Congenital Limb Malformations Research Articles

Cycasin derivative: a potential embryotoxic component of Atractylodes macrocephala rhizome for limb malformation.

The rhizome of Atractylodes macrocephala Koidz. (Asteraceae), called Atractylodes macrocephala rhizome (AMR) and known by its traditional name Bai Zhu, is a prominent Chinese herbal medicine employed for preventing miscarriage. However, our previous study revealed that high dosages of AMR administered during pregnancy could cause embryotoxicity but the specific embryotoxic components and their underlying mechanisms remain unclear. This study aimed to screen and identify the potential embryotoxic components of AMR. The AMR extracts and sub-fractions were analyzed by thin layer chromatography and subsequently screened by in vitro mouse limb bud micromass and mouse whole embryo culture bioassays. The embryotoxic fractions from AMR were further evaluated in vivo using a pregnant mouse model. The structures of the potential embryotoxic components were analyzed using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). In vitro and in vivo bioassays revealed that AMR glycoside-enriched sub-fractions (AMR-A-IIa and AMR-A-IIb) exhibited potential embryotoxicity. These sub-fractions, when administered to pregnant animals, increased the incidence of stillbirth and congenital limb malformations. MS spectrometry analysis identified cycasin derivatives in both sub-fractions, suggesting their possible role in the observed limb malformations. However, further experiments are necessary to validate this hypothesis and to elucidate the underlying mechanisms. Our study provides significant scientific evidence on the pharmacotoxicity of AMR, which is important for the safe clinical application of commonly used Chinese herbal medicines during pregnancy.

STIGMA: Single-cell tissue-specific gene prioritization using machine learning

Clinical exome and genome sequencing have revolutionized the understanding of human disease genetics. Yet many genes remain functionally uncharacterized, complicating the establishment of causal disease links for genetic variants. While several scoring methods have been devised to prioritize these candidate genes, these methods fall short of capturing the expression heterogeneity across cell subpopulations within tissues. Here, we introduce single-cell tissue-specific gene prioritization using machine learning (STIGMA), an approach that leverages single-cell RNA-seq (scRNA-seq) data to prioritize candidate genes associated with rare congenital diseases. STIGMA prioritizes genes by learning the temporal dynamics of gene expression across cell types during healthy organogenesis. To assess the efficacy of our framework, we applied STIGMA to mouse limb and human fetal heart scRNA-seq datasets. In a cohort of individuals with congenital limb malformation, STIGMA prioritized 469 variants in 345 genes, with UBA2 as a notable example. For congenital heart defects, we detected 34 genes harboring nonsynonymous de novo variants (nsDNVs) in two or more individuals from a set of 7,958 individuals, including the ortholog of Prdm1, which is associated with hypoplastic left ventricle and hypoplastic aortic arch. Overall, our findings demonstrate that STIGMA effectively prioritizes tissue-specific candidate genes by utilizing single-cell transcriptome data. The ability to capture the heterogeneity of gene expression across cell populations makes STIGMA a powerful tool for the discovery of disease-associated genes and facilitates the identification of causal variants underlying human genetic disorders.

Identification of the first homozygous intragenic deletion in the YY1AP1 gene in a consanguineous family: New insights into the phenotypic variability associated with Grange syndrome.

Grange syndrome (GRNG-MIM#135580) is a rare recessive disorder associating variable features including diffuse vascular stenosis, brachysyndactyly, osteopenia with increased bone fragility, cardiac malformations, and variable developmental delay. Since its first description in 1998, only 15 individuals from 10 families have been reported, carrying homozygous or compound heterozygous frameshift or nonsense variants in YY1AP1. In a patient with cutaneous and bone syndactyly and a hemorrhagic stroke at the age of 16 months, consistent with a clinical diagnosis of GRNG, we performed exome sequencing after negative array-CGH and congenital limb malformation panel results. Copy number variant analysis from exome data identified a homozygous intragenic out-of-frame deletion of 1.84 kb encompassing exons seven and eight of YY1AP1, confirming a molecular diagnosis of GRNG. Genetic counseling led to the identification of additional family members compatible with GRNG. Here, we provide new insights into the phenotypic variability associated with GRNG and highlight the utility of the detection of small copy number variants to identify the molecular causes of heterogeneous malformative genetic disorders.

Mothers of disabled infants had higher cortisol levels in a free-ranging group of Japanese macaques (Macacafuscata).

Glucocorticoids (GCs) are hormones released in response to stressors and can provide insight into an organism's physiological well-being. Experiencing chronic challenges to homeostasis is associated with significant deviations from baseline fecal GCs (fGCs) in many species, providing a noninvasive biomarker for assessing stress. In the group of free-ranging Japanese macaques (Macaca fuscata) at the Awajishima Monkey Center in Japan, ~17% have congenital limb malformations. We collected 646 fecal samples from 27 females over three consecutive birth seasons (May-August)and analyzed them using enzyme immunoassay to extract fGCs. We explored the relationship between fGC levels and individual (physical impairment and reproductive status), social (dominance rank and availability of kin for social support), and ecological variables (exposure to potential predators, rainfall, and wild fruit availability). A disabled infant was associated significantly with higher fGC in the mother; however, physical impairment in adult females was not significantly related to fGC levels. Females with higher dominance rank had significantly lower fGC levels than lower ranking females. Other factors did not relate significantly to fGC. These results suggest that providing care that meets the support needs of disabled infants poses a physiological challenge for mothersand suggests that physically impaired adults are able to effectively compensate for their disabilities with behavioral plasticity. Once an individual with congenital limb malformations survives infancy through their mother's care, physical impairment does not appear to influence fGC values, while social variables like dominance rank significantly influenced cortisol values in free-ranging female Japanese macaques.

To the fin and back again: expanding our understanding of congenital limb malformation

Fins are not limbs. However, findings from comparative developmental and genetic analyses show that many of the processes essential in establishing a limb are similar for fin formation. Nevertheless, due to their structural differences, fins are not often used as models to understand the causes or potential treatment of limb disorders.Split hand/foot malformation (SHFM) presents with loss or mis-patterning of elements of hands or feet leading to a ‘clefting-like’ phenotype, often with joining of the digits. This rare disorder is genetically and phenotypically heterogeneous, and knowledge of its etiology remains largely incomplete. Recent work by Truong et al. provides new insight into SHFM by identifying new genetic variants within three different families, predicted to cause changes in function of the PRDM1/Blimp1 gene. PRDM1 is a transcription factor known to be essential for early appendage development in both zebrafish and mice. However, PRDM1 action is nuanced, in part due to its multifaceted roles in integrating signaling, transcription and chromatin state. This complicates the functional characterization of variants in this gene that have unknown significance in SHFM patients. Truong et al. demonstrate the utility of the zebrafish fin as an assay to directly address the function of domain-specific mutations in prdm1a by using the ability of the mutant alleles to rescue fin phenotypes caused by loss of Prdm1a function or, in the case of putative dominant variants, to suppress wildtype zebrafish fin development.A fin may not, or more likely will not, have the complete genetic networks underlying limb development and patterning. However, core mechanisms remain active and, at times, latent in fin development, allowing experimental dissection of mechanisms and gene function. Truong et al. harnessed the experimental malleability of zebrafish to isolate pectoral fin cells in early development and detail Prdm1a-dependent regulation of genes. These data establish aspects of the downstream regulatory network of fin development regulated by Prdm1a, providing insight into the broader array of changes involved in the etiology of this disorder.Core mechanisms and common evolutionary foundations allow for the establishment of defined genetic models of limb disorders in zebrafish and, importantly, accessible experimental models to formulate and test hypotheses on how these disease states are attained. Conversely, identification of disease-associating alleles provides insight into molecular action of genes in development and reveals details of the specificity of their regulation. Fin-in-hand, leading towards new discoveries for congenital limb malformations.

Combinatorial effects on gene expression at the Lbx1/Fgf8 locus resolve split-hand/foot malformation type 3

Split-Hand/Foot Malformation type 3 (SHFM3) is a congenital limb malformation associated with tandem duplications at the LBX1/FGF8 locus. Yet, the disease patho-mechanism remains unsolved. Here we investigate the functional consequences of SHFM3-associated rearrangements on chromatin conformation and gene expression in vivo in transgenic mice. We show that the Lbx1/Fgf8 locus consists of two separate, but interacting, regulatory domains. Re-engineering of a SHFM3-associated duplication and a newly reported inversion in mice results in restructuring of the chromatin architecture. This leads to ectopic activation of the Lbx1 and Btrc genes in the apical ectodermal ridge (AER) in an Fgf8-like pattern induced by AER-specific enhancers of Fgf8. We provide evidence that the SHFM3 phenotype is the result of a combinatorial effect on gene misexpression in the developing limb. Our results reveal insights into the molecular mechanism underlying SHFM3 and provide conceptual framework for how genomic rearrangements can cause gene misexpression and disease.

Sequence Variants in MEGF8 and GJA1 Underlying Syndactyly

Introduction: Syndactyly is a common congenital limb malformation. It occurs due to embryological failure of digit separation during limb development. Syndactyly often runs in families with an incidence of about one out of every 2,500–3,000 live births. Methods: Here, we have reported two families presenting features of severe forms of syndactyly. The disorder segregated in autosomal recessive in one and in autosomal dominant manner in the second family. Search for the causative variants was carried out using whole-exome sequencing in family A and candidate gene sequencing in family B. Results: Analysis of the sequencing data revealed two novel missense variants, including p.(Cys1925Arg) in MEGF8 in family A and p.(Thr89Ile) in GJA1 in family B. Conclusion: In conclusion, the novel findings, presented here, not only expand the mutation spectrum in the genes MEGF8 and GJA1, but this will also facilitate screening other families carrying similar clinical features in the Pakistani population.

Lower Extremity Malformations.

Lower Extremity Malformations.

Congenital Limb Defects: A Retrospective Cohort Study and Overview of the Literature.

Congenital limb defects are common malformations that are often associated with other organ defects and genetic disorders. Since prenatal detection is challenging and classification is often complex, the aim of this study was to describe a large cohort of fetuses with congenital limb defects and to identify characteristics that are essential for prenatal evaluation, counselling, and management. In this retrospective cohort study, all cases of confirmed fetal limb defects from two centers for prenatal ultrasound between 2001 and 2021 were evaluated. Cases with skeletal dysplasia were excluded from this study. Demographic data, association with genetic disorders, and correlation with maternal parameters were analyzed statistically. 170 fetuses were included in this study. 60% were diagnosed with a reduction anomaly and 40% with a duplication anomaly. The majority of fetuses were male, and in 73.5% of all cases, additional malformations were present. Among the genetic causes, trisomy 13 and 18 were the most common in this cohort. Congenital limb malformations are important markers for complex fetal disorders that warrant referral to specialists in prenatal ultrasound. To improve prenatal detection, care should be taken to visualize all fetal extremities already in early pregnancy.

Developmental genomics of limb malformations: Allelic series in association with gene dosage effects contribute to the clinical variability.

SummaryGenetic heterogeneity, reduced penetrance, and variable expressivity, the latter including asymmetric body axis plane presentations, have all been described in families with congenital limb malformations (CLMs). Interfamilial and intrafamilial heterogeneity highlight the complexity of the underlying genetic pathogenesis of these developmental anomalies. Family-based genomics by exome sequencing (ES) and rare variant analyses combined with whole-genome array-based comparative genomic hybridization were implemented to investigate 18 families with limb birth defects. Eleven of 18 (61%) families revealed explanatory variants, including 7 single-nucleotide variant alleles and 3 copy number variants (CNVs), at previously reported “disease trait associated loci”: BHLHA9, GLI3, HOXD cluster, HOXD13, NPR2, and WNT10B. Breakpoint junction analyses for all three CNV alleles revealed mutational signatures consistent with microhomology-mediated break-induced replication, a mechanism facilitated by Alu/Alu-mediated rearrangement. Homozygous duplication of BHLHA9 was observed in one Turkish kindred and represents a novel contributory genetic mechanism to Gollop-Wolfgang Complex (MIM: 228250), where triplication of the locus has been reported in one family from Japan (i.e., 4n = 2n + 2n versus 4n = 3n + 1n allelic configurations). Genes acting on limb patterning are sensitive to a gene dosage effect and are often associated with an allelic series. We extend an allele-specific gene dosage model to potentially assist, in an adjuvant way, interpretations of interconnections among an allelic series, clinical severity, and reduced penetrance of the BHLHA9-related CLM spectrum.

89Zr-3,2-HOPO-Mesothelin Antibody PET Imaging Reflects Tumor Uptake of Mesothelin-Targeted 227Th-Conjugate Therapy in Mice.

The mesothelin (MSLN)-targeted 227Th conjugate is a novel α-therapy developed to treat MSLN-overexpressing cancers. We radiolabeled the same antibody-chelator conjugate with 89Zr to evaluate whether PET imaging with 89Zr-MSLN matches 227Th-MSLN tumor uptake, biodistribution, and antitumor activity. Methods: Serial PET imaging with protein doses of 4, 20, or 40 μg of 89Zr-MSLN and 89Zr-control was performed up to 168 h after tracer injection in human tumor-bearing nude mice with high (HT29-MSLN) and low (BxPc3) MSLN expression. 89Zr-MSLN and 227Th-MSLN ex vivo tumor uptake and biodistribution were compared at 6 time points in HT29-MSLN and in medium-MSLN-expressing (OVCAR-3) tumor-bearing mice. 89Zr-MSLN PET imaging was performed before 227Th-MSLN treatment in HT29-MSLN and BxPc3 tumor-bearing mice. Results: 89Zr-MSLN PET imaging showed an SUVmean of 2.2 ± 0.5 in HT29-MSLN tumors. Ex vivo tumor uptake was 10.6% ± 2.4% injected dose per gram at 168 h. 89Zr-MSLN tumor uptake was higher than uptake of 89Zr-control (P = 0.0043). 89Zr-MSLN and 227Th-MSLN showed comparable tumor uptake and biodistribution in OVCAR-3 and HT29-MSLN tumor-bearing mice. Pretreatment SUVmean was 2.2 ± 0.2 in HT29-MSLN tumors, which decreased in volume on 227Th-MSLN treatment. BxPc3 tumors showed an SUVmean of 1.2 ± 0.3 and remained similar in size after 227Th-MSLN treatment. Conclusion: 89Zr-MSLN PET imaging reflected MSLN expression and matched 227Th-MSLN tumor uptake and biodistribution. Our data support the clinical exploration of 89Zr-MSLN PET imaging together with 227Th-MSLN therapy, both using the same antibody-chelator conjugate.

Hnrnpul1 controls transcription, splicing, and modulates skeletal and limb development in vivo.

Mutations in RNA-binding proteins can lead to pleiotropic phenotypes including craniofacial, skeletal, limb, and neurological symptoms. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are involved in nucleic acid binding, transcription, and splicing through direct binding to DNA and RNA, or through interaction with other proteins in the spliceosome. We show a developmental role for Hnrnpul1 in zebrafish, resulting in reduced body and fin growth and missing bones. Defects in craniofacial tendon growth and adult-onset caudal scoliosis are also seen. We demonstrate a role for Hnrnpul1 in alternative splicing and transcriptional regulation using RNA-sequencing, particularly of genes involved in translation, ubiquitination, and DNA damage. Given its cross-species conservation and role in splicing, it would not be surprising if it had a role in human development. Whole-exome sequencing detected a homozygous frameshift variant in HNRNPUL1 in 2 siblings with congenital limb malformations, which is a candidate gene for their limb malformations. Zebrafish Hnrnpul1 mutants suggest an important developmental role of hnRNPUL1 and provide motivation for exploring the potential conservation of ancient regulatory circuits involving hnRNPUL1 in human development.

Two nonsense GLI3 variants are associated with polydactyly and syndactyly in two families by affecting the sonic hedgehog signaling pathway.

BackgroundPolydactyly and syndactyly are congenital limb deformities, segregating in an autosomal‐dominant fashion. The variants in the GLI3 gene are closely related to congenital limb malformations. However, the causes underlying polydactyly and syndactyly are not well understood.MethodsWe conducted a whole‐exome sequencing on two four‐generation Chinese families with polydactyly and syndactyly. Then c.2374C>T and c.1728C>A mutant plasmids were transfected to HEK293T cells and mice limb bud cells to explore the functional consequences of these variants. Western blot and real‐time quantitative PCR were used to analyze the expression of GLI3 and Shh.ResultsIn these two families, the known GLI3 variant (NM_000168.6:c.2374C>T) and the novel GLI3 variant (NM_000168.6:c.1728C>A) contributed to polydactyly and syndactyly. Additionally, the GLI3 c.2374C>T mutant plasmid led to truncated GLI3 protein, and the GLI3 c.1728C>A mutant plasmid led to degraded GLI3 protein. Simultaneously, we demonstrated that the GLI3‐mutant plasmids led to decreased Shh expression in mice limb bud cells.ConclusionWe demonstrated that the novel GLI3 variant (c.1728C>A) and known GLI3 variant (c.2374C>T) contributed to the malformations in two four‐generation pedigrees with polydactyly and syndactyly by affecting SHH signaling.

Long-read whole genome sequencing reveals HOXD13 alterations in synpolydactyly.

Synpolydactyly 1, also called syndactyly type II (SDTY2), is a genetic limb malformation characterized by polydactyly with syndactyly involving the webbing of the third and fourth fingers, and the fourth and fifth toes. It is caused by heterozygous alterations in HOXD13 with incomplete penetrance and phenotypic variability. In our study, a five-generation family with an SPD phenotype was enrolled in our Rare Disease Genomics Protocol. A comprehensive examination of three generations using Illumina short-read whole-genome sequencing (WGS) did not identify any causative variants. Subsequent WGS using Pacific Biosciences (PacBio) long-read HiFi Circular Consensus Sequencing (CCS) revealed a heterozygous 27-bp duplication in the polyalanine tract of HOXD13. Sanger sequencing of all available family members confirmed that the variant segregates with affected individuals. Reanalysis of an unrelated family with a similar SPD phenotype uncovered a 21-bp (7-alanine) duplication in the same region of HOXD13. Although ExpansionHunter identified these events in most individuals in a retrospective analysis, low sequence coverage due to high GC content in the HOXD13 polyalanine tract makes detection of these events challenging. Our findings highlight the value of long-read WGS in elucidating the molecular etiology of congenital limb malformation disorders.

A Novel Missense Variant of HOXD13 Caused Atypical Synpolydactyly by Impairing the Downstream Gene Expression and Literature Review for Genotype-Phenotype Correlations.

Synpolydactyly (SPD) is a hereditary congenital limb malformation with distinct syndactyly designated as SPD1, SPD2, and SPD3. SPD1 is caused by mutations of HOXD13, which is a homeobox transcription factor crucial for limb development. More than 143 SPD patients have been reported to carry HOXD13 mutations, but there is a lack of genotype–phenotype correlation. We report a novel missense mutation of c. 925A > T (p.I309F) in an individual with atypical synpolydactyly inherited from her father with mild clinodactyly and three other different alanine insertion mutations in HOXD13 identified by whole exome sequencing (WES) in 12 Chinese SPD families. Unlike polyalanine extension, which tends to form α-helix and causes protein aggregation in the cytoplasm as shown by molecular simulation and immunofluorescence, the c. 925A > T mutation impairs downstream transcription of EPHA7. We compiled literature findings and analyzed genotype–phenotype features in 173 SPD individuals of 53 families, including 12 newly identified families. Among the HOXD13-related individuals, mutations were distributed in three regions: polyalanine, homeobox, and non-homeobox. Polyalanine extension was the most common variant (45%), followed by missense mutations (32%) mostly in the homeobox compared with the loss-of-function (LOF) variants more likely in non-homeobox. Furthermore, a more severe degree and classic SPD were associated with polyalanine mutations although missense variants were associated with brachydactyly and syndactyly in hands and feet and LOF variants with clinodactyly in hands. Our study broadens the HOXD13 mutation spectrum and reveals the profile of three different variants and their severity of SPD, the genotype–phenotype correlation related to the HOXD13 mutation site provides clinical insight, including for genetic counseling.

Position effects at the FGF8 locus are associated with femoral hypoplasia

Copy-number variations (CNVs) are a common cause of congenital limb malformations and are interpreted primarily on the basis of their effect on gene dosage. However, recent studies show that CNVs also influence the 3D genome chromatin organization. The functional interpretation of whether a phenotype is the result of gene dosage or a regulatory position effect remains challenging. Here, we report on two unrelated families with individuals affected by bilateral hypoplasia of the femoral bones, both harboring de novo duplications on chromosome 10q24.32. The ∼0.5 Mb duplications include FGF8, a key regulator of limb development and several limb enhancer elements. To functionally characterize these variants, we analyzed the local chromatin architecture in the affected individuals' cells and re-engineered the duplications in mice by using CRISPR-Cas9 genome editing. We found that the duplications were associated with ectopic chromatin contacts and increased FGF8 expression. Transgenic mice carrying the heterozygous tandem duplication including Fgf8 exhibited proximal shortening of the limbs, resembling the human phenotype. To evaluate whether the phenotype was a result of gene dosage, we generated another transgenic mice line, carrying the duplication on one allele and a concurrent Fgf8 deletion on the other allele, as a control. Surprisingly, the same malformations were observed. Capture Hi-C experiments revealed ectopic interaction with the duplicated region and Fgf8, indicating a position effect. In summary, we show that duplications at the FGF8 locus are associated with femoral hypoplasia and that the phenotype is most likely the result of position effects altering FGF8 expression rather than gene dosage effects.

Genome sequencing in families with congenital limb malformations

The extensive clinical and genetic heterogeneity of congenital limb malformation calls for comprehensive genome-wide analysis of genetic variation. Genome sequencing (GS) has the potential to identify all genetic variants. Here we aim to determine the diagnostic potential of GS as a comprehensive one-test-for-all strategy in a cohort of undiagnosed patients with congenital limb malformations. We collected 69 cases (64 trios, 1 duo, 5 singletons) with congenital limb malformations with no molecular diagnosis after standard clinical genetic testing and performed genome sequencing. We also developed a framework to identify potential noncoding pathogenic variants. We identified likely pathogenic/disease-associated variants in 12 cases (17.4%) including four in known disease genes, and one repeat expansion in HOXD13. In three unrelated cases with ectrodactyly, we identified likely pathogenic variants in UBA2, establishing it as a novel disease gene. In addition, we found two complex structural variants (3%). We also identified likely causative variants in three novel high confidence candidate genes. We were not able to identify any noncoding variants. GS is a powerful strategy to identify all types of genomic variants associated with congenital limb malformation, including repeat expansions and complex structural variants missed by standard diagnostic approaches. In this cohort, no causative noncoding SNVs could be identified.

Deciphering the mutational signature of congenital limb malformations

Congenital limb malformations (CLMs) affect 1 in 500 live births. However, the value of exome sequencing (ES) for CLM is lacking. The purpose of this study was to decipher the mutational signature of CLM on an exome level. We enrolled a cohort of 66 unrelated probands (including 47 families) with CLM requiring surgical correction. ES was performed for all patients and available parental samples. A definite molecular diagnosis was achieved in 21 out of 66 (32%) patients. We identified 19 pathogenic or likely pathogenic single-nucleotide variants and three copy number variants, of which 11 variants were novel. We identified four variants of uncertain significance. Additionally, we identified RPL9 and UBA2 as novel candidate genes for CLM. By comparing the detailed phenotypic features, we expand the phenotypic spectrum of diastrophic dysplasia and chromosome 6q terminal deletion syndrome. We also found that the diagnostic rate was significantly higher in patients with a family history of CLM (p = 0.012) or more than one limb affected (p = 0.034). Our study expands our understanding of the mutational and phenotypic spectrum of CLM and provides novel insights into the genetic basis of these syndromes.

Novel Use of Pennig External Fixation for the Treatment of Pediatric Syndactyly.

Syndactyly is one of the most common congenital limb malformations, second only to polydactyly. Syndactyly is treated with surgical intervention. During separation, free skin grafting or skin flap transplantation is required to allow for adequate skin closure. We introduce a novel application method of the Pennig external fixation device for the management of pediatric syndactyly without skin grafting. The Pennig minifixator was used in syndactyly to expand the web space soft tissue in order to allow a Z-plasty to be performed in a second operation. All patients had developed sufficient skin tissue from the external fixation. No infection, cutaneous necrosis or nonunion was noted in any of the patients. All patients had a satisfactory outcome in terms of final appearance and digit function. On the basis of the principle of strain-stress, use of the Penning external minifixator allows development of sufficient tissue in the web space for a second phase of reconstruction in syndactyly. The need for skin grafting and risk of blood vessel damage were avoided in this study, and, the incidence of skin necrosis and scar formation was reduced. Both the patients’ families and our research members expressed high satisfaction with the postoperative function and appearance. Overall, application of the Pennig external fixation frame in our novel method is effective for the management of syndactyly and should be studied further. Level of Evidence: Level IV, case series.

Non-coding deletions identify Maenli lncRNA as a limb-specific En1 regulator.

Long non-coding RNAs (lncRNAs) can be important components in gene-regulatory networks1, but the exact nature and extent of their involvement in human Mendelian disease is largely unknown. Here we show that genetic ablation of a lncRNA locus on human chromosome 2 causes a severe congenital limb malformation. We identified homozygous 27-63-kilobase deletions located 300kilobases upstream of the engrailed-1 gene (EN1) in patients with a complex limb malformation featuring mesomelic shortening, syndactyly and ventral nails (dorsal dimelia). Re-engineering of the human deletions in mice resulted in a complete loss of En1 expression in the limb and a double dorsal-limb phenotype that recapitulates the human disease phenotype. Genome-wide transcriptome analysis in the developing mouse limb revealed a four-exon-long non-coding transcript within the deleted region, which we named Maenli. Functional dissection of the Maenli locus showed that its transcriptional activity is required for limb-specific En1 activation in cis, thereby fine-tuning the gene-regulatory networks controlling dorso-ventral polarity in the developing limb bud. Its loss results in the En1-related dorsal ventral limb phenotype, a subset of the full En1-associated phenotype. Our findings demonstrate that mutations involving lncRNA loci can result in human Mendelian disease.