A variant in the LDL receptor-related protein encoding gene LRP4 underlying polydactyly and phalangeal synostosis in a family of Pakistani origin.

A family of Pakistani origin, segregating polydactyly, and phalangeal synostosis in an autosomal dominant manner, has been investigated and presented in the present report. Whole-exome sequencing (WES), followed by segregation analysis using Sanger sequencing, revealed a heterozygous missense variant [c.G1696A, p.(Gly566Ser)] in the LRP4 gene located on human chromosome 11p11.2. Homology protein modeling revealed the mutant Ser566 generated new interactions with at least four other amino acids and disrupted protein folding and function. Our findings demonstrated the first direct evidence of involvement of LRP4 in causing polydactyly and phalangeal synostosis in the same family. This study highlighted the importance of inclusion of LRP4 gene in screening individuals presenting polydactyly in hands and feet, and phalangeal synostosis in the same family.

A De Novo 8q22.2q22.3 Interstitial Microdeletion in a Girl with Developmental Delay and Congenital Defects.

Background and Objectives: Only nine patients with interstitial de novo 8q22.2q22.3 microdeletions have been reported to date. The objective of this report is to present clinical features of a new patient with an 8q22.2q22.3 microdeletion, to compare her phenotype to other previously reported patients, and to further expand the phenotype associated with this microdeletion. Materials and Methods: We describe an 8½-year-old girl with developmental delay, congenital hip dysplasia, a bilateral foot deformity, bilateral congenital radioulnar synostosis, a congenital heart defect, and minor facial anomalies. Results: Chromosomal microarray analysis revealed a 4.9 Mb deletion in the 8q22.2q22.3 region. De novo origin was confirmed by real-time PCR analysis. Conclusions: Microdeletions in the 8q22.2q22.3 region are characterized by moderate to severe intellectual disability, seizures, distinct facial features and skeletal abnormalities. In addition to one already reported individual with an 8q22.2q22.3 microdeletion and unilateral radioulnar synostosis, this report of a child with bilateral radioulnar synostosis provides additional evidence, that radioulnar synostosis is not an incidental finding in individuals with an 8q22.2q22.3 microdeletion. Additional patients with similar microdeletions would be of a great importance for more accurate phenotypic description and further analysis of the genotypic-phenotypic relationship.

Fetal hydrops caused by a novel pathogenic MECOM variant.

We report a fetus with hydrops, congenital heart disease and bilateral radioulnar synostosis caused by a novel pathogenic MECOM variant. The female fetus was referred for post-mortem examination after fetal hydrops and intrauterine death was diagnosed at 20 weeks gestation. Post-mortem examination confirmed fetal hydrops, pallor, truncus arteriosus and bilateral radioulnar synostosis. Trio whole genome sequencing analysis detected a novel de novo heterozygous pathogenic loss-of-function variant in MECOM (NM_004991), associated with a diagnosis of Radioulnar Synostosis with Amegakaryocytic Thrombocytopenia 2 (RUSAT-2). RUSAT-2 is a variable condition associated postnatally with bone marrow failure, radioulnar synostosis and congenital anomalies. RUSAT-2 is not currently associated with a prenatal phenotype or fetal demise, and was not present on diagnostic NHS prenatal gene panels at time of diagnosis. This case highlights the diagnostic value of detailed phenotyping with post-mortem examination, and of using a broad sequencing approach.

Defective Joint Development and Maintenance in GDF6-Related Multiple Synostoses Syndrome.

Multiple synostoses syndromes (SYNS) are a group of rare genetic bone disorders characterized by multiple joint fusions. We previously reported an SYNS4-causing GDF6 c.1330 T > A (p.Tyr444Asn) mutation, which reduced Noggin-induced GDF6 inhibition and enhanced SMAD1/5/8 signaling. However, the mechanisms by which GDF6 gain-of-function mutation alters joint formation and the comprehensive molecular portraits of SYNS4 remain unclear. Herein, we introduce the p.Tyr443Asn (orthologous to the human GDF6 p.Tyr444Asn) mutation into the mouse Gdf6 locus and report the results of extensive phenotype analysis, joint development investigation, and transcriptome profiling of Gdf6 p.Tyr443Asn limb buds. Gdf6 p.Tyr443Asn knock-in mice recapitulated the morphological features of human SYNS4, showing joint fusion in the wrists, ankles, phalanges, and auditory ossicles. Analysis of mouse embryonic forelimbs demonstrated joint interzone formation defects and excess chondrogenesis in Gdf6 p.Tyr443Asn knock-in mice. Further, RNA sequencing of forelimb buds revealed enhanced bone formation and upregulated bone morphogenetic protein (BMP) signaling in mice carrying the Gdf6 p.Tyr443Asn mutation. Because tightly regulated BMP signaling is critical for skeletal development and joint morphogenesis, our study shows that enhancing GDF6 activity has a significant impact on both prenatal joint development and postnatal joint maintenance. © 2023 American Society for Bone and Mineral Research (ASBMR).

Bi-allelic MYH3 loss-of-function variants cause a lethal form of contractures, pterygia, and spondylocarpotarsal fusion syndrome 1B.

Arthrogryposis is a consequence of reduced fetal movements and arises due to environmental factors or underlying genetic defects, with extensive genetic heterogeneity. In many instances, the genes responsible are involved in neuromuscular function. Missense variants in the gene encoding embryonic myosin heavy chain (MYH3) usually cause distal arthrogryposis. Recently, mono-allelic or bi-allelic MYH3 variants have been associated with contractures, pterygia, and spondylocarpotarsal fusion syndrome 1 (CPSFS1A and CPSFS1B). Here we describe three fetuses presenting in the second trimester with a lethal form of arthrogryposis and pterygia and harbouring bi-allelic variants in MYH3. One proband was compound heterozygous for a missense change and an extended splice site variant, a second proband had a homozygous frameshift variant, and a third proband was homozygous for a nonsense variant. Minigene assays performed on the first fetus showed that the missense and extended splice site variants resulted in aberrant splicing, likely resulting in near complete loss of full-length MYH3 transcript. This study shows that loss of MYH3 is associated with a lethal arthrogryposis phenotype and highlights the utility of minigene assays to assess splicing.

Clinical observation and genetic analysis of a SYNS1 family caused by novel NOG gene mutation.

OBJECTIVE: Analyze the clinical and genetic characteristics of a rare Chinese family with Multiple synostoses syndrome and identify the causative variant with the high-throughput sequencing approach. METHODS: The medical history investigation, physical examination, imaging examination, and audiological examination of the family members were performed. DNA samples were extracted from the family members. The candidate variant was identified by performing whole-exome sequencing of the proband, then verified by Sanger sequencing in the family. RESULTS: The family named HBSY-018 from Hubei province had 18 subjects in three generations, and six subjects were diagnosed with conductive or mixed hearing loss. Meanwhile, characteristic features including short philtrum, hemicylindrical nose, and hypoplastic alae nasi were noticed among those patients. Symptoms of proximal interdigital joint adhesion and inflexibility were found. The family was diagnosed as Multiple synostoses syndrome type 1 (SYNS1).The inheritance pattern of this family was autosomal dominant. A novel mutation in the NOG gene c.533G>A was identified by performing whole-exome sequencing of the proband. The substitution of cysteine encoding 178th position with tyrosine (p.Cys178Tyr) was caused by this mutation, which was conserved across species. Co-segregation of disease phenotypes was demonstrated by the family verification. CONCLUSION: The family diagnosed as SYNS1 was caused by the novel mutation (c.533G>A) of NOG. The combination of clinical diagnosis and molecular diagnosis had improved the understanding of this rare disease and provided a scientific basis for genetic counseling in the family.

Novel FGF9 variant contributes to multiple synostoses syndrome 3.

Multiple synostoses syndromes (SYNS) are autosomal dominant syndromes characterized by multiple joint fusions commonly involving the carpal-tarsal, interphalangeal, humeroradial, and cervical spine joints. They display genetic heterogeneity with pathogenic variants reported in four separate genes (NOG, GDF5, FGF9, and GDF6) defining four different SYNS forms. FGF9 variants have been reported in SYNS3, a SYNS with multiple synostoses, normal cognition, normal hearing, and craniosynostosis. Here, we report a novel FGF9 c.569G > C p.(Arg190Thr) variant identified by whole-exome sequencing in a patient with multiple bony abnormalities. The patient initially presented with elbow instability and decreased range of motion. Imaging revealed bilateral radial head deformities, carpal-tarsal fusions, brachydactyly, and osteoarthritis of the sacroiliac joints. In silico protein modeling of the identified FGF9 variant predicts decreased stability of ligand-receptor binding supporting the pathogenicity of this finding. This finding expands the repertoire of FGF9 variants and phenotypic information reported for SYNS3 and suggest that genotype phenotype correlations due to localization seem less likely and more so due to the consequence of the pathogenic variant on the receptor. This is useful in the counseling in families as more de novo variants emerge.

Bioinformatics Analysis and Experimental Verification Identify Downregulation of COL27A1 in Poor Segmental Congenital Scoliosis.

BACKGROUND: Congenital scoliosis (CS) represents the congenital defect disease, and poor segmental congenital scoliosis (PSCS) represents one of its types. Delayed intervention can result in disability and paralysis. In this study, we would identify the core biomarkers for PSCS progression through bioinformatics analysis combined with experimental verification. METHODS: This work obtained the GSE11854 expression dataset associated with somite formation in the GEO database, which covers data of 13 samples. Thereafter, we utilized the edgeR of the R package to obtain DEGs in this dataset. Then, GO annotation, KEGG analyses, and DO annotation of DEGs were performed by "clusterProfiler" of the R package. This study performed LASSO regression for screening the optimal predicting factors for somite formation. Through RNA sequencing based on peripheral blood samples from healthy donors and PSCS cases, we obtained the RNA expression patterns and screen out DEGs using the R package DESeq2. The present work analyzed COL27A1 expression in PSCS patients by the RT-PCR assay. RESULTS: A total of 443 genes from the GSE11854 dataset were identified as DEGs, which were involved in BP associated with DNA replication, CC associated with chromosomal region, and MF associated with ATPase activity. These DEGs were primarily enriched in the TGF-ß signaling pathway and spinal deformity. Further, LASSO regression suggested that 9 DEGs acted as the signature markers for somite formation. We discovered altogether 162 DEGs in PSCS patients, which were involved in BP associated with cardiac myofibril assembly and MF associated with structural constituent of muscle. However, these 162 DEGs were not significantly correlated with any pathways. Finally, COL27A1 was identified as the only intersected gene between the best predictors for somite formation and PSCS-related DEGs, which was significantly downregulated in PSCS patients. CONCLUSION: This work sheds novel lights on DEGs related to the PSCS pathogenic mechanism, and COL27A1 is the possible therapeutic target for PSCS. Findings in this work may contribute to developing therapeutic strategies for PSCS.

MECOM-related disorder: Radioulnar synostosis without hematological aberration due to unique variants.

PURPOSE: The etiology for a considerable proportion of patients with congenital radioulnar synostosis (RUS) remains unclear. This study aimed to investigate the genetic cause of RUS without a known cause. METHODS: Patients with RUS were investigated. Exome sequencing and/or Sanger sequencing was performed. Bioinformatics analysis was also performed. Pathogenicity was evaluated for variants of interest. RESULTS: We identified unique missense variants in MECOM (encodes EVI1) associated with RUS in 8 families. Of them, 6 families had variants in residue R781, including 3 families with R781C (c.2341C>T), 2 families with R781H (c.2342G>A), and 1 family with R781L (c.2342G>T). Another 2 variants included I783T (c.2348T>C) in 1 family and Q777E (c.2329C>G) in 1 family. All these variants were clustered within the ninth zinc finger motif of EVI1. Phenotype evaluation identified that most of these patients with RUS harboring mutant MECOM had finger malformations, but none of them had identifiable hematological abnormalities. Functional experiments showed that MECOM R781C led to alterations in TGF-ß-mediated transcriptional responses. CONCLUSION: This study examined MECOM variants by focusing on RUS instead of hematological abnormalities. The R781 residue in EVI1 is a hotspot for human RUS variants. Mutant MECOM is the second most common cause for familial RUS.

A genotype and phenotype analysis of SMAD6 mutant patients with radioulnar synostosis.

BACKGROUND: SMAD6 variants have been reported in patients with radioulnar synostosis (RUS). This study aimed to investigate the genotypes and phenotypes for a large cohort of patients with RUS having mutant SMAD6. METHODS: Genomic DNA samples were isolated from 251 RUS sporadic patients (with their parents) and 27 RUS pedigrees. Sanger sequencing was performed for the SMAD6 coding regions. For positive probands, co-segregation and parental-origin analysis of SMAD6 variants and phenotypic re-evaluation were performed for their family members. RESULTS: We identified 50 RUS probands with SMAD6 variants (13 co-segregated with RUS in pedigrees and 37 in RUS-sporadic patients). Based on the new and previous data, we identified SMAD6 mutated in 16/38 RUS pedigrees and 61/393 RUS sporadic patients, respectively. Overall, 93 SMAD6 mutant patients with RUS were identified, among which 29 patients had unilateral RUS, where the left side was more involved than the right side (left:right = 20:9). Female protective effects and non-full penetrance were observed, in which only 6.90% mothers (vs. ~50% fathers) of SMAD6 mutant RUS probands had RUS. Pleiotropy was observed as a re-evaluation of SMAD6 mutant families identified: (a) three families had axial skeletal malformations; (b) two families had polydactyly; and (c) eight families had other known malformations. CONCLUSION: SMAD6 was mutated in 42.11% RUS pedigrees and 15.52% RUS sporadic patients. The RUS patients with SMAD6 variants exhibit both non-full-penetrance, variable expressivity, pleiotropy, female protective effects, and the left side is more susceptible than the right side.

GDF6 Knockdown in a Family with Multiple Synostosis Syndrome and Speech Impairment.

Multiple synostoses syndrome type 4 (SYNS4; MIM 617898) is an autosomal dominant disorder characterized by carpal-tarsal coalition and otosclerosis-associated hearing loss. SYSN4 has been associated with GDF6 gain-of-function mutations. Here we report a five-generation SYNS4 family with a reduction in GDF6 expression resulting from a chromosomal breakpoint 3' of GDF6. A 30-year medical history of the family indicated bilateral carpal-tarsal coalition in ~50% of affected family members and acquired otosclerosis-associated hearing loss in females only, whereas vertebral fusion was present in all affected family members, most of whom were speech impaired. All vertebral fusions were acquired postnatally in progressive fashion from a very early age. Thinning across the 2nd cervical vertebral interspace (C2-3) in the proband during infancy progressed to block fusion across C2-7 and T3-7 later in life. Carpal-tarsal coalition and pisiform expansion were bilaterally symmetrical within, but varied greatly between, affected family members. This is the first report of SYNS4 in a family with reduced GDF6 expression indicating a prenatal role for GDF6 in regulating development of the joints of the carpals and tarsals, the pisiform, ears, larynx, mouth and face and an overlapping postnatal role in suppression of aberrant ossification and synostosis of the joints of the inner ear (otosclerosis), larynx and vertebrae. RNAseq gene expression analysis indicated >10 fold knockdown of NOMO3, RBMXL1 and NEIL2 in both primary fibroblast cultures and fresh white blood cells. Together these results provide greater insight into the role of GDF6 in skeletal joint development.

siRNA Mediate RNA Interference Concordant with Early On-Target Transient Transcriptional Interference.

Exogenous siRNAs are commonly used to regulate endogenous gene expression levels for gene function analysis, genotype-phenotype association studies and for gene therapy. Exogenous siRNAs can target mRNAs within the cytosol as well as nascent RNA transcripts within the nucleus, thus complicating siRNA targeting specificity. To highlight challenges in achieving siRNA target specificity, we targeted an overlapping gene set that we found associated with a familial form of multiple synostosis syndrome type 4 (SYSN4). In the affected family, we found that a previously unknown non-coding gene TOSPEAK/C8orf37AS1 was disrupted and the adjacent gene GDF6 was downregulated. Moreover, a conserved long-range enhancer for GDF6 was found located within TOSPEAK which in turn overlapped another gene which we named SMALLTALK/C8orf37. In fibroblast cell lines, SMALLTALK is transcribed at much higher levels in the opposite (convergent) direction to TOSPEAK. siRNA targeting of SMALLTALK resulted in post transcriptional gene silencing (PTGS/RNAi) of SMALLTALK that peaked at 72 h together with a rapid early increase in the level of both TOSPEAK and GDF6 that peaked and waned after 24 h. These findings indicated the following sequence of events: Firstly, the siRNA designed to target SMALLTALK mRNA for RNAi in the cytosol had also caused an early and transient transcriptional interference of SMALLTALK in the nucleus; Secondly, the resulting interference of SMALLTALK transcription increased the transcription of TOSPEAK; Thirdly, the increased transcription of TOSPEAK increased the transcription of GDF6. These findings have implications for the design and application of RNA and DNA targeting technologies including siRNA and CRISPR. For example, we used siRNA targeting of SMALLTALK to successfully restore GDF6 levels in the gene therapy of SYNS4 family fibroblasts in culture. To confidently apply gene targeting technologies, it is important to first determine the transcriptional interference effects of the targeting reagent and the targeted gene.

Multiple synostoses syndrome: Radiological findings and orthopedic management in a single institution cohort.

PURPOSE: Multiple synostoses syndrome (MSS) is a rare genetic condition. Classical features consist of joint fusions which notably start at the distal phalanx of the hands and feet with symphalangism progressing proximally to carpal, tarsal, radio-ulnar, and radio-humeral joints, as well as the spine. Usually, genetic testing reveals a mutation of the NOG gene with variable expressivity. The goal was to present the anatomical, functional, and radiological presentations of MSS in a series of patients followed since childhood. METHODS: Patients with more than 3 synostoses affecting at least one hand joint were included. When possible, genetic screening was offered. RESULTS: A retrospective study was performed from 1972 to 2017 and included 14 patients with a mean follow-up of 18.6 years. Mutation of the NOG protein coding gene was seen in 3 patients. All presented with tarsal synostoses including 9 carpal, 7 elbow, and 2 vertebral fusions. Facial dysmorphia was seen in 6 patients and 3 were hearing-impaired. Surgical treatment of tarsal synostosis was performed in 4 patients. Progressing joint fusions were invariably seen on x-rays amongst adults. CONCLUSION: Long radiological follow-up allowed the assessment of MSS progression. Feet deformities resulted in a severe impact on quality of life, and neurological complications secondary to spine fusions warranted performing at least one imaging study in childhood. As there is no treatment of ankylosis, physiotherapy is not recommended. However, surgical arthrodesis for the treatment of pain may have reasonable outcomes.

Nonlethal presentations of CYP26B1-related skeletal anomalies and multiple synostoses syndrome.

Retinoic acid exposures as well as defects in the retinoic acid-degrading enzyme CYP26B1 have teratogenic effects on both limb and craniofacial skeleton. An initial report of four individuals described a syndrome of fetal and infantile lethality with craniosynostosis and skeletal anomalies caused by homozygous pathogenic missense variants in CYP26B1. In contrast, a 22-year-old female was reported with a homozygous missense pathogenic variant in CYP26B1 with complex multisuture craniosynostosis and intellectual disability, suggesting that in some cases, biallelic pathogenic variants of CYP26B1 may be compatible with life. Here we describe four additional living individuals from two families with compound heterozygous pathogenic missense variants in CYP26B1. Structural assessment of these additional missense variants places them further from the catalytic site and supports a model consistent with milder nonlethal disease. In addition to previously reported findings of multisuture craniosynostosis, conductive hearing loss, joint contractures, long slender fingers, camptodactly, broad fingertips, and developmental delay/intellectual disability, skeletal imaging in our cases also revealed gracile long bones, gracile ribs, radioulnar synostosis, and carpal and/or tarsal fusions. These individuals broaden the phenotypic range of biallelic pathogenic variants in CYPB26B1 and most significantly clarify that mortality can range from perinatal lethality to survival into adulthood.

Genetic Heterogeneity and Core Clinical Features of NOG-Related-Symphalangism Spectrum Disorder.

OBJECTIVES: To better distinguish NOG-related-symphalangism spectrum disorder (NOG-SSD) from chromosomal 17q22 microdeletion syndromes and to inform surgical considerations in stapes surgery for patients with NOG-SSD. BACKGROUND: Mutations in NOG cause a variety of skeletal syndromes that often include conductive hearing loss. Several microdeletions of chromosome 17q22 lead to severe syndromes with clinical characteristics that overlap NOG-SSD. Isolated deletion of NOG has not been described, and therefore the contribution of NOG deletion in these syndromes is unknown. METHODS: Two families with autosomal dominant NOG-SSD exhibited stapes ankylosis, facial dysmorphisms, and skeletal and joint anomalies. In each family, NOG was evaluated by genomic sequencing and candidate mutations confirmed as damaging by in vitro assays. Temporal bone histology of a patient with NOG-SSD was compared with temporal bones of 40 patients diagnosed with otosclerosis. RESULTS: Family 1 harbors a 555 kb chromosomal deletion encompassing only NOG and ANKFN1. Family 2 harbors a missense mutation in NOG leading to absence of noggin protein. The incus-footplate distance of the temporal bone was significantly longer in a patient with NOG-SSD than in patients with otosclerosis. CONCLUSION: The chromosomal microdeletion of family 1 led to a phenotype comparable to that due to a NOG point mutation and much milder than the phenotypes due to other chromosome 17q22 microdeletions. Severe clinical findings in other microdeletion cases are likely due to deletion of genes other than NOG. Based on temporal bone findings, we recommend that surgeons obtain longer stapes prostheses before stapes surgery in individuals with NOG-SSD stapes ankylosis.

Spondylocarpotarsal synostosis syndrome due to a novel loss of function FLNB variant: a case report.

BACKGROUND: Loss of function or gain of function variants of Filamin B (FLNB) cause recessive or dominant skeletal disorders respectively. Spondylocarpotarsal synostosis syndrome (SCT) is a rare autosomal recessive disorder characterized by short stature, fused vertebrae and fusion of carpal and tarsal bones. We present a novel FLNB homozygous pathogenic variant and present a carrier of the variant with short height. CASE PRESENTATION: We describe a family with five patients affected with skeletal malformations, short stature and vertebral deformities. Exome sequencing revealed a novel homozygous frameshift variant c.2911dupG p.(Ala971GlyfsTer122) in FLNB, segregating with the phenotype in the family. The variant was absent in public databases and 100 ethnically matched control chromosomes. One of the heterozygous carriers of the variant had short stature. CONCLUSION: Our report expands the genetic spectrum of FLNB pathogenic variants. It also indicates a need to assess the heights of other carriers of FLNB recessive variants to explore a possible role in idiopathic short stature.

Musculoskeletal abnormalities in a large international cohort of boys with 49,XXXXY.

49,XXXXY is the rarest X and Y chromosomal variation, with an incidence of 1 in 80,000-100,000 live male births and has been associated with numerous musculoskeletal abnormalities. Data was collected from an international cohort of boys with 49,XXXXY over 10 years. Children were evaluated by a multidisciplinary team consisting of a pediatric orthopedist, a neurogeneticist, a neurodevelopmentalist, and two physical therapists. Increased rates of torticollis (32.4%), hamstring tightness (42%), radioulnar synostosis (67.6%), pes planus (65.2%), and other foot abnormalities (86.9%) were observed. Several anomalies increased with age, specifically hamstring tightness, kyphosis, and scoliosis. The elucidation of the orthopedic profile of this population is necessary in order to provide healthcare providers with current medical information. This research further supports the necessity for the comprehensive multidisciplinary treatment of boys with 49,XXXXY.

A novel heterozygous variant in FGF9 associated with previously unreported features of multiple synostosis syndrome 3.

Human multiple synostoses syndrome 3 is an autosomal dominant disorder caused by pathogenic variants in FGF9. Only two variants have been described in FGF9 in humans so far, and one in mice. Here we report a novel missense variant c.566C > G, p.(Pro189Arg) in FGF9. Functional studies showed this variant impairs FGF9 homodimerization, but not FGFR3c binding. We also review the findings of cases reported previously and report on additional features not described previously.

Identification of the third FGF9 variant in a girl with multiple synostosis-comparison of the genotype:phenotype of FGF9 variants in humans and mice.

Multiple synostosis syndrome (SYNS) is a heterogeneous group of genetic disorders mainly characterized by multiple joint synostosis due to variants in either NOG, GDF5, FGF9 or GDF6. To date, only two FGF9 variants have been associated with SYNS, characterized with hand and feet joint synostosis and fusion of the elbow and vertebral lumbar joints. Craniosynostosis was also observed in one family. Here, we report the clinical and radiological description of a young girl with a third heterozygous FGF9 variant, NM_002010.2:c.427A>T;p.(Asn143Tyr), which interestingly, is located at the same amino acid as the well characterized spontaneous Eks mouse variant. We also compare the genotype: phenotypes observed between humans and mice with SYNS.