Delayed diagnosis and underreporting of congenital anomalies associated with oral clefts in the Netherlands: a national validation study.

OBJECTIVE: Since 1997, the 15 Dutch cleft palate teams have reported their patients with oral clefts to the national oral cleft registry (NVSCA). During the first visit of the patient to the team - which is usually within the first year of life - the oral cleft and associated congenital anomalies are recorded through a unique recording form by a plastic surgeon/orthodontist/paediatrician. In this study, we evaluated the quality of data on congenital anomalies associated with clefts. METHODS: We drew a random sample of 250 cases registered in the national database with oral clefts from 1997 through 2003; of these, 13 were excluded. Using two independent reregisters derived from two-phased medical data review, we analysed whether associated anomalies were correctly diagnosed and recorded. RESULTS: The agreement on associated anomalies between the NVSCA and medical data ranged from moderate to poor (kappa 0.59 to 0). Seventy-seven percent of the craniofacial anomalies were underreported in the NVSCA: 30% due to delayed diagnosis and 47% due to deficient recording. Additionally, 80% of the associated anomalies of other organ systems were underreported: 52% due to delayed diagnosis and 28% due to deficient recording. The reporting of final diagnoses was somewhat better; however, 54% were still underreported (24% delayed diagnosis and 30% deficient recording). The rate of overreporting was 1.6% or lower. CONCLUSION: Congenital anomalies associated with clefts are underreported in the NVSCA because they are under diagnosed and deficiently recorded during the first consultations with the cleft palate teams. Our results emphasise the need for routine and thorough examination of patients with clefts. Team members should be more focussed on co-occurring anomalies, and early genetic counselling seems warranted in most cases. Additionally, our findings underline the need for postnatal follow-up and ongoing registration of associated anomalies; reregistration in the NVSCA at a later age is recommended.

Clubbed fingers: the claws we lost?

Clubbed digits resemble the human embryonic fingers and toes, which look like the digits of a claw. Clubbed digits, thus, may represent the return of the embryonic claw and may even represent the claws man has lost during evolution, if ontogenesis really recapitulates phylogenesis. We put forward the hypothesis that secondary clubbing, like gynecomastia, is caused by a pathologic condition, which alters hormone levels in the blood, leading to the activation of 'dormant' genes, resulting in the development of an organ. However, the nature of the diseases that cause clubbing suggests that these hormones may actually be cytokines, acting as hormones. The nature of these cytokines is not known. They may be identified by comparing their blood levels or the combination of their blood levels to the presence or absence of clubbing, but also to the degree of clubbing and its disappearance after treatment of the primary disease.

Detection of apoptosis in ovarian cells in vitro and in vivo using the annexin v-affinity assay.

The ability of a cell to undergo apoptosis is crucial during development, tissue homeostasis, and in the pathogenesis and treatment of disease (1). To study apoptosis, it is important to be able to detect apoptotic cells reliably. Here we describe a method to detect apoptosis in vitro and in vivo on basis of the changes in phospholipid distribution in the plasma membrane that occur during this process. In healthy cells, phosphatidylserine (PS) is maintained predominantly in the inner plasma membrane surface by an aminophospholipid translocase (2). However, early during apoptosis, PS is translocated from the inner to the outer membrane surface and serves as a trigger for adjacent phagocytes to remove the dying cell (3-5). Exposure of PS can be detected in vitro and in vivo with fluorochrome- or biotin-labeled annexin V, a protein that binds to negatively charged phospholipids in the presence of calcium ions (6,7). In cells that are cultured in suspension, detection of apoptosis on the basis of PS exposure is relatively easy (8). However, sample handling of adherent cell lines, such as the ovarian cell line PA-1, might interfere with reliable detection of PS exposure. Therefore, we developed a method to detect PS exposure in adherent cell lines by labeling cells in a monolayer with annexin V and harvesting the cells afterwards by mechanical scraping (9) (Figs. 1 and 2). Fixation of annexin V-labeled cells also allows the study of the relationship between PS exposure and expression of intracellular antigens (10). We also present a method to detect apoptosis in vivo during follicular maturation in the mouse Fig. 3). This method is based on in vivo studies of viable mouse embryos, which indicate that PS exposure is a pancellular phenomenon of apoptosis during mammalian development (11,12). Fig. 1. Confocal scanning laser microscopy analysis of PA-1 ovary teratocarcinoma cells. Apoptosis was induced by treating the cells for 6 h with 50 µM roscovitine, a cyclin-dependent kinase inhibitor. Cells were labeled with annexin V-Oregon green to detect PS exposure, harvested by mechanical scraping, and labeled with propidium iodide (PI) to detect plasma membrane integrity. A and B show a linear projection of a stack of confocal images of early apoptotic cells after labeling with annexin V. At this stage, the plasma membrane integrity is preserved and, therefore, PI cannot enter the cell. C shows a secondary necrotic PA-1 cell, with clear annexin V staining at the plasma membrane (C1) and with PI staining of the condensed and fragmented chromatin (C2). Fig. 2. Dotplot of bivariate PI/annexin V flow cytometric analysis of adherent ovary cell line PA-1. Plasma membrane integrity is shown on the X-axis and annexin V immunofluorescence is shown on the Y-axis. Cells were treated with 50 µM roscovitine to induce apoptosis. 6 h after roscovitine treatment, cells were labeled with annexin V-Oregon green, harvested by scraping, and labeled with PI. Four populations of cells can be identified: region R1: vital cells (annexin V negative/PI negative), region R2: apoptotic cells (annexin V positive/PI negative), region R3: dead cells (annexin V positive/ PI positive); and region R4: damaged cells (annexin V negative/PI positive). For technical details, see ref. 9. Fig. 3. Micrographs of paraffin sections through mice ovaries that were perfused with biotinylated annexin V (A-F) or HEPES-buffer only (G and H). In A, annexin V labeled early apoptotic cells (arrowhead) and late apoptotic-pyknotic (arrow) granulosa cells are shown. During follicle maturation, initially apoptosis is absent (B). At later phases, annexin V labeled apoptotic granulosa cells (C, arrow) were observed in the primary (C) and secondary (D) follicles. Unstained pyknotic cells were also observed (C, arrowhead), presumably these cells were already located in the phagosomes before perfusion with annexin V. Also in the Graafian follicle, apoptotic cells were present in large numbers (E). F shows an enlargement of the boxed area in E. Labeled apoptotic and postapoptotic necrotic cells that have been shed into the antrum are clearly visible (asterisk), as well as unlabeled late postapoptotic necrotic cells. Labeling of ingested (arrowhead) and noningested (arrow) apoptotic cells was absent in ovaries of specimen that were perfused with HEPES-buffer only (G: overview, H: detail of boxed area in G). Scale bars equal 10 µm (A), 25 µm (C, F, and H), 50 µm (B and D) and 100 µm (E and G).

Classification of congenital anomalies of the upper limb.

Six hundred and ninety-four patients with 993 anomalies of the upper limbs were classified according to the classification of Swanson et al. (1983). The data from these patients were compared with previous studies, and similar discrepancies were found. One explanation for these discrepancies is a lack of uniformity in the classification of Swanson et al., which may be caused by out-dated knowledge of the pathogenesis of congenital limb anomalies. Therefore, it seems necessary to describe the anomalies instead of the diagnoses. A descriptive method is being validated in our outpatient department that records all anomalies of the upper limb.

Simultaneous induction of apoptosis, collagen type I expression and mineralization in the developing coronal suture following FGF4 and FGF2 application.

This study aimed to evaluate the disturbances in normal coronal suture development resulting in craniosynostosis, a congenital disorder in which the calvarial sutures close prematurely. Craniosynostosis syndromes can be caused by mutations in the genes encoding for the fibroblast growth factor receptors (FGFRs) 1, 2, and 3. These gain-of-function mutations cause the transcribed receptor to be constitutively activated. To mimic this genetic defect, fibroblast growth factor (FGF) 2 or 4 was administered near the developing coronal suture in normal mouse embryos through ex utero surgery. The effect on apoptosis and bone differentiation, as collagen type I expression and mineralization, within the FGF-exposed coronal suture was investigated through (immuno)histochemical staining. An increase in the number of apoptotic cells together with ectopic collagen type I expression within the suture and accelerated mineralization followed FGF application. Macroscopically, this presented as a synostotic coronal suture. These results suggest that both apoptosis and differentiation are two processes that are simultaneously implicated in synostosis of the coronal suture in case of a FGFR-related craniosynostosis.

Symmetry and morbidity assessment of unilateral complete cleft lip nose corrected with or without primary nasal correction.

OBJECTIVE: Unilateral complete cleft lip patients treated with or without a primary nasal correction at the time of cleft lip repair were compared to evaluate the relevance of early surgical correction of the nose by using two assessments: nasal symmetry and morbidity. DESIGN, SETTING, PATIENTS: The no nasal correction group (NNC, n = 19) was operated by surgeon A using the Millard technique. The primary nasal correction group (PNC, n = 9) was operated by surgeon B combining the modified Millard technique with a columellar lift and alar mobilization. Symmetry was assessed on two sets of standardized photographs at 9 years of age using a computer-assisted analysis. Both cleft groups were compared with normal controls (NC, n = 20). The computer method included area and angular measurements. Morbidity was assessed by the number of procedures on the vermilion, the lip, and/ or nose for revisional surgery up to the age of 9 (NNC, n = 26; PNC, n = 12). RESULTS: No significant differences in symmetry were found between the NNC and PNC groups regarding the area and angular measurements. With regard to the area measurements, both cleft groups produced a significant asymmetry when compared to the NC group. Concerning the angular measurements, however, the NNC group differed significantly from the NC group, whereas such a difference could not be noted between the PNC group and NC group. With respect to morbidity, no revisional procedures were performed in the PNC group. The number of revisional procedures in the NNC group was 16 in 10 patients. CONCLUSION: Results are presented that favor, up to the age of 9 years, a primary nasal correction at the time of cleft lip repair.

Full monosomy 21, prenatally diagnosed by fluorescent in situ hybridization.

We describe a case of full monosomy 21 which was prenatally diagnosed in chorionic villi by fluorescent in situ hybridization (FISH). Because of intrauterine fetal death, a curettage was performed and cytogenetic analysis of skin fibroblasts confirmed the presence of monosomy 21 in fetal cells. DNA investigations showed a paternal origin of the single chromosome 21. Inspection and autopsy of the fetus revealed several congenital malformations. Some of them have been reported in earlier studies of monosomy 21; others concern new observations. Regarding the eye, the following abnormalities were microscopically observed: absence of the anterior and posterior eye chambers, aniridy, a hypoplastic ciliary body, Peter's anomaly, and a double retina with secondary dysplasia. In addition, malformations of the extremities were seen: partial, proximal syndactyly of digits 3 and 4 of the right hand; pes varus position of the right foot; and transverse reduction defect at the tarsals of the left foot. To our knowledge, this is the first case in which full monosomy 21 has been proven.

Maternoembryonic transfusion and congenital malformations.

There is an increasing number of reports relating chorionic villus sampling (CVS) to transverse limb reduction defects or the oromandibular limb hypogenesis complex. In addition, a correlation has been established between the severity of the defect and the gestational age when CVS is performed. Several hypotheses have been proposed for the increased incidence of congenital malformations after CVS including vascular disruption. Recently, it has been suggested that maternoembryonic transfusion can occur after CVS and that this can lead to a local antibody-mediated reaction, followed by local pathogenetic cell degeneration, i.e., apoptotic cell death, due to vascular disruption. This increased apoptotic cell death will ultimately result in congenital malformations. This paper describes an experimental model that can explain the pathogenesis of congenital malformations after CVS. The model designed uses a whole rat embryo culture technique and intracardiac injection of antisera, mimicking transplacental transfusion after CVS. Injection of antibodies directed against blood group antigens is capable of inducing increased apoptotic cell death. Immunological staining gives evidence of involvement of antibody-mediated reactions in the occurrence of apoptotic cell death. The dorsal aortae in 10-day-old rat embryos of 10-somite stages of development consist of a continuous endothelial cell layer. The effect of intracardiac injection of antisera on the dorsal aortae is only transient. Smaller vessels such as the pharyngeal arch arteries or arteries of the limbs still have fenestrated endothelium and are therefore more vulnerable to the pathogenetic effect of the reaction after transplacental transfusion causing vascular disruption. Development of the vascular pattern and differentiation of the vascular wall reduce the risk of severe malformations later on in pregnancy, although the risk of malformations remains throughout pregnancy. Thus, intracardiac injection of antisera simulating maternoembryonic transfusion such as after CVS can lead to an antibody-mediated reaction with vascular disruption early in pregnancy inducing apoptotic cell death. Increased cell death may ultimately result in congenital malformations, such as transverse limb defects or the oromandibular limb hypogenesis complex.

Chorionic villus sampling and end-artery disruption of the fetus.

A fetus with transverse limb reduction defects and jejunal atresia after exposure to chorionic villus sampling (CVS) at 9 weeks of amenorrhoea is described. A theory involving disruption of end-arteries due to the CVS procedure is suggested.

First-trimester diagnosis of Blomstrand lethal osteochondrodysplasia.

Blomstrand chondrodysplasia is a rare lethal skeletal dysplasia with presumed autosomal-recessive inheritance. A family with 2 affected fetuses was studied. One fetus demonstrated a severe skeletal dysplasia at routine transabdominal ultrasound examination at 18.5 weeks of gestation. The pregnancy was terminated and the diagnosis of Blomstrand chondrodysplasia was made at autopsy. A second affected fetus was identified by first-trimester transvaginal ultrasound at 12 weeks of gestation. In this case the diagnosis was confirmed by posttermination radiography and histopathology. From these observations, Blomstrand chondrodysplasia seems like a lethal rhizo/mesomelic short-limb, early-onset dysplasia with autosomal-recessive inheritance. Easy detectability by transvaginal ultrasound is demonstrated, but general applicability awaits further studies on the intra- and interfamilial variability of this disorder.

Apoptotic cell death during normal embryogenesis of the coronal suture: early detection of apoptosis in mice using annexin V.

Regulation of programmed cell death (apoptosis) is crucial for normal development and growth, both prenatally and postnatally. If its role during normal embryogenesis of a given structure is established, a number of related congenital disorders can be explained by a (local) deregulation of apoptosis. In this study, apoptotic cell death patterns during normal development of the murine coronal suture were investigated. Detection of apoptotic cells was undertaken by labeling with Annexin V. Results showed apoptosis occurring at the same time and place as suture initiation. Apoptotic cells are located along the entire established part of the suture and its developing part. Because apoptosis is shown to be highly associated with sutural genesis, the theory of craniosynostosis being the equivalent of deregulation at this locus seems in line with these findings.

The role of bone centers in the pathogenesis of craniosynostosis: an embryologic approach using CT measurements in isolated craniosynostosis and Apert and Crouzon syndromes.

This paper describes the role of the displacement of bone centers, i.e., the tubers, in the pathogenesis of craniosynostosis. This displacement was studied in 54 patients with isolated or syndromic craniosynostosis in the form of CT scans as well as in two dry neonate skulls with Apert syndrome. For comparison, 49 fetal and 8 normal infant dry skulls were studied. Our investigation was restricted to the coronal and metopic sutures. The results showed a significantly more occipital localization of the frontal bone center and a more frontal localization of the parietal bone center at the side of a synostotic coronal suture in the isolated form as well as in Apert syndrome. In contrast, this was not the case in Crouzon syndrome, thus showing that these two syndromes have a different pathogenesis. For trigonocephaly, a more anteromedial localization of the frontal bone centers was found.

Embryonic development of the ventral body wall and its congenital malformations.

Congenital abdominal wall defects, frequently associated with other anomalies, are found in many forms. Consequently, there is still controversy in the literature concerning nomenclature, classification, and pathogenesis. Recently, we proposed a new nomenclature and classification of abdominal wall defects based on the early development of the umbilical cord and of the ventral body wall. According to this classification the complete spectrum of abdominal wall defects, including cloacal exstrophy, bladder exstrophy, and epispadias, can be subdivided into four types: primary (thoraco-)abdominoschisis, omphalocele, body wall dysplasia, and secondary (thoraco-)abdominoschisis. Each type is characterized by its specific configuration of the placenta, the membranes, the umbilical cord, and the fetus. Anomalies such as urachal remnants and omphalomesenteric duct malformations can be explained by disturbances during later stages of umbilical cord development.

Chorionic villus sampling and materno-fetal transfusions: an immunological pathogenesis of vascular disruptive syndromes?

Experimental materno-embryonic transfusions with serum that is immunologically active against blood group antigens cause congenital malformations in the rat embryo. In view of the possible increased incidence of vascular disruptive syndromes after chorionic villus sampling (CVS), we investigated the occurrence of materno-fetal transfusions (MFTs) in this procedure. In 18 pregnant women experiencing two needle introductions at CVS, we looked immunohistochemically at the presence of haemoglobin A1-containing maternal erythrocytes in the fetal circulation of the separately collected first and second chorionic villus samples. In 4 of 18 patients (22 per cent), a significant increase of maternal cells was observed in the second sample compared with the first sample, indicating the occurrence of MFT by CVS. On the rare occasion of maternal immunization against fetal antigens, a CVS-associated MFT might provoke immunological damage to the fetus.

Median cleft of the lower lip and mandible: case reports, a new embryologic hypothesis, and subdivision.

Median clefts of the lower lip and mandible are rare. In the literature so far, about 62 cases have been described. In addition, three more patients are presented here. These cases show a broad variation in the severity of this deformity, ranging from a simple notch in the vermillion to a complete cleft of the lip involving the tongue, the chin, the mandible, the supporting structures of the median of the neck, and the manubrium sterni. Several hypotheses concerning the pathogenesis of median clefts of the lip and mandible have been proposed. Most authors consider it to be a failure of fusion of the first pair of branchial arches or failure of mesodermal penetration into the midline. From our embryologic point of view, however, instead of paired branchial arches, only one first branchial arch develops during the early embryonic period (< or = 17 mm crown-rump length). Within this first branchial arch, two mandibular processes grow out, separated by a groove in the median. These mandibular processes do not fuse but merge during the late embryonic period (> or = 17 mm to < or = 60 mm crown-rump length). In the same developmental period, there is formation of the lip and the alveolar process and the anlage and outgrowth of one membrane bone center in each mandibular process, resulting in the formation of the mandible with its symphysis. As a consequence of the preceding, we propose the following subdivision of the median clefts of the lip and/or mandible: Hypoplasia of the mandibular processes during the early embryonic period will lead to the severest cleft of the mandible extending into the neck. During the late embryonic period, the less severe median clefts will develop. Disturbances of the outgrowth of bone centers of the mandible, resulting in nonformation of its symphysis, cause clefting of the mandible with involvement of all related soft tissues. Defects in the merging process produce just a notch of the vermilion or a higher cleft of the lower lip with or without involvement of the alveolar process of the mandible. In conclusion, the variety of the clefts in the median of the lower lip and/or mandible as well as the low rate of incidence can be explained by the embryologic hypothesis proposed here.

Intracranial teratoma with multiple fetuses: pre- and post-natal appearance.

An intracranial teratoma in which six distinct dysmorphic fetuses were included was studied at autopsy. Karyotopic studies showed a normal chromosomal number in the child and the same karyotype in the three tumoral parts were examined. This is the second tumor of this type reported.

The genetic background of craniosynostosis syndromes.

Craniosynostosis syndromes are developmental disorders that cause an abnormal shape of the skull due to the premature fusion of cranial sutures. Enormous progress has been made recently in understanding the genetic background of these disorders and a classification of syndromes on a genetic basis is beginning to emerge. Members of at least three gene families that play an important role in vertebrate development are associated with different craniosynostosis syndromes. Here we review the genetic aspects of this fast-moving field.

The arterial pattern at the base of arhinencephalic and holoprosencephalic brains.

The mechanisms by which the anatomical variations of the circle of Willis develop is considered to be related to haemodynamic factors, i.e. the differential growth of the various parts of the brain will continuously change the haemodynamic demands and consequently the flow patterns in the cerebral arteries. It is therefore to be expected that, if a selected part of the brain does not develop, the change in the haemodynamic demand will affect the development of some cerebral arteries. Consequently the arteries at the base of 2 arhinencephalic and 8 holoprosencephalic brains were studied in conjunction with the brain malformations. The defects of holoprosencephaly are believed to arise from a failure of the prosencephalon to separate fully into the telencephalon and diencephalon and become manifest at the time that the prosencephalon normally starts to separate into the hemispheres, i.e. 28-34 d p.c. Arhinencephalic brains are fully diverticulated. There is only a partial or complete agenesis of the olfactory tracts and bulbs. The defect causing arhinencephaly starts at 43 d p.c. In the arhinencephalic brains no particular vascular abnormalities were found. However, at the base of the holoprosencephalic brains no complete circle of Willis was present; the anterior part was lacking and was replaced by anterior branches which emerged unilaterally or bilaterally from the internal carotid artery. The choroidal arteries were of very large calibre and ran to the highly vascularised wall of the dorsal cyst which is usually present in holoprosencephalic brains. In contrast to the anterior part, the posterior arterial pattern was almost identical to the posterior part of the circle of Willis of normal brains. The basic vascular patterns found in the holoprosencephalic brains displayed the features of Padget's developmental stages 2 and 3 of the cerebral vasculature, i.e. the pattern that has normally developed within 28-40 d p.c. The further modification of this pattern could largely be understood from the functional demand imposed on the circulation by the enlarged anterior choroidal arteries. Because the development of the anterior part of the circle of Willis precedes the developmental derangement causing arhinencephaly, a complete circle was found in these brains.

Embryonic development of the mammalian caudal neural tube.

In the literature, some controversy still exists about the embryonic developmental processes involved in the formation of the caudal neural tube. Therefore, a three-dimensional and histological study concerning the normal development of the caudal neural tube was performed on both mouse and human embryos. Three developmental processes can be distinguished in caudal neural tube development: caudal neuropore closure, secondary neurulation, and degeneration and differentiation of the secondary neural tube. Caudal neuropore closure occurs at the level of somite 32-34 in both species. Therefore, primary neurulation leads to the formation of all spinal cord segments and ganglia. Secondary neurulation involves cell deposition from a cluster of neurectodermal cells at the caudal end of the closed neural tube, directly around a lumen, the lumen always in contact with the lumen of the primary neural tube. This process leads only to the formation of the primordia of the filum terminale and ventriculus terminalis and, possibly, part of the conus medullaris. Secondary neurulation is followed by a period characterized by degeneration and differentiation of the secondary neural tube. Its lumen and neural tissue will disappear, whereas part of the secondary neurectodermal cells differentiate to a fibrous layer comparable and continuous with the marginal layer of the primary neural tube. This fibrous layer represents the future filum terminale. The embryological processes indicated above can be helpful in the interpretation of congenital anomalies affecting the caudal spinal cord and spine.