Refining the 9q34.3 microduplication syndrome reveals mild neurodevelopmental features associated with a distinct global DNA methylation profile.

Precise regulation of gene expression is important for correct neurodevelopment. 9q34.3 deletions affecting the EHMT1 gene result in a syndromic neurodevelopmental disorder named Kleefstra syndrome. In contrast, duplications of the 9q34.3 locus encompassing EHMT1 have been suggested to cause developmental disorders, but only limited information has been available. We have identified 15 individuals from 10 unrelated families, with 9q34.3 duplications <1.5 Mb in size, encompassing EHMT1 entirely. Clinical features included mild developmental delay, mild intellectual disability or learning problems, autism spectrum disorder, and behavior problems. The individuals did not consistently display dysmorphic features, congenital anomalies, or growth abnormalities. DNA methylation analysis revealed a weak DNAm profile for the cases with 9q34.3 duplication encompassing EHMT1, which could segregate the majority of the affected cases from controls. This study shows that individuals with 9q34.3 duplications including EHMT1 gene present with mild non-syndromic neurodevelopmental disorders and DNA methylation changes different from Kleefstra syndrome.

New classification and surgical strategy for work type I congenital first branchial cleft anomalies in children.

PURPOSE: To investigate the anatomical relationships between the structures adjacent to the cartilaginous portion of the ear canal in children with Work type I congenital branchial cleft anomalies (CFBCAs) and to develop new classifications and surgical strategies. METHODS: Retrospective analysis was performed on 50 children with Work type I CFBCAs admitted between December 2018 and December 2022. RESULTS: Among the 50 children, total parotidectomy was performed on 49 sides. In 44 cases (88%), the main body of the lesion was closely associated with the cartilage of the inferior ear canal wall. Among these cases, the lesions in 40 cases occurred within the space enclosed by the dorsal inferior wall cartilage, mastoid process, and parotid gland, while in the remaining four cases, the lesions were located between the anterior inferior wall cartilage and parotid gland. Based on the preoperative imaging observations, clinical manifestations, and intraoperative findings, the cases were classified into 6 subtypes (a to f) including 21 cases (42%) of Type Ia (inferior wall of EAC), 7 cases (14%) of Type Ib (bottom wall of EAC), 12 cases (24%) of Type Ic (posterior-inferior wall of EAC), 4 cases (8%) of Type Id (anterior-inferior wall of EAC), 4 cases (8%) of Type Ie (anterior ear wall of EAC), and 2 cases (4%) of Type If (isolated from parotid). CONCLUSION: Surgical intervention is the only treatment for first branchial cleft anomalies and a comprehensive understanding of the classifications will help with the precise localisation and excision of the lesions.

POLR1A variants underlie phenotypic heterogeneity in craniofacial, neural, and cardiac anomalies.

Heterozygous pathogenic variants in POLR1A, which encodes the largest subunit of RNA Polymerase I, were previously identified as the cause of acrofacial dysostosis, Cincinnati-type. The predominant phenotypes observed in the cohort of 3 individuals were craniofacial anomalies reminiscent of Treacher Collins syndrome. We subsequently identified 17 additional individuals with 12 unique heterozygous variants in POLR1A and observed numerous additional phenotypes including neurodevelopmental abnormalities and structural cardiac defects, in combination with highly prevalent craniofacial anomalies and variable limb defects. To understand the pathogenesis of this pleiotropy, we modeled an allelic series of POLR1A variants in vitro and in vivo. In vitro assessments demonstrate variable effects of individual pathogenic variants on ribosomal RNA synthesis and nucleolar morphology, which supports the possibility of variant-specific phenotypic effects in affected individuals. To further explore variant-specific effects in vivo, we used CRISPR-Cas9 gene editing to recapitulate two human variants in mice. Additionally, spatiotemporal requirements for Polr1a in developmental lineages contributing to congenital anomalies in affected individuals were examined via conditional mutagenesis in neural crest cells (face and heart), the second heart field (cardiac outflow tract and right ventricle), and forebrain precursors in mice. Consistent with its ubiquitous role in the essential function of ribosome biogenesis, we observed that loss of Polr1a in any of these lineages causes cell-autonomous apoptosis resulting in embryonic malformations. Altogether, our work greatly expands the phenotype of human POLR1A-related disorders and demonstrates variant-specific effects that provide insights into the underlying pathogenesis of ribosomopathies.

[Effect analysis of individualized operation for congenital preauricular fistula].

Objective:To investigate the effect and influencing factors of individualized operation for congenital preauricular fistula in children. Methods:The clinical data of 98 cases （109 ears） of congenital preauricular fistula treated in Department of Otolaryngology，Fuzhou Children's Hospital of Fujian Medical University from July 2016 to December 2020 were retrospectively analyzed. According to the characteristics and infection of preauricular fistula，they were divided into common type and variant type，static period of inflammation and period of infection.Individual surgical methods such as classical fistula resection, double fusiform incision and fistula location resection were used respectively.The efficacy，complication and influencing factors of different surgical methods were analyzed. Results:The operation time of classical fistula resection was shorter, and the difference was statistically significant（t = －2.905 and－3.005 respectively, all P<0.05）. According to the stages and types of fistulas, the selection of individualized surgical methods had achieved good results. There was no significant difference in incision complications and fistula recurrence among different surgical methods （all P>0.05）. Conclusion:Once infection occurs in congenital preauricular fistula, surgical resection should be performed as soon as possible after infection control, or as early as possible after infection maximum control if infection cannot completely subside. Surgical incision design should be individualized, complete resection of fistulas and lesions, minimally invasive and aesthetic.

Augmentation of bone morphogenetic protein signaling in cranial neural crest cells in mice deforms skull base due to premature fusion of intersphenoidal synchondrosis.

Craniofacial anomalies (CFAs) are a diverse group of disorders affecting the shapes of the face and the head. Malformation of the cranial base in humans leads CFAs, such as midfacial hypoplasia and craniosynostosis. These patients have significant burdens associated with breathing, speaking, and chewing. Invasive surgical intervention is the current primary option to correct these structural deficiencies. Understanding molecular cellular mechanism for craniofacial development would provide novel therapeutic options for CFAs. In this study, we found that enhanced bone morphogenetic protein (BMP) signaling in cranial neural crest cells (NCCs) (P0-Cre;caBmpr1a mice) causes premature fusion of intersphenoid synchondrosis (ISS) resulting in leading to short snouts and hypertelorism. Histological analyses revealed reduction of proliferation and higher cell death in ISS at postnatal day 3. We demonstrated to prevent the premature fusion of ISS in P0-Cre;caBmpr1a mice by injecting a p53 inhibitor Pifithrin-α to the pregnant mother from E15.5 to E18.5, resulting in rescue from short snouts and hypertelorism. We further demonstrated to prevent premature fusion of cranial sutures in P0-Cre;caBmpr1a mice by injecting Pifithrin-α through E8.5 to E18.5. These results suggested that enhanced BMP-p53-induced cell death in cranial NCCs causes premature fusion of ISS and sutures in time-dependent manner.

Obsessive-compulsive symptoms in ACTG1-associated Baraitser-Winter cerebrofrontofacial syndrome.

Symptoms of obsessive-compulsive disorder (OCD) may rarely occur in the context of genetic syndromes. So far, an association between obsessive-compulsive symptoms (OCS) and ACTG1-associated Baraitser-Winter cerebrofrontofacial syndrome has not been described as yet. A thoroughly phenotyped patient with OCS and ACTG1-associated Baraitser-Winter cerebrofrontofacial syndrome is presented. The 25-year-old male patient was admitted to in-patient psychiatric care due to OCD. A whole-exome sequencing analysis was initiated as the patient also showed an autistic personality structure, below average intelligence measures, craniofacial dysmorphia signs, sensorineural hearing loss, and sinus cavernoma as well as subtle cardiac and ophthalmological alterations. The diagnosis of Baraitser-Winter cerebrofrontofacial syndrome type 2 was confirmed by the detection of a heterozygous likely pathogenic variant in the ACTG1 gene [c.1003C > T; p.(Arg335Cys), ACMG class 4]. The automated analysis of magnetic resonance imaging (MRI) revealed changes in the orbitofrontal, parietal, and occipital cortex of both sides and in the right mesiotemporal cortex. Electroencephalography (EEG) revealed intermittent rhythmic delta activity in the occipital and right temporal areas. Right mesiotemporal MRI and EEG alterations could be caused by a small brain parenchymal defect with hemosiderin deposits after a cavernomectomy. This paradigmatic case provides evidence of syndromic OCS in ACTG1-associated Baraitser-Winter cerebrofrontofacial syndrome. The MRI findings are compatible with a dysfunction of the cortico-striato-thalamo-cortical loops involved in OCD. If a common pathophysiology is confirmed in future studies, corresponding patients with Baraitser-Winter cerebrofrontofacial syndrome type 2 should be screened for OCS. The association may also contribute to a better understanding of OCD pathophysiology.

A recessive variant in SIM2 in a child with complex craniofacial anomalies and global developmental delay.

Rare deletions and duplications on the long arm of Chromosome 21 have previously been reported in many patients with craniofacial and developmental phenotypes. However, this Down Syndrome Critical Region (DSCR) contains multiple genes, making identifying a single causative gene difficult. Here, we report a case of a boy with bicoronal craniosynostosis, facial dysmorphism, developmental delay, and intellectual impairment who was found by whole genome sequencing to have a homozygous missense mutation in the Single-Minded Homolog 2 (SIM2) gene (c.461 A > G, p.Tyr154Cys) within the DSCR. SIM2 encodes an essential bHLH and PAS domain transcription factor expressed during fetal brain development and acts as a master regulator of neurogenesis. This variant is globally very rare, segregates in the family, and is predicted to be highly deleterious by in silico analysis, 3D molecular modeling of protein structure, and functional analysis of zebrafish models. Zebrafish expressing the human SIM2p.Y154C variant displayed a progressed microcephaly-like phenotype and head shape abnormalities. When combined with careful phenotyping of the patient vis-à-vis previously reported cases harboring structural variants in this critical 21q22 region, the data support a pathogenic role of SIM2 in this complex syndrome and demonstrates the utility of next-generation sequencing in prioritizing genes in contiguous deletions/duplications syndromes and diagnosing microarray-negative patients in the craniofacial clinic.

Deep learning is widely applicable to phenotyping embryonic development and disease.

Genome editing simplifies the generation of new animal models for congenital disorders. However, the detailed and unbiased phenotypic assessment of altered embryonic development remains a challenge. Here, we explore how deep learning (U-Net) can automate segmentation tasks in various imaging modalities, and we quantify phenotypes of altered renal, neural and craniofacial development in Xenopus embryos in comparison with normal variability. We demonstrate the utility of this approach in embryos with polycystic kidneys (pkd1 and pkd2) and craniofacial dysmorphia (six1). We highlight how in toto light-sheet microscopy facilitates accurate reconstruction of brain and craniofacial structures within X. tropicalis embryos upon dyrk1a and six1 loss of function or treatment with retinoic acid inhibitors. These tools increase the sensitivity and throughput of evaluating developmental malformations caused by chemical or genetic disruption. Furthermore, we provide a library of pre-trained networks and detailed instructions for applying deep learning to the reader's own datasets. We demonstrate the versatility, precision and scalability of deep neural network phenotyping on embryonic disease models. By combining light-sheet microscopy and deep learning, we provide a framework for higher-throughput characterization of embryonic model organisms. This article has an associated 'The people behind the papers' interview.

Phenotypic Characterization of Immortalized Chondrocytes from a Desbuquois Dysplasia Type 1 Mouse Model: A Tool for Studying Defects in Glycosaminoglycan Biosynthesis.

The complexity of skeletal pathologies makes use of in vivo models essential to elucidate the pathogenesis of the diseases; nevertheless, chondrocyte and osteoblast cell lines provide relevant information on the underlying disease mechanisms. Due to the limitations of primary chondrocytes, immortalized cells represent a unique tool to overcome this problem since they grow very easily for several passages. However, in the immortalization procedure the cells might lose the original phenotype; thus, these cell lines should be deeply characterized before their use. We immortalized primary chondrocytes from a Cant1 knock-out mouse, an animal model of Desbuquois dysplasia type 1, with a plasmid expressing the SV40 large and small T antigen. This cell line, based on morphological and biochemical parameters, showed preservation of the chondrocyte phenotype. In addition reduced proteoglycan synthesis and oversulfation of glycosaminoglycan chains were demonstrated, as already observed in primary chondrocytes from the Cant1 knock-out mouse. In conclusion, immortalized Cant1 knock-out chondrocytes maintained the disease phenotype observed in primary cells validating the in vitro model and providing an additional tool to further study the proteoglycan biosynthesis defect. The same approach might be extended to other cartilage disorders.

Two new cases of interstitial 7q35q36.1 deletion including CNTNAP2 and KMT2C.

BACKGROUND: Terminal deletions of the long arm of chromosome 7 are well known and frequently associated with syndromic holoprosencephaly due to the involvement of the SHH (aliases HHG1, SMMCI, TPT, TPTPS, and MCOPCB5) gene region. However, interstitial deletions including CNTNAP2 (aliases Caspr2, KIAA0868, and NRXN4) and excluding the SHH region are less common. METHODS: We report the clinical and molecular characterization associated with pure 7q35 and 7q35q36.1 deletion in two unrelated patients as detected by oligonucleotide-based array-CGH analysis. RESULTS: The common clinical features were abnormal maternal serum screening during first-trimester pregnancy, low occipitofrontal circumference at birth, hypotonia, abnormal feet, developmental delay, impaired language development, generalized seizures, hyperactive behavior, friendly personality, and cranio-facial dysmorphism. Both deletions occurred de novo and sequencing of CNTNAP2, a candidate gene for epilepsy and autism showed absence of mutation on the contralateral allele. CONCLUSION: Combined haploinsufficiency of GALNTL5 (alias GalNAc-T5L), CUL1, SSPO (aliases SCO-spondin, KIAA0543, and FLJ36112), AOC1 (alias DAO), RHEB, and especially KMT2C (alias KIAA1506 and HALR) with monoallelic disruption of CNTNAP2 may explain neurologic abnormalities, hypotonia, and exostoses. Haploinsufficiency of PRKAG2 (aliases AAKG, AAKG2, H91620p, WPWS, and CMH6) and KCNH2 (aliases Kv11.1, HERG, and erg1) genes may be responsible of long QT syndrome observed for one patient.

Rare and de novo variants in 827 congenital diaphragmatic hernia probands implicate LONP1 as candidate risk gene.

Congenital diaphragmatic hernia (CDH) is a severe congenital anomaly that is often accompanied by other anomalies. Although the role of genetics in the pathogenesis of CDH has been established, only a small number of disease-associated genes have been identified. To further investigate the genetics of CDH, we analyzed de novo coding variants in 827 proband-parent trios and confirmed an overall significant enrichment of damaging de novo variants, especially in constrained genes. We identified LONP1 (lon peptidase 1, mitochondrial) and ALYREF (Aly/REF export factor) as candidate CDH-associated genes on the basis of de novo variants at a false discovery rate below 0.05. We also performed ultra-rare variant association analyses in 748 affected individuals and 11,220 ancestry-matched population control individuals and identified LONP1 as a risk gene contributing to CDH through both de novo and ultra-rare inherited largely heterozygous variants clustered in the core of the domains and segregating with CDH in affected familial individuals. Approximately 3% of our CDH cohort who are heterozygous with ultra-rare predicted damaging variants in LONP1 have a range of clinical phenotypes, including other anomalies in some individuals and higher mortality and requirement for extracorporeal membrane oxygenation. Mice with lung epithelium-specific deletion of Lonp1 die immediately after birth, most likely because of the observed severe reduction of lung growth, a known contributor to the high mortality in humans. Our findings of both de novo and inherited rare variants in the same gene may have implications in the design and analysis for other genetic studies of congenital anomalies.

Kleefstra syndrome: Recurrence in siblings due to a paternal mosaic mutation.

Kleefstra syndrome (KS) is a rare autosomic dominant genetic disorder caused by euchromatic histone methyltransferase 1 (EHMT1) alterations. Patients mainly present with moderate to severe intellectual disability, a severe delay in/or absence of speech, autism spectrum disorder, childhood hypotonia, neuropsychiatric anomalies, and distinctive dysmorphic features. Here, we report the cases of a male and a female, two younger siblings of three, with asymptomatic parents. An EHMT1 new mutation was identified. Both presented with a typical core phenotype. Some specific features were noted, such as macrocephaly (previously reported) and enuresis (not yet described). Parental analysis identified the mutation in the mosaic state in the father. Reverse phenotyping enabled us to highlight the pauci phenotype features of inguinal hernia, azoospermia, and possible behavioral disorders. This allowed us to adapt his follow-up and genetic counseling for the family. Our three reported cases provide a new description of KS with an intragenic EHMT1 mutation, whereas in the literature most reported cases have EHMT1 deletions. Moreover, in the areas of next-generation sequencing and trio techniques with parental segregation, it is important to remain cautious about disregarding variants based on an autosomal recessive hypothesis.

Extended phenotypes of PIEZO1-related lymphatic dysplasia caused by two novel compound heterozygous variants.

Defects in the PIEZO1 gene cause lymphatic dysplasia in an autosomal recessive manner, mostly by loss-of-function variants. Moreover, since 2019, the role of PIEZO1 in bone formation has been established, but there have been no PIEZO1-related cases presenting definite skeletal involvement to date. A 21-year-old male with primary lymphatic dysplasia had some other distinctive clinical features, including multiple fracture history during infancy, thoracolumbar scoliosis, short stature, and left-sided facial bone hypoplasia. We analyzed the whole exome of the patient and found two novel pathogenic variants of PIEZO1 in trans: a 93.7 kb heterozygous deletion (chr16:88,782,477-88,876,207; exon 1-50) and c.2858G>A (p.Arg953His). Sanger sequencing validated the deletion with breakpoints, and each variant was inherited from a different parent. This study presented an extremely rare case of a patient with lymphatic dysplasia caused by compound heterozygous variants of PIEZO1, along with additional clinical manifestations including several skeletal phenotypes.

Prenatal diagnosis of Baraitser - Winter syndrome using exome sequencing: Clinical report and review of literature.

Baraitser - Winter Cerebrofrontofacial Syndrome (BWCFF) is a rare disorder characterized by facial dysmorphism and mental retardation of varying grades. The clinical phenotype of BWCFF indicates variable phenotypic expression involving various congenital malformations such as cardiac, renal and musculoskeletal abnormalities. Nevertheless, the prenatal presentation of BWCFF is rarely described, making prenatal diagnosis challenging. This report describes a prenatal diagnosis of BWCFF syndrome to date; a case of a fetus with intrauterine growth restriction, increased nuchal fold, bilateral hydronerphosis, rocker bottom foot and clubfoot detected on Anomaly Scan is outlined. Molecular karyotype failed to detect any abnormality. Assessment with Next Generation Sequencing was then performed, revealing a heterozygous de novo mutation in ACTB gene setting the diagnosis of BWCFF.

A ZFHX4 mutation associated with a recognizable neuropsychological and facial phenotype.

Several patients with chromosomal deletions including ZFHX4 gene have been described, whereas point mutations are very rare. This gene encodes for a transcription factor involved in the development of several embryonal processes, including brain differentiation. Patients with 8q21.11 deletions usually show intellectual disability, short stature, peculiar facial features, and severe eye abnormalities. We describe a female patient with mild intellectual disability, autism spectrum disorder, strabismus, ptosis, low-set and prominent ears, high-arched palate, microretrognathia. Clinical Exome Sequencing revealed the presence of a de novo heterozygous variant in ZFHX4. Therefore, we further investigate the different phenotypes of ZFHX4 mutations and 8q21.11 deletions.

Phenotypic heterogeneity and mosaicism in Xia-Gibbs syndrome: Five Danish patients with novel variants in AHDC1.

Xia-Gibbs syndrome (XGS) is a neurodevelopmental disorder characterized by intellectual disability, developmental delay, seizures, hypotonia, obstructive sleep apnoea and mild facial dysmorphism. Heterozygosity for loss-of-function variants in AHDC1, encoding the AT-hook DNA binding motif containing protein 1, were discovered in 2014 as the likely genetic cause of Xia-Gibbs syndrome. We present five patients with Xia-Gibbs syndrome caused by previously unreported variants in AHDC1. Two of the patients share a frameshift variant: c.2849del (p.(Pro950Argfs*192)) in AHDC1. Despite sharing this variant, the two patients show remarkable phenotypic differences underscoring the clinical heterogeneity of Xia-Gibbs syndrome. In addition, we present a case of Xia-Gibbs syndrome caused by mosaicism for an AHDC1 variant.

SETBP1 accumulation induces P53 inhibition and genotoxic stress in neural progenitors underlying neurodegeneration in Schinzel-Giedion syndrome.

The investigation of genetic forms of juvenile neurodegeneration could shed light on the causative mechanisms of neuronal loss. Schinzel-Giedion syndrome (SGS) is a fatal developmental syndrome caused by mutations in the SETBP1 gene, inducing the accumulation of its protein product. SGS features multi-organ involvement with severe intellectual and physical deficits due, at least in part, to early neurodegeneration. Here we introduce a human SGS model that displays disease-relevant phenotypes. We show that SGS neural progenitors exhibit aberrant proliferation, deregulation of oncogenes and suppressors, unresolved DNA damage, and resistance to apoptosis. Mechanistically, we demonstrate that high SETBP1 levels inhibit P53 function through the stabilization of SET, which in turn hinders P53 acetylation. We find that the inheritance of unresolved DNA damage in SGS neurons triggers the neurodegenerative process that can be alleviated either by PARP-1 inhibition or by NAD + supplementation. These results implicate that neuronal death in SGS originates from developmental alterations mainly in safeguarding cell identity and homeostasis.

Clinical Manifestations in a Girl with NAA10-Related Syndrome and Genotype-Phenotype Correlation in Females.

Since 2011, eight males with an X-linked recessive disorder (Ogden syndrome, MIM #300855) associated with the same missense variant p.(Ser37Pro) in the NAA10 gene have been described. After the advent of whole exome sequencing, many NAA10 variants have been reported as causative of syndromic or non-syndromic intellectual disability in both males and females. The NAA10 gene lies in the Xq28 region and encodes the catalytic subunit of the major N-terminal acetyltransferase complex NatA, which acetylates almost half the human proteome. Here, we present a young female carrying a de novo NAA10 [NM_003491:c.247C > T, p.(Arg83Cys)] variant. The 18-year-old girl has severely delayed motor and language development, autistic traits, postnatal growth failure, facial dysmorphisms, interventricular septal defect, neuroimaging anomalies and epilepsy. Our attempt is to expand and compare genotype-phenotype correlation in females with NAA10-related syndrome. A detailed clinical description could have relevant consequences for the clinical management of known and newly identified individuals.

Conservation of Zebrafish MicroRNA-145 and Its Role during Neural Crest Cell Development.

The neural crest is a multipotent cell population that develops from the dorsal neural fold of vertebrate embryos in order to migrate extensively and differentiate into a variety of tissues. A number of gene regulatory networks coordinating neural crest cell specification and differentiation have been extensively studied to date. Although several publications suggest a common role for microRNA-145 (miR-145) in molecular reprogramming for cell cycle regulation and/or cellular differentiation, little is known about its role during in vivo cranial neural crest development. By modifying miR-145 levels in zebrafish embryos, abnormal craniofacial development and aberrant pigmentation phenotypes were detected. By whole-mount in situ hybridization, changes in expression patterns of col2a1a and Sry-related HMG box (Sox) transcription factors sox9a and sox9b were observed in overexpressed miR-145 embryos. In agreement, zebrafish sox9b expression was downregulated by miR-145 overexpression. In silico and in vivo analysis of the sox9b 3'UTR revealed a conserved potential miR-145 binding site likely involved in its post-transcriptional regulation. Based on these findings, we speculate that miR-145 participates in the gene regulatory network governing zebrafish chondrocyte differentiation by controlling sox9b expression.

Interstitial duplication of 20q11.22q13.11: A case report and review of literature.

BACKGROUND: Reports of interstitial duplication of chromosome 20q11 are rare with only nine published patients to date. METHODS: We performed karyotype and chromosomal microarray analysis on a peripheral blood sample for our patient and reviewed the genes in the region to provide genotype-phenotype correlation. RESULTS: Clinical features of the patient include minor dysmorphic facial features, shorthands and feet, bilateral conductive hearing loss, global developmental delay, and behavioral issues with attention deficit hyperactivity disorder. Together with previously published cases of 20q11 duplication, we show that patients with overlapping duplications share a similar clinical phenotype of dysmorphic craniofacial features and developmental delay. CONCLUSION: We report an 8-year-old girl with a 9.1 Mb interstitial duplication of chromosome 20q11.22q13.11. Our observations suggest that a novel duplication syndrome and documentation of similar cases will further help clarify the phenotype.