The circadian molecular clock in the suprachiasmatic nucleus is necessary but not sufficient for fear entrainment.

We show that nocturnal aversive stimuli presented to mice while they are eating and drinking outside of their safe nest can entrain circadian behaviors, leading to a shift toward daytime activity. We also show that the canonical molecular circadian clock is necessary for fear entrainment and that an intact molecular clockwork in the suprachiasmatic nucleus, the site of the central circadian pacemaker, is necessary but not sufficient to sustain fear entrainment of circadian rhythms. Our results demonstrate that entrainment of a circadian clock by cyclic fearful stimuli can lead to severely mistimed circadian behavior that persists even after the aversive stimulus is removed. Together, our findings support the interpretation that circadian and sleep symptoms associated with fear and anxiety disorders are, in part, the output of a fear-entrained clock, and provide a mechanistic insight into this clock.

Customizing carrier screening in the Chinese population: Insights from a 334-gene panel.

OBJECTIVE: This study aimed to evaluate the yield and applicability of expanded carrier screening and propose carrier rate screening thresholds suitable for the Chinese population by comparing the current screening panel with the American College of Medical Genetics and Genomics recommended panel of 113 genes. METHODS: Using targeted next-generation sequencing, a customized panel with 334 genes was performed on 2168 individuals without clinical phenotypes for expanded carrier screening purpose. Variant interpretation followed the American College of Medical Genetics and Genomics guidelines. Carrier rates were calculated for each identified variant and each gene. At-risk couple rates were also assessed. The yield of expanded carrier screening was evaluated through calculating cumulative carrier rate. RESULTS: Overall, 65.87% of the individuals were found to be carriers of at least 1 disease causing variants. The overall at-risk couple rate was 11.76%, of which the GJB2:c.109G > A related at-risk couple rate was 5.78%. The cumulative carrier rate of 334-panel was 65.53%. When screened genes with gene carrier rate ≥1/1000, the expanded carrier screening can cover over 90% of the cumulative carrier rate and at-risk couples. A total of 86 genes overlapped with American College of Medical Genetics and Genomics Tier-3 genes and were attributed to the cumulative carrier rate of 47.33%. CONCLUSION: Expanded carrier screening using the 334-gene panel showed high screening efficiency. A threshold of gene carrier rate ≥1/1000 is recommended for selecting carrier screening genes in the Chinese Han population. This study highlights the importance of customizing screening panels based on the ACMG Tier-3 genes in conjunction with population-specific carrier frequencies to improve the accuracy and effectiveness of expanded carrier screening.

Clinical predictors of pulmonary embolism for inpatients: are computed tomography pulmonary angiograms being requested appropriately?

BACKGROUND: The heterogeneity of inpatient pulmonary embolism (PE) presentations may lead to computed tomography pulmonary angiograms (CTPA) being over-requested. Current clinical predictors for PE, including Wells criteria and Pulmonary Embolism Rule-out Criteria (PERC), have predominantly focussed on outpatient and emergency department populations. AIM: To determine the clinical indicators for ordering inpatient CTPA and the predictors of positive scans for PE. METHODS: Consecutive inpatient CTPA (performed >24 h after admission) from January 2017 to December 2017 were retrospectively reviewed. Variables including baseline characteristics, vital signs and risk factors for PE were extracted. RESULTS: A total of 312 CTPA was reviewed (average patient age 67 years; 46% male) and 36 CTPA were positive for PE (11.5%). The average time to inpatient CTPA request was 7 days. Clinical indicators associated with positive scans were hypoxia (odds ratio (OR) 2.4; 95% confidence interval (CI) 1.1-5.6), tachypnoea (OR 2.5; 95% CI 1.2-6.0), recent surgery or immobilisation (OR 2.7; 95% CI 1.2-6.4), S1Q3T3 pattern on electrocardiogram (ECG; OR 7.2; 95% CI 1.4-35.7) and right bundle branch block pattern on ECG (OR 4.7; 95% CI 1.6-13.1). Hypotension, fever and malignancy were not significant. Both PERC and Wells criteria had poor positive predictive value (12% and 27% respectively), but the negative predictive value for PERC and Wells was 100% and 95.8% respectively. CONCLUSION: Inpatient CTPA appear to be over-requested and can potentially be rationalised based on a combination of clinical predictors and Wells criteria and/or PERC rule. Further prospective studies are needed to develop accurate clinical decision tools targeted towards inpatients.

Phononic Crystal Made of Silicon Ridges on a Membrane for Liquid Sensing.

We propose the design of a phononic crystal to sense the acoustic properties of a liquid that is constituted by an array of silicon ridges on a membrane. In contrast to other concepts, the ridges are immersed in the liquid. The introduction of a suitable cavity in the periodic array gives rise to a confined defect mode with high localization in the cavity region and strong solid-liquid interaction, which make it sensitive to the acoustic properties of the liquid. By using a finite element method simulation, we theoretically study the transmission and cavity excitation of an incident flexural wave of the membrane. The observation of the vibrations of this mode can be achieved either outside the area of the phononic crystal or just above the cavity. We discuss the existence of the resonant modes, as well as its quality factor and sensitivity to liquid properties as a function of the geometrical parameters. The performance of the proposed sensor has then been tested to detect the variation in NaI concentration in a NaI-water mixture.

Mosaicism in tuberous sclerosis complex: Lowering the threshold for clinical reporting.

Tuberous sclerosis complex (TSC) is a multi-system genetic disorder. Most patients have germline mutations in TSC1 or TSC2 but, 10%-15% patients do not have TSC1/TSC2 mutations detected on routine clinical genetic testing. We investigated the contribution of low-level mosaic TSC1/TSC2 mutations in unsolved sporadic patients and families with TSC. Thirty-one sporadic TSC patients negative on routine testing and eight families with suspected parental mosaicism were sequenced using deep panel sequencing followed by droplet digital polymerase chain reaction. Pathogenic variants were found in 22/31 (71%) unsolved sporadic patients, 16 were mosaic (median variant allele fraction [VAF] 6.8% in blood) and 6 had missed germline mutations. Parental mosaicism was detected in 5/8 families (median VAF 1% in blood). Clinical testing laboratories typically only report pathogenic variants with allele fractions above 10%. Our findings highlight the critical need to change laboratory practice by implementing higher sensitivity assays to improve diagnostic yield, inform patient management and guide reproductive counseling.

Accurate Identification of Breakpoints in a Cryptic Reciprocal Translocation by Whole-Genome Sequencing.

Chromosomal abnormalities are a common cause of spontaneous abortions, but conventional detection methods (karyotype, FISH, and chromosomal microarray [CMA]) have limitations, and many cryptic balanced chromosomal rearrangements are difficult to detect. We describe a couple who experienced a missed abortion, studied by CMA. CMA of the abortion tissue detected a 1.62-Mb duplication at 14q11.2 and a 5.09-Mb deletion at 21q11.2q21.1, while the couple seemed to have a normal karyotype. Combining the results of CMA, whole-genome sequencing (WGS) breakpoint analysis, Sanger sequencing, and FISH, we found that the father was a 46,XY,t(14;21)(q11.2;q21.1) balanced translocation carrier. Our results indicate that WGS is an efficient and accurate approach to map breakpoints of cryptic reciprocal balanced translocations undetectable by standard karyotype.

Mesenchymal stem cells from different sources show distinct therapeutic effects in hyperoxia-induced bronchopulmonary dysplasia in rats.

Mesenchymal stem cells (MSCs) have been shown as an effective medicinal means to treat bronchopulmonary dysplasia (BPD). The widely used MSCs were from Wharton's jelly of umbilical cord (UC-MSCs) and bone marrow (BM-MSCs). Amniotic fluid MSCs (AF-MSCs) may be produced before an individual is born to treat foetal diseases by autoplastic transplantation. We evaluated intratracheal (IT) MSCs as an approach to treat an hyperoxia-induced BPD animal model and compared the therapeutic effects between AF-, UC- and BM-MSCs. A BPD animal model was generated by exposing newborn rats to 95% O2 . The continued stress lasted 21 days, and the treatment of IT MSCs was conducted for 4 days. The therapeutic effects were analysed, including lung histology, level of inflammatory cytokines, cell death ratio and state of angiogenesis, by sacrificing the experimental animal at day 21. The lasting hyperoxia stress induced BPD similar to the biological phenotype. The treatment of IT MSCs was safe without deaths and normal organ histopathology. Specifically, the treatment was effective by inhibiting the alveolar dilatation, reducing inflammatory cytokines, inducing angiogenesis and lowering the cell death ratio. AF-MSCs had better therapeutic effects compared with UC-MSCs in relieving the pulmonary alveoli histological changes and promoting neovascularization, and UC-MSCs had the best immunosuppressive effect in plasma and lung lysis compared with AF-MSCs and BM-MSCs. This study demonstrated the therapeutic effects of AF-, UC- and BM-MSCs in BPD model. Superior treatment effect was provided by antenatal MSCs compared to BM-MSC in a statistical comparison.

De Novo Mutations of CCNK Cause a Syndromic Neurodevelopmental Disorder with Distinctive Facial Dysmorphism.

Neurodevelopment is a transcriptionally orchestrated process. Cyclin K, a regulator of transcription encoded by CCNK, is thought to play a critical role in the RNA polymerase II-mediated activities. However, dysfunction of CCNK has not been linked to genetic disorders. In this study, we identified three unrelated individuals harboring de novo heterozygous copy number loss of CCNK in an overlapping 14q32.3 region and one individual harboring a de novo nonsynonymous variant c.331A>G (p.Lys111Glu) in CCNK. These four individuals, though from different ethnic backgrounds, shared a common phenotype of developmental delay and intellectual disability (DD/ID), language defects, and distinctive facial dysmorphism including high hairline, hypertelorism, thin eyebrows, dysmorphic ears, broad nasal bridge and tip, and narrow jaw. Functional assay in zebrafish larvae showed that Ccnk knockdown resulted in defective brain development, small eyes, and curly spinal cord. These defects were partially rescued by wild-type mRNA coding CCNK but not the mRNA with the identified likely pathogenic variant c.331A>G, supporting a causal role of CCNK variants in neurodevelopmental disorders. Taken together, we reported a syndromic neurodevelopmental disorder with DD/ID and facial characteristics caused by CCNK variations, possibly through a mechanism of haploinsufficiency.

Exome-based preconception carrier testing for consanguineous couples in China.

OBJECTIVE: To evaluate the utility of clinical exome sequencing (ES)-based carrier screening in Chinese consanguineous couples. METHODS: Consanguineous couples were screened for autosomal recessive (AR) disorders using the clinical ES of 5000 genes associated with human diseases. RESULTS: We recruited 14 couples who elected to have sequencing. One couple was related as first cousins and 13 as second cousins. Both partners carrying the same pathogenic variant were detected in four couples. One couple was found in which one partner carried a splice variant, and the other had a missence variant of the same gene. These five couples were identified as being at risk of having a child affected by an AR disorder. CONCLUSION: Our study demonstrates that ES-based preconception screening yields a clinical value for Chinese consanguineous couples. It enables to detect at-risk couples for rare AR diseases.

Clinical and laboratory interpretation of mitochondrial mRNA variants.

Interpretation of mitochondrial protein-encoding (mt-mRNA) variants has been challenging due to mitochondrial characteristics that have not been addressed by American College of Medical Genetics and Genomics guidelines. We developed criteria for the interpretation of mt-mRNA variants via literature review of reported variants, tested and refined these criteria by using our new cases, followed by interpreting 421 novel variants in our clinical database using these verified criteria. A total of 32 of 56 previously reported pathogenic (P) variants had convincing evidence for pathogenicity. These variants are either null variants, well-known disease-causing variants, or have robust functional data or strong phenotypic correlation with heteroplasmy levels. Based on our criteria, 65.7% (730/1,111) of variants of unknown significance (VUS) were reclassified as benign (B) or likely benign (LB), and one variant was scored as likely pathogenic (LP). Furthermore, using our criteria we classified 2, 12, and 23 as P, LP, and LB, respectively, among 421 novel variants. The remaining stayed as VUS (91.2%). Appropriate interpretation of mt-mRNA variants is the basis for clinical diagnosis and genetic counseling. Mutation type, heteroplasmy levels in different tissues of the probands and matrilineal relatives, in silico predictions, population data, as well as functional studies are key points for pathogenicity assessments.

Correction: Interpretation of mitochondrial tRNA variants.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Correction: Interpretation of mitochondrial tRNA variants.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Interpretation of mitochondrial tRNA variants.

PURPOSE: To develop criteria to interpret mitochondrial transfer RNA (mt-tRNA) variants based on unique characteristics of mitochondrial genetics and conserved structural/functional properties of tRNA. METHODS: We developed rules on a set of established pathogenic/benign variants by examining heteroplasmy correlations with phenotype, tissue distribution, family members, and among unrelated families from published literature. We validated these deduced rules using our new cases and applied them to classify novel variants. RESULTS: Evaluation of previously reported pathogenic variants found that 80.6% had sufficient evidence to support phenotypic correlation with heteroplasmy levels among and within families. The remaining variants were downgraded due to the lack of similar evidence. Application of the verified criteria resulted in rescoring 80.8% of reported variants of uncertain significance (VUS) to benign and likely benign. Among 97 novel variants, none met pathogenic criteria. A large proportion of novel variants (84.5%) remained as VUS, while only 10.3% were likely pathogenic. Detection of these novel variants in additional individuals would facilitate their classification. CONCLUSION: Proper interpretation of mt-tRNA variants is crucial for accurate clinical diagnosis and genetic counseling. Correlations with tissue distribution, heteroplasmy levels, predicted perturbations to tRNA structure, and phenotypes provide important evidence for determining the clinical significance of mt-tRNA variants.

Glucocorticoids and activity in free-living arctic ground squirrels: Interrelationships between weather, body condition, and reproduction.

The dynamic relationship between glucocorticoids and behavior are not well understood in wild mammals. We investigated how weather, body condition, and reproduction interact to affect cortisol levels and activity patterns in a free-living population of arctic ground squirrels (Urocitellus parryii). As a proxy for foraging and escape behaviors, collar-mounted accelerometers and light loggers were used to measure above-ground activity levels and the amount of time squirrels spent below the surface, respectively. Fecal cortisol metabolites (FCMs) were quantified to assess glucocorticoid secretion in squirrels. Male and female squirrels differed in above-ground activity levels and time spent below-ground across the active season, with males being most active during mating and females most active during lactation. We also found that female, but not male, squirrels exhibited seasonal variation in FCM levels, with concentrations highest during mid-lactation and lowest after the lactation period. In female squirrels, the seasonal relationships between breeding stage, activity, and FCM levels were also consistent with changes in maternal investment and the preparative role that glucocorticoids are hypothesized to play in energy mobilization. Body condition was not associated with FCM levels in squirrels. As predicted, deteriorating weather also influenced FCM levels and activity patterns in squirrels. FCM concentrations were affected by an interaction between temperature and wind speed when seasonal temperatures were lowest. In addition, above-ground activity, but not time spent below-ground, positively correlated with FCM levels. These results suggest that, although ground squirrels avoid inclement weather by remaining below-ground, activation of the stress axis may stimulate foraging activity.

Rapid prenatal diagnosis of skeletal dysplasia using medical trio exome sequencing: Benefit for prenatal counseling and pregnancy management.

OBJECTIVE: The aim of this study is to explore the utility of rapid medical trio exome sequencing (ES) for prenatal diagnosis using the skeletal dysplasia as an exemplar. METHOD: Pregnant women who were referred for genetic testing because of ultrasound detection of fetal abnormalities suggestive of a skeletal dysplasia were identified prospectively. Fetal samples (amniocytes or cord blood), along with parental blood, were send for rapid copy number variations testing and medical trio ES in parallel. RESULTS: Definitive molecular diagnosis was made in 24/27 (88.9%) cases. Chromosomal abnormality (partial trisomy 18) was detected in one case. Sequencing results had explained the prenatal phenotype enabling definitive diagnoses to be made in 23 cases. There were 16 de novo dominant pathogenic variants, four dominant pathogenic variants inherited maternally or paternally, two recessive conditions with pathogenic variants inherited from unaffected parents, and one X-linked condition. The turnaround time from receipt of samples in the laboratory to reporting sequencing results was within 2 weeks. CONCLUSION: Medical trio ES can yield very timely and high diagnostic rates in fetuses presenting with suspected skeletal dysplasia. These definite diagnoses aided parental counseling and decision making in most of cases.

Novel SPEG variant cause centronuclear myopathy in China.

BACKGROUND: Centronuclear myopathy (CNM), a subtype of congenital myopathy (CM), is a group of clinical and genetically heterogeneous muscle disorders. Centronuclear myopathy is a kind of disease difficult to diagnose due to its genetic diversity. Since the discovery of the SPEG gene and disease-causing variants, only a few additional patients have been reported. METHODS: A radiograph test, ultrasonic test, and biochemical tests were applied to clinical diagnosis of CNM. We performed trio medical exome sequencing of the family and conservation analysis to identify variants. RESULTS: We report a pair of severe CNM twins with the same novel homozygous SPEG variant c. 8710A>G (p.Thr2904Ala) identified by clinical trio medical exome sequencing of the family and conservation analysis. The twins showed clinical symptoms of facial weakness, hypotonia, arthrogryposis, strephenopodia, patent ductus arteriosus, and pulmonary arterial hypertension. CONCLUSIONS: Our report expands the clinical and molecular repertoire of CNM and enriches the variant spectrum of the SPEG gene in the Chinese population and helps us further understand the pathogenesis of CNM.

The nuclear background influences the penetrance of the near-homoplasmic m.1630 A > G MELAS variant in a symptomatic proband and asymptomatic mother.

In this study, we report the metabolic consequences of the m.1630 A > G variant in fibroblasts from the symptomatic proband affected with the mitochondrial encephalomyopathy lactic acidosis and stroke-like episode Syndrome and her asymptomatic mother. By long-range PCR followed by massively parallel sequencing of the mitochondrial genome, we accurately measured heteroplasmy in fibroblasts from the proband (89.6%) and her mother (94.8%). Using complementary experimental approaches, we show a functional correlation between manifestation of clinical symptoms and bioenergetic potential. Our mitochondrial morphometric analysis reveals a link between defects of mitochondrial cristae ultrastructure and symptomatic status. Despite near-homoplasmic level of the m.1630A > G variant, the mother's fibroblasts have a normal OXPHOS metabolism, which stands in contrast to the severely impaired OXPHOS response of the proband's fibroblasts. The proband's fibroblasts also exhibit glycolysis at near constitutive levels resulting in a stunted compensatory glycolytic response to offset the severe OXPHOS defect. Whole exome sequencing reveals the presence of a heterozygous nonsense VARS2 variant (p.R334X) exclusively in the proband, which removes two thirds of the VARS2 protein containing key domains interacting with the mt-tRNAval and may play a role in modulating the penetrance of the m.1630A > G variant despite similar near homoplasmic levels. Our transmission electron microscopy study also shows unexpected ultrastructural changes of chromatin suggestive of differential epigenomic regulation between the proband and her mother that may explain the differential OXPHOS response between the proband and her mother. Future study will decipher by which molecular mechanisms the nuclear background influences the penetrance of the m.1630 A > G variant causing MELAS.

Mutations in both SAMD9 and SLC19A2 genes caused complex phenotypes characterized by recurrent infection, dysphagia and profound deafness - a case report for dual diagnosis.

BACKGROUND: Phenotypic difference is general in Mendelian disease. Due to the extremely low incidence for a single disease, phenotype spectrum needs to be expanded. Meanwhile, earlier knowledge says patients who suffered from two kinds of different Mendelian disease are very rare. CASE PRESENTATION: We describe a case of neonatal male with genital anomalies, growth delay, skin hyperpigmentation, chronic lung disease with recurrent infection, anemia, and severe deafness. Without any clear etiology after routine workflow, whole exome sequencing was carried on. A pathogenic de novo SAMD9 mutation and compound heterozygous likely-pathogenic variants in SLC19A2 were identified. Some symptoms were improved after the patient was treated with vitamin B1. Unfortunately, the boy died from sepsis and multiple organ failure before 1 year old. CONCLUSION: Combining the phenotype and clinical progress of treatment, we report that it is the first case of a patient with both MIRAGE syndrome and TRMA syndrome.

Improving two-way satellite time and frequency transfer with redundant links for UTC generation.

Two-way satellite time and frequency transfer (TWSTFT) is a primary technique for the generation of coordinated universal time (UTC). At present, more than 12 timing laboratories around the world use SAtellite Time and Ranging Equipment (SATRE) modems in TWSTFT operations and contribute data for the realization of UTC. The advantages of TWSTFT are its small calibration uncertainty (≤1.0 ns if the link is calibrated with a TWSTFT mobile station) and its long-term link stability. However, the precision of SATRE TWSTFT in the operational networks is degraded by a daily variation pattern (diurnal) in the TWSTFT results. The diurnal with varying amplitude appears virtually in all SATRE TWSTFT links. The observed peak-topeak variation of the diurnals can reach 2.0 ns in some cases. So far, studies on the sources of the diurnal have not provided conclusive understanding of the diurnal's dominant origin. Therefore, efforts have been made to reduce the impact of the diurnal variation in TWSTFT for UTC computation. The BIPM has been using the combination of SATRE TWSTFT results and GPS carrier-phase precise point positioning solutions (GPSPPP) for UTC computation since 2010. The combination adjusts the GPSPPP results to long-term averages of TWSTFT and is effectively free from the diurnal variations because the GPSPPP results contain almost no diurnal. Lately, the use of software-defined radio receivers (SDR) in TWSTFT has shown one way of how to reduce the diurnal variations by a factor of two to three in most of the inner-continental SATRE TWSTFT links, and furthermore, how the short-term stability for all UTC SDR TWSTFT links can be improved. In addition, there has been research on the full use of the redundancy in the TWSTFT network to improve the TWSTFT link stability. Recent studies on evaluating indirect links revealed that it is possible to apply a simplified procedure to use the redundancy, in a most effective way, to reduce the diurnal variations in the Europeto-Europe SATRE TWSTFT links by a factor of two to three. Based on these findings, we gained new insights about the diurnals and its dominant origin(s) which are discussed in this paper. The methods of the combination of SATRE TWSTFT and GPSPPP as well as the indirect SATRE TWSTFT links utilize the redundancy in the UTC time transfer network. SDR TWSTFT can largely reduce the diurnal in SATRE TWSTFT, but noticeable residual diurnal remains. In this paper, we provide the analyses of using the combination of SDR TWSTFT and GPSPPP results, as well as using the indirect SDR TWSTFT links. This paper concludes that the use of SDR TWSTFT redundant links can further improve the stabilities of UTC TWSTFT links. In addition, the use of SDR TWSTFT indirect links is a pure TWSTFT solution. The independence of the TWSTFT results to GPS results can improve the robustness of UTC computation.

Identification of a novel DNMT1 mutation in a Chinese patient with hereditary sensory and autonomic neuropathy type IE.

BACKGROUND: DNA methyltransferase 1 (EC 2.1.1.37), encoded by DNMT1 gene, is one of key enzymes in maintaining DNA methylation patterns of the human genome. It plays a crucial role in embryonic development, imprinting and genome stability, cell differentiation. The dysfunction of this group of enzymes can lead to a variety of human genetic disorders. Until now, mutations in DNMT1 have been found to be associated with two distinct phenotypes. Mutations in exon 20 of this gene leads to hereditary sensory and autonomic neuropathy type IE, and mutations in exon 21 cause autosomal dominant cerebellar ataxia, deafness and narcolepsy. CASE PRESENTATION: Here we report a novel DNMT1 mutation in a sporadic case of a Chinese patient with cerebellar ataxia, multiple motor and sensory neuropathy, hearing loss and psychiatric manifestations. Furthermore, we elucidated its pathogenic effect through molecular genetics studies and revealed that this defective DNMT1 function is responsible for the phenotypes in this individual. CONCLUSION: Our findings expand the spectrum of DNMT1-related disorders and provide a good example of precision medicine through the combination of exome sequencing and clinical testing.