2003年，Karkpatrick提出BDA1存在第三个致病基因位点，新致病基因位点的定位表明BDA1具有遗传异质性。我们在山东省济宁市发现了一个大的短指家系，临床特征分析和家系调查表明该家系中患者的表型特征符合典型BDA1。为确定该BDA1家系的致病基因，我们采用候选基因分析方法对该家系进行了基因定位和基因突变分析。
一、家系调查
该短指家系共有三代26名家系成员纳入到本研究中，包括14名患者和12名正常个体。根据知情同意原则，在征得所有家系成员同意后，所有受试对象测量身高，拍摄手、足部照片，由骨科医生进行体检，选择6个患者进行手、足部X-线照相，另对Ⅲ-3，Ⅲ-4进行全身各部位X-线照相。
体检显示该家系中患者的异常仅限定于手、足部，患者的手短而宽，表现为所有的手指都缩短，部分患者的中指比食指和无名指还短。X-线照片显示，所有受检患者均有手、足部的骨骼异常，异常主要表现为中节指骨和掌骨(跖骨)的缩短。但是，表型的严重程度在家系内不同患者之间变化很大。Ⅲ-4是最严重的患者之一，他双手的中节指骨都缺失或与远端指骨发生融合，而且他双手的第三和第四掌骨均发生不同程度缩短。另一名患者Ⅲ-3的所有中节指骨也全部缺失或与远端指骨发生融合，掌骨同样发生缩短，但是她缩短的掌骨限定于第一和第三掌骨，同时，她的第一近端指骨也缩短。以前报道的BDA1患者中没有出现过严重的第一掌骨缩短，这是第一次发现第一掌骨和第一近端指骨同时缩短的BDA1患者。
二、连锁分析与突变检测
因为已知的BDA1基因位点分别位于2q35-2q36和5p13.2-p13.3区域，因此，我们首先从这两个区域内选择多态位点进行连锁分析。家系中各成员微卫星多态位点D5S1470的基因分型结果显示家系中患者携带不同的等位基因，表明本家系致病基因不在5p13.2-p13.3区域。利用所选择的IHH基因两侧微卫星多态位点对该家系患者进行基因型分析，各位点连锁分析结果显示致病基因与多态位点D2S2250、D2S433、D2S163和D2S2359紧密连锁，家系中所有患者都携带4-3-2-3单倍型；但对于DS2434位点，患者Ⅲ-13及其患病儿子Ⅳ-9携带等位基因3，而家系中其余患者携带等位基因4，提示D2S434与D2S2250之间发生了重组，致病基因位于D2S434下游。以上结果提示IHH基因是该BDA1家系的最佳候选基因。
对该家系中一名患者(Ⅳ-3)与其正常的同胞兄弟(Ⅳ-2)IHH基因所有外显子和外显子／内含子接头序列进行PCR扩增，PCR产物的电泳结果显示IHH基因无大的插入和缺失突变发生。对PCR产物测序发现在IHH基因第一外显子第298位核苷酸发生了G→A杂合错义突变，这一突变导致IHH多肽链第100位的氨基酸由天冬氨酸转变为天冬酰胺(D100N)。为确定是否该突变在家系中与患者表型共分离，我们采用修饰引物引入酶切位点的聚合酶链式反应—限制性片段长度多态性(ploymerase chain reaction-restriction fragment length polymorphism，PCR-RFLP)方法对该家系中的所有成员和100名正常中国人进行了IHH基因G298A基因型分析。酶切结果显示家系中所有正常个体的两个等位基因均被切开，是野生型等位基因的纯合子，而家系中所有患者只有一个等位基因被切开，是野生型和突变型等位基因的杂合子。对100名正常中国人200条染色体的分析没有发现298A等位基因存在。这些结果充分证明中国山东的短指家系患者是由于IHH基因G298A杂合突变所致。
三、单倍型分析
因为已经在6个白种人BDA1家系中发现了IHH基因G298A杂合错义突变，并且IHH基因G298A突变被认为可能是一个建立者突变。为确定本研究发现的BDA1家系是新发突变还是与白种人BDA1家系突变来源相同，我们选择4个与IHH基因紧密连锁的微卫星多态位点和IHH基因内5个SNPs位点构建了疾病相关单倍型(haplotype)，并与以前报道的Farabee-Drinkwater家系疾病相关单倍型进行了比较。结果显示在中国山东BDA1家系的单倍型和Farabee-Drinkwater家系单倍型之间有三个微卫星位点(D2S2250、D2S433和D2S163)和两个SNP位点(SNPe和SNPd)携带不同等位基因，由此可以认为中国山东BDA1家系的IHH基因G298A突变与白种人BDA1家系的IHH基因G298A突变具有不同的起源，IHH基因G298A突变可能发生过不只一次。
综上所述，通过家系分析和临床调查，我们确定一个新发现的中国短指家系其表型属于A1型短指。通过连锁分析，我们排除了5p13.2-p13.3区域位点与本家系致病基因连锁，发现该家系致病基因位于2q35-2q36。候选基因分析结合突变检测发现IHH基因的G298A杂合错义突变导致该家系BDA1，并且利用疾病相关单倍型比较证实该家系中检测到的G298A突变是新发突变而非来自建立者突变(founder mutation)。对突变位点附近核苷酸序列分析发现，该突变可能是由于非编码链上5' CpG 3'二核苷酸位点的5-甲基胞嘧啶容易发生自发的脱氨基作用所致突变热点效应造成的。作为IHH基因的一个突变热点，我们推测G298A杂合错义突变还会出现在其它BDA1家系中。
第二部分Lrp5成骨作用相关基因筛选
骨质疏松(osteoporosis)是以骨量减少、微结构异常为特征的、致使骨脆性增加而易于发生骨折的一种全身性代谢性疾病。骨质疏松是由于个体在生长发育时未能达到最佳峰值骨量(peak bone mass)和／或晚年骨量维持异常所造成的。骨量的维持是通过成骨细胞和破骨细胞共同作用的结果。
低密度脂蛋白受体相关蛋白5(low-density lipoprotein receptor related protein 5，LRP5)是细胞表面的受体蛋白，属于低密度脂蛋白受体家族，可通过与相应配体结合参与受体介导的细胞内吞作用而实现多方面的功能。研究表明LRP5基因的丧失功能突变导致骨质疏松—假性神经胶质瘤综合征，携带突变的杂合子个体骨密度显著低于正常对照，且易发生成年型骨质疏松，而LRP5基因的获得功能突变导致高骨密度和骨胳石化症(大理石样骨病)(osteosclerosis)。
为理解LRP5在成骨细胞发生发展中的作用，本研究以小鼠骨髓基质细胞系ST2为实验对象，分别转染Lrp5基因获得功能突变(G171V)的表达载体和抑制Lrp5基因表达的Lrp5-RNAi表达载体，观察增强和抑制Lrp5基因功能对ST2细胞增殖和分化的影响；并在此基础上，采用抑制性消减杂交(SuppressionSubtractive Hybridization，SSH)技术，对比分析稳定表达获得功能突变Lrp5基因(G171V)和Lrp5-RNAi细胞的基因表达差异。取得了以下结果：
一、Lrp5基因G171V获得功能突变表达载体构建与表达
采用融合PCR技术对野生型Lrp5基因进行定点突变，通过片段置换构建带有Lrp5基因G171V获得功能突变的表达载体pcDNA3.1C-GFLrp5。经限制性内切酶酶切和测序证实。用pcDNA3.1C-GFLrp5表达载体转染ST2细胞，细胞免疫荧光技术和Western Blot技术证实pcDNA3.1C-GFLrp5可以在细胞内表达并与运输到细胞膜上发挥功能，同时通过共转染，证明mu6／LRP5表达载体可以有效抑制与其共转染的pcDNA3.1C-GFLrp5的表达，间接证明mu6／LRP5表达载体可以有效抑制细胞内源性Lrp5基因的表达。
二、通过ST2细胞增殖能力和von Kossa染色实验确定两种表达载体的作用效果
ST2是小鼠骨髓基质细胞来源的细胞系，具有多向分化的潜能，在有β-甘油磷酸钠存在的条件下，L-抗坏血酸可以诱导ST2细胞向成骨细胞样细胞分化。ST2细胞分别转染pcDNA3.1C-GFLrp5表达载体与抑制Lrp5基因的RNAi表达载体mu6／LRP5后，经过G418筛选稳定转染的细胞分别标记为S-LGF和S-LLF。MTT法检测S-LGF和S-LLF细胞增殖能力的结果表明，细胞接种后24小时、48小时和72小时三个时间点，S-LGF的细胞增值能力都显著高于S-LLF细胞，表明Lrp5基因G171V获得功能突变对ST2细胞的增殖能力具有很强的促进作用。对向成骨细胞诱导分化培养28天的S-LGF和S-LLF细胞进行von Kossa染色，结果显示S-LGF细胞比S-LLF细胞可以更早、形成更多、染色更深的骨样结节。对连续培养达到40天的S-LGF和S-LLF细胞进行von Kossa染色的结果差别更加明显，表明与对细胞内的Lrp5基因表达进行抑制相比，向细胞内转染Lrp5基因G171V获得功能突变对ST2细胞向成骨细胞分化和增殖能力都有明显促进作用。
三、S-LGF和S-LLF细胞消减cDNA文库的构建及筛选
为分离和鉴定Lrp5成骨相关基因，我们采用抑制性消减杂交技术，以S-LGF细胞的mRNA为实验方、S-LLF细胞的mRNA为驱动方构建了正向消减cDNA文库，同时以S-LLF细胞的mRNA为实验方、S-LGF细胞的mRNA为驱动方构建了反向消减cDNA文库。利用菌液做模板进行PCR扩增，我们对构建的正向和反向消减cDNA文库进行了筛选。在随机选择的各200个克隆中，从正向消减cDNA文库中筛选出105个有插入片段的克隆，从反向消减cDNA文库中筛选出97个有插入片段的克隆，结合不同限制性内切酶的酶切图谱，选择正向消减cDNA文库的60个克隆和反向消减cDNA文库的61个克隆进行测序。
四、cDNA测序与同源性分析初步结果
将测序结果与GenBank中的核酸序列比较后获得了在S-LGF中差异表达的cDNA片段51个，其中已知基因44个，RIKEN cDNA基因3，另有4个片段在GenBank中无对应的已知基因，可能为新基因。在S-LLF细胞中差异表达的cDNA片段58个，其中已知基因43个，RIKEN cDNA基因8个，另有7个片段在GenBank中无对应的已知基因，可能为新基因。下一步我们将在证实这些基因差异表达的基础上进一步分析这些基因在成骨细胞发生发展中的作用。
Brachydactylies (BDs) are a group of inherited malformations characterized by shortening of the digits due to abnormal development of the phalanges and/or the metacarpals. They have been classified on anatomic and genetic basis into five groups, A-E, by Bell in 1951. Group A is further divided into three subgroups, A1, A2, and A3, and usually manifests as autosomal dominant traits. Brachydactyly type A1 (BDA1, MIM 112500) is the first autosomal dominant Mendelian trait in human and first described by Farabee in 1903. According to the reclassified criteria which Fitch used in 1979, BDA1 is characterized by hypoplasia/aplasia of all middle phalanges, sometimes with joint fusions between the middle and the proximal phalanx. In some individuals, the metacarpals were also short. Affected family members might have short stature compared to normal family members. BDA1 often occurred as an isolated malformation. However, others also experienced musculoskeletal abnormalities, scoliosis, nystagmus, and/or developmental delay.
Yang et al. mapped the BDA1 locus to chromosome 2q35-36 through linkage analysis of two Chinese families in 2000. Analysis of the function of genes located in this region suggested that Indian Hedgehog gene (IHH) be a good candidate gene for BDA1. Subsequent sequences analysis identified that mutations in the IHH gene were the causal mutations for three Chinese BDA1 families by Gao in 2001. However, IHH mutations account for only a subset of BDA1 cases. Armour et al. found BDA1 is linked to an 11cM critical region on chromosome 5p13.2-13.3 in a four generation BDA1 family of Canadian descent. Kirkpatrick et al. provided evidence that support the existence of a third locus for BDA1. These data suggested that the BDA1 is genetically heterogeneous.
We found a large brachydactyly family in Jining city, Shandong province. Clinical and family analysis revealed that the characterization of the cases in this family were consistent with the typical BDA1. To determine the causal gene of this BDA1 family, we carried out linkage analysis and mutation detection.
1 Family Study
A large family from Shandong province, China, was recruited in this study. Twenty six individuals (14 affected, 12 unaffected) were included in this study. Affected and unaffected individuals were physically examined, and six selected individuals (II-5, III-3, III-4, ffl-9, 111-12(?)111-13) were radiographed.
The defects found in the affected individuals of this family were confined to the hands and feet. The hands of the affected family members appeared to be broad and showed shortening of all digits. X-ray examination revealed anomalies of hand and foot bones, but the defects were mainly confined to the middle phalanges and metacarpals. The severity of BDA1 was varied among the affected members of the family. Individual III-4 is one of the most severe case, whose middle phalanges were all absent or fused to the distal phalanges and both the third and forth metacarpals were shortened in both hands. Another affected individual, III-3, also has all the middle phalanges missing and shows shortened metacarpals. But her shortened metacarpals were limited to the first and third metacarpals, and her proximal 1~(st) phalange was shortened, too. To our knowledge, the previously reported BDA1 patients were not known to have severely shortened first metacarpals. Other affected members examined by radiography had shortened middle phalanges, though with varying numbers.
2 Linkage Analysis and Mutation Detection
Two known disease loci located in 2q35-2q36 and 5pl3.2-pl3.3, respectively. So we selected polymorphic microsatellite markers from these two regions for linkage analysis. Result of genotyping D5S1470 located in 5p13.2-p13.3 showed that patients in this BDA1 family carried different alleles and excluded that the causal gene for this BDA1 family is in this region. Using a set of markers spanning the IHH gene, we were able to demonstrate that BDA1 was linked to chromosome 2q35-q36. Complete linkage was shown for markers D2S2250, D2S433, D2S163, and D2S2359, and recombination event was observed between D2S434 and D2S2250 in individual 111-13. These results suggested IHH gene may be the best candidated gene for this BDA1 family.
Four pairs of primers were designed to amplify all three exons and exon/intron boundaries of the IHH gene. Agarose gel analysis showed the sizes of PCR products were consistent with the expectation and indicated that there were no large insert or deletion mutitons of IHH gene. The mutation screen limited to the DNA from one affected (IV-3) and one unaffected individual (IV-2) with the direct sequencing of PCR products identified a heterozygous G-to-A transition (G298A) in exon 1, which results in an aspartate to asparagine substitution (D100N). To detect the G298A mutation in all family members and controls, a modified primer was used to create a restriction endonuclease (Sal 1) site to distinguish 298G and 298A alleles. By using PCR-RFLP analysis, the G298A transition was identified in all affected individuals. Moreover, the G298A allele was not detected among 200 chromosomes from unrelated normal Chinese controls.
3 Haplotype Analysis
The IHH G298A mutation had previously been identified in six Caucasian families with BDA1, and McCready et al. provided the evidence that support the existence of a common ancestral allele between Farabee's family and Drinkwater's families.To discriminate either a common founder mutation or a de novo mutation contributed to this Chinese kindred, we constituted the disease-associated haplotype with microsatellite markers D2S2250, D2S433, D2S163, and D2S2359 that are tightly linked to the IHH gene and five intragenic SNPs described by McCready of this Chinese family. Then the disease-related haplotype of this Chinese BDA1 family was compared with the common haplotype reported for the Farabee-Drinkwater families.
A comparison of the haplotypes from different families indicated our patients carried different alleles compared to the patients from Farabee-Drinkwater's families at three microsatellite markers (D2S2250, D2S433, and D2S163) and two intragenic SNPs (SNPe and SNPd). Another three intragenic SNPs (SNPa, SNPb, and SNPc) were not informative in our family and ethnic-matched controls. We then concluded that the IHH gene G298A mutation in this Chinese BDA1 family had different origin from those in Caucasian families. It looked like the IHH gene G298A mutation occurred more than once historical.
In conclusion, we identified a BDA1 family through clinical research and family analysis. By using linkage analysis, we excluded the linkage of the causal gene with 5p13.2-p13.3 and confirmed the linkage with 2q35-2q36. Combined candidate gene analysis and mutation detection, we identified a heterozygous missense mutation G298A in exon 1 of IHH gene, which results in an aspartate to asparagine substitution (D100N) was the causal mutation of this BDA1 family. Comparision of disease-related haplotypes from this family with the Farabee-Drinkwater families' revealed that a de novo G298A mutation, not a fouder mutation, caused the BDA1 of this family. Sequence analysis around IHH gene 298G showed that it maybe a mutational hot spot due to spontaneous deamination of a 5-methylcytosine on the noncoding strand of the gene. As a mutational hot spot, we predict that the IHH G298A mutation will be identified in additional BDA1 families.
Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. Osteoporosis results from a failure to acquire optimal peak bone mass during growth and/or to maintain bone mass in later years, which means a disruption of the fine equilibrium between the activity of osteoblasts responsible for bone deposition and osteoclasts for bone resorption.
Low-density lipoprotein receptor related protein 5, LRP5, is a member of low-density lipoprotein receptor family. LRP5 have multiple functions by banding and internalizing different ligands. Recent studies have indicated that the loss-of-function mutations in LRP5 gene cause Osteoporosis-Pseudoglioma syndrome, an autosomal recessive disorder characterized by severe juvenile-onset osteoporosis and congenital or juvenile-onset blindness. The gain-of-function mutations in LRP5 gene lead to increased bone mass and osteopetroses.
To further understand the role of LRP5 in bone formation, we used mouse bone marrow stromal cell line ST2 as a study model. We transfected ST2 cells with the constructs expressing Lrp5 gain-of-function muatation and LRP5-RNAi, respectively. We analyzed the effect of LRP5 status on ST2 cells proliferation and differentiation in the stable-transfected cells. With Suppression Subtractive Hybridization (SSH) technique, we constructed the subtractive cDNA libraries of ST2 cells stable-expressed Lrp5 gene with gain-of-function muatation and Lrp5-RNAi construct, respectively. The LRP5-related genes could be screened from these subtractive cDNA libraries.
1. Generation and transfection of the construct expressing the Lrp5 gene with G171V mutation
Fusion-PCR method was used to introduce G171V mutation using the wild Lrp5 expression constructs as the template. The resulting fragments containing the G171V mutation was used to replace the corresponding fragment from the wild type cDNA, and resulting insert was cloned into pcDNA3.1C. vector and named as pcDNA3.1C-GFLrp5. The construct was confirmed by PCR, restriction enzyme digestion and direct sequencing. The pcDNA3.1C-GFLrp5 Construct was transfected into ST2 cells. Cell-based immunofluorescence and Western Blotting analysis showed that pcDNA3.1C-GFLrp5 expressed in ST2 cells and transported to the membrane.
To inhibit the Lrp5 expression, we transfected the Lrp5-RNAi constructs, mu6/LRP5 to ST2 cells. The efficiency RNAi targeting was confirmed by cotransfectiong cells with mu6/LRP5 and pcDNA3.1C-GFLrp5. The decreasing in the expression of the pcDNA3.1C-GFLrp5 construct was observed with the anti-myc antibody by cell-based immunofluorescence and Western Blot.
2. Effects of stable expressing pcDNA3.1C-GFLrp5 construct and Lrp5-RNAi construct mu6/LRP5 on proliferation and differentiation of ST2 cells
ST2 Cells, a clone of stromal cells, were isolated from the bone marrow of BC8 mice, capable of the potential ability to differentiate into various mesenchymal cell lineages. However, when subcultured with medium supplemented with ascorbic acid, the ST2 cells developed an osteoblastic phenotype. We transfected pcDNA3.1C-GFLrp5 construct and the Lrp5 gene RNAi mu6/LRP5 construct into the cultured ST2 and selected with G418. The cells stable-expressiong Lrp5 G171V mutation expression construct and Lrp5 RNAi expression construct were obtained, and named as S-LGF and S-LLF, respectively.
MTT assays revealed that the cells proliferation of S-LGF was significantly higher than that of S-LLF cells measured at different time points: cultured for 24 hours, 48 hours and 72 hours. This suggested that the exogenous Lrp5 G171V mutant protein can notably enhance the proliferation of ST2 cells compared with ST2 cells in which Lrp5 were silenced. The more bone-like nodules were observed in the S-LGF than S-LLF cells by von Kossa's staining when cells cultured for 28 days under conditioned medium supplemented with ascorbic acid. The difference was much more significantly when cells cultured for 40 days.
3. Construction and screening of the S-LGF and S-LLF subtractive cDNA libraries
Suppression subtractive hybridization (SSH) is the combination of suppression PCR and subtractive hybridization technique. This technique could clone the unknown sequence of differentially expressed genes. The method is characterized with high efficiency, low false positive rate, targeting sequence enrichment, and low complexity of the experimental results. Using the S-LGF mRNA as the tester and the S-LLF mRNA as driver, we constructed the forward subtracted cDNA library. The reverse subtracted cDNA library was constructed by using the S-LLF mRNA as the tester and the S-LGF mRNA as driver.
Combining PCR amplification with cultured E. coli as template and the restriction endonuclease digestion, we screened the forward and reverse subtracted cDNA libraries. We found 105 clones with insert fragment out of 200 random selected clones from the forward subtracted cDNA library and 97 out of 200 from the reverse subtracted cDNA library. According to the patterns of restriction endonuclease digestion, 60 and 61 clones with different inserts from the forward and reverse subtracted cDNA libraries were selected for sequencing, respectively.
4. The differentially expressed sequences
We obtained 51 cDNA fragments differentially expressed in the S-LGF cells by comparison analysis with nucleotide sequences in GenBank. There are 44 cDNA fragments which were highly homologous with the known genes. Three cDNA fragments belong to the RIKEN cDNA gene. While other 4 cDNA fragments were not homologous with any known genes, which may be from novel genes. Similarly, 58 differentially expressed cDNA fragments in the S-LLF cells were obtained. Among them, 43 cDNA fragments were from known homologous genes and eight were RIKEN cDNA genes. Also, seven differentially expressed cDNA fragments were not matched to known homologous sequence.
These differentially expressed sequences will be confirmed by RT-PCR and real-time PCR. Further analysis of these differentially expressed genes may lead to identification of new genes related with Lrp5 controlling osteoblast proliferation and function and eventually shed new light on the molecular mechanisms and the new drugs development for the treatment of osteoporosis.
短指(趾)(Brachydactylies，BDs)是一组以指(趾)畸形为主要特征的手(足)遗传病。1951年，Bell根据畸形发生部位和患者受累程度的不同，将遗传性短指畸形分为A、B、C、D、E五种类型。并将A型进一步细分为A1、A2、A3三种亚型。A1型短指最早是在1903年由Farabee在他的博士论文中描述的一种常染色体显性遗传病，这也是在人类中确定的第一种常染色体显性遗传病。1979年，Fitch对短指畸形进行了重新分类。根据Fitch的重新分类，A1亚型(Brachydactyly type A1，BDA1)的特征主要包括：患者所有指(趾)中节指骨短小、缺失或与远端指骨发生融合，拇指(趾)的近端指骨一般变短；有些个体的掌骨和跖骨也变短；有些家系患者还表现为身材矮小。部分患者会伴有肌肉骨骼的异常、脊柱侧凸、眼球震颤和发育迟缓等症状。
2000年，Yang等通过对两个中国BDA1家系进行连锁分析，成功地将BDA1致病基因定位于2q35-2q36间8.1cM的区域内，对区域内的基因进行功能分析的结果提示Indian Hedgehog(IHH)基因有可能是BDA1的致病基因。2001年，Gao等在三个无血缘关系的中国BDA1大家系的患者中发现了IHH基因三个杂合错义突变，这些突变都位于IHH基因编码N-端功能区域内，从而确定IHH基因是BDA1的致病基因。但是，IHH基因错义突变并非导致BDA1的唯一致病基因。2002年，Armour发现一个加拿大A1型短指家系致病基因位于5p13.2-p13.3之间一个11cM区域内。2003年，Karkpatrick提出BDA1存在第三个致病基因位点，新致病基因位点的定位表明BDA1具有遗传异质性。我们在山东省济宁市发现了一个大的短指家系，临床特征分析和家系调查表明该家系中患者的表型特征符合典型BDA1。为确定该BDA1家系的致病基因，我们采用候选基因分析方法对该家系进行了基因定位和基因突变分析。
一、家系调查
该短指家系共有三代26名家系成员纳入到本研究中，包括14名患者和12名正常个体。根据知情同意原则，在征得所有家系成员同意后，所有受试对象测量身高，拍摄手、足部照片，由骨科医生进行体检，选择6个患者进行手、足部X-线照相，另对Ⅲ-3，Ⅲ-4进行全身各部位X-线照相。
体检显示该家系中患者的异常仅限定于手、足部，患者的手短而宽，表现为所有的手指都缩短，部分患者的中指比食指和无名指还短。X-线照片显示，所有受检患者均有手、足部的骨骼异常，异常主要表现为中节指骨和掌骨(跖骨)的缩短。但是，表型的严重程度在家系内不同患者之间变化很大。Ⅲ-4是最严重的患者之一，他双手的中节指骨都缺失或与远端指骨发生融合，而且他双手的第三和第四掌骨均发生不同程度缩短。另一名患者Ⅲ-3的所有中节指骨也全部缺失或与远端指骨发生融合，掌骨同样发生缩短，但是她缩短的掌骨限定于第一和第三掌骨，同时，她的第一近端指骨也缩短。以前报道的BDA1患者中没有出现过严重的第一掌骨缩短，这是第一次发现第一掌骨和第一近端指骨同时缩短的BDA1患者。
二、连锁分析与突变检测
因为已知的BDA1基因位点分别位于2q35-2q36和5p13.2-p13.3区域，因此，我们首先从这两个区域内选择多态位点进行连锁分析。家系中各成员微卫星多态位点D5S1470的基因分型结果显示家系中患者携带不同的等位基因，表明本家系致病基因不在5p13.2-p13.3区域。利用所选择的IHH基因两侧微卫星多态位点对该家系患者进行基因型分析，各位点连锁分析结果显示致病基因与多态位点D2S2250、D2S433、D2S163和D2S2359紧密连锁，家系中所有患者都携带4-3-2-3单倍型；但对于DS2434位点，患者Ⅲ-13及其患病儿子Ⅳ-9携带等位基因3，而家系中其余患者携带等位基因4，提示D2S434与D2S2250之间发生了重组，致病基因位于D2S434下游。以上结果提示IHH基因是该BDA1家系的最佳候选基因。
对该家系中一名患者(Ⅳ-3)与其正常的同胞兄弟(Ⅳ-2)IHH基因所有外显子和外显子／内含子接头序列进行PCR扩增，PCR产物的电泳结果显示IHH基因无大的插入和缺失突变发生。对PCR产物测序发现在IHH基因第一外显子第298位核苷酸发生了G→A杂合错义突变，这一突变导致IHH多肽链第100位的氨基酸由天冬氨酸转变为天冬酰胺(D100N)。为确定是否该突变在家系中与患者表型共分离，我们采用修饰引物引入酶切位点的聚合酶链式反应—限制性片段长度多态性(ploymerase chain reaction-restriction fragment length polymorphism，PCR-RFLP)方法对该家系中的所有成员和100名正常中国人进行了IHH基因G298A基因型分析。酶切结果显示家系中所有正常个体的两个等位基因均被切开，是野生型等位基因的纯合子，而家系中所有患者只有一个等位基因被切开，是野生型和突变型等位基因的杂合子。对100名正常中国人200条染色体的分析没有发现298A等位基因存在。这些结果充分证明中国山东的短指家系患者是由于IHH基因G298A杂合突变所致。
三、单倍型分析
因为已经在6个白种人BDA1家系中发现了IHH基因G298A杂合错义突变，并且IHH基因G298A突变被认为可能是一个建立者突变。为确定本研究发现的BDA1家系是新发突变还是与白种人BDA1家系突变来源相同，我们选择4个与IHH基因紧密连锁的微卫星多态位点和IHH基因内5个SNPs位点构建了疾病相关单倍型(haplotype)，并与以前报道的Farabee-Drinkwater家系疾病相关单倍型进行了比较。结果显示在中国山东BDA1家系的单倍型和Farabee-Drinkwater家系单倍型之间有三个微卫星位点(D2S2250、D2S433和D2S163)和两个SNP位点(SNPe和SNPd)携带不同等位基因，由此可以认为中国山东BDA1家系的IHH基因G298A突变与白种人BDA1家系的IHH基因G298A突变具有不同的起源，IHH基因G298A突变可能发生过不只一次。
综上所述，通过家系分析和临床调查，我们确定一个新发现的中国短指家系其表型属于A1型短指。通过连锁分析，我们排除了5p13.2-p13.3区域位点与本家系致病基因连锁，发现该家系致病基因位于2q35-2q36。候选基因分析结合突变检测发现IHH基因的G298A杂合错义突变导致该家系BDA1，并且利用疾病相关单倍型比较证实该家系中检测到的G298A突变是新发突变而非来自建立者突变(founder mutation)。对突变位点附近核苷酸序列分析发现，该突变可能是由于非编码链上5' CpG 3'二核苷酸位点的5-甲基胞嘧啶容易发生自发的脱氨基作用所致突变热点效应造成的。作为IHH基因的一个突变热点，我们推测G298A杂合错义突变还会出现在其它BDA1家系中。
第二部分Lrp5成骨作用相关基因筛选
骨质疏松(osteoporosis)是以骨量减少、微结构异常为特征的、致使骨脆性增加而易于发生骨折的一种全身性代谢性疾病。骨质疏松是由于个体在生长发育时未能达到最佳峰值骨量(peak bone mass)和／或晚年骨量维持异常所造成的。骨量的维持是通过成骨细胞和破骨细胞共同作用的结果。
低密度脂蛋白受体相关蛋白5(low-density lipoprotein receptor related protein 5，LRP5)是细胞表面的受体蛋白，属于低密度脂蛋白受体家族，可通过与相应配体结合参与受体介导的细胞内吞作用而实现多方面的功能。研究表明LRP5基因的丧失功能突变导致骨质疏松—假性神经胶质瘤综合征，携带突变的杂合子个体骨密度显著低于正常对照，且易发生成年型骨质疏松，而LRP5基因的获得功能突变导致高骨密度和骨胳石化症(大理石样骨病)(osteosclerosis)。
为理解LRP5在成骨细胞发生发展中的作用，本研究以小鼠骨髓基质细胞系ST2为实验对象，分别转染Lrp5基因获得功能突变(G171V)的表达载体和抑制Lrp5基因表达的Lrp5-RNAi表达载体，观察增强和抑制Lrp5基因功能对ST2细胞增殖和分化的影响；并在此基础上，采用抑制性消减杂交(SuppressionSubtractive Hybridization，SSH)技术，对比分析稳定表达获得功能突变Lrp5基因(G171V)和Lrp5-RNAi细胞的基因表达差异。取得了以下结果：
一、Lrp5基因G171V获得功能突变表达载体构建与表达
采用融合PCR技术对野生型Lrp5基因进行定点突变，通过片段置换构建带有Lrp5基因G171V获得功能突变的表达载体pcDNA3.1C-GFLrp5。经限制性内切酶酶切和测序证实。用pcDNA3.1C-GFLrp5表达载体转染ST2细胞，细胞免疫荧光技术和Western Blot技术证实pcDNA3.1C-GFLrp5可以在细胞内表达并与运输到细胞膜上发挥功能，同时通过共转染，证明mu6／LRP5表达载体可以有效抑制与其共转染的pcDNA3.1C-GFLrp5的表达，间接证明mu6／LRP5表达载体可以有效抑制细胞内源性Lrp5基因的表达。
二、通过ST2细胞增殖能力和von Kossa染色实验确定两种表达载体的作用效果
ST2是小鼠骨髓基质细胞来源的细胞系，具有多向分化的潜能，在有β-甘油磷酸钠存在的条件下，L-抗坏血酸可以诱导ST2细胞向成骨细胞样细胞分化。ST2细胞分别转染pcDNA3.1C-GFLrp5表达载体与抑制Lrp5基因的RNAi表达载体mu6／LRP5后，经过G418筛选稳定转染的细胞分别标记为S-LGF和S-LLF。MTT法检测S-LGF和S-LLF细胞增殖能力的结果表明，细胞接种后24小时、48小时和72小时三个时间点，S-LGF的细胞增值能力都显著高于S-LLF细胞，表明Lrp5基因G171V获得功能突变对ST2细胞的增殖能力具有很强的促进作用。对向成骨细胞诱导分化培养28天的S-LGF和S-LLF细胞进行von Kossa染色，结果显示S-LGF细胞比S-LLF细胞可以更早、形成更多、染色更深的骨样结节。对连续培养达到40天的S-LGF和S-LLF细胞进行von Kossa染色的结果差别更加明显，表明与对细胞内的Lrp5基因表达进行抑制相比，向细胞内转染Lrp5基因G171V获得功能突变对ST2细胞向成骨细胞分化和增殖能力都有明显促进作用。
三、S-LGF和S-LLF细胞消减cDNA文库的构建及筛选
为分离和鉴定Lrp5成骨相关基因，我们采用抑制性消减杂交技术，以S-LGF细胞的mRNA为实验方、S-LLF细胞的mRNA为驱动方构建了正向消减cDNA文库，同时以S-LLF细胞的mRNA为实验方、S-LGF细胞的mRNA为驱动方构建了反向消减cDNA文库。利用菌液做模板进行PCR扩增，我们对构建的正向和反向消减cDNA文库进行了筛选。在随机选择的各200个克隆中，从正向消减cDNA文库中筛选出105个有插入片段的克隆，从反向消减cDNA文库中筛选出97个有插入片段的克隆，结合不同限制性内切酶的酶切图谱，选择正向消减cDNA文库的60个克隆和反向消减cDNA文库的61个克隆进行测序。
四、cDNA测序与同源性分析初步结果
将测序结果与GenBank中的核酸序列比较后获得了在S-LGF中差异表达的cDNA片段51个，其中已知基因44个，RIKEN cDNA基因3，另有4个片段在GenBank中无对应的已知基因，可能为新基因。在S-LLF细胞中差异表达的cDNA片段58个，其中已知基因43个，RIKEN cDNA基因8个，另有7个片段在GenBank中无对应的已知基因，可能为新基因。下一步我们将在证实这些基因差异表达的基础上进一步分析这些基因在成骨细胞发生发展中的作用。
Brachydactylies (BDs) are a group of inherited malformations characterized by shortening of the digits due to abnormal development of the phalanges and/or the metacarpals. They have been classified on anatomic and genetic basis into five groups, A-E, by Bell in 1951. Group A is further divided into three subgroups, A1, A2, and A3, and usually manifests as autosomal dominant traits. Brachydactyly type A1 (BDA1, MIM 112500) is the first autosomal dominant Mendelian trait in human and first described by Farabee in 1903. According to the reclassified criteria which Fitch used in 1979, BDA1 is characterized by hypoplasia/aplasia of all middle phalanges, sometimes with joint fusions between the middle and the proximal phalanx. In some individuals, the metacarpals were also short. Affected family members might have short stature compared to normal family members. BDA1 often occurred as an isolated malformation. However, others also experienced musculoskeletal abnormalities, scoliosis, nystagmus, and/or developmental delay.
Yang et al. mapped the BDA1 locus to chromosome 2q35-36 through linkage analysis of two Chinese families in 2000. Analysis of the function of genes located in this region suggested that Indian Hedgehog gene (IHH) be a good candidate gene for BDA1. Subsequent sequences analysis identified that mutations in the IHH gene were the causal mutations for three Chinese BDA1 families by Gao in 2001. However, IHH mutations account for only a subset of BDA1 cases. Armour et al. found BDA1 is linked to an 11cM critical region on chromosome 5p13.2-13.3 in a four generation BDA1 family of Canadian descent. Kirkpatrick et al. provided evidence that support the existence of a third locus for BDA1. These data suggested that the BDA1 is genetically heterogeneous.
We found a large brachydactyly family in Jining city, Shandong province. Clinical and family analysis revealed that the characterization of the cases in this family were consistent with the typical BDA1. To determine the causal gene of this BDA1 family, we carried out linkage analysis and mutation detection.
1 Family Study
A large family from Shandong province, China, was recruited in this study. Twenty six individuals (14 affected, 12 unaffected) were included in this study. Affected and unaffected individuals were physically examined, and six selected individuals (II-5, III-3, III-4, ffl-9, 111-12(?)111-13) were radiographed.
The defects found in the affected individuals of this family were confined to the hands and feet. The hands of the affected family members appeared to be broad and showed shortening of all digits. X-ray examination revealed anomalies of hand and foot bones, but the defects were mainly confined to the middle phalanges and metacarpals. The severity of BDA1 was varied among the affected members of the family. Individual III-4 is one of the most severe case, whose middle phalanges were all absent or fused to the distal phalanges and both the third and forth metacarpals were shortened in both hands. Another affected individual, III-3, also has all the middle phalanges missing and shows shortened metacarpals. But her shortened metacarpals were limited to the first and third metacarpals, and her proximal 1~(st) phalange was shortened, too. To our knowledge, the previously reported BDA1 patients were not known to have severely shortened first metacarpals. Other affected members examined by radiography had shortened middle phalanges, though with varying numbers.
2 Linkage Analysis and Mutation Detection
Two known disease loci located in 2q35-2q36 and 5pl3.2-pl3.3, respectively. So we selected polymorphic microsatellite markers from these two regions for linkage analysis. Result of genotyping D5S1470 located in 5p13.2-p13.3 showed that patients in this BDA1 family carried different alleles and excluded that the causal gene for this BDA1 family is in this region. Using a set of markers spanning the IHH gene, we were able to demonstrate that BDA1 was linked to chromosome 2q35-q36. Complete linkage was shown for markers D2S2250, D2S433, D2S163, and D2S2359, and recombination event was observed between D2S434 and D2S2250 in individual 111-13. These results suggested IHH gene may be the best candidated gene for this BDA1 family.
Four pairs of primers were designed to amplify all three exons and exon/intron boundaries of the IHH gene. Agarose gel analysis showed the sizes of PCR products were consistent with the expectation and indicated that there were no large insert or deletion mutitons of IHH gene. The mutation screen limited to the DNA from one affected (IV-3) and one unaffected individual (IV-2) with the direct sequencing of PCR products identified a heterozygous G-to-A transition (G298A) in exon 1, which results in an aspartate to asparagine substitution (D100N). To detect the G298A mutation in all family members and controls, a modified primer was used to create a restriction endonuclease (Sal 1) site to distinguish 298G and 298A alleles. By using PCR-RFLP analysis, the G298A transition was identified in all affected individuals. Moreover, the G298A allele was not detected among 200 chromosomes from unrelated normal Chinese controls.
3 Haplotype Analysis
The IHH G298A mutation had previously been identified in six Caucasian families with BDA1, and McCready et al. provided the evidence that support the existence of a common ancestral allele between Farabee's family and Drinkwater's families.To discriminate either a common founder mutation or a de novo mutation contributed to this Chinese kindred, we constituted the disease-associated haplotype with microsatellite markers D2S2250, D2S433, D2S163, and D2S2359 that are tightly linked to the IHH gene and five intragenic SNPs described by McCready of this Chinese family. Then the disease-related haplotype of this Chinese BDA1 family was compared with the common haplotype reported for the Farabee-Drinkwater families.
A comparison of the haplotypes from different families indicated our patients carried different alleles compared to the patients from Farabee-Drinkwater's families at three microsatellite markers (D2S2250, D2S433, and D2S163) and two intragenic SNPs (SNPe and SNPd). Another three intragenic SNPs (SNPa, SNPb, and SNPc) were not informative in our family and ethnic-matched controls. We then concluded that the IHH gene G298A mutation in this Chinese BDA1 family had different origin from those in Caucasian families. It looked like the IHH gene G298A mutation occurred more than once historical.
In conclusion, we identified a BDA1 family through clinical research and family analysis. By using linkage analysis, we excluded the linkage of the causal gene with 5p13.2-p13.3 and confirmed the linkage with 2q35-2q36. Combined candidate gene analysis and mutation detection, we identified a heterozygous missense mutation G298A in exon 1 of IHH gene, which results in an aspartate to asparagine substitution (D100N) was the causal mutation of this BDA1 family. Comparision of disease-related haplotypes from this family with the Farabee-Drinkwater families' revealed that a de novo G298A mutation, not a fouder mutation, caused the BDA1 of this family. Sequence analysis around IHH gene 298G showed that it maybe a mutational hot spot due to spontaneous deamination of a 5-methylcytosine on the noncoding strand of the gene. As a mutational hot spot, we predict that the IHH G298A mutation will be identified in additional BDA1 families.
Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. Osteoporosis results from a failure to acquire optimal peak bone mass during growth and/or to maintain bone mass in later years, which means a disruption of the fine equilibrium between the activity of osteoblasts responsible for bone deposition and osteoclasts for bone resorption.
Low-density lipoprotein receptor related protein 5, LRP5, is a member of low-density lipoprotein receptor family. LRP5 have multiple functions by banding and internalizing different ligands. Recent studies have indicated that the loss-of-function mutations in LRP5 gene cause Osteoporosis-Pseudoglioma syndrome, an autosomal recessive disorder characterized by severe juvenile-onset osteoporosis and congenital or juvenile-onset blindness. The gain-of-function mutations in LRP5 gene lead to increased bone mass and osteopetroses.
To further understand the role of LRP5 in bone formation, we used mouse bone marrow stromal cell line ST2 as a study model. We transfected ST2 cells with the constructs expressing Lrp5 gain-of-function muatation and LRP5-RNAi, respectively. We analyzed the effect of LRP5 status on ST2 cells proliferation and differentiation in the stable-transfected cells. With Suppression Subtractive Hybridization (SSH) technique, we constructed the subtractive cDNA libraries of ST2 cells stable-expressed Lrp5 gene with gain-of-function muatation and Lrp5-RNAi construct, respectively. The LRP5-related genes could be screened from these subtractive cDNA libraries.
1. Generation and transfection of the construct expressing the Lrp5 gene with G171V mutation
Fusion-PCR method was used to introduce G171V mutation using the wild Lrp5 expression constructs as the template. The resulting fragments containing the G171V mutation was used to replace the corresponding fragment from the wild type cDNA, and resulting insert was cloned into pcDNA3.1C. vector and named as pcDNA3.1C-GFLrp5. The construct was confirmed by PCR, restriction enzyme digestion and direct sequencing. The pcDNA3.1C-GFLrp5 Construct was transfected into ST2 cells. Cell-based immunofluorescence and Western Blotting analysis showed that pcDNA3.1C-GFLrp5 expressed in ST2 cells and transported to the membrane.
To inhibit the Lrp5 expression, we transfected the Lrp5-RNAi constructs, mu6/LRP5 to ST2 cells. The efficiency RNAi targeting was confirmed by cotransfectiong cells with mu6/LRP5 and pcDNA3.1C-GFLrp5. The decreasing in the expression of the pcDNA3.1C-GFLrp5 construct was observed with the anti-myc antibody by cell-based immunofluorescence and Western Blot.
2. Effects of stable expressing pcDNA3.1C-GFLrp5 construct and Lrp5-RNAi construct mu6/LRP5 on proliferation and differentiation of ST2 cells
ST2 Cells, a clone of stromal cells, were isolated from the bone marrow of BC8 mice, capable of the potential ability to differentiate into various mesenchymal cell lineages. However, when subcultured with medium supplemented with ascorbic acid, the ST2 cells developed an osteoblastic phenotype. We transfected pcDNA3.1C-GFLrp5 construct and the Lrp5 gene RNAi mu6/LRP5 construct into the cultured ST2 and selected with G418. The cells stable-expressiong Lrp5 G171V mutation expression construct and Lrp5 RNAi expression construct were obtained, and named as S-LGF and S-LLF, respectively.
MTT assays revealed that the cells proliferation of S-LGF was significantly higher than that of S-LLF cells measured at different time points: cultured for 24 hours, 48 hours and 72 hours. This suggested that the exogenous Lrp5 G171V mutant protein can notably enhance the proliferation of ST2 cells compared with ST2 cells in which Lrp5 were silenced. The more bone-like nodules were observed in the S-LGF than S-LLF cells by von Kossa's staining when cells cultured for 28 days under conditioned medium supplemented with ascorbic acid. The difference was much more significantly when cells cultured for 40 days.
3. Construction and screening of the S-LGF and S-LLF subtractive cDNA libraries
Suppression subtractive hybridization (SSH) is the combination of suppression PCR and subtractive hybridization technique. This technique could clone the unknown sequence of differentially expressed genes. The method is characterized with high efficiency, low false positive rate, targeting sequence enrichment, and low complexity of the experimental results. Using the S-LGF mRNA as the tester and the S-LLF mRNA as driver, we constructed the forward subtracted cDNA library. The reverse subtracted cDNA library was constructed by using the S-LLF mRNA as the tester and the S-LGF mRNA as driver.
Combining PCR amplification with cultured E. coli as template and the restriction endonuclease digestion, we screened the forward and reverse subtracted cDNA libraries. We found 105 clones with insert fragment out of 200 random selected clones from the forward subtracted cDNA library and 97 out of 200 from the reverse subtracted cDNA library. According to the patterns of restriction endonuclease digestion, 60 and 61 clones with different inserts from the forward and reverse subtracted cDNA libraries were selected for sequencing, respectively.
4. The differentially expressed sequences
We obtained 51 cDNA fragments differentially expressed in the S-LGF cells by comparison analysis with nucleotide sequences in GenBank. There are 44 cDNA fragments which were highly homologous with the known genes. Three cDNA fragments belong to the RIKEN cDNA gene. While other 4 cDNA fragments were not homologous with any known genes, which may be from novel genes. Similarly, 58 differentially expressed cDNA fragments in the S-LLF cells were obtained. Among them, 43 cDNA fragments were from known homologous genes and eight were RIKEN cDNA genes. Also, seven differentially expressed cDNA fragments were not matched to known homologous sequence.
These differentially expressed sequences will be confirmed by RT-PCR and real-time PCR. Further analysis of these differentially expressed genes may lead to identification of new genes related with Lrp5 controlling osteoblast proliferation and function and eventually shed new light on the molecular mechanisms and the new drugs development for the treatment of osteoporosis.
引文
1. Gong Y, Chitayat D, Kerr B, Chen T, Babul-Hirji R, Pal A, Reiss M, Warman ML. Brachydactyly type B: clinical description, genetic mapping to chromosome 9q, and evidence for a shared ancestral mutation. Am J Hum Genet 1999; 64: 570-577.
2. Farabee WC. Hereditary and sexual influence in meristic variation: a study of malformations in man. PhD thesis, Harvard University, 1903.
3. Bell J. On brachydactyly and symphalangism: introduction and classification of cases. Treasury of Human Inheritance 1951; 5:1-31.
4. Haws DV. Inherited brachydactyly and hypoplasia of the bones of the extremities. Ann Hum Genet 1963; 26: 201-212.
5. Thomsen O. Hereditary growth anomaly of the thumb. Hereditas 1928; 10: 261-273.
6. Villaverde MM, DiSilva JA. Distal brachyphalangy of the thumb in mental retardation. J Med Genet 1975; 12: 401-404.
7. McKusick V. Mendelian Inheritance in Man. Johns Hopkins University Press 1975.
8. Fitch N. Classification and identification of inherited brachydactylies. J Med Genet 1979; 16: 36-44.
9. Osebold WR, Remondini DJ, Lester EL, Spranger JW, Opitz JM. An autosomal dominant syndrome of short stature with mesomelic shortness of limbs, abnormal carpal and tarsal bones, hypoplastic middle phalanges, and bipartite calcanei. Am J Med Genet 1985; 22: 791-809.
10. Drinkwater H. An account of a brachydactylous family. Proc R Soc Edin 1908; 28: 35-57.
11. Drinkwater H. Account of a family showing minor brachydactyly. J Genet 1912; 2: 21-40.
12. Drinkwater H. A second brachydactylous family. J Genet 1915; 4: 323-339.
13. Temtamy SA, McKusick VA. The genetics of hand malformations. Birth Defects Orig Artic Ser 1978; 14: 1-619.
14. Laporte G, Serville F, Peant J. Type A1 branchydactyly. Study of one family. Nouv Presse Med 1979; 8: 4095-4907.
15. Piussan C, Lenaerts C, Mathieu M, Boudailliez B. Regular dominance of thumb ankylosis with mental retardation transmitted over 3 generations. J Genet Hum 1983; 31:107-114.
16. Tsukahara M, Azuno Y, Kajii T. Type A1 brachydactyly, dwarfism, ptosis, mixed partial hearing loss, microcephaly, and mental retardation. Am J Med Genet 1989; 33: 7-9.
17. Fukushima Y, Ohashi H, Wakui K, et al. De novo apparently balanced reciprocal translocation between 5q11.2 and 17q23 associated with Klippel-Feil anomaly and type A1 brachydactyly. Am J Med Genet 1995; 57: 447-449.
18. Mastrobattista JM, Dolle P, Blanton SH, Northrup H. Evaluation of candidate genes for familial brachydactyly. J Med Genet 1995; 32:851-854.
19. Raff ML, Leppig KA, Rutledge JC, Weinberger E, Pagon RA. Brachydactyly type A1 with abnormal menisci and scoliosis in three generations. Clin Dysmorphol 1998; 7: 29-34.
20. Yang X, She C, Guo J, et al. A locus for brachydactyly type A-1 maps to chromosome 2q35-q36. Am J Hum Genet 2000; 66: 892-903.
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23. Giordano N, Germari L, Bruttini M, et al. Mild brachydactyly type A1 maps to chromosome 2q35-q36 and is caused by a novel IHH mutation in a three generation family. J Med Genet 2003; 40:132-135.
24. Kirkpatrick TJ, Au KS, Mastrobattista JM, McCready ME, Bulman DE, Northrup H. Identification of a mutation in the Indian Hedgehog (IHH) gene causing brachydactyly type A1 and evidence for a third locus. J Med Genet 2003; 40: 42-44.
25. McCready ME, Grimsey A, Styer T, Nikkel SM, Bulman DE. A century later Farabee has his mutation. Hum Genet 2005; 117: 285-287.
26. Liu M, Wang X, Cai Z, et al: A novel heterozygous mutation in the Indian hedgehog gene (IHH) is associated with brachydactyly type A1 in a Chinese family. J Hum Genet 2006; 51:727-731.
27. Vortkamp A, Lee K, Lanske B, et al. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 1996; 273: 613-622.
28. St-Jacques B, Hammerschmidt M, McMahon AP. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev 1999; 13: 2072-2086.
29. Armour CM, McCready ME, Balg A, Hunter AG, Bulman DE. A novel locus for brachydactyly type A1 on chromosome 5p13.3-p13.2. J Med Genet 2002; 39: 186-188.
30. McCready ME, Sweeney E, Fryer AE, et al: A novel mutation in the IHH gene causes brachydactyly type A1: a 95-year-old mystery resolved. Hum Genet 2002; 111: 368-375.
31. Hellemans J, Coucke PJ, Giedion A, et al. Homozygous mutations in IHH cause acrocapitofemoral dysplasia, an autosomal recessive disorder with cone-shaped epiphyses in hands and hips. Am J Hum Genet 2003; 72: 1040-1046.
32. Gao B, He L. Answering a century old riddle:brachydactyly type A1. Cell Res 2004; 14: 179-187.
33. Ingham PW, McMahon AP. Hedgehog signaling in animal development: paradigms and principles. Genes Dev 2001; 15: 3059-3087.
34. Porter JA, yon Kessler DP, Ekker SC, et al. The product of hedgehog autoproteolytic cleavage active in local and long-range signalling. Nature 1995; 374: 363-366.
35. Porter JA, Young KE, Beachy PA: Cholesterol modification of hedgehog signaling proteins in animal development. Science 1996; 274: 255-259.
36. Lee JJ, Ekker SC, von Kessler DP, Porter JA, Sun BI, Beachy PA. Autoproteolysis in hedgehog protein biogenesis. Science 1994; 266:1528-1537.
37. Holliday R, Grigg G. DNA methylation and mutation. Murat Res 1993; 285: 61-67.
38. Bottema CD, Bottema MJ, Ketterling RP, et al. Why does the human factor Ⅸ gene have a G+C content of 40%? Am J Hum Genet 1991; 49: 839-850.
39. Fahsold R, Hoffmeyer S, Mischung C, et al. Minor lesion mutational spectrum of the entire NF1 gene does not explain its high mutability but points to a functional domain upstream of the GAP-related domain. Am J Hum Genet 2000; 66: 790-818.
40. Murphy BC, Scriver CR, Singh SM. CpG methylation accounts for a recurrent mutation (c.1222C>T) in the human PAH gene. Hum Mutat 2006; 27: 975.
41. Pfeifer GP. Mutagenesis at methylated CpG sequences. Curr Top Microbiol Immunol 2006; 301:259-281.
1. Osteoporosis prevention, diagnosis, and therapy. NIH Consensus Development Panel on Osteoporosis Prevention, Diagnosis and Therapy. JAMA 2001; 285: 785-795.
2. Bonjour, JP, Theintz, G, Law, F, Slosman, D, Rizzoli, R. Peak bone mass. Osteoporos Int 1994; 4: 7-13.
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4. Krall EA, Dawson-Hughes B: Heritable and life-style determinants of bone mineral density. J Bone Miner Res 1993, 8: 1-9.
5. Gueguen R, Jouanny P, Guillemin F, Kuntz C, Pourel J, Siest G: Segregation analysis and variance components analysis of bone mineral density in healthy families. J Bone Miner Res 1995, 12:2017-2022.
6. Gong G, Stern HS, Cheng SC, et al. The association of bone mineral density with vitamin D receptor gene polymorphisms. Osteoporos Int, 1999; 9: 55-64.
7. Thakkinstian A, D_Este C, Eisman J, et al. Meta-analysis of molecular association studies: vitamin D receptor gene polymorphisms and BMD as a case study. J Bone Miner Res, 2004; 19: 419-428.
8. Albagha OM, McGuigan FE, Reid DM, et al .Estrogen receptor α gene polymorphisms and bone mineral density: haplotype analysis in women from the United Kingdom. J Bone Miner Res, 2002; 16: 128-134.
9. Becherini L, Gennari L, Masi L, et al. Evidence of a linkage disequilibrium between polymorphisms in the human estrogen receptor α gene and their relationship to bone mass variation in post menopausal Italian women. Hum Mol Genet, 2000; 9: 2043-2050.
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11. Mann V, Hobson EE, LIB, et al. A COL1A1 Spl binding site polymorphism predisposes to osteoporotic fracture by affecting bone density and quality. J Clin Invest, 2001; 107: 899-907.
12. McGuigan FE, ArmbrechtG, SmithR, et al. Prediction of osteoporotic fractures by bone densitometry and COL1A1 genotyping: a prospective, population-based study in men and women. Osteoporos Int, 2001; 12: 91-96.
13. Garnero P, Borel O, Somay-Rendu E, et al. Association between a functional interleukin-6 gene polymorphism and peak bone mineral density and postmenopausal bone loss in women: the OFELY study. Bone, 2002; 31: 43-50.
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2. Farabee WC. Hereditary and sexual influence in meristic variation: a study of malformations in man. PhD thesis, Harvard University, 1903.
3. Bell J. On brachydactyly and symphalangism: introduction and classification of cases. Treasury of Human Inheritance 1951; 5:1-31.
4. Haws DV. Inherited brachydactyly and hypoplasia of the bones of the extremities. Ann Hum Genet 1963; 26: 201-212.
5. Thomsen O. Hereditary growth anomaly of the thumb. Hereditas 1928; 10: 261-273.
6. Villaverde MM, DiSilva JA. Distal brachyphalangy of the thumb in mental retardation. J Med Genet 1975; 12: 401-404.
7. McKusick V. Mendelian Inheritance in Man. Johns Hopkins University Press 1975.
8. Fitch N. Classification and identification of inherited brachydactylies. J Med Genet 1979; 16: 36-44.
9. Osebold WR, Remondini DJ, Lester EL, Spranger JW, Opitz JM. An autosomal dominant syndrome of short stature with mesomelic shortness of limbs, abnormal carpal and tarsal bones, hypoplastic middle phalanges, and bipartite calcanei. Am J Med Genet 1985; 22: 791-809.
10. Drinkwater H. An account of a brachydactylous family. Proc R Soc Edin 1908; 28: 35-57.
11. Drinkwater H. Account of a family showing minor brachydactyly. J Genet 1912; 2: 21-40.
12. Drinkwater H. A second brachydactylous family. J Genet 1915; 4: 323-339.
13. Temtamy SA, McKusick VA. The genetics of hand malformations. Birth Defects Orig Artic Ser 1978; 14: 1-619.
14. Laporte G, Serville F, Peant J. Type A1 branchydactyly. Study of one family. Nouv Presse Med 1979; 8: 4095-4907.
15. Piussan C, Lenaerts C, Mathieu M, Boudailliez B. Regular dominance of thumb ankylosis with mental retardation transmitted over 3 generations. J Genet Hum 1983; 31:107-114.
16. Tsukahara M, Azuno Y, Kajii T. Type A1 brachydactyly, dwarfism, ptosis, mixed partial hearing loss, microcephaly, and mental retardation. Am J Med Genet 1989; 33: 7-9.
17. Fukushima Y, Ohashi H, Wakui K, et al. De novo apparently balanced reciprocal translocation between 5q11.2 and 17q23 associated with Klippel-Feil anomaly and type A1 brachydactyly. Am J Med Genet 1995; 57: 447-449.
18. Mastrobattista JM, Dolle P, Blanton SH, Northrup H. Evaluation of candidate genes for familial brachydactyly. J Med Genet 1995; 32:851-854.
19. Raff ML, Leppig KA, Rutledge JC, Weinberger E, Pagon RA. Brachydactyly type A1 with abnormal menisci and scoliosis in three generations. Clin Dysmorphol 1998; 7: 29-34.
20. Yang X, She C, Guo J, et al. A locus for brachydactyly type A-1 maps to chromosome 2q35-q36. Am J Hum Genet 2000; 66: 892-903.
21. den Hollander NS, Hoogeboom AJ, Niermeijer MF, Wladimiroff JW. Prenatal diagnosis of type A1 brachydactyly. Ultrasound Obstet Gynecol. 2001; 17: 29-30.
22. Gao B, Guo J, She C, et al. Mutations in IHH, encoding Indian hedgehog, cause brachydactyly type A-1. Nat Genet 2001; 28: 386-388.
23. Giordano N, Germari L, Bruttini M, et al. Mild brachydactyly type A1 maps to chromosome 2q35-q36 and is caused by a novel IHH mutation in a three generation family. J Med Genet 2003; 40:132-135.
24. Kirkpatrick TJ, Au KS, Mastrobattista JM, McCready ME, Bulman DE, Northrup H. Identification of a mutation in the Indian Hedgehog (IHH) gene causing brachydactyly type A1 and evidence for a third locus. J Med Genet 2003; 40: 42-44.
25. McCready ME, Grimsey A, Styer T, Nikkel SM, Bulman DE. A century later Farabee has his mutation. Hum Genet 2005; 117: 285-287.
26. Liu M, Wang X, Cai Z, et al: A novel heterozygous mutation in the Indian hedgehog gene (IHH) is associated with brachydactyly type A1 in a Chinese family. J Hum Genet 2006; 51:727-731.
27. Vortkamp A, Lee K, Lanske B, et al. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 1996; 273: 613-622.
28. St-Jacques B, Hammerschmidt M, McMahon AP. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev 1999; 13: 2072-2086.
29. Armour CM, McCready ME, Balg A, Hunter AG, Bulman DE. A novel locus for brachydactyly type A1 on chromosome 5p13.3-p13.2. J Med Genet 2002; 39: 186-188.
30. McCready ME, Sweeney E, Fryer AE, et al: A novel mutation in the IHH gene causes brachydactyly type A1: a 95-year-old mystery resolved. Hum Genet 2002; 111: 368-375.
31. Hellemans J, Coucke PJ, Giedion A, et al. Homozygous mutations in IHH cause acrocapitofemoral dysplasia, an autosomal recessive disorder with cone-shaped epiphyses in hands and hips. Am J Hum Genet 2003; 72: 1040-1046.
32. Gao B, He L. Answering a century old riddle:brachydactyly type A1. Cell Res 2004; 14: 179-187.
33. Ingham PW, McMahon AP. Hedgehog signaling in animal development: paradigms and principles. Genes Dev 2001; 15: 3059-3087.
34. Porter JA, yon Kessler DP, Ekker SC, et al. The product of hedgehog autoproteolytic cleavage active in local and long-range signalling. Nature 1995; 374: 363-366.
35. Porter JA, Young KE, Beachy PA: Cholesterol modification of hedgehog signaling proteins in animal development. Science 1996; 274: 255-259.
36. Lee JJ, Ekker SC, von Kessler DP, Porter JA, Sun BI, Beachy PA. Autoproteolysis in hedgehog protein biogenesis. Science 1994; 266:1528-1537.
37. Holliday R, Grigg G. DNA methylation and mutation. Murat Res 1993; 285: 61-67.
38. Bottema CD, Bottema MJ, Ketterling RP, et al. Why does the human factor Ⅸ gene have a G+C content of 40%? Am J Hum Genet 1991; 49: 839-850.
39. Fahsold R, Hoffmeyer S, Mischung C, et al. Minor lesion mutational spectrum of the entire NF1 gene does not explain its high mutability but points to a functional domain upstream of the GAP-related domain. Am J Hum Genet 2000; 66: 790-818.
40. Murphy BC, Scriver CR, Singh SM. CpG methylation accounts for a recurrent mutation (c.1222C>T) in the human PAH gene. Hum Mutat 2006; 27: 975.
41. Pfeifer GP. Mutagenesis at methylated CpG sequences. Curr Top Microbiol Immunol 2006; 301:259-281.
1. Osteoporosis prevention, diagnosis, and therapy. NIH Consensus Development Panel on Osteoporosis Prevention, Diagnosis and Therapy. JAMA 2001; 285: 785-795.
2. Bonjour, JP, Theintz, G, Law, F, Slosman, D, Rizzoli, R. Peak bone mass. Osteoporos Int 1994; 4: 7-13.
3. Ferrari, S, Rizzoli, R, Bonjour, JP. Heritable and nutritional influences on bone mineral mass. Aging (Milano) 1998; 10:205-213.
4. Krall EA, Dawson-Hughes B: Heritable and life-style determinants of bone mineral density. J Bone Miner Res 1993, 8: 1-9.
5. Gueguen R, Jouanny P, Guillemin F, Kuntz C, Pourel J, Siest G: Segregation analysis and variance components analysis of bone mineral density in healthy families. J Bone Miner Res 1995, 12:2017-2022.
6. Gong G, Stern HS, Cheng SC, et al. The association of bone mineral density with vitamin D receptor gene polymorphisms. Osteoporos Int, 1999; 9: 55-64.
7. Thakkinstian A, D_Este C, Eisman J, et al. Meta-analysis of molecular association studies: vitamin D receptor gene polymorphisms and BMD as a case study. J Bone Miner Res, 2004; 19: 419-428.
8. Albagha OM, McGuigan FE, Reid DM, et al .Estrogen receptor α gene polymorphisms and bone mineral density: haplotype analysis in women from the United Kingdom. J Bone Miner Res, 2002; 16: 128-134.
9. Becherini L, Gennari L, Masi L, et al. Evidence of a linkage disequilibrium between polymorphisms in the human estrogen receptor α gene and their relationship to bone mass variation in post menopausal Italian women. Hum Mol Genet, 2000; 9: 2043-2050.
10. Grant SF, Reid DM, Blake G, et al. Reduced bone density and osteoporosis associated with a polymorphic Spl site in the collagen typel α1 gene. Nat Genet, 1996; 14: 203-205.
11. Mann V, Hobson EE, LIB, et al. A COL1A1 Spl binding site polymorphism predisposes to osteoporotic fracture by affecting bone density and quality. J Clin Invest, 2001; 107: 899-907.
12. McGuigan FE, ArmbrechtG, SmithR, et al. Prediction of osteoporotic fractures by bone densitometry and COL1A1 genotyping: a prospective, population-based study in men and women. Osteoporos Int, 2001; 12: 91-96.
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