Correction to: A zebrafish model of foxe3 deficiency demonstrates lens and eye defects with dysregulation of key genes involved in cataract formation in humans.

The authors noticed that Fig. 5A and B aspect ratios appeared sub-optimal in the online published version. This has now been changed.

A zebrafish model of foxe3 deficiency demonstrates lens and eye defects with dysregulation of key genes involved in cataract formation in humans.

The Forkhead box E3 (FOXE3) gene encodes a transcription factor with a forkhead/winged helix domain that is critical for development of the lens and anterior segment of the eye. Monoallelic and biallelic deleterious sequence variants in FOXE3 cause aphakia, cataracts, sclerocornea and microphthalmia in humans. We used clustered regularly interspaced short palindromic repeats/Cas9 injections to target the foxe3 transcript in zebrafish in order to create an experimental model of loss of function for this gene. Larvae that were homozygous for an indel variant, c.296_300delTGCAG, predicting p.(Val99Alafs*2), demonstrated severe eye defects, including small or absent lenses and microphthalmia. The lenses of the homozygous foxe3 indel mutants showed more intense staining with zl-1 antibody compared to control lenses, consistent with increased lens fiber cell differentiation. Whole genome transcriptome analysis (RNA-Seq) on RNA isolated from wildtype larvae and larvae with eye defects that were putative homozygotes for the foxe3 indel variant found significant dysregulation of genes expressed in the lens and eye whose orthologues are associated with cataracts in human patients, including cryba2a, cryba1l1, mipa and hsf4. Comparative analysis of this RNA-seq data with iSyTE data identified several lens-enriched genes to be down-regulated in foxe3 indel mutants. We also noted upregulation of lgsn and crygmxl2 and downregulation of fmodb and cx43.4, genes that are expressed in the zebrafish lens, but that are not yet associated with an eye phenotype in humans. These findings demonstrate that this new zebrafish foxe3 mutant model is highly relevant to the study of the gene regulatory networks conserved in vertebrate lens and eye development.

Association of the missense variant p.Arg203Trp in PACS1 as a cause of intellectual disability and seizures.

Graphical abstract key: ADHD, attention deficit hyperactivity disorder; ASD, atrial septal defect; DD, developmental delay; EEG, electroencephalogram; Ht, height; ID, intellectual disability; OCD, obsessive-compulsive disorder; OFC, open fontanelle; PDA, patent ductus arteriosis; PFO, patent foramen ovale; VSD, ventricular septal defect; Wt, weight.

Two missense mutations in SALL4 in a patient with microphthalmia, coloboma, and optic nerve hypoplasia.

To investigate the genetic etiology of anophthalmia and microphthalmia, we used exome sequencing in a Caucasian female with unilateral microphthalmia and coloboma, bilateral optic nerve hypoplasia, ventricular and atrial septal defects, and growth delays. We found two sequence variants in SALL4 - c.[575C>A], predicting p.(Ala192Glu), that was paternally inherited, and c.[2053G>C], predicting p.(Asp685His), that was maternally inherited. Haploinsufficiency for SALL4 due to nonsense or frameshift mutations has been associated with acro-renal ocular syndrome that is characterized by eye defects including Duane anomaly and coloboma, in addition to radial ray malformations and renal abnormalities. Our report is the first description of structural eye defects associated with two missense variants in SALL4 inherited in trans; the absence of reported findings in both parents suggests that both sequence variants are hypomorphic mutations and that both are needed for the ocular phenotype. SALL4 is expressed in the developing lens and regulates BMP4, leading us to speculate that altered BMP4 expression was responsible for the eye defects, but we could not demonstrate altered BMP4 expression in vitro after using small interfering RNAs (siRNAs) to reduce SALL4 expression. We conclude that SALL4 hypomorphic variants may influence eye development.

Expanding the Clinical Spectrum Associated With GLIS3 Mutations.

CONTEXT: GLIS3 (GLI-similar 3) is a member of the GLI-similar zinc finger protein family encoding for a nuclear protein with 5 C2H2-type zinc finger domains. The protein is expressed early in embryogenesis and plays a critical role as both a repressor and activator of transcription. Human GLIS3 mutations are extremely rare. OBJECTIVE: The purpose of this article was determine the phenotypic presentation of 12 patients with a variety of GLIS3 mutations. METHODS: GLIS3 gene mutations were sought by PCR amplification and sequence analysis of exons 1 to 11. Clinical information was provided by the referring clinicians and subsequently using a questionnaire circulated to gain further information. RESULTS: We report the first case of a patient with a compound heterozygous mutation in GLIS3 who did not present with congenital hypothyroidism. All patients presented with neonatal diabetes with a range of insulin sensitivities. Thyroid disease varied among patients. Hepatic and renal disease was common with liver dysfunction ranging from hepatitis to cirrhosis; cystic dysplasia was the most common renal manifestation. We describe new presenting features in patients with GLIS3 mutations, including craniosynostosis, hiatus hernia, atrial septal defect, splenic cyst, and choanal atresia and confirm further cases with sensorineural deafness and exocrine pancreatic insufficiency. CONCLUSION: We report new findings within the GLIS3 phenotype, further extending the spectrum of abnormalities associated with GLIS3 mutations and providing novel insights into the role of GLIS3 in human physiological development. All but 2 of the patients within our cohort are still alive, and we describe the first patient to live to adulthood with a GLIS3 mutation, suggesting that even patients with a severe GLIS3 phenotype may have a longer life expectancy than originally described.

Exome sequencing in 32 patients with anophthalmia/microphthalmia and developmental eye defects.

Anophthalmia/microphthalmia (A/M) is a genetically heterogeneous birth defect for which the etiology is unknown in more than 50% of patients. We used exome sequencing with the ACE Exome(TM) (Personalis, Inc; 18 cases) and UCSF Genomics Core (21 cases) to sequence 28 patients with A/M and four patients with varied developmental eye defects. In the 28 patients with A/M, we identified de novo mutations in three patients (OTX2, p.(Gln91His), RARB, p.Arg387Cys and GDF6, p.Ala249Glu) and inherited mutations in STRA6 in two patients. In patients with developmental eye defects, a female with cataracts and cardiomyopathy had a de novo COL4A1 mutation, p.(Gly773Arg), expanding the phenotype associated with COL4A1 to include cardiomyopathy. A male with a chorioretinal defect, microcephaly, seizures and sensorineural deafness had two PNPT1 mutations, p.(Ala507Ser) and c.401-1G>A, and we describe eye defects associated with this gene for the first time. Exome sequencing was efficient for identifying mutations in pathogenic genes for which there is no clinical testing available and for identifying cases that expand phenotypic spectra, such as the PNPT1 and COL4A1-associated disorders described here.

Sequence variants in the HLX gene at chromosome 1q41-1q42 in patients with diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a common birth defect for which few causative genes have been identified. Several candidate regions containing genes necessary for normal diaphragm development have been identified, including a 4-5 Mb deleted region at chromosome 1q41-1q42 from which the causative gene(s) has/have not been cloned. We selected the HLX gene from this interval as a candidate gene for CDH, as the Hlx homozygous null mouse has been reported to have diaphragmatic defects and the gene was described as being expressed in the murine diaphragm. We re-sequenced HLX in 119 CDH patients and identified four novel single nucleotide substitutions that predict amino acid changes: p.S12F, p.S18L, p.D173Y and p.A235V. These sequence alterations were all present in patients with isolated CDH, although patients with both isolated CHD and CDH with additional anomalies were studied. The single-nucleotide substitutions were absent in more than 186 control chromosomes. In-situ hybridization studies confirmed expression of Hlx in the developing murine diaphragm at the site of the junction of the diaphragm and the liver. Although functional studies to determine if these novel sequence variants altered the inductive activity of Hlx on the alpha-smooth muscle actin and SM22alpha promoters showed no significant differences between the variants and wild-type Hlx, sequence variants in HLX may still be relevant in the pathogenesis of CDH in combination with additional genetic and environmental factors.

Phenotypic expansion and further characterisation of the 17q21.31 microdeletion syndrome.

BACKGROUND: The recognition of the 17q21.31 microdeletion syndrome has been facilitated by high resolution microarray technology. Recent clinical delineation of this condition emphasises a typical facial appearance, cardiac and renal defects, and speech delay in addition to intellectual disability, hypotonia and seizures. METHODS AND RESULTS: We describe 11 previously unreported patients expanding the phenotypic spectrum to include aortic root dilatation, recurrent joint subluxation, conductive hearing loss due to chronic otitis media, dental anomalies, and persistence of fetal fingertip pads. Molecular analysis of the deletions demonstrates a critical region spanning 440 kb involving either partially or wholly five genes, CRHR1, IMP5, MAPT, STH, and KIAA1267. CONCLUSION: These data have significant implications for the clinical diagnosis and management of other individuals with 17q21.31 deletions.

Of brain and bone: the unusual case of Dr. A.

Frontotemporal dementia (FTD) is a clinical syndrome characterized by progressive decline in social conduct and a focal pattern of frontal and temporal lobe damage. Its biological basis is still poorly understood but the focality of the brain degeneration provides a powerful model to study the cognitive and anatomical basis of social cognition. Here, we present Dr. A, a patient with a rare hereditary bone disease (hereditary multiple exostoses) and FTD (pathologically characterized as Pick's disease), who presented with a profound behavioral disturbance characterized by acquired sociopathy. We conducted a detailed genetic, pathological, neuroimaging and cognitive study, including a battery of tests designed to investigate Dr. A's abilities to understand emotional cues and to infer mental states and intentions to others (theory of mind). Dr. A's genetic profile suggests the possibility that a mutation causing hereditary multiple exostoses, Ext2, may play a role in the pattern of neurodegeneration in frontotemporal dementia since knockout mice deficient in the Ext gene family member, Ext1, show severe CNS defects including loss of olfactory bulbs and abnormally small cerebral cortex. Dr. A showed significant impairment in emotion comprehension, second order theory of mind, attribution of intentions, and empathy despite preserved general cognitive abilities. Voxel-based morphometry on structural MRI images showed significant atrophy in the medial and right orbital frontal and anterior temporal regions with sparing of dorsolateral frontal cortex. This case demonstrates that social and emotional dysfunction in FTD can be dissociated from preserved performance on classic executive functioning tasks. The specific pattern of anatomical damage shown by VBM emphasizes the importance of the network including the superior medial frontal gyrus as well as temporal polar areas, in regulation of social cognition and theory of mind. This case provides new evidence regarding the neural basis of social cognition and suggests a possible genetic link between bone disease and FTD.

Subtypes of frontonasal dysplasia are useful in determining clinical prognosis.

Frontonasal dysplasia [FND; OMIM 136760] results from abnormal morphogenesis of the frontonasal process with disruption of the formation of the facial midline. Craniofacial anomalies in FND include anterior cranium bifidum, ocular hypertelorism, orofacial clefting and notching or clefting of the alae nasi. The majority of FND cases are sporadic and discordance has been demonstrated in monozygotic twin pairs, arguing against a strong inherited component in pathogenesis. However, pedigrees with Mendelian inheritance and non-recurrent chromosome aberrations in FND patients demonstrate that gene mutations or cytogenetic imbalance can also be important in the etiology of this phenotype. We classified 101 reported cases of FND from the medical literature and three new cases into seven separate FND phenotypic subtypes in addition to isolated FND. Our aim was to evaluate FND patients for distinct phenotypes within the FND spectrum and to determine the evidence for a genetic etiology in each of the different subtypes. Our analysis showed significant differences in the severity of the accompanying malformations and the rates of learning disabilities in the FND subtypes, although the small patient numbers and method of patient ascertainment may have influenced the data. The results suggest that cases with FND should be evaluated for additional anomalies, as these may help to determine prognosis.

Candidate genes for congenital diaphragmatic hernia from animal models: sequencing of FOG2 and PDGFRalpha reveals rare variants in diaphragmatic hernia patients.

Congenital diaphragmatic hernia (CDH) is a common, life threatening birth defect. Although there is strong evidence implicating genetic factors in its pathogenesis, few causative genes have been identified, and in isolated CDH, only one de novo, nonsense mutation has been reported in FOG2 in a female with posterior diaphragmatic eventration. We report here that the homozygous null mouse for the Pdgfralpha gene has posterolateral diaphragmatic defects and thus is a model for human CDH. We hypothesized that mutations in this gene could cause human CDH. We sequenced PDGFRalpha and FOG2 in 96 patients with CDH, of which 53 had isolated CDH (55.2%), 36 had CDH and additional anomalies (37.5%), and 7 had CDH and known chromosome aberrations (7.3%). For FOG2, we identified novel sequence alterations predicting p.M703L and p.T843A in two patients with isolated CDH that were absent in 526 and 564 control chromosomes respectively. These altered amino acids were highly conserved. However, due to the lack of available parental DNA samples we were not able to determine if the sequence alterations were de novo. For PDGFRalpha, we found a single variant predicting p.L967V in a patient with CDH and multiple anomalies that was absent in 768 control chromosomes. This patient also had one cell with trisomy 15 on skin fibroblast culture, a finding of uncertain significance. Although our study identified sequence variants in FOG2 and PDGFRalpha, we have not definitively established the variants as mutations and we found no evidence that CDH commonly results from mutations in these genes.

KBG syndrome: report of twins, neurological characteristics, and delineation of diagnostic criteria.

KBG syndrome is a multiple congenital anomaly (MCA) syndrome comprising developmental delay, postnatal short stature, and delayed bone age. Many physical anomalies involving the face, hands, and costovertebral axis have been described in this syndrome. We present twin males with KBG syndrome and a review of 50 published case reports, with particular emphasis on the neurological aspects of KBG syndrome, including seizures, MRI findings, and behavior difficulties. It is argued that diagnostic criteria for KBG syndrome should include neurological involvement, that is, global developmental delay, seizures, and/or mental retardation (MR). The characteristic facial changes and representative hand and costovertebral anomalies are also defined. These diagnostic criteria were obtained from 50 publications and appeared to support the diagnosis in 43 cases. They will be helpful to pediatricians, geneticists, and neurologists in evaluating patients for this condition.

Mutation analysis of the FRAS1 gene demonstrates new mutations in a propositus with Fraser syndrome.

Fraser syndrome (OMIM 219000) is a rare, autosomal recessive condition with classical features of cryptophthalmos, syndactyly, ambiguous genitalia, laryngeal, and genitourinary malformations, oral clefting and mental retardation. Mutations causing loss of function of the FRAS1 gene have been demonstrated in five patients with Fraser syndrome. However, no phenotype-genotype correlation was established and there was evidence for genetic heterogeneity. Fraser syndrome is rare and the FRAS1 gene has 75 exons, complicating mutation screening in affected patients. We have screened two patients who fulfilled the diagnostic criteria for Fraser syndrome and three patients with related phenotypes (two patients with Manitoba oculotrichoanal syndrome and one patient with unilateral cryptophthalmos and labial fusion) for mutations in FRAS1 to increase the molecular genetic data in patients with Fraser syndrome and related conditions. We report two new mutations in a patient with Fraser syndrome, a frameshift mutation and a deletion of two amino acids that we consider pathogenic as both alter the NG2-like domain of the protein. Although we are still unable to clarify a phenotype-genotype relationship in Fraser syndrome, our data add to the list of mutations associated with this syndrome.

Genomic microarray analysis identifies candidate loci in patients with corpus callosum anomalies.

Absence of the corpus callosum is often associated with cognitive deficits, autism, and epilepsy. Using a genomic microarray, the authors analyzed DNA from 25 patients with radiographically confirmed callosal anomalies and identified three patients with de novo copy number changes in chromosome regions 2q37, 6qter, and 8p. Chromosomal deletions and duplications may be a relatively common cause of cerebral malformations.

Fryns syndrome phenotype caused by chromosome microdeletions at 15q26.2 and 8p23.1.

BACKGROUND: Fryns syndrome (FS) is the commonest autosomal recessive syndrome in which congenital diaphragmatic hernia (CDH) is a cardinal feature. It has been estimated that 10% of patients with CDH have FS. The autosomal recessive inheritance in FS contrasts with the sporadic inheritance for the majority of patients with CDH and renders the correct diagnosis critical for accurate genetic counselling. The cause of FS is unknown. METHODS: We have used array comparative genomic hybridisation (array CGH) to screen patients who have CDH and additional phenotypic anomalies consistent with FS for cryptic chromosome aberrations. RESULTS: We present three probands who were previously diagnosed with FS who had submicroscopic chromosome deletions detected by array CGH after normal karyotyping with G-banded chromosome analysis. Two female infants were found to have microdeletions involving chromosome band 15q26.2 and one male had a deletion of chromosome band 8p23.1. CONCLUSIONS: We conclude that phenotypes similar to FS can be caused by submicroscopic chromosome deletions and that high resolution karyotyping, including array CGH if possible, should be performed prior to the diagnosis of FS to provide an accurate recurrence risk in patients with CDH and physical anomalies consistent with FS.