Clinical heterogeneity of NADSYN1-associated VCRL syndrome.

The NADSYN1 gene [MIM*608285] encodes the NAD synthetase 1 enzyme involved in the final step of NAD biosynthesis, crucial for cell metabolism and organ embryogenesis. Perturbating the role of NAD biosynthesis results in the association of vertebral, cardiac, renal, and limb anomalies (VCRL). This condition was initially characterized as severe with perinatal lethality or developmental delay and complex malformations in alive cases. Sixteen NADSYN1-associated patients have been published so far. This study illustrates the wide phenotypic variability in NADSYN1-associated NAD deficiency disorder. We report the clinical and molecular findings in three novel cases, two of them being siblings with the same homozygous variant and presenting with either a very severe prenatal lethal or a mild phenotypic form. In addition to an exhaustive literature, we validate the expansion of the spectrum of NAD deficiency disorder. Our findings indicate that NAD deficiency disorder should be suspected not only in the presence of the full spectrum of VCRL, but even a single of the aforementioned organs is affected. Decreased plasmatic levels of NAD should then strongly encourage the screening for any of the genes responsible for a NAD deficiency disorder.

Identification of bi-allelic LFNG variants in three patients and further clinical and molecular refinement of spondylocostal dysostosis 3.

Spondylocostal dysostosis (SCD), a condition characterized by multiple segmentation defects of the vertebrae and rib malformations, is caused by bi-allelic variants in one of the genes involved in the Notch signaling pathway that tunes the "segmentation clock" of somitogenesis: DLL3, HES7, LFNG, MESP2, RIPPLY2, and TBX6. To date, seven individuals with LFNG variants have been reported in the literature. In this study we describe two newborns and one fetus with SCD, who were found by trio-based exome sequencing (trio-ES) to carry homozygous (c.822-5C>T) or compound heterozygous (c.[863dup];[1063G>A]) and (c.[521G>T];[890T>G]) variants in LFNG. Notably, the c.822-5C>T change, affecting the polypyrimidine tract of intron 5, is the first non-coding variant reported in LFNG. This study further refines the clinical and molecular features of spondylocostal dysostosis 3 and adds to the numerous investigations supporting the usefulness of trio-ES approach in prenatal and neonatal settings.

Congenital cervical spine malformation due to bi-allelic RIPPLY2 variants in spondylocostal dysostosis type 6.

RIPPLY2 is an essential part of the formation of somite patterning during embryogenesis and in establishment of rostro-caudal polarity. Here, we describe three individuals from two families with compound-heterozygous variants in RIPPLY2 (NM_001009994.2): c.238A > T, p.(Arg80*) and c.240-4 T > G, p.(?), in two 15 and 20-year-old sisters, and a homozygous nonsense variant, c.238A > T, p.(Arg80*), in an 8 year old boy. All patients had multiple vertebral body malformations in the cervical and thoracic region, small or absent rib involvement, myelopathies, and common clinical features of SCDO6 including scoliosis, mild facial asymmetry, spinal spasticity and hemivertebrae. The nonsense variant can be classified as likely pathogenic based on the ACMG criteria while the splice variants must be classified as a variant of unknown significance. With this report on two further families, we confirm RIPPLY2 as the gene for SCDO6 and broaden the phenotype by adding myelopathy with or without spinal canal stenosis and spinal spasticity to the symptom spectrum.

Congenital posterior cervical spine malformation due to biallelic c.240-4T>G RIPPLY2 variant: A discrete entity.

The clinical and radiological spectrum of spondylocostal dysostosis syndromes encompasses distinctive costo-vertebral anomalies. RIPPLY2 biallelic pathogenic variants were described in two distinct cervical spine malformation syndromes: Klippel-Feil syndrome and posterior cervical spine malformation. RIPPLY2 is involved in the determination of rostro-caudal polarity and somite patterning during development. To date, only four cases have been reported. The current report aims at further delineating the posterior malformation in three new patients. Three patients from two unrelated families underwent clinical and radiological examination through X-ray, 3D computed tomography and brain magnetic resonance imaging. After informed consent was obtained, family-based whole exome sequencing (WES) was performed. Complex vertebral segmentation defects in the cervico-thoracic spine were observed in all patients. WES led to the identification of the homozygous splicing variant c.240-4T>G in all subjects. This variant is predicted to result in aberrant splicing of Exon 4. The current report highlights a subtype of cervical spine malformation with major atlo-axoidal malformation compromising spinal cord integrity. This distinctive mutation-specific pattern of malformation differs from Klippel-Feil syndrome and broadens the current classification, defining a sub-type of RIPPLY2-related skeletal disorder. Of note, the phenotype of one patient overlaps with oculo-auriculo-vertebral spectrum disorder.

Bi-allelic loss of function variants of TBX6 causes a spectrum of malformation of spine and rib including congenital scoliosis and spondylocostal dysostosis.

BACKGROUND: Congenital scoliosis (CS) is a common vertebral malformation. Spondylocostal dysostosis (SCD) is a rare skeletal dysplasia characterised by multiple vertebral malformations and rib anomalies. In a previous study, a compound heterozygosity for a null mutation and a risk haplotype composed by three single-nucleotide polymorphisms in TBX6 have been reported as a disease-causing model of CS. Another study identified bi-allelic missense variants in a SCD patient. The purpose of our study is to identify TBX6 variants in CS and SCD and examine their pathogenicity. METHODS: We recruited 200 patients with CS or SCD and investigated TBX6 variants. We evaluated the pathogenicity of the variants by in silico prediction and in vitro experiments. RESULTS: We identified five 16p11.2 deletions, one splice-site variant and five missense variants in 10 patients. In vitro functional assays for missense variants identified in the previous and present studies demonstrated that most of the variants caused abnormal localisation of TBX6 proteins. We confirmed mislocalisation of TBX6 proteins in presomitic mesoderm cells induced from SCD patient-derived iPS cells. In induced cells, we found decreased mRNA expressions of TBX6 and its downstream genes were involved in somite formation. All CS patients with missense variants had the risk haplotype in the opposite allele, while a SCD patient with bi-allelic missense variants did not have the haplotype. CONCLUSIONS: Our study suggests that bi-allelic loss of function variants of TBX6 cause a spectrum of phenotypes including CS and SCD, depending on the severity of the loss of TBX6 function.

Diagnostic strategy in segmentation defect of the vertebrae: a retrospective study of 73 patients.

BACKGROUND: Segmentation defects of the vertebrae (SDV) are non-specific features found in various syndromes. The molecular bases of SDV are not fully elucidated due to the wide range of phenotypes and classification issues. The genes involved are in the Notch signalling pathway, which is a key system in somitogenesis. Here we report on mutations identified in a diagnosis cohort of SDV. We focused on spondylocostal dysostosis (SCD) and the phenotype of these patients in order to establish a diagnostic strategy when confronted with SDV. PATIENTS AND METHODS: We used DNA samples from a cohort of 73 patients and performed targeted sequencing of the five known SCD-causing genes (DLL3, MESP2, LFNG, HES7 and TBX6) in the first 48 patients and whole-exome sequencing (WES) in 28 relevant patients. RESULTS: Ten diagnoses, including four biallelic variants in TBX6, two biallelic variants in LFNG and DLL3, and one in MESP2 and HES7, were made with the gene panel, and two diagnoses, including biallelic variants in FLNB and one variant in MEOX1, were made by WES. The diagnostic yield of the gene panel was 10/73 (13.7%) in the global cohort but 8/10 (80%) in the subgroup meeting the SCD criteria; the diagnostic yield of WES was 2/28 (8%). CONCLUSION: After negative array CGH, targeted sequencing of the five known SCD genes should only be performed in patients who meet the diagnostic criteria of SCD. The low proportion of candidate genes identified by WES in our cohort suggests the need to consider more complex genetic architectures in cases of SDV.

Spondylocostal Dysostosis Associated with Split Spinal Cord and Other Malformations.

Spondylocostal dysostosis is a very rare combination of complex vertebra and rib malformations, accompanied occasionally by other disorders. A 3-year-old girl presented kyphoscoliosis, foot deformities, gate disturbance, and urinary incontinence. The CT and MRI examination revealed kyphosis and scoliosis with a double curve, some absent, broadened, bifurcating and fused ribs, hemivertebrae, butterfly and cleft vertebrae in thoracic and lumbar region, sporadic cleft or absent vertebral arches or pedicles, and hypoplastic sacrum with a cleft of the S2 vertebra. Spina bifida occulta extended from T10 to T11, and from L3 to the end of the sacrum. Two hemicords, separated by a bony septum and surrounded by their own dural tubes (type I), were present from the level of T9 to the conus medullaris. Filum terminale was thick and duplicated. Syringomyelia was present in the thoracic cord from T5 to T8. Finally, a small meningocele was seen at the T10-T11 level, and a subcutaneous lipoma in the thoracolumbar region. To our knowledge, such a combination of vertebra, rib, and cord malformations, including the mentioned additional disorders, has never been reported.

Homozygous DMRT2 variant associates with severe rib malformations in a newborn.

Spondylocostal dysostosis (SCD) is a rare disorder characterized by vertebral segmentation defects and malformations of the ribs. SCD patients have some degree of (kypho)scoliosis, short stature and suffer from respiratory impairment due to the reduced size of their thoracic cage. Mutations in DLL3, MESP2, LFNG, HES7, TBX6, and RIPPLY2 are known to cause different subtypes of SCD. Here, we report on a male neonate with an apparent distinct SCD-like phenotype only partly overlapping the previously described SCD subtypes. The proband presented with severe rib malformations (missing, fused, bifid, and hypoplastic ribs), vertebral malformations (intervertebral fusions of the laminae and irregular ossification of the vertebral bodies), and a mild scoliosis. Clear segmentation defects of the vertebral bodies were lacking. Other dysmorphic features were present as well. Severe respiratory insufficiency was present from birth. Whole exome sequencing identified a homozygous start-loss variant in DMRT2 (NM_006557.6: c.1A > T p.[Met1?]) being a likely cause of the SCD-like phenotype in the proband. Mutations in DMRT2 (OMIM#604935) have not been described in relation to SCD-related phenotypes in humans before. However, Dmrt2 knock-out mice exhibit severe rib and vertebral defects that strikingly overlap with the radiological phenotype of the proband reported here. Therefore, it seems plausible that mutations in DMRT2 are associated with a different (novel) subtype of SCD mainly characterized by severe rib anomalies but lacking clear segmentation defects of the vertebral bodies.

Compound Heterozygosity for Null Mutations and a Common Hypomorphic Risk Haplotype in TBX6 Causes Congenital Scoliosis.

Congenital scoliosis (CS) occurs as a result of vertebral malformations and has an incidence of 0.5-1/1,000 births. Recently, TBX6 on chromosome 16p11.2 was reported as a disease gene for CS; about 10% of Chinese CS patients were compound heterozygotes for rare null mutations and a common haplotype defined by three SNPs in TBX6. All patients had hemivertebrae. We recruited 94 Japanese CS patients, investigated the TBX6 locus for both mutations and the risk haplotype, examined transcriptional activities of mutant TBX6 in vitro, and evaluated clinical and radiographic features. We identified TBX6 null mutations in nine patients, including a missense mutation that had a loss of function in vitro. All had the risk haplotype in the opposite allele. One of the mutations showed dominant negative effect. Although all Chinese patients had one or more hemivertebrae, two Japanese patients did not have hemivertebra. The compound heterozygosity of null mutations and the common risk haplotype in TBX6 also causes CS in Japanese patients with similar incidence. Hemivertebra was not a specific type of spinal malformation in TBX6-associated CS (TACS). A heterozygous TBX6 loss-of-function mutation has been reported in a family with autosomal-dominant spondylocostal dysostosis, but it may represent a spectrum of the same disease with TACS.

Autosomal recessive variations of TBX6, from congenital scoliosis to spondylocostal dysostosis.

Proximal 16p11.2 microdeletions are recurrent microdeletions with an overall prevalence of 0.03%. In patients with segmentation defects of the vertebra (SDV), a burden of this microdeletion was observed with TBX6 as a candidate gene for SDV. In a published cohort of patients with congenital scoliosis (CS), TBX6 haploinsufficiency was compound heterozygous with a common haplotype. Besides, a single three-generation family with spondylocostal dysostosis (SCD) was reported with a heterozygous stop-loss of TBX6. These observations questioned both on the inheritance mode and on the variable expressivity associated with TBX6-associated SDV. Based on a national recruitment of 56 patients with SDV, we describe four patients with variable SDV ranging from CS to SCD associated with biallelic variations of TBX6. Two patients with CS were carrying a proximal 16p11.2 microdeletion associated with the previously reported haplotype. One patient with extensive SDV was carrying a proximal 16p11.2 microdeletion associated with a TBX6 rare missense change. One patient with a clinical diagnosis of SCD was compound heterozygous for two TBX6 rare missense changes. The three rare variants were affecting the chromatin-binding domain. Our data illustrate the variable expressivity of recessive TBX6 ranging from CS to SCD.

Chest wall hypoplasia--principles and treatment.

The chest is a dynamic structure. For normal movement it relies on a coordinated movement of the multiple bones, joints and muscles of the respiratory system. While muscle weakness can have clear impact on respiration by decreasing respiratory motion, so can conditions that cause chest wall hypoplasia and produce an immobile chest wall. These conditions, such as Jarcho-Levin and Jeune syndrome, present significantly different challenges than those faced with early onset scoliosis in which chest wall mechanics and thoracic volume may be much closer to normal. Because of this difference more aggressive approaches to clinical and surgical management are necessary.

Spondylocostal dysostosis with type II split cord malformation: A report of a rare case and brief review of the literature.

Spondylocostal dysostosis (Jarcho Levin syndrome) is a rare costovertebral malformation syndrome that will result in restrictive pulmonary physiology. It manifests its major components at birth. Split cord malformation, together with spondylocostal dysostosis, is even rarer. We hereby report our experience with diagnosing 1 infant with spondylocostal dysostosis and type II split cord malformation using computed tomography and magnetic resonance imaging. We also present a concise summary of previously published case reports and case series involving patients with concurrent spondylocostal dysostosis and split cord malformations.

Molecular landscape of congenital vertebral malformations: recent discoveries and future directions.

Vertebral malformations (VMs) pose a significant global health problem, causing chronic pain and disability. Vertebral defects occur as isolated conditions or within the spectrum of various congenital disorders, such as Klippel-Feil syndrome, congenital scoliosis, spondylocostal dysostosis, sacral agenesis, and neural tube defects. Although both genetic abnormalities and environmental factors can contribute to abnormal vertebral development, our knowledge on molecular mechanisms of numerous VMs is still limited. Furthermore, there is a lack of resource that consolidates the current knowledge in this field. In this pioneering review, we provide a comprehensive analysis of the latest research on the molecular basis of VMs and the association of the VMs-related causative genes with bone developmental signaling pathways. Our study identifies 118 genes linked to VMs, with 98 genes involved in biological pathways crucial for the formation of the vertebral column. Overall, the review summarizes the current knowledge on VM genetics, and provides new insights into potential involvement of biological pathways in VM pathogenesis. We also present an overview of available data regarding the role of epigenetic and environmental factors in VMs. We identify areas where knowledge is lacking, such as precise molecular mechanisms in which specific genes contribute to the development of VMs. Finally, we propose future research avenues that could address knowledge gaps.

Mutation of HES7 in a large extended family with spondylocostal dysostosis and dextrocardia with situs inversus.

Spondylocostal dysotosis (SCD) is a rare developmental congenital abnormality of the axial skeleton. Mutation of genes in the Notch signaling pathway cause SCD types 1-5. Dextrocardia with situs inversus is a rare congenital malformation in which the thoracic and abdominal organs are mirror images of normal. Such laterality defects are associated with gene mutations in the Nodal signaling pathway or cilia assembly or function. We investigated two distantly related individuals with a rare combination of severe segmental defects of the vertebrae (SDV) and dextrocardia with situs inversus. We found that both individuals were homozygous for the same mutation in HES7, and that this mutation caused a significant reduction of HES7 protein function; HES7 mutation causes SCD4. Two other individuals with SDV from two unrelated families were found to be homozygous for the same mutation. Interestingly, although the penetrance of the vertebral defects was complete, only 3/7 had dextrocardia with situs inversus, suggesting randomization of left-right patterning. Two of the affected individuals presented with neural tube malformations including myelomeningocele, spina bifida occulta and/or Chiari II malformation. Such neural tube phenotypes are shared with the originally identified SCD4 patient, but have not been reported in the other forms of SCD. In conclusion, it appears that mutation of HES7 is uniquely associated with defects in vertebral, heart and neural tube formation, and this observation will help provide a discriminatory diagnostic guide in patients with SCD, as well as inform molecular genetic testing.

Sprengel's deformity and spinal dysraphism: connecting the shoulder and the spine.

BACKGROUND AND PURPOSE: Sprengel's deformity, a rare congenital malformation of the scapula, may be observed in combination with spinal dysraphism. The co-occurrence of these malformations suggests an unknown shared etiology. Therefore, we reviewed the medical records of eight children presenting with both malformations and performed a review of the literature. PATIENTS AND METHODS: Databases from four university medical centers were searched for children presenting between 1992 and 2012 with spinal dysraphism and a Sprengel's deformity. CONCLUSION: The combination of spinal dysraphism and Sprengel's deformity is rare, and is associated with segmentation defects of the spine and ribs. Although the etiology of both spinal dysraphism and Sprengel's deformity remains unclear, all deformities of the spine, ribs, and shoulder might result from a common genetic defect affecting somitogenesis.

Case report: Abdominal hernia repair using a surgical wire and an autologous omental graft in a Japanese Black calf.

Background: Hernia formation is a well-known abdominal wall disorder in calves and most often occurs in the umbilical region. In addition, it occurs in the abdominal wall outside the umbilical region. It has been reported to involve acquired factors, such as external force to the lower or lateral abdominal wall, trauma, muscle weakness, and, although rare, congenital or hereditary factors. Although there have been reports on the repair of abdominal wall hernias caused by abnormal muscle formation, there have been no reports on the treatment of abdominal wall hernias caused by abnormal vertebral and rib formation in cattle or other ruminants. In this study, for the first time, we describe a case of successful closure of a hernia in the left flank caused by malformation of the vertebrae and ribs. The repair was performed by narrowing the ribs using a surgical wire and covering the defect with a free omental graft. Case presentation: A male Japanese Black calf showed a mild bulge of the left abdominal wall and abnormal morphology of the left ribs immediately after birth. At 3 months of age, computed tomography revealed fusion of the 9th, 10th, and 11th thoracic vertebrae and missing formation of the 10th and 11th left ribs at the thoracic vertebral fusion site. Additionally, a 15.2 × 24.4 cm abdominal hernia had formed in the abdominal wall. During surgery, the ribs forming the hernia were narrowed with a surgical wire, and the hernia was covered with a large autologous free omental graft to prevent intra-abdominal organ prolapse. At the monthly follow-up (11 months after surgery), the calf had developed similarly to other calves of the same age on the farm, and no complications were noted. Conclusions: The current case shows that a combination of rib correction using a surgical wire combined with transplantation of a free autologous greater omentum graft was shown to be a possible treatment option for surgical repair of large abdominal wall hernias caused by rib malformation.

The role of Notch signaling pathway in metabolic bone diseases.

Metabolic bone diseases is the third most common endocrine diseases after diabetes and thyroid diseases. More than 500 million people worldwide suffer from metabolic bone diseases. The generation and development of bone metabolic diseases is a complex process regulated by multiple signaling pathways, among which the Notch signaling pathway is one of the most important pathways. The Notch signaling pathway regulates the differentiation and function of osteoblasts and osteoclasts, and affects the process of cartilage formation, bone formation and bone resorption. Genetic mutations in upstream and downstream of Notch signaling genes can lead to a series of metabolic bone diseases, such as Alagille syndrome, Adams-Oliver syndrome and spondylocostal dysostosis. In this review, we analyzed the mechanisms of Notch ligands, Notch receptors and signaling molecules in the process of signal transduction, and summarized the progress on the pathogenesis and clinical manifestations of bone metabolic diseases caused by Notch gene mutation. We hope to draw attention to the role of the Notch signaling pathway in metabolic bone diseases and provide new ideas and approaches for the diagnosis and treatment of metabolic bone diseases.

Identification of a Novel Nonsense Variant in the DLL3 Gene Underlying Spondylocostal Dysostosis in a Consanguineous Pakistani Family.

Introduction: Spondylocostal dysostosis (SCD) is characterized by multiple vertebral abnormalities associated with abnormalities of the ribs. Five genes causative for the disease have been identified. These include DLL3 (OMIM *602768), MESP2 (OMIM #608681), LFNG (OMIM #609813), TBX6 (OMIM *602427), and HES7 (OMIM *608059). Methods: In the current study, we investigated a Pakistani consanguineous family segregating spondylocostal dysotosis. Whole-exome sequencing (WES) followed by Sanger sequencing was performed using DNA of affected and unaffected individuals to identify pathogenic variant(s). The identified variant was interpreted using ACMG classification. Literature review was performed to summarize currently known mutated alleles of DLL3 and the underlying clinical phenotypes. Results: Clinical examination using anthropometric measurements and radiographs diagnosed the patients to be afflicted with SCD. Pedigree analysis of the affected family showed an autosomal recessive inheritance pattern of the disease. WES followed by Sanger sequencing identified a novel homozygous nonsense variant (DLL3(NM_016941.4): c.535G>T; p.Glu179Ter) in the DLL3 gene located on chromosome 19q13.2. Conclusion: The study will be helpful in carrier testing and genetic counseling to prevent segregation of the disease to the next generations within this family. It also provides knowledge for clinicians and researchers in search of a better understanding of SCD anomalies.

A novel homozygous HES7 splicing variant causing spondylocostal dysostosis 4: a case report.

Background: Spondylocostal dysostosis 4 (SCDO4) is characterized by short stature (mainly short trunk), dyspnea, brain meningocele, and spina bifida occulta, which is caused by homozygous or compound heterozygous HES7 (HES family bHLH transcription factor 7) variants. The incidence of SCDO4 remains unknown due to the extremely low number of cases. This study reveals a novel homozygous HES7 splicing variant causing SCDO4 and reviews all the previously reported HES7 variants and corresponding symptoms, providing a comprehensive overview of the phenotypes and genotypes of HES7 variants. Case presentation: This case report focuses on a Chinese neonate who was first hospitalized for tachypnea, cleft palate, and short trunk. After a series of auxiliary examinations, the patient was also found to have deformities of vertebrae and rib, left hydronephrosis, and patent foramen ovale. He underwent surgery for congenital hydronephrosis at 5 months old and underwent cleft palate repair when he was 1 year old. After two and half years of follow-up, the boy developed normally. A novel homozygous HES7 splicing variant (c.226+1G>A, NM_001165967.2) was identified in the proband by whole-exome sequencing and verified by Sanger sequencing. The variant was inherited from both parents and minigene assays demonstrated that this variant resulted in the retention of intron3 in the HES7 transcript. Including this case, a total of six HES7 variants and 13 patients with SCDO4 have been reported. Conclusions: Our findings expand the genotype-phenotype knowledge of SCDO4 and provide new evidence for genetic counseling.