Association analysis between four vitamin D receptor gene polymorphisms and developmental dysplasia of the hip

Developmental dysplasia of the hip (DDH) is a congenital condition characterized by abnormality in acetabulum size and/or shape. The incidence rate of DDH differs between different populations with risk factors including positive family history, breech presentation, sex, firstborn status, side of the hip, mode of delivery and oligohydramnios. It is recognized that DDH has a genetic component that exhibit autosomal dominant patterns. Many candidate genes have been studied and found to be associated with the disease; most of them are normally involved in cartilage development and joint metabolism. In this study, the association of four single-nucleotide polymorphisms (SNPs) (rs731236, rs1544410, rs7975232 and rs2228570) in the vitamin D receptor (VDR) gene was studied by a case–control analysis. The study sample involves 50 cases with confirmed DDH presentation and 50 nonDDH controls. SNPs were genotyped using conventional polymerase chain reaction (PCR) and restriction fragment-length polymorphism (RFLP) techniques. Genotype and allele frequencies were analysed using SPSS software. No significant associations were found between the VDR polymorphisms analysed and DDH. Further work need to be performed using genomewide analysis to elucidate the genetic basis of DDH.     

转化生长因子5在发育性髋关节发育不良中的作用研究进展

发育性髋关节发育不良(developmental dysplasia of the hip,DDH)是一种主要因髋臼、股骨近端和关节囊等存在结构性畸形而导致的不稳定关节病变,进而发展成为髋关节的脱位。髋关节内软骨发育不良、骨骼及肌腱的异常均可导致髋关节结构的畸形,最终造成DDH。因此,早期预防与诊断是DDH治疗的关键。研究表明,DDH具有遗传基础,其易感基因包括GDF5、HOXD9、COL2AL、PAPPA2等,其中遗传因子转化生长因子5(growth differentiation factor 5,GDF5)对软骨细胞的增殖、分化具有重要作用,是当前研究治疗DDH的热点之一。因而,了解GDF5基因对软骨发育及分化的影响,对于DDH的发病机制和治疗具有重要意义。基于此,综述了国内外近期探讨的GDF5在基因层面上对DDH的影响,以及通过关节内注射重组人GDF5等基于GDF5的DDH治疗方案,以期为DDH的临床治疗提供新的策略。     

Smooth,rough and upside-down neocortical development

Lissencephaly, which means 'smooth cortex', is caused by defective neuronal migration during development of the cerebral cortex and has devastating clinical consequences. 'Classical' lissencephaly seems to reflect mutations in regulators of the microtubule cytoskeleton, whereas 'cobblestone' lissencephaly is caused by mutations in genes needed for the integrity of the basal lamina of the central nervous system. Reelin, which is mutated in a third type of lissencephaly, may represent a unifying link because it encodes an extracellular protein that regulates neuronal migration and may also regulate the microtubule cytoskeleton.     

Distinct roles for the actin and microtubule cytoskeletons in the morphogenesis of epidermal hairs during wing development in Drosophila

We have found that the actin and microtubule cytoskeletons have overlapping, but distinct roles in the morphogenesis of epidermal hairs during Drosophila wing development. The function of both the actin and microtubule cytoskeletons appears to be required for the growth of wing hairs, as treatment of cultured pupal wings with either cytochalasin D or vinblastine was able to slow prehair extension. At higher doses a complete blockage of hair development was seen. The microtubule cytoskeleton is also required for localizing prehair initiation to the distalmost part of the cell. Disruption of the microtubule cytoskeleton resulted in the development of multiple prehairs along the apical cell periphery. The multiple hair cells were a phenocopy of mutations in the inturned group of tissue polarity genes, which are downstream targets of the frizzled signaling/signal transduction pathway. The actin cytoskeleton also plays a role in maintaining prehair integrity during prehair development as treatment of pupal wings with cytochalasin D, which inhibits actin polymerization, led to branched prehairs. This is a phenocopy of mutations in crinkled, and suggests mutations that cause branched hairs will be in genes that encode products that interact with the actin cytoskeleton.     

高通量测序技术在罕见病分子诊疗中的 应用及临床实例分析

罕见病病种繁多,且表型复杂多样,不仅仅体现在疾病间的不同,同一种疾病的不同患者在表型上也可能大相径庭。这种普遍存 在的遗传异质性和临床异质性,使罕见病的诊疗极具挑战。近年来,在后人类基因组计划时代,各种测序技术快速发展,使得大规模测 序如疾病目标基因集测序、全外显子组测序、全基因组测序等成为了现实。高通量测序技术可实现对多个靶基因进行高通量平行测序, 有效节约了成本与时间,越来越广泛地应用到临床疾病分子诊疗领域。分析传统测序技术与高通量测序技术的优缺点,介绍罕见病诊疗 中常用的高通量测序策略,并结合临床实例,综述高通量测序技术在罕见病诊疗中的应用。     

European Society of Biomechanics S.M. Perren Award 2018: Altered biomechanical stimulation of the developing hip joint in presence of hip dysplasia risk factors

Fetal kicking and movements generate biomechanical stimulation in the fetal skeleton, which is important for prenatal musculoskeletal development, particularly joint shape. Developmental dysplasia of the hip (DDH) is the most common joint shape abnormality at birth, with many risk factors for the condition being associated with restricted fetal movement. In this study, we investigate the biomechanics of fetal movements in such situations, namely fetal breech position, oligohydramnios and primiparity (firstborn pregnancy). We also investigate twin pregnancies, which are not at greater risk of DDH incidence, despite the more restricted intra-uterine environment. We track fetal movements for each of these situations using cine-MRI technology, quantify the kick and muscle forces, and characterise the resulting stress and strain in the hip joint, testing the hypothesis that altered biomechanical stimuli may explain the link between certain intra-uterine conditions and risk of DDH. Kick force, stress and strain were found to be significantly lower in cases of breech position and oligohydramnios. Similarly, firstborn fetuses were found to generate significantly lower kick forces than non-firstborns. Interestingly, no significant difference was observed in twins compared to singletons. This research represents the first evidence of a link between the biomechanics of fetal movements and the risk of DDH, potentially informing the development of future preventative measures and enhanced diagnosis. Our results emphasise the importance of ultrasound screening for breech position and oligohydramnios, particularly later in pregnancy, and suggest that earlier intervention to correct breech position through external cephalic version could reduce the risk of hip dysplasia.     

Brief communication: developmental dysplasia of the hip in medieval London

Developmental dysplasia of the hip (DDH) is a spectrum of disease starting in childhood and in many cases persisting into adulthood. The spectrum ranges from acetabular dysplasia, through hip subluxation to dislocation. The aim of this research was to determine the prevalence and pathoanatomy of acetabular dysplasia and subluxation in excavated human skeletal remains, to complement past research on dislocation in DDH. The material under study was the medieval cemetery of St. Mary Spital in London, in use from c.1100 to 1539 AD. A series of 572 adults with both hips preserved were analyzed. Acetabular dysplasia was indicated by a shallow acetabulum with upward sloping roof. Subluxation was suggested by degenerative change along the margin of the acetabulum suggestive of labral tears, and degenerative change in the outer part of the acetabular roof suggestive of osteoarthritis. The prevalence of DDH (acetabular dysplasia, subluxation, or dislocation) was 1.7%. Because this a congenital musculoskeletal disorder of relatively high frequency, with significant variation in prevalence between populations around the world, it is a topic that warrants targeted research from physical anthropologists studying past populations.     

Pavlik吊带治疗早期发育性髋关节脱位的研究进展

发育性髋关节脱位(Developmental dysplasia of the hip,DDH)是指一系列髋关节结构异常的疾病,病变严重程度涵盖轻度髋臼发育不良到不可逆的髋关节脱位。DDH的治疗手段进展迅速,但早期闭合复位使用最多的仍是Pavlik吊带。通过超声可监测髋关节复位情况从而评估Pavlik吊带的治疗效果。同时,随着超生髋关节检查技术在我国的推广,使得早期诊断DDH成为可能,这也促进了Pavlik吊带在临床的应用。未来研究的方向在于制定基于询证医学证据的Pavlik吊带使用规范和开发出效果更好、并发症更少的新型支具。     

The identification of novel genes required for Drosophila anteroposterior axis formation in a germline clone screen using GFP-Staufen

The anteroposterior axis of Drosophila is defined during oogenesis, when the polarisation of the oocyte microtubule cytoskeleton directs the localisation of bicoid and oskar mRNAs to the anterior and posterior poles, respectively. Although maternal-effect lethal and female-sterile screens have identified many mutants that disrupt these processes, these screens could not recover mutations in essential genes. Here we describe a genetic screen in germline clones for mutants that disrupt the localisation of GFP-Staufen in living oocytes, which overcomes this limitation. As Staufen localises to the posterior with oskar mRNA and to the anterior with bicoid mRNA, it acts as a marker for both poles of the oocyte, allowing the identification of mutants that affect the localisation of either mRNA, as well as mutants that disrupt oocyte polarity. Using this approach, we have identified 23 novel complementation groups on chromosome 3R that disrupt anteroposterior axis formation. Analyses of new alleles of spn-E and orb show that both SPN-E and ORB proteins are required to organise the microtubule cytoskeleton at stage 9, and to prevent premature cytoplasmic streaming. Furthermore, yps mutants partially suppress the premature cytoplasmic streaming of orb mutants. As orb, yps and spn-E encode RNA-binding proteins, they may regulate the translation of unidentified RNAs necessary for the polarisation of the microtubule cytoskeleton.     

The rational strategies for detecting developmental dysplasia of the hip at the age of 4-6 months old infants: a prospective study

Using ultrasound in evaluation of infant's hip development can reduce surgical procedures, hospitalization and late presentation of developmental dysplasia of the hip (DDH). The increasing incidence of DDH after ultrasound examination is observed and published by many authors. In a prospective study, radiograph of every single ultrasonographic positive hip in infants older than three months, was taken and analyzed in order to see whether it affects infants splintage rate in treating DDH. In a period of 30 months, clinical and simple static ultrasonographic examinations according to Graf were performed on 1430 consecutive infant hips in patients aged between 4 and 6 months. Sonographic positive hips were radiographed and acetabular index (AI) values on simple AP radiographs were analyzed. The sonographic DDH incidence was 51.75 per 1000 hips (51.75 per thousand). After X-ray examination of all 74 ultrasonographic positive hips, only 44 remained abnormal and required treatment indicating a true DDH incidence of 30.77 per 1000 hips (30.77 per thousand). The difference in incidence per ultrasonographic and X-ray positive hips is statistically significant p < 0.01 (t = 5,536). The rational approach in detection of DDH in a child more than 3 months old is to do radiographic assessment of every sonographic positive hip.     

Association of interleukin-6 and transforming growth factor-β1 gene polymorphisms with developmental hip dysplasia and severe adult hip osteoarthritis: a preliminary study

Developmental hip dysplasia (DDH) greatly contributes to occurrence of severe hip osteoarthritis (OA) in adulthood, but the association between the two is not a perfect one. Both conditions are known to have a strong genetic component. Transforming growth factor β1 (TGF-β1) and interleukin-6 (IL-6) are two pro-inflammatory cytokines included in pathogenesis of OA, bone remodeling and development of bone and joint tissues. TGF-β1 gene has a polymorphic site in the signal sequence ((29)T→C) and "C allele carriage" is associated with higher circulating TGF-β1 levels. IL-6 gene has several polymorphic sites in the promoter region including -572T→C transition associated with higher circulating IL-6 levels. As a preliminary investigation on possible association between these polymorphisms and severe adult hip OA secondary to DDH, 28 consecutive patients and 20 healthy controls were genotyped at these loci. With adjustment for sex, "C allele carriage" in the TGF-β1 signal sequence and CC genotype ("transition homozygous") at locus -572 in the IL-6 promoter were each associated with severe OA secondary to DDH (OR=13.4, p=0.016 and OR=6.2, p=0.024, respectively). The combination of these genotypes was particularly strongly associated with the disease (OR=11.1, p<0.001). Data support feasibility of larger-scale studies on potential association between TGF-β1 signal sequence and IL-6 promoter polymorphisms and occurrence of DDH and (un)related severe OA.     

The fission yeast ran GTPase is required for microtubule integrity

The microtubule cytoskeleton plays a pivotal role in cytoplasmic organization, cell division, and the correct transmission of genetic information. In a screen designed to identify fission yeast genes required for chromosome segregation, we identified a strain that carries a point mutation in the SpRan GTPase. Ran is an evolutionarily conserved eukaryotic GTPase that directly participates in nucleocytoplasmic transport and whose loss affects many biological processes. Recently a transport-independent effect of Ran on spindle formation in vitro was demonstrated, but the in vivo relevance of these findings was unclear. Here, we report the characterization of a Schizosaccharomyces pombe Ran GTPase partial loss of function mutant in which nucleocytoplasmic protein transport is normal, but the microtubule cytoskeleton is defective, resulting in chromosome missegregation and abnormal cell shape. These abnormalities are exacerbated by microtubule destabilizing drugs, by loss of the spindle checkpoint protein Mph1p, and by mutations in the spindle pole body component Cut11p, indicating that SpRan influences microtubule integrity. As the SpRan mutant phenotype can be partially suppressed by the presence of extra Mal3p, we suggest that SpRan plays a role in microtubule stability.     

The rho-GAP encoded by BEM2 regulates cytoskeletal structure in budding yeast.

Microfilaments are required for polarized growth and morphogenesis in Saccharomyces cerevisiae. To accomplish this, actin cables and patches are redistributed during the cell cycle to direct secretory components to appropriate sites for cell growth. A major component of actin cables is tropomyosin I, encoded by TPM1, that determines or stabilizes these structures. Disruption of TPM1 is not lethal but results in the loss of actin cables and confers a partial defect in polarized secretion. Using a synthetic lethal screen, we have identified seven mutations residing in six genes whose products are required in the absence of Tpm1p. Each mutant exhibited a morphological defect, suggesting a functional link to the actin cytoskeleton. Complementation cloning of one mutation revealed that it lies in BEM2, which encodes a GTPase-activating protein for the RHO1 product. bem2 mutations also show synthetic lethality with rho1 and mutations in certain other cytoskeletal genes (ACT1, MYO1, MYO2, and SAC6) but not with mutations in several noncytoskeletal genes. These data therefore provide a genetic link between the GAP encoded by BEM2 and the functional organization of microfilaments. In addition, we show that bem2 mutations confer benomyl sensitivity and have abnormal microtubule arrays, suggesting that the BEM2 product may also be involved directly or indirectly in regulating microtubule function.     

Whole-exome re-sequencing in a family quartet identifies POP1 mutations as the cause of a novel skeletal dysplasia

Recent advances in DNA sequencing have enabled mapping of genes for monogenic traits in families with small pedigrees and even in unrelated cases. We report the identification of disease-causing mutations in a rare, severe, skeletal dysplasia, studying a family of two healthy unrelated parents and two affected children using whole-exome sequencing. The two affected daughters have clinical and radiographic features suggestive of anauxetic dysplasia (OMIM 607095), a rare form of dwarfism caused by mutations of RMRP. However, mutations of RMRP were excluded in this family by direct sequencing. Our studies identified two novel compound heterozygous loss-of-function mutations in POP1, which encodes a core component of the RNase mitochondrial RNA processing (RNase MRP) complex that directly interacts with the RMRP RNA domains that are affected in anauxetic dysplasia. We demonstrate that these mutations impair the integrity and activity of this complex and that they impair cell proliferation, providing likely molecular and cellular mechanisms by which POP1 mutations cause this severe skeletal dysplasia.     

Identification of sequence variants in genetic disease-causing genes using targeted next-generation sequencing

Background

Identification of gene variants plays an important role in research on and diagnosis of genetic diseases. A combination of enrichment of targeted genes and next-generation sequencing (targeted DNA-HiSeq) results in both high efficiency and low cost for targeted sequencing of genes of interest.

Methodology/Principal Findings

To identify mutations associated with genetic diseases, we designed an array-based gene chip to capture all of the exons of 193 genes involved in 103 genetic diseases. To evaluate this technology, we selected 7 samples from seven patients with six different genetic diseases resulting from six disease-causing genes and 100 samples from normal human adults as controls. The data obtained showed that on average, 99.14% of 3,382 exons with more than 30-fold coverage were successfully detected using Targeted DNA-HiSeq technology, and we found six known variants in four disease-causing genes and two novel mutations in two other disease-causing genes (the STS gene for XLI and the FBN1 gene for MFS) as well as one exon deletion mutation in the DMD gene. These results were confirmed in their entirety using either the Sanger sequencing method or real-time PCR.

Conclusions/Significance

Targeted DNA-HiSeq combines next-generation sequencing with the capture of sequences from a relevant subset of high-interest genes. This method was tested by capturing sequences from a DNA library through hybridization to oligonucleotide probes specific for genetic disorder-related genes and was found to show high selectivity, improve the detection of mutations, enabling the discovery of novel variants, and provide additional indel data. Thus, targeted DNA-HiSeq can be used to analyze the gene variant profiles of monogenic diseases with high sensitivity, fidelity, throughput and speed.     

An indication of major genes affecting hip and elbow dysplasia in four Finnish dog populations

The aim of the study was to assess the possible existence of major genes influencing hip and elbow dysplasia in four dog populations. A Bayesian segregation analysis was performed separately on each population. In total, 34 140 dogs were included in the data set. Data were analysed with both a polygenic and a mixed inheritance model. Polygenic models included fixed and random environmental effects and additive genetic effects. To apply mixed inheritance models, the effect of a major gene was added to the polygenic models. The major gene was modelled as an autosomal biallelic locus with Mendelian transmission probabilities. Gibbs sampling and a Monte Carlo Markov Chain algorithm were used. The goodness-of-fit of the different models were compared using the residual sum-of-squares. The existence of a major gene was considered likely for hip dysplasia in all the breeds and for elbow dysplasia in one breed. Several procedures were followed to exclude the possible false detection of major genes based on non-normality of data: permuted datasets were analysed, data-transformations were applied, and residuals were judged for normality. Allelic effects at the major gene locus showed nearly to complete dominance, with a recessive, unfavourable allele in both traits. Relatively high estimates of the frequencies of unfavourable alleles in each breed suggest that considerable genetic progress would be possible by selection against major genes. However, the major genes that are possibly affecting hip and elbow dysplasia in these populations will require further study.     

Convergence of mutation and epigenetic alterations identifies common genes in cancer that predict for poor prognosis

Background

The identification and characterization of tumor suppressor genes has enhanced our understanding of the biology of cancer and enabled the development of new diagnostic and therapeutic modalities. Whereas in past decades, a handful of tumor suppressors have been slowly identified using techniques such as linkage analysis, large-scale sequencing of the cancer genome has enabled the rapid identification of a large number of genes that are mutated in cancer. However, determining which of these many genes play key roles in cancer development has proven challenging. Specifically, recent sequencing of human breast and colon cancers has revealed a large number of somatic gene mutations, but virtually all are heterozygous, occur at low frequency, and are tumor-type specific. We hypothesize that key tumor suppressor genes in cancer may be subject to mutation or hypermethylation.

Methods and Findings

Here, we show that combined genetic and epigenetic analysis of these genes reveals many with a higher putative tumor suppressor status than would otherwise be appreciated. At least 36 of the 189 genes newly recognized to be mutated are targets of promoter CpG island hypermethylation, often in both colon and breast cancer cell lines. Analyses of primary tumors show that 18 of these genes are hypermethylated strictly in primary cancers and often with an incidence that is much higher than for the mutations and which is not restricted to a single tumor-type. In the identical breast cancer cell lines in which the mutations were identified, hypermethylation is usually, but not always, mutually exclusive from genetic changes for a given tumor, and there is a high incidence of concomitant loss of expression. Sixteen out of 18 (89%) of these genes map to loci deleted in human cancers. Lastly, and most importantly, the reduced expression of a subset of these genes strongly correlates with poor clinical outcome.

Conclusions

Using an unbiased genome-wide approach, our analysis has enabled the discovery of a number of clinically significant genes targeted by multiple modes of inactivation in breast and colon cancer. Importantly, we demonstrate that a subset of these genes predict strongly for poor clinical outcome. Our data define a set of genes that are targeted by both genetic and epigenetic events, predict for clinical prognosis, and are likely fundamentally important for cancer initiation or progression.     

An interaction type of genetic screen reveals a role of the Rab11 gene in oskar mRNA localization in the developing Drosophila melanogaster oocyte.

Abdomen and germ cell development of Drosophila melanogaster embryo requires proper localization of oskar mRNA to the posterior pole of the developing oocyte. oskar mRNA localization depends on complex cell biological events like cell-cell communication, dynamic rearrangement of the microtubule network, and function of the actin cytoskeleton of the oocyte. To investigate the cellular mechanisms involved, we developed a novel interaction type of genetic screen by which we isolated 14 dominant enhancers of a sensitized genetic background composed of mutations in oskar and in TropomyosinII, an actin binding protein. Here we describe the detailed analysis of two allelic modifiers that identify Drosophila Rab11, a gene encoding small monomeric GTPase. We demonstrate that mutation of the Rab11 gene, involved in various vesicle transport processes, results in ectopic localization of oskar mRNA, whereas localization of gurken and bicoid mRNAs and signaling between the oocyte and the somatic follicle cells are unaffected. We show that the ectopic oskar mRNA localization in the Rab11 mutants is a consequence of an abnormally polarized oocyte microtubule cytoskeleton. Our results indicate that the internal membranous structures play an important role in the microtubule organization in the Drosophila oocyte and, thus, in oskar RNA localization.     

Allopatric divergence and hybridization within Cupressus chengiana (Cupressaceae), a threatened conifer in the northern Hengduan Mountains of western China

Having a comprehensive understanding of population structure, genetic differentiation and demographic history is important for the conservation and management of threatened species. High‐throughput sequencing (HTS) provides exciting opportunities to address a wide range of factors for conservation genetics. Here, we generated HTS data and identified 266,884 high‐quality single nucleotide polymorphisms from 82 individuals of Cupressus chengiana, to assess population genomics across the species' full range, comprising the Daduhe River (DDH), Minjiang River (MJR) and Bailongjiang River (BLJ) catchments in western China. admixture , principal components analysis and phylogenetic analyses indicated that each region contains a distinct lineage, with high levels of differentiation between them (DDH, MJR and BLJ lineages). MJR was newly distinguished compared to previous surveys, and evidence including coalescent simulations supported a hybrid origin of MJR during the Quaternary. Each of these three lineages should be recognized as an evolutionarily significant unit (ESU), due to isolation, differing genetic adaptations and different demographic history. Currently, each ESU faces distinct threats, and will require different conservation strategies. Our work shows that population genomic approaches using HTS can reconstruct the complex evolutionary history of threatened species in mountainous regions, and hence inform conservation efforts, and contribute to the understanding of high biodiversity in mountains.