José Ignacio Rodríguez

Cardiovascular malformations in congenital diaphragmatic hernia: Human and experimental studies

BACKGROUND/PURPOSE Cardiovascular malformations (CVM) associated with congenital diaphragmatic hernia (CDH) account in part for the high mortality caused by this defect. The aim of this study is to examine the nature of these malformations in a large series of autopsies and to assess if similar defects are also present in rat fetuses with experimental CDH. METHODS The incidence of CVM and their nature were examined in the autopsy records of 136 stillborns and neonates with CDH admitted to our institution in the last 30 years. Experimental CDH was induced in rat fetuses by giving 100 mg of nitrofen to their mothers on gestational day 9.5, and the fetuses were harvested on day 21 (near full term). The presence of CDH and the anatomy of the heart and great vessels were studied under dissecting microscope after formalin fixation. Unexposed fetuses were used as controls. RESULTS Thirty-three newborns with CDH (24%) had CVM, either isolated or associated with other defects, and 7 had heart hypoplasia. Most CVM (ventricular septal defect, tetralogy of Fallot, transposition of the great vessels, double-outlet right ventricle) involved the outflow tract. In our animal experiments, no malformations were found in 21 control pups. Conversely, 80 of 130 nitrofen-exposed fetuses (61%) had CDH, and 59 of them (74%) had CVM. A significant association (Fishers Exact test, P<.01) was found between CDH and CVM because only 25 of the 50 exposed animals without CDH (50%) had CVM. Again, most defects involved the outflow tract and were similar to those seen in human CDH (tetralogy of Fallot, persistent truncus, ventricular septal defect, double-outlet right ventricle, aberrant right subclavian artery, agenetic ductus, and interrupted aortic arch). Animals with CDH had significantly decreased heart weight to fetal weight ratio in comparison with controls and with those without CDH. CONCLUSIONS The similar nature of the cardiovascular defects found in babies succumbing to CDH and in nitrofen-exposed rats suggests that a similar disturbance of the regional organogenesis related to the neural crest might be involved in both settings, and further validates the use of this animal model for clarifying the cellular and molecular pathogenetic mechanisms.

Vascular and connective tissue anomalies associated with X-linked periventricular heterotopia due to mutations in Filamin A

Mutations conferring loss of function at the FLNA (encoding filamin A) locus lead to X-linked periventricular nodular heterotopia (XL-PH), with seizures constituting the most common clinical manifestation of this disorder in female heterozygotes. Vascular dilatation (mainly the aorta), joint hypermobility and variable skin findings are also associated anomalies, with some reports suggesting that this might represents a separate syndrome allelic to XL-PH, termed as Ehlers-Danlos syndrome-periventricular heterotopia variant (EDS-PH). Here, we report a cohort of 11 males and females with both hypomorphic and null mutations in FLNA that manifest a wide spectrum of connective tissue and vascular anomalies. The spectrum of cutaneous defects was broader than previously described and is inconsistent with a specific type of EDS. We also extend the range of vascular anomalies associated with XL-PH to included peripheral arterial dilatation and atresia. Based on these observations, we suggest that there is little molecular or clinical justification for considering EDS-PH as a separate entity from XL-PH, but instead propose that there is a spectrum of vascular and connective tissues anomalies associated with this condition for which all individuals with loss-of-function mutations in FLNA should be evaluated. In addition, since some patients with XL-PH can present primarily with a joint hypermobility syndrome, we propose that screening for cardiovascular manifestations should be offered to those patients when there are associated seizures or an X-linked pattern of inheritance.

Lethal neonatal Hutchinson-Gilford progeria syndrome

We report on a 35-week gestation female fetus with Hutchinson-Gilford progeria (HGP). This patient, who is the first reported with neonatal HGP in the English literature but is the fourth, counting three previous French cases, supports the existence of a more severe prenatal form of progeria. She died 7 hours after birth and presented with intrauterine growth retardation, premature aging, absence of subcutaneous fat, brachydactyly, absent nipples, hypoplastic external genitalia, and abnormal ear lobes. The childs combination of clinical and skeletal manifestations differentiates this form of HGP from other progeroid syndromes with neonatal presentation. We also report previously undescribed autopsy findings including premature loss of hair follicles, premature regression of the renal nephrogenic layer, and premature closure of the growth plates in the distal phalanges that may be related to the aging processes in this condition. We could not find any histological data to support acro-osteolysis, which is the radiographic sign of brachydactyly. The terminal phalanges in HGP seem to be underdeveloped rather than osteolytic.

A prenatally diagnosed patient with full monosomy 21: ultrasound, cytogenetic, clinical, molecular, and necropsy findings.

We report on a patient with a full monosomy 21 (FM21) prenatally diagnosed in cord fetal blood, and subsequently confirmed in other tissues. Subtle chromosomal translocations of chromosome 21, were ruled‐out by FISH using both painting and 21q telomeric probes. Microsatellites analysis demonstrated the paternal origin of the single chromosome. The propositus showed at 32 weeks of gestation a severe intrauterine growth retardation and microcephaly. He was born with multiple congenital malformations, hypotonia, microcephaly, bilateral microphthalmia (more severe on the left), facial dysmorphism, agenesis of the external auditory meatus, redundant skin in the neck, narrow chest, flat scrotum, cryptorchydism, hypospadias, micropene, camptodactyly, nail hypoplasia, and abnormal palmar and plantar creases. The patient died in the first day of life. At necropsy, micrencephaly, semilobar holoprosencephaly, polimicrogyria, ocular abnormalities, skeletal anomalies, congenital heart disease, and agenesis of right kidney were also observed. To our best knowledge, this case is one of the most completely patient studied with FM21.

Neural crest-derived defects in experimental esophageal atresia.

Esophageal atresia (EA) is often associated with cardiovascular and other malformations that are likely neural crest derived. The present study tests the hypothesis that the heart and great vessels and the thymus and parathyroids may be abnormal in the rat model of EA as a result of disturbed neural crest development. Time-mated pregnant rats received intraperitoneally on d 8 and 9 of gestation either 2 mg/kg adriamycin or vehicle. Esophageal, heart, and thymic malformations were sought under the microscope in term fetuses. The parathyroids were histologically investigated. Control fetuses had no malformations, whereas 69 of 109 fetuses exposed to adriamycin had EA and 45 of 69 had 15 right aortic arches, nine aberrant right subclavia, eight ventricular septal defects, six narrow pulmonary outflow tracts, five tetralogies of Fallot, three double outflow right ventricles, three double aortic arches, three atrial septal defects, three right ductus arteriosus, and two truncus. The thymus was absent in 19, hypoplastic in 12, and ectopic in five out of 36 fetuses with EA in which it was studied, whereas the parathyroid glands were absent in 16, single in four, and ectopic in one of the 23 fetuses with EA in which they were studied. In conclusion, the nature of the cardiovascular, thymic, and parathyroid malformations associated with EA in rats is consistent with the hypothesis of neural crest participation in their pathogenesis. Mechanisms simultaneously disturbing foregut septation, somitic segmentation, and neural crest development should be sought to explain the combined occurrence of malformations in EA.

Transverse Bone Growth and Cortical Bone Mass in the Human Prenatal Period

The prenatal development of the normal diaphysis of the human long bone was studied through postmortem radiographs in 60 stillborns and 86 newborns of 20-41 weeks gestational age. Quantitative parameters were determined for the tibia, femur, and humerus. In all three long bones, significant positive correlations were found between the diaphyseal diameter, medullary diameter, cortical thickness, and cortical area, and the gestational age, body weight, body height, and bone length. No significant differences in any studied parameters were observed between males and females. In the tibia, the diaphyseal diameter grew more than in the femur and humerus. In these two latter bones, the medullary diameter growth rate was greater than the diaphyseal diameter growth rate, and thus the Barnett-Nordin index and percentage of cortical area showed mild but significant negative correlations with gestational age, body weight, body height, and bone length. The velocity of medullary diameter growth was similar (0.05 mm/week) in all three long bones; however, the velocity of diaphyseal diameter growth was greater in the tibia (0.161 mm/week) than in the femur (0.142 mm/week) or humerus (0.129 mm/week). The diaphyseal growth rate decreased in the second half of the period studied in all three bones. This decrease was more striking in the humerus. Observed differences in diaphyseal growth among the long bones studied may be genetically determined in relation to its different postnatal functions.

Skeletal malformations associated with esophageal atresia: clinical and experimental studies.

BACKGROUND/PURPOSE Patients with esophageal atresia (EA) often have skeletal malformations. The purpose of this study is to examine if similar defects occur in rat fetuses prenatally exposed to Adriamycin, a chemical capable of causing EA in these animals. METHODS The charts of 443 babies with EA were reviewed to assess the incidence and nature of these defects in them. Time-mated female rats were given either 2 mg/kg intraperitoneal Adriamycin (experimental group, n = 16) or no treatment (control group, n = 4) on gestational days 8 and 9, and the fetuses were removed near term. Skeletal anatomy was studied after alcian blue and alizarin red staining. RESULTS A total of 528 skeletal malformations, mainly abnormal segmentation and vertebral identity (extra or defective bodies or ribs), mishaped vertebral bodies, and limb malformations like radial aplasia or hypoplasia were found in 245 babies (55%). Costal fusion and sternal anomalies were present in 17 and 4 babies, respectively. In the animal study, all control fetuses were normal, whereas 83 of 134 experimental fetuses (62%) had EA accompanied by other malformations. No segmentation or vertebral identity anomalies were seen, but butterfly, wedged, and asymmetric vertebral bodies were found at various levels in all animals with EA and in about half of those without it. Three fetuses had rib anomalies, and 3 more had sternal malformations. Ossification of limbs was delayed in treated fetuses and short, thick, and crooked bones were seen in 4 of 31 fetuses with EA and in none of the Adriamycin-exposed ones without EA. CONCLUSIONS Adriamycin exposure induces in fetal rats, in addition to esophageal, duodenal, and anorectal atresias, high proportions of vertebral malformations and some limb defects of nature not identical but quite similar to that of babies with EA. This further validates this model for investigating the nature of the processes leading to EA and its associated malformations.

Cystic dysplasia of the epididymis: a disorder of mesonephric differentiation associated with renal maldevelopment

The occurrence of congenital epididymal malformations with a cystic component has not been fully characterized. Most epididymal cysts occur later in life and are likely acquired. In addition, congenital malformations of the male excretory system are extremely uncommon in fetuses and neonates, and epididymal dysplastic changes have not been reported in these cases. In this study, we report 20 cases (including 19 fetal/neonatal autopsies and one surgical specimen from an older child) showing the same spectrum of histological findings in the epididymis, characterized by cystic ductal dilation with dysplastic ducts of variable diameters and irregular shapes, with ill-defined walls. Efferent ductules also showed dysplastic features. In addition, 18 cases had either renal and/or urinary tract anomalies, including renal dysplasia (eight), pelvicaliceal dilation (eight), renal agenesis (four) and hypoplasia (one), ureteral agenesis (two) and hypoplasia (one), urethra and bladder agenesis (two), prostate agenesis (two), and autosomal recessive polycystic renal disease (two). Our observations led to the recognition of a peculiar, not previously described congenital lesion of the epididymis, and we propose the term cystic dysplasia of the epididymis for this anomaly. Similar to what is observed in other male genital system anomalies (including malformations of the rete testis, vas deferens, and seminal vesicles), most lesions occurred in association with renal and/or urinary tract malformations, suggesting a spectrum of congenital malformations. The shared embryological origin of these structures may explain their simultaneous occurrence, possibly related to disrupted mesonephric duct development.

Prenatal growth of the human mandibular condylar cartilage

The question of whether the condylar cartilage possesses a growth potential like that of the long bone growth plates has been the subject of contrasting viewpoints. We have recently established that the thickness of the human tibial growth plate progressively decreases during the second half of the fetal period, but that the changes in the total human condylar thickness do not correlate with fetal age or weight. The present study examined the change in the thickness of the human mandibular condyle layers during the fetal growth of the mandible. Mandibles were obtained from autopsy of 19 human fetuses ranging in fetal age from 18 to 41 weeks. The total length of the mandible, the lengths of the mandibular body and of the ramus were measured, as well as the gonial angles. The total thickness of the condyle, and the thickness of the articular, progenitor, cartilage, chondroblast, and hypertrophic chondrocyte layers were measured on the central segment of central sagittal sections of the mandibular condylar cartilage. The total mandible, the corpus and the ramus lengths increased linearly with the age of the fetus and they all correlated strongly with fetal weight. However, changes in the total condylar thickness and in the thickness of the cartilage layer (chondroblast plus hypertrophic chondrocytes) did not correlate with fetal weight or mandibular length. The thickness of the articular layer increased with weight, but changes in the progenitor layer were independent of corporal and mandibular growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormal intrinsic esophageal innervation in congenital diaphragmatic hernia: a likely cause of motor dysfunction

PURPOSE Patients with congenital diaphragmatic hernia (CDH) often have dilated esophagus and gastroesophageal reflux. Sparse intrinsic esophageal innervation has been described in rats with CDH, but this issue has not been investigated in patients with CDH. The present study tests the hypothesis that innervatory anomalies could account for motor dysfunction in human CDH. METHODS The esophagi of CDH (n = 6) and control babies dead of other causes (n = 6) were included in paraffin, transversally sectioned, and immunostained with antineurofilament and anti-S-100 antibodies. The proportion of the section surface occupied by neural structures, the ganglionar surface, and the number of neurons per ganglion were measured in 2 to 5 low-power fields from the proximal and distal esophagus with the assistance of image analysis software. Mann-Whitney tests were used for comparing the results using a threshold of significance of P < .05. RESULTS The percentage of neural/muscle surface was similar in the upper esophagus in both groups, but it was significantly decreased in the lower esophagus of patients with CDH in comparison with controls. There was a relative scarcity of neural tissue in the intermuscular plexus of the lower esophagus. On the other hand, the ganglionar surface and the number of neurons per ganglion were identical in both groups. These results were similar with both immunostainings. CONCLUSION Intrinsic innervation of the lower esophagus in CDH is abnormal in terms of decreased density of neural structures in the intermuscular plexus. These neural crest-derived anomalies could explain in part the esophageal dysfunction in survivors of CDH.