Monoallelic variation in DHX9, the gene encoding the DExH-box helicase DHX9, underlies neurodevelopment disorders and Charcot-Marie-Tooth disease.

DExD/H-box RNA helicases (DDX/DHX) are encoded by a large paralogous gene family; in a subset of these human helicase genes, pathogenic variation causes neurodevelopmental disorder (NDD) traits and cancer. DHX9 encodes a BRCA1-interacting nuclear helicase regulating transcription, R-loops, and homologous recombination and exhibits the highest mutational constraint of all DDX/DHX paralogs but remains unassociated with disease traits in OMIM. Using exome sequencing and family-based rare-variant analyses, we identified 20 individuals with de novo, ultra-rare, heterozygous missense or loss-of-function (LoF) DHX9 variant alleles. Phenotypes ranged from NDDs to the distal symmetric polyneuropathy axonal Charcot-Marie-Tooth disease (CMT2). Quantitative Human Phenotype Ontology (HPO) analysis demonstrated genotype-phenotype correlations with LoF variants causing mild NDD phenotypes and nuclear localization signal (NLS) missense variants causing severe NDD. We investigated DHX9 variant-associated cellular phenotypes in human cell lines. Whereas wild-type DHX9 was restricted to the nucleus, NLS missense variants abnormally accumulated in the cytoplasm. Fibroblasts from an individual with an NLS variant also showed abnormal cytoplasmic DHX9 accumulation. CMT2-associated missense variants caused aberrant nucleolar DHX9 accumulation, a phenomenon previously associated with cellular stress. Two NDD-associated variants, p.Gly411Glu and p.Arg761Gln, altered DHX9 ATPase activity. The severe NDD-associated variant p.Arg141Gln did not affect DHX9 localization but instead increased R-loop levels and double-stranded DNA breaks. Dhx9-/- mice exhibited hypoactivity in novel environments, tremor, and sensorineural hearing loss. All together, these results establish DHX9 as a critical regulator of mammalian neurodevelopment and neuronal homeostasis.

Phenotypic spectrum of the recurrent TRPM3 p.(Val837Met) substitution in seven individuals with global developmental delay and hypotonia.

TRPM3 encodes a transient receptor potential cation channel of the melastatin family, expressed in the central nervous system and in peripheral sensory neurons of the dorsal root ganglia. The recurrent substitution in TRPM3: c.2509G>A, p.(Val837Met) has been associated with syndromic intellectual disability and seizures. In this report, we present the clinical and molecular features of seven previously unreported individuals, identified by exome sequencing, with the recurrent p.(Val837Met) variant and global developmental delay. Other shared clinical features included congenital hypotonia, dysmorphic facial features (broad forehead, deep-set eyes, and down turned mouth), exotropia, and musculoskeletal issues (hip dysplasia, hip dislocation, scoliosis). Seizures were observed in two of seven individuals (febrile seizure in one and generalized tonic-clonic seizures with atonic drops in another), and epileptiform activity was observed in an additional two individuals. This report extends the number of affected individuals to 16 who are heterozygous for the de novo recurrent substitution p.(Val837Met). In contrast with the initial report, epilepsy was not a mandatory feature observed in this series. TRPM3 pathogenic variation should be considered in individuals with global developmental delays, moderate-severe intellectual disability with, or without, childhood-onset epilepsy.

ZTTK syndrome: Clinical and molecular findings of 15 cases and a review of the literature.

Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is caused by de novo loss-of-function variants in the SON gene (MIM #617140). This multisystemic disorder is characterized by intellectual disability, seizures, abnormal brain imaging, variable dysmorphic features, and various congenital anomalies. The wide application and increasing accessibility of whole exome sequencing (WES) has helped to identify new cases of ZTTK syndrome over the last few years. To date, there have been approximately 45 cases reported in the literature. Here, we describe 15 additional individuals with variants in the SON gene, including those with missense variants bringing the total number of known cases to 60. We have reviewed the clinical and molecular data of these new cases and all previously reported cases to further delineate the most common as well as emerging clinical findings related to this syndrome. Furthermore, we aim to delineate any genotype-phenotype correlations specifically for a recurring pathogenic four base pair deletion (c.5753_5756del) along with discussing the impact of missense variants seen in the SON gene.

Germline De Novo Mutations in GNB1 Cause Severe Neurodevelopmental Disability, Hypotonia, and Seizures.

Whole-exome sequencing of 13 individuals with developmental delay commonly accompanied by abnormal muscle tone and seizures identified de novo missense mutations enriched within a sub-region of GNB1, a gene encoding the guanine nucleotide-binding protein subunit beta-1, Gß. These 13 individuals were identified among a base of 5,855 individuals recruited for various undiagnosed genetic disorders. The probability of observing 13 or more de novo mutations by chance among 5,855 individuals is very low (p = 7.1 × 10(-21)), implicating GNB1 as a genome-wide-significant disease-associated gene. The majority of these 13 mutations affect known Gß binding sites, which suggests that a likely disease mechanism is through the disruption of the protein interface required for Gα-Gßγ interaction (resulting in a constitutively active Gßγ) or through the disruption of residues relevant for interaction between Gßγ and certain downstream effectors (resulting in reduced interaction with the effectors). Strikingly, 8 of the 13 individuals recruited here for a neurodevelopmental disorder have a germline de novo GNB1 mutation that overlaps a set of five recurrent somatic tumor mutations for which recent functional studies demonstrated a gain-of-function effect due to constitutive activation of G protein downstream signaling cascades for some of the affected residues.

Histologic Funisitis and Likelihood of Intrauterine Inflammation or Infection: A Case-Control Study.

OBJECTIVE: The objective of this study was to ascertain the likelihood of isolated maternal fever and suspected intrauterine inflammation or infection or both (Triple I) among cases of histologic chorioamnionitis with funisitis (HCF) at term. STUDY DESIGN: In this case-control study, placental pathology records were reviewed to identify term singleton laboring patients with HCF. Controls (1:1) were matched for gestational age. RESULTS: During the 6-month period, there were 2,399 term deliveries of laboring women. Of 1,552 (65%) term placentas examined, 4% (n = 60) had HCF.Features of Triple I were significantly more common among cases than controls: (1) isolated maternal fever of ≥100.4°F, twice, at least 30 minutes apart (p = 0.014); (2) fever with fetal tachycardia (p = 0.029); 3) fever with either fetal tachycardia or white blood cell count greater than 15,000 per mm3 (p = 0.034). The feature of Triple I with the highest sensitivity at 10% (95% confidence intervals [CI] 4-21%) was isolated maternal fever using ≥100.4°F on two occasions. The specificity for all features was consistently 100% (95% CI 91-100%). CONCLUSION: To our knowledge, this is the first report on HCF and Triple I features. Though the sensitivity of Triple I to identify HCF is low, specificity is excellent.

Antagonistic action on NMDA/GluN2B mediated currents of two peptides that were conantokin-G structure-based designed.

BACKGROUND: The GluN2B subunit of the N-methyl-D-aspartate receptor (NMDAr) modulates many physiological processes including learning, memory, and pain. Excessive increase in NMDAr/GluN2B activity has been associated with various disorders such neuropathic pain and neuronal death following hypoxia. Thus there is an interest in identifying NMDAr antagonists that interact specifically with the GluN2B subunit. Recently based on structural analysis between the GluN2B subunit and conantokin-G, a toxin that interacts selectively with the GluN2B subunit, we designed various peptides that are predicted to act as NMDAr antagonists by interacting with the GluN2B subunit. In this study we tested this prediction for two of these peptides EAR16 and EAR18. RESULTS: The effects of EAR16 and EAR18 in NMDA-evoked currents were measured in cultured rat embryonic hippocampal neurons and in HEK-293 cells expressing recombinant NMDAr comprised of GluN1a-GluN2A or GluN1a-GluN2B subunits. In hippocampal neurons, EAR16 and EAR18 reduced the NMDA-evoked calcium currents in a dose-dependent and reversible manner with comparable IC50 (half maximal inhibitory concentration) values of 241 and 176 µM, respectively. At 500 µM, EAR16 blocked more strongly the NMDA-evoked currents mediated by the GluN1a-GluN2B (84%) than those mediated by the GluN1a-GluN2A (50%) subunits. At 500 µM, EAR18 blocked to a similar extent the NMDA-evoked currents mediated by the GluN1a-GluN2B (62%) and the GluN1a-GluN2A (55%) subunits. CONCLUSIONS: The newly designed EAR16 and EAR18 peptides were shown to block in reversible manner NMDA-evoked currents, and EAR16 showed a stronger selectivity for GluN2B than for GluN2A.

FTO variant associated with malformation syndrome.

Common FTO variants are associated with obesity. However, it has recently been shown that homozygous FTO c.947G>A variant, which predicts p.R316Q, and c.956C>T, which predicts p.S319F, are associated with a malformation syndrome inherited in an autosomal recessive pattern. We present a similar homozygous FTO c.965G>A variant that predicts p.R322Q, associated with a lethal malformation syndrome in a consanguineous Yemeni family. Functional studies showed that the p.R316Q, p.S219F, and p.R322Q variants render the FTO protein inactive. We further expand on the phenotype of homozygous FTO loss-of-function mutations to include eye abnormalities, gingival overgrowth, craniosynostosis, and cutaneous photosensitivity.

The usefulness of whole-exome sequencing in routine clinical practice.

PURPOSE: Reports of the use of whole-exome sequencing in clinical practice are limited. We report our experience with whole-exome sequencing in 115 patients in a single center and evaluate its feasibility and clinical usefulness in clinical care. METHODS: Whole-exome sequencing was utilized based on the judgment of three clinical geneticists. We describe age, gender, ethnicity, consanguinity, indication for testing, family history, insurance, laboratory results, clinician interpretation of results, and impact on patient care. RESULTS: Most patients were children (78.9%). The most common indications for testing were birth defects (24.3%) and developmental delay (25.2%). We identified four new candidate human disease genes and possibly expanded the disease phenotypes associated with five different genes. Establishing a diagnosis led to discontinuation of additional planned testing in all patients, screening for additional manifestations in eight, altered management in fourteen, novel therapy in two, identification of other familial mutation carriers in five, and reproductive planning in six. CONCLUSION: Our results show that whole-exome sequencing is feasible, has clinical usefulness, and allows timely medical interventions, informed reproductive choices, and avoidance of additional testing. Our results also suggest phenotype expansion and identification of new candidate disease genes that would have been impossible to diagnose by other targeted testing methods.

Pathogenic heterozygous TRPM7 variants and hypomagnesemia with developmental delay.

Background: Heterozygous variants in Transient receptor potential melastatin type 7 (TRPM7), encoding an essential and ubiquitously expressed cation channel, may cause hypomagnesemia, but current evidence is insufficient to draw definite conclusions and it is unclear whether any other phenotypes can occur. Methods: Individuals with unexplained hypomagnesemia underwent whole-exome sequencing which identified TRPM7 variants. Pathogenicity of the identified variants was assessed by combining phenotypic, functional and in silico analyses. Results: We report three new heterozygous missense variants in TRPM7 (p.Met1000Thr, p.Gly1046Arg, p.Leu1081Arg) in individuals with hypomagnesemia. Strikingly, autism spectrum disorder and developmental delay, mainly affecting speech and motor skills, was observed in all three individuals, while two out of three also presented with seizures. The three variants are predicted to be severely damaging by in silico prediction tools and structural modeling. Furthermore, these variants result in a clear loss-of-function of TRPM7-mediated magnesium uptake in vitro, while not affecting TRPM7 expression or insertion into the plasma membrane. Conclusions: This study provides additional evidence for the association between heterozygous TRPM7 variants and hypomagnesemia and adds developmental delay to the phenotypic spectrum of TRPM7-related disorders. Considering that the TRPM7 gene is relatively tolerant to loss-of-function variants, future research should aim to unravel by what mechanisms specific heterozygous TRPM7 variants can cause disease.

A central role of plasmin in cardiac injury initiated by fetal exposure to maternal anti-Ro autoantibodies.

OBJECTIVE: Cardiac neonatal lupus (cardiac-NL), initiated by surface binding of anti-Ro60 autoantibodies to apoptotic cardiocytes during development, activates the urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) system. Subsequent accumulation of apoptotic cells and plasmin generation facilitates increased binding of anti-Ro60 by disrupting and cleaving circulating ß2-glycoprotein I (ß2GPI) thereby eliminating its protective effect. The association of soluble levels of components of the uPA/uPAR system with cardiac-NL was examined. METHODS: Levels of the uPA/uPAR system were assessed by ELISA in cord blood and immunohistological evaluation of autopsies. RESULTS: uPA, uPAR and plasminogen levels were each significantly higher in cord blood from cardiac-NL (n = 35) compared with non-cardiac-NL (n = 26) anti-Ro-exposed neonates: 3.3 ± 0.1 vs 1.9 ± 0.05 ng/ml (P < 0.0001), 6.6 ± 0.3 vs 2.1 ± 0.2 ng/ml (P < 0.0001) and 435 ± 34 vs 220 ± 19 ng/ml (P < 0.0001), respectively. In three twin pairs discordant for cardiac-NL, the twin with cardiac-NL had higher levels of uPA, uPAR and plasminogen than the unaffected twin (3.1 ± 0.1 vs 1.9 ± 0.05 ng/ml; P = 0.0086, 6.2 ± 1.4 vs 2.2 ± 0.7 ng/ml; P = 0.147 and 412 ± 61 vs 260 ± 27 ng/ml; P = 0.152, respectively). Immunohistological evaluation of three hearts from fetuses dying with cardiac-NL revealed macrophages and giant cells expressing uPA and plasminogen in the septal region. CONCLUSION: Increased soluble uPA, uPAR and plasminogen in cord blood and expression in affected tissue of fetuses with cardiac-NL supports the hypothesis that fetal cardiac injury is in part mediated by plasmin generation initiated by anti-Ro binding to the apoptotic cardiocyte.

Simazine transport in undisturbed soils from a vineyard at the Casablanca valley, Chile.

Simazine is a soil-active herbicide that has been applied worldwide in agricultural soils, being the second most commonly detected herbicide in groundwater and surface waters. Although its use has been restricted in many countries of Europe, it is still applied in many locations around the world in orchards, vineyards and forestry. Therefore, it is important to study its fate and transport in the environment. This paper investigates simazine transport in undisturbed bare soils from a vineyard at the Casablanca valley, Chile. In the study site, shallow groundwater tables (<1.0 m depth) and high simazine levels (>15 µg L(-1)) in the groundwater were observed and thus, there is potential for simazine to be transported further away through the saturated zone. The soils from the study site were characterized and the hydrodynamic transport parameters were determined. Column leaching experiments showed that the two-site chemical non-equilibrium model correctly represented simazine transport. It was found that 36.3% of the adsorption sites achieve instantaneous equilibrium and that the first-order kinetic rate of the non-equilibrium sites was 6.2 × 10(-3) h(-1). Hydrus 2D was used to predict the transport of simazine in the study site under natural field conditions. Simulation results showed that simazine concentrations at depths shallower than 2.1 m are above the maximum contaminant level of 4 µg L(-1) (defined by the U.S. Environmental Protection Agency). The timing of herbicide application was found to be important on simazine leaching and the main processes involved in simazine transport were degradation and adsorption, which accounted for 95.78 and 4.19% of the simulated mass of pesticide, respectively. A qualitative agreement in the timing and magnitude of simazine concentration was obtained between the simulations and the field data. Therefore, the model utilized in this investigation can be used to predict simazine transport and is a valuable tool to assess agricultural practices to minimize environmental impacts of simazine.

Does midtrimester cervical length ≥25 mm predict preterm birth in high-risk women?

OBJECTIVE: We sought to assess pregnancy outcome along a continuum of cervical lengths (CLs) ≥25 mm. STUDY DESIGN: We conducted planned secondary analysis of a randomized cerclage trial of women with prior spontaneous preterm birth 17(0)-34(6/7) weeks. Outcomes of women who maintained CLs ≥25 mm were analyzed. Women with CLs <25 mm randomized to no cerclage comprised an internal comparison group. RESULTS: Of 1014 screened, 153 had CL <25 mm, and 672 had CL ≥25 mm. Birth <35 weeks occurred in 16% of the ≥25 mm cohort. The relationship between CLs ≥25 mm and birth gestational age was null (P = .15). In the <25 mm group, progressively shorter CLs predicted birth <35 weeks (P < .001); this relationship was null in the ≥25 mm group (P = .17). CONCLUSION: The continuum of CLs ≥25 mm measured between 16(0/7)-22(6/7) weeks does not predict gestational length in women with prior spontaneous preterm birth.

Cervical funneling: effect on gestational length and ultrasound-indicated cerclage in high-risk women.

OBJECTIVE: The purpose of this study was to assess funnel type and pregnancy duration in women with previous spontaneous preterm birth and cervical length <25 mm. STUDY DESIGN: We performed a secondary analysis of a multicenter randomized trial of cerclage. At the randomization scan that documented short cervix, the presence and type of funnel (U or V) were recorded. RESULTS: One hundred forty-seven of 301 women (49%) had funneling: V-shaped funnel, 99 women; U-shaped funnel, 48 women. U-shaped funnel was associated significantly with preterm birth at <24, <28, <35, and <37 weeks of gestation. In multivariable models that controlled for randomization cervical length and cerclage, women with U-shaped funnel delivered earlier than women with either V-shaped funnel or no funnel. Interaction between cerclage and U-shaped funnel was observed, and analyses that were stratified by cerclage showed that women with a U-shaped funnel and cerclage delivered at a mean of 33.8 +/- 6.6 weeks of gestation, compared with women who did not receive cerclage (28.9 +/- 6.9 weeks of gestation). CONCLUSION: U-shaped funnels in high-risk women with a short cervix are associated with earlier birth.

17-alpha-hydroxyprogesterone caproate for the prevention of preterm birth in women with prior preterm birth and a short cervical length.

OBJECTIVE: We sought to evaluate 17-alpha-hydroxyprogesterone caproate (17P) for prevention of preterm birth (PTB) in women with prior spontaneous PTB (SPTB) and cervical length (CL) <25 mm. STUDY DESIGN: We conducted planned secondary analysis of the Eunice Kennedy Shriver National Institute of Child Health and Human Development-sponsored randomized trial evaluating cerclage for women with singleton gestations, prior SPTB (17-33 6/7 weeks), and CL <25 mm between 16-22 6/7 weeks. Women were stratified at randomization to intent to use or not use 17P. The effect of 17P was analyzed separately for cerclage and no-cerclage groups. Primary outcome was PTB <35 weeks. RESULTS: In 300 women, 17P had no effect on PTB <35 weeks in either cerclage (P = .64) or no-cerclage (P = .51) groups. Only PTB <24 weeks (odds ratio, 0.08) and perinatal death (odds ratio, 0.14) were significantly lower for those with 17P in the no-cerclage group. CONCLUSION: 17P had no additional benefit for prevention of PTB in women who had prior SPTB and got ultrasound-indicated cerclage for CL <25 mm. In women who did not get cerclage, 17P reduced previable birth and perinatal mortality.

Gestational age at previous preterm birth does not affect cerclage efficacy.

OBJECTIVE: The purpose of this study was to evaluate the effect of earliest previous spontaneous preterm birth (SPTB) gestational age on cervical length, pregnancy duration, and ultrasound-indicated cerclage efficacy in a subsequent gestation. STUDY DESIGN: Planned secondary analysis of the National Institute of Child Health and Human Development-trial of cerclage for cervical length of <25 mm. Women with at least 1 previous SPTB between 17-33 weeks 6 days of gestation underwent serial vaginal ultrasound screening between 16 and 23 weeks 6 days of gestation; cervical length at qualifying randomization evaluation was used. RESULTS: We observed a significant correlation (P = .0008) between previous SPTB gestational age and qualifying cervical length. In a linear regression model that was controlled for cervical length and cerclage, neither previous SPTB gestational age nor the interaction between cerclage and previous birth gestational age was significant predictor of subsequent birth gestational age. CONCLUSION: Although there is an association between previous SPTB gestational age and cervical length in women with a mid-trimester cervical length of <25 mm, there does not appear to be a disproportionate benefit of cerclage in women with earlier previous SPTB.

Impact of patient education videos on genetic counseling outcomes after exome sequencing.

OBJECTIVE: Growing use of clinical exome sequencing (CES) has led to an increased burden of genomic education. Self-guided educational tools can minimize the educational burden for genetic counselors (GCs). The effectiveness of these tools must be evaluated. METHODS: Parents of patients offered CES were randomized to watch educational videos before their visit or to receive routine care. Parents and GCs were surveyed about their experiences following the sessions. The responses of the video (n = 102) and no-video (n = 105) groups were compared. RESULTS: GCs reported no significant differences between parents in the video and no-video groups on genetics knowledge or CES knowledge. In contrast, parents' scores on genetics knowledge questions were lower in the video than no-video group (p = 0.007). Most parents reported the videos were informative, and the groups did not differ in satisfaction with GCs or decisions to have CES. CONCLUSION: GCs and parents perceived the videos to be beneficial. However, lower scores on genetics knowledge questions highlight the need for careful development of educational tools. PRACTICE IMPLICATIONS: Educational tools should be developed and assessed for effectiveness with the input of all stakeholders before widespread implementation. Better measures of the effectiveness of these educational tools are needed.

The diagnostic dilemma of thrombotic thrombocytopenic purpura/hemolytic uremic syndrome in the obstetric triage and emergency department: lessons from 4 tertiary hospitals.

OBJECTIVE: We report a series of occurrences of thrombotic thrombocytopenic purpura (TTP)/hemolytic uremic syndrome (HUS) in pregnancy that emphasizes early diagnosis. STUDY DESIGN: Fourteen pregnancies with TTP (n = 12) or HUS (n = 2) were studied. Analysis focused on clinical and laboratory findings on examination, initial diagnosis, and treatment. RESULTS: There were 14 pregnancies in 12 patients; 2 cases of TTP were diagnosed as recurrent. Five women were admitted to the emergency department (ED), and 7 patients were admitted to an obstetrics triage. Patients who were evaluated by an obstetrician were treated initially for hemolysis, elevated liver enzymes and low platelets syndrome/preeclampsia, whereas patients who were seen in the ED had a diagnosis that is commonplace in the ED (panic attack, domestic violence, gastroenteritis). Latency from the onset of symptoms to diagnosis ranged from 1-7 days. Plasmapheresis treatments in early gestation resulted in favorable maternal-neonatal outcome. Maternal and perinatal mortality rates were 25% each. CONCLUSION: TTP/HUS is a challenging diagnosis in obstetric triage and ED areas. We propose a management scheme that suggests how to triage patients for early diagnosis in pregnancy.

Multicenter randomized trial of cerclage for preterm birth prevention in high-risk women with shortened midtrimester cervical length.

OBJECTIVE: The objective of the study was to assess cerclage to prevent recurrent preterm birth in women with short cervix. STUDY DESIGN: Women with prior spontaneous preterm birth less than 34 weeks were screened for short cervix and randomly assigned to cerclage if cervical length was less than 25 mm. RESULTS: Of 1014 women screened, 302 were randomized; 42% of women not assigned and 32% of those assigned to cerclage delivered less than 35 weeks (P = .09). In planned analyses, birth less than 24 weeks (P = .03) and perinatal mortality (P = .046) were less frequent in the cerclage group. There was a significant interaction between cervical length and cerclage. Birth less than 35 weeks (P = .006) was reduced in the less than 15 mm stratum with a null effect in the 15-24 mm stratum. CONCLUSION: In women with a prior spontaneous preterm birth less than 34 weeks and cervical length less than 25 mm, cerclage reduced previable birth and perinatal mortality but did not prevent birth less than 35 weeks, unless cervical length was less than 15 mm.

Homozygous noncanonical splice variant in LSM1 in two siblings with multiple congenital anomalies and global developmental delay.

Two siblings, one male and one female, ages 6 and 13 yr old, have similar clinical features of global developmental delay, multiple congenital anomalies affecting the cardiac, genitourinary, and skeletal systems, and abnormal eye movements. Whole-genome sequencing revealed a homozygous splice variant (NM_014462.3:c.231+4A>C) in LSM1 that segregated with the phenotype in the family. LSM1 has a role in pre-mRNA splicing and degradation. Expression studies revealed absence of expression of the canonical isoform in the affected individuals. The Lsm1 knockout mice have a partially overlapping phenotype that affects the brain, heart, and eye. To our knowledge, LSM1 has not been associated with any human disorder; however, the tissue expression pattern, gene constraint, and the similarity of the phenotype in our patients and the knockout mice models suggest it has a role in the development of multiple organ systems in humans.