DEFECTS OF BLASTOGENESIS

Ever more frequent and closer involvement by clinical geneticists and counselors in the prenatal assessment of development mandates a better understanding of all stages of human ontogeny, but especially those of earliest development during which most of the lethal and all of the gross, multiple and complex defects of morphogenesis arise. Because of the phenomenon of universality, i.e., identical molecular inductive mechanisms involved in the process of embryonic patternformation in all vertebrates, experimental animals indeed are a most valuable approach to an understanding of the causal and formal aspects of development and are beginning to forge essential, strong bonds between molecular biologists and clinicians in a mutually supportive discipline of developmental biology.

However, to grieving parents of a stillborn fetus with, say, Pentalogy of Cantrell, sirenomelia or otocephaly, mouse data offer little comfort or reassurance about recurrence; thus, it is imperative to make ever more effective a science of human teratology (sensu lato) with participating reproductive geneticists, obstetricians, neonatologists, ultrasonographers, pediatric/fetal pathologists, cytogeneticists and pediatric geneticists to generate the diagnostic, pathogenetic and causal data necessary to counsel and to comfort the parents. Few molecular data exist on causes of blastogenetic defects in humans; however, the phenomenon of parsimony, whereby the same “morphogenetic” molecule, say, sonic hedgehog (SHH), is “deployed” simultaneously or sequentially during the morphogenesis (and even the histogenesis) of several/many embryonic primordia, makes it likely that a genetic/epigenetic disturbance of such an inductive system will have multiple effects on blastogenetic, organogenetic and perhaps also histogenetic events in the embryo. If causally defined, such a pattern of anomalies constitutes pleiotropy, and the embryo/fetus can be said to have a syndrome. If cause is unknown, the presumption of pleiotropy is less certain, and the fetus/infant may be said to have an “association” with low empiric recurrence risk.

NEUROPATHOLOGY OF FAMILIAL SCHIZENCEPHALY

W. Squier

Radcliffe Infirmary, Oxford

M. Rutherford and F. Cowan

Imperial College School of Medicine, Hammersmith Hospital London

Schizencephaly is defined as a full thickness cleft in the brain wall which may be malformative or destructive in nature. Rare familial and syndromic cases are described: mutations in the EMX2 gene have been implicated.

A 24 week gestation male infant died shortly after birth. Cranial ultrasound imaging suggested bilateral schizencephaly. At post-mortem the brain had multiple bilateral defects in middle cerebral artery territory. The cortex was replaced by a membrane. Histology showed brain destruction, dense calcification and widespread reactive changes in the ventricular lining including gliosis and haemosiderin laden macrophages. Heterotopic neuronal nodules were seen adjacent to the defects and in their lining. No viral inclusions were identified. The findings are characteristic of haemorrhagic infarction occurring before 28 weeks of gestation.

The mother has communication difficulties. Two older female children initially appeared normal. The younger had IUGR at term presented by 6 months with left hemiplegia and global developmental delay. Brain MR imaging showed bilateral schizencephalic (narrow, open on the right, closed on the left) clefts at the junction of the frontal, parietal and temporal lobes and abnormal signal in the right internal capsule. The father has idiopathic cataracts.

This family provides further evidence for vascular or ischaemic damage underlying some familial brain malformations. Genetically determined thrombophilic variants e.g. Factor V Leiden mutations, described in some infants with perinatal stroke, are being sought.

We wish to thank Dr. V. Rudran for referring the older sibling and Dr. A King for the postmortem.

ACROCALLOSAL SYNDROME : 2 OBSERVATIONS WITH DETAILED NEUROPATHOLOGICAL DATA AND REVIEW OF THE LITTERATURE

C. Fernandez and C. Fallet-Bianco

Service d'Anatomie Pathologique Centre Hospitalier Sainte-Anne Paris 75014 France

M. Soulier, A. Liprandi and D. Figarella-Branger

Service d'Anatomie Pathologique Hôpital de la Timone Marseille 13000 France

We report 2 cases of acrocallosal syndrome observed in 2 male fetuses of 33- and 25 –gestational weeks. The first one showed a dolichocephaly and a broad nasal bridge. His left hand displayed a broad thumb and a 4–5 syndactyly. The second fetus had also craniofacial and extremities abnormalities, with bilateral syndactyly of the 4^th and 5^th fingers and preaxial polydactyly of the left foot. Moreover, an extra-bone was present within the anterior fontanelle. In both cases, an agenesis of the corpus callosum was associated with one or several interhemispheric cysts. In front of the cysts the cerebral cortex showed a nodular aspect. Brain stem and cerebellum were hypoplastic. Microscopic analysis revealed that the nodular masses corresponded to large dysplastic areas in which the normal cortical plate was absent whereas numerous clusters of immature neurons were scattered in the white matter. The cysts wall was lined by ependymal cells and choroids plexus suggesting a developmental abnormality of the ventricles. In the hypoplastic cerebellum the dentate nuclei were fragmented in both cases. In one case, numerous neuronal heterotopia associated with ectopic ependymal cavities were observed in the vermis. The pons was hypoplastic with nearly complete lack of pontine nuclei. Inferior olivary nuclei were severely dysplastic.

These cases represent the two first reports of acrocallosal syndrome in fetus with complete neuropathological study. The relationship between the acrocallosal syndrome and the Greig cephalosyndactyly syndrome associated with GLI 3 gene mutations will be discussed.

NEONATAL CASE OF CONGENITAL DEFECT OF GLYCOSYLATION (CDG) TYPE IA

V. Cusí and J. Vila

Pathology S. Hospital Universitari Sant Joan de Déu. Esplugues de Llobregat. Barcelona

B. Perez-Dueñas and J. CampistoL

Neurology S. Hospital Universitari Sant Joan de Déu. Esplugues de Llobregat. Barcelona

M.A. Vilaseca

Biochemical S. Hospital Universitari Sant Joan de Déu. Esplugues de Llobregat. Barcelona

I. Lizarraga

Neonatology S. Hospital Universitari Sant Joan de Déu. Esplugues de Llobregat. Barcelona

Aim: We present a case with neonatal manifestations of a CDG type Ia.

Case report: Newborn female weight 3000 g, presenting from birth failure to thrive, hepatic failure, protein-loosing enteropathy, axial hypotony, deaffness, anaemia and coagulopathy. Physical examination showed facial dysmorphy, inverted mamiles and lipodystrophy. She died 29 days after the birth. Post-mortem examination disclosed severe pericardial and pleural haematic effusion, hepatic fatty changes and bilateral nephromegaly with cystic tubular cortical changes. The brain showed small cerebellum and pons with severe neuronal loss at the cerebellar cortex and diffuse astrocytosis in the white and grey cerebellar matter and lack of myelinization of the transverse pontine fibers. The encephalon showed cortical changes of hypoxia-ischemia.

Discussion: The pontocerebellar atrophy is a congenital alteration of diverse aetiology. Among them we have to consider the Glycosylation defects. One of the most frequent is the phosphomannomutase deficiency that explains the 83% of the known cases of CDG. The genetic abnormality is linked to chromosome 16p13 and several mutations have been described. They can present with neurological symptoms or multisystemic form. Atrophy of the cerebellum is a constant finding in CDG type Ia. Enzymatic or molecular studies are necessary to the genetic counselling and prenatal diagnosis.

ELEVEN CASES OF PERINATAL SIALIC ACID STORAGE DISEASE

R. Froissart and Maire

Laboratoire de Biochime Pédiatrique, Hôpital Debrousse, Lyon, France

R. Bouvier

Laboratoire d'Anatomie Pathologique, Hôpital E. Herriot, Lyon, France

A. M. Beaufrère

Laboratoire d'Anatomie Pathologique, Hôtel-Dieu, Clermont Ferrand

Sialic acid storage disease (SSD) is a rare autosomal recessive disease. The disorder is due to the deficiency of the membrane transporter SLC17A5 or sialin, leading to the storage of free sialic acid within lysosomes. Clinical presentations are very heterogeneous ranging from in utero severe ascites to the milder Salla disease (common in Finland).

We have diagnosed 11 perinatal cases: 7 in utero and 4 in the neonatal period (for 3 of them, echographic signs were evocative of a storage disease). The most prominent ultrasound signs consisted of ascites discovered between 15 to 34 W.A. Foetopathological examination showed ascites and edema. Hepatomegaly, dysmorphy and dysostosis multiplex were often found in utero or progressively appeared after birth and increased with time. Histopathological examination of the placenta showed extensive vacuolization of syncytiotrophoblatic and Hofbauer cells.

In the fetus the storage was present in most organs but better seen in podocytes and highlighted by colloidal iron staining. Study of the sialin gene allowed to identify 14 different gene alterations including 6 novel. The identification of the mutations allows an early and reliable prenatal diagnosis in further pregnancies.

This series shows that the perinatal presentation of SSD is the most common one in France as we diagnosed only 1 Salla disease during the same period. The high frequency of the fetal presentation of lysosomal diseases involving sialic acid metabolism (sialidosis, galactosialidosis and SSD) suggest a critical importance of this compound during the fetal life.

(This series was collected thanks to the Société Franc¸aise de Foetopathologie).

PILI CANACLICULI : A FAMILIAL STUDY

Giordano G. and Gnetti l.

Department of Pathology and Laboratory Medicine, Section of Pathological Anatomy and Histology, University of Parma.

Zendri E. and Boccaletti V.

Department of Surgery, Section of Dermatology, University of Parma.

Neri T.

Department of Medicine, Section of Medical Genetic, University of Parma.

The pili canaliculi is a rare defect, first described in 1973 by Dupre’ et al. as “le syndrome des cheveux incoiffables” (1). The condition, characterized by dry, frizzy, unruly, rough and blond hair probably is due to a keratinization defect (2). The disorder may be autosomal dominant, recessive with variable levels of penetrance or sporadic (3).

Sometimes it is associated with ectodermal dysplasia (4,5), retinal dysplasia (1), dental anomalies (6), ichthyosis vulgaris (7), atopic eczema (8,15), progressive alopecia areata (3,15), hamartomas (9,15), lichen sclerosus (10), woolly hair nevus (11), brachydactyly (12), pili torti (13,15), digit and nail abnormalities (14,15). We report a three years old child with thin, blond, uncombable hairs, atopic dermatitis and onychoschizia. The maternal grandmother were said to have the same hairs in infancy. The mother had normal hairs. A study with scanning electron microscopy revealed the presence of typical triangular cross-sectional shapes with indentation, longitudinal grooving and flattening of the hair surfaces. These morphologic features were discontinuous and found on different surfaces along the length of the hair. The overlying cuticle of the hair was instead normal. These findings were present in many hairs of child and grandmother, but in few hairs of the mother. The repeating of this peculiar aspect in the hairs within this family suggests that inheritance pattern is autosomal dominant. Moreover, the characteristic hair morphology can be observed in relatives, without hair abnormality. Since, the disorder could be associated to other extracutaneous abnormalities, it is indispensable a follow-up of the affected patient.

REFERENCES

1. Dupre’ A, Rochiccioli P, Bonafe JL “Cheveaux incoiffables”: anomalie congenitale des cheveaux. Bull Soc Fr Dermatol Syphiligr. 1973;80:111–11.

2. Hicks J, Meltry DW, Barrish J, Levy M. Uncombable hair (cheveaux incoiffables, pili trianguli et canaliculi) Syndrome: brief review and role of scanning electron microscopy in diagnosis Ultrastruct Pathol. 2001; 25:99–103.

3. Hebert AA, Charrow J, Esterly NB; Fretzin DF.Uncombable hair (pili trianguli et canaliculi); evidence for dominant inheritance with complete penetrance with complete penetrance based on scanning electron microscopy. Am J Med Genet. 1987;28:185–193.

4. Shelley WB, Shelley ED.Uncombable hair syndrome:observation on response to biotin and occurrence in siblings with ectodermal dysplasia.J Am Acad Dermatol. 1985;13:97–102.

5. Stroud JP, Mehregan AH.“Spun glass hair”: a clinicopathologic study of an unusual hair defect. The first human symposium NewYork, Medcorn Press,1973:103–107.

6. Kuhn CA, Helm TN, Bergfeld WF, Mc Mahon JT.Acquired uncombable hair. Arch Dermatol. 1993; 129:1061–1062.

7. Rest QB, Fretzin DF, Quantitative assessment of scanning electron microscope defects in uncombable-hair syndrome.Pediatr Dermatol. 1990;7:93–96.

8. Ravella A, Puiol RM, Noguera X, de Moragas JM Localized pili canaliculi and trianguli J Am Acad Dermatol 1987;17:377–380.

9. Garty B, Metzker a, Mimouni M, Varsono I. Uncombable hair: a condition with autosomal domina nt inheritance. Arch Dis Child. 1982;57:710–712.

10. De Luna MM, Rubinson R, De Kohan ZB.Pili canaliculi : uncombable hair syndrome in a family with apparent autosomal dominant inheritance. Pediatr Dermatol. 1985;2:324–327.

11. Mc Cullum N, Sperling LC, Vidmar D: The uncombable hair syndrome. Cutis. 1990;46:479–483.

12. Bork K, Stender E, Schimdt D, et al. Familiare Kongenitale Hypotricose mit “unkammbaren haaren”, retina- pigmentblatt- dystrophie,juvenilen katarakt und brachymetakarpie:eine weitere entitat ausder gruppe der ektodermalen dysplasien.Hautarzt 1987,38:342–347.

13. Agular a, Sobrinto-Simoes MM, Finseth I, Johannessen JV, Nesland JM.Uncombable hair. Arch Dis Child. 1984; 59:92–93.

14. Rogers M. Hair shaft abnormalities. Part I Australas Dermatol. 1996; 37: 1–11.

15. Braun-Falco O. Ryckmanns F. Heilgemeir GP. Ring J. A syndrome : uncombable hair.Observations on 6 members of family with pili canaliculi associated with pili torti, progressive alopecia, atopic eczema and hamartomas. Hautarzt;1982;33:366–372.

GENETIC AND CYTOGENETIC ABNORMALITIES IN FETAL IUGR. MOSAICISMS, UPD AND ROLE OF SPECIFIC GENES IN “IDIOPATHIC” IUGR

Monica Miozzo, Francesca Grati, Barbara Cassani, Valeria Barresi, and Gaetano Bulfamante

Speaker: Valeria Barresi

The fetal intrauterine growth restriction (IUGR) is the final result of different etiologies and pathogenesis. In the past a great relevance was given to some maternal diseases or conditions (e.g. malnutrition, vascular/renal diseases, congenital or acquired thrombophilic disorders, drugs/life style) as causes of foetal growth abnormalities. More recently fetal genetic and cytogenetic defects have been highlighted as causes of IUGR or gigantism. In particular these diseases can be the result of fetal and placental chromosomal mosaicism or of abnormal expression of imprinted genes as it happens in the uniparental disomy (UPD).

In the last two years our group studied two series of IUGR pregnancies to verify the number of chromosomal mosaicisms or UPD (study n. 1) and the imprinting status of ESX1L (the human orthologous Esx1 gene, imprinted in the mouse) (study n. 2).

Briefly, the study n. 1 considered 12 pregnancies with unexplained (“idiopathic”) severe IUGR. We applied a combined cytogenetic/molecular approach improved by a new application of QF-PCR method to reveal and quantify mosaicisms involving a trisomic cell line. This technique allowed us to unmask and quantify the level of mosaicism even in cases with less than 5% of trisomic cells. The tissues sampling included parental peripheral blood lymphocytes, cord blood, umbilical cord fragment and four placental cotyledons. We observed three pregnancies with a mosaic trisomy for chromosomes 7, 2 and 14 respectively. The former cases presented also matUPD7, as results of a trisomic zygotic rescue. It is intriguing that the chromosomes 7, 2, 14 are known or suspected to harbour imprinted genes so that unbalanced gene dosage in a subset of cells during embryonic development could lead to an early impairment of placental function.

The study n. 2 analyzed 13 pregnancies carrying an “idiopathic” IUGR and 4 full term normal pregnancies. Since the ESX1L gene is located in X chromosome, we considered only IUGR pregnancies with a female fetus. The studied tissues included parental peripheral blood lymphocytes, cord blood, umbilical cord fragment and four placental cotyledons. The methodology of the research included cytogenetic analysis, X chromosome inactivation (XCI) assay, ESX1L expression study with RT-PCR and ESX1L methylation specific PCR (MSP). Our findings demonstrated that IUGR as well as normal full term placentas display XCI heterogeneity, thus indicating that IUGR phenotype is not correlated with preferential pattern of XCI in placentas. ESX1L was homogeneously expressed in IUGR and normal placentas and showed the same methylation pattern in those with random XCI, as well as in those with preferential X^P and X^M inactivation. Taken together these findings provide evidence that in human placenta ESX1L is not imprinted and there is not a parent-of-origin effect of chromosome X associated with placental function.

A MULTIMODAL APPROACH TO HUMAN BRAIN DEVELOPMENT

Lambot M-A, Noël J-C, Libert F, and Vanderhaeghen P

Despite numerous animal studies, many questions remain unanswered regarding the in-utero development of the human brain both molecularly and anatomically.

To address these issues, we developed a system based on whole brain cryosections which provides us not only with numerous “identical” sections allowing the simultaneous study of different gene or protein expression patterns but also allows us to reconstruct the three-dimensional anatomy of the brain as well.

First we used this method to study the expression of the ephrin gene family in the developing human brain. Eph receptors and ephrins are involved in some of the most important steps during the development of the central nervous system, including cell migration, axon guidance, topographic mapping and synapse formation. To achieve their role in the mapping of cortical areas, they are often expressed in gradients. After initial determination of ephrin/Eph gene expression by in-situ hybridisation, we are now applying the observed cortical expression to 3D-reconstructed brain models for better spatial assessment of the potential ephrin gradients.

Since the use of fresh sample cryosections allows us ARN extraction we are also performing gene profiling experiments in selected cortical areas using microarray hybridisation. We have currently arrayed over 20000 genes to this end and comparison of selected cortical area is in progress. This will enable us to determine new candidate genes involved in early cortical development.

DOUBLE-OUTLET RIGHT VENTRICLE AND CHROMOSOME 22: A NEW DELETION

A. Coulomb and D. Dal Soglio

Service d'anatomie pathologique, GHU-sud, hôpital Antoine Béclère, Clamart, AP-HP

L. Houyel

Service de chirurgie des cardiopathies congénitales, hôpital Marie Lannelongue, Le Plessis-Robinson

A. Aboura and G. Tachdjian

Service de Génétique et Reproduction, GHU-sud, hôpital Antoine Béclère, Clamart, AP-HP

F. Audibert

Service de Gynécologie Obstétrique, GHU-sud, hôpital Antoine Béclère, Clamart, AP-HP

The great anatomic heterogeneity of double-outlet right ventricle (DORV) makes very difficult all attempts of classification.

We report a case of congenital heart defect suggesting a hypoplasic left heart syndrome on the 24 WG cardiac ultrasounds leading to fetal karyotype and termination of pregnancy. The anatomic diagnosis of the cardiopathy was a complex form of DORV with absent left ventricle and mitral atresia. Conventional and molecular cytogenetics showed a female karyotype with a de novo pericentric inversion of one chromosome 22 associated with a chromosomal region 22q12 deletion leading to a partial monosomy 22q12.

Group I DORV with conotruncal anomalies, involve one segment of the heart (great arteries), are due to abnormal wedging only and are associated with interstitial 22q11.2 deletion. Group II DORV, as we observed in this report, are associated with malformation of the atrioventricle canal and ventricle, are due to early looping anomalies and involve two segments (great arteries and ventricles). Group III DORV are observed in the setting of heterotaxy sequences and involves three segments (great arteries, ventricle, atria) and are related to anomalies of the laterality genes.

This observation provides a better understanding of the classification of DORV and suggests that one or several genes implicated in early looping stage may reside in chromosomal region 22q12. Search of 22q12 microdeletion region should be performed in patients with DORV.

NEU-LAXOVA SYNDROME

E. Geán

Genetics Section

V. Cusi

Pathology Service

E. Tamayo and M. Iriondo

Neonatology Service

A male foetus with multiple congenital anomalies was delivered vaginally at 38 weeks of gestation to a 31-year-old gravida 5, para 2 woman. Weight 2.450 g; length 45 cm and head circumference 32 cm. Apgar 7/9/10. He died at 25 hours after delivery. Mother past medical history was remarkable for three spontaneous abortions. Parents were non consanguineous.

Prenatal ultrasound at 20 weeks of gestation was normal. At 34 and 36 weeks, ultrasonography discovered bilateral Dandy-Walker malformation, agenesis of corpus callosum hypoplastic right heart, short femur and single umbilical artery. Congenital bilateral cataract was suspected.

Neonatal examination showed generalized oedema. Hypoplastic right heart with pulmonary and tricuspid atresia. Single umbilical artery. Limb grossly swollen hands and feet and small nails. Microrretrognathia. Microtia. Ectropion. Microcephaly and Dandy-Walker malformation. Agenesis of corpus callosum. Unspecified ophthalmic pathology. Collodion skin. Epispadias.

Post mortem examination showed male newborn, weight 2320 g according developmental of 36-37 weeks. Ichthyosis: collodion skin. Congenital heart defect: severe right ventricle hypoplasia, tricuspid and pulmonary atresia and minimal subaortic ventricular septal defect. Dandy-Walker malformation. Hydrocephaly, focal cortical cerebral dysplasia and corpus callosum hypoplasia.

Based on the clinical and pathological features, a diagnosis of Neu-Laxova Syndrome was made. Differential diagnosis will be discussed.

ACROFACIAL DYSOSTOSIS, TYPE RODRIGUEZ. ANOTHER CASE REPORT AND LITERATURE REVIEW

Connie Schrander-Stumpel

Department of Clinical Genetics, academic hospital Maastricht, Maastricht

Connie Schrander-Stumpel

Research Institute Growth & Development (GROW), Maastricht University, Maastricht

Jos Offermans

Department of Obstetrics/Gynaecology, academic hospital Maastricht, Maastricht

Nathalie Vandevijver

Department of Pathology, Atrium Medical Center, Heerlen, the Netherlands

Jean-Pierre Fryns

Center for Human Genetics, University Hospital, Leuven, Belgium

The female proband died during pregnancy at 31 weeks gestation. She was the first child of healthy non-consanguineous parents. Family history was negative for congenital anomalies or miscarriages. Cardiac ultrasound revealed a suggestion of situs inversus. The mandible was noted to be small.

The macerated proband (figure) had a normal weight and length for gestation; OFC was27 cm (P10). The mandible was extreme small; the mouth was broad with thin lips. The external ears were very low-implanted, rudimentary and without an ear canal visible. The palate had a palpable V-formed rigde. The palpebral fissures were mildly downslanted. Both hands showed camptodactyly of fingers II-IV. Thumbs were low-implanted. Postnatally a total body X-ray was made and autopsy was performed (data pending).

Differential diagnosis focused on acrofacial dysostosis and, because of the dysmorphism of both hands, the Rodriguez type. Facial anomalies are characteristic for this condition, and most cases reported have been lethal. Skeletal abnormalities are variable, include pre- and postaxial anomalies, and appear rather mild in this patient. Heart defects have been reported. The condition is very rare; an autosomal recessive mode of inheritance cannot be excluded.

FETAL CARDIAC ANOMALIES : ORIGINAL DATABASE FOR REGISTRATION AND ENCODING

Gilda Caruso, Andrea Marzullo, Simona Parisi, Anna Scattone, and Gabriella Serio

Cardiovascular pathology. University of Bari

Riccardo Murari and Pietro Gallo

Cardiovascular pathology. University of Rome “La Sapienza”

In the last few years there has been a relevant improvement in the prenatal diagnosis of congenital heart anomalies. In particular the recent literature about prenatal echocardiographic diagnosis and genetics demonstrated that more than 50% of congenital heart disease are syndromic i.e. associated with other extracardiac malformations

Aim of this study is to present an original database recently developed in our Institutions for registration and encoding of congenital heart anomalies detected in utero, both in syndromic and non syndromic cases. The software is based on the registration of all clinically relevant, genetic and morphological data, regarding the cardiac and extracardiac pathologies of fetuses in which a complete autopsy has been performed. For each case the pertinent data are recorded in an individual report that is part of the general database that collects all the cases and from which any possible query is then eventually obtained. For the anomalies of the heart we have chosen to use the method of sequential analysis. By this system, already well known for more than twenty years, the heart is described in terms of anatomical description of the atrial situs, atrio-ventricular and ventriculo-arterial connections. To any of the anomaly of the heart and other organs a specific code number is given. The specificity of our database is that all anatomical and pathological options are already inside the program, and one has only to choose from a menu. For example for the atrial situs the string shows the terms of solitus, inversus, right isomerism and left isomerism, i.e. all the anatomical variants. Is therefore very fast and easy to record any anomaly, even in the most complicated cases. This system allows standardisation of the records because of the flexibility of the multiple choice menu. Our database also permits complete statistical analysis and generation of graphics of the registered data for anatomo-clinical correlation

PLACENTAL MESENCHYMAL DYSPLASIA: VARIATION IN CLINICAL PRESENTATION AS ILLUSTRATED BY 2 PREGNANCIES

G. de Jong

Divisions of Human Genetics, Dept. Obstetrics and Gynecology, Faculty of Health Sciences of University of Stellenbosch, Tygerberg

M. Robertson

Division of Ultrasound, Dept. Obstetrics and Gynecology, Faculty of Health Sciences of University of Stellenbosch, Tygerberg

H. Wainwright

Pathology, University of Cape Town, Observatory; South Africa

Mesenchymal dysplasia(md) is a rare placental condition, which has often, but not exclusively, been described with Beckwith-Wiedemann syndrome (BWS).

PATIENTS

First patient presented with pre-eclampsia (PET) and ultrasound showed typical signs of BWS. Follow-up showed placentomegaly with cystic changes. The mother developed eclampsia at 25 weeks gestation, necessitating TOP. Clinical examination and autopsy (intrapartum death) confirmed BWS and md.

Second patient had a spontaneous twin pregnancy, which at 16 weeks gestation showed normal twins and a large cystic placenta. Normal chromosome analysis and normal βHCG excluded partial mole. Spontaneous intra-uterine death resulted at 23 weeks gestation. The mother developed mild PET shortly after delivery of short duration. Autopsy showed (md), some macroglossia of both fetuses, but no other signs of BWS.

DISCUSSION

BWS is associated with anomalies at 11p15; on chromosomal or gene level and shows variable clinical expression. Most are associated with a duplication of the paternal IGF2 gene causing increased growth of the fetoplacental unit. Other imprinted growth promoting/inhibiting genes in the area may be involved.

Md is often associated with early onset PET, but triploidy and trisomy 13 should be excluded. Placental md has a high risk of fetal, perinatal and post partum complications and fetal demise is common.

PRIMARY ANOPHTHALMIA IN A FETUS WITH A 3q26-q28 INTERSTITIAL DELETION INVOLVING SOX2

Dominique Bonneau and Agnès Guichet

Service de génétique médicale, CHU Angers, France

StéphaneTriau

Laboratoire d'anatomie pathologique, CHU Angers, France

Catherine Lépinard and Florence Biquard

Service de gynécologie-obstétrique, CHU Angers, France

Chantal Esculapavit and Férechté Encha-Razavi

Unité de pathologie fœtale et placentaire, CHU Necker Enfants Malades, Paris, France

True anophthalmia, or complete absence of ocular tissue in the orbit, is caused either by complete failure of outgrowth or complete degeneration of the primary optic vesicle. Six cases of anophthalmia/micropthalmia have been associated with deletion or translocation involving the terminal long arm of chromosome 3. Recently, mutations of the SOX2 gene, located at 3q27-q28, have been reported in individuals with anophthalmia. We report here an interstitial deletion of chromosome 3q26-q28 involving SOX2 in a fetus in which anophthalmia had been detected antenatally. After termination of pregnancy, histological examination of the orbits showed complete absence of neural epithelium derivatives. Neuropathological examination disclosed a microcephalic brain, absence of optic nerve, chiasm and optic tracts and absence of right olfactory tract. A high resolution karyotype evidenced a de novo interstitial deletion of the telomeric end of the long arm of chromosome 3 (q26.3q28). FISH analyses using BAC clones encompassing the SOX2 locus showed that BAC clone RP11-43F17 containing SOX2 spanned the deletion breakpoint. SOX2 is an HMG-box transcription factor which, together with RX and PAX6, is specifically required for the induction of eye development. Truncating mutations of SOX2 have been identified 4 of 35 individuals affected with anophthalmia (Fantes et al. Nat Genet 2003;33:461-463). The case reported here confirms that haploinsufficiency for SOX2 plays a specific and crucial role in human eye development and emphasizes the necessity of careful chromosomal analysis, including FISH analysis of the 3q region, in case of prenatal discovery of anophthalmia on ultrasound examination.

BARDET-BIEDL SYNDROME, TYPE 6

Nina Canki-Klain

Croatian Institute for Brain Research and Department of Neurology, Zagreb University Medical School, Croatia

Thirtheen years ago we reported (Canki-Klain et al. Clin Genet 1991:40:135-6) on one unrelated healthy young Slovene couple whose first premature female infant born at 33 gestation had vaginal and urethral atresia, primary unrecognized polycystic kidneys, bilateral hydronephrosis and a rudimentary extra digit on the left hand. This child died at the age of 15 weeks because of urosepsis. Subsequent two pregnancies have been monitored by ultrasound that has detected the existence of bilateral polycystic kidneys at 24 and 21 weeks's gestation, respectively. The parents decided to terminate both pregnancies. The first fetus, a male, had postaxial polydactily on hands and feet, atrial septal defect and large polycystic kidneys. The second fetus had in addition to polydactyly and polycystic kidneys hydrometrocolpos. The presence of polydactyly together with urethral and vaginal atresia in two girls and congenital heart defect in male infant associated with hexadactyly oriented us to diagnosis of McKusick-Kaufman syndrome. Disturbing symptom in all affected siblings were polycystic kidneys, that was explained at time either as association of two independent recessives genes in the family or as a rare part of the syndrome.

Recent evidence that the McKusick-Kaufman syndrome is caused by mutation in the same gene on chromosome 20p12(MIM *604b896) encoding a protein with similarity to members of the chaperonin family and the fact that mutations in the same gene causes Bardet-Biedel syndrome, type 6(BBS6) oriented the author to the latter diagnosis.

X-LINKED CEREBELLAR MALFORMATIONS

Ginevra Zanni

Institut Cochin de Génétique Moléculaire INSERM U567, Paris Ospedale Pediatrico Bambin Gesù, Laboratorio di Medicina Molecolare, Roma

Although the developmental and molecular basis of human cerebellar development are not yet fully understood, the growing number of genes implicated in cerebellar malformations can provide certain clues. We have focused our attention on X-linked cerebellar malformations, either syndromic or isolated (congenital ataxia). Several loci have been mapped and a number of genes have been identified.

The X chromosome thus seems to harbour many genes involved cerebellar development.

OPHN1, an X-linked mental retardation gene involved in neuronal development has been recently implicated in cerebellar hypoplasia, predominantly in the vermis. Mutations of genes implicated in laterality and midline formation and patterning (ZIC3, MID1) responsible for X-linked heterotaxy and Opitz/GBBB syndrome respectively, can result in vermis hypoplasia or agenesis.

Mutations of genes involved in neuronal migration (Doublecortin, Filamin A) are responsible for cerebellar hypoplasia with associated lissencephaly or periventricular heterotopia

The description and classification of cerebellar phenotypes is not easy, at the boundary between early degenerative disease and congenital non progressive anomalies, due to the persistence of cerebellar development well beyond birth. The integration of clinical-neuroradiological, neuropathological and genetic data will help the identification of new genes involved in cerebellar pathology, thus enhancing our understanding of normal and abnormal cerebellum development.

MUTATIONAL SPECTRUM IN THE OPITZ SYNDROME GENE MID1 AND EXPRESSION DURING HUMAN DEVELOPMENT

Expression and mutations of MID1

L. Pinson, J. Augé, S. Audollent, G. Mattéi, H. Etchevers, N. Gigarel, F. Razavi, M. Le Merrer, J. Amiel, A. Munnich, S. Lyonnet, M. Vekemans, and T. Attié-Bitach

Département de Génétique et Unité INSERM U-393, Hôpital Necker-Enfants Malades, Paris, France

D. Lacombe

Service de Génétique Médicale, Hôpital Pellegrin Enfants, Bordeaux, France

S. Odent

Service de Génétique Médicale, Hôpital Pontchaillou, Rennes, France

G. Meroni

Telethon Institute of Genetics and Medicine, Naples, Italy

Opitz syndrome (OS, G/BBB syndrome, MIM145410 and MIM300000) is a midline congenital malformation characterised by hypertelorism, hypospadias and esolaryngotracheal defects leading to swallowing difficulties and hoarse voice. This condition is genetically heterogeneous with a X-linked recessive form mapped to Xp22.3 and at least one autosomal dominant form mapped to chromosome 22q11.2. Recently, mutations in MID1, a member of the B-box protein family have been identified in the X-linked form of the disease but the gene for the autosomal dominant form on 22q11 remains unknown.

Here we report on six MID1 mutations in a series of 14 unrelated OS patients including five familial forms and one sporadic case. All mutations identified in our series were novel except the R495X mutation previously reported in three unrelated patients. Interestingly, a vermis hypoplasia/agenesis was frequently present in our patients with a MID1 mutation (4/9). We also report on heart and hindbrain expression of MID1 during early human development using in situ hybridization that further supports the view that heart defects and vermis hypoplasia/agenesis are important clinical features of the syndrome. Finally, the study of X inactivation pattern in obligate carrier mothers does not help discriminating between X-linked and autosomal form of the disease in our series.

SIRENOMELIA ASSOCIATED WITH ECTROMELIA OF THE UPPER LIMB, VACTERL ASSOCIATION, AND SPINA BIFIDA. TWO CASE REPORTS OF SINGLE PREGNANCIES

Gaetano Bulfamante

Unit of Pathology, D.M.C.O., Medical School, Milan University, Italy

Giovanna Giordano

Department of Pathology and Laboratory Medicine, Medical School, Parma University, Italy

Speaker: Giovanna Giordano

Sirenomelia sequence, a rare congenital malformation⁽¹⁾, is characterized by fusion of the lower limbs and severe visceral anomalies. Here, we report the simultaneous occurrence of sirenomelia, ectromelia (radial hypoplasia), amelia of the upper limbs, VACTERAL association and systemic vascular abnormalities in two singleton foetuses, selected from among more than 2200 consecutive foetal/neonatal autopsies. The association between sirenomelia and amelia ⁽²⁾ or ectromelia (radial hypoplasia)⁽³⁾, reported only in twin pregnancies ^(4–6), casts doubt on the hypothesis that the former may be the result of direct damage to the caudal region of the embryo. Other doubts about this theory were raised by the discovery of foetuses with sirenomelia and other malformations, including omphalocele, meningomyelocele,⁽⁷⁾ and malformations of the central nervous system^(8,9). The present cases of sirenomelia with malformations of organs which are distant and embryologically different could support the theory, known as “primary midline developmental field theory”^(10,11). According to this theory multiple malformations could result from abnormalities of a part of the embryo which responds as a coordinated unit to embryonic induction resulting complex or multiple anatomic structures ⁽¹²⁾. Anatomic definitive structures resulting from a “developmental field”, in fact, may be located in anatomical regions which are distant from each other. The molecular basis of “developmental field defect” is still obscure. We suppose that the damage to “ middle developmental field” could be due to mutations of genes which simultaneously regulate the development of different organs and it is accompanied by a rearrangement process of the embryonal vascular system.

REFERENCES

1. Nyberg, D.A., Mahoney, B.S. and Pretorius D.H. (eds) (1989) Diagnostic Ultrasound of Fetal Anomalies: Text and Atlas. Year Book Medical Publishers, Chicago, pp. 187–190.

2. Biswas, B.P., Dawn, T.K, Biswas, S., (1985) Combined sirerenomelus and upper limb amelia in a uniovular twin. J Indian MA, 83(7), 245–246.

3. Managoli, S., Chaturvedi, P., Vilhekar, K.Y., Iyenger, J., Dutta, V., Gagane, N. (2002) Sirenomelia dipus in a dizygotic twin. Indian J Pediatr, 69(12), 1093–1095.

4. Di Lorenzo, M., Brandt, M.L., Veilleux, A., (1991) Sirenomelia in an identical twin: a case report. J Pediatr Surg, 11, 1334–1336.

5. Duncan, P.A., Shapiro, L.R. (1993) Interrelationship of the hemifacial microsomia: VATER, VATER, and sirenomelia phenotypes. Am J Med Genet, 47, 75–84.

6. Stocker, J.T., Heifetz, S.A. (1987) Sirenomelia. A morphological study of 33 cases and review of the literature. Perspect. Pediatr. Pathol. 10, 7–50.

7. McCoy, MC, Chescheir, NC, Kuller, JA, Altman, GC, Flannagan (1994) A fetus with sirenomelia, omphalocele,and meningomyelocele, but normal kidneys. Teratology, 50: 168–171.

8. Chen, CP, Shih, SL, Liu, FF, Jan SW (1997) Cebocephaly, alobar holoprosencephaly, spina bifida, and sirenomelia in a stillbirth. J Med Genet, 34: 252–255.

9. Martìnez-Frias, ML, Bermejo, E, Garcia, A, Galan, E., Prieto, L. (1994) Holoprosencephaly associated with caudal dysgenesis: a clinical-epidemiological analysis. Am J Med Genet, 53: 46–51.

10. Lubinsky, MS (1987) Midline developmental “weakness” as a consequence of determinative field properties. Am J Med Genet Suppl, 3:23–28.

11. Opitz, JM, Gilbert, EF (1982) Editorial comment. CNS anomalies and the midline as a “developmental fields”. Am J Med Genet, 12: 443–455.

12. Spranger, J, Bernirscke, K, Hall, JG, Lenz, W, Lowry, RB, Opitz JM, Pinsky, L, Schwarzacher, HG, Smith, DW (1982) Errors of morphogenesis : Concepts and terms. J Pediatr, 100: 160–165.

ISOLATED INFERIOR CEREBELLAR VERMIS DEFECT UNRELATED TO JOUBERT OR DANDY-WALKER MALFORMATIONS: POSTMORTEM STUDY OF 5 CASES

R. Russo, C. Fallet-Bianco, H. B. Sarnat

The isolated inferior cerebellar vermis agenesis or hypoplasia seems to be an early developmental defect of the cerebellar primordium which mainly involve structures related to the roof of the fourth ventricle. Morphological study of midsagittal cerebellar vermis in four 21–24 weeks fetuses and 28 wks newborn with Beckwith-Weidemann syndrome showed the same anomaly. The pathological features included: the roof of IV ventricle showed an abnormally flat profile, lacking the median unpair lobule X with hypercellularity and an abnormally located germinal matrix at the posterior end of the vermis. External granular cells precursors exhibited abnormal migration from the germinal matrix, running along and inside the meningeal tissue. Focal subependymal gliosis was also present in the posterior area of the roof. Posterior vermian lobules appeared small with delayed foliation, mainly involving lobule IX. The cerebellar cortex showed normal cytoarchitecture but a paucity of Bergmann glia was present; Purkinje cells and deep cerebellar nuclei were normally preserved.The lateral cerebellar hemispheres were microscopically unremarkable. Brainstem, diencephalic and telencephalic structures appeared normal. The morphology of the roof of the fourth ventricle indicates a developmental arrest at 12 weeks of gestation before median unpair lobule X appearance so that a failure of the development or early fusion of paired rudiments of the lobule X could be take in consideration. The lack of median lobule X might to some extent cause the abnormal germinal matrix location and afterwards the abnormal migration pattern of EG cells precursors. Delayed posterior vermian lobules growth and foliation could be a secondary related event. The involvement of posterior vermis may correspond to both rostrocaudal and mediolateral gradients of genetic expression and growth in the axes of the neural tube.

PRENATAL DIAGNOSIS OF JUBERG-HAYWARD SYNDROME

Nicole Laurent

Service d'Anatomie et de Cytologie Pathologiques, Centre Hospitalier Universitaire, Dijon, France

Stéphanie Couvreur-Lionnais, Thierry Rousseau, Eve Senet-Lacombe, and Paul Sagot

Clinique Gynécologique et Obstétricale, Centre Hospitalier Universitaire, Dijon, France.

Christel Thauvin-Robinet and Laurence Faivre

Centre de Génétique, Centre Hospitalier Universitaire, Dijon, France.

Anne Lise Delezoïde

Service d'Anatomo-Pathologie, Centre Hospitalier Universitaire Robert Debré, Paris, France.

Francine Mugneret

Service de Cytogénétique, Centre Hospitalier Universitaire, Dijon, France.

Christine Durand

Département de Radiologie et d'Imagerie Médicale, Centre Hospitalier Universitaire, Dijon, France.

Marie Gonzales

Service de Cytogénétique, Centre Hospitalier Universitaire, Hôpital Saint Antoine, Paris, France.

Ferechte Encha-Razavi

Service d'Histologie Embryologie Cytogénétique, Centre Hospitalier Universitaire Necker-Enfants Malades, Paris, France.

Juberg-Hayward is a rare autosomal recessive syndrome characterised by the association of growth retardation, microcephaly, cleft lip and palate, and thumbs and radial ray abnormalities. To date, no prenatal cases has been reported.

Here we report on the first prenatal case of Juberg-Hayward syndrome. The diagnosis was established following foetopathological study.

Besides to the cardinal features of the syndrome, this prenatal case was remarkable by the severity of the short arm malformation and by the finding of big toe agenesis and cerebral abnormalities including hydrocephaly, agenesis of corpus callosum, and cerebellar hypoplasia.

We conclude that the diagnosis of Juberg-Haywar syndrome can be discussed prenatally following ultrasound diagnosis of the associatio of intra-uterine growth retardation, microcephaly, thumbs/radial anomalies, cleft lip/palate.

MOLECULAR ANALYSIS OF THE HLXB9 GENE IN CURRARINO TRIAD FAMILIAL CASES

Célia Crétolle, Tania Attié-Bitach, Stanislas Lyonnet, Arnold Munnich, Michel Zérah, and Claire Nihoul-Fékété

Service de Génétique et de Chirurgie Infantile, Necker-Enfants Malades Hospital

Mutations of the HLXB9 homeobox gene, involved in sacral and anorectal development, were recently identified as disease causing the Currarino triad, a hereditary autosomal dominant syndrome. Currarino triad is a variant of caudal regression syndrome with hemisacrum and normal first sacral vertebra (type IV sacral anomaly), anorectal malformation and presacral mass (anterior meningocele or teratoma). Currarino triad often displays a broad phenotypic variability both interfamilial and intrafamilial.

The HLXB9 gene has been mapped to the terminal end (q36) of human chromosome 7; HLXB9 encodes the protein HB9, a 403 amino acid transcription factor, containing three significant domains: a polyalanine repeat region variable in length, a homeodomain, and a very highly conserved region of 82-amino acids of unknown function, upstream of the homeodomain.

Here, we report our first results of a clinical and genetic investigation started at the Necker-Enfants Malades hospital in 2003. The three exons of HLXB9 gene were analysed by DNA sequencing. Four patients belonging to Currarino families were tested and harboured, heterozygous point mutations of the the coding sequence, that would predict for deleterious changes in the protein, namely : two frameshifts and two missenses mutations. Both missense mutations involved the homeodomain, suggesting that amino acid changes in that region are relevant to the normal function of the protein.

The data futher support the involvement of HLXB9 in anorectal and sacral malformations. Our study will be extended to 30 sporadic Currarino patients and their tumors. We hope to study the correlation between phenotype and genotype, as well as information regarding HLXB9 expression pattern in early stages of embryological tail bud development.

ANTENATAL AND NEONATAL INTRACRANIAL TERATOMAS. CASE REPORT AND REVIEW OF 169 CASES IN THE LITERATURE

D. Satgé Tulle

France

P. Dechelotte

France

M. Nishi Tobetsu

Japan

M-P. Chenard

France

CH. Rickert Munster

Germany

A gestation induced with citrate clomifene was interrupted at 33 weeks, due to a large immature intracranial teratoma and hypoplasia of the left leg. A review of the literature including this observation gathers 170 cases of fetal/neonatal (less than 28 days) intracranial teratomas; the most frequent brain tumor in the fetus. Associated conditions were: one trisomy 21, one t(1;19) inherited balanced translocation, one immature ovarian teratoma of the mother, and 3 familial history of twinning. Maternal medications were rarely reported. Associated anomalies were mainly loco-regional. Usually the tumors were very large. The smaller ones were seen mostly in the midline and the anterior part of the cranial cavity. One fourth (42 cases) extended outside, into the orbit (16), oropharyngeal region (13), face (2), neck (2) or several sites (9). Malignancy was diagnosed in 18 cases based on foci of yolk sac tumor in 3, highly immature neural tissue in 10, and not indicated in 5 others. One benign immature teratoma recurred as an embryonal carcinoma. Surgical resection was performed in 48 cases. 26 did not survive, for 6 the evolution is not given. 18 infants and children are alive up to 10 years; 7 of them with moderate anomalies, corresponding mainly to the smallest tumors.

Conclusion: Intracranial teratomas are similar to other teratomas of the newborn. Due to the localization most part of them are rapidly fatal. However 10%, the smallest may be surgically cured.

RISE IN ALPHA-FOETOPROTEIN REVEALING CONGENITAL NEPHRITIC SYNDROME OF THE FINNISH TYPE (CNF)

P. Loget, S. Visée, E. Julien, and Martin D.

Centre de diagnostic prénatal, Hôpital du Mans, Paris. France

M.C. Gubler, and C. Antignac

Centre de diagnostic prénatal, Hôpital Necker, Paris, France

F. Muller

Centre de diagnostic prénatal, Hôpital Robert Debré, Paris, France

CNF is a rare autosomal recessive disorder due to mutations in the NPHS1 gene encoding Nephrin, the main protein of the slit diaphragm in the renal glomeruli.

It was the first gestation of a 21 years old mother without familial history characterized by normal nuchal translucency and a 10 more a Fetoprotein maternal blood level. The biochemical analysis of amniotic fluid focused on renal abnormality (20 W.G).with high levels of Fetoprotein and proteins (Albumin, Globulin, β2 µ Globulin). Ultra sound scan (22 W.G.) revealed a thick placenta, normal doppler, large kidney with a lack of cortico-medullary differentiation, abdominal calcifications and normal neuronal tube.

The high foetus blood level of β2 µ Globulin led to termination of pregnancy (26 WG).

The thick placenta was normal. The 95th percentile weight and size, non ambiguous male foetus (46 XY) showed dolichocephaly and low set ears. There was no visceral malformation expect hypertrophic kidneys with normal macroscopic sections. Light microscopy showed features of CNF with hypertrophic glomeruli and some tubular dilatations. NPHS1 mutation screening by direct sequencing of the 29 coding exons of the gene disclosed two heterozygous mutations, a missense mutation in exon 16 (S724C) and a splice site mutation in exon 15 (1931-1G > A), proving the diagnosis of CNF in the foetus and allowing an early prenatal diagnosis in a future pregnancy.

The CNF is a rare aetiology of rise in fetoprotein which can also reveal neuronal tube defect, placenta pathologies (as maternal floor infarction), and even less inherited epidermolysis bullosa.

DEVELOPMENT OF THE HUMAN ADRENAL MEDULLA FROM 14 TO 38 WEEKS OF GESTATION

K. Folligan, F. Targe, and J. Trouillas

Laboratoire d'Histologie et Embryologie Moléculaires, Faculté de Médecine Lyon - RTH Laennec, Université Claude Bernard Lyon I. Lyon - France

R. Bouvier

Laboratoire Central d'Anatomie et de Cytologie Pathologiques, Hôpital Edouard Herriot. Lyon - France

The development of the human adrenal gland has been studied in169 fetuses without any obvious pathology, obtained from spontaneous or medical abortions. The age varied from 14 to 38 weeks of gestation. We focused here on the fetal adrenal medulla about which little is known, except that it derived from neuroectoblastic neural crest cells. We evaluated the cell proliferation by immunohistochemical method using two nuclear markers (Ki-67 and PCNA) and studied the neuroendocrine differentiation by expression of chromogranine A (CgA).

From 14 to 17 weeks of gestation many nests of migrating neuroblasts were observed in the definitive and fetal adrenal cortex. The percentage of Ki-67 and PCNA positive nuclei decreased from 20 and 30% respectively at the 14^th week to 5% at the 21^th week. No proliferation was observed after 23 weeks of gestation.

The cells expressing CgA were observed from 14 to 38 weeks of gestation. In the fetal zone, until the 17^th week, only few positive cells were found in the nests of CgA-negative migrating neuroblasts. No CgA positive cells were observed in the definitive zone. After the 17^th week of gestation, these cells were organized in the center of the gland where many large cells expressing CgA were mingled with small CgA-negative neuroblasts.

In this study concerning the developing human adrenal medulla, we demonstrate for the first time, that the migrating neuroblasts proliferate from 14 to 23 weeks of gestation. Then, from 17 to 38 weeks, they stop their proliferation and differentiate in mature chromaffin cells in the center of the gland.

HYPERECHOGENIC FETAL BOWEL: DILEMMAS IN COUNSELING

D. Marcus-Soekarman, N. Muntjewerff, and C. Schrander-Stumpel

Department of Human Genetics

J. Offermans and M. Vossen

Department of Gynecology and Obstetrics, Academic Hospital, Maastricht, The Netherlands

D. Halley, W. Kleijer, and D. Dooijes

Department of Cell Biology and Genetics Erasmus, Medical Center, Rotterdam, The Netherlands

A. L. M. Mulder

Department of Pediatrics, Academic Hospital, Maastricht, The Netherlands

D. Marcus-Soekarman and C. Schrander-Stumpel

Institute for Growth and Development, University of Maastricht, The Netherlands

Dense fetal bowels, detected by ultrasound examination in the second trimester of pregnancy, may be a harmless observation or a sign of a pathologic condition. We report the case of a 24-year-old woman who came for counselling. An amniocentesis was offered. The karyotype of the fetus was normal, however one mutation for Cystic Fibrosis was identified. Dilemmas in counselling this particular case are discussed and follow-up results are presented.

FIRST-TRIMESTER FETAL EXAMINATION: A 3-D METHOD FOR RECONSTRUCTION AND MORPHOLOGICAL EVALUATION OF HUMAN HEART.

L. Loeuillet, J-M. Schleich, J-L. Dillenseger

Département d'Anatomie Pathologique, Hôpital de Pontchaillou, CHR Rennes 35033. France

Recent advances in ultrasound technology made it possible to identify fetal structural abnormalities and genetic syndromes in the first trimester of pregnancy. Pathologic embryo examinations are necessary to confirm the prenatal diagnosis and to determine the etiology and the pathogenetic mechanism of the anomaly. There is usually no great difficulty in performing a “ micro-autopsy ” using a dissecting microscope to confirm the obvious abnormalities. But some complex internal malformations including congenital heart defects are still difficult to identify.

We propose a simple and inexpensive method to analyze human embryo hearts, based on a spatial three-dimensional (3D) reconstruction of histological sections. This study was approved by the ethical comity of our hospital.

Embryos and small fetuses were fixed in 4% paraformaldehyde for several days. After microdissection, hearts were post-fixed in the same fixative, then dehydrated and embedded in paraffin according to standard procedures. They were entirely cut from the apex into 10 µm-thick slices, organized serially. One slice out of 10 has been stained with haematoxylin-eosin.

Digital images were obtained by optical microscopy. A 3D model of the heart was reconstructed after alignments and processing (noise reduction and semi-automatic tissue recognition) of the images. 3D imaging methods allows to study and analyze the internal cardiac structures.

We demonstrated that this novel 3-D reconstruction and visualization method ensures a correct interpretation of the cardiac morphology with a high level of resolution.

AUTOSOMAL RECESSIVE DISTAL SPINAL MUSCULAR ATROPHY (CHRONIC DSMA) WITH CONGENITAL ONSET : CLINICAL PICTURE AND GENETIC STUDY OF 4 CASES

M. Zarhrate and A. Munnich

INSERM U393, Hopital Necker Enfants Malades, 149 rue de Sèvres, 75743 PARIS, Cedex 15

L. Viollet

Service de Pediatrie et Reeducation Neuro-respiratoire, Hopital Raymond Poincare, 92380 Garches

Autosomal recessive distal spinal muscular atrophy (Chronic DSMA), is a neuromuscular disorder characterized by progressive anterior horn cell degeneration, leading to motor weakness and muscular atrophy predominating in the distal part of the limbs. Chronic DSMA – also called Distal Hereditary Motor Neuronopathy type III (A Harding) - is a rare condition, accounting for less than 10% of all spinal muscular atrophy cases. Since year 1999, we collected a series of 12 European unrelated patients showing the clinical picture and the electrophysiological criteria for Chronic DSMA, with a broad range of age at onset, extending from 0 to 12 years. By the mean of linkage analysis in the most informative pedigrees, we mapped the gene for Chronic DSMA in a short genetic interval on chromosome 11q13.3. We showed evidence of a common haplotype located at the 11q13.3 locus in 8 of the 12 patients, suggesting a partial linkage disequilibrium in the European population.

Among the 4 Chronic DSMA cases with congenital onset, the clinical presentation at birth was characterized by a generalized hypotonia with bilateral equinus varus feet and flexed fingers attitude. No neonatal respiratory distress was present. Outcome was marked by delayed motor milestones, a low progression of the paralyses during childhood and survival at adult age. Progressive restrictive respiratory insufficiency was observed, due to diaphragmatic paralysis. Neurophysiological assays showed a severe muscle denervation with normal nerve conduction velocities. Detection of distal sensory potentials confirmed the absence of sensory track involvement, as observed in spinal muscular atrophies. Genetic study revealed that all of the four patients harbored the common “European” haplotype at locus 11q13.3, strongly supporting the view that this congenital phenotype is linked to the Chronic DSMA locus on chromosome 11q13.3.

UNCOMMON FETAL PHENOTYPES OF BECKWITH-WIEDEMANN SYNDROME (BWS)

D. Gaillard, M. Mozelle-Nivoix, F. Carre-Pigeon and M. Doco-Fenzy

Service de Génétique CHU Reims

A. Alanio-Detton

Laboratoire Pol Bouin CHU Reims

S. Mehaut and N. Brissart

Anatomie pathologique et maternité CH Troyes

O. Graesslin

Maternité CHU Reims

M. Sinico

Foetopathologie CHIC Créteil

S. Rossignol and C. Gicquel

Biologie moléculaire Hôpital Trousseau Paris

The major features of BWS are macroglossia, pre-and/or postnatal overgrowth and abdominal wall defects which can be detected by prenatal ultrasonography. BWS is a model imprinting disorder in the 11p15.5 chromosomal region. Early misleading anomalies are reported in 3 fetuses without macroglossia, overgrowth and family history or assisted reproductive technologies.

• Ultrasonography detected cervical hygroma with a small umbilical hernia, intestinal hyperechogenicity, growth retardation (5°p) and hydrops at 18 weeks. These anomalies and the fetal karyotype: 45,X led to termination. The enlarged cellular placenta and detection of bilateral adrenal cytomegaly suggested BWS.

• Megacystis was detected at 13 weeks in the fetus of a fat woman with increased α fetoprotein (AFP) level and no diabetes. Sonographic follow-up showed persisting megacystis, ascites and normal amniotic fluid and growth. Peritoneal fluid (400 ml) could be punctured. These features suggested Prune belly syndrome (PBS) and the pregnancy was terminated. The karyotype was 46,XY and no metabolic disorder could be detected in the fluids. Microscopic examination showed adrenal cytomegaly, Leydig cell and pancreatic endocrine cell hyperplasia with large islets.

• Large exomphalos and megacystis was detected at 13 weeks and pregnancy was terminated at 16 weeks. Fetal examination showed thin distended abdominal wall, megacystis (PBS), organomegaly, adrenocortical cytomegaly and normal karyotype.

Isolated demethylation of the KCNQ1OT gene was found in the cells collected from the amniotic or peritoneal fluid in the 3 cases.

Early findings can be unusual in BWS. PBS was already reported in 3 cases, Turner syndrome was never associated. Overgrowth in rare before midgestation. Increased AFP and adrenal cytomegaly must lead to 11p15 chromosomal imprinting analysis.

PONTOCEREBELLAR HYPOPLASIA/ATROPHY (PCH/PCA): A HETEROGENEOUS GROUP OF NEURODEGENERATIVE DISORDERS WITH PRENATAL ONSET. A NEUROPATHOLOGICAL STUDY OF SEVEN CASES

C. Fallet-Bianco, B. Bessières, F. Ménez, C. Fernandez, A. Probst

Pontocerebellar hypoplasia-atrophy (PCH-PCA) is a group of congenital disorders characterized by an impaired development and a degenerative process of prenatal onset affecting the neopontocerebellar pathways. PCH is distinct from cerebellar hypoplasia and from olivopontocerebellar hypoplasia-atrophy (OPCA). In 1993, Barth delineated two entities: PCH1 characterized by spinal anterior horn degeneration and a lethal phenotype of spinal muscular atrophy, PCH2 characterized by progressive microcephaly, abnormal movements and normal spinal cord. Subsequently, several authors have reported familial cases of PCH with clinical and neuropathological features distinct from both PCH1 and PCH2 suggesting different subtypes. PCH-PCA has been associated with both a carbohydrate deficient glycoprotein syndrome and respiratory chain defects.

We examined 7 cases of PCH in a detailed neuropathological study. All cases displayed microcephaly with poorly developed gyration. One case was typically PCH1. Two cases showed PCH2 with distinct neuropathological data. Four cases displayed, in addition to PCH, a more severe phenotype including different degrees of degeneration of the extra pyramidal and pyramidal pathways closely related with cerebellar pathways and spinal anterior horn atrophy demonstrating a multisystemic degenerative process. This was associated, in one case with respiratory chain defects.

We suggest that PCH is a heterogeneous group of multisystemic neurodegenerative disorders with a large spectrum of phenotypic expression and different aetiologies. Recently, a linkage to chromosome 7q11.21 has been identified, but the molecular basis of PCH remains largely unknown. Since genetic markers are not yet available, more detailed and systematic neuropathological studies are likely provide progress in the understanding of the pathogenesis of PCH.

X-LINKED LISSENCEPHALY WITH ABSENT CORPUS CALLOSUM AND AMBIGUOUS GENITALIA (XLAG). CLINICAL, MRI, NEURO-PATHOLOGICAL AND MOLECULAR FINDINGS

Dominique Bonneau

Service de Génétique Médicale, CHU Angers

Annick Toutain

Service de Génétique, CHU Tours, France

Annie Laquerrière

Laboratoire de Neuropathologie, CHU Rouen, France

Stéphane Triau

Laboratoire d'Anatomie pathologique, CHU Angers

Antoinette Gélot

Unité de Neuropathologie et Service de Neuropédiatrie, Hôpital Saint Vincent de Paul, Paris

Williams B. Dobbyns

Department of Human Genetics, The University of Chicago, Chicago, USA

Mutations in the ARX gene are responsible for X-linked Lissencephaly with absent corpus callosum and ambiguous Genitalia (XLAG), a newly recognized syndrome consisting in a severe neurological disorder of neonatal onset in boys. We report here clinical, MRI, neuropathological and molecular findings in four cases affected with XLAG. In affected boys, consistent clinical features of XLAG are intractable epilepsy of neonatal onset, severe hypotonia, poor responsiveness, genital abnormalities and early death. On MRI, a gyration defect consisting of anterior pachygyria and posterior agyria with a moderately thickened brain cortex, dysplastic basal ganglia and complete agenesis of the corpus callosum are consistently found. Neuropathological examination of the brain shows a tri-layered cortex containing exclusively pyramidal neurons, a neuronal migration defect, a disorganization of the basal ganglia and a gliotic and spongy white matter. In addition, two mutations (G66-c562del and E78X) were evidenced in the ARX gene resulting in deletion or premature termination of the gene. Finally, females related to affected boys may have mental retardation and epilepsy, and often display agenesis of the corpus callosum.

SPINA BIFIDA AND THYMUS AGENESIS: DELETION 22q11.2

M. J. Perez, M. Marechaud, V. Bellec, G. Magnin

Unité de fœtopathologie-CHU POITIERS (86)

We report the case of severe spina bifida with neurological effects revealed by prenatal ultra sound with normal amniocentesis-46 XX-. The post mortem examination shows the absence of thymus and permits the diagnosis of 22q11.2 deletion.

The -25–year–old mother, gravida 1, has a cousin with anencephaly.

The Prenatal ultrasound examination reveals a severe spina bifida without meningomyelocele. The defect extends lower thoracic and lumbosacral region with neurological effects ( ventriculomegaly, Arnold-Chiari II malformation, equinovarus feet). Amniocentesis demonstrates a normal fetal karyotype 46 XX.

In relation with the poor predicted prognosis for this fetus, a termination of pregnancy was performed at 22 weeks.

The post mortem examination confirmes the large schisis extending from sixth thoracic vertebrae at first sacral vertebrae with necrotic cord tissue.

This female fetus has a normal growth, she presents facial anomalies hypertelorism, micrognathia, low-set and posteriorly rotated ears and equinovarus feet. The thymus is absent, she has not conotroncal abnormality. The neuropathological study is not possible.

We realize a fluorescence in situ hybridation ( FISH) on amniotic cells, the analysis confirmes a deletion 22q 11.2.

The micro deletion 22q 11 phenotype and prognosis is variable. The majority of deletion are de novo with 10% or less inherited from affected parents.

The familial genetic study is in progress.

ASSOCIATON BETWEEN ROBIN SEQUENCE AND CONGENITAL HYDROCEPHALUS IN A FETUS, WITH NORMAL MALE KARIOTYPE.

Fabrizio Ambrosetti

Unit of Human Pathology, Italy

Elvio Della Giustina

Unit of Child Neuropsychiatry, Italy

Valeria Barresi and Gaetano Bulfamante

Unit of Human Pathology, DMCO, University of Milan, Italy

The Robin sequence (RS) and the congenital hydrocephalus (CI) may have different etiologies and pathogenesis. The RS, characterized by micrognathia, cleft palate and glossoptosis, may result from oligohydramnios, neurogenic hypotonia, growth deficiency or connective tissue disorder. The CI may be caused by a post-haemorrhagic or a post-inflammatory obstruction in the flow of CSF or by brain malformations and genetically transmitted disorders, such as autosomal recessive or dominant syndromes, chromosome aberrations, X-linked stenosis of the aqueduct of Sylvius. The association between RS and CI may be present in many syndromes (e.g. trisomy 13, trisomy 18, trisomy 9p + , Meckel-Gruber syndrome, Roberts syndrome, Smith-Lemli-Opitz syndrome, Toriello-Carey X-linked syndrome, Silverman-Handmaker syndrome, etc.).

We described a case of RS and CI association, in a male fetus, spontaneously aborted on the 31st week of gestation. The abortion was consequent to abruptio placentae.The autopsy and X-ray examination did not show any other fetal malformation, thus excluding most of syndromes previously considered. The normal male kariotype (46,XY) of our fetus excluded the hypothesis of a syndrome related to chromosome aberrations, but not the hypotesis of an X-linked syndrome. The observed CI was associated with forking and dysplasia of the aqueduct of Sylvius and the immunohistochemical analysis with GFAP revealed a marked periaqueductal glyosis.

These findings suggest an early secondary CNS damage (Post-haemorrhagic? Post-inflammatory?). The related neurogenic hypotonia, producing lack of mandibular exercise, may produce the phenotype of the Robin sequence.

RAINE SYNDROME A PROPOS DEUX CAS

Siala S. Gaigi, J. Chneina, A. Masmoudi, S. Jabnoun, N. Khrouf

Centre De Maternite Et De Neonatologie De Tunis

Nous rapportons le cas de deux frères décédés en période néonatale, issus d'une consanguinité de 1^er degré, portant la même dysmorphie faciale :(microcéphalie, yeux exorbités, nez enfoncé, philtrum long, implantation basse des oreilles).

La radiographie du squelette met en évidence une exagération de l'ossification de la base de craˆne avec une hypoplasie des cavités orbitaires ainsi qu'une irrégularité des métaphyses des os longs sans fracture ni déformation.

L'échographie cérébrale a objectivé une importante hyper échogénicité péri ventriculaire sans autre malformation visible.

L'examen foetopathologique a mis en évidence:

• Une fente palatine et une atrésie bilatérale des choanes

• Au niveau craˆnien une hypoplasie de l'étage antérieur et moyen de la base du craˆne, dont les os sont épaissis, poussant les lobes temporaux en haut et les globes oculaires en avant avec quasi absence de cavité orbitaire, les petites ailles sphénoïdales sont droites, absence de cartilage nasal.

• Le reste de l'examen est normal.

Le syndrome de RAINE a été fortement évoqué devant:

• La récidive

• La dysmorphie faciale

• L'atteinte de la base du craˆne

AN OVERVIEW OF CNS EMBRYOGENESIS

K. K. Sulik

Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill NC, USA

Scanning electron microscopic analyses of human embryos ranging from presomite-stages through the time of posterior neuropore closure (Stages 8–13), have revealed striking similarities as well as differences relative to animal models from which much of our information regarding CNS embryogenesis is commonly extrapolated. As also observed in mouse embryos, at presomite stages, ciliated cells populate the human notochordal plate. While a neurenteric canal is present in human embryos at this stage, this structure is not identifiable in the mouse. As segments become morphologically evident in the developing human brain, it is noteworthy that the midbrain segment initially appears very small relative to the forebrain and hindbrain. Comparable to the mouse, rhombomere 4 is initially much larger than the flanking rhombomeres 3 and 5. When examined at the light microscopic level, the relatively large size of rhombomere 4, which bulges toward the midline prior to neural tube closure, creates the misimpression that an intermittent closure site may be present. In contrast to the mouse, however, anterior neural tube closure progresses in an uninterrupted manner from the caudal hindbrain through the forebrain. At approximately the 13 somite stage, the forebrain neural folds appear everted. As neural tube closure proceeds through the level of the midbrain in embryos having 15–16 somite pairs, the folds invert so that the margins of the right and left forebrain hemispheres can unite. Human anterior neuropore closure is completed by approximately 24 days post-fertilization. At approximately 26 days, the posterior neuropore closes at lower sacral levels, i.e. caudal to the developing lower limb. Following closure of the neural tube the hindbrain roofplate becomes very thin; a condition that is most likely involved in the genesis of occipital encephalocele. Understanding the basis for this, and other CNS developmental abnormalities, is greatly facilitated by a thorough understanding of its morphogenesis.

FOETOPATHOLOGICAL DIAGNOSIS OF A NEONATAL MARFAN SYNDROME REVEALED BY AKINESIA SEQUENCE

S. Blesson, C. l. Moraine, and A. Toutain

Genetics Department, University Hospital Tours, France

C. Paillet

Ultrasonography Department, University Hospital Tours, France

M. C. Vaillant

Cardiology Department, University Hospital Tours, France

F. Dijoud

Anatomopathology Laboratory, University Hospital Lyon, France

M. T. Zabot

Cellular Biotechnology Center, University Hospital Lyon, France

C. Boileau

Molecular and Hormonology Laboratory, Ambroise Paré Hospital, Boulogne

We describe a male foetus with neonatal Marfan syndrome revealed by fetal akinesia sequence. The pregnancy was terminated at 30 weeks of gestation because of foetal immobility and ultrasonographic suspicion of aortic arch interruption.

The chromosomal studies were normal and there were no deletion of 22q11 locus.

The foetus had facial dysmorphic features and tall and slender limbs, severe arachnodactyly camptodactyly, flexion contractures of elbows, hips and knees, and club feet. Necropsy showed severe dilatation of dysplastic aorta and pulmonary artery, dysplastic heart valves with abnormaly inserted chordae tendinae and myxoid degeneration of the sigmoids valves.

We report also the results of fibrilline I immunoreactivity in the extracellular matrix, as well as fibrilline expression in fibroblast cultures and genomic DNA screening.

INBORN ERRORS OF CHOLESTEROL BIOSYNTHESIS: WINDOWS ON THE ROLE OF STEROLS IN EMBRYOGENESIS

R. I. Kelley

Kennedy Krieger Institute, Johns Hopkins Medical Institutions, Baltimore, MD, USA

Although the pioneering studies in the 1960s of Roux and colleagues first showed that inhibition of cholesterol biosynthesis could cause congenital malformations in rats, it was not until 30 years later in 1992 that Smith-Lemli-Opitz syndrome (SLOS) came to be recognized as the first human disorder of cholesterol biosynthesis, caused by a deficiency of 7-dehydrocholesterol reductase. Since then however, progress has been more rapid, and human malformation syndromes now have been linked to genetic deficiencies of every major step in the post-squalene cholesterol biosynthetic pathway. Many of the characteristic malformations of SLOS closely parallel those described in the rat model of Roux et al. However, with each newly identified disorder of cholesterol biosynthesis, quite different malformations have been added to the list of embryological defects associated with abnormal cholesterol biosynthesis. Important among these more recently identified sterol-related malformations are chondrodysplasia punctata and ichthyosis in Conradi-Hünermann syndrome, limb deficiencies and ichthyosiform nevus in CHILD syndrome, Pelger-Huët anomaly and severe skeletal dysgenesis in Greenberg dysplasia, and craniosynostosis and humeroradial synostosis in Antley-Bixler syndrome. In addition to these cardinal malformations, disorders of cholesterol biosynthesis as a group manifest a remarkably wide variety of other malformations, including holoprosencephaly, microcephaly, polydactyly, endocardial cushion defects, renal dysgenesis, Hirschsprung disease, rhizomelic dwarfism, cataracts, and genital malformations. Mendelian animal models for more than half of the sterol deficiency syndromes now have been found or created, permitting numerous experimental studies to identify the embryological mechanisms responsible for these malformations.

Since the rediscovery of SLOS as a disorder of cholesterol biosynthesis, much has been learned about the diverse roles of cholesterol and its precursors in normal and abnormal embryogenesis. Most important has been the recognition that impaired function of the three known vertebrate hedgehog proteins–Sonic, Desert, and Indian Hedgehog–can explain almost all of the characteristic malformations of SLOS. Because Hedgehog proteins are distinguished from other nuclear signaling proteins by covalent binding of a molecule of cholesterol, early speculation was that the low levels of cholesterol or high levels of 7-dehydrocholesterol characteristic of SLOS caused malformations by interfering with the binding of cholesterol to Hedgehog proteins. Elegant studies by Beachy and Cooper, however, clearly showed that abnormal cholesterol biosynthesis impairs Sonic Hedgehog (SHH) function not by interfering with cholesterol attachment to SHH, but by impairing the function of Smoothened, a more distal protein in the hedgehog signaling cascade that interacts directly with Patched, the receptor for SHH and related Hedgehog proteins. Their studies also showed that it is the intracellular deficiency of cholesterol, not the accumulation of any specific sterol precursor, that impairs Smoothened signaling activity. These findings are consistent with the clinical observation that all of the sterol disorders associated with low cellular cholesterol levels–SLOS, lathosterolosis, desmosterolosis and severe CDPX2–share Hedgehog-related malformations, whereas more proximal defects in the pathway, usually associated with normal cholesterol levels, usually lack such malformations. However, because all genetic defects of cholesterol biosynthesis are readily distinguished from one another clinically, almost certainly some malformations are determined by the accumulation of abnormal sterols or by impairment of possible non-sterol functions of the mutant enzymes, such as the intranuclear structural function of the Lamin B receptor in Greenberg dysplasia.

As further support for a link between abnormal cholesterol metabolism and hedgehog-related malformations, evidence from animal models of sterol dysmetabolism and from studies of isolated human malformations indicates that the maternal supply of cholesterol to the developing embryo can modify the frequency and severity of Hedgehog-related malformations. This association was first shown by Roux, Barbu, and others in an animal model in which pregnant rats were treated with AY-9944, an inhibitor of 7-dehydrocholesterol reductase. In what we now recognize is an animal model for SLOS, these investigators found that raising the maternal cholesterol level at a critical time early in gestation protected against the teratogenic effect of AY-9944-lowered fetal cholesterol levels. More recently, in a study of isolated holoprosencephaly (HPE), we found a significantly lowered mean cholesterol level in mothers but not fathers of HPE children. Similar anecdotal correlations with maternal cholesterol levels have been made in SLOS children with HPE, and Witsch-Baumgärtner and colleagues have recently found a strong correlation between clinical severity in SLOS and the maternal apoE genotype, which has an important role in the regulation of intracellular cholesterol trafficking.

Whereas the role of hypocholesterolemia in impairing hedgehog function now seems clear, the identification of mechanisms of embryonic malformation among the more proximal defects of cholesterol biosynthesis has been more difficult. Because low blood or tissue levels of cholesterol seem to be rare among the proximal defects, and because many of their characteristic malformations are not explained by abnormal hedgehog function, it is possible that the various methylsterols and 14-unsaturated sterols that accumulate in these disorders have toxic actions or other direct effects on the unfolding of the embryonic body plan. The characteristic and striking hyperplastic nevus of CHILD syndrome caused by NSDHL deficiency is evidence that some intermediate sterols may have a growth-inductive, hyperproliferative effect. Moreover, the discovery that two sterols that accumulate behind a block in sterol 4-demethylase, 4,4′-dimethyl-5□-cholesta-8,24-dien-3B-ol and 4,4′-dimethyl-5□-cholesta-8,14,24-trien-3B-ol, have intrinsic nuclear signaling activity as “meiosis activating sterols” provides further evidence that some sterols act as growth-inducing signaling molecules. Because the phylogeny of steroid dehydrogenases suggests that the synthesis of endocrine steroids evolved from pathways of cholesterol biosynthesis, it is possible that other signaling sterols important in embryogenesis will be discovered though the study of malformations processes among disorders of cholesterol biosynthesis. An important evolutionary link between sterol and steroid metabolism is also illustrated in the recent discovery that an autosomal recessive form of Antley-Bixler syndrome is caused by the deficiency of a P450 oxidoreductase that is an essential cofactor for enzymes of both sterol and steroid biosynthesis.

The study of human and animal models of disorders of cholesterol biosynthesis has provided us with many remarkable lessons about the role of cholesterol metabolism in normal and abnormal embryogenesis. Moreover, the evidence linking variations in maternal cholesterol metabolism to certain of these malformations has important public health implications with regard to the role of maternal nutrition in the prevention of birth defects. Perhaps even more important is the evidence that variations in maternal cholesterol metabolism may influence even “normal” brain growth and development in utero and that a better understanding of this relationship may be important in the effort to understand and prevent nonsyndromic mental retardation.

HOLO…ENCEPHALY: A NEW ENTITY?

P. Marcorelles, M. Gonzalès, J. Martinovic, J. Tantau, N. Joyé, N. Morichon, and F. Encha-Razavi

We are reporting on 2 fetal cases of an unusual syngenetic central nervous system (CNS) phenotype, including telencephalosynapsis, mésencephalosynapsis and rhombencephalosynapsis associated with midline dysmorphic features. No particular maternal disorder had been pointed out. Only two similar cases have been published in a context of maternal diabetes, allowing a teratogenic mechanism to be suspected.

During CNS development, a well-kept balance between ventralization and dorsalization is compulsary not only in forebrain but also in midbrain and hindbrain patterning. This combined malformation of forebrain, midbrain and hindbrain may be linked to a severe dorsoventral patterning defect of the entire CNS. Several dorsoventral patterning pathways are now identified. They may be a possible target of toxic agents, which may mimic genetic causes of malformations.

LINK BETWEEN CNS OVERGROWTH/EXENCEPHALY AND ABNORMAL APOPTOSIS- LEARNING FROM MOUSE MODELS

A. Laquerriere and S. Patrier

Pathology Laboratory,University hospital, Rouen- France

P. Marcorelles

Pathology Laboratory, University Hospital, Brest- France

F. Razavi

Unit of Embryology, Histology and Cytogenetics, Necker Hospital for sick children, Paris- France

We report on five unrelated male fetuses with normal karyotype, presenting exencephaly/megalencephaly discovered by ultrasound, at the beginning of the second trimester. Fetal examination disclosed absence of calvaria and a protruding cerebral masse with irregular shape and a “cobblestone” surface, without any associated visceral malformation. CNS abnormalities were mainly confined to the telencephalon. The cerebral mantle showed a chaotic organization with several infoldings, multiple “rosette-like” formations and a severely disorganized cortical plate. Immunostains revealed a strong positivity for the proliferative marker Ki67, mainly in the rosette like formations and in migrating cells, expressing Neu-N. Interestingly, null mice for caspase3 and 9 share some strong phenotypic similarities with our cases. In -/- mutants, abnormal apoptosis related to dysfunction of cytochrome -c mediated caspase pathway has been considered as responsible for the overgrowth of the brain and exencephaly. We hypothesize that in our cases, the overgrowth of neural tissue and exencephaly may be caused by the persistence of mitotic activity presumably associated to a defect of programmed cell death. Further studies are required to confirm this hypothesis.

ASSOCIATION OF AGNATHIA AND CRANIO-RACHISCHISIS IN A 12 WEEKS HUMAN EMBRYO

Gerard-Blanluet Marion

Genetics, Neonatalogy, Centre Hospitalier Intercommunal, Créteil, France

Sinico Martine, and Encha-Razavi Ferechte

Obstetrics, Centre Hospitalier Intercommunal, Créteil, France

Touboul Claudine, Gour Joelle, Haddad Bassam, Abd Alsamad Issam, Paniel Bernard-Jean

Foetopathology, Centre Hospitalier Intercommunal, Créteil, France

Amselem Serge

Molecular genetics, Hôpital Henri Mondor, Créteil, France

Agnathia with otocephaly is an exceptional condition in human reproduction described in 1822 by Geoffroy Saint Hilaire. The suggested embryologic sequence is the underdevelopment of the median forebrain (mesencephalon) and part of the rhombencephalon, with abnormal placement of otic placodes fused on midline, abortion of mandibular processes and of the second branchial arc. Association of agnathia and holoprosencephaly as been described (OMIM number 20650).

One consanguineous couple (first cousins), mother 26 years old, father 37 years old, consulted in genetic counseling because of recurrence of anencephaly, early diagnosed in pregnancy. No familial history of neural tube defect was present. These pregnancies were preceded by 3 spontaneous abortions, with antinuclear factor slightly elevated at 1/160. Karyotype of the first anencephalic fetus and parents were normal. No anatomo-pathologic study was possible on the first fetus (aspiration). Foldine treatment was proposed, and followed two months only. For the next pregnancy, under aspirin and corticoids, the 11 weeks ultrasonography revealed recurrence of cranioschisis with abnormal face shape, leading to interruption of the pregnancy.

Fetal examination at 12 weeks showed agnathia with no oral orifice, midline placed ears, with probable otic orifice in medial part of maxilla. A protruding midface was evident, as an overgrowth of medial and lateral facial swellings. Protruding normal eyes were present, without hypotelorism. Skull was absent. The brain was abnormal, dysplastic, without convincing evidence of holoprosencephaly. Osseous basal structures were normal, with semicircular canals. An iniencephaly was present, with high spina-bifida, and rectitude of rachis. No other malformations were found in the female fetus (limbs, genitals, heart and kidneys).

This observation of recurrent cranioschisis in first cousins, without teratogens nor history or chromosomal abnormalities, could indicate the presence of a major gene responsible for otocephaly in human. In the mouse, a recessive lethal mutation (Oto), responsible of otocephaly, was localized on the murine chromosome 1 (Juriloff, 1985). Chordin / Noggin double-null murine embryos have the association of holoprosencephaly and agnathia (Bachiller, 2000), leading to the molecular study of the human genes (Chordin, 3q27, Noggin 17q22).

THE SER351CYS FGFR2 MUTATION DOES NOT CAUSE ANTLEY-BIXLER SYNDROME

M. Gonzales and S. Delahaye

Service de foetopathologie, Hôpital St Antoine Paris 12

S. Heuertz, M. Le Merrer, and J. Bonaventure

INSERM U393 Hôpital Necker, Paris 15

J. Martinovic and A. Bazin

Pasteur-CERBA, Pontoise

P. Loget

CHU Rennes

C. Wolf

INSERM U538 Hôpital St Antoine Paris 12

The report by Chun et al in 1998 (Am J Med Genet 77: 219–224) that Antley-Bixler syndrome (ABS) could be an autosomal dominant condition caused by an FGFR2 missense mutation (S351C) has led to controversy with regard to the diagnosis of ABS and its possible clinical overlap with Pfeiffer syndrome type III.

We report here on a series of five fetuses with craniosynostosis, midface hypoplasia, frontal bossing, proptosis and variable skeletal anomalies. Two of these patients had dysplastic ears, slender fingers with arachnodactyly, marked femoral bowing and radiohumeral synostosis. No FGFR2 mutations were identified in those cases but a 10-fold increase in lanosterol level was detected in one case, suggesting sterol metabolism defect and further supporting the diagnosis of ABS.

Out of the three other patients, only two had elbow ankylosis, and none of them exhibited bowing of the femurs. Broad halluces were visible in two cases in the absence of arachnodactyly. Interestingly, all three foetuses had tracheal cartilaginous sleeve and vertebral anomalies including vertebral fusions and segmentation of some cervical vertebrae. These clinical defects were associated with the presence of the S351C FGFR2 mutation in the extracellular domain of the receptor. We suggest that these patients do not have Antler-Bixler syndrome but would be better diagnosed as severe Pfeiffer syndrome (type III).

Since segmental spinal defects were reproducibly observed in patients carrying the S351C FGFR2 mutation but absent in typical ABS, we propose that vertebral anomalies represent a cardinal sign for the differential diagnosis of Pfeiffer type III versus Antley-Bixler syndrome.

CHROMOSOME NON DISJONCTION, TRISOMY 21 AND MEIOSIS

M. O. North, L. Telvi, and J. P. Barbet

Laboratoire d'Histologie, Embryologie, Cytogénétique, Faculté de Médecine Cochin Port Royal Paris

M. Gonzales

Laboratoire de Foetopathologie, Hôpital Saint Antoine, Paris

Trisomy 21 appears after chromosome 21 meiotic nondisjunction (ND) of maternal origin in approximately 90% of the cases either after the first meiotic division (67.5%), or after the second (22.5%). Recent molecular and cytogenetic approaches start to explain the ND mechanisms leading to aneuploïdy. Several studies of meiotic recombination by techniques of molecular biology identified various patterns of crossing-overs (co) increasing the risk of ND: (1),absence of co; (2), reduction in the number of co with co more distal; (3) increase in the number of co with very proximal co. The absence of recombination leads to ND after the first meiotic division and excess, the ND of the second meiotic division. At the diakinesis stage of the first meiotic division, the chiasmas form the visible cytogenetic consequence of co. Thus by direct counting of the chiasmas, it is possible to study meiotic disturbancess of the exchanges leading to trisomy 21. And these studies of fetal ovaries noncarrying karyotypic anomalies are all the more important as it was now shown that any event appearing during the first meiotic division influences the events later proceeding several tens of years (like the segregation at the time of the second meiotic division). In addition, the trisomic 21women are fertile and it was shown that the supernumerary chromosome can be divided into two chromatides with the first meiotic division, thus increasing the risk of aneuploïdy. We thus also undertook the study of ovaries of trisomic 21 fetuses. We present here, the preliminary results of our group after in situ Hybridation with probes of chromosome painting of the germinal cells nuclei in the first meiotic division from ovaries of fetus with normal somatic karyotype and of trisomic 21fetus.

REPORT OF TWO SEVERE FETAL CASES OF X-LINKED DOMINANT CHONDRODYSPLASIA PUNCTATA (CDPX2)

A. Toutain and S. Blesson

Genetics Department, University Hospital, Tours, France

C. Vibert-Guigue

Obstetrics and Gynecology Department, Sud Francilien Hospital, Evry, France

R. Grigorescu and M. Gonzales

Embryology, Fetopathology and Cytogenetics laboratory, Saint-Antoine University Hospital, Paris, France

Ph. Hervé

Ultrasonography Department, University Hospital, Tours, France

P. Vabres

Dermatology Department, University Hospital, Poitiers, France

B. Hermelin and C. Wolf

Molecular Biology Laboratory, Saint-Antoine University Hospital, Paris, France

C. Wolf

Spectrometry Laboratory, Saint-Antoine University Hospital, Paris, France

Chondrodysplasia punctata (CDP) is a heterogeneous group of skeletal dysplasias characterized by stippled epiphyses. A subtype of CDP, Conradi-Hünermann syndrome or CDPX2 is characterized by short stature, rhizomesomelia, asymmetry of limbs, cataracts, ichthyosis and X-linked dominant transmission with lethality in males. CDPX2 belongs to the extending group of cholesterol metabolism disorders and is caused by mutations in the emopamil binding protein (EBP) gene. The EBP gene encodes a sterol-delta 8 isomerase which catalyses an intermediate step in the conversion of lanosterol to cholesterol. The phenotype of CDPX2 ranges from stillborn to mildly affected individuals identified in adulthood with intra- and inter-familial variability.

We report two female fetal cases of CDPX2, detected prenatally on ultrasound scan and confirmed by cholesterol metabolism and EBP gene analysis, which illustrate the variable expression of the condition. One case was a sporadic case, probably occurring by new mutation, with a phenotype of lethal case. In the second case, the mother was mildly affected but the fetus had skeletal abnormalities suggesting a severe phenotype.

TYPE III LISSENCEPHALY : THE COMMON DENOMINATOR BETWEEN NEU-LAXOVA AND OMIM N°601160 SD

J Martinovic, M. Gonzalès, J. Roume, C. Fallet, C. Esculpavit, J. Chelly, and F. Encha-Razavi

Lissencephaly is absence or rarefaction of cerebral convolutions. Two major groups, type I and type II are classically described. A third type (LIS III) with distinct neuropathological pattern was first described in Neu-Laxova syndrome (NLS) and recently reported by our group in an apparently different context of Fetal Akinesia Deformation Sequence (FADS), with thick and/or fragile skin, but no ichthyosis (OMIM N° 601160).

In a Turkish, consanguineous family we found LISIII in 4 fetuses ( male and female) presenting with FADS, microcephaly/lissencephaly, delayed cerebral cortex maturation, lack of callosal and cerebrospinal tracts, neuronal depletion of the germinal zones, basal ganglia, brain stem nuclei and spinal cord. Changes suggestive of a neurodegenerative process, such as balloon shaped neurons with chromatolysis and TUNEL positive fragmentation were found. Absence of adrenal glands and thick and fragile skin without ichthyosis were noticed.

Our hypothesis is that LIS III is the common denominator between NLS and OMIM syndrome N° 601160. A detailed analysis of NLS discloses evident similarities between the two entities, that may be both linked to a “neuro-ectodermal dysplasia”. Polyhydramnios, flexion deformity of the limbs, and oedema constantly reported in NLS are part of FADS. On the other hand, skin abnormalities although less severe are found in OMIM syndrome N° 601160. Further studies are needed to clarify the frontier between the two entities and the possibility of a lesional continuum between them.

MALFORMATION AND RARE DISEASE DATABASE. NEW TECHNOLOGY TOOLS FOR ARCHIVES, DIAGNOSIS AND TELECOUNSULING

G. Bulfamante

The macroscopic and the microscopic pictures are the principle diagnostic elements in human pathology. Actually the modern technique of digital photographs and the possibility to send the images via internet, have multiplied their usefulness and possibilities.

In order to use these images in a diagnosis, in didactic and database of pathology, it must have standard criteria of quality and must be easily findable in the files.

Regarding the quality of the photos, these must represent in the true original manner (morphology proportions, and colors). This is particularly important when the images reflect dimorphisms, which are small or uncertain. The filing of the images must respond to the level of quality and efficiency. In fact an image must always be easily to found even after a long period, even in very large archives. The digitalization of images has made it possible for them to be easily filed in the computer. Unfortunately not all software files and handling of images in this moment available are of quality, because rarely did any one think it could be useful for specific medical necessities. In Italy I coordinated a working group of the G.I. APEFA, which has been dedicated in regulating standards of taking photographs, with a particular attention to the problems in photographing macroscopic of the fetus, the neonatal, and the placenta. Never the less there is now a software specifically made for filing and handling of images and information, dedicated to prenatal pathology, obstetrics and pediatrics and genetic medicine (3SSS-ARPA©, SPHAERA, Torino, Italy).

Once the images are standardized on a level of quality and organized from efficient software they may be shared. They will enhance medical files; permit diagnostic consultants or a second opinion diagnosis. It may also permit the didactic activization and professional education at a distance. To help with the realization in applying digital images (acquired originally in digital or with the digitalization from photographs or X-rays) there has been a website created (from the italian group of fetal pathology, placenta and pediatrics) and a web platform (www.ppp-g.org) dedicated to the prenatal and pediatric pathology and genetics. Using these instruments it will be possible to pass also in the medical field of philosophy communications “point to point” (tele-pathology and tele-consultant classics) to those of the web-community.

ALTERED GLYCOLYSATION AND CNS DISORDERS

Nathalie Seta

Laboratoire de Biochimie A, Hôpital Bichat-Claude Bernard, AP-HP, 75877 Paris Cedex 18, France

Walker-Warburg syndrome (WWS; OMIM#236670) is an autosomal recessive CNS disorder characterized by severe brain malformations, including the cobblestone complex, caused by altered neuronal migration, associated with congenital muscular dystrophy, structural eye abnormalities. It shares with Fukuyama congenital muscular dystrophy (FCMD; OMIM#25800) and muscle-eye-brain disease (MEB; OMIM#253280) clinical features, although WWS is more severe than the two others and even is usually lethal in the first year of life. All three have also in common the presence of muscle abnormal glycosylated □-dystroglycan. □-dystroglycan is a O-mannosylated glycoprotein, present in the muscle, peripheral nerves, and brain, and involved in fetal neuronal migration. The glycan chain of O-mannosylated glycoproteins synthesis is performed owing to specific glycosyltransferases. The genes underlying the two latter disorders have been implicated in O-mannosylation: fukutin (FCMD), POMGnT1 (MEB). Recently, mutations in another glycosyltransferase gene, POMT1, have been found in some of the WWS patients. Puzzling results, such as mutations in fukutin in a patients with a WWS phenotype, and an overlap between the clinical features might lead to revisit the classification of these SNC disorders related to O-mannosylation alteration.

These diseases are part of the growing group of congenital disorders related to glycosylation, such as the presently rather well defined Congenital Disorders of Glycosylation (CDG) with defects in N-glycosylation. Most of the CDG subtypes are also severe multi-systemic disorders with central and peripheral nervous involvement. Congenital altered glycosylation is associated with SNC disorders.

PRENATAL DIAGNOSIS OF MOSAIC TRISOMY : TWO CASES WITH BODY ASYMMETRY.

S. Delahaye, M. Gonzales, N. Joyé, V. Koubi, and M. H. Saint Frison

Laboratoire d'Embryologie Pathologique et de Cytogénétique, Hôpital Saint-Antoine, Paris

F. Stampf

Service de Gynécologie Obstétrique, Hôpital de Villeneuve Saint Georges

C. Fallet-Bianco

Hôpital Sainte Anne, Paris

F. Encha-Razavi

Unité de Pathologie Fœtale et Placentaire, Hôpital Necker-Enfants Malades, Paris

A. Bazin

Laboratoire Pasteur Cerba, Cergy Pontoise

We report two cases of prenatal diagnosis of mosaic trisomy and foetal examination. In the first case, amniocentesis performed at 18 WG for creased risk of Down syndrome revealed a 47, XX, + 16 [4]/46, XX [14]. karyotype. Ultrasound disclosed minor anomalies. After genetic counselling, parents opted for termination of pregnancy at 20 WG. The fetus had slight lateral asymmetry with a left malformed foot associated to visceral asymmetry. Lateral asymmetry was also found in the CNS. Karyotypes on foetal tissue samples revealed different degrees of mosaicism.

In the second case, amniocentesis performed at 17 WG for increased nuchal translucency on first trimester screening scan revealed a 47, XX, + 15 karyotype in all amniocytes (13). Ultrasound revealed a severe IUGR ( < 5th Percentile), single umbilical artery, cardiac malformation, skinny limbs and talipes equinovarus which were confirmed at foetal autopsy. Considering the foetal phenotype, karyotypes were performed on different foetal tissue samples and surprisingly revealed a 69, XXX/47, XX, + 15 karyotype.

Hypothesis of the mechanism of these chromosomal aberrations had to be clarified by further molecular studies. Body asymmetry has been associated with various chromosomal mosaicism but to our knowledge these are the first cases involving foetuses.

FETAL PHENOTYPE OF SMITH-LEMLI-OPITZ SYNDROME : REPORT OF A SERIE OF SIX “MALE” FETUSES

P. Loget

Unités de Foetopathologie: Hôpital le Mans

A. Bazin

Unités de Foetopathologie: Laboratoire Pasteur Cerba, Cergy Pontoise

J. Martinovic and F. Encha-Razavi

Unités de Foetopathologie: Hôpital Necker-Enfants Malades, Paris

M. B. Leger-Ravet, C. Fredouille, M. Gonzales

Unités de Foetopathologie: Hôpital Saint-Antoine, Paris

C. Fallet-Bianco

Unités de Foetopathologie: Hôpital Sainte Anne, Paris

B. Hermelin and C. Wolf

Unités de Foetopathologie: Laboratoire de biologie moléculaire et de spectrométrie de masse, CHU Saint-Antoine, Paris

Smith-Lemli-Opitz Syndrome (SLOS) is a rare autosomal recessive multiple malformational syndrome ascribed to 7-dehydrocholesterol reductase deficiency in the cholesterol biosynthesis pathway.

We report antenatal manifestations and post-mortem findings with biochemical and molecular analysis, in 6 fetuses with 46, XY karyotype, after pregnancy termination between 21 to 26 WG. Antenatal diagnosis of SLOS was made in one case, suspected in 2 cases. In 3 cases diagnosis followed post-mortem examination. Antenatal US manifestations of SLOS are multiple : increased nuchal translucency, IUGR, polydactyly, ambiguous genitalia, and various visceral malformations (renal, cardiac and cerebral).

In our series, 5 fetuses showed ambiguous genitalia and one had complete sex reversal. Fetal examination revealed in two cases unusual manifestations of SLOS: Skeletal anomalies in one case and hypothalamic hamartoma in another case. Fetal manifestations of SLOS include a large spectrum of abnormalities. This report emphasizes on diagnosis criteria of SLOS, which should be considered in fetuses having with IUGR one or more associated malformation(s).

SIRENOMELIA ASSOCIATED WITH TRACHEOESOPHAGEAL FISTULA, PART OF VATER SEQUENCE

M. Sinico, F. Encha-Razavi

Foetopathologie, CHIC, Créteil, France

C. Touboul, B. Haddad, JM. Levaillant, A. Vergnaud

Obstetrique, CHIC, Créteil, France

M. Gerard-Blanluet

Génétique Médicale, CHIC, Créteil, France

Sirenomelia is a rare fatal condition characterized by fusion of the lower extremities. It has been suggested that sirenomelia is a severe form of caudal regression syndrome complex for which the pathogenesis is controversial. Review of the literature indicates that the basic defect in sirenomelia and the VATER association lies in the formation and differentiation of mesodermal tissue and that sirenomelia, the VATER association, and monozygotic twinning show a complex etiological interrelationship. Here, we report the second case, at our knowledge of sirenomelia, with associated tracheo-esophageal fistula.

One young nonconsanguineous couple were referred for genetic counseling because of the discovery of sirenomelia, bilateral renal agenesis and sacral spina bifida at 11 weeks ultrasonography. No family history of malformations, nor spontaneous abortions were found. Terminaison of pregnancy (TOP) was proposed and accepted. Fetal karyotype was normal. Pathology done at 14 weeks shows complete sirenomelia with a single lower limb, ended by a single finger with a nail. No external genital organs, nor anal orifice were present. In sacral region, epidermized spina bifida was confirmed, meningocele at aperture. No external anomalies were found on the upper part of the fetus (head, upper limbs, hands, thorax). Pathology confirmed classical bilateral kidneys agenesis, and absence of bladder. Testes were localized under the adrenals. A tracheoesophageal atresia was discovered, with a upper segment closed, and a tracheoesophageal fistula issued from the lower segment. There was a single umbilical chord. X-rays showed a complete disorganization of lumbo-sacral vertebrae, mostly normal dorsal vertebrae but with hemivertebrae in D7. X-rays of the lower limb showed a single normal femur, and a single distal bone structure, probably a rudimentary tibiae.

Association of sirenomelia and VATER signs has already been described (Duncan et al., 1993; Harika et al., 1995), and the association with tracheoesophageal atresia has been confirmed recently (Sozubir et al., 2000). Pathogenesis is controversial, with a frequent association with a single umbilical artery, leading to the conclusion of a precocious blood vascular steal. Another hypothesis is the anomaly of a major gene, responsible for the formation of mesoderm, and explaining both the vertebral defects and tracheoesophageal anomalies. A collaborative study of major developmental genes is proposed in sirenomelia, with or without VATER association, with direct sequencing of major genes such as TBX19 or brachyury.