The morbid genome of ciliopathies: an update.

PURPOSE: Ciliopathies are highly heterogeneous clinical disorders of the primary cilium. We aim to characterize a large cohort of ciliopathies phenotypically and molecularly. METHODS: Detailed phenotypic and genomic analysis of patients with ciliopathies, and functional characterization of novel candidate genes. RESULTS: In this study, we describe 125 families with ciliopathies and show that deleterious variants in previously reported genes, including cryptic splicing variants, account for 87% of cases. Additionally, we further support a number of previously reported candidate genes (BBIP1, MAPKBP1, PDE6D, and WDPCP), and propose nine novel candidate genes (CCDC67, CCDC96, CCDC172, CEP295, FAM166B, LRRC34, TMEM17, TTC6, and TTC23), three of which (LRRC34, TTC6, and TTC23) are supported by functional assays that we performed on available patient-derived fibroblasts. From a phenotypic perspective, we expand the phenomenon of allelism that characterizes ciliopathies by describing novel associations including WDR19-related Stargardt disease and SCLT1- and CEP164-related Bardet-Biedl syndrome. CONCLUSION: In this cohort of phenotypically and molecularly characterized ciliopathies, we draw important lessons that inform the clinical management and the diagnostics of this class of disorders as well as their basic biology.

Use of the Foetal Myocardial Performance Index in monochorionic, diamniotic twin pregnancy: a prospective cohort and nested case-control study.

AIMS: Assess clinical utility of the foetal Myocardial Performance Index (MPI) in evaluation and management of monochorionic, diamniotic twin (MCDA) pregnancies. METHODS: Prospective cohort of (a) initially uncomplicated MCDA (b) Complicated MCDA, including twin-twin transfusion syndrome (TTTS), selective intrauterine growth restriction (sIUGR), and liquor and/or growth discordance (L/GD) not meeting TTTS or sIUGR criteria. TTTS and sIUGR were case-control matched. Routine Dopplers and MPI were taken and correlated to diagnosis and final outcome. RESULTS: Twenty-six always uncomplicated pairs, 51 always complicated pairs, and seven uncomplicated to pathological pairs were included. TTTS recipient (n = 25) left and right MPI and intertwin difference (ITD) were significantly elevated, however, were already elevated in Stage I (n = 10), and did not predict progression or pregnancy outcome. sIUGR MPI (n = 11) did not differ significantly from control. Of 15-L/GD pairs, two that progressed to TTTS had significantly higher left and right MPI values in the future recipient (0.61 and 0.72) versus future sIUGR larger twins (0.48 and 0.51) or stable L/GD (0.47 and 0.52): p < .01 for all comparisons. CONCLUSIONS: In this cohort, MPI did not add substantial diagnostic/prognostic information to current routine evaluation in established TTTS or sIUGR though potentially differentiated L/GD cases progressing to TTTS.

GWAS signals revisited using human knockouts.

PurposeGenome-wide association studies (GWAS) have been instrumental to our understanding of the genetic risk determinants of complex traits. A common challenge in GWAS is the interpretation of signals, which are usually attributed to the genes closest to the polymorphic markers that display the strongest statistical association. Naturally occurring complete loss of function (knockout) of these genes in humans can inform GWAS interpretation by unmasking their deficiency state in a clinical context.MethodsWe exploited the unique population structure of Saudi Arabia to identify novel knockout events in genes previously highlighted in GWAS using combined autozygome/exome analysis.ResultsWe report five families with homozygous truncating mutations in genes that had only been linked to human disease through GWAS. The phenotypes observed in the natural knockouts for these genes (TRAF3IP2, FRMD3, RSRC1, BTBD9, and PXDNL) range from consistent with, to unrelated to, the previously reported GWAS phenotype.ConclusionWe expand the role of human knockouts in the medical annotation of the human genome, and show their potential value in informing the interpretation of GWAS of complex traits.

Fetal cardiac function: Feasibility in obtaining the right modified myocardial performance index in a single Doppler waveform.

OBJECTIVE: We aimed to assess the feasibility of assessing the fetal right Myocardial Performance Index (RMPI) using single waveform and to compare absolute values with dual technique. METHODS: We studied 145 morphologically normal appropriately grown fetuses at 16-28 weeks' gestation with local Ethics Committee approval using fixed machine settings: Doppler sweep velocity at 15 cm/s; angle of insonation <150; wall motion filter 300 Hz. Doppler gate was 3 mm, increased to 4-5 mm if needed. RMPI was obtained twice in the same fetus; using 'dual-image' and 'single-image' techniques. Dual images were acquired as previously described. Single images were taken from the tip or just below the tricuspid valve towards the ventricular septum in the apical four-chamber view. RMPI was calculated using two-value (a-b/b) or three-value (ICT+IRT/ET) formulae where 'a', 'b' or (ET) represent the isovolumetric and ejection times, and ICT and IRT represent the isovolumetric contraction and relaxation times. RESULTS: Dual image was accessible in 100% of fetuses. Single-image acquisition was 100%, 92.3% and 76.5% at 16+0-24+0, 24+1-27+0, and 27+1-28+0 weeks respectively (95.2% overall). Doppler gate increased in 23 cases (16.6%); 8/17 (47%) at 27+1-28+0 weeks' gestation. Mean and standard deviation for 'dual image' and 'single image' were: RMPI 0.46 ± 0.09 and 0.49 ± 0.07; 'a' 249.06 ± 11.50 and 249.11 ± 11.93; 'b' 170.85 ± 8.95 and 167.62 ± 8.39. CONCLUSIONS: Single-image acquisition RMPI is highly feasible from 16 to 26 weeks gestation. Difference in mean values may represent overestimation of ejection time in the 'dual-image' technique.

Novel spatial-temporal image correlation derived indices of tissue vascular impedance: A variability study.

OBJECTIVES: 4D (3D + time) indices of tissue impedance using power Doppler (PD) ultrasound (US) can be measured with spatial-temporal image correlation (STIC) imaging. We wished to evaluate their repeatability and their influence under changes to US machine settings and regional differences within the placenta. METHODS: A total of 46 healthy women were recruited at 20-34 weeks of gestation. A total of 9940 3D frames from 644 4D data sets were analysed providing both 3D and 4D indices of vascularity. 4D vs. 3D indices were compared with different machine settings, across the cardiac cycle and in the different placenta regions to assess regional variability. RESULTS: 3D and 4D indices significantly decreased as wall motion filter (WMF) was increased (P < 0.001). Repeatability decreased as WMF increased (ICC; low1 = 0.80; high1 = 0.60). Indices were significantly lower at the maternal aspect (P = 0.002-0.009) of the placenta and showed less repeatability (ICC; 0.42-0.79) than the fetal aspect (ICC 0.49-0.88). 4D repeatability was good in the central region (ICC 0.80-0.81) but poor in the periphery (ICC 0.45-0.59), while 3D indices were good and comparable between regions (ICC; 0.80 central; 0.81 peripheral). CONCLUSIONS: This study supports the future use of WMF 'low1' and PD to generate more reliable 4D indices values. For 3D indices, HD Flow may improve Doppler signal sensitivity. Regarding placental regional variability, the fetal plate and the central region demonstrated more repeatable 4D indices. 4D PD indices have potential to overcome the limitations of VOCAL™ indices and provide an internally standardised measure of localised impedance in vascular beds.

Characterizing the morbid genome of ciliopathies.

BACKGROUND: Ciliopathies are clinically diverse disorders of the primary cilium. Remarkable progress has been made in understanding the molecular basis of these genetically heterogeneous conditions; however, our knowledge of their morbid genome, pleiotropy, and variable expressivity remains incomplete. RESULTS: We applied genomic approaches on a large patient cohort of 371 affected individuals from 265 families, with phenotypes that span the entire ciliopathy spectrum. Likely causal mutations in previously described ciliopathy genes were identified in 85% (225/265) of the families, adding 32 novel alleles. Consistent with a fully penetrant model for these genes, we found no significant difference in their "mutation load" beyond the causal variants between our ciliopathy cohort and a control non-ciliopathy cohort. Genomic analysis of our cohort further identified mutations in a novel morbid gene TXNDC15, encoding a thiol isomerase, based on independent loss of function mutations in individuals with a consistent ciliopathy phenotype (Meckel-Gruber syndrome) and a functional effect of its deficiency on ciliary signaling. Our study also highlighted seven novel candidate genes (TRAPPC3, EXOC3L2, FAM98C, C17orf61, LRRCC1, NEK4, and CELSR2) some of which have established links to ciliogenesis. Finally, we show that the morbid genome of ciliopathies encompasses many founder mutations, the combined carrier frequency of which accounts for a high disease burden in the study population. CONCLUSIONS: Our study increases our understanding of the morbid genome of ciliopathies. We also provide the strongest evidence, to date, in support of the classical Mendelian inheritance of Bardet-Biedl syndrome and other ciliopathies.

Audit of immediate outcomes for MCDA twins following laser therapy for twin-twin transfusion syndrome at the NSW Fetal Therapy Centre.

OBJECTIVE: To audit immediate pregnancy and neonatal outcomes of selective laser photocoagulation of communicating vessels (SLPCV) for twin-twin transfusion syndrome (TTTS) at the New South Wales Fetal Therapy Centre. METHODS: Retrospective cohort study of 151 TTTS cases undergoing SLPCV between July 2003 and May 2013, evaluating procedural details, delivery and perinatal outcomes. RESULTS: The majority of cases were Stage III at SLPCV (56.9%), although proportion of Stage II SLPCV increased over time (P = 0.03). Survival to hospital discharge of at least one baby was 85.6% and dual survival was 52.5%. Median gestational age at delivery was 32.6 weeks (IQR 29.0-35.0 weeks) with a median of 11.4 weeks (IQR 8.3-14.7) from laser to delivery. Median birthweight was 1792 g (IQR 1288-2233 g), with 75% of babies admitted to the nursery, predominantly secondary to prematurity. Immediate SLPCV complications were in utero fetal demise <1 week postprocedure in 27 fetuses (19.6%) and/or ruptured membranes <1 week postprocedure in 9 fetuses (6.6%). CONCLUSIONS: This Australian series shows that local outcomes after SLPCV for stages II-IV TTTS remain equal to the international published literature and have remained stable after an initial learning curve. Women were more likely to be Stage II rather than III in the more recent years. However, this does not appear to be attributable to altered referral patterns.

The (Pulsed-Wave) Doppler Fetal Myocardial Performance Index: Technical Challenges, Clinical Applications and Future Research.

Functional cardiovascular assessment is becoming an increasingly important tool in the study of fetal pathology. The myocardial performance index (MPI) is a parameter measuring global myocardial function. Since its introduction, several studies have proposed methods to improve its reproducibility and have constructed normative reference ranges. Fetal heart evaluation using the MPI is technically challenging, requiring specific training and expertise, and a consensus has yet to be reached on the method of delineating the time periods used to calculate the index. Despite these limitations, it has been shown to be a useful and highly sensitive parameter of dysfunction in a number of fetal pathologies. Further research is warranted into the effect of pathology on MPI, parameters of unilateral cardiac strain that utilise MPI, and automation of the MPI to encourage incorporation of the MPI as a useful tool in clinical practice.

Is There a Measurable Difference between the Left and Right Modified Myocardial Performance Indices, and Does This Change to Reflect Unilateral Myocardial Dysfunction in Pathology?

INTRODUCTION: Fetal cardiac dysfunction may manifest itself unilaterally as right and left ventricles differing in design, function and load, measurable as differing in myocardial performance indices (MPIs). We wished to define this difference ('delta-MPI' or DMPI), present its normal range and pilot its use in pathological pregnancy. MATERIAL AND METHODS: Prospective cross-sectional study of 324 normal singleton fetuses (16-38 weeks of gestation). Left and right modified MPI (LMPI and RMPI) were performed during a single examination using the 'peak' valve click technique. Thirty-seven pathological singleton and monochorionic diamniotic twin pregnancies were compared as pilot data. RESULTS: Modified MPIs (mean ± SD) were 0.45 ± 0.06 (LMPI) and 0.47 ± 0.09 (RMPI), being similar at 18 weeks' gestation with DMPI increasing slightly throughout pregnancy (0.02 ± 0.08). Both singleton intrauterine growth restriction (IUGR) and recipient twin-twin transfusion syndrome (TTTS) showed significantly elevated RMPI, LMPI and DMPI, most pronounced for DMPI (450 and 500% increase, respectively; p < 0.01). DMPI acquisition rates were 83.3% normal and 87.0% pathological. DISCUSSION: We demonstrate for the first time differing intrafetal LMPI and RMPI in a large gestational cohort, with this difference increasing with gestational age. Pilot data confirm the potential for DMPI as a tool to assess unilateral myocardial function in singleton IUGR and recipient twins in TTTS, and further studies are under way to evaluate its clinical utility.

Development of normal gestational ranges for the right myocardial performance index in the Australian population with three alternative caliper placements.

OBJECTIVES: To construct gestational age-adjusted reference ranges for the right fetal modified myocardial performance index (RMPI) in an Australian population and to assess the influence of valve click caliper position on constituent time intervals and the RMPI. METHODS: A prospective cross-sectional study of RMPI from 235 normal fetuses at 17-38 weeks of gestation was performed. Two Doppler waveforms were obtained: tricuspid and pulmonary valves for 'a' and 'b' readings, respectively. The ultrasound machine settings were: Doppler sweep velocity 15 cm/s, angle of insonation <15°, minimal gain, and wall motion filter 300 Hz. The 'a' and 'b' intervals were measured at three different caliper positions in each fetus: at the beginning of the original valve clicks ('original'), at the beginning of the reflected tricuspid and pulmonary closure clicks ('reflected') and at the peak of valve clicks ('peak'). RMPI was calculated as (a - b)/b. The three readings were obtained and averaged per examination, with intraobserver repeatability assessed by intraclass correlation coefficient (ICC) and 95% CI. RESULTS: For 'original', 'reflected' and 'peak' RMPI, mean ± SD, ICC (95% CI) were: 0.53 ± 0.10, 0.86 (0.83-0.89); 0.48 ± 0.10, 0.84 (0.81-0.87) and 0.48 ± 0.10, 0.89 (0.87-0.91), respectively. The RMPI increased by approximately 15% as gestation increased and decreased slightly with increasing heart rate. CONCLUSION: This is the first publication of reference ranges for RMPI based on caliper position. All methods showed good ICC, including the 'peak' method which we have previously proposed for routine use based on its repeatability and ease of identification when measuring the myocardial performance index.

Selection of the sub-noise gain level for acquisition of VOCAL data sets: a reliability study.

This study was aimed at assessing the intra-observer and inter-observer repeatability of selecting the sub-noise gain (SNG) level when acquiring placental volumes with 3-D power Doppler for analysis using virtual organ computer-aided analysis (VOCAL). Sixty women with uncomplicated singleton pregnancies between 20 and 38 wk of gestation were recruited. Two women were excluded for flash artifact noted during image analysis. Two blinded observers independently adjusted gain to their perceived SNG level before acquiring a static 3-D volume of the placenta at the cord insertion; observers alternated after each acquisition until each had acquired two volumes. A single observer operated the probe at all times. During offline analysis, SNG levels were recorded and VOCAL indices were calculated. SNG exhibited excellent intra-observer and inter-observer reliability. Intra-observer intra-class correlation coefficients (95% confidence intervals) were 0.98 (0.97-0.99) and 0.98 (0.98-0.99) for observers 1 and 2, respectively. The inter-observer intra-class correlation coefficient was 0.96 (0.93-0.98). Despite its perceived inherent subjectivity, the excellent intra-class correlation coefficients obtained in this study support SNG as a promising tool for future research using 3-D power Doppler.

Use of four-dimensional analysis of power Doppler perfusion indices to demonstrate cardiac cycle pulsatility in fetoplacental flow.

The aim of this study is to quantify fetoplacental cardiac cycle variation in virtual organ computer-aided analysis (VOCAL) power Doppler (PD) indices by novel application of spatio-temporal imaging correlation (STIC). We recruited 25 healthy women (20-34 weeks gestation) with uncomplicated, viable singleton pregnancies with anterior placentae. Three four-dimensional (4-D) STIC PD datasets of the fetoplacental circulation were obtained above the placental cord insertion. The vascularization index (VI), flow index (FI) and vascularization-flow index (VFI) were calculated offline using a standardized spherical sonobiopsy technique for all frames of the cardiac cycle. Clear maximum (systole) and minimum (diastole) values with progressive fluctuation were seen in the majority of datasets (VI 66/75 [88%]; FI 58/75 [77%]; VFI 68/75 [91%]). Variation from mean was: VI ± 3.33% (0.34%-9.69%); VFI ± 3.46% (0.27%-10.02%); FI ± 0.74% (0.14%-1.60%). All indices were significantly higher in systole than diastole (p < 0.001). Mean systolic:diastolic ratios were: VI 1.07 (SD 0.06), FI 1.01 (SD 0.01) and VFI 1.07 (SD 0.06). Intraclass correlation coefficients (ICCs) for the frames ascribed to systole and diastole and to the mean value across the cardiac cycle of the indices (95% confidence interval [CI]) were: systole VI 0.91 (0.83-0.96), FI 0.85 (0.72-0.92), VFI 0.92 (0.85-0.96); diastole VI 0.91 (0.84-0.96), FI 0.84 (0.71-0.92), VFI 0.92 (0.86-0.96); mean VI 0.91 (0.84-0.96), FI 0.84 (0.72-0.92), VFI 0.92 (0.86-0.96). There is clear cardiac cycle variation in VOCAL indices of fetoplacental blood flow, establishing the need to control for phase of the cardiac cycle, and raising the possibility of future 4-D evaluation of vascular flow change or impedance.

Evaluation of neonatal regional cerebral perfusion using power Doppler and the index fractional moving blood volume.

BACKGROUND: The high cerebral morbidity of premature neonates is thought to be related to changes in tissue perfusion in vulnerable areas of the brain. Quantification of power Doppler (PD) images using the index fractional moving blood volume (FMBV) may allow measurement of regional cerebral perfusion. OBJECTIVE: To evaluate the reproducibility of calculating FMBV using PD ultrasound images to estimate cerebral perfusion. METHODS: Two experienced clinicians performed head ultrasounds on 24 normally-grown neonates at less than 33 weeks' gestation. Both clinicians independently acquired and stored three PD images in two different coronal planes. FMBV was calculated offline after selecting two predefined regions of interest within these planes (basal ganglia and subependymal regions). Reproducibility was evaluated by calculating the intraclass correlation coefficient (intraCC) and the interclass correlation coefficient (interCC). RESULTS: FMBV was successfully evaluated in 24/24 neonates by both clinicians. The intraCC for repeatability for observer A was 1.00 (95% CI 1.00-1.00) for the basal ganglia and 0.99 (95% CI 0.99-1.00) for the subependymal region, and for observer B was 0.99 (95% CI 0.99-1.00) for the basal ganglia and 0.96 (95% CI 0.92-0.98) for the subependymal region. The interCC was 0.86 (95% CI 0.68-0.94) for the basal ganglia and 0.93 (95% CI 0.86-0.97) for the subependymal region. CONCLUSION: Using standardised settings and a well-defined region of interest, the calculation of FMBV using PD images is a reproducible method of estimating neonatal regional cerebral perfusion.