Craniofacial studies in chicken embryos confirm the pathogenicity of human FZD2 variants associated with Robinow syndrome.

Robinow syndrome is a rare disease caused by variants of seven WNT pathway genes. Craniofacial features include widening of the nasal bridge and jaw hypoplasia. We used the chicken embryo to test whether two missense human FZD2 variants (1301G>T, p.Gly434Val; 425C>T, p.Pro142Lys) were sufficient to change frontonasal mass development. In vivo, the overexpression of retroviruses with wild-type or variant human FZD2 inhibited upper beak ossification. In primary cultures, wild-type and variant human FZD2 significantly inhibited chondrogenesis, with the 425C>T variant significantly decreasing activity of a SOX9 luciferase reporter compared to that for the wild type or 1301G>T. Both variants also increased nuclear shuttling of ß-catenin (CTNNB1) and increased the expression of TWIST1, which are inhibitory to chondrogenesis. In canonical WNT luciferase assays using frontonasal mass cells, the variants had dominant-negative effects on wild-type FZD2. In non-canonical assays, the 425C>T variant failed to activate the reporter above control levels and was unresponsive to exogenous WNT5A. This is the first single amino acid change to selectively alter ligand binding in a FZD receptor. Therefore, FZD2 missense variants are pathogenic and could lead to the altered craniofacial morphogenesis seen in Robinow syndrome.

Prenatal diagnosis of a severe form of frontonasal dysplasia with severe limb anomalies, hydrocephaly, a hypoplastic corpus callosum, and a ventricular septal defect using 3D ultrasound: a case report and literature review.

BACKGROUND: Frontonasal dysplasia (FND) is a rare congenital anomaly resulting from the underdevelopment of the frontonasal process, and it can be syndromic or nonsyndromic. The typical features of FND include a deformed nose and ocular hypertelorism, which are sometimes associated with cleft lip and/or palate. Only approximately 10 cases of prenatally diagnosed nonsyndromic FND have been reported in the past 30 years. CASE PRESENTATION: A 33-year-old woman (G2P1) was referred to our center at 20 gestational weeks for bilateral hydrocephaly. We detected typical features of FND, including severe hypertelorism, median nasal bifidity, a minor cleft lip, and multiple limb anomalies using three-dimensional (3D) ultrasound. A hypoplastic corpus callosum, unilateral microtia, and a ventricular septal defect were also detected. Genetic testing, including karyotype analysis, copy number variation (CNV) analysis, trio-whole exome sequencing (trio-WES), and trio-whole-gene sequencing (trio-WGS), was performed; however, we did not find any de novo gene variants in the fetus as compared to the parents. Postmortem examination confirmed the prenatal diagnosis of FND. CONCLUSION: The present case expands the wide phenotypic spectrum of prenatal FND patients. 3D ultrasound is a useful tool for detecting facial and limb deformities.

Microdeletion on Xq27.1 in a Chinese VACTERL-Like Family with Kidney and Anal Anomalies.

Objective: VATER/VACTERL-like association is associated with adverse pregnancy outcomes. Genetic evidence of this disorder is sporadic. In this study, we aimed to provide genetic insights to improve the diagnosis of VACTERL. Methods: We have described a Chinese family in which four members were affected by renal defects or agenesis, anal atresia, and anovaginal fistula, which is consistent with the diagnosis of a VACTERL-like association. Pedigree and genetic analyses were conducted using genome and exome sequencing. Results: Segregation analysis revealed the presence of a recessive X-linked microdeletion in two living affected individuals, harboring a 196-380 kb microdeletion on Xq27.1, which was identified by familial exome sequencing. Genome sequencing was performed on the affected male, confirming a -196 kb microdeletion in Xq27.1, which included a 28% loss of the CDR-1 gene. Four family members were included in the co-segregation analysis, and only VACTERL-like cases with microdeletions were reported in X27.1. Conclusion: These results suggest that the 196-380 kb microdeletion in Xq27.1 could be a possible cause of the VATER/VACTERL-like association. However, further genetic and functional analyses are required to confirm or rule out genetic background as the definitive cause of the VACTERL association.

Prenatal diagnosis of a skeletal disorder characterized by rhizomelic shortening of limbs caused by compound heterozygous variants in the PKDCC gene: Case report and literature review.

BACKGROUND: The protein kinase domain containing cytoplasmic (PKDCC) gene (OMIM#618821) is associated with bone development. Biallelic variants in the PKDCC gene can cause rhizomelic limb shortening with dysmorphic features. CASE REPORT: A fetus was found to be rhizomelic limb shortening at 16 weeks of gestation and amniocentesis was performed at 19 weeks of gestation. Genomic DNA extracted from the amniotic fluid was subjected to chromosomal microarray analysis (CMA), and Trio-total whole-exome sequencing (Trio-WES). Sanger sequencing was used to verify the candidate pathogenic variants. CMA was normal, while Trio-WES identified two compound heterozygous variants in the PKDCC gene, namely c.417_c.423delCGGCGCG insTCATGGGCTCAGTACAC(p.G140fs*35) and c.345G>A (p.W115*,379). Then the fetus was aborted and the development of its bone cells were compared with that of a normal fetus of similar gestational age by histopathological examination. Clinical findings of the fetus were shortening humerus and femur, synophrys, much hair on the side face, simian line on the right palm, etc. Histopathological examination showed that the affected fetus had increased proliferative chondrocytes, widened proliferative bands, and delayed bone mineralization. CONCLUSIONS: We reported a prenatal case of rhizomelic shortening of limbs caused by compound heterozygous variants in the PKDCC gene, which emphasized the important role of Trio-WES for diagnosis of skeletal dysplasia in fetuses.

Expanding the phenotypic spectrum of NOTCH1 variants: clinical manifestations in families with congenital heart disease.

Pathogenic variants in NOTCH1 are associated with non-syndromic congenital heart disease (CHD) and Adams-Oliver syndrome (AOS). The clinical presentation of individuals with damaging NOTCH1 variants is characterized by variable expressivity and incomplete penetrance; however, data on systematic phenotypic characterization are limited. We report the genotype and phenotype of a cohort of 33 individuals (20 females, 13 males; median age 23.4 years, range 2.5-68.3 years) from 11 families with causative NOTCH1 variants (9 inherited, 2 de novo; 9 novel), ascertained from a proband with CHD. We describe the cardiac and extracardiac anomalies identified in these 33 individuals, only four of whom met criteria for AOS. The most common CHD identified was tetralogy of Fallot, though various left- and right-sided lesions and septal defects were also present. Extracardiac anomalies identified include cutis aplasia (5/33), cutaneous vascular anomalies (7/33), vascular anomalies of the central nervous system (2/10), Poland anomaly (1/33), pulmonary hypertension (2/33), and structural brain anomalies (3/14). Identification of these findings in a cardiac proband cohort supports NOTCH1-associated CHD and NOTCH1-associated AOS lying on a phenotypic continuum. Our findings also support (1) Broad indications for NOTCH1 molecular testing (any familial CHD, simplex tetralogy of Fallot or hypoplastic left heart); (2) Cascade testing in all at-risk relatives; and (3) A thorough physical exam, in addition to cardiac, brain (structural and vascular), abdominal, and ophthalmologic imaging, in all gene-positive individuals. This information is important for guiding the medical management of these individuals, particularly given the high prevalence of NOTCH1 variants in the CHD population.

Characterization of a New Variant in ARHGAP31 Probably Involved in Adams-Oliver Syndrome in a Family with a Variable Phenotypic Spectrum.

Adams-Oliver syndrome is a rare inherited condition characterized by scalp defects and limb abnormalities. It is caused by variants in different genes such as ARHGAP31. Here, we used an interdisciplinary approach to study a family with lower limb anomalies. We identified a novel variant in the ARHGAP31 gene that is predicted to result in a truncated protein with a constitutively activated catalytic site due to the loss of 688 amino acids involved in the C-terminal domain, essential for protein auto-inhibition. Pathogenic variants in ARHGAP31 exon 12, leading to a premature protein termination, are associated with Adams-Oliver syndrome. Bioinformatic analysis was useful to elucidate the impact of the identified genetic variant on protein structure. To better understand the impact of the identified variant, 3D protein models were predicted for the ARHGAP31 wild type, the newly discovered variant, and other pathogenetic alterations already reported. Our study identified a novel variant probably involved in Adams-Oliver syndrome and increased the evidence on the phenotypic variability in patients affected by this syndrome, underlining the importance of translational research, including experimental and bioinformatics analyses. This strategy represents a successful model to investigate molecular mechanisms involved in syndrome occurrence.

VACTERL Association in Patients With Metopic Synostosis: Is There a Link?

VACTERL association is diagnosed based on the non-random co-occurrence of at least 3 out of 6 congenital malformations. The prevalence is thought to be less than 1 in 10,000 to 1 in 40,000. There is no known link between VACTERL association and metopic synostosis in the literature. There were 122 operated cases of metopic synostosis at our institution from 1999 to 2023, with a 2.3:1 male-to-female ratio. The authors describe the co-occurrence of VACTERL association and metopic synostosis in 3 female patients with no identifiable genetic variants. Given that VACTERL association is a diagnosis of exclusion, other rare syndromes were considered but ultimately excluded. This suggests that the co-occurrence of VACTERL association and metopic synostosis is a potentially rare finding, and underlying pathogenic variants are yet to be identified.

The pathogenic mechanism of syndactyly type V identified in a Hoxd13Q50R knock-in mice.

Syndactyly type V (SDTY5) is an autosomal dominant extremity malformation characterized by fusion of the fourth and fifth metacarpals. In the previous publication, we first identified a heterozygous missense mutation Q50R in homeobox domain (HD) of HOXD13 in a large Chinese family with SDTY5. In order to substantiate the pathogenicity of the variant and elucidate the underlying pathogenic mechanism causing limb malformation, transcription-activator-like effector nucleases (TALEN) was employed to generate a Hoxd13Q50R mutant mouse. The mutant mice exhibited obvious limb malformations including slight brachydactyly and partial syndactyly between digits 2-4 in the heterozygotes, and severe syndactyly, brachydactyly and polydactyly in homozygotes. Focusing on BMP2 and SHH/GREM1/AER-FGF epithelial mesenchymal (e-m) feedback, a crucial signal pathway for limb development, we found the ectopically expressed Shh, Grem1 and Fgf8 and down-regulated Bmp2 in the embryonic limb bud at E10.5 to E12.5. A transcriptome sequencing analysis was conducted on limb buds (LBs) at E11.5, revealing 31 genes that exhibited notable disparities in mRNA level between the Hoxd13Q50R homozygotes and the wild-type. These genes are known to be involved in various processes such as limb development, cell proliferation, migration, and apoptosis. Our findings indicate that the ectopic expression of Shh and Fgf8, in conjunction with the down-regulation of Bmp2, results in a failure of patterning along both the anterior-posterior and proximal-distal axes, as well as a decrease in interdigital programmed cell death (PCD). This cascade ultimately leads to the development of syndactyly and brachydactyly in heterozygous mice, and severe limb malformations in homozygous mice. These findings suggest that abnormal expression of SHH, FGF8, and BMP2 induced by HOXD13Q50R may be responsible for the manifestation of human SDTY5.

Heterozygous MAP3K20 variants cause ectodermal dysplasia, craniosynostosis, sensorineural hearing loss, and limb anomalies.

Biallelic pathogenic variants in MAP3K20, which encodes a mitogen-activated protein kinase, are a rare cause of split-hand foot malformation (SHFM), hearing loss, and nail abnormalities or congenital myopathy. However, heterozygous variants in this gene have not been definitively associated with a phenotype. Here, we describe the phenotypic spectrum associated with heterozygous de novo variants in the linker region between the kinase domain and leucine zipper domain of MAP3K20. We report five individuals with diverse clinical features, including craniosynostosis, limb anomalies, sensorineural hearing loss, and ectodermal dysplasia-like phenotypes who have heterozygous de novo variants in this specific region of the gene. These individuals exhibit both shared and unique clinical manifestations, highlighting the complexity and variability of the disorder. We propose that the involvement of MAP3K20 in endothelial-mesenchymal transition provides a plausible etiology of these features. Together, these findings characterize a disorder that both expands the phenotypic spectrum associated with MAP3K20 and highlights the need for further studies on its role in early human development.

[A Uncommon Case: Kasabach-Merritt syndrome with VACTERL Association]. / Ein seltener Fall: Kasabach-Merrit-Syndrom bei VACTERL-Assoziation.

The Kasabach-Merrit syndrome is characterized as the association of a vascular tumor, typically a caposiform hemangioendothelioma and rarely a tufted hemangioma, and a severe consumptive coagulopathy with potentially life-threatening thrombocytopenia. The severe coagulopathy with increased bleeding tendency must be considered before invasive procedures and often requires repeated platelet concentrate substitutions. We present a case of a mature male neonate with Kasabach-Merritt- Syndrome as well as VACTERL association. The VACTERL association describes a group of malformations. Our patient presented with anal atresia combined with tethered cord, and left renal agenesis. The VACTERL association as well as Kasabach-Merritt syndrome were found to be independent pathologies within this patient. A common occurrence or an association with each other has not been described in the literature so far. The challenging coagulation setting due to severe thrombocytopenia complicated the surgical management so far. Finally, mTOR-inhibitor sirolimus was successful in terms of tumor reduction and especially reduction of platelet consumption.

[Comparative study of external fixator combined with Kirschner wire fixation and Kirschner wires fixation in treatment of cubitus varus deformity in children].

Objective: To analyze the effectiveness comparison of external fixator combined with Kirschner wire fixation and Kirschner wire fixation in the treatment of cubitus varus deformity in children. Methods: A retrospective case-control study was conducted to collect 36 children of postoperative supracondylar humerus fracture complicating cubitus varus deformity between January 2018 and July 2022. Among them, 17 cases were treated with distal humeral wedge osteotomy external fixation combined with Kirschner wire fixation (observation group), and 19 cases were treated with distal humeral wedge osteotomy and Kirschner wire fixation (control group). The baseline data including age, gender, deformity side, time from fracture to operation, carrying angle of the healthy side and preoperative carrying angle of the affected side, elbow flexion and extension range of motion, and lateral condylar prominence index (LCPI) showed no significant difference between the two groups ( P>0.05). The operation time, hospitalization cost, healing time of osteotomy, postoperative complications, and the carrying angle, LCPI, and elbow flexion and extention range of motion were recorded and compared between the two groups. The elbow function was assessed by Oppenheim score at 3 months after operation and at last follow-up. Results: The children in both groups were followed up 13-48 months, with an average of 26.7 months. There was 1 case of needle tract infection in the observation group and 2 cases in the control group, and no nerve injury occurred, the difference in the incidence of complication (5.88% vs 10.53%) between the two groups was not significant ( χ 2=0.502, P=0.593). There was no significant difference in the operation time and fracture healing time between the two groups ( P>0.05); the hospitalization cost of the observation group was significantly higher than that of the control group ( P<0.05). The Oppenheim score of the observation group was significantly better than that of the control group at 3 months after operation ( P<0.05), but there was no significant difference in the Oppenheim score between the two groups at last follow-up ( P>0.05). At last follow-up, the carrying angle of affected side significantly improved in both groups when compared with preoperative ones ( P<0.05); the differences of the pre- and post-operative carrying angle of affected side and elbow flexion and extension range of motion showed no significant differences between the two groups ( P>0.05), but the difference in pre- and post-operative LCPI of the observation group was significantly better than that of the control group ( P<0.05). Conclusion: External fixator combined with Kirschner wire fixation and Kirschner wire fixation both can achieve satisfactory correction of cubitus varus deformity in children, and the former can achieve better short-term functional recovery of elbow joint and reduce the incidence of humeral lateral condyle protrusion.

Tracheal agenesis versus tracheal atresia: anatomical conditions, pathomechanisms and causes with a possible link to a novel MAPK11 variant in one case.

BACKGROUND: In this study we aimed to describe the morphological and pathogenetic differences between tracheal agenesis and tracheal atresia, which are not clearly distinguished from each other in the literature, and to contribute thereby to the understanding and management of these conditions. Both tracheal agenesis and tracheal atresia represent rare disorders of still unknown aetiology that cannot be detected by prenatal ultrasound. If the affected foetuses survive until birth these conditions result in respiratory failure and in futile attempts to rescue the infant's life. RESULTS: Autopsies and genetic analyses, including singleton or trio exome sequencing, were performed on five neonates/foetuses with tracheal agenesis and three foetuses with tracheal atresia. Tracheal agenesis was characterized by absence of the sublaryngeal trachea and presence of a bronchooesophageal fistula and by pulmonary isomerism and occurred as an isolated malformation complex or as part of a VACTERL association. Special findings were an additional so-called 'pig bronchus' and a first case of tracheal agenesis with sirenomelia. Tracheal atresia presenting with partial obliteration of its lumen and persistence of a fibromuscular streak resulted in CHAOS. This condition was associated with normal lung lobulation and single, non-VACTERL type malformations. Trio ES revealed a novel variant of MAPK11 in one tracheal agenesis case. Its involvement in tracheooesophageal malformation is herein discussed, but remains hypothetical. CONCLUSION: Tracheal agenesis and tracheal atresia represent different disease entities in terms of morphology, pathogenesis and accompanying anomalies due to a primary developmental and secondary disruptive possibly vascular disturbance, respectively.

Pathogenic variants affecting the TB5 domain of the fibrillin-1 protein: not only in geleophysic/acromicric dysplasias but also in Marfan syndrome.

BACKGROUND: Marfan syndrome (MFS) is a multisystem disease with a unique combination of skeletal, cardiovascular and ocular features. Geleophysic/acromicric dysplasias (GPHYSD/ACMICD), characterised by short stature and extremities, are described as 'the mirror image' of MFS. The numerous FBN1 pathogenic variants identified in MFS are located all along the gene and lead to the same final pathogenic sequence. Conversely, in GPHYSD/ACMICD, the 28 known heterozygous FBN1 pathogenic variants all affect exons 41-42 encoding TGFß-binding protein-like domain 5 (TB5). METHODS: Since 1996, more than 5000 consecutive probands have been referred nationwide to our laboratory for molecular diagnosis of suspected MFS. RESULTS: We identified five MFS probands carrying distinct heterozygous pathogenic in-frame variants affecting the TB5 domain of FBN1. The clinical data showed that the probands displayed a classical form of MFS. Strikingly, one missense variant affects an amino acid that was previously involved in GPHYSD. CONCLUSION: Surprisingly, pathogenic variants in the TB5 domain of FBN1 can lead to two opposite phenotypes: GPHYSD/ACMICD and MFS, suggesting the existence of different pathogenic sequences with the involvement of tissue specificity. Further functional studies are ongoing to determine the precise role of this domain in the physiopathology of each disease.

Limb-body wall complex: Literature review and case report.

INTRODUCTION: Body wall anomalies comprise a wide range of malformations. Limb-Body wall complex (LBWC) represents the most severe presentation of this group, with life threatening malformations in practically all the cases, including craniofacial, body wall defects, and limb anomalies. There is no consensus about its etiology and folding and gastrulation defects have been involved. Also, impaired angiogenesis has been proposed as a causative process. CASE REPORT: We present the case of a masculine stillborn, product of the first pregnancy in a 15-year-old, apparently healthy mother. He was delivered at 31 weeks of gestation due to an early rupture of membranes. He presented with multiple malformations including a wide body wall defect with multiple organ herniation and meromelia of the lower right limb. DISCUSSION AND CONCLUSIONS: LBWC represents a severe and invariably fatal pathology. There are no described risk factors, nevertheless, this case presented in a teenage mother, a well-described risk factor for other body wall anomalies. Its diagnosis allows us to discriminate between other pathologies that require prenatal or postnatal specialized treatment.

[Clinical phenotype and genetic analysis of six Chinese patients affected with Acromicric dysplasia due to variants of FBN1 gene].

OBJECTIVE: To retrospectively analyze the clinical and genetic characteristics of six patients with Acromicric dysplasia due to variants of the FBN1 gene. METHODS: Six patients who had visited the Affiliated Hospital of Qingdao University between February 2018 and October 2020 were selected as the study subjects. Clinical data of the patients were collected. High-throughput sequencing was carried out. And candidate variants were verified by Sanger sequencing. RESULTS: All of the six patients had presented with severe short stature (< 3s), brachydactyly, short and broad hands and feet. Other manifestations included joint stiffness, facial dysmorphism, delayed bone age, liver enlargement, coracoid femoral head, and lumbar lordosis. Genetic testing revealed that all had harbored heterozygous variants of the FBN1 gene. Patient 1 had harbored a c.5183C>T (p.A1728V) missense variant in exon 42, which had derived from his father (patient 2). Patient 3 had harbored a c.5284G>A (p.G1762S) missense variant in exon 43, which had derived from her mother (patient 4). Patient 5 had harbored a c.5156G>T (p.C1719F) missense variant in exon 42, which was de novo in origin. Patient 6 had harbored a c.5272G>T (p.D1758Y) missense variant in exon 43, which was also de novo in origin. The variants carried by patients 1, 3 and 6 were known to be pathogenic. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the FBN1: c.5156G>T was rated as a pathogenic variant (PS2+PM1+PM2_Supporting +PM5+PP3). CONCLUSION: All of the six patients had severe short stature and a variety of other clinical manifestations, which may be attributed to the variants of the FBN1 gene.

Prenatal diagnosis of ROR-2 related Robinow syndrome presenting with fetal ultrasound findings of mesomelia, vertebral, digital and genital abnormalities.

Autosomal recessive ROR2-Robinow syndrome is caused by pathogenic variants in the ROR2 gene. Fetal ultrasound done on our patient at 24 + 3/7 weeks gestation showed macrocephaly, brachycephaly, flat face, prominent forehead, mild frontal bossing, lower thoracic hemivertebrae, digital abnormalities and micropenis. Fetal trio whole exome sequencing done on amniocytes showed two pathogenic compound heterozygous variants in the ROR2 gene, c.1324 C > T; p.(Arg442*) maternally inherited and c.1366dup; p.(Leu456Profs*3) apparently de novo. c.1324 C > T; p.(Arg442*) is a nonsense variant resulting in protein truncation reported to be associated with RRS3. c.1366dup; p.(Leu456Profs*3) is a frameshift variant predicted to result in protein truncation reported to segregate with the disease in multiple affected individuals from a single large family with distal symphalangism of the fourth finger. Fetal autopsy following pregnancy termination showed a large head with low-set ears, facial abnormalities, mesomelic bone shortening, hemivertebra, fused S3 and S4 vertebral bodies, several fused rib heads and short penis with buried shaft.

Bovine Aortic Arch with an Aberrant Left Vertebral Artery in a 3-Year-Old Boy with VACTERL Association: A Case Report.

BACKGROUND The VACTEREL association is an acronym that includes vertebral malformations (V), anal atresia (A), cardiac defects (C), tracheoesophageal fistula (TE), renal defects (R), and limb malformations (L). The aortic arch is the section between the ascending aorta and the descending aorta, where some variants have been described, such as the right aortic arch and bovine aortic arch, among others. A rare presentation in the Natsis classification is the "type X" where a bovine aortic arch and anomalous origin of the left vertebral artery are present. Several structural cardiac malformations have been described in the VACTEREL association. Still, there is no bovine arch or an anomalous left vertebral artery. CASE REPORT Our patient was a 3-year-old boy with a diagnosis of VACTEREL association (type III esophageal atresia, congenital hip dislocation, scoliosis, bilateral clubfoot, and grade IV biliary ureteral reflux). Echocardiographic findings showed changes in the aortic arch, and angiotomography and magnetic resonance angiography showed a bovine aortic arch and an anomalous left vertebral artery. At the time of diagnosis, there were no clinical manifestations or complications due to the anomalous origin of the left vertebral artery. CONCLUSIONS This is the first description of a bovine type X arch according to the Natsis classification in a VACTEREL association. In general, knowledge of the anatomical variants of the aortic arch and the origin and course of the vertebral arteries is of great clinical and interventional importance, mainly because of the risk of cerebral ischemia.

SALL4 deletion and kidney and cardiac defects associated with VACTERL association.

Congenital anomalies of the kidney and urinary tract (CAKUT) can be a part of the VACTERL association, which represents the non-random combination of the following congenital anomalies: vertebral anomalies, anal anomalies, cardiac anomalies, tracheal-esophageal anomalies, kidney anomalies, and limb anomalies. VACTERL association is generally considered to be a non-genetic condition. Exceptions include a patient with a heterozygous nonsense SALL4 variant and anal stenosis, tetralogy of Fallot, sacro-vertebral fusion, and radial and thumb anomalies. SALL4 encodes a transcription factor that plays a critical role in kidney morphogenesis. Here, we report a patient with VACTERL association and a heterozygous 128-kb deletion spanning SALL4 who presented with renal hypoplasia, radial and atrio-septal defects, and patent ductus arteriosus. The present report of SALL4 deletion, in addition to a previously reported patient with VACTERL association phenotype and SALL4 nonsense mutation, further supports the notion that SALL4 haploinsufficiency can lead to VACTERL association.