Artifacts and main lesions in fetal ocular histology.

Histological artifacts in fetal eyes can involve different tissues and can be related to mechanical or autolytic lesions, fixation, the cause of death or the cutting technique. Knowing the causes, effects and appearance of artifacts allow the minimization of the risk of avoidable artifacts and help distinguish them from "true" pathological lesions. We describe these different types of artifacts and specifically analyze their involvement in different tissue structures of the eye. We compare them with primary fetal ocular lesions. Given the wide variety of artifacts, the identification of lesions in fetal eyes must be done with caution, since differentiating true lesions from artifacts requires some experience and relies on both macroscopic examination and microscopic analyses, with ideally comparisons with references' images of normal tissues of the same gestational age.

Re-focusing on Agnathia-Otocephaly complex.

OBJECTIVES: Agnathia-otocephaly complex is a rare condition characterized by mandibular hypoplasia or agnathia, ear anomalies (melotia/synotia) and microstomia with aglossia. This severe anomaly of the first branchial arch is most often lethal. The estimated incidence is less than 1 in 70.000 births, with etiologies linked to both genetic and teratogenic factors. Most of the cases are sporadic. To date, two genes have been described in humans to be involved in this condition: OTX2 and PRRX1. Nevertheless, the overall proportion of mutated cases is unknown and a significant number of patients remain without molecular diagnosis. Thus, the involvement of other genes than OTX2 and PRRX1 in the agnathia-otocephaly complex is not unlikely. Heterozygous mutations in Cnbp in mice are responsible for mandibular and eye defects mimicking the agnathia-otocephaly complex in humans and appear as a good candidate. Therefore, in this study, we aimed (i) to collect patients presenting with agnathia-otocephaly complex for screening CNBP, in parallel with OTX2 and PRRX1, to check its possible implication in the human phenotype and (ii) to compare our results with the literature data to estimate the proportion of mutated cases after genetic testing. MATERIALS AND METHODS: In this work, we describe 10 patients suffering from the agnathia-otocephaly complex. All of them benefited from array-CGH and Sanger sequencing of OTX2, PRRX1 and CNBP. A complete review of the literature was made using the Pubmed database to collect all the patients described with a phenotype of agnathia-otocephaly complex during the 20 last years (1998-2019) in order (i) to study etiology (genetic causes, iatrogenic causes…) and (ii), when genetic testing was performed, to study which genes were tested and by which type of technologies. RESULTS: In our 10 patients' cohort, no point mutation in the three tested genes was detected by Sanger sequencing, while array-CGH has allowed identifying a 107-kb deletion encompassing OTX2 responsible for the agnathia-otocephaly complex phenotype in 1 of them. In 4 of the 70 cases described in the literature, a toxic cause was identified and 22 out the 66 remaining cases benefited from genetic testing. Among those 22 patients, 6 were carrying mutation or deletion in the OTX2 gene and 4 in the PRRX1 gene. Thus, when compiling results from our cohort and the literature, a total of 32 patients benefited from genetic testing, with only 34% (11/32) of patients having a mutation in one of the two known genes, OTX2 or PRRX1. CONCLUSIONS: From our work and the literature review, only mutations in OTX2 and PRRX1 have been found to date in patients, explaining around one third of the etiologies after genetic testing. Thus, agnathia-otocephaly complex remains unexplained in the majority of the patients, which indicates that other factors might be involved. Although involved in first branchial arch defects, no mutation in the CNBP gene was found in this study. This suggests that mutations in CNBP might not be involved in such phenotype in humans or that, unlike in mice, a compensatory effect might exist in humans. Nevertheless, given that agnathia-otocephaly complex is a rare phenotype, more patients have to be screened for CNBP mutations before we definitively conclude about its potential implication. Therefore, this work presents the current state of knowledge on agnathia-otocephaly complex and underlines the need to expand further the understanding of the genetic bases of this disorder, which remains largely unknown. CLINICAL RELEVANCE: We made here an update and focus on the clinical and genetic aspects of agnathia-otocephaly complex as well as a more general review of craniofacial development.

[Linear lesions: A key dermatological feature of X-linked ectodermal dysplasia in a young girl]. / Lésions linéaires : un signe dermatologique clé de la dysplasie ectodermique liée à l'X chez la fille.

INTRODUCTION: X-linked hypo/anhidrotic ectodermal dysplasia (AED) is the most common form of AED. It is manifested in boys by involvement of the adnexa, teeth and sweat glands. In girls, signs are usually minor and may include linear lesions that are poorly known since they are reported infrequently or overlooked. Herein we report 3 cases. PATIENTS AND METHODS: There were two female patients who had been followed for several years, as well as the mother of one of the patients. Both of the younger patients had early diagnosis of DEA in childhood based on severe dental abnormalities, i.e. hypodontia and conical teeth, a typical facies, and cutaneous xerosis. The mother had milder signs and the diagnosis was made at the time of her daughter's diagnosis. All 3 had hypopigmented linear skin lesions (arms, buttocks or back), associated with a decrease in hair in one of them. Genetic analysis showed the R156H missense mutation at exon 3 of the EDA gene in all 3 patients. CONCLUSION: These hypopigmentation linear lesions, sometimes with hair loss, are well known to pediatric clinicians and dermatologists concerning early diagnosis of AED in girls, especially where the other signs are mild. Early diagnosis enables appropriate therapeutic management and genetic counseling regarding future pregnancy.

Genetics of anophthalmia and microphthalmia. Part 1: Non-syndromic anophthalmia/microphthalmia.

Eye formation is the result of coordinated induction and differentiation processes during embryogenesis. Disruption of any one of these events has the potential to cause ocular growth and structural defects, such as anophthalmia and microphthalmia (A/M). A/M can be isolated or occur with systemic anomalies, when they may form part of a recognizable syndrome. Their etiology includes genetic and environmental factors; several hundred genes involved in ocular development have been identified in humans or animal models. In humans, around 30 genes have been repeatedly implicated in A/M families, although many other genes have been described in single cases or families, and some genetic syndromes include eye anomalies occasionally as part of a wider phenotype. As a result of this broad genetic heterogeneity, with one or two notable exceptions, each gene explains only a small percentage of cases. Given the overlapping phenotypes, these genes can be most efficiently tested on panels or by whole exome/genome sequencing for the purposes of molecular diagnosis. However, despite whole exome/genome testing more than half of patients currently remain without a molecular diagnosis. The proportion of undiagnosed cases is even higher in those individuals with unilateral or milder phenotypes. Furthermore, even when a strong gene candidate is available for a patient, issues of incomplete penetrance and germinal mosaicism make diagnosis and genetic counseling challenging. In this review, we present the main genes implicated in non-syndromic human A/M phenotypes and, for practical purposes, classify them according to the most frequent or predominant phenotype each is associated with. Our intention is that this will allow clinicians to rank and prioritize their molecular analyses and interpretations according to the phenotypes of their patients.

Implication of non-coding PAX6 mutations in aniridia.

There is an increasing implication of non-coding regions in pathological processes of genetic origin. This is partly due to the emergence of sophisticated techniques that have transformed research into gene expression by allowing a more global understanding of the genome, both at the genomic, epigenomic and chromatin levels. Here, we implemented the analysis of PAX6, whose coding loss-of-function variants are mainly implied in aniridia, by studying its non-coding regions (untranslated regions, introns and cis-regulatory sequences). In particular, we have taken advantage of the development of high-throughput approaches to screen the upstream and downstream regulatory regions of PAX6 in 47 aniridia patients without identified mutation in the coding sequence. This was made possible through the use of custom targeted resequencing and/or CGH array to analyze the entire PAX6 locus on 11p13. We found candidate variants in 30 of the 47 patients. 9/30 correspond to the well-known described 3' deletions encompassing SIMO and other enhancer elements. In addition, we identified numerous different variants in various non-coding regions, in particular untranslated regions. Among these latter, most of them demonstrated an in vitro functional effect using a minigene strategy, and 12/21 are thus considered as causative mutations or very likely to explain the phenotypes. This new analysis strategy brings molecular diagnosis to more than 90% of our aniridia patients. This study revealed an outstanding mutation pattern in non-coding PAX6 regions confirming that PAX6 remains the major gene for aniridia.

FOXE3 mutations: genotype-phenotype correlations.

Microphthalmia and anophthalmia (MA) are severe developmental eye anomalies, many of which are likely to have an underlying genetic cause. More than 30 genes have been described, each of which is responsible for a small percentage of these anomalies. Among these, is the FOXE3 gene, which was initially described in individuals with dominantly inherited anterior segment dysgenesis and, subsequently, associated with recessively inherited primary aphakia, sclerocornea and microphthalmia. In this work, we describe 8 individuals presenting with an MA phenotype. Among them, 7 are carrying biallelic recessive FOXE3 mutations and 2 of these have novel mutations: p.(Ala78Thr) and p.(Arg104Cys). The last of our patients is carrying in the heterozygous state the recessive p.(Arg90Leu) mutation in the FOXE3 gene. To further understand FOXE3 involvement in this wide spectrum of ocular anomalies with 2 different patterns of inheritance, we reviewed all individuals with ocular abnormalities described in the literature for which a FOXE3 mutation was identified. This review demonstrates that correlations exist between the mutation type, mode of inheritance and the phenotype severity. Furthermore, understanding the genetic basis of these conditions will contribute to overall understanding of eye development, improve the quality of care, genetic counseling and, in future, gene-based therapies.

4q25 microdeletion encompassing PITX2: A patient presenting with tetralogy of Fallot and dental anomalies without ocular features.

Axenfeld-Rieger syndrome (ARS) is a heterogeneous clinical entity transmitted in an autosomal dominant manner. The main feature, Axenfeld-Rieger Anomaly (ARA), is a malformation of the anterior segment of the eye that can lead to glaucoma and impair vision. Extra-ocular defects have also been reported. Point mutations of FOXC1 and PITX2 are responsible for about 40% of the ARS cases. We describe the phenotype of a patient carrying a deletion encompassing the 4q25 locus containing PITX2 gene. This child presented with a congenital heart defect (Tetralogy of Fallot, TOF) and no signs of ARA. He is the first patient described with TOF and a complete deletion of PITX2 (arr[GRCh37]4q25(110843057-112077858)x1, involving PITX2, EGF, ELOVL6 and ENPEP) inherited from his ARS affected mother. In addition, to our knowledge, he is the first patient reported with no ocular phenotype associated with haploinsufficiency of PITX2. We compare the phenotype and genotype of this patient to those of five other patients carrying 4q25 deletions. Two of these patients were enrolled in the university hospital in Toulouse, while the other three were already documented in DECIPHER. This comparative study suggests both an incomplete penetrance of the ocular malformation pattern in patients carrying PITX2 deletions and a putative association between TOF and PITX2 haploinsufficiency.

Dental and extra-oral clinical features in 41 patients with WNT10A gene mutations: A multicentric genotype-phenotype study.

WNT10A gene encodes a canonical wingless pathway signaling molecule involved in cell fate specification as well as morphogenetic patterning of the developing ectoderm, nervous system, skeleton, and tooth. In patients, WNT10A mutations are responsible for ectodermal-derived pathologies including isolated hypo-oligodontia, tricho-odonto-onycho-dermal dysplasia and Schöpf-Schulz-Passarge syndrome (SSPS). Here we describe the dental, ectodermal, and extra-ectodermal phenotypic features of a cohort of 41 patients from 32 unrelated families. Correlations with WNT10A molecular status (heterozygous carrier, compound heterozygous, homozygous) and patient's phenotypes were performed. Mild to severe oligodontia was observed in all patients bearing biallelic WNT10A mutations. However, patients with compound heterozygous mutations presented no significant difference in phenotypes compared with homozygous individuals. Anomalies in tooth morphology were frequently observed with heterozygous patients displaying hypodontia. No signs of SSPS, especially eyelids cysts, were detected in our cohort. Interestingly, extra-ectodermal signs consisted of skeletal, neurological and vascular anomalies, the latter suggesting a wider phenotypic spectrum associated with WNT10A mutations. Indeed, the Wnt pathway plays a crucial role in skeletal development, lipid metabolism, and neurogenesis, potentially explaining patient's clinical manifestations.

Genetic counselling difficulties and ethical implications of incidental findings from array-CGH: a 7-year national survey.

Microarray-based comparative genomic hybridization (aCGH) is commonly used in diagnosing patients with intellectual disability (ID) with or without congenital malformation. Because aCGH interrogates with the whole genome, there is a risk of being confronted with incidental findings (IF). In order to anticipate the ethical issues of IF with the generalization of new genome-wide analysis technologies, we questioned French clinicians and cytogeneticists about the situations they have faced regarding IF from aCGH. Sixty-five IF were reported. Forty corresponded to autosomal dominant diseases with incomplete penetrance, 7 to autosomal dominant diseases with complete penetrance, 14 to X-linked diseases, and 4 were heterozygotes for autosomal recessive diseases with a high prevalence of heterozygotes in the population. Therapeutic/preventive measures or genetic counselling could be argued for all cases except four. These four IF were intentionally not returned to the patients. Clinicians reported difficulties in returning the results in 29% of the cases, mainly when the question of IF had not been anticipated. Indeed, at the time of the investigation, only 48% of the clinicians used consents mentioning the risk of IF. With the emergence of new technologies, there is a need to report such national experiences; they show the importance of pre-test information on IF.

Molecular findings and clinical data in a cohort of 150 patients with anophthalmia/microphthalmia.

Anophthalmia and microphthalmia (AM) are the most severe malformations of the eye, corresponding respectively to reduced size or absent ocular globe. Wide genetic heterogeneity has been reported and different genes have been demonstrated to be causative of syndromic and non-syndromic forms of AM. We screened seven AM genes [GDF6 (growth differentiation factor 6), FOXE3 (forkhead box E3), OTX2 (orthodenticle protein homolog 2), PAX6 (paired box 6), RAX (retina and anterior neural fold homeobox), SOX2 (SRY sex determining region Y-box 2), and VSX2 (visual system homeobox 2 gene)] in a cohort of 150 patients with isolated or syndromic AM. The causative genetic defect was identified in 21% of the patients (32/150). Point mutations were identified by direct sequencing of these genes in 25 patients (13 in SOX2, 4 in RAX, 3 in OTX2, 2 in FOXE3, 1 in VSX2, 1 in PAX6, and 1 in GDF6). In addition eight gene deletions (five SOX2, two OTX2 and one RAX) were identified using a semi-quantitative multiplex polymerase chain reaction (PCR) [quantitative multiplex PCR amplification of short fluorescent fragments (QMPSF)]. The causative genetic defect was identified in 21% of the patients. This result contributes to our knowledge of the molecular basis of AM, and will facilitate accurate genetic counselling.

Otocephaly-Dysgnathia Complex: Description of Four Cases and Confirmation of the Role of OTX2.

Otocephaly-dysgnathia complex is characterized by mandibular hypo- or aplasia, ear abnormalities, microstomia, and microglossia. Mutations in the orthodenticle homeobox 2 (OTX2) and paired related homeobox 1 (PRRX1) genes have recently been identified in some cases. We screened 4 otocephalic cases for these 2 genes and identified OTX2 mutations in 2 of them, thus confirming OTX2 is implicated in otocephaly. No PRRX1 mutation was identified. Interestingly, ocular involvement is not a constant feature in otocephalic cases with an OTX2 mutation. In one case, the mutation was inherited from a microphthalmic mother. The mechanism underlying this intrafamilial phenotypic variability remains unclear, but other genetic factors are likely to be necessary for the manifestation of the otocephalic phenotype.

Mutation analysis of the STRA6 gene in isolated and non-isolated anophthalmia/microphthalmia.

PDAC syndrome [Pulmonary hypoplasia/agenesis, Diaphragmatic hernia/eventration, Anophthalmia/microphthalmia (A/M) and Cardiac Defect] is a condition associated with recessive mutations in the STRA6 gene in some of these patients. Recently, cases with isolated anophthalmia have been associated with STRA6 mutations. To determine the minimal findings associated with STRA6 mutations, we performed mutation analysis of the STRA6 gene in 28 cases with anophthalmia. In 7 of the cases the anophthalmia was isolated, in 14 cases it was associated with one of the major features included in PDAC and 7 had other abnormalities. Mutations were identified in two individuals: one with bilateral anophthalmia and some features included in PDAC, who was a compound heterozygote for a missense mutation and a large intragenic deletion, and the second case with all the major features of PDAC and who had a homozygous splicing mutation. This study suggests that STRA6 mutations are more likely to be identified in individuals with A/M and other abnormalities included in the PDAC spectrum, rather than in isolated A/M cases.

X-linked and autosomal recessive Hypohidrotic Ectodermal Dysplasia: genotypic-dental phenotypic findings.

Hypohidrotic ectodermal dysplasia (HED) is characterized by abnormal development of ectodermal structures and its molecular etiology corresponds to mutations of EDA-EDAR genes. The aim of this study was first to investigate the genotype and dental phenotype associated with HED and second, to explore possible correlations between dental features and molecular defects. A total of 27 patients from 24 unrelated families exhibiting clinical signs of HED (22 XLHED males, 5 autosomal recessive forms) were retrospectively included. In the sample, 25 different mutations on EDA and EDAR genes were detected; 10 were not previously described. EDA and EDAR mutations corresponded respectively to 80.0% and 20.0% of the mutations. The dental phenotype analysis revealed a mean number of primary and permanent missing teeth ranging respectively from 14.5 (4-20) to 22.5 (10-28); the majority of the patients exhibited dysmorphic teeth. Overall, no differential expression in the degree of oligodontia according to either the mutated gene, the mutated functional sub-domains, or the mutation type, could be observed. Nevertheless, the furin group exhibited severe phenotypes unobserved in the TNF group. Significant differences in the number of some primary missing teeth (incisor and canine) related to EDA-EDAR genes defects were detected for the first time between XLHED and autosomal recessive HED, suggesting differential local effects of EDA-EDAR genes during odontogenesis. The present genotypic-phenotypic findings may add to the knowledge of the consequences of the molecular dysfunction of EDA-NF-kB in odontogenesis, and could be helpful in genetic counseling to distinguish autosomal forms from other HED syndromes.

Mutations in EDARADD account for a small proportion of hypohidrotic ectodermal dysplasia cases.

BACKGROUND: Hypohidrotic ectodermal dysplasia (HED) is characterized by abnormal development of the eccrine sweat glands, hair and teeth. The X-linked form of the disease, caused by mutations in the EDA gene, represents the majority of HED cases. Autosomal dominant and recessive forms occasionally occur and result from mutations in at least two other genes: EDAR and EDARADD. EDARADD interacts with the TAB2/TRAF6/TAK1 complex, which is necessary for NF-kappaB activation by EDAR. OBJECTIVES: To determine frequency of EDARADD, TRAF6, TAB2 and TAK1 mutations in HED. MATERIALS AND METHODS: We have screened 28 familial or sporadic HED cases with no mutations in the EDA and EDAR genes for EDARADD, TRAF6, TAB2 and TAK1 mutations. RESULTS: We identified one EDARADD 6-bp homozygous in-frame deletion (c.402-407del, p.Thr135-Val136del) in a patient born to consanguineous parents. Functional studies showed that the p.Thr135-Val136del impaired the EDAR-EDARADD interaction and then severely inhibited NF-kappaB activity. In the remaining 27 patients, we failed to find causative mutations in EDARADD, or in TRAF6, TAB2 or TAK1. CONCLUSIONS: Our study demonstrates that EDARADD mutations are not a frequent cause of HED, while mutations in TRAF6, TAB2 and TAK1 may not be implicated in this disease.

An unusual severe vascular case of pseudoxanthoma elasticum presenting as generalized arterial calcification of infancy.

Pseudoxanthoma elasticum (PXE) is an autosomal recessive disease affecting tissues rich in elastic fibers such as the skin, retina, and cardiovascular system. Mutations in the ABCC6 gene are known to be causative in most patients. Generalized arterial calcification of infancy (GACI) is characterized by extensive hydroxyapatite deposits in the internal elastic laminae in large and medium-sized arteries, leading to arterial stenoses and early and severe myocardial ischemia. GACI has been found to be primarily caused by mutations in the ENPP1 gene. We report two brothers born to unrelated parents. The elder developed uncomplicated PXE in adolescence and harbored mutations in the ABCC6 gene. The younger child died of a condition strikingly reminiscent of GACI at 15 months of age. This case of GACI was independent of mutations in the ENPP1 gene but was probably related to ABCC6 mutations. We demonstrate that matrix Gla protein and fetuin-A, involved in PXE, are also expressed in this case of GACI. These proteins could act as local and systemic inhibitors to limit the extension of mineralization. This report emphasizes concurrently that ABCC6 may be a relevant candidate gene in some cases of GACI with no mutations in the ENPP1 gene, and that GACI may be an atypical and severe end of the vascular phenotype spectrum of PXE.

Manifestations of pseudoxanthoma elasticum in childhood.

BACKGROUND: Pseudoxanthoma elasticum (PXE) affects the skin, retina and cardiovascular system. Most cases are related to mutations in the ABCC6 gene. The diagnosis is most often made late in the second or third decade of life. OBJECTIVES: To describe the manifestations of PXE before the age of 15 years. METHODS: Children under age 15 years with definite PXE were evaluated at a PXE referral centre, as were adult patients in whom serious manifestations of PXE had occurred before the age of 15 years. RESULTS: Our series included 96 patients; 15 (16%) had paediatric onset of the disease. Nine children were diagnosed at a mean age of 10 years, a mean of 2.5 years after the presenting symptoms. Cutaneous lesions were the presenting symptoms in eight. None had cardiovascular or ophthalmological symptoms. Six adult patients had had severe cutaneous and/or cardiovascular manifestations before the age of 15 years. Both adult patients with early extensive skin lesions had the PXE-like condition related to the GGCX gene. No ocular symptoms were recorded during childhood. CONCLUSIONS: Cutaneous manifestations of PXE are the same in children as in young adults. Absence of complications is common in childhood, but severe complications are unpredictable. The frequency of complications was retrospectively estimated to be 7% in the adults of our series, although this figure was probably an overestimate because of the recruitment bias in a referral centre. It is, however, important to consider PXE in the paediatric setting, as early diagnosis may be important to provide accurate information and discuss lifestyle adjustments in order to improve the prognosis of the disease.

Confirmation of RAX gene involvement in human anophthalmia.

Microphthalmia and anophthalmia are at the severe end of the spectrum of abnormalities in ocular development. Mutations in several genes have been involved in syndromic and non-syndromic anophthalmia. Previously, RAX recessive mutations were implicated in a single patient with right anophthalmia, left microphthalmia and sclerocornea. In this study, we report the findings of novel compound heterozygous RAX mutations in a child with bilateral anophthalmia. Both mutations are located in exon 3. c.664delT is a frameshifting deletion predicted to introduce a premature stop codon (p.Ser222ArgfsX62), and c.909C>G is a nonsense mutation with similar consequences (p.Tyr303X). This is the second report of a patient with anophthalmia caused by RAX mutations. These findings confirm that RAX plays a major role in the early stages of eye development and is involved in human anophthalmia.