Intragenic rearrangements in LARGE and POMGNT1 genes in severe dystroglycanopathies.

Dystroglycanopathies are a heterogeneous group of muscular dystrophies with autosomal recessive inheritance characterized by abnormal glycosylation of alpha-dystroglycan. The most severe phenotypes are Walker-Warburg Syndrome (WWS) and muscle-eye-brain disease (MEB) presenting with lissencephaly type II (LIS II) and in which muscular dystrophy is associated with mental retardation and eye abnormalities. To date, six distinct genes, POMT1, POMT2, POMGNT1, FKTN, FKRP, LARGE and recently in one case DPM3, have been shown to be involved in dystroglycanopathies. Genomic sequencing alone is still frequently used for diagnosis purpose, not allowing detection of intragenic rearrangements at the heterozygous state contrarily to RNA analysis, quantitative PCR and CGH array analysis. These latter methods enabled us to identify four new intragenic rearrangements in the LARGE gene in three fetuses with WWS, born to two unrelated families: deletion of exons 9-10 and duplication of introns 1-4 for the first family and deletion of exons 4 and 7 for the second one; and a deletion of the last six exons of the POMGNT1 gene in two unrelated MEB patients. Genomic dosage studies using emerging tools such as CGH array should be included in routine molecular analysis of dystroglycanopathies, not only for the screening of the LARGE gene in which this kind of mutation seems to be more frequent than point mutations, but also for the other involved genes, especially in severe clinical cases.

Refining the clinicopathological pattern of cerebral proliferative glomeruloid vasculopathy (Fowler syndrome): report of 16 fetal cases.

Cerebral proliferative glomeruloid vasculopathy (PGV) is a severe disorder of brain angiogenesis, resulting in abnormally thickened and aberrant perforating vessels, forming glomeruloids with inclusion-bearing endothelial cells. This peculiar vascular malformation was delineated by Fowler in 1972 as a stereotyped lethal fetal phenotype associating hydranencephaly-hydrocephaly with limb deformities, called Fowler syndrome (FS) or "proliferative vasculopathy and hydranencephaly-hydrocephaly" or "encephaloclastic proliferative vasculopathy" (OMIM#225790). In PGV, the disruptive impact of vascular malformation on the developing central nervous system (CNS) is now well admitted. However, molecular mechanisms of abnormal angiogenesis involving the CNS vasculature exclusively remain unknown, as no genes have been localized nor identified to date. We observed the pathognomonic FS vascular malformation in 16 fetuses, born to eight families, four consanguineous and four non-consanguineous. A diffuse form of PGV affecting the entire CNS and resulting in classical FS in 14 cases, can be contrasted to two cases with focal forms, confined to restricted territories of the CNS. Interestingly in PGV, immunohistological response to a marker of pericytes (SMA, Smooth in PGV Muscle Actin), was drastically reduced as compared to a match control. Our studies has expanded the description of FS to additional phenotypes, that could be called Fowler-like syndromes and suggest that the pathogenesis of PGV may be related to abnormal pericyte-dependent remodelling of the CNS vasculature, during CNS angiogenesis. Gene identification will determine the molecular basis of PGV and will help to know whether the Fowler-like phenotypes are due to the same underlying molecular mechanisms.

Molecular heterogeneity in fetal forms of type II lissencephaly.

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

Stability of the m.8993T->G mtDNA mutation load during human embryofetal development has implications for the feasibility of prenatal diagnosis in NARP syndrome.

BACKGROUND: Mitochondrial DNA (mtDNA) mutations cause a wide range of serious genetic diseases with maternal inheritance. Because of the high transmission risk and the absence of therapy in these disorders, at-risk couples often ask for prenatal diagnosis (PND). However, because heteroplasmy load (coexistence of mutant and wild-type mtDNA) may vary among tissues and with time, the possibility that a single fetal sample may not reflect the whole neonate impedes prenatal diagnosis of mtDNA diseases. METHODS: We performed 13 prenatal diagnoses for the NARP (neurogenic weakness, ataxia, retinitis pigmentosa) m.8993T-->G mtDNA mutation (p.Leu156Arg) in the ATP synthase subunit 6 gene. Analyses were performed on chorionic villous (CVS) and/or amniocyte samples carried out at various stages of pregnancy, using a method enabling quantification of low DNA amounts. RESULTS: Maternal mutant loads ranged from 0 to 75% in blood and had no predictive value for the fetus status, except for women with no detectable mutant DNA, whose fetuses were consistently mutation-free. In 8/13 PND, mutant load was <30%. These children are healthy at 2-7 years of age. In 5/13 PND, mutant load ranged from 65 to 100%, and parents preferred to terminate the pregnancies (15-22 weeks of gestation). Single-cell analysis of 20 trophoblastic cells and 21 amniocytes isolated from two affected fetuses found an average mutant load close to the overall CVS and amniocyte mutant load, despite striking intercellular variation. The m.8993T-->G mutant loads, assessed in 7, 17, 11, and 5 different tissues from 4 terminations, respectively, were identical in all tissues from a given individual (mean (SD) 78 (1.2)%, 91 (0.7)%, 74 (2)%, and 63 (1.6)% for the 4 fetuses, respectively). CONCLUSIONS: Our results indicate that the placental/amniotic mutant loads do reflect the NARP mutant mtDNA load in the whole fetus, even when the sample amount is small, and suggest that heteroplasmy level remains stable during pregnancy, at least after 10 weeks of gestation. Although these data establish the feasibility of PND for this mutation, assessing more precisely the correlation between mutant load and disease severity should further help in interpreting PND results.

Currarino syndrome shown by prenatal onset ventriculomegaly and spinal dysraphism.

The most frequent cause of ventriculomegaly is spina bifida when associated with Arnold-Chiari type II malformation. We report on a prenatal diagnosis of severe ventriculomegaly in association with spinal dysraphism that was indicative of a Currarino syndrome (CS) due to a c.584delA, p.H195fsX28 truncated mutation within the HLXB9 gene. Physiopathology of the ventriculomegaly is discussed in reference to the fetopathological examination and CS embryopathology. In the present case, prognosis was poor and pregnancy termination was authorized. However, such a decision may be controversial in fetuses with less severe malformations on sonographic examination, since mutations in the HLXB9 gene can predict neither the severity nor the long-term prognosis of the disease. Due to a lack of genotype-phenotype correlation and the broad variability of phenotype in heterozygotes, clinical and genetic investigations among relatives are mandatory in all HLXB9 gene mutation cases, to detect asymptomatic CS cases and to prevent the occurrence of severe complications.

De novo subtelomeric deletion additional to an inherited apparently balanced reciprocal translocation.

OBJECTIVE: We describe the analysis of an apparently balanced inherited reciprocal translocation in a fetus presenting with multiple congenital abnormalities, characterize the structural chromosome rearrangement, and report an unexpected additional imbalance to the inherited rearrangement. METHODS: DNA microarray was used to screen for genomic imbalance in subtelomeric and interstitial critical regions. High-resolution comparative genomic hybridization was used to screen for genomic imbalance at a genome-wide level. Fluorescence in situ hybridization using whole-chromosome painting and specific probes was used to characterize the inherited translocation, and the size of the de novoadditional deletion. RESULTS: An unexpected additional deletion was found in 7qter on derivative 10 of the inherited maternal reciprocal translocation t(7;10)(q11.23; p14). CONCLUSIONS: We show the usefulness of genome-wide and specific molecular cytogenetic techniques to explore apparently balanced rearrangements.

Specific osseous spurs in a lethal form of hypophosphatasia correlated with 3D prenatal ultrasonographic images.

BACKGROUND: Hypophosphatasia is an osseous dysplasia with highly variable clinical expression, ranging from a recessive lethal prenatal type to late onset dominant short stature with premature shedding of teeth. Lethal forms of hypophosphatasia include short limb dwarfism with lack of ossification, especially on the vertebral bodies, very slender ribs and clavicles, and bowed, short lower extremities, with a bifid aspect of the diaphyses. Alkaline phosphatase is abnormally low in liver, bone, kidney and plasma. METHODS: We present here the prenatal images of a lethal form of hypophosphatasia, diagnosed precociously because of specific osseous spurs in a context of recurrent short limb dwarfism. RESULTS: Prenatal 3D ultrasonography has shown these spurs as early as 18 weeks. Molecular biology found compound heterozygous mutations in the gene TNSALP. CONCLUSION: In a context of short limb dwarfism, the search for these specific osseous spurs orient strongly toward the diagnosis of lethal hypophosphatasia.

Detection of an Alu insertion in the POMT1 gene from three French Walker Warburg syndrome families.

Walker Warburg syndrome (WWS) is the most severe of a group of multiple congenital disorders known as lissencephaly type II ( LIS Type II) associated with congenital muscular dystrophy and eye abnormalities. The POMT1 gene is the most frequently affected found in 20% of patients with WWS. We describe five fetuses with WWS in three non-related families carrying a same mutation in the POMT1 gene. All fetuses presented with tetra ventricular hydrocephaly, and arachnoidal neuroglial ectopia and cortical dysplasia characteristic of LIS type II. We performed sequencing of the POMT1 gene on fetal DNA. The five fetuses were found to share an insertion of an inversed Alu repeated DNA element within exon 3 of the POMT1 gene, all at the heterozygous state except one at the homozygous state. This mutation was associated with a common transition c.2203 C > T (p.Arg735Cys) in exon 20 on the same allele and similar intragenic haplotype, suggesting that the three families could be related or indicating a possible founder effect in France. Insertions of Alu sequences, which are rarely found in coding regions, have occasionally been reported to cause other genetic diseases. However, this is the first report of a retrotransposon insertion in the POMT1 gene associated with WWS.

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

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

Pathophysiology of syndromic combined pituitary hormone deficiency due to a LHX3 defect in light of LHX3 and LHX4 expression during early human development.

The pathophysiology of combined pituitary hormone deficiency (CPHD) is just beginning to be elucidated, with mutations in genes encoding transcription factors expressed at different stages of pituitary development. Among them, the two closely related genes, LHX3 and LHX4, are believed to share redundant biological properties. The patients with a LHX3 mutation display a CPHD phenotype, associated with a rigid cervical spine. This latter feature, not reported in Lhx3-/- and Lhx4-/- mice nor in patients with a LHX4 defect, prompted us to study the molecular consequences of a previously identified LHX3 23-bp deletion and to determine the LHX3 and LHX4 expression patterns during early human development. This deletion, which results in the skipping of one coding exon, would lead to a protein with no transcriptional capability. Using in situ hybridization, we show that LHX3 and LHX4 are expressed in the developing human pituitary and along the rostro-caudal length of the spinal cord; here, both transcripts are detected in the ventral part giving rise to motorneurons and interneurons. However, whereas LHX3 is expressed at all stages studied, LHX4 expression is transient, and, at 6 weeks of development, is stronger at the caudal than at the cervical level.

Prenatal diagnosis and characterization of an analphoid marker chromosome 16.

We report on a fetus with intrauterine growth retardation and multiple malformations diagnosed on ultrasound at 32 weeks. Examination of amniotic fluid cells in culture showed a 47,XY, i(16)(q10), +mar karyotype. Chromosome analysis of both parents was normal. Using spectral karyotyping, we identified the marker chromosome as a mitotically stable acentric marker chromosome derived from chromosome 16. Further studies using subtelomeric fluorescent probes confirmed the presence of an isochromosome for the long arm of chromosome 16 and showed that the acentric marker chromosome derived from the short arm of chromosome 16 leading to a trisomy for the long arm of chromosome 16. After genetic counseling, the parents decided to terminate the pregnancy. Fetal autopsy showed a male fetus with ambiguous external genitalia, cardiac malformation, megacystis and limbs anomalies as observed in other cases of trisomy for the long arm of chromosome 16. In addition, fetal brain examination showed vermian and olfactory bulb hypoplasia.

[Ultrasonographic and pathological correlation in a fetal intracranial cyst: a case of "diencephalo-synapsis"]. / Corrélation écho-anatomique d'une image kystique cérébrale foetale: une observation de "diencéphalo-synapsis".

Brain imaging now provides exquisite images of the central nervous system (CNS) enabling identification of CNS malformations early during pregnancy. However, pathogenical evaluation, necessary for genetic counselling, requires a detailed neuropathological analysis. Brain imaging of a female fetus at 27 weeks gestation disclosed a paramedial cystic formation, considered to be a porencephalic lesion. Neuropathological correlation after pregnancy termination disclosed partial atresia of the third ventricle, responsible for lateral ventricle dilatation and corpus callosum lamination. Atresia of the third ventricle, that we suggest could be called "diencephalo-synapsis", is a rare CNS malformation due to an unknown cause. Further neuropathological studies and phenotype-genotype correlations are necessary for the delineation of the entity and the comprehension of its cause and pathogenesis.

Arthrogryposis multiplex congenita and cerebellopontine ischemic lesions in sibs: recurrence of prenatal disruptive brain lesions with different patterns of expression?

Arthrogryposis multiplex congenita (AMC) is a heterogeneous group of disorders in which prolonged decrease or absence of fetal movements results in a series of deformational anomalies. The rate of recurrence ranges from 25% in some recessive forms of myogenic arthrogryposis or of primary anterior horn cell loss, to less than 1% in anoxic-ischaemic damage. Cerebral clastic processes are considered as sporadic. We report on a non-consanguineous family in which the first child was affected by AMC and the following pregnancy was terminated because cerebellum hypoplasia was suspected at ultrasound and confirmed by fetal magnetic resonance imaging. Post-mortem findings demonstrated pontocerebellar ischaemic-haemorrhagic injuries. The occurrence of these neurologic abnormalities in the same family suggests a common mechanism, which might correspond to a same genetic defect with different patterns of expression. This is the first prenatal report suggesting that an 'ischaemic' process, usually recognised as sporadic could in fact be due to an inherited abnormality. Careful prenatal follow-up of third-trimester fetal brain development may be required in pregnant women with a family history of AMC.

Lissencephaly type III, stippled epiphyses and loose, thick skin: a new recessively inherited syndrome.

We report on two new cases of syndromic lissencephaly in two consanguineous sibs, with skeletal abnormality, born to young, healthy, second cousin parents with healthy children. In Case 1, fetal ultrasound screening at 32 weeks of gestation showed microcephaly, skin infiltration and equinovarus feet. MRI disclosed cerebral agyria, hypoplastic cerebral mantle and posterior agenesis of the corpus callosum. The propositus, a boy, died soon after birth at term. In Case 2, fetal ultrasound study performed at 16 weeks of gestation disclosed skin infiltration. MRI at 22 weeks of gestation showed microcephaly with agenesis of corpus callosum and cerebellar hypoplasia. Pregnancy was terminated at 22 weeks of gestation. The fetus had normal 46, XY karyotype and similar anomalies found in the index case, with cranio-facial edema and arthrogryposis. X-ray films showed epiphyseal stippling of cervical vertebrae, feet and sacrum. Metacarpal bones were shortened with hypoplastic distal phalanges. Neuropathological findings were concordant with the pattern described in type III lissencephaly: an agyric brain with hypoplastic brain stem and cerebellum, severe neuronal loss of the cortical plate, matrix zone, basal ganglia, brainstem nuclei and spinal cord with axonal swelling and microcalcification. This entity seems to be a new syndromic lissencephaly type III, because of epiphyseal calcifications and metacarpophalangeal bone dysplasia.

JAGGED1 gene expression during human embryogenesis elucidates the wide phenotypic spectrum of Alagille syndrome.

Mutations of the JAGGED1 gene, encoding a NOTCH receptor ligand, cause Alagille syndrome (AGS), a complex malformative disorder affecting mainly the liver, heart, vertebrae, eye, and face. Minor and occasional features involving kidney, pharynx, systemic arteries, skeleton, and ear are in some cases associated with the syndrome. To describe the expression of JAGGED1 during human embryogenesis and to study its relationship with all the features of AGS, we performed in situ hybridization studies on human embryos and fetal tissue sections. JAGGED1 was mainly expressed in the cardiovascular system. In the liver, JAGGED1 transcripts were only detected in blood vessels. JAGGED1 was also expressed in other structures of mesenchymal origin (distal mesenchyme of limb buds; mesonephric and metanephric tubules of the kidney) and in epithelial structures including the ciliary margin of the retina and the posterior part of the lens, the ventral epithelium of the otic vesicle, the neurosensory epithelium of the ear vestibule, the epithelium of pharyngeal arches, and the developing central nervous system. The strong JAGGED1 expression during human embryo- and feto-genesis both in the vascular system and in other mesenchymal and epithelial tissues implicates abnormal angiogenesis in the pathogenesis of Alagille syndrome and particularly the paucity of interlobular bile ducts. However, it is probably not the only mechanism of the disease. Except for the central nervous system, there is a strong correlation between JAGGED1 expression and all the features of AGS. This implies that the features occasionally associated with the syndrome are not coincidental.

Expression of the PAX2 gene in human embryos and exclusion in the CHARGE syndrome.

The CHARGE syndrome comprises ocular coloboma, heart malformation, choanal atresia, retarded growth and development, central nervous system malformations, genital hypoplasia, ear abnormalities, or deafness. The cause of the CHARGE syndrome remains unknown. In the present study, we analyzed the distribution pattern of the PAX2 gene in human embryos and found that PAX2 gene expression occurs in the primordia affected in the CHARGE syndrome. These data prompted us to consider the PAX2 gene a candidate gene in the CHARGE "association." We analyzed the PAX2 gene in 34 patients fulfilling the diagnostic criteria of the CHARGE syndrome for deletion and nucleotidic variations of the coding sequence and identified only polymorphisms. Our data suggest that mutation of the PAX2 gene is not a cause of the CHARGE association. However, the pattern of expression of PAX2 suggests that genes encoding downstream targets effectors could be candidate genes for the CHARGE syndrome.