Postnatal outcome of children with antenatal colonic hyperechogenicity.

OBJECTIVE: To evaluate the postnatal outcome of children with antenatal colonic hyperechogenicity, currently considered as a sign of lysinuria-cystinuria, but which may also be a sign of other disorders with a more severe prognosis. METHOD: We carried out a French multi-centric retrospective study via 15 Multidisciplinary Center for Prenatal Diagnosis from January 2011 to January 2021. We included pregnancies for which fetal colonic hyperechogenicity had been demonstrated. We collected the investigations performed during pregnancy and at birth as well as the main clinical features of the mother and the child. We then established the prevalence of pathologies such as lysinuria-cystinuria (LC), hypotonia-cystinuria syndrome (HC), or lysinuric protein intolerance (LPI). RESULTS: Among the 33 cases of colonic hyperechogenicity collected, and after exclusion of those lost to follow-up, we identified 63% of children with lysinuria-cystinuria, 8% with lysinuric rotein intolerance, and 4% with hypotonia-cystinuria syndrome. CONCLUSION: Management of prenatal hyperechoic colon should include a specialized consultation with a clinical geneticist to discuss further investigations, which could include invasive amniotic fluid sampling for molecular diagnosis. A better understanding of diagnoses and prognosis should improve medical counseling and guide parental decision making.

Prenatal diagnosis by trio exome sequencing in fetuses with ultrasound anomalies: A powerful diagnostic tool.

Introduction: Prenatal ultrasound (US) anomalies are detected in around 5%-10% of pregnancies. In prenatal diagnosis, exome sequencing (ES) diagnostic yield ranges from 6% to 80% depending on the inclusion criteria. We describe the first French national multicenter pilot study aiming to implement ES in prenatal diagnosis following the detection of anomalies on US. Patients and methods: We prospectively performed prenatal trio-ES in 150 fetuses with at least two US anomalies or one US anomaly known to be frequently linked to a genetic disorder. Trio-ES was only performed if the results could influence pregnancy management. Chromosomal microarray (CMA) was performed before or in parallel. Results: A causal diagnosis was identified in 52/150 fetuses (34%) with a median time to diagnosis of 28 days, which rose to 56/150 fetuses (37%) after additional investigation. Sporadic occurrences were identified in 34/56 (60%) fetuses and unfavorable vital and/or neurodevelopmental prognosis was made in 13/56 (24%) fetuses. The overall diagnostic yield was 41% (37/89) with first-line trio-ES versus 31% (19/61) after normal CMA. Trio-ES and CMA were systematically concordant for identification of pathogenic CNV. Conclusion: Trio-ES provided a substantial prenatal diagnostic yield, similar to postnatal diagnosis with a median turnaround of approximately 1 month, supporting its routine implementation during the detection of prenatal US anomalies.

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.

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.

Hypolacrimia and Alacrimia as Diagnostic Features for Genetic or Congenital Conditions.

As part of the lacrimal apparatus, the lacrimal gland participates in the maintenance of a healthy eye surface by producing the aqueous part of the tear film. Alacrimia and hypolacrimia, which are relatively rare during childhood or young adulthood, have their origin in a number of mechanisms which include agenesia, aplasia, hypoplasia, or incorrect maturation of the gland. Moreover, impaired innervation of the gland and/or the cornea and alterations of protein secretion pathways can lead to a defective tear film. In most conditions leading to alacrimia or hypolacrimia, however, the altered tear film is only one of numerous defects that arise and therefore is commonly disregarded. Here, we have systematically reviewed all of those genetic conditions or congenital disorders that have alacrimia or hypolacrimia as a feature. Where it is known, we describe the mechanism of the defect in question. It has been possible to clearly establish the physiopathology of only a minority of these conditions. As hypolacrimia and alacrimia are rare features, this review could be used as a tool in clinical genetics to perform a quick diagnosis, necessary for appropriate care and counseling.

Rapid exome sequencing in critically ill infants: implementation in routine care from French regional hospital's perspective.

This monocentric study included fifteen children under a year old in intensive care with suspected monogenic conditions for rapid trio exome sequencing (rES) between April 2019 and April 2021. The primary outcome was the time from blood sampling to rapid exome sequencing report to parents. All results were available within 16 days and were reported to parents in or under 16 days in 13 of the 15 individuals (86%). Six individuals (40%) received a diagnosis with rES, two had a genetic condition not diagnosed by rES. Eight individuals had their care impacted by their rES results, four were discharged or died before the results. This small-scale study shows that rES can be implemented in a regional University hospital with rapid impactful diagnosis to improve care in critically ill infants.

Extending the prenatal Noonan's phenotype by review of ultrasound and autopsy data.

OBJECTIVES: The antenatal phenotypic spectrum of Noonan Syndrome (NS) requires better characterization. METHODS: This multicenter retrospective observational included 16 fetuses with molecularly confirmed NS admitted for fetopathological examination between 2009 and 2016. RESULTS: Among 12 pathogenic variants (PV) in PTPN11 (80%), 5 (42%) fell between position c.179 and c.182. Ultrasound showed increased nuchal translucency (n = 13/16, 93%), increased nuchal fold after 15 weeks of gestation (n = 12/16, 75%), pleural effusions (n = 11/16, 69%), polyhydramnios (n = 9/16, 56%), hydrops (n = 7/16, 44%), cardiovascular (n = 6/16, 38%) and cerebral (n = 4/16, 25%) anomalies. Fetopathological examination found dysmorphic features in all cases, cardiovascular anomalies (n = 12/15, 80%), pulmonary hypoplasia (n = 10/15, 67%), effusions (n = 7/15, 47%) and neuropathological anomalies (n = 5/15, 33%). Hydrops was significantly (p = 0.02) more frequent in the four fetuses with RIT1, NRAS and RAF1 PV versus the 12 fetuses with PTPN11 PV. CONCLUSIONS: Increased nuchal translucency and nuchal fold is common in NS. Noonan Syndrome antenatal phenotype showed high in utero fetal death, hydrops, prenatal pleural effusion and pulmonary hypoplasia, although the inclusion of only deceased fetuses will have selected more severe phenotypes. Non-specific cardiovascular and neurological abnormalities should be added to NS antenatal phenotype. Next generation sequencing will help detect more genotypes, clarifying the prenatal phenotype and identifying genotype-phenotype correlations.

Use of Remifentanil Associated with Lidocaine for Feticides in Late Terminations of Pregnancy: A Randomized Clinical Trial.

INTRODUCTION: In France, performance of a termination of pregnancy is legally possible without any gestational age limit. After 22 weeks of gestation, a feticide is ethically performed using usually sufentanil and lidocaine. The aim of this study was to compare the use of remifentanil, a fast-acting morphine-derivating product, instead of sufentanil. METHODS: This 2-center randomized, controlled, single-blinded phase-III treatment trial had 2 parallel arms: an experimental group using remifentanil with lidocaine versus a control group receiving sufentanil associated with lidocaine. This trial took place over a 40-month period. The primary outcome was time to fetal asystole after lidocaine injection. The secondary outcome measures were the procedure's success rate, the rate of serious maternal side effects, and the presence of cellular or tissue modifications. RESULTS: The study included 66 women, randomized into 2 groups of similar size and characteristics. Time to fetal asystole did not differ significantly between the groups, with a delay of 4 min (Q1-Q3, 2-11) in the sufentanil group and 4 min (Q1-Q3, 1-10) in the remifentanil group (p = 0.84). Similarly, the success rate of the procedure did not differ significantly. Fetal asystole was procured in <2 min and persisted >1 min for 16 (25.8%) women in our total population: 7 (22.5%) in the sufentanil group and 9 (29.0%) in the remifentanil group, p = 0.77. No severe maternal side effects were observed. Among the 49 fetopathological examinations performed, the few tissue and cell modifications observed did not cause any interpretation difficulties in either group. DISCUSSION/CONCLUSION: Use of remifentanil instead of sufentanil for feticide procedure did not improve time to fetal asystole. No harmful effect was observed for either maternal tolerance or interpretation of the histologic slides.

Prenatal ultrasound diagnosis of cleft palate without cleft lip, the new ultrasound semiology.

OBJECTIVES: The aim of this study was to define the prenatal ultrasound semiology of cleft palate without cleft lip using 3D visualization of the fetal palate. METHODS: A prospective longitudinal study was performed in our University Hospital Center from 2011 to 2018. The fetal secondary palate was studied in 3D, starting with 2D axial transverse ultrasound view. We defined a cleft palate as a disruption of the horizontal plate of the palatine bone of the secondary palate. Prenatal findings were correlated to anatomic postnatal examinations performed by a paediatric plastic surgeon. RESULTS: Forty-three cases of cleft palate without cleft lip were prenatally diagnosed, of whom 34 were associated with malformations. We defined four types of disruptive appearances: isolated nonvisualization of the posterior nasal spine; partial-disruption or cleft velum; complete disappearance or V-shaped cleft palate; and complete disappearance or U-shaped cleft palate. The adjusted kappa coefficient, between prenatal and postnatal evaluation, was 0.88 (95% CI: 0.79-0.97), corresponding to an excellent agreement. CONCLUSIONS: Using a strictly axial transverse ultrasound view, visualization of the secondary fetal palate enables to diagnose a cleft palate without cleft lip. This method offers a prenatal anatomic classification of cleft palate with a high level of concordance to postnatal findings.

Further delineation of the clinical spectrum of KAT6B disorders and allelic series of pathogenic variants.

PURPOSE: Genitopatellar syndrome and Say-Barber-Biesecker-Young-Simpson syndrome are caused by variants in the KAT6B gene and are part of a broad clinical spectrum called KAT6B disorders, whose variable expressivity is increasingly being recognized. METHODS: We herein present the phenotypes of 32 previously unreported individuals with a molecularly confirmed diagnosis of a KAT6B disorder, report 24 new pathogenic KAT6B variants, and review phenotypic information available on all published individuals with this condition. We also suggest a classification of clinical subtypes within the KAT6B disorder spectrum. RESULTS: We demonstrate that cerebral anomalies, optic nerve hypoplasia, neurobehavioral difficulties, and distal limb anomalies other than long thumbs and great toes, such as polydactyly, are more frequently observed than initially reported. Intestinal malrotation and its serious consequences can be present in affected individuals. Additionally, we identified four children with Pierre Robin sequence, four individuals who had increased nuchal translucency/cystic hygroma prenatally, and two fetuses with severe renal anomalies leading to renal failure. We also report an individual in which a pathogenic variant was inherited from a mildly affected parent. CONCLUSION: Our work provides a comprehensive review and expansion of the genotypic and phenotypic spectrum of KAT6B disorders that will assist clinicians in the assessment, counseling, and management of affected individuals.

Disruption of chromatin organisation causes MEF2C gene overexpression in intellectual disability: a case report.

BACKGROUND: Balanced structural variants are mostly described in disease with gene disruption or subtle rearrangement at breakpoints. CASE PRESENTATION: Here we report a patient with mild intellectual deficiency who carries a de novo balanced translocation t(3;5). Breakpoints were fully explored by microarray, Array Painting and Sanger sequencing. No gene disruption was found but the chromosome 5 breakpoint was localized 228-kb upstream of the MEF2C gene. The predicted Topologically Associated Domains analysis shows that it contains only the MEF2C gene and a long non-coding RNA LINC01226. RNA studies looking for MEF2C gene expression revealed an overexpression of MEF2C in the lymphoblastoid cell line of the patient. CONCLUSIONS: Pathogenicity of MEF2C overexpression is still unclear as only four patients with mild intellectual deficiency carrying 5q14.3 microduplications containing MEF2C are described in the literature. The microduplications in these individuals also contain other genes expressed in the brain. The patient presented the same phenotype as 5q14.3 microduplication patients. We report the first case of a balanced translocation leading to an overexpression of MEF2C similar to a functional duplication.

Report on three additional patients and genotype-phenotype correlation in SLC25A22-related disorders group.

Early infantile epileptic encephalopathy (EIEE) is a heterogeneous group of severe forms of age-related developmental and epileptic encephalopathies with onset during the first weeks or months of life. The interictal electroencephalogram (EEG) shows a "suppression burst" (SB) pattern. The prognosis is usually poor and most children die within the first two years or survive with very severe intellectual disabilities. EIEE type 3 is caused by variants affecting function, in SLC25A22, which is also responsible for epilepsy of infancy with migrating focal seizures (EIMFS). We report a family with a less severe phenotype of EIEE type 3. We performed exome sequencing and identified two unreported variants in SLC25A22 in the compound heterozygous state: NM_024698.4: c.[813_814delTG];[818 G>A] (p.[Ala272Glnfs*144];[Arg273Lys]). Functional studies in cultured skin fibroblasts from a patient showed that glutamate oxidation was strongly defective, based on a literature review. We clustered the 18 published patients (including those from this family) into three groups according to the severity of the SLC25A22-related disorders. In an attempt to identify genotype-phenotype correlations, we compared the variants according to the location depending on the protein domains. We observed that patients with two variants located in helical transmembrane domains presented a severe phenotype, whereas patients with at least one variant outside helical transmembrane domains presented a milder phenotype. These data are suggestive of a continuum of disorders related to SLC25A22 that could be called SLC25A22-related disorders. This might be a first clue to enable geneticists to outline a prognosis based on genetic molecular data regarding the SLC25A22 gene.

Correlation between RB1germline mutations and second primary malignancies in hereditary retinoblastoma patients treated with external beam radiotherapy.

Retinoblastoma (Rb) results from biallelic inactivation of the RB1 gene. Hereditary Rb patients i. e germline carriers of a RB1 mutation also have a risk of developing subsequent malignant neoplasms (SMN) such as osteosarcomas. This SMN risk is maximized by external beam radiotherapy treatments (EBRT), which is why these treatments are now avoided. Nevertheless, EBRT is still a matter of great concern, as EBRT-treated patients are in their adulthood and SMNs remain the major cause of death for patients. To decipher the relationship between RB1 genotype and SMN development in EBRT treated patients, we conducted a retrospective study in a cohort of 160 irradiated hereditary Rbs with fully resolved RB1 mutational status. Median follow-up was 22 years [1-51] and median age of patients was 27 years old [7-53]. Among these 160 Rb patients, 120 did not develop any SMN (75%) and 40 developed SMNs (25%). The age at which EBRT is given (i.e. before or after the age of 12 months) was not correlated to SMN development (p = 0.6). We didn't find any difference in RB1 mutation type between patients with or without SMN, neither could we detect any linkage between mutation type and SMN location, SMN type and age at diagnosis. Interestingly, among 13 carriers of a RB1 low penetrance mutation, 3 of them developed sarcomas, a rare tumor that cannot be attributed to the general population. Our study cannot explain why a RB1 mutation leads or not to a SMN but demonstrated that EBRT patients with a low penetrance mutation remain at risk of SMN and should be cautiously monitored.

Recessive Spondylocarpotarsal Synostosis Syndrome Due to Compound Heterozygosity for Variants in MYH3.

Spondylocarpotarsal synostosis syndrome (SCTS) is characterized by intervertebral fusions and fusion of the carpal and tarsal bones. Biallelic mutations in FLNB cause this condition in some families, whereas monoallelic variants in MYH3, encoding embryonic heavy chain myosin 3, have been implicated in dominantly inherited forms of the disorder. Here, five individuals without FLNB mutations from three families were hypothesized to be affected by recessive SCTS on account of sibling recurrence of the phenotype. Initial whole-exome sequencing (WES) showed that all five were heterozygous for one of two independent splice-site variants in MYH3. Despite evidence indicating that three of the five individuals shared two allelic haplotypes encompassing MYH3, no second variant could be located in the WES datasets. Subsequent genome sequencing of these three individuals demonstrated a variant altering a 5' UTR splice donor site (rs557849165 in MYH3) not represented by exome-capture platforms. When the cohort was expanded to 16 SCTS-affected individuals without FLNB mutations, nine had truncating mutations transmitted by unaffected parents, and six inherited the rs557849165 variant in trans, an observation at odds with the population allele frequency for this variant. The rs557849165 variant disrupts splicing in the 5' UTR but is still permissive of MYH3 translational initiation, albeit with reduced efficiency. Although some MYH3 variants cause dominant SCTS, these data indicate that others (notably truncating variants) do not, except in the context of compound heterozygosity for a second hypomorphic allele. These observations make genetic diagnosis challenging in the context of simplex presentations of the disorder.

WDR26 Haploinsufficiency Causes a Recognizable Syndrome of Intellectual Disability, Seizures, Abnormal Gait, and Distinctive Facial Features.

We report 15 individuals with de novo pathogenic variants in WDR26. Eleven of the individuals carry loss-of-function mutations, and four harbor missense substitutions. These 15 individuals comprise ten females and five males, and all have intellectual disability with delayed speech, a history of febrile and/or non-febrile seizures, and a wide-based, spastic, and/or stiff-legged gait. These subjects share a set of common facial features that include a prominent maxilla and upper lip that readily reveal the upper gingiva, widely spaced teeth, and a broad nasal tip. Together, these features comprise a recognizable facial phenotype. We compared these features with those of chromosome 1q41q42 microdeletion syndrome, which typically contains WDR26, and noted that clinical features are consistent between the two subsets, suggesting that haploinsufficiency of WDR26 contributes to the pathology of 1q41q42 microdeletion syndrome. Consistent with this, WDR26 loss-of-function single-nucleotide mutations identified in these subjects lead to nonsense-mediated decay with subsequent reduction of RNA expression and protein levels. We derived a structural model of WDR26 and note that missense variants identified in these individuals localize to highly conserved residues of this WD-40-repeat-containing protein. Given that WDR26 mutations have been identified in ∼1 in 2,000 of subjects in our clinical cohorts and that WDR26 might be poorly annotated in exome variant-interpretation pipelines, we would anticipate that this disorder could be more common than currently appreciated.

A case of mild CHARGE syndrome associated with a splice site mutation in CHD7.

CHARGE syndrome (MIM#214800) (Coloboma, Heart defect, Atresia of choanae, Retarded growth and development, Genital hypoplasia, Ear abnormalities/deafness) is caused by heterozygous mutation of CHD7 transmitted in an autosomal dominant manner. In this report, we describe a patient with bilateral hearing impairment, unusually-shaped ears, no intellectual disability and a patent ductus arteriosus. Further investigation showed abnormal semicircular canals and the presence of olfactory bulbs. He does not fulfill the Blake or the Verloes criteria for CHARGE. A de novo mutation at the donor splice site of intron 33 was identified (c.7164 + 1G > A). It is of importance to diagnose mildly affected patients for appropriate genetic counselling and to better understand the mild end of the phenotypic spectrum of CHARGE syndrome.

First fetal case of the 8q24.3 contiguous genes syndrome.

Molecular cytogenetics, particularly array-CGH, opened the way to the « genotype first approach ¼ and for the discovery of new micro rearrangement syndromes. This was the case for the 8q24.3 microdeletion syndrome. Here, we describe the phenotype of a fetus with a 8q24.3 deletion. This rare condition has to be considered as a contiguous genes syndrome because its phenotype is generated by the SCRIB and PUF60 adjacent gene endophenotypes. The fetus presented atrioventricular septal defect and hypoplastic aortic arch, facial dysmorphism, microretrognathia, dysmorphic ears, clinodactyly of the 5th digit on both hands, mild rocker bottom feet and abnormal third sacral vertebra. This fetus is the first case where the endophenotype produced by SCRIB gene is absent. This case is compared with the previous published cases.