Genotype-first in a cohort of 95 fetuses with multiple congenital abnormalities: when exome sequencing reveals unexpected fetal phenotype-genotype correlations.

PURPOSE: Molecular diagnosis based on singleton exome sequencing (sES) is particularly challenging in fetuses with multiple congenital abnormalities (MCA). Indeed, some studies reveal a diagnostic yield of about 20%, far lower than in live birth individuals showing developmental abnormalities (30%), suggesting that standard analyses, based on the correlation between clinical hallmarks described in postnatal syndromic presentations and genotype, may underestimate the impact of the genetic variants identified in fetal analyses. METHODS: We performed sES in 95 fetuses with MCA. Blind to phenotype, we applied a genotype-first approach consisting of combined analyses based on variants annotation and bioinformatics predictions followed by reverse phenotyping. Initially applied to OMIM-morbid genes, analyses were then extended to all genes. We complemented our approach by using reverse phenotyping, variant segregation analysis, bibliographic search and data sharing in order to establish the clinical significance of the prioritised variants. RESULTS: sES rapidly identified causal variant in 24/95 fetuses (25%), variants of unknown significance in OMIM genes in 8/95 fetuses (8%) and six novel candidate genes in 6/95 fetuses (6%). CONCLUSIONS: This method, based on a genotype-first approach followed by reverse phenotyping, shed light on unexpected fetal phenotype-genotype correlations, emphasising the relevance of prenatal studies to reveal extreme clinical presentations associated with well-known Mendelian disorders.

Risk factors of clinical dysimmune manifestations in a cohort of 86 children with 22q11.2 deletion syndrome: A retrospective study in France.

In this study, we describe the biological immune profiles and clinical dysimmune manifestations (infections, autoimmune diseases, and allergies) of patients with 22q11.2 deletion syndrome with the aim of determining risk factors for clinical events. This retrospective study concerned all the patients with 22q11 deletion syndrome attending the Montpellier University Hospital from January 1, 1992, to December 31, 2014 who had at least one immune investigation before the age of 18. We analyzed the clinical features, biological tests and the course of infections, autoimmunity, and allergy of 86 children. Among these 86 children, 48 (59%) had a low T lymphocyte level. Twenty-nine patients (34%) had a severe infection. The only risk factor for severe infection was the low level of CD4+ T-cells (OR: 3.3; 95% confidence interval (CI) [1.020-11.108]). Eleven patients (13%) developed an autoimmune disease; the only risk factor was an antecedent of severe infection (OR: 4.1; 95% CI [1.099-15.573]). Twenty-three patients (27%) had allergic episodes. A low level of CD8+ T-cells (OR: 3.2; 95% CI [1.07-9.409]) was significantly associated with allergy manifestations. Patients with 22q11 deletion syndrome have a high rate of dysimmune manifestations. We found statistic correlations among CD4+ T-cell count, infectious manifestations, and autoimmunity.

Treacher Collins syndrome: a clinical and molecular study based on a large series of patients.

PURPOSE: Treacher Collins/Franceschetti syndrome (TCS; OMIM 154500) is a disorder of craniofacial development belonging to the heterogeneous group of mandibulofacial dysostoses. TCS is classically characterized by bilateral mandibular and malar hypoplasia, downward-slanting palpebral fissures, and microtia. To date, three genes have been identified in TCS:,TCOF1, POLR1D, and POLR1C. METHODS: We report a clinical and extensive molecular study, including TCOF1, POLR1D, POLR1C, and EFTUD2 genes, in a series of 146 patients with TCS. Phenotype-genotype correlations were investigated for 19 clinical features, between TCOF1 and POLR1D, and the type of mutation or its localization in the TCOF1 gene. RESULTS: We identified 92/146 patients (63%) with a molecular anomaly within TCOF1, 9/146 (6%) within POLR1D, and none within POLR1C. Among the atypical negative patients (with intellectual disability and/or microcephaly), we identified four patients carrying a mutation in EFTUD2 and two patients with 5q32 deletion encompassing TCOF1 and CAMK2A in particular. Congenital cardiac defects occurred more frequently among patients with TCOF1 mutation (7/92, 8%) than reported in the literature. CONCLUSION: Even though TCOF1 and POLR1D were associated with extreme clinical variability, we found no phenotype-genotype correlation. In cases with a typical phenotype of TCS, 6/146 (4%) remained with an unidentified molecular defect.

A balance between activating and repressive histone modifications regulates cystic fibrosis transmembrane conductance regulator (CFTR) expression in vivo.

The genetic mechanisms that regulate CFTR, the gene responsible for cystic fibrosis, have been widely investigated in cultured cells. However, mechanisms responsible for tissue-specific and time-specific expression are not completely elucidated in vivo. Through the survey of public databases, we found that the promoter of CFTR was associated with bivalent chromatin in human embryonic stem (ES) cells. In this work, we analyzed fetal (at different stages of pregnancy) and adult tissues and showed that, in digestive and lung tissues, which expressed CFTR, H3K4me3 was maintained in the promoter. Histone acetylation was high in the promoter and in two intronic enhancers, especially in fetal tissues. In contrast, in blood cells, which did not express CFTR, the bivalent chromatin was resolved (the promoter was labeled by the silencing mark H3K27me3). Cis-regulatory sequences were associated with lowly acetylated histones. We also provide evidence that the tissue-specific expression of CFTR is not regulated by dynamic changes of DNA methylation in the promoter. Overall, this work shows that a balance between activating and repressive histone modifications in the promoter and intronic enhancers results in the fine regulation of CFTR expression during development, thereby ensuring tissue specificity.

Integrin alpha 8 recessive mutations are responsible for bilateral renal agenesis in humans.

Renal hypodysplasia (RHD) is a heterogeneous condition encompassing a spectrum of kidney development defects including renal agenesis, hypoplasia, and (cystic) dysplasia. Heterozygous mutations of several genes have been identified as genetic causes of RHD with various severity. However, these genes and mutations are not associated with bilateral renal agenesis, except for RET mutations, which could be involved in a few cases. The pathophysiological mechanisms leading to total absence of kidney development thus remain largely elusive. By using a whole-exome sequencing approach in families with several fetuses with bilateral renal agenesis, we identified recessive mutations in the integrin α8-encoding gene ITGA8 in two families. Itga8 homozygous knockout in mice is known to result in absence of kidney development. We provide evidence of a damaging effect of the human ITGA8 mutations. These results demonstrate that mutations of ITGA8 are a genetic cause of bilateral renal agenesis and that, at least in some cases, bilateral renal agenesis is an autosomal-recessive disease.

Mutations in CNTNAP1 and ADCY6 are responsible for severe arthrogryposis multiplex congenita with axoglial defects.

Non-syndromic arthrogryposis multiplex congenita (AMC) is characterized by multiple congenital contractures resulting from reduced fetal mobility. Genetic mapping and whole exome sequencing (WES) were performed in 31 multiplex and/or consanguineous undiagnosed AMC families. Although this approach identified known AMC genes, we here report pathogenic mutations in two new genes. Homozygous frameshift mutations in CNTNAP1 were found in four unrelated families. Patients showed a marked reduction in motor nerve conduction velocity (<10 m/s) and transmission electron microscopy (TEM) of sciatic nerve in the index cases revealed severe abnormalities of both nodes of Ranvier width and myelinated axons. CNTNAP1 encodes CASPR, an essential component of node of Ranvier domains which underlies saltatory conduction of action potentials along the myelinated axons, an important process for neuronal function. A homozygous missense mutation in adenylate cyclase 6 gene (ADCY6) was found in another family characterized by a lack of myelin in the peripheral nervous system (PNS) as determined by TEM. Morpholino knockdown of the zebrafish orthologs led to severe and specific defects in peripheral myelin in spite of the presence of Schwann cells. ADCY6 encodes a protein that belongs to the adenylate cyclase family responsible for the synthesis of cAMP. Elevation of cAMP can mimic axonal contact in vitro and upregulates myelinating signals. Our data indicate an essential and so far unknown role of ADCY6 in PNS myelination likely through the cAMP pathway. Mutations of genes encoding proteins of Ranvier domains or involved in myelination of Schwann cells are responsible for novel and severe human axoglial diseases.

Neuropathological review of 138 cases genetically tested for X-linked hydrocephalus: evidence for closely related clinical entities of unknown molecular bases.

L1 syndrome results from mutations in the L1CAM gene located at Xq28. It encompasses a wide spectrum of diseases, X-linked hydrocephalus being the most severe phenotype detected in utero, and whose pathophysiology is incompletely understood. The aim of this study was to report detailed neuropathological data from patients with mutations, to delineate the neuropathological criteria required for L1CAM gene screening in foetuses by characterizing the sensitivity, specificity and positive predictive value of the cardinal signs, and to discuss the main differential diagnoses in non-mutated foetuses in order to delineate closely related conditions without L1CAM mutations. Neuropathological data from 138 cases referred to our genetic laboratory for screening of the L1CAM gene were retrospectively reviewed. Fifty-seven cases had deleterious L1CAM mutations. Of these, 100 % had hydrocephalus, 88 % adducted thumbs, 98 % pyramidal tract agenesis/hypoplasia, 90 % stenosis of the aqueduct of Sylvius and 68 % agenesis/hypoplasia of the corpus callosum. Two foetuses had L1CAM mutations of unknown significance. Seventy-nine cases had no L1CAM mutations; these were subdivided into four groups: (1) hydrocephalus sometimes associated with corpus callosum agenesis (44 %); (2) atresia/forking of the aqueduct of Sylvius/rhombencephalosynapsis spectrum (27 %); (3) syndromic hydrocephalus (9 %), and (4) phenocopies with no mutations in the L1CAM gene (20 %) and in whom family history strongly suggested an autosomal recessive mode of transmission. These data underline the existence of closely related clinical entities whose molecular bases are currently unknown. The identification of the causative genes would greatly improve our knowledge of the defective pathways involved in these cerebral malformations.

Exon skipping as a therapeutic strategy applied to an RYR1 mutation with pseudo-exon inclusion causing a severe core myopathy.

Central core disease is a myopathy often arising from mutations in the type 1 ryanodine receptor (RYR1) gene, encoding the sarcoplasmic reticulum calcium release channel RyR1. No treatment is currently available for this disease. We studied the pathological situation of a severely affected child with two recessive mutations, which resulted in a massive reduction in the amount of RyR1. The paternal mutation induced the inclusion of a new in-frame pseudo-exon in RyR1 mRNA that resulted in the insertion of additional amino acids leading to the instability of the protein. We hypothesized that skipping this additional exon would be sufficient to restore RyR1 expression and to normalize calcium releases. We therefore developed U7-AON lentiviral vectors to force exon skipping on affected primary muscle cells. The efficiency of the exon skipping was evaluated at the mRNA level, at the protein level, and at the functional level using calcium imaging. In these affected cells, we observed a decreased inclusion of the pseudo-exon, an increased RyR1 protein expression, and a restoration of calcium releases of normal amplitude either upon direct RyR1 stimulation or in response to membrane depolarization. This study is the first demonstration of the potential of exon-skipping strategy for the therapy of central core disease, from the molecular to the functional level.

Antenatal spectrum of CHARGE syndrome in 40 fetuses with CHD7 mutations.

BACKGROUND: CHARGE syndrome is a rare, usually sporadic disorder of multiple congenital anomalies ascribed to a CHD7 gene mutation in 60% of cases. Although the syndrome is well characterised in children, only one series of 10 fetuses with CHARGE syndrome has been reported to date. Therefore, we performed a detailed clinicopathological survey in our series of fetuses with CHD7 mutations, now extended to 40 cases. CHARGE syndrome is increasingly diagnosed antenatally, but remains challenging in many instances. METHOD: Here we report a retrospective study of 40 cases of CHARGE syndrome with a CHD7 mutation, including 10 previously reported fetuses, in which fetal or neonatal clinical, radiological and histopathological examinations were performed. RESULTS: Conversely to postnatal studies, the proportion of males is high in our series (male to female ratio 2.6:1) suggesting a greater severity in males. Features almost constant in fetuses were external ear anomalies, arhinencephaly and semicircular canal agenesis, while intrauterine growth retardation was never observed. Finally, except for one, all other mutations identified in our antenatal series were truncating, suggesting a possible phenotype-genotype correlation. CONCLUSIONS: Clinical analysis allowed us to refine the clinical description of CHARGE syndrome in fetuses, describe some novel features and set up diagnostic criteria in order to help the diagnosis of CHARGE syndrome after termination of pregnancies following the detection of severe malformations.

NEK1 and DYNC2H1 are both involved in short rib polydactyly Majewski type but not in Beemer Langer cases.

BACKGROUND: The lethal short rib polydactyly syndromes (SRP type I-IV) are characterised by notably short ribs, short limbs, polydactyly, multiple anomalies of major organs, and autosomal recessive mode of inheritance. Among them, SRP type II (Majewski; MIM 263520) is characterised by short ovoid tibiae or tibial agenesis and is radiographically closely related to SRP type IV (Beemer-Langer; MIM 269860) which is distinguished by bowed radii and ulnae and relatively well tubulated tibiae. NEK1 mutations have been recently identified in SRP type II. Double heterozygosity for mutations in both NEK1 and DYNC2H1 in one SRP type II case supported possible digenic diallelic inheritance. METHODS: The aim of this study was to screen DYNC2H1 and NEK1 in 13 SRP type II cases and seven SRP type IV cases. It was not possible to screen DYNC2H1 in two patients due to insufficient amount of DNA. RESULTS: The study identified homozygous NEK1 mutations in 5/13 SRP type II and compound heterozygous DYNC2H1 mutations in 4/12 cases. Finally, NEK1 and DYNC2H1 were excluded in 3/12 SRP type II and in all SRP type IV cases. The main difference between the mutation positive SRP type II group and the mutation negative SRP type II group was the presence of holoprosencephaly and polymycrogyria in the mutation negative group. CONCLUSION: This study confirms that NEK1 is one gene causing SRP type II but also reports mutations in DYNC2H1, expanding the phenotypic spectrum of DYNC2H1 mutations. The exclusion of NEK1 and DYNC2H1 in 3/12 SRP type II and in all SRP type IV cases further support genetic heterogeneity.

Cobblestone lissencephaly: neuropathological subtypes and correlations with genes of dystroglycanopathies.

Cobblestone lissencephaly represents a peculiar brain malformation with characteristic radiological anomalies, defined as cortical dysplasia combined with dysmyelination, dysplastic cerebellum with cysts and brainstem hypoplasia. Cortical dysplasia results from neuroglial overmigration into the arachnoid space, forming an extracortical layer, responsible for agyria and/or 'cobblestone' brain surface and ventricular enlargement. The underlying mechanism is a disruption of the glia limitans, the outermost layer of the brain. Cobblestone lissencephaly is pathognomonic of a continuum of autosomal recessive diseases with cerebral, ocular and muscular deficits, Walker-Warburg syndrome, muscle-eye-brain and Fukuyama muscular dystrophy. Mutations in POMT1, POMT2, POMGNT1, LARGE, FKTN and FKRP genes attributed these diseases to α-dystroglycanopathies. However, studies have not been able to identify causal mutations in the majority of patients and to establish a clear phenotype/genotype correlation. Therefore, we decided to perform a detailed neuropathological survey and molecular screenings in 65 foetal cases selected on the basis of histopathological criteria. After sequencing the six genes of α-dystroglycanopathies, a causal mutation was observed in 66% of cases. On the basis of a ratio of severity, three subtypes clearly emerged. The most severe, which we called cobblestone lissencephaly A, was linked to mutations in POMT1 (34%), POMT2 (8%) and FKRP (1.5%). The least severe, cobblestone lissencephaly C, was linked to POMGNT1 mutations (18%). An intermediary type, cobblestone lissencephaly B, was linked to LARGE mutations (4.5%) identified for the first time in foetuses. We conclude that cobblestone lissencephaly encompasses three distinct subtypes of cortical malformations with different degrees of neuroglial ectopia into the arachnoid space and cortical plate disorganization regardless of gestational age. In the cerebellum, histopathological changes support the novel hypothesis that abnormal lamination arises from a deficiency in granule cells. Our studies demonstrate the positive impact of histoneuropathology on the identification of α-dystroglycanopathies found in 66% of cases, while with neuroimaging criteria and biological values, mutations are found in 32-50% of patients. Interestingly, our morphological classification was central in the orientation of genetic screening of POMT1, POMT2, POMGNT1, LARGE and FKRP. Despite intensive research, one-third of our cases remained unexplained; suggesting that other genes and/or pathways may be involved. This material offers a rich resource for studies on the affected neurodevelopmental processes of cobblestone lissencephaly and on the identification of other responsible gene(s)/pathway(s).

Decreased full breastfeeding, altered practices, perceptions, and infant weight change of prepregnant obese women: a need for extra support.

OBJECTIVE: The purpose of this work was to compare breastfeeding practices, perceptions, and infant weight change of prepregnant obese versus normal-weight mothers in the first 3 months postpartum. PATIENTS AND METHODS: For the prospective case-control study, obese mothers (prepregnant BMI > or = 30 kg/m(2)) were matched with normal-weight mothers (18.5 < or = prepregnant BMI < 25 kg/m(2)) according to initial infant feeding, parity, maternal age, ethnicity, and education. Participants completed an oral questionnaire in the hospital and a telephone interview at 1 and 3 months postpartum. RESULTS: Of 1432 mothers who had given birth at a university hospital in France, 10% were obese. Breastfeeding initiation was lower for obese (48%) versus normal-weight (64%) mothers. A total of 111 of 141 obese mothers were paired with 111 normal-weight mothers. Infant birth weight was similar for newborns of obese and normal-weight mothers. Among mothers who initiated breastfeeding, infant weight gain from 0 to 1 month was lower in breastfed infants of obese mothers compared to normal-weight mothers. Obese mothers were less likely to maintain full breastfeeding at 1 month and 3 months. The percentage of mothers breastfeeding to any extent did not differ between obese and reference women. Obese mothers more often felt uncomfortable breastfeeding in public at 3 months. Fewer obese mothers perceived that their milk supply was sufficient at 1 month and 3 months. Despite greater breastfeeding difficulties, obese mothers were less likely to seek support for breastfeeding in the first 3 months postpartum. CONCLUSIONS: Pediatricians and health professionals should recognize that obese mothers have different breastfeeding practices and perceptions. Extra support and intervention are needed among obese mothers during prenatal and early postnatal periods so that their children can benefit from breastfeeding.

Epithelial phenotypes in the developing human prostate.

An intermediate population has been identified among prostate glands called transiently amplifying (TA) cells, which are characterized by coexpression of basal and luminal cytokeratins (CKs), high proliferation, and lack of p27 expression. These cells are rare in the normal adult prostate and increase in pretumoral conditions, but their importance in the developing gland remains unknown. We analyzed fetal prostates for the expression of CKs (5/6, 18, 19) and factors involved in proliferation and apoptosis: p63, Ki67, p27, epidermal growth factor (EGFR), Bcl2, androgen receptor (AR). Immunostaining was performed on a tissue microarray, including 40 prostates from fetuses aged 13-42 weeks and normal prostate tissue from 10 adults. In both solid buds and the basal compartment of canalized glands, cells expressed p63, CK5/6, CK19, CK18, BCL2, EGFR and were p27 negative. Luminal cells of fetal canalized glands continue to express CK19, EGFR, and BCL2, without p27 expression. In contrast, adult epithelial luminal cells showed diffuse AR and p27 expression, without CK19, BCL2, and EGFR staining. Proliferation was high and diffuse in fetal glands and rare and restricted to basal cells in adult glands. These results indicate that most fetal epithelial prostatic cells exhibit the phenotype of TA cells, suggesting their regulatory function in prostate development.