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1.  Genome-wide identification of signaling center enhancers in the developing limb 
Development (Cambridge, England)  2014;141(21):4194-4198.
The limb is widely used as a model developmental system and changes to gene expression patterns in its signaling centers, notably the zone of polarizing activity (ZPA) and the apical ectodermal ridge (AER), are known to cause limb malformations and evolutionary differences in limb morphology. Although several genes that define these limb signaling centers have been described, the identification of regulatory elements that are active within these centers has been limited. By dissecting mouse E11.5 limbs that fluorescently mark the ZPA or AER, followed by fluorescence-activated cell sorting and low-cell H3K27ac ChIP-seq, we identified thousands of specific signaling-center enhancers. Our ChIP-seq datasets show strong correlation with ZPA- and AER-expressed genes, previously characterized functional ZPA and AER enhancers and enrichment for relevant biological terms related to limb development and malformation for the neighboring genes. Using transgenic assays, we show that several of these sequences function as ZPA and AER enhancers. Our results identify novel ZPA and AER enhancers that could be important regulators of genes involved in the establishment of these specialized regions and the patterning of tetrapod limbs.
doi:10.1242/dev.110965
PMCID: PMC4302890  PMID: 25273087
Enhancer; AER; ZPA; Limb; Mouse
2.  A Novel ZRS Mutation Leads to Preaxial Polydactyly Type 2 in a Heterozygous Form and Werner Mesomelic Syndrome in a Homozygous Form 
Human mutation  2014;35(8):945-948.
Point mutations in the zone of polarizing activity regulatory sequence (ZRS) are known to cause human limb malformations. Although most mutations cause preaxial polydactyly (PPD), triphalangeal thumb (TPT) or both, a mutation in position 404 of the ZRS causes more severe Werner mesomelic syndrome (WMS) for which malformations include the distal arm or leg bones in addition to the hands and/or feet. Of more than 15 reported families with ZRS mutations, only one homozygous individual has been reported, with no change in phenotype compared with heterozygotes. Here, we describe a novel point mutation in the ZRS, 402C>T (AC007097.4:g.105548C>T), that is transmitted through two Mexican families with one homozygous individual. The homozygous phenotype for this mutation, WMS, is more severe than the numerous heterozygous individuals genotyped from both families who have TPT and PPD. A mouse transgenic enhancer assay shows that this mutation causes an expansion of the enhancer’s expression domain in the developing mouse limb, confirming its pathogenicity. Combined, our results identify a novel ZRS mutation in the Mexican population, 402C>T, and suggest that a dosage effect exists for this ZRS mutation.
doi:10.1002/humu.22581
PMCID: PMC4110103  PMID: 24777739
ZRS; enhancer; SHH; preaxial polydactyly; Werner mesomelic syndrome
3.  A Novel ZRS Mutation in a Balochi Tribal Family with Triphalangeal Thumb, Pre-axial Polydactyly, Postaxial Polydactyly and Syndactyly 
Limb malformations are one of the most common types of human congenital malformations. Mutations in the ZRS enhancer of Sonic Hedgehog are thought to be responsible for preaxial polydactyly in multiple independent families. Here, we describe a large Balochi tribal family from Southern Punjab, Pakistan, with a variable set of limb malformations and a novel ZRS mutation. The family has a limb phenotype characterized by triphalangeal thumb, preaxial polydactyly and postaxial polydactyly. There is also a high degree of phenotypic heterogeneity with less common clinical findings in the affected family members that include osseous syndactyly of forth-fifth fingers, clinodactyly, hypoplasia of mesoaxial fingers, and bifid halluces. The presentation in most of the affected patients was bilateral and symmetrical. A heterozygous C>A mutation at position 287 of the ZRS enhancer (chr7:156,584,283; hg19) was detected in all affected subjects and is absent from four unaffected family members, 42 unrelated samples and multiple databases of human variation. Combined, these results identify a novel ZRS287 C>A mutation which leads to a variable spectrum of limb phenotypes.
doi:10.1002/ajmg.a.35473
PMCID: PMC3402602  PMID: 22786669
Polydactyly; triphalangeal thumb; syndactyly; ZRS
4.  A Novel 13 Base Pair Insertion in the Sonic Hedgehog ZRS Limb Enhancer (LMBR1) Causes Preaxial Polydactyly with Triphalangeal Thumb 
Human Mutation  2012;33(7):1063-1066.
Mutations in the Sonic Hedgehog limb enhancer, the zone of polarizing activity regulatory sequence (LMBR1, commonly called the ZRS), cause limb malformations. In humans, three classes of mutations have been proposed based on the limb phenotype; single base changes throughout the region cause preaxial polydactyly, single base changes at one specific site cause Werner mesomelic syndrome and large duplications cause polysyndactyly. This study presents a novel mutation– a small insertion. In a Swedish family with autosomal dominant preaxial polydactyly, we found a 13 base pair insertion within the ZRS, ZRS603ins13 (NG-009240.1:g.106934_106935ins13). Computational transcription factor binding site predictions suggest that this insertion creates new binding sites and a mouse enhancer assay shows that this insertion causes ectopic gene expression. This study is the first to discover a small insertion in an enhancer that causes a human limb malformation and suggests a potential mechanism that could explain the ectopic expression caused by this mutation.
doi:10.1002/humu.22097
PMCID: PMC3370115  PMID: 22495965
enhancer; limb; polydactyly; SHH; LMBR1; ZRS
5.  Human Developmental Enhancers Conserved between Deuterostomes and Protostomes 
PLoS Genetics  2012;8(8):e1002852.
The identification of homologies, whether morphological, molecular, or genetic, is fundamental to our understanding of common biological principles. Homologies bridging the great divide between deuterostomes and protostomes have served as the basis for current models of animal evolution and development. It is now appreciated that these two clades share a common developmental toolkit consisting of conserved transcription factors and signaling pathways. These patterning genes sometimes show common expression patterns and genetic interactions, suggesting the existence of similar or even conserved regulatory apparatus. However, previous studies have found no regulatory sequence conserved between deuterostomes and protostomes. Here we describe the first such enhancers, which we call bilaterian conserved regulatory elements (Bicores). Bicores show conservation of sequence and gene synteny. Sequence conservation of Bicores reflects conserved patterns of transcription factor binding sites. We predict that Bicores act as response elements to signaling pathways, and we show that Bicores are developmental enhancers that drive expression of transcriptional repressors in the vertebrate central nervous system. Although the small number of identified Bicores suggests extensive rewiring of cis-regulation between the protostome and deuterostome clades, additional Bicores may be revealed as our understanding of cis-regulatory logic and sample of bilaterian genomes continue to grow.
Author Summary
Flies and worms have long served as valuable model organisms for the study of human development and health. Despite the great morphological and evolutionary distance between them, humans, flies, and worms share many commonalities. Each develops from three major germ layers and is patterned along the two major spatial axes. At the molecular level, development in these widely diverged species is often controlled by the same signaling pathways activating members of the same transcription factor and target gene families, shared since the common ancestor of humans, flies, and worms. And yet, at the gene regulatory level, humans and flies or worms seem starkly different, with not a single regulatory region shared across the phyla. Here we discover the first two examples of developmental enhancers conserved between deuterostomes (ranging from human to sea urchins) and protostomes (a large clade that includes flies and worms). We show evidence that these ancient regulatory loci retain the capacity to respond to the same signaling pathways in these widely diverged organisms, and we show that they have been co-opted, along with the molecular pathways that control them, to pattern the vertebrate nervous systems. Our screen supports large scale regulatory rewiring, while offering the first intriguing outliers.
doi:10.1371/journal.pgen.1002852
PMCID: PMC3410860  PMID: 22876195
6.  Evaluation of common genetic variants in 82 candidate genes as risk factors for neural tube defects 
BMC Medical Genetics  2012;13:62.
Background
Neural tube defects (NTDs) are common birth defects (~1 in 1000 pregnancies in the US and Europe) that have complex origins, including environmental and genetic factors. A low level of maternal folate is one well-established risk factor, with maternal periconceptional folic acid supplementation reducing the occurrence of NTD pregnancies by 50-70%. Gene variants in the folate metabolic pathway (e.g., MTHFR rs1801133 (677 C > T) and MTHFD1 rs2236225 (R653Q)) have been found to increase NTD risk. We hypothesized that variants in additional folate/B12 pathway genes contribute to NTD risk.
Methods
A tagSNP approach was used to screen common variation in 82 candidate genes selected from the folate/B12 pathway and NTD mouse models. We initially genotyped polymorphisms in 320 Irish triads (NTD cases and their parents), including 301 cases and 341 Irish controls to perform case–control and family based association tests. Significantly associated polymorphisms were genotyped in a secondary set of 250 families that included 229 cases and 658 controls. The combined results for 1441 SNPs were used in a joint analysis to test for case and maternal effects.
Results
Nearly 70 SNPs in 30 genes were found to be associated with NTDs at the p < 0.01 level. The ten strongest association signals (p-value range: 0.0003–0.0023) were found in nine genes (MFTC, CDKN2A, ADA, PEMT, CUBN, GART, DNMT3A, MTHFD1 and T (Brachyury)) and included the known NTD risk factor MTHFD1 R653Q (rs2236225). The single strongest signal was observed in a new candidate, MFTC rs17803441 (OR = 1.61 [1.23-2.08], p = 0.0003 for the minor allele). Though nominally significant, these associations did not remain significant after correction for multiple hypothesis testing.
Conclusions
To our knowledge, with respect to sample size and scope of evaluation of candidate polymorphisms, this is the largest NTD genetic association study reported to date. The scale of the study and the stringency of correction are likely to have contributed to real associations failing to survive correction. We have produced a ranked list of variants with the strongest association signals. Variants in the highest rank of associations are likely to include true associations and should be high priority candidates for further study of NTD risk.
doi:10.1186/1471-2350-13-62
PMCID: PMC3458983  PMID: 22856873
Neural tube defects; Spina bifida; Folic acid; One-carbon metabolism; Candidate gene
7.  cis-Regulatory Mutations Are a Genetic Cause of Human Limb Malformations 
The underlying mutations that cause human limb malformations are often difficult to determine, particularly for limb malformations that occur as isolated traits. Evidence from a variety of studies shows that cis-regulatory mutations, specifically in enhancers, can lead to some of these isolated limb malformations. Here, we provide a review of human limb malformations that have been shown to be caused by enhancer mutations and propose that cis-regulatory mutations will continue to be identified as the cause of additional human malformations as our understanding of regulatory sequences improves.
doi:10.1002/dvdy.22535
PMCID: PMC3174732  PMID: 21509892
cis-regulatory; enhancer; human limb malformation

Results 1-7 (7)