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.

Smooth muscle of the male pelvic floor: An anatomic study.

Knowledge of the anatomy of the male pelvic floor is important to avoid damaging the pelvic floor muscles during surgery. We set out to explore the structure and innervation of the smooth muscle (SM) of the whole pelvic floor using male fetuses. We removed en-bloc the entire pelvis of three male fetuses. The specimens were serially sectioned before being stained with Masson's trichrome and hematoxylin and eosin, and immunostained for SMs, and somatic, adrenergic, sensory and nitrergic nerve fibers. Slides were digitized for three-dimensional reconstruction. We individualized a middle compartment that contains SM cells. This compartment is in close relation with the levator ani muscle (LAM), rectum, and urethra. We describe a posterior part of the middle compartment posterior to the rectal wall and an anterior part anterior to the rectal wall. The anterior part is split into (1) a centro-levator area of SM cells localized between the right and left LAM, (2) an endo-levator area that upholsters the internal aspect of the LAM, and (3) an infra-levator area below the LAM. All these areas are innervated by autonomic nerves coming from the inferior hypogastric plexus. The core and the infra-levator area receive the cavernous nerve and nerves supplying the urethra. We thus demonstrate that these muscular structures are smooth and under autonomic influence. These findings are relevant for the pelvic surgeon, and especially the urologist, during radical prostatectomy, abdominoperineal resection and intersphincteric resection. Clin. Anat., 2019. © 2019 Wiley Periodicals, Inc.

A series of 38 novel germline and somatic mutations of NIPBL in Cornelia de Lange syndrome.

Cornelia de Lange syndrome is a multisystemic developmental disorder mainly related to de novo heterozygous NIPBL mutation. Recently, NIPBL somatic mosaicism has been highlighted through buccal cell DNA study in some patients with a negative molecular analysis on leukocyte DNA. Here, we present a series of 38 patients with a Cornelia de Lange syndrome related to a heterozygous NIPBL mutation identified by Sanger sequencing. The diagnosis was based on the following criteria: (i) intrauterine growth retardation and postnatal short stature, (ii) feeding difficulties and/or gastro-oesophageal reflux, (iii) microcephaly, (iv) intellectual disability, and (v) characteristic facial features. We identified 37 novel NIPBL mutations including 34 in leukocytes and 3 in buccal cells only. All mutations shown to have arisen de novo when parent blood samples were available. The present series confirms the difficulty in predicting the phenotype according to the NIPBL mutation. Until now, somatic mosaicism has been observed for 20 cases which do not seem to be consistently associated with a milder phenotype. Besides, several reports support a postzygotic event for those cases. Considering these elements, we recommend a first-line buccal cell DNA analysis in order to improve gene testing sensitivity in Cornelia de Lange syndrome and genetic counselling.

Detailed in utero ultrasound description of 30 cases of congenital cytomegalovirus infection.

OBJECTIVES: The aim of this research was to describe precisely prenatal ultrasound (US) features in congenital cytomegalovirus (CMV) infection. METHODS: We retrospectively evaluated the US descriptions of cases of congenital CMV infection between 2004 and 2013. RESULTS: In 69 congenital CMV infections, related US abnormalities were reported in 30 cases (43.5%). There were both extracerebral and cerebral abnormalities in 16 cases, purely abnormal brain features in ten, and purely extracerebral features in two. About 19/30 cases presented extracerebral features of 11 different sorts of abnormalities, mainly hyperechogenic bowel (ten cases) and intrauterine growth retardation (nine cases). About 24/30 cases presented cerebral features of 13 different sorts, mainly brain calcifications (12 cases) and occipital horn cavity (11 cases). The main US findings in our series are not specific to CMV infection. However, a frequent finding attracted our attention: the anechogenic cavity located on the extremity of the occipital horn, a region which contains numerous proliferating and differentiating germinal cells. CONCLUSIONS: By improving knowledge of US findings linked to CMV infection, US sensitivity may be improved. Understanding why CMV leads to lesions of the occipital horn may help clarify the pathophysiology of congenital infection.

The fetal safety of cetirizine: an observational cohort study and meta-analysis.

Cetirizine, a second-generation antihistamine, is an active metabolite of hydroxyzine used in the treatment of allergies, but the data on fetal safety are inconclusive. Pregnant women who were counselled by the 'Motherisk Program' regarding cetirizine exposure were enrolled in a cohort study and compared with pregnant women counselled for non-teratogenic exposures. The objective was to measure the rate of adverse pregnancy outcomes. Subsequently, we also conducted a meta-analysis of cohort studies that examined the pregnancy outcomes of women exposed to hydroxyzine or cetirizine during pregnancy. In the cohort study, there were no significant differences in the rates of major malformations between the cetirizine exposed and comparison group. In the meta-analysis, cetirizine was not associated with increased teratogenic risk. In contrast, a meta-analysis of cetirizine and hydroxyzine studies showed a marginal association with major malformations. Cetirizine is not associated with a clinically important increase in risk of adverse fetal outcomes.

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.

Three-dimensional helical computed tomography in prenatal diagnosis of fetal skeletal dysplasia.

OBJECTIVES: (1) To study the use and diagnostic value, as a complement to ultrasound, of helical computed tomography (helical CT) to differentiate normal fetuses from cases of skeletal dysplasia; (2) to define the most relevant indications for helical CT; and (3) to evaluate its diagnostic performance with respect to radiological criteria considered discriminatory. METHODS: This was a retrospective study from 2005 to 2008 in 67 pregnant women who underwent helical CT after 26 weeks of gestation for suspected fetal skeletal dysplasia due to fetal shortened long bones on ultrasound (≤ 10(th) percentile), either alone or associated with other bone abnormalities. The results were compared with pediatric examinations in 41 cases and with fetal autopsy findings after elective termination of pregnancy in the others. RESULTS: Helical CT had a sensitivity of 82%, specificity of 91% and positive and negative predictive values of 90% and 83%, respectively, for diagnosis of fetal skeletal dysplasia. An etiological diagnosis that had not been suspected at ultrasound was specified in 15% of cases and diagnoses suspected at ultrasound were confirmed in 24% and discounted in 43% of cases. The prevalence of skeletal dysplasia was increased in cases of micromelia < 3(rd) percentile or if there was a combination of bone signs. Helical CT showed 69% sensitivity in identifying individual predefined pathological bone signs which were confirmed on fetal autopsy findings. CONCLUSION: Helical CT is a key examination, in combination with ultrasound, in the diagnosis of fetal skeletal dysplasia from 26 weeks of gestation. It should be reserved for cases with severe micromelia below the 3(rd) percentile and for those with micromelia ≤ 10(th) percentile associated with another bone sign. A checklist of discriminatory signs is proposed.

Antenatal ultrasound prediction of pulmonary hypoplasia in congenital diaphragmatic hernia: correlation with pathology.

OBJECTIVE: To examine the relationship between observed to expected (o/e) lung to head circumference ratio (LHR) and lung-to-body weight ratio (LBWR) in fetuses with congenital diaphragmatic hernia (CDH). METHODS: All consecutive fetuses with CDH and termination of pregnancy for which a postmortem examination was available, examined at three institutions between 2000 and 2010, were included in the study. Contralateral fetal lung area was measured by two-dimensional ultrasonography using the longest axis method and the o/e-LHR was calculated based on the appropriate normal mean for gestational age (GA). Regression analysis was used to determine the significance of association between the LBWR and the o/e-LHR for left and right-sided cases, and subsequently the predicted LBWR in left-sided CDH was calculated using the regression equation. Regression analysis was used to investigate the effect on the proportional difference between the predicted and observed LBWR of GA at o/e-LHR, time gap between o/e-LHR and LBWR measurement, proportional weight of the ipsilateral compared with total lung weight, presence of associated anomalies and intrathoracic herniation of the liver. RESULTS: There were 23 fetuses with left-sided and seven fetuses with right-sided CDH. In left-sided CDH, the LBWR and the o/e-LHR correlated significantly, following the linear equation: LBWR = 0.0043 + (0.0134 × o/e-LHR) (r = 0.52, P = 0.012), but this was not the case for right-sided CDH, for which LBWR followed the equation: LBWR = 0.0107 - (0.0014 × o/e-LHR) (r = 0.08, P = 0.862), where o/e-LHR is expressed as percentage. Regression analysis showed that the proportional difference between predicted and observed LBWR in left-sided CDH was significantly and independently associated with GA at o/e-LHR measurement and proportional weight of ipsilateral vs. total lung weight. CONCLUSION: In left-sided CDH, o/e-LHR correlates well with LBWR irrespective of the length of time between o/e-LHR and LBWR measurement, presence of associated anomalies and intrathoracic herniation of the liver. Inconsistencies between the two measurements are mainly attributable to the contribution of the ipsilateral lung to the total lung weight. In right-sided CDH, o/e-LHR does not correlate with LBWR.

Factors associated with partial and complete regression of fetal lung lesions.

OBJECTIVE: A decrease in the volume of congenital pulmonary malformations (CPM) can be observed on prenatal ultrasonography, but the underlying mechanism for this phenomenon is unknown. Our objective was to identify factors associated with the prenatal reduction in size of cystic and/or hyperechoic lung lesions. METHODS: This was a retrospective study of cases with a prenatal diagnosis of hyperechoic and/or cystic lung lesion. The extent of reduction in lesion size was calculated from ultrasound measurements. Clinical, ultrasound, radiological and histological data were tested for their relationship with prenatal CPM reduction. RESULTS: In a 4-year period, 36 patients were referred with a cystic and/or hyperechoic fetal lung lesion diagnosed at a mean gestational age of 23.4 weeks. The lesions were cystic in 16 cases (44%), hyperechoic in 12 (33%) and both in eight (22%). The malformation was no longer visible before birth (apparent disappearance) in nine cases (25%), shrank by 18-90% in 15 (42%) and did not reduce in 12 (33%). Findings on postnatal computed tomography were always abnormal. Isolated hyperechoic lesions were significantly more likely to shrink in utero. The mean reductions were 79%, 35% and 19%, for isolated hyperechoic, cystic and mixed lesions, respectively (P=0.001). Only 8% of hyperechoic lesions demonstrated no volume reduction, as compared to 50% and 42% of cystic and mixed lesions, respectively (P=0.03). Greater gestational age at birth was also associated with a decrease in the incidence of malformations (P=0.02). In cases that underwent surgery, hyperechoic lesions were linked to a variety of pathological diagnoses, whereas cystic lesions were all described histologically as congenital cystic adenomatoid malformations. CONCLUSIONS: Prenatal size reduction of fetal lung malformations is associated with isolated hyperechogenicity and greater gestational age at birth. This might result from the resumption of normal lung development after local disruption of lung growth.

Gender-specific oxidative stress parameters.

The aim of the present study was to examine the association of proteins that regulate iron transport/storage content and acute phase response with oxidative stress in male and female athletes. Serum ferritin, transferrin, soluble transferrin receptor, C-reactive protein, interleukin-6 and oxidative stress parameters (reactive oxygen metabolites, superoxide anion, advanced oxidation protein products, lipid hydroperoxides, superoxide-dismutase and pro-oxidant-antioxidant balance) were determined in 138 athletes (73 females and 65 males). A general linear model indicated significant gender differences between athletes in terms of reactive oxygen metabolites (307.48 ± 61.02 VS. 276.98 ± 50.08; P=0.030), superoxide-dismutase (114.60 ± 41.64 VS. 101.42 ± 38.76; P=0.001), lipid hydroperoxides (149.84 ± 38.95 VS. 101.43 ± 39.26; P<0.001), pro-oxidant-antioxidant balance (512.40 ± 148.67 VS. 413.09 ± 120.30; P=0.002), advanced oxidation protein products (1.49 ± 0.30 VS. 0.91 ± 0.25; P<0.001) and superoxide (2.61 ± 0.36 VS. 2.22 ± 0.35; P=0.001), which were all significantly higher in females. Multivariate analysis of covariance indicated gender (P<0.001), training experience (P=0.004), C-reactive protein (P=0.002), soluble transferrin receptor (P=0.004) and transferrin (P<0.001) as significant covariates. Gender accounted for the largest proportion of variability for all oxidative stress parameters (46.3%) and female athletes were more susceptible to oxidative stress. Iron transport and storage proteins (transferrin and ferritin), but also acute phase reactants, were negatively related factors for oxidative stress. In conclusion, variation in the ferritin level may contribute to the different oxidative stress level between the sexes.

Proxidant* antioxidant balance in supplemented elite female volleyball athletes during a six week training period.

AIM: The objectives of this study were: 1) to evaluate pro-oxidant-antioxidant balance (PAB) associations with the commonly measured parameters of oxidative stress and antioxidatve defence in elite female volleyball athletes; 2) to investigate changes in the parameters of oxidative stress during a period of intense training and dietary antioxidant supplementation. METHODS: Twenty-seven female volleyball players participated in this study. Blood samples were collected the day before the pre-competitive mesocycle training period began. After the first blood sample donation and during the next six weeks fourteen players (supplemented group) received a cocktail of antioxidants while thirteen of them (control group) received no dietary supplementation. The following parameters were measured: reactive oxygen metabolites (ROMs), superoxide anion (O2-), malondialdehyde (MDA), advanced oxidation protein products (AOPP), lipid hydroperoxides (LOOH), biological antioxidative potential (BAP), paraoxonase activity toward paraoxon (POase) and diazoxon (DZOase), superoxide dismutase (SOD), total sulphydryl group concentration (SH groups) and pro-oxidant-antioxidant balance. RESULTS: Significant associations were observed between biomarkers of oxidative damage with PAB in multiple linear regression model in the supplemented and the control groups (82.3% vs. 83.1%) before training and in the control group (82.1%) after training. Significant associations between antioxidative defence parameters and PAB values were found in the supplemented group after six-weeks of training (57%). CONCLUSION: In the absence of antioxidant supplementation, PAB values were dependent on the association with biomarkers of oxidative damage before and after training. After a six-week training period and the applied antioxidant supplementation, PAB values were under the influence of non-enzymatic anti-oxidative defence.

Genotype-phenotype correlations in fetuses and neonates with autosomal recessive polycystic kidney disease.

The prognosis of autosomal recessive polycystic kidney disease is known to correlate with genotype. The presence of two truncating mutations in the PKHD1 gene encoding the fibrocystin protein is associated with neonatal death while patients who survive have at least one missense mutation. To determine relationships between genotype and renal and hepatic abnormalities we correlated the severity of renal and hepatic histological lesions to the type of PKHD1 mutations in 54 fetuses (medical pregnancy termination) and 20 neonates who died shortly after birth. Within this cohort, 55.5% of the mutations truncated fibrocystin. The severity of cortical collecting duct dilatations, cortical tubule and glomerular lesions, and renal cortical and hepatic portal fibrosis increased with gestational age. Severe genotypes, defined by two truncating mutations, were more frequent in patients of less than 30 weeks gestation compared to older fetuses and neonates. When adjusted to gestational age, the extension of collecting duct dilatation into the cortex and cortical tubule lesions, but not portal fibrosis, was more prevalent in patients with severe than in those with a non-severe genotype. Our results show the presence of two truncating mutations of the PKHD1 gene is associated with the most severe renal forms of prenatally detected autosomal recessive polycystic kidney disease. Their absence, however, does not guarantee survival to the neonatal period.

Early second-trimester diagnosis of intracranial teratoma.

Congenital brain tumors are rare and, whether diagnosed prenatally or postnatally, the most frequent type is teratoma. Prenatal diagnosis relies on sonography and magnetic resonance imaging, and is usually achieved during the second or third trimester. We report a case of an intracranial tumor diagnosed in the early second trimester. The diagnosis had been suspected at first-trimester routine sonography, which showed a compressive intracranial mass with mild vascularization. Because of the poor prognosis, termination of pregnancy was discussed with the parents and was carried out at 14 weeks of gestation. Postmortem examination provided a diagnosis of right frontal immature teratoma.

Prenatal diagnosis of cardiac rhabdomyomas: incidence of associated cerebral lesions of tuberous sclerosis complex.

OBJECTIVES: To determine the prevalence of specific cerebral lesions of tuberous sclerosis complex (TSC) and neurological outcome in cases diagnosed prenatally with cardiac rhabdomyomas. METHODS: We reviewed all fetuses diagnosed prenatally with cardiac rhabdomyomas which had undergone detailed ultrasound evaluation and cerebral magnetic resonance imaging (MRI) and which were recorded in the database of a single institution covering the period January 1992 to December 2005. RESULTS: Fifty-one fetuses were included in the study. MRI was performed at a mean +/- SD gestational age of 30 +/- 3 gestational weeks and showed specific lesions of TSC in 49% of cases. Termination of pregnancy was chosen by the parents in 26 cases. Neurological development was studied in 20 cases, follow-up lasting 4.8 +/- 2.9 years. Neurodevelopmental events occurred during the follow-up period in 45% of cases. Neurological complications occurred in 67% of patients who had cerebral lesions at MRI and in 33% of patients with normal MRI results. There was no significant difference between the two groups of patients (P = 0.2). CONCLUSION: In fetuses with cardiac rhabdomyomas detailed ultrasound examination and third-trimester cerebral MRI are able to diagnose most TSC cerebral lesions, but fail to determine neurological outcome.

Long-term effects of oxidative stress in volleyball players.

The aim of this study was to determine the impact of long-term training on elite female volleyball players and to determine parameters that could discriminate them according to the level of oxidative stress-associated adaptation. Fifty-four elite female volleyball players were divided into 3 groups (1: below-average training experience <8.0 years, 2: average training experience between 8.0 and 10.5 years and 3: above-average training experience >10.5 years). The measured parameters were reactive oxygen metabolites, biological anti-oxidative potential, superoxide anion, malondialdehyde, advanced oxidation protein products, lipid hydroperoxides, paraoxonase activity, superoxide-dismutase activity and sulphydryl groups. Multiple discriminant analysis of the oxidative stress status parameters between the three groups of athletes indicated a statistically significant difference (Wilks' lambda=0.458, X(2)=35.898, p=0.031). The most important discriminant variables, superoxide-dismutase and superoxide anion, were the best indicators of differences between groups with different training experience. The significantly higher values were found in Group 3 compared with Group 1 in superoxide-dismutase activity (141+/-32 vs. 86+/-46; p=0.002), sulphydryl groups (p=0.031), and reactive oxygen metabolites (p=0.042). The significantly lower superoxide anion was found between Group 3 and Group 1 (377+/-187 vs. 1183+/-905; p=0.001). Oxidative stress status parameters adequately discriminated 68.5% of athletes with different training experience.

Molecular heterogeneity in fetal forms of type II lissencephaly.

Type II lissencephaly (type II LIS) is a group of autosomal recessive congenital muscular dystrophies (CMD) associated with defects in alpha-DG O-glycosylation, which comprises Walker-Warburg syndrome, Fukuyama cerebral and muscular dystrophy, or muscle-eye-brain disease. The most severe forms of these diseases often have a fetal presentation and lead to a pregnancy termination. We report here the first molecular study on fetal type II LIS in a series of 47 fetuses from 41 unrelated families. Sequencing of the different genes known to be involved in alpha-DG O-glycosylation allowed the molecular diagnosis in 22 families: involvement of POMT1 was demonstrated in 32% of cases, whereas POMGNT1 and POMT2 were incriminated in 15% and in 7% of cases, respectively. We found 30 different mutations in these three genes, 25 were described herein for the first time, 15 in POMT1, and five in POMT2 and POMGNT1. Despite sequencing of FKRP, FCMD, and LARGE, no definitive molecular diagnosis could be made for the other half of our cases. Preliminary results concerning genotype-phenotype correlations show that the choice of the first gene sequenced should depend on the clinical severity of the type II LIS; POMT1 and POMT2 for severest clinical picture and POMGNT1 for milder disease. The other genes, FKRP, FCMD, and LARGE, seem not to be implicated in the fetal form of CMD.