SRSF1 haploinsufficiency is responsible for a syndromic developmental disorder associated with intellectual disability.

SRSF1 (also known as ASF/SF2) is a non-small nuclear ribonucleoprotein (non-snRNP) that belongs to the arginine/serine (R/S) domain family. It recognizes and binds to mRNA, regulating both constitutive and alternative splicing. The complete loss of this proto-oncogene in mice is embryonically lethal. Through international data sharing, we identified 17 individuals (10 females and 7 males) with a neurodevelopmental disorder (NDD) with heterozygous germline SRSF1 variants, mostly de novo, including three frameshift variants, three nonsense variants, seven missense variants, and two microdeletions within region 17q22 encompassing SRSF1. Only in one family, the de novo origin could not be established. All individuals featured a recurrent phenotype including developmental delay and intellectual disability (DD/ID), hypotonia, neurobehavioral problems, with variable skeletal (66.7%) and cardiac (46%) anomalies. To investigate the functional consequences of SRSF1 variants, we performed in silico structural modeling, developed an in vivo splicing assay in Drosophila, and carried out episignature analysis in blood-derived DNA from affected individuals. We found that all loss-of-function and 5 out of 7 missense variants were pathogenic, leading to a loss of SRSF1 splicing activity in Drosophila, correlating with a detectable and specific DNA methylation episignature. In addition, our orthogonal in silico, in vivo, and epigenetics analyses enabled the separation of clearly pathogenic missense variants from those with uncertain significance. Overall, these results indicated that haploinsufficiency of SRSF1 is responsible for a syndromic NDD with ID due to a partial loss of SRSF1-mediated splicing activity.

Impaired glucose-1,6-biphosphate production due to bi-allelic PGM2L1 mutations is associated with a neurodevelopmental disorder.

We describe a genetic syndrome due to PGM2L1 deficiency. PGM2 and PGM2L1 make hexose-bisphosphates, like glucose-1,6-bisphosphate, which are indispensable cofactors for sugar phosphomutases. These enzymes form the hexose-1-phosphates crucial for NDP-sugars synthesis and ensuing glycosylation reactions. While PGM2 has a wide tissue distribution, PGM2L1 is highly expressed in the brain, accounting for the elevated concentrations of glucose-1,6-bisphosphate found there. Four individuals (three females and one male aged between 2 and 7.5 years) with bi-allelic inactivating mutations of PGM2L1 were identified by exome sequencing. All four had severe developmental and speech delay, dysmorphic facial features, ear anomalies, high arched palate, strabismus, hypotonia, and keratosis pilaris. Early obesity and seizures were present in three individuals. Analysis of the children's fibroblasts showed that glucose-1,6-bisphosphate and other sugar bisphosphates were markedly reduced but still present at concentrations able to stimulate phosphomutases maximally. Hence, the concentrations of NDP-sugars and glycosylation of the heavily glycosylated protein LAMP2 were normal. Consistent with this, serum transferrin was normally glycosylated in affected individuals. PGM2L1 deficiency does not appear to be a glycosylation defect, but the clinical features observed in this neurodevelopmental disorder point toward an important but still unknown role of glucose-1,6-bisphosphate or other sugar bisphosphates in brain metabolism.

NCKAP1 Disruptive Variants Lead to a Neurodevelopmental Disorder with Core Features of Autism.

NCKAP1/NAP1 regulates neuronal cytoskeletal dynamics and is essential for neuronal differentiation in the developing brain. Deleterious variants in NCKAP1 have been identified in individuals with autism spectrum disorder (ASD) and intellectual disability; however, its clinical significance remains unclear. To determine its significance, we assemble genotype and phenotype data for 21 affected individuals from 20 unrelated families with predicted deleterious variants in NCKAP1. This includes 16 individuals with de novo (n = 8), transmitted (n = 6), or inheritance unknown (n = 2) truncating variants, two individuals with structural variants, and three with potentially disruptive de novo missense variants. We report a de novo and ultra-rare deleterious variant burden of NCKAP1 in individuals with neurodevelopmental disorders which needs further replication. ASD or autistic features, language and motor delay, and variable expression of intellectual or learning disability are common clinical features. Among inherited cases, there is evidence of deleterious variants segregating with neuropsychiatric disorders. Based on available human brain transcriptomic data, we show that NCKAP1 is broadly and highly expressed in both prenatal and postnatal periods and demostrate enriched expression in excitatory neurons and radial glias but depleted expression in inhibitory neurons. Mouse in utero electroporation experiments reveal that Nckap1 loss of function promotes neuronal migration during early cortical development. Combined, these data support a role for disruptive NCKAP1 variants in neurodevelopmental delay/autism, possibly by interfering with neuronal migration early in cortical development.

Comprehensive prenatal diagnostics: Exome versus genome sequencing.

OBJECTIVE: This study aimed to assess the diagnostic yield of prenatal genetic testing using trio whole exome sequencing (WES) and trio whole genome sequencing (WGS) in pregnancies with fetal anomalies by comparing the results with conventional chromosomal microarray (CMA) analysis. METHODS: A total of 40 pregnancies with fetal anomalies or increased nuchal translucency (NT ≥ 5 mm) were included between the 12th and 21st week of gestation. Trio WES/WGS and CMA were performed in all cases. RESULTS: The trio WES/WGS analysis increased the diagnostic yield by 25% in cases with negative CMA results. Furthermore, all six chromosomal aberrations identified by CMA were independently detected by WES/WGS analysis. In total, 16 out of 40 cases obtained a genetic sequence variant, copy number variant, or aneuploidy explaining the phenotype, resulting in an overall WES/WGS diagnostic yield of 40%. WES analysis provided a more reliable identification of mosaic sequence variants than WGS because of its higher sequencing depth. CONCLUSIONS: Prenatal WES/WGS proved to be powerful diagnostic tools for fetal anomalies, surpassing the diagnostic yield of CMA. They have the potential to serve as standalone methods for prenatal diagnosis. The study highlighted the limitations of WGS in accurately detecting mosaic variants, which is particularly relevant when analyzing chorionic villus samples.

Variants in ADD1 cause intellectual disability, corpus callosum dysgenesis, and ventriculomegaly in humans.

PURPOSE: Adducins interconnect spectrin and actin filaments to form polygonal scaffolds beneath the cell membranes and form ring-like structures in neuronal axons. Adducins regulate mouse neural development, but their function in the human brain is unknown. METHODS: We used exome sequencing to uncover ADD1 variants associated with intellectual disability (ID) and brain malformations. We studied ADD1 splice isoforms in mouse and human neocortex development with RNA sequencing, super resolution imaging, and immunoblotting. We investigated 4 variant ADD1 proteins and heterozygous ADD1 cells for protein expression and ADD1-ADD2 dimerization. We studied Add1 functions in vivo using Add1 knockout mice. RESULTS: We uncovered loss-of-function ADD1 variants in 4 unrelated individuals affected by ID and/or structural brain defects. Three additional de novo copy number variations covering the ADD1 locus were associated with ID and brain malformations. ADD1 is highly expressed in the neocortex and the corpus callosum, whereas ADD1 splice isoforms are dynamically expressed between cortical progenitors and postmitotic neurons. Human variants impair ADD1 protein expression and/or dimerization with ADD2. Add1 knockout mice recapitulate corpus callosum dysgenesis and ventriculomegaly phenotypes. CONCLUSION: Our human and mouse genetics results indicate that pathogenic ADD1 variants cause corpus callosum dysgenesis, ventriculomegaly, and/or ID.

Loss-of-function variants in SRRM2 cause a neurodevelopmental disorder.

PURPOSE: SRRM2 encodes the SRm300 protein, a splicing factor of the SR-related protein family characterized by its serine- and arginine-enriched domains. It promotes interactions between messenger RNA and the spliceosome catalytic machinery. This gene, predicted to be highly intolerant to loss of function (LoF) and very conserved through evolution, has not been previously reported in constitutive human disease. METHODS: Among the 1000 probands studied with developmental delay and intellectual disability in our database, we found 2 patients with de novo LoF variants in SRRM2. Additional families were identified through GeneMatcher. RESULTS: Here, we report on 22 patients with LoF variants in SRRM2 and provide a description of the phenotype. Molecular analysis identified 12 frameshift variants, 8 nonsense variants, and 2 microdeletions of 66 kb and 270 kb. The patients presented with a mild developmental delay, predominant speech delay, autistic or attention-deficit/hyperactivity disorder features, overfriendliness, generalized hypotonia, overweight, and dysmorphic facial features. Intellectual disability was variable and mild when present. CONCLUSION: We established SRRM2 as a gene responsible for a rare neurodevelopmental disease.

Mono-allelic loss of YTHDF3 and neurodevelopmental disorder: clinical features of four individuals with 8q12.3 deletions.

The YTH domain family member 3 gene (YTHDF3) encodes a reader of the abundant N6-methyladenosine (m6 A) modification of eukaryotic mRNA, which plays an essential role in regulating mRNA stability and is necessary to achieve normal development of the central nervous system in animal models. YTHDF3 has not previously been implicated in Mendelian disease despite a high probability of loss of function intolerance and statistical evidence of enrichment for gene-disruptive de novo variants in large-scale studies of individuals with intellectual disability and/or developmental delay. We report four individuals with deletion of 8q12.3, deletion size 1.38-2.60 Mb, encompassing YTHDF3, three of them were de novo, and in one case, the inheritance was unknown. Common features of the individuals (age range, 4-22 years) were developmental delay and/or intellectual disability. Two individuals underwent squint surgery. We suggest that haploinsufficiency of YTHDF3 causes a neurodevelopmental disorder with developmental delay and intellectual disability of variable degree.

ZMYND11 variants are a novel cause of centrotemporal and generalised epilepsies with neurodevelopmental disorder.

ZMYND11 is the critical gene in chromosome 10p15.3 microdeletion syndrome, a syndromic cause of intellectual disability. The phenotype of ZMYND11 variants has recently been extended to autism and seizures. We expand on the epilepsy phenotype of 20 individuals with pathogenic variants in ZMYND11. We obtained clinical descriptions of 16 new and nine published individuals, plus detailed case history of two children. New individuals were identified through GeneMatcher, ClinVar and the European Network for Therapies in Rare Epilepsy (NETRE). Genetic evaluation was performed using gene panels or exome sequencing; variants were classified using American College of Medical Genetics (ACMG) criteria. Individuals with ZMYND11 associated epilepsy fell into three groups: (i) atypical benign partial epilepsy or idiopathic focal epilepsy (n = 8); (ii) generalised epilepsies/infantile epileptic encephalopathy (n = 4); (iii) unclassified (n = 8). Seizure prognosis ranged from spontaneous remission to drug resistant. Neurodevelopmental deficits were invariable. Dysmorphic features were variable. Variants were distributed across the gene and mostly de novo with no precise genotype-phenotype correlation. ZMYND11 is one of a small group of chromatin reader genes associated in the pathogenesis of epilepsy, and specifically ABPE. More detailed epilepsy descriptions of larger cohorts and functional studies might reveal genotype-phenotype correlation. The epileptogenic mechanism may be linked to interaction with histone H3.3.

National data on the early clinical use of non-invasive prenatal testing in public and private healthcare in Denmark 2013-2017.

INTRODUCTION: In Denmark, non-invasive prenatal testing (NIPT) has been used since 2013. We aimed to evaluate the early clinical use of NIPT in Danish public and private healthcare settings before NIPT became an integrated part of the national guidelines on prenatal screening and diagnosis in 2017. MATERIAL AND METHODS: NIPT data were collected between March 2013 and June 2017 from national public registries and private providers. Results from follow-up samples (chorionic villi, amniotic fluid, postnatal blood or fetal tissue) were included from The Danish Cytogenetics Central Registry and indications and outcome from The Danish Fetal Medicine Database. RESULTS: A total of 3936 NIPT results were included in the study from public hospitals (n = 3463, 88.0%) and private clinics (n = 473, 12.0%). The total number of prenatal tests was 19 713 during the study period: 20% were NIPT analyses (n = 3936) and 80% invasive procedures (n = 15 777). Twenty-five percent of NIPTs in the private clinics were performed before gestational week 11+0 , whereas NIPT in public settings was used only after combined first trimester screening (P < .001). Regardless of indication, the national public sensitivity was 96.9% (95% CI 82.0%-99.8%) for trisomy 21, 100% (95% CI 46.3%-100%) for trisomy 18, 100% (95% CI 5.5%-100%) for trisomy 13, and 87.0% (95% CI 74.5%-92.4%) for any fetal chromosomal aberration. Forty-seven true-positive NIPT results included cases of common aneuplodies (trisomy 21, n = 31; trisomy 18, n = 5; and trisomy 13, n = 1), sex chromosomal aberrations (n = 7) and atypical chromosomal aberrations (n = 3). One false-negative NIPT result occurred (trisomy 21). Of 47 cases, 21 (45%) cases with a true-positive NIPT result resulted in live births by choice; 11 of these children had Down and 4 had Edwards syndrome. CONCLUSIONS: The total number of NIPT analyses was low compared with the number of invasive procedures in the implementation period. In contrast to the generally high termination rate after a positive result following invasive testing in Denmark, a high proportion of true-positive NIPT results from the public setting resulted in live births. NIPT may be an important risk-free alternative to invasive testing for a minority of women in the public setting who wish to use prenatal genetic testing for information only and not for reproductive decision-making.

YY1 Haploinsufficiency Causes an Intellectual Disability Syndrome Featuring Transcriptional and Chromatin Dysfunction.

Yin and yang 1 (YY1) is a well-known zinc-finger transcription factor with crucial roles in normal development and malignancy. YY1 acts both as a repressor and as an activator of gene expression. We have identified 23 individuals with de novo mutations or deletions of YY1 and phenotypic features that define a syndrome of cognitive impairment, behavioral alterations, intrauterine growth restriction, feeding problems, and various congenital malformations. Our combined clinical and molecular data define "YY1 syndrome" as a haploinsufficiency syndrome. Through immunoprecipitation of YY1-bound chromatin from affected individuals' cells with antibodies recognizing both ends of the protein, we show that YY1 deletions and missense mutations lead to a global loss of YY1 binding with a preferential retention at high-occupancy sites. Finally, we uncover a widespread loss of H3K27 acetylation in particular on the YY1-bound enhancers, underscoring a crucial role for YY1 in enhancer regulation. Collectively, these results define a clinical syndrome caused by haploinsufficiency of YY1 through dysregulation of key transcriptional regulators.

A new 1p36.13-1p36.12 microdeletion syndrome characterized by learning disability, behavioral abnormalities, and ptosis.

Two 1p36 contiguous gene deletion syndromes are known so far: the terminal 1p36 deletion syndrome and a 1p36 deletion syndrome with a critical region located more proximal at 1p36.23-1p36.22. We present even more proximally located overlapping deletions from seven individuals, with the smallest region of overlap comprising 1 Mb at 1p36.13-1p36.12 (chr1:19077793-20081292 (GRCh37/hg19)) defining a new contiguous gene deletion syndrome. The characteristic features of this new syndrome are learning disability or mild intellectual disability, speech delay, behavioral abnormalities, and ptosis. The genes UBR4 and CAPZB are considered the most likely candidate genes for the features of this new syndrome.

Estimating the effect size of the 15Q11.2 BP1-BP2 deletion and its contribution to neurodevelopmental symptoms: recommendations for practice.

BACKGROUND: The 15q11.2 deletion is frequently identified in the neurodevelopmental clinic. Case-control studies have associated the 15q11.2 deletion with neurodevelopmental disorders, and clinical case series have attempted to delineate a microdeletion syndrome with considerable phenotypic variability. The literature on this deletion is extensive and confusing, which is a challenge for genetic counselling. The aim of this study was to estimate the effect size of the 15q11.2 deletion and quantify its contribution to neurodevelopmental disorders. METHODS: We performed meta-analyses on new and previously published case-control studies and used statistical models trained in unselected populations with cognitive assessments. We used new (n=241) and previously published (n=150) data from a clinically referred group of deletion carriers. 15q11.2 duplications (new n=179 and previously published n=35) were used as a neutral control variant. RESULTS: The deletion decreases IQ by 4.3 points. The estimated ORs and respective frequencies in deletion carriers for intellectual disabilities, schizophrenia and epilepsy are 1.7 (3.4%), 1.5 (2%) and 3.1 (2.1%), respectively. There is no increased risk for heart malformations and autism. In the clinically referred group, the frequency and nature of symptoms in deletions are not different from those observed in carriers of the 15q11.2 duplication suggesting that most of the reported symptoms are due to ascertainment bias. CONCLUSIONS: We recommend that the deletion should be classified as 'pathogenic of mild effect size'. Since it explains only a small proportion of the phenotypic variance in carriers, it is not worth discussing in the developmental clinic or in a prenatal setting.

Deletion of T-type calcium channels Cav3.1 or Cav3.2 attenuates endothelial dysfunction in aging mice.

Impairment of endothelial function with aging is accompanied by reduced nitric oxide (NO) production. T-type Cav3.1 channels augment nitric oxide and co-localize with eNOS. Therefore, the hypothesis was that T-type channels contribute to the endothelial dysfunction of aging. Endothelial function was determined in mesenteric arteries (perfusion) and aortae (isometric contraction) of young and old wild-type (WT), Cav3.1, and Cav3.2 knockout mice. NO production was measured by fluorescence imaging in mesenteric arteries. With age, endothelium-dependent subsequent dilatation (following depolarization with KCl) of mesenteric arteries was diminished in the arteries of WT mice, unchanged in Cav3.2-/- preparations but increased in those of Cav3.1-/- mice. NO synthase inhibition abolished the subsequent dilatation in mesenteric arteries and acetylcholine-induced relaxations in aortae. NO levels were significantly reduced in mesenteric arteries of old compared to young WT mice. In Cav3.1-/- and Cav3.2-/- preparations, NO levels increased significantly with age. Relaxations to acetylcholine were significantly smaller in the aortae of old compared to young WT mice, while such responses were comparable in preparations of young and old Cav3.1-/- and Cav3.2-/- mice. The expression of Cav3.1 was significantly reduced in aortae from aged compared to young WT mice. The level of phosphorylated eNOS was significantly increased in aortae from aged Cav3.1-/- mice. In conclusion, T-type calcium channel-deficient mice develop less age-dependent endothelial dysfunction. Changes in NO levels are involved in this phenomenon in WT and Cav3.1-/- mice. These findings suggest that T-type channels play an important role in age-induced endothelial dysfunction.

17q12 deletion and duplication syndrome in Denmark-A clinical cohort of 38 patients and review of the literature.

17q12 deletions and duplications are two distinct, recurrent chromosomal aberrations usually diagnosed by chromosomal microarray analysis (CMA). The aberrations encompass the genes, HNF1B, LHX1, and ACACA, among others. We here describe a large national cohort of 12 phenotyped patients with 17q12 deletions and 26 phenotyped patients with 17q12 duplications. The total cohort includes 19 index patients and 19 family members. We also reviewed the literature in order to further improve the basis for the counseling. We emphasize that renal disease, learning disability, behavioral abnormalities, epilepsy, autism, schizophrenia, structural brain abnormalities, facial dysmorphism, and joint laxity are features seen in both the 17q12 deletion syndrome and the reciprocal 17q12 duplication syndrome; and we extend the list of features seen in both patient categories to include strabismus, esophageal defects, and duodenal atresia. Delayed language development, learning disability, kidney involvement, and eye dysmorphism and strabismus were the most consistently shared features among patients with 17q12 deletion. Patients with 17q12 duplications were characterized by an extremely wide phenotypic spectrum, including a variable degree of learning disabilities, delayed language development, delayed motor milestones, and a broad range of psychiatric and neurological features. This patient group also included adults achieving an academic degree. Assessing index patients and non-index patients separately, our observations illustrate that an overall milder disease burden is seen, in particular in patients with 17q12 duplications who are ascertained on the duplication rather than the phenotype. This evidence may be useful in prenatal counseling. © 2016 Wiley Periodicals, Inc.

S100A14 is a novel independent prognostic biomarker in the triple-negative breast cancer subtype.

Triple-negative breast cancer (TNBC) represents a heterogeneous subgroup with generally poor outcome and lack of an effective targeted therapy. Prognostic or predictive biomarkers to guide treatment decisions for this group of patients are needed. To evaluate the potential of S100A14 protein as a novel biomarker in TNBC, the protein expression of S100A14 was correlated with clinical outcomes in a Pilot Sample set and a Danish cohort of predominantly TNBC patients. Kaplan-Meier analysis identified a prognostic impact of S100A14 on disease-free survival and overall survival, showing that tumors with high S100A14 protein expression levels were significantly correlated with poor outcome in TNBC patients (p = 0.017; p = 0.038), particularly those in the basal-like subgroup (p = 0.006; p = 0.037). Importantly, TNBC patients with high S100A14 expression, but tumor-negative axillary lymph nodes (N-), had equally poor outcomes as those with tumor-positive axillary lymph nodes (N+), while TNBC/N- patients with low S100A14 expression had a significantly better disease free survival (p = 0.013). Multivariate analysis revealed that S100A14 is an independent prognostic factor for TNBC patients (p = 0.024; p = 0.05). At the cellular level, S100A14 was found to be expressed in epithelial-like, but not in mesenchymal-like, TNBC cells in vitro. S100A14 is an independent prognostic factor in TNBC and a novel potential therapeutic target in TNBC.

Quantification of morphine, morphine 6-glucuronide, buprenorphine, and the enantiomers of methadone by enantioselective mass spectrometric chromatography in whole blood.

PURPOSE: Deaths among drug addicts are frequently caused by intoxication with methadone and/or morphine. These drugs are often used in combination with other drugs, such as buprenorphine. In addition, methadone is generally used as a mixture of R- and S-enantiomers. To date, a method for separation and quantitation of these specific drugs has not been developed. The aim of this study was to develop a sensitive enantioselective method for quantitation of morphine, its active metabolite morphine 6-glucuronide, buprenorphine, and R- and S-methadone, in a single analytical run. METHODS: Whole blood samples were diluted with 0.5 mol/L ammonium carbonate buffer and extracted on a Bond Elut C18 solid-phase extraction column with an automatic solid-phase extraction system. Chromatographic separation was performed on a chiral alpha-1-acid glycoprotein column with an acetonitrile/ammonium acetate buffer (10 mmol/L, pH 7.0, 22:78 v/v) mobile phase. The whole blood concentrations of the drugs were quantified by mass spectrometry using their stable isotope-labeled compounds as internal standards. RESULTS: The method was validated with respect to specificity, linearity, precision, limits of detection, and quantification and matrix effects. The precision (coefficient of variation) was below 15%, and the accuracy was between 90 and 115%. CONCLUSIONS: This method will be useful for routine analyses in forensic laboratories where blood samples are frequently analyzed for drugs of abuse. In some cases, sudden death from methadone overdose is caused by the enantiomeric form of the methadone, which makes the enantiomer separation capability of this method important.

Frail inner limiting membrane maculopathy suggested to describe a new retinal Alport-like condition with two variants in three generations of females.

BACKGROUND: We report a three-generation family with isolated Alport-like retinal abnormalities in the absence of lenticonus, hearing loss, kidney disease, and detectable molecular genetic defects in known Alport-related genes. METHODS: Clinical examination includes ocular biomicroscopy, fundus photography, optical coherence tomography, dipstick urinalysis, serum creatinine assessment, and molecular genetic analysis. RESULTS: The proband, her mother, and her maternal grandmother had normal best-corrected visual acuity and normal visual fields in both eyes. The macula presented a petaloid stair-case profile with scarce vessels in both eyes of the proband and a flat temporal macula lacking a foveal avascular zone in her mother and her grandmother. No family member had renal symptoms, unexplained subnormal hearing, or lenticonus. Sequencing and MLPA found no defect in COL4A3, COL4A4, and COL4A5. Common SNPs around the genes ± 1Mb showed no segregation. Furthermore, none of the variants shared between the affected individuals in genes from a gene panel of genes relevant for ophthalmopathy nor whole exome- and genome sequencing explained the phenotype. CONCLUSION: A new condition with two retinal Alport-like phenotypes was found. No abnormalities of the kidneys and lens were found, neither abnormalities of the type IV collagen genes related to Alport syndrome. Homology with retinal abnormalities seen in patients after surgical removal of the inner limiting membrane of the retina suggests that this is where the defect is located. We therefore suggest that the new retinal phenotypes and similar phenotypes can be described with the new definition "frail inner limiting membrane maculopathy."