Weaver syndrome, first described in 1974, is characterized by tall stature, a typical facial appearance, and variable intellectual disability. In 2011, mutations in the histone methyltransferase, EZH2, were shown to cause Weaver syndrome. To date, we have identified 48 individuals with EZH2 mutations. The mutations were primarily missense mutations occurring throughout the gene, with some clustering in the SET domain (12/48). 2972Truncating mutations were uncommon (4/48) and only identified in the final exon, after the SET domain. Through analyses of clinical data and facial photographs of EZH2 mutation-positive individuals, we have shown that the facial features can be subtle and the clinical diagnosis of Weaver syndrome is thus challenging, especially in older individuals. However, tall stature is very common, reported in >90% of affected individuals. Intellectual disability is also common, present in $80%, but is highly variable and frequently mild. Additional clinical features which may help in stratifying individuals to EZH2 mutation testing include camptodactyly, soft, doughy skin, umbilical hernia, and a low, hoarse cry. Considerable phenotypic overlap between Sotos and Weaver syndromes is also evident. The identification of an EZH2 mutation can therefore provide an objective means of confirming a subtle presentation of Weaver syndrome and/or distinguishing Weaver and Sotos syndromes. As mutation testing becomes increasingly accessible and larger numbers of EZH2 mutationpositive individuals are identified, knowledge of the clinical spectrum and prognostic implications of EZH2 mutations should improve.

In recent studies on prenatal testing for Noonan syndrome (NS) in fetuses with an increased nuchal translucency (NT) and a normal karyotype, mutations have been reported in 9-16% of cases. In this study, DNA of 75 fetuses with a normal karyotype and abnormal ultrasound findings was tested in a diagnostic setting for mutations in (a subset of) the four most commonly mutated NS genes. A de novo mutation in either PTPN11, KRAS or RAF1 was detected in 13 fetuses (17.3%). Ultrasound findings were increased NT, distended jugular lymphatic sacs (JLS), hydrothorax, renal anomalies, polyhydramnios, cystic hygroma, cardiac anomalies, hydrops fetalis and ascites. A second group, consisting of anonymized DNA of 60 other fetuses with sonographic abnormalities, was tested for mutations in 10 NS genes. In this group, five possible pathogenic mutations have been identified (in PTPN11 (n ¼ 2), RAF1, BRAF and MAP2K1 (each n ¼ 1)). We recommend prenatal testing of PTPN11, KRAS and RAF1 in pregnancies with an increased NT and at least one of the following additional features: polyhydramnios, hydrops fetalis, renal anomalies, distended JLS, hydrothorax, cardiac anomalies, cystic hygroma and ascites. If possible, mutation analysis of BRAF and MAP2K1 should be considered.

BackgroundThis study evaluates 6 years of prenatal rasopathy testing in the Netherlands, updates on previous data and gives recommendations for prenatal rasopathy testing.Methods424 fetal samples, sent in for prenatal rasopathy testing in 2011–2016, were collected. Cohort 1 included 231 samples that were sequenced for 1–5 rasopathy genes. Cohort 2 included 193 samples that were analysed with a 14-gene next generation sequencing (NGS) panel. For all mutation-positive samples in both cohorts, the referring physician provided detailed ultrasound findings and postnatal follow-up. For 168 mutation-negative samples in cohort 2, solely clinical information on the requisition form was collected.ResultsIn total, 40 (likely) pathogenic variants were detected (9.4%). All fetuses showed a variable degree of involvement of prenatal findings: increased nuchal translucency (NT)/cystic hygroma, distended jugular lymph sacs (JLS), hydrops fetalis, polyhydramnios, pleural effusion, ascites, cardiac defects and renal anomalies. An increased NT was the most common finding. Eight fetuses showed solely an increased NT/cystic hygroma, which were all larger than 5.5 mm. Ascites and renal anomalies appeared to be poor predictors of pathogenic outcome.ConclusionFetuses with a rasopathy show in general multiple ultrasound findings. The larger the NT and the longer it persists, the more likely it is to find a pathogenic variant. Rasopathy testing is recommended when the fetus shows an isolated increased NT ≥5.0 mm or when NT of ≥3.5 mm and at least one of the following ultrasound anomalies is present: distended JLS, hydrops fetalis, polyhydramnios, pleural effusion, ascites, cardiac defects and renal anomalies.

Genomic sequence interpretation can miss clinically relevant missense variants for several reasons. Rare missense variants are numerous in the exome and difficult to prioritise. Affected genes may also not have existing disease association. To improve variant prioritisation, we leverage population exome data to identify intragenic missense-depleted regions (MDRs) genome-wide that may be important in disease. We then use missense depletion analyses to help prioritise undiagnosed disease exome variants. We demonstrate application of this strategy to identify a novel gene association for human brain malformation. We identified de novo missense variants that affect the GDP/GTP-binding site of ARF1 in three unrelated patients. Corresponding functional analysis suggests ARF1 GDP/GTP-activation is affected by the specific missense mutations associated with heterotopia. These findings expand the genetic pathway underpinning neurologic disease that classically includes FLNA. ARF1 along with ARFGEF2 add further evidence implicating ARF/GEFs in the brain. Using functional ontology, top MDR-containing genes were highly enriched for nucleotide-binding function, suggesting these may be candidates for human disease. Routine consideration of MDR in the interpretation of exome data for rare diseases may help identify strong genetic factors for many severe conditions, infertility/reduction in reproductive capability, and embryonic conditions contributing to preterm loss.

Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation.