Fetal phenotypes in otopalatodigital spectrum disorders.

Otopalatodigital spectrum disorders (OPDSD) include OPD syndromes types 1 and type 2 (OPD1, OPD2), Melnick-Needles syndrome (MNS), and frontometaphyseal dysplasia (FMD). These conditions are clinically characterized by variable skeletal dysplasia associated in males, with extra-skeletal features including brain malformations, cleft palate, cardiac anomalies, omphalocele and obstructive uropathy. Mutations in the FLNA gene have been reported in most FMD and OPD2 cases and in all instances of typical OPD1 and MNS. Here, we report a series of 10 fetuses and a neonatally deceased newborn displaying a multiple congenital anomalies syndrome suggestive of OPDSD and in whom we performed FLNA analysis. We found a global mutation rate of 44%. This series allows expanding the clinical and FLNA mutational spectrum in OPDSD. However, we emphasize difficulties to correctly discriminate OPDSD based on clinical criteria in fetuses due to the major overlap between these conditions. Molecular analyses may help pathologists to refine clinical diagnosis according to the type and the location of FLNA mutations. Discriminating the type of OPDSD is of importance in order to improve the genetic counseling to provide to families.

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.

Maternal transmission of interstitial 8p23.1 deletion detected during prenatal diagnosis.

We report on the first prenatally diagnosed interstitial 8p23.1 maternally inherited deletion. At 20 weeks of gestation (WG) the fetus was diagnosed with a complete atrioventricular canal. In infancy, the mother underwent a two-step cardiac surgery for an interrupted aortic arch type A associated to an inlet ventricular septal defect (VSD). A straddling of the tricuspid valve type B was confirmed during surgery. The outcome showed no cardiac failure or conduction anomalies. However, she presented with moderate intellectual disability. Classical and molecular cytogenetic studies on amniotic and maternal lymphocytes cells showed a nearly identical interstitial deletion of the 8p23.1 region encompassing the GATA4 gene locus (Mother: nt 6,913,337-12,580,828, fetus: nt 7,074,449-12,580,828) with no modification of the telomeric region. The relevance of our report is not only the maternal syndromic interstitial 8p23.1 deletion, but also maternal transmission which has never been reported before. The maternal and fetal phenotypes were not identical, however, even though they had the same cellular and molecular background: an alteration of the epithelial mesenchymal transition of the atrioventricular valvulo-septal complex where GATA4 plays a positive role in the regulation. We reviewed all cases of interstitial 8p23.1 deletions diagnosed either prenatally or postnatally.

Nucleus basalis magnocellularis and hippocampus are the major sites of FMR-1 expression in the human fetal brain.

The expression of the FMR-1 gene, which is implicated in fragile-X syndrome was investigated in human fetuses by in situ hybridization. In 8 and 9 week-old fetuses, FMR-1 mRNAs are expressed in proliferating and migrating cells of the nervous system, in the retina, and in several non-nervous tissues. In the brain of 25 week-old fetuses, FMR-1 mRNAs are produced in all nearly differenciated structures, with the highest level in cholinergic neurons of the nucleus basalis magnocellularis and in pyramidal neurons of hippocampus. The early transcription of FMR-1 gene and the distribution of FMR-1 mRNAs in human fetuses suggest that alterations of FMR-1 gene expression may contribute to the pathogenesis of fragile-X syndrome and especially the mental retardation.

SLC25A12 expression is associated with neurite outgrowth and is upregulated in the prefrontal cortex of autistic subjects.

Autism is a neurodevelopmental disorder with a strong genetic component, probably involving several genes. Genome screens have provided evidence of linkage to chromosome 2q31-q33, which includes the SLC25A12 gene. Association between autism and single-nucleotide polymorphisms in SLC25A12 has been reported in various studies. SLC25A12 encodes the mitochondrial aspartate/glutamate carrier functionally important in neurons with high-metabolic activity. Neuropathological findings and functional abnormalities in autism have been reported for Brodmann's area (BA) 46 and the cerebellum. We found that SLC25A12 was expressed more strongly in the post-mortem brain tissues of autistic subjects than in those of controls, in the BA46 prefrontal cortex but not in cerebellar granule cells. SLC25A12 expression was not modified in brain subregions of bipolar and schizophrenic patients. SLC25A12 was expressed in developing human neuronal tissues, including neocortical regions containing excitatory neurons and neocortical progenitors and the ganglionic eminences that generate neocortical inhibitory interneurons. At mid-gestation, when gyri and sulci start to develop, SLC25A12 molecular gradients were identified in the lateral prefrontal and ventral temporal cortex. These fetal structures generate regions with abnormal activity in autism, including the dorsolateral prefrontal cortex (BA46), the pars opercularis of the inferior frontal cortex and the fusiform gyrus. SLC25A12 overexpression or silencing in mouse embryonic cortical neurons also modified dendrite length and the mobility of dendritic mitochondria. Our findings suggest that SLC25A12 overexpression may be involved in the pathophysiology of autism, modifying neuronal networks in specific subregions, such as the dorsolateral prefrontal cortex and fusiform gyrus, at both pre- and postnatal stages.

Phenotypic spectrum of CHARGE syndrome in fetuses with CHD7 truncating mutations correlates with expression during human development.

BACKGROUND: The acronym CHARGE refers to a non-random cluster of malformations including coloboma, heart malformation, choanal atresia, retardation of growth and/or development, genital anomalies, and ear anomalies. This set of multiple congenital anomalies is frequent, despite rare patients with normal intelligence, and prognosis remains poor. Recently, CHD7 gene mutations have been identified in CHARGE patients; however, the function of CHD7 during development remains unknown. METHODS: We studied a series of 10 antenatal cases in whom the diagnosis of CHARGE syndrome was suspected, considering that a careful pathological description would shed light on the CHD7 function during development. CHD7 sequence analysis and in situ hybridisation were employed. RESULTS: The diagnosis of CHARGE syndrome was confirmed in all 10 fetuses by the identification of a CHD7 heterozygous truncating mutation. Interestingly, arhinencephaly and semi-circular canal agenesis were two constant features which are not included in formal diagnostic criteria so far. In situ hybridisation analysis of the CHD7 gene during early human development emphasised the role of CHD7 in the development of the central nervous system, internal ear, and neural crest of pharyngeal arches, and more generally showed a good correlation between specific CHD7 expression pattern and the developmental anomalies observed in CHARGE syndrome. CONCLUSIONS: These results allowed us to further refine the phenotypic spectrum of developmental anomalies resulting from CHD7 dysfunction.

C21orf5, a human candidate gene for brain abnormalities and mental retardation in Down syndrome.

Mental retardation represents the more invalidating pathological aspect of trisomy 21 and has a hard impact on public health. The dosage imbalance of chromosome 21 genes could be the cause of neurological alterations and mental retardation seen in Down syndrome. We studied C21orf5 that we have demonstrated to be overexpressed in Down syndrome tissues, as a candidate gene for trisomy 21. A new optical technology (Rachidi et al., 2000) was used to compare signal intensity and cell density in presumptive embryonic brain compartments, at their boundaries and in higher specialized brain centres during fetal lifespan. We showed a developmentally regulated transcriptional activity of C21orf5 and a regional and cellular specific distribution of gene transcripts during human embryonic and fetal development. A wide but differential expression was detected in the nervous system during embryogenesis with a relatively lower level in the forebrain than in the midbrain and hindbrain and the highest transcription intensity in the future cerebellum. This developmentally regulated expression is maintained during post-embryogenesis and evolves selectively in fetal cerebral, hippocampal and cerebellar areas. Differential and cellular specificity were detected in hippocampus with higher C21orf5 mRNA level in the pyramidal cells compared to granular cells of the dentate gyrus. The expression pattern detected in cortical and cerebellar structures correlates well to the altered cortical lamination and to the lower size of the cerebellum observed in Down syndrome patients. In addition, the patterned differential expression detected in the medial temporal-lobe system, including hippocampal formation and perirhinal cortex, working as control centres of the memory circuits and involved in cognitive processes and memory storage, also corresponds to abnormal brain regions seen in Down syndrome patients. The C21orf5 selective expression in the key brain structures for learning and memory suggests that C21orf5 overexpression could participate in mental retardation pathogenesis in Down syndrome patients.

Developmentally regulated expression of the murine ortholog of the potassium channel KIR4.2 (KCNJ15).

The gene KIR4.2 (K(+) inwardly rectifying channel 4.2) has been recently identified in the Down syndrome Chromosome Region 1. We have cloned the mouse ortholog of KIR4.2 and characterized its expression pattern. In situ hybridization showed a restricted and developmentally regulated pattern of expression. The expression is starting at E12.5 and expands at E14.5 in different tissues and organs, which may be affected in Down syndrome: heart, thymus, thyroid gland, and perichondrium. At E17.5, additional epithelia (kidney, bladder, stomach, lung) expressed also strongly the gene.

Regional and cellular specificity of the expression of TPRD, the tetratricopeptide Down syndrome gene, during human embryonic development.

The TPRD gene (tetratricopeptide (TPR) containing Down syndrome gene) is one of the candidate genes in the Down syndrome chromosomal region-1. Duplication of this gene may be the cause of major phenotypic features of Down syndrome. Here we show that the TPRD expression is developmentally regulated during human embryogenesis. At the earliest stages of development (Carnegie 8-12) TPRD expression is ubiquitous. At later developmental stages (Carnegie stages 14, 16 and 18), it becomes restricted to the nervous system, as is the case for the mtprd gene during mouse development. We extended our analysis of TPRD expression during fetal development of the human nervous system (13, 22 and 24 weeks). A new oblique illumination technique was used to compare signal intensity and cell density. Some regions of the nervous system such as the external cortical layers of the brain, and the inner neuroblastic layer of the eye, strongly express the TPRD gene.

Spatio-temporal expression of FGFR 1, 2 and 3 genes during human embryo-fetal ossification.

Mutations in FGFR 1-3 genes account for various human craniosynostosis syndromes, while dwarfism syndromes have been ascribed exclusively to FGFR 3 mutations. However, the exact role of FGFR 1-3 genes in human skeletal development is not understood. Here we describe the expression pattern of FGFR 1-3 genes during human embryonic and fetal endochondral and membranous ossification. In the limb bud, FGFR 1 and FGFR 2 are initially expressed in the mesenchyme and in epidermal cells, respectively, but FGFR 3 is undetectable. At later stages, FGFR 2 appears as the first marker of prechondrogenic condensations. In the growing long bones, FGFR 1 and FGFR 2 transcripts are restricted to the perichondrium and periosteum, while FGFR 3 is mainly expressed in mature chondrocytes of the cartilage growth plate. Marked FGFR 2 expression is also observed in the periarticular cartilage. Finally, membranous ossification of the skull vault is characterized by co-expression of the FGFR 1-3 genes in preosteoblasts and osteoblasts. In summary, the simultaneous expression of FGFR 1-3 genes in cranial sutures might explain their involvement in craniosynostosis syndromes, whereas the specific expression of FGFR 3 in chondrocytes does correlate with the involvement of FGFR 3 mutations in inherited defective growth of human long bones.

[Ductus venosus agenesis]. / Agénésie du canal d'Arantius.

OBJECTIVE: Congenital absence of the ductus venosus is a rare anomaly in the fetus. The aim of our study was to evaluate the clinical and ultrasonographic features and outcome of the fetuses with ductus venosus agenesis. STUDY DESIGN: We describe 12 cases in the period between 1992 and 2004. The umbilical vein drained either into the right atrium directly (2 cases) or by the coronary sinus (1 case), or in the inferior vena cava (5 cases), or in the azygos vein (1 case), or in the portal vein (3 cases). Our data where analyzed with the cases published in the literature. Two groups of anastomoses where defined on the basis of the hemodynamic consequences: the group of extrahepatic anastomoses (53 cases) and the group of intrahepatic anastomoses (22 cases). RESULTS: In the group of extra hepatic anastomoses, cardiomegaly was the most common antenatal finding (39%), while in the intra hepatic group hydrops fetalis occurred most frequently (23%). Malformation rate was high in both groups (56% and 45%) and chromosomal anomalies where present in 9% of cases. CONCLUSION: Careful assessment of the umbilical venous return and the ductus venosus should be a part of examination of every fetus with cardiomegaly, polyhydramnios, ascites or hydrops. In case of absence of the ductus venosus a referral scan, a fetal echocardiography and a karyotype should be performed.

EXT 1 gene mutation induces chondrocyte cytoskeletal abnormalities and defective collagen expression in the exostoses.

Hereditary multiple exostoses (HME), an autosomal skeletal disorder characterized by cartilage-capped excrescences, has been ascribed to mutations in EXT 1 and EXT 2, two tumor suppressor-related genes encoding glycosyltransferases involved in the heparan sulfate proteoglycan (HSPG) biosynthesis. Taking advantage of the availability of three different exostoses from a patient with HME harboring a premature termination codon in the EXT 1 gene, morphological, immunologic, and biochemical analyses of the samples were carried out. The cartilaginous exostosis, when compared with control cartilage, exhibited alterations in the distribution and morphology of chondrocytes with abundant bundles of actin filaments indicative of cytoskeletal defects. Chondrocytes in the exostosis were surrounded by an extracellular matrix containing abnormally high amounts of collagen type X. The unexpected presence of collagen type I unevenly distributed in the cartilage matrix further suggested that some of the hypertrophic chondrocytes detected in the cartilaginous caps of the exostoses underwent accelerated differentiation. The two mineralized exostoses presented lamellar bone arrangement undergoing intense remodeling as evidenced by the presence of numerous reversal lines. The increased electrophoretic mobility of chondroitin sulfate and dermatan sulfate proteoglycans (PGs) extracted from the two bony exostoses was ascribed to an absence of the decorin core protein. Altogether, these data indicate that EXT mutations might induce a defective endochondral ossification process in exostoses by altering actin distribution and chondrocyte differentiation and by promoting primary calcification through decorin removal.

Absence of functional type 1 parathyroid hormone (PTH)/PTH-related protein receptors in humans is associated with abnormal breast development and tooth impaction.

Recent studies in transgenic mice have demonstrated that PTH-related protein (PTHrP), signaling through the type 1 PTH/PTHrP receptor (PTHR1), regulates endochondral bone development and epithelial-mesenchymal interactions during the formation of the mammary glands and teeth. Recently, it has been shown that loss-of-function mutations in the PTHR1 gene result in a rare, lethal form of dwarfism known as Blomstrand chondrodysplasia. These patients suffer from severe defects in endochondral bone formation, but abnormalities in breast and tooth development have not been reported. To ascertain whether PTHrP signaling was important to human breast and tooth development, we studied two fetuses with Blomstrand chondrodysplasia. These fetuses lack nipples and breasts. Developing teeth were present, but they were severely impacted within the surrounding alveolar bone, leading to distortions in their architecture and orientation. Compatible with the involvement of PTHR1 and PTHrP in human breast and tooth morphogenesis, both were expressed within the developing breasts and teeth of normal human fetuses. Therefore, impairment of the PTHrP/PTHR1 signaling pathway in humans is associated with severe abnormalities in tooth and breast development. In addition to regulating human bone formation, this signaling pathway is also necessary for the normal development of the human breast and tooth.

Calpain3 expression during human cardiogenesis.

Transcripts of calpain3, the gene involved in limb girdle muscular dystrophy type 2A, appear in organs other than the skeletal muscle during human development, the first of which being the early embryonic heart. We examined more precisely the spatio-temporal transcription pattern of calpain3 during human cardiogenesis and the appearance of its protein in fetal tissues, and correlated it to titin expression. Different events of the heart's maturation can be recognized: (i) the presence of titin RNA or protein constitute very precocious developmental cardiac markers appearing before the fusion of the two lateral endocardial tubes; (ii) the disappearance of calpain3 RNA from the ventricular compartment later in the embryonic heart. Finally, although calpain3 transcripts are present in the heart, the corresponding protein is not detected elsewhere than in skeletal muscle.

Familial Blomstrand chondrodysplasia with advanced skeletal maturation: further delineation.

We report on two sibs with a rare lethal chondrodysplasia born to a non-consanguineous couple. The hallmarks of this affection, also called Blomstrand chondrodysplasia, are short limbs, polyhydramnios, hydrops fetalis, facial anomalies, increased bone density, and a remarkable advance in skeletal maturation. We describe the radiologic and pathologic manifestations in these two cases. This recurrence affecting a male and a female fetus, born to the same couple, suggests autosomal recessive inheritance.

Congenital hypothalamic hamartoma syndrome: nosological discussion and minimum diagnostic criteria of a possibly familial form.

We report on congenital hypothalamic hamartomas, discovered at autopsy in 3 unrelated fetuses. In the first 2 patients, the tumor was associated with skeletal dysplasia only. In the third patient, it was part of a non-random congenital malformation association, suggestive of Meckel syndrome. In one family, a previous boy died soon after birth with similar craniofacial and skeletal abnormalities. As far as we know, the association between isolated skeletal dysplasia and congenital hypothalamic hamartomas has not yet been documented in the literature. Nevertheless, a spectrum of skeletal abnormalities has been described in association with congenital hypothalamic "hamartoblastoma" and a constellation of variable visceral malformations under the eponym of "Pallister-Hall syndrome" (PHS). A detailed analysis of the PHS reported cases shows that only skeletal dysplasia and oro-facial abnormalities are present constantly. They show similarities with those found in our first 2 cases. These findings prompt us to consider skeletal dysplasia and oro-facial abnormalities as common denominator and minimum criteria required to define a nosologically distinct, possibly familial entity, which we suggest calling "congenital hypothalamic hamartoma syndrome" (CHHS).

Clinical homogeneity of the Stüve-Wiedemann syndrome and overlap with the Schwartz-Jampel syndrome type 2.

The Stüve-Wiedemann syndrome (SWS) is a rare disorder characterized by respiratory distress, hyperthermic episodes, and early lethality and radiologically by bowing of the long bones with internal cortical thickening and large metaphyses. We report findings in 8 new patients suggesting that this syndrome is clinically homogeneous. All patients had feeding and swallowing difficulties, respiratory insufficiency, abnormal appearance, muscle hypotonia, and postnatal short stature. Recurrent episodes of unexplained fever occurred in all and were the cause of death in 6 of 8 cases. Parental consanguinity and sib recurrence suggest autosomal recessive inheritance. The clinical, radiological, and histological similarities between our patients with SWS and those with the recently delineated "neonatal" Schwartz-Jampel syndrome (SJS type 2) lead us to suggest that SWS and SJS type 2 may be a single entity.