Neuropathological hallmarks of antenatal mitochondrial diseases with a corpus callosum defect.

Corpus callosum defects are frequent congenital cerebral disorders caused by mutations in more than 300 genes. These include genes implicated in corpus callosum development or function, as well as genes essential for mitochondrial physiology. However, in utero corpus callosum anomalies rarely raise a suspicion of mitochondrial disease and are characterized by a very large clinical heterogeneity. Here, we report a detailed pathological and neuro-histopathological investigation of nine foetuses from four unrelated families with prenatal onset of corpus callosum anomalies, sometimes associated with other cerebral or extra-cerebral defects. Next generation sequencing allowed the identification of novel pathogenic variants in three different nuclear genes previously reported in mitochondrial diseases: TIMMDC1, encoding a Complex I assembly factor never involved before in corpus callosum defect; MRPS22, a protein of the small mitoribosomal subunit; and EARS2, the mitochondrial tRNA-glutamyl synthetase. The present report describes the antenatal histopathological findings in mitochondrial diseases and expands the genetic spectrum of antenatal corpus callosum anomalies establishing OXPHOS function as an important factor for corpus callosum biogenesis. We propose that, when observed, antenatal corpus callosum anomalies should raise suspicion of mitochondrial disease and prenatal genetic counselling should be considered.

Discovery of pathogenic variants in EFEMP2 and RAG1 and undetectable fetal phenotype: A challenge of prenatal exome sequencing.

BACKGROUND: Exome sequencing in prenatal context confronts with pathogenic variants associated with phenotypes that are not detectable prenatally. MATERIALS AND METHODS: A consanguineous couple was referred at 24 weeks of gestation for prenatal genetic investigations after ultrasonography findings including decreased fetal movements, hypoplastic male external genitalia, retrognathia, prefrontal edema, anomalies of the great vessels with pulmonary atresia and dilated tortuous aorta. RESULT: Prenatal trio exome sequencing identified two homozygous likely pathogenic variants, i.e. a missense in EFEMP2 involved in cutis laxa and a nonsense in RAG1 involved in several types of severe combined immunodeficiency. DISCUSSION: The fetal ultrasonographic phenotype was partially compatible with previously reported prenatal presentations secondary to EFEMP2 biallelic variants, but prenatal presentations have never been reported for RAG1 related disorders because the RAG1 phenotype is undetectable during pregnancy. CONCLUSION: Both EFEMP2 and RAG1 variants were disclosed to the couple because the EFEMP2 variant was considered causative for the fetal ultrasonographic phenotype and the RAG1 variant was considered a finding of strong interest for genetic counselling and monitoring of future pregnancies following the American College of Medical Genetics and Genomics recommendations about the discovery of incidental findings in fetal exome sequencing in prenatal diagnosis.

Biallelic truncating variants in VGLL2 cause syngnathia in humans.

BACKGROUND: Syngnathia is an ultrarare craniofacial malformation characterised by an inability to open the mouth due to congenital fusion of the upper and lower jaws. The genetic causes of isolated bony syngnathia are unknown. METHODS: We used whole exome and Sanger sequencing and microsatellite analysis in six patients (from four families) presenting with syngnathia. We used CRISPR/Cas9 genome editing to generate vgll2a and vgll4l germline mutant zebrafish, and performed craniofacial cartilage analysis in homozygous mutants. RESULTS: We identified homozygous truncating variants in vestigial-like family member 2 (VGLL2) in all six patients. Two alleles were identified: one in families of Turkish origin and the other in families of Moroccan origin, suggesting a founder effect for each. A shared haplotype was confirmed for the Turkish patients. The VGLL family of genes encode cofactors of TEAD transcriptional regulators. Vgll2 is regionally expressed in the pharyngeal arches of model vertebrate embryos, and morpholino-based knockdown of vgll2a in zebrafish has been reported to cause defects in development of pharyngeal arch cartilages. However, we did not observe craniofacial anomalies in vgll2a or vgll4l homozygous mutant zebrafish nor in fish with double knockout of vgll2a and vgll4l. In Vgll2 -/- mice, which are known to present a skeletal muscle phenotype, we did not identify defects of the craniofacial skeleton. CONCLUSION: Our results suggest that although loss of VGLL2 leads to a striking jaw phenotype in humans, other vertebrates may have the capacity to compensate for its absence during craniofacial development.

An unusual presentation of de novo RAC3 variation in prenatal diagnosis.

Pathogenic variants in RAC3 cause a neurodevelopmental disorder with brain malformations and craniofacial dysmorphism, called NEDBAF. This gene encodes a small GTPase, which plays a critical role in neurogenesis and neuronal migration. We report a 31 weeks of gestation fetus with triventricular dilatation, and temporal and perisylvian polymicrogyria, without cerebellar, brainstem, or callosal anomalies. Trio whole exome sequencing identified a RAC3 (NM_005052.3, GRCh38) probably pathogenic de novo variant c.276 T>A p.(Asn92Lys). Eighteen patients harboring 13 different and essentially de novo missense RAC3 variants were previously reported. All the patients presented with corpus callosum malformations. Gyration disorders, ventriculomegaly (VM), and brainstem and cerebellar malformations have frequently been described. The only previous prenatal case associated with RAC3 variant presented with complex brain malformations, mainly consisting of midline and posterior fossa anomalies. We report the second prenatal case of NEDBAF presenting an undescribed pattern of cerebral anomalies, including VM and polymicrogyria, without callosal, cerebellar, or brainstem malformations. All neuroimaging data were reviewed to clarify the spectrum of cerebral malformations.

Clinical heterogeneity of NADSYN1-associated VCRL syndrome.

The NADSYN1 gene [MIM*608285] encodes the NAD synthetase 1 enzyme involved in the final step of NAD biosynthesis, crucial for cell metabolism and organ embryogenesis. Perturbating the role of NAD biosynthesis results in the association of vertebral, cardiac, renal, and limb anomalies (VCRL). This condition was initially characterized as severe with perinatal lethality or developmental delay and complex malformations in alive cases. Sixteen NADSYN1-associated patients have been published so far. This study illustrates the wide phenotypic variability in NADSYN1-associated NAD deficiency disorder. We report the clinical and molecular findings in three novel cases, two of them being siblings with the same homozygous variant and presenting with either a very severe prenatal lethal or a mild phenotypic form. In addition to an exhaustive literature, we validate the expansion of the spectrum of NAD deficiency disorder. Our findings indicate that NAD deficiency disorder should be suspected not only in the presence of the full spectrum of VCRL, but even a single of the aforementioned organs is affected. Decreased plasmatic levels of NAD should then strongly encourage the screening for any of the genes responsible for a NAD deficiency disorder.

Exome sequencing as a first-tier test for copy number variant detection: retrospective evaluation and prospective screening in 2418 cases.

BACKGROUND: Despite the availability of whole exome (WES) and genome sequencing (WGS), chromosomal microarray (CMA) remains the first-line diagnostic test in most rare disorders diagnostic workup, looking for copy number variations (CNVs), with a diagnostic yield of 10%-20%. The question of the equivalence of CMA and WES in CNV calling is an organisational and economic question, especially when ordering a WGS after a negative CMA and/or WES. METHODS: This study measures the equivalence between CMA and GATK4 exome sequencing depth of coverage method in detecting coding CNVs on a retrospective cohort of 615 unrelated individuals. A prospective detection of WES-CNV on a cohort of 2418 unrelated individuals, including the 615 individuals from the validation cohort, was performed. RESULTS: On the retrospective validation cohort, every CNV detectable by the method (ie, a CNV with at least one exon not in a dark zone) was accurately called (64/64 events). In the prospective cohort, 32 diagnoses were performed among the 2418 individuals with CNVs ranging from 704 bp to aneuploidy. An incidental finding was reported. The overall increase in diagnostic yield was of 1.7%, varying from 1.2% in individuals with multiple congenital anomalies to 1.9% in individuals with chronic kidney failure. CONCLUSION: Combining single-nucleotide variant (SNV) and CNV detection increases the suitability of exome sequencing as a first-tier diagnostic test for suspected rare Mendelian disorders. Before considering the prescription of a WGS after a negative WES, a careful reanalysis with updated CNV calling and SNV annotation should be considered.

Bi-allelic pathogenic variations in DNAJB11 cause Ivemark II syndrome, a renal-hepatic-pancreatic dysplasia.

DNAJB11 (DnaJ Heat Shock Protein Family (Hsp40) Member B11) heterozygous loss of function variations have been reported in autosomal dominant cystic kidney disease with extensive fibrosis, associated with maturation and trafficking defect involving both the autosomal dominant polycystic kidney disease protein polycystin-1 and the autosomal dominant tubulointerstitial kidney disease protein uromodulin. Here we show that biallelic pathogenic variations in DNAJB11 lead to a severe fetal disease including enlarged cystic kidneys, dilation and proliferation of pancreatic duct cells, and liver ductal plate malformation, an association known as Ivemark II syndrome. Cysts of the kidney were developed exclusively from uromodulin negative tubular segments. In addition, tubular cells from the affected kidneys had elongated primary cilia, a finding previously reported in ciliopathies. Thus, our data show that the recessive disease associated with DNAJB11 variations is a ciliopathy rather than a disease of the autosomal dominant tubulointerstitial kidney disease spectrum, and prompt screening of DNAJB11 in fetal hyperechogenic/cystic kidneys.

Hydrothorax in fetal cases of Opitz G/BBB diagnosis: Extending the phenotype?

We report two fetal cases carrying a de novo MID1 mutation and presenting with severe hydrothorax, suggesting the expansion of the phenotype of Opitz GBBB syndrome.

Fetal megacystis-microcolon: Genetic mutational spectrum and identification of PDCL3 as a novel candidate gene.

Megacystis-microcolon-intestinal-hypoperistalsis syndrome (MMIHS) is a severe congenital visceral myopathy characterized by an abdominal distension due to a large non-obstructed urinary bladder, a microcolon and intestinal hypo- or aperistalsis. Most of the patients described to date carry a sporadic heterozygous variant in ACTG2. More recently, recessive forms have been reported and mutations in MYH11, LMOD1, MYLK and MYL9 have been described at the molecular level. In the present report, we describe five patients carrying a recurrent heterozygous variant in ACTG2. Exome sequencing performed in four families allowed us to identify the genetic cause in three. In two families, we identified variants in MMIHS causal genes, respectively a nonsense homozygous variant in MYH11 and a previously described homozygous deletion in MYL9. Finally, we identified compound heterozygous variants in a novel candidate gene, PDCL3, c.[143_144del];[380G>A], p.[(Tyr48Ter)];[(Cys127Tyr)]. After cDNA analysis, a complete absence of PDLC3 expression was observed in affected individuals, indicating that both mutated transcripts were unstable and prone to mediated mRNA decay. PDCL3 encodes a protein involved in the folding of actin, a key step in thin filament formation. Presumably, loss-of-function of this protein affects the contractility of smooth muscle tissues, making PDCL3 an excellent candidate gene for autosomal recessive forms of MMIHS.

Severe and progressive neuronal loss in myelomeningocele begins before 16 weeks of pregnancy.

BACKGROUND: Despite undisputable benefits, midtrimester prenatal surgery is not a cure for myelomeningocele (MMC): residual intracranial and motor deficits leading to lifelong handicap question the timing of prenatal surgery. Indeed, the timing and intensity of intrauterine spinal cord injury remains ill defined. OBJECTIVE: We aimed to describe the natural history of neuronal loss in MMC in utero based on postmortem pathology. STUDY DESIGN: Pathology findings were analyzed in 186 cases of myelomeningocele with lesion level between S1 and T1. Using a case-control, cross-sectional design, we investigated the timewise progression and topographic extension of neuronal loss between 13 and 39 weeks. Motor neurons were counted on histology at several spinal levels in 54 isolated MMC meeting quality criteria for cell counting. These were expressed as observed-to-expected ratios, after matching for gestational age and spinal level with 41 controls. RESULTS: Chiari II malformation increased from 30.7% to 91.6% after 16 weeks. The exposed spinal cord displayed early, severe, and progressive neuronal loss: the observed-to-expected count dropped from 17% to ≤2% after 16 weeks. Neuronal loss extended beyond the lesion to the upper levels: in cases <16 weeks, the observed-to-expected motor neuron count was 60% in the adjacent spinal cord, decreasing at a rate of 16% per week. Progressive loss was also found in the upper thoracic cord, but in much smaller proportions. The observed-over-expected ratio of motor neurons was not correlated with the level of myelomeningocele. CONCLUSIONS: Significant neuronal loss is present ≤16 weeks in the exposed cord and progressively extends cranially. Earlier prenatal repair (<16 weeks) could prevent Chiari II malformation in 69.3% of cases, rescue the 17% remaining motor neurons in the exposed cord, and prevent the extension to the upper spinal cord.

Postmortem Diagnosis of Heart-hand Syndrome Associated With a 7p22.1p22.3 Deletion in a 16-week-old Fetus.

We report a male fetus with a 6.8 Mb deletion on chromosome 7p22.1p22.3 at 16 weeks of gestation. The fetus presented a heart-hand syndrome with great artery malposition, bilateral radial ray deficiency, a single pelvic kidney, and growth retardation. This deletion involves a minimal deleted region for cardiac malformation and the RAC1 gene, previously described in limb anomalies in mice. This fetus is the third human case with limb defects and RAC1 deletion.

Clinical and molecular findings in 39 patients with KBG syndrome caused by deletion or mutation of ANKRD11.

KBG syndrome, due to ANKRD11 alteration is characterized by developmental delay, short stature, dysmorphic facial features, and skeletal anomalies. We report a clinical and molecular study of 39 patients affected by KBG syndrome. Among them, 19 were diagnosed after the detection of a 16q24.3 deletion encompassing the ANKRD11 gene by array CGH. In the 20 remaining patients, the clinical suspicion was confirmed by the identification of an ANKRD11 mutation by direct sequencing. We present arguments to modulate the previously reported diagnostic criteria. Macrodontia should no longer be considered a mandatory feature. KBG syndrome is compatible with autonomous life in adulthood. Autism is less frequent than previously reported. We also describe new clinical findings with a potential impact on the follow-up of patients, such as precocious puberty and a case of malignancy. Most deletions remove the 5'end or the entire coding region but never extend toward 16q telomere suggesting that distal 16q deletion could be lethal. Although ANKRD11 appears to be a major gene associated with intellectual disability, KBG syndrome remains under-diagnosed. NGS-based approaches for sequencing will improve the detection of point mutations in this gene. Broad knowledge of the clinical phenotype is essential for a correct interpretation of the molecular results. © 2016 Wiley Periodicals, Inc.

Fraser syndrome: features suggestive of prenatal diagnosis in a review of 38 cases.

OBJECTIVE: Fraser syndrome (FS) is a rare malformation recessive disorder. Major criteria are cryptophtalmos, syndactyly, respiratory, genital and urinary tract anomalies. Few prenatal presentations have been reported. METHOD: We analyzed the prenatal and postnatal fetal phenotype in 38 cases of FS, including 25 pregnancy termination cases, 8 intra-uterine death cases and 4 cases that died after birth. RESULTS: Including both prenatal and postnatal fetal phenotypic evaluation, all cases presented dysmorphic features with nose and ear dysplasia. Renal anomalies and syndactyly were present in 37/38 cases, cryptophtalmos in 36/38, airways anomalies in 30/37 and genital anomalies in 30/35 cases. Anomalies of the abdominal wall such as low set umbilicus and omphalocele were found in 31 cases. Among the 26 cases for which ultrasound data were available, detectable anomalies included oligohydramnios (22), ascites/hydrops (9), renal anomalies (20), evidence for high airways obstruction (11), ophthalmologic anomalies (4), ear dysplasia (2) and syndactyly (2). CONCLUSION: This study shows that the postnatal phenotype of FS is very specific, whereas oligohydramnios hampers the prenatal recognition of the cardinal FS diagnosis criteria. Association of oligohydramnios, kidney agenesis and CHAOS should lead to consider this diagnosis. © 2016 John Wiley & Sons, Ltd.

Bi-allelic variations in CRB2, encoding the crumbs cell polarity complex component 2, lead to non-communicating hydrocephalus due to atresia of the aqueduct of sylvius and central canal of the medulla.

Congenital hydrocephalus is a common condition caused by the accumulation of cerebrospinal fluid in the ventricular system. Four major genes are currently known to be causally involved in hydrocephalus, either isolated or as a common clinical feature: L1CAM, AP1S2, MPDZ and CCDC88C. Here, we report 3 cases from 2 families with congenital hydrocephalus due to bi-allelic variations in CRB2, a gene previously reported to cause nephrotic syndrome, variably associated with hydrocephalus. While 2 cases presented with renal cysts, one case presented with isolated hydrocephalus. Neurohistopathological analysis allowed us to demonstrate that, contrary to what was previously proposed, the pathological mechanisms underlying hydrocephalus secondary to CRB2 variations are not due to stenosis but to atresia of both Sylvius Aqueduct and central medullar canal. While CRB2 has been largely shown crucial for apico-basal polarity, immunolabelling experiments in our fetal cases showed normal localization and level of PAR complex components (PKCι and PKCζ) as well as of tight (ZO-1) and adherens (ß-catenin and N-Cadherin) junction molecules indicating a priori normal apicobasal polarity and cell-cell adhesion of the ventricular epithelium suggesting another pathological mechanism. Interestingly, atresia but not stenosis of Sylvius aqueduct was also described in cases with variations in MPDZ and CCDC88C encoding proteins previously linked functionally to the Crumbs (CRB) polarity complex, and all 3 being more recently involved in apical constriction, a process crucial for the formation of the central medullar canal. Overall, our findings argue for a common mechanism of CRB2, MPDZ and CCDC88C variations that might lead to abnormal apical constriction of the ventricular cells of the neural tube that will form the ependymal cells lining the definitive central canal of the medulla. Our study thus highlights that hydrocephalus related to CRB2, MPDZ and CCDC88C constitutes a separate pathogenic group of congenital non-communicating hydrocephalus with atresia of both Sylvius aqueduct and central canal of the medulla.

Human neuropathology confirms projection neuron and interneuron defects and delayed oligodendrocyte production and maturation in FOXG1 syndrome.

The Forkhead transcription factor FOXG1 is a prerequisite for telencephalon development in mammals and is an essential factor controlling expansion of the dorsal telencephalon by promoting neuron and interneuron production. Heterozygous FOXG1 gene mutations cause FOXG1 syndrome characterized by severe intellectual disability, motor delay, dyskinetic movements and epilepsy. Neuroimaging studies in patients disclose constant features including microcephaly, corpus callosum dysgenesis and delayed myelination. Currently, investigative research on the underlying pathophysiology relies on mouse models only and indicates that de-repression of FOXG1 target genes may cause premature neuronal differentiation at the expense of the progenitor pool, patterning and migration defects with impaired formation of cortico-cortical projections. It remains an open question to which extent this recapitulates the neurodevelopmental pathophysiology in FOXG1-haploinsufficient patients. To close this gap, we performed neuropathological analyses in two foetal cases with FOXG1 premature stop codon mutations interrupted during the third trimester of the pregnancy for microcephaly and corpus callosum dysgenesis. In these foetuses, we observed cortical lamination defects and decreased neuronal density mainly affecting layers II, III and V that normally give rise to cortico-cortical and inter-hemispheric axonal projections. GABAergic interneurons were also reduced in number in the cortical plate and persisting germinative zones. Additionally, we observed more numerous PDGFRα-positive oligodendrocyte precursor cells and fewer Olig2-positive pre-oligodendrocytes compared to age-matched control brains, arguing for delayed production and differentiation of oligodendrocyte lineage leading to delayed myelination. These findings provide key insights into the human pathophysiology of FOXG1 syndrome.

Huntington's disease alters human neurodevelopment.

Although Huntington's disease is a late-manifesting neurodegenerative disorder, both mouse studies and neuroimaging studies of presymptomatic mutation carriers suggest that Huntington's disease might affect neurodevelopment. To determine whether this is actually the case, we examined tissue from human fetuses (13 weeks gestation) that carried the Huntington's disease mutation. These tissues showed clear abnormalities in the developing cortex, including mislocalization of mutant huntingtin and junctional complex proteins, defects in neuroprogenitor cell polarity and differentiation, abnormal ciliogenesis, and changes in mitosis and cell cycle progression. We observed the same phenomena in Huntington's disease mouse embryos, where we linked these abnormalities to defects in interkinetic nuclear migration of progenitor cells. Huntington's disease thus has a neurodevelopmental component and is not solely a degenerative disease.