DNA methylation episignature for Witteveen-Kolk syndrome due to SIN3A haploinsufficiency.

PURPOSE: Witteveen-Kolk syndrome (WITKOS) is a rare, autosomal dominant neurodevelopmental disorder caused by heterozygous loss-of-function alterations in the SIN3A gene. WITKOS has variable expressivity that commonly overlaps with other neurodevelopmental disorders. In this study, we characterized a distinct DNA methylation epigenetic signature (episignature) distinguishing WITKOS from unaffected individuals as well as individuals with other neurodevelopmental disorders with episignatures and described 9 previously unpublished individuals with SIN3A haploinsufficiency. METHODS: We studied the phenotypic characteristics and the genome-wide DNA methylation in the peripheral blood samples of 20 individuals with heterozygous alterations in SIN3A. A total of 14 samples were used for the identification of the episignature and building of a predictive diagnostic biomarker, whereas the diagnostic model was used to investigate the methylation pattern of the remaining 6 samples. RESULTS: A predominantly hypomethylated DNA methylation profile specific to WITKOS was identified, and the classifier model was able to diagnose a previously unresolved test case. The episignature was sensitive enough to detect individuals with varying degrees of phenotypic severity carrying SIN3A haploinsufficient variants. CONCLUSION: We identified a novel, robust episignature in WITKOS due to SIN3A haploinsufficiency. This episignature has the potential to aid identification and diagnosis of individuals with WITKOS.

Evaluation of the impact of the 2021 revised Neurofibromatosis type 1 diagnostic criteria on time to diagnosis.

Neurofibromatosis type 1 (NF1) has historically been diagnosed clinically based on the NIH Consensus Conference diagnostic criteria. The molecular and clinical knowledge of NF1 has subsequently improved, and an international group of experts published revised diagnostic criteria in 2021, incorporating new diagnostic criteria such as pathogenic variants in NF1. This study aimed to investigate the impact of these new diagnostic criteria on time to diagnosis (TTD) of NF1. A retrospective chart review of individuals evaluated for a diagnosis of NF1 at the Medical Genetics Clinic at Stanford Children's Health was performed. The TTD was determined by calculating the days between their first visit with a medical geneticist for NF1 and the date they would have received a diagnosis based on the previous NF1 diagnostic criteria and the 2021 updated diagnostic criteria. The revised diagnostic criteria for NF1 decreased TTD. The mean difference in TTD was 113 days shorter for the new criteria (p-value = 1.306x-05 ). This study highlights that the revised 2021 NF1 diagnostic criteria can decrease the TTD. The addition of a heterozygous pathogenic variant in NF1 as a criterion was the change that decreased TTD.

Anteroposterior axis patterning by early canonical Wnt signaling during hemichordate development.

The Wnt family of secreted proteins has been proposed to play a conserved role in early specification of the bilaterian anteroposterior (A/P) axis. This hypothesis is based predominantly on data from vertebrate embryogenesis as well as planarian regeneration and homeostasis, indicating that canonical Wnt (cWnt) signaling endows cells with positional information along the A/P axis. Outside of these phyla, there is strong support for a conserved role of cWnt signaling in the repression of anterior fates, but little comparative support for a conserved role in promotion of posterior fates. We further test the hypothesis by investigating the role of cWnt signaling during early patterning along the A/P axis of the hemichordate Saccoglossus kowalevskii. We have cloned and investigated the expression of the complete Wnt ligand and Frizzled receptor complement of S. kowalevskii during early development along with many secreted Wnt modifiers. Eleven of the 13 Wnt ligands are ectodermally expressed in overlapping domains, predominantly in the posterior, and Wnt antagonists are localized predominantly to the anterior ectoderm in a pattern reminiscent of their distribution in vertebrate embryos. Overexpression and knockdown experiments, in combination with embryological manipulations, establish the importance of cWnt signaling for repression of anterior fates and activation of mid-axial ectodermal fates during the early development of S. kowalevskii. However, surprisingly, terminal posterior fates, defined by posterior Hox genes, are unresponsive to manipulation of cWnt levels during the early establishment of the A/P axis at late blastula and early gastrula. We establish experimental support for a conserved role of Wnt signaling in the early specification of the A/P axis during deuterostome body plan diversification, and further build support for an ancestral role of this pathway in early evolution of the bilaterian A/P axis. We find strong support for a role of cWnt in suppression of anterior fates and promotion of mid-axial fates, but we find no evidence that cWnt signaling plays a role in the early specification of the most posterior axial fates in S. kowalevskii. This posterior autonomy may be a conserved feature of early deuterostome axis specification.

KMT2B-related disorders: expansion of the phenotypic spectrum and long-term efficacy of deep brain stimulation.

Heterozygous mutations in KMT2B are associated with an early-onset, progressive and often complex dystonia (DYT28). Key characteristics of typical disease include focal motor features at disease presentation, evolving through a caudocranial pattern into generalized dystonia, with prominent oromandibular, laryngeal and cervical involvement. Although KMT2B-related disease is emerging as one of the most common causes of early-onset genetic dystonia, much remains to be understood about the full spectrum of the disease. We describe a cohort of 53 patients with KMT2B mutations, with detailed delineation of their clinical phenotype and molecular genetic features. We report new disease presentations, including atypical patterns of dystonia evolution and a subgroup of patients with a non-dystonic neurodevelopmental phenotype. In addition to the previously reported systemic features, our study has identified co-morbidities, including the risk of status dystonicus, intrauterine growth retardation, and endocrinopathies. Analysis of this study cohort (n = 53) in tandem with published cases (n = 80) revealed that patients with chromosomal deletions and protein truncating variants had a significantly higher burden of systemic disease (with earlier onset of dystonia) than those with missense variants. Eighteen individuals had detailed longitudinal data available after insertion of deep brain stimulation for medically refractory dystonia. Median age at deep brain stimulation was 11.5 years (range: 4.5-37.0 years). Follow-up after deep brain stimulation ranged from 0.25 to 22 years. Significant improvement of motor function and disability (as assessed by the Burke Fahn Marsden's Dystonia Rating Scales, BFMDRS-M and BFMDRS-D) was evident at 6 months, 1 year and last follow-up (motor, P = 0.001, P = 0.004, and P = 0.012; disability, P = 0.009, P = 0.002 and P = 0.012). At 1 year post-deep brain stimulation, >50% of subjects showed BFMDRS-M and BFMDRS-D improvements of >30%. In the long-term deep brain stimulation cohort (deep brain stimulation inserted for >5 years, n = 8), improvement of >30% was maintained in 5/8 and 3/8 subjects for the BFMDRS-M and BFMDRS-D, respectively. The greatest BFMDRS-M improvements were observed for trunk (53.2%) and cervical (50.5%) dystonia, with less clinical impact on laryngeal dystonia. Improvements in gait dystonia decreased from 20.9% at 1 year to 16.2% at last assessment; no patient maintained a fully independent gait. Reduction of BFMDRS-D was maintained for swallowing (52.9%). Five patients developed mild parkinsonism following deep brain stimulation. KMT2B-related disease comprises an expanding continuum from infancy to adulthood, with early evidence of genotype-phenotype correlations. Except for laryngeal dysphonia, deep brain stimulation provides a significant improvement in quality of life and function with sustained clinical benefit depending on symptoms distribution.

Inhibiting NF-κB-inducing kinase (NIK): discovery, structure-based design, synthesis, structure-activity relationship, and co-crystal structures.

The discovery, structure-based design, synthesis, and optimization of NIK inhibitors are described. Our work began with an HTS hit, imidazopyridinyl pyrimidinamine 1. We utilized homology modeling and conformational analysis to optimize the indole scaffold leading to the discovery of novel and potent conformationally constrained inhibitors such as compounds 25 and 28. Compounds 25 and 31 were co-crystallized with NIK kinase domain to provide structural insights.

Synthesis and optimization of substituted furo[2,3-d]-pyrimidin-4-amines and 7H-pyrrolo[2,3-d]pyrimidin-4-amines as ACK1 inhibitors.

Two classes of ACK1 inhibitors, 4,5,6-trisubstituted furo[2,3-d]pyrimidin4-amines and 4,5,6-trisubstituted 7H-pyrrolo[2,3-d]pyrimidin-4-amines, were discovered and evaluated as ACK1 inhibitors. Further structural refinement led to the identification of potent and selective dithiolane inhibitor 37.

Genetic counseling for prenatal testing: where is the discussion about disability?

There are little data revealing how genetic counselors talk about disability in the prenatal setting. We performed a qualitative analysis of 93 existing transcripts from simulated patient (SP) genetic counseling sessions conducted in 2003­4 through the Genetic Counseling Video Project. We found that most genetic counselors (95%) focused on the physical aspects of disability while fewer (27%) discussed the social aspects. In addition, few genetic counselors (38%) asked patients about personal experiences with disability. When discussing options available if a pregnancy were diagnosed with a disability, most genetic counselors mentioned termination (86%) while fewer mentioned the continuation of the pregnancy (37%) or adoption (13%). Only half of the genetic counselors asked the SP if she had thought about how she might use the results of prenatal screening. To better facilitate informed decision-making that is consistent with patient values, we recommend genetic counselors engage prenatal patients in a deeper discussion about their ability and willingness to parent a child with a disability.

Stress and Coping in Caregivers of Children with RASopathies: Assessment of the Impact of Caregiver Conferences.

The study aimed to assess baseline stress and coping mechanisms among caregivers of children with RASopathies (i.e., cardiofaciocutaneous and Costello's syndrome) and the impact of attending biennial caregiver conferences. Caregivers completed the Perceived Stress Scale, Coping Health Inventory for Parents, and demographic surveys prior to family conferences, and 1- and 6-month postconferences. Baseline stress was increased and associated with child age, parental age, and number of conferences attended. After 1 month, caregiver stress was lowered among men and caregivers attending ≥2 support conferences.

Identification and optimization of N3,N6-diaryl-1H-pyrazolo[3,4-d]pyrimidine-3,6-diamines as a novel class of ACK1 inhibitors.

A new series of pyrazolo[3,4-d]pyrimidine-3,6-diamines was designed and synthesized as potent and selective inhibitors of the nonreceptor tyrosine kinase, ACK1. These compounds arose from efforts to rigidify an earlier series of N-aryl pyrimidine-5-carboxamides. The synthesis and structure-activity relationships of this new series of inhibitors are reported. The most promising compounds were also profiled for their pharmacokinetic properties.

Mutations in MAST1 Cause Mega-Corpus-Callosum Syndrome with Cerebellar Hypoplasia and Cortical Malformations.

Corpus callosum malformations are associated with a broad range of neurodevelopmental diseases. We report that de novo mutations in MAST1 cause mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations (MCC-CH-CM) in the absence of megalencephaly. We show that MAST1 is a microtubule-associated protein that is predominantly expressed in post-mitotic neurons and is present in both dendritic and axonal compartments. We further show that Mast1 null animals are phenotypically normal, whereas the deletion of a single amino acid (L278del) recapitulates the distinct neurological phenotype observed in patients. In animals harboring Mast1 microdeletions, we find that the PI3K/AKT3/mTOR pathway is unperturbed, whereas Mast2 and Mast3 levels are diminished, indicative of a dominant-negative mode of action. Finally, we report that de novo MAST1 substitutions are present in patients with autism and microcephaly, raising the prospect that mutations in this gene give rise to a spectrum of neurodevelopmental diseases.

Exome sequencing identifies de novo pathogenic variants in FBN1 and TRPS1 in a patient with a complex connective tissue phenotype.

Here we describe a patient who presented with a history of congenital diaphragmatic hernia, inguinal hernia, and recurrent umbilical hernia. She also has joint laxity, hypotonia, and dysmorphic features. A unifying diagnosis was not identified based on her clinical phenotype. As part of her evaluation through the Undiagnosed Diseases Network, trio whole-exome sequencing was performed. Pathogenic variants in FBN1 and TRPS1 were identified as causing two distinct autosomal dominant conditions, each with de novo inheritance. Fibrillin 1 (FBN1) mutations are associated with Marfan syndrome and a spectrum of similar phenotypes. TRPS1 mutations are associated with trichorhinophalangeal syndrome types I and III. Features of both conditions are evident in the patient reported here. Discrepant features of the conditions (e.g., stature) and the young age of the patient may have made a clinical diagnosis more difficult in the absence of exome-wide genetic testing.

Phenotypic and molecular characterisation of CDK13-related congenital heart defects, dysmorphic facial features and intellectual developmental disorders.

BACKGROUND: De novo missense variants in CDK13 have been described as the cause of syndromic congenital heart defects in seven individuals ascertained from a large congenital cardiovascular malformations cohort. We aimed to further define the phenotypic and molecular spectrum of this newly described disorder. METHODS: To minimise ascertainment bias, we recruited nine additional individuals with CDK13 pathogenic variants from clinical and research exome laboratory sequencing cohorts. Each individual underwent dysmorphology exam and comprehensive medical history review. RESULTS: We demonstrate greater than expected phenotypic heterogeneity, including 33% (3/9) of individuals without structural heart disease on echocardiogram. There was a high penetrance for a unique constellation of facial dysmorphism and global developmental delay, as well as less frequently seen renal and sacral anomalies. Two individuals had novel CDK13 variants (p.Asn842Asp, p.Lys734Glu), while the remaining seven unrelated individuals had a recurrent, previously published p.Asn842Ser variant. Summary of all variants published to date demonstrates apparent restriction of pathogenic variants to the protein kinase domain with clustering in the ATP and magnesium binding sites. CONCLUSIONS: Here we provide detailed phenotypic and molecular characterisation of individuals with pathogenic variants in CDK13 and propose management guidelines based upon the estimated prevalence of anomalies identified.

An ultrasensitive high-throughput electrochemiluminescence immunoassay for the Cdc42-associated protein tyrosine kinase ACK1.

Several drugs inhibiting protein kinases have been launched successfully, demonstrating the attractiveness of protein kinases as therapeutic targets. Functional genomics research within both academia and industry has led to the identification of many more kinases as potential drug targets. Although a number of well-known formats are used for measuring protein kinase activity, some less well-characterized protein kinases identified through functional genomics present particular challenges for existing assay formats when there is limited knowledge of the endogenous substrates or activation mechanisms for these novel kinase targets. This is especially the case when a very sensitive assay is required to differentiate often highly potent inhibitors developed by late-stage medicinal chemistry programs. ACK1 is a non-receptor tyrosine kinase that has been shown to be involved in tumorigenesis and metastasis. Here we describe the development of an extremely sensitive high-throughput assay for ACK1 capable of detecting 240 fmol per well of the kinase reaction product employing a BV-tag-based electrochemiluminescence assay. This assay is universally applicable to protein tyrosine kinases using a BV-tag-labeled monoclonal antibody against phosphotyrosine. Furthermore, this assay can be extended to the evaluation of Ser/Thr kinases in those cases where an antibody recognizing the phospho-product is available.

Secretion of MUC5AC mucin from pancreatic cancer cells in response to forskolin and VIP.

MUC5AC mucin is not expressed in normal pancreas but is expressed in tumors. Little is known about the mechanisms that lead to this atypical expression. In this study, we demonstrate that stimulation of adenylyl cyclase and the protein kinase A (PKA) pathway by forskolin and vasoactive intestinal peptide (VIP) increased MUC5AC antigen expression and release from pancreatic cancer cells. Stimulation of the PKA pathway also increased MUC5AC mRNA. When SW1990 pancreatic cancer cells were grown on porous membranes they released MUC5AC mucins apically in response to VIP (10(-7) M) applied to their basolateral surfaces. SW1990 cells, as have been reported for other pancreatic cancer cells, have high affinity (<10(-7) M) VIP receptors and low affinity (>10(-6) M) secretin receptors. We also showed that four antibodies (CLH2, 21M1, 45M1, and Nd2) react with MUC5AC antigen in different cellular compartments of both tissues and cultured cells. In conclusion, the PKA pathway may contribute to the up-regulation of MUC5AC expression seen in pancreatic tumors.