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- Subjects and Methods
- Supporting Information
We have identified and retrieved DNA from one of the largest documented families showing intergenerational transmission of DDH. The goal of this study is to identify the molecular basis of the disease in this family using the approach of genomewide linkage analysis together with whole-exome sequencing, as illustrated schematically in Fig. 4. By performing genome-wide linkage analysis, which makes no assumptions about where a mutation might reside, a 2.61-Mb candidate region on chromosome 3p22.2 has been identified with a high degree of certainty. However, classical linkage analysis requires unambiguous knowledge of who is affected in a given pedigree. Because this disorder is complex, with both environmental and genetic causes, and shows incomplete penetrance, not all individuals who appear unaffected will, in fact, be free of the disease allele. One method that we have chosen to address the problem of unknown, incomplete penetrance in our analysis is to define as affected only those family members whose diagnosis is certain (ie, those having three or more signs of DDH). Penetrance for these affected individuals is irrelevant and unaffected individuals are scored as having an unknown phenotype. Using this “affected only” model-based analysis assuming an autosomal dominant mode of inheritance, linkage analysis was performed and the results compared to model-free linkage analysis in which no assumption about the mode of inheritance was made. In both analyses the same candidate region on chromosome 3 resulted, with no other genomic region producing LOD scores higher than 0 in the model-based analysis or 2.055 in the model-free analysis.
The maximum LOD score of 3.31 was found in an interval spanning 38.91 to 40.66 Mb from the p-terminal end of chromosome 3 (Fig. 2). This 2.61-Mb region has within it several candidate genes that might explain many of the signs seen in individuals affected with DDH. To further scrutinize these candidates, exomes of four severely affected family members were sequenced. Results of sequencing for the chromosome 3 candidate region shown in Table 1 reflect a spectrum of variants including synonymous and nonsynonymous SNPs, variants that mapped to the untranslated region of various genes, deletions, and SNPs in intronic and intergenic regions, as well as seven novel mutations. Novel mutations are those that appear in Supplementary Table S1 without a dbSNP designation and are not found in the single nucleotide database 135. One of these novel mutations, an indel in the DLEC gene, was located in a part of the coding sequence found to be highly variable in evolution. The six other novel mutations occurred in intronic regions not related to mRNA splicing or in untranslated regions of their respective genes. None of these were in conserved genomic regions and it is difficult to evaluate their significance at this time.
In order to predict whether shared nonsynonymous variants might be deleterious to the function of the respective genes, they were analyzed by both PolyPhen-2 and SIFT. Among the subset of nine shared nonsynonymous variants, only one was predicted to be detrimental to protein function. The variant rs3732378 causes a C to T transition in the coding region of the chemokine receptor (CX3CR1). This missense mutation converts a threonine (polar) to methionine (non-polar) at position 280 in the transmembrane domain. PolyPhen-2, which uses structural features of the protein as well as information on evolutionary conservation, predicted that this mutation was “possibly damaging.” SIFT predicts that this mutation is damaging (score of 0.01, with a range of 0 = damaging to 0.05 = tolerated) (Supplementary Table S1).
Sequence conservation in evolution is often considered a measure of relative biologic importance. GERP is a method for producing position-specific estimates of evolutionary constraint using maximum likelihood evolutionary rate estimation. None of the candidate region variants (with the exception of rs3732378) were found to localize in regions of the genome that were highly evolutionarily conserved. At position 280 in CX3CR1, the threonine to methionine mutation, shared by all affected individuals in this family, has a GERP score of 3.98; any positive GERP score >2 is considered evidence of evolutionary constraint. Threonine 280 is conserved in all mammals, the chicken, and the D. rerio species of fish, lending support to this analysis.
The presence of this SNP was validated by Sanger sequencing in the four affected individuals and in the DNA of all 13 other affected individuals and obligate heterozygotes within the family. Sanger sequencing was also performed to discover the presence of this SNP in individuals with fewer signs of DDH as well as in seemingly unaffected married-in individuals (see Fig. 3). Individuals 8, 18, 32, 38, 44, 45, and 59, who appeared to have fewer signs of DDH, were found to carry this variant. Interestingly, only one questionable child (individual 59) was found to have the disease variant, whereas in the DNA of the other adolescents who had been thought to have signs of DDH (individuals 27, 30, 31, 62, and 63), the mutation was missing. Five married in individuals (14, 21, 46, 58, 64) were also found to have this mutation in their DNA, supporting the fact that the prevalence of this allele is very high in the indigenous Utah population from which this family originates. Analysis of genomewide SNPs did not show any evidence of high levels of homozygosity in the genomes of the family members, which would have been suggestive of inbreeding, and none of the spouses appeared to be related by SNP analysis.
The incomplete penetrance of DDH seen in some members of this family is understandable in light of the fact that DDH is a complex disorder with multiple genetic, epigenetic, and environmental causes. Indeed, the allele frequency of this relatively common variant varies from 3% in the Han Chinese to 8% in whites (Caucasians) and is higher in other populations, including this one (Utah residents with Northern and Western European ancestry), whose allele frequency is 0.173, one of the highest recorded in the United States. As found in most other multifactorial disorders, a number of individuals including the obligate heterozygotes and some “married-in” individuals in this family carry this allele but manifest no signs of DDH. This supports the idea that in this family this variant may be necessary but not sufficient by itself to cause the disorder. We are vigorously pursuing other etiologic factors that might explain the severity of DDH in this family and plan to address this issue at a later point in this continuing study.
Epigenetic factors that may cause altered gene expression of CX3CR1 include the histone modifications H3k4Me1 and Me2. These methylation sites, which are often located near regulatory elements, span this variant sequence in seven cell lines. This DNA site also includes a binding site for the transcription factor Jun D in the K562 cell line. Jun D is required for bone morphogenic protein 4 (BMP4)-induced hematopoiesis in Xenopus. The significance of the presence of a PABPC1 spanning this DNA segment is not clear.
Although this study appears to have identified a significant genetic risk factor shared by all affected members of this family, it is not known how prevalent this variant will be in the overall DDH patient population. We are in the process of validating this mutation in our DNA databank of over 30 dysplastics. Additionally, exome sequencing does not detect mutations that could exist in intergenic regions within the chromosome 3 candidate region. Important regulatory sequences that affect gene expression could reside there. Also not known is whether the association of the CX3CR1 variant with DDH susceptibility will prove to be an etiologic factor. In vitro and in vivo experiments are currently underway to answer this question. Finally, pleiotropy with age-related macular degeneration (AMD) and cardiovascular disease cannot be ruled out or confirmed in this family.
This receptor, CX3CR1, which is involved in cell adhesion and migration, is known to play a role in chondrocyte maturation, being expressed at higher levels in mesenchymal stem cells and becoming downregulated during the process of chondrocyte development. Recently in human mesenchymal stem cells that were induced to differentiate to chondrocytes, Djouad and colleagues observed that CX3CR1 was present at higher levels in the stem cell and not expressed in the differentiated chondrocyte. Additionally, Cristino and colleagues, using a three-dimensional hyaluronic acid scaffold, have found that the chemokine receptors CXCR4 and CXCR5 were modulated during in vitro chondrogenic differentiation, suggesting a role for the CXC class of chemokines in the differentiation and maturation of a cartilage-like structure in vitro. The ability of mesenchymal cells to migrate and to receive regulatory signals from their environment via chemokine receptors may play a role in the developing structure of the cartilage anlage of the acetabulum.
The specific variant found in the DNA of all affected family members in this study is known to have significant biologic effects in a number of organ systems, all related to CX3Cr1's capacity to regulate cell migration or adhesion. In 2007, Combadière and colleagues found that homozygosity for the CX3CR1 M280 allele was consistently more frequent in AMD patients compared to controls, observing that impaired migration of microglial cells occurred in affected patients. They also found that chemotaxis of monocytes from individuals with homozygous M280 was impaired in the presence of bound Cx3CR1. Cx3CR1 is a human immunodeficiency virus (HIV) coreceptor as well as a leukocyte chemotactic/adhesion receptor for fractalkine and is overexpressed in the lymph nodes of HIV patients. Individuals with the M280 variant are more susceptible to HIV infection resulting from compromised migration of immune cells. Finally, in cardiovascular disease, the M280 variant provided a protective effect against carotid and coronary artery disease and stroke by decreasing cellular adhesion.
All severely and moderately affected individuals were heterozygous for the M280 variant with a 50% complement of unmutated CX3CR1. The effect of this possibly-decreased amount of fully functional receptor on the development of the acetabular labrum is unknown and is currently being investigated in animal models. The lack of effect of the variant on other organ systems in which it is present could be a result of a lack of sensitivity during a particular phase of development and the fact that they can function within their biological limits on a possibly lower concentration of this protein. Additionally, the unique morphology of the developing human hip might account for the tissue-specific phenotypic effect of this heterozygous mutant.
To explain the severity DDH in some individuals but not in others, we hypothesized that severely affected individuals might have a second aggravating mutation. To determine which variants these might be, we searched throughout the human exome for variants shared by four severely affected individuals that caused various kinds of mutations (see Subjects and Methods) and that resided in pathways related to bone formation. The variants described in Table 2 map outside the chromosome 3 candidate region and for this reason are unlikely to be shared by all affecteds and by obligate heterozygotes because linkage analysis has shown that there is likely to be only one co-inherited genetic locus. One rare variant (rs77857664 allele frequency 0.361%), a T to C transition, was found in intron 1 of BMP8B, a gene known to be involved in development of the skeletal system. OXCT2 is a relatively small gene located entirely within intron 1 of BMP8B and has a function unrelated to bone metabolism. This T to C transition, which also causes a glutamic acid to glycine amino acid change in the OXCT2 protein, is located in a transcriptionally active region, one with possible regulatory elements, as shown by a DNase I hypersensitivity cluster and an H3k27 acetylation mark found within this region by the ENCODE consortium. SIFT analysis predicts this variant to have the potential to increase reproductive fitness.
Three of the observed variants shared by four severely affected family members involve insertion or deletions of repetitive amino acid(s). One of these in-frame variants in the transcription factor E2F4, on chromosome 16q22.1, is a novel mutation involving the insertion of the nucleotide triplet CAG in a highly conserved DNA sequence. This transcription factor participates in the transforming growth factor-beta (TGF-beta) superfamily mediated signaling pathway, and deficiency of the E2F4 transcript leads to abnormal turbinate bone formation. Another variant that causes an in-frame insertion of two leucines is latent transforming growth factor beta binding protein (LTBP3) on chromosome 11q12. This protein, which forms a complex with TGF-beta, may be involved in their subcellular localization. Dabovic and colleagues created an Ltbp3-null mutation in the mouse by gene targeting. Mice homozygous for the mutation developed craniofacial malformations by day 10. At 2 months, there was a pronounced rounding of the cranial vault, extension of the mandible beyond the maxilla, and kyphosis. Between 6 and 9 months of age, mutant mice also developed osteosclerosis and osteoarthritis. The pathologic changes were consistent with perturbed TGF-beta signaling in the skull and long bones. The third variant causes an in-frame deletion of glutamine in retinoic acid inducible gene 1 (RAI 1) on chromosome 17p11.2. Inactivation of RAI1 in the mouse recapitulates phenotypes seen in Smith Magenis syndrome. Bone-related signs of this syndrome are deficient rib and nose cartilage formation.
The last two variants shared by four severely affected family members are potentially more disruptive. A deleted G in exon 2 of the RAI1 gene causes a frameshift mutation at position 280 in the protein. Two individuals (3 and 9) are homozygous for this deletion. Finally, the last variant found is a novel mutation, a deletion of a 12-bp segment in the promoter of the TMEM99 gene on chromosome 17q21.2. This segment of chromosome 17 located within exon 6 of the KRT10 gene co-encodes the repetitive amino acid motif glycine-histidine in the protein sequence of connective tissue protein, Keratin 10. Individual 1 is homozygous for this mutation the other three family members are heterozygous. Although the function of TMEM99 is unknown, it (and many other members of the TMEM gene family) are strongly expressed in cartilage. This deleted promoter sequence encodes at least three predicted CpG methylation sites. These CpG ”islands” are often methylated in promoters of genes that are inactivated. This finding might provide insight into the mechanism of incomplete penetrance often seen in familial DDH. This TMEM promoter is also the binding site of C/EBP beta, a transcription factor known to be involved in bone regulation. Last, and interestingly, this novel promoter mutation in TMEM maps to chromosome 17q21 very near the proximal border of the candidate region of another smaller DDH family analyzed by Feldman and colleagues. Further validation of these novel mutations/variants is ongoing.
In summary, a novel role is being proposed for an existing polymorphic nucleotide that is known to be biologically significant, causing both beneficial and detrimental effects in the cardiovascular and ocular systems, respectively. Gene dosage, timing of expression, and the unique morphology of the developing hip all create the microenvironment in which this variant acts, possibly explaining the phenotype of those with deficient labrum formation seen in DDH. Variants and new mutations that may contribute to the DDH phenotype severity have been found. Along with other genetic risk factors that we expect to be found in other families (because hip formation is a complex process involving the timed interaction of many proteins), these findings might illuminate the molecular pathways affecting chondrocyte maturation and bone formation.