Structural brain abnormalities in Pallister-Killian syndrome: a neuroimaging study of 31 children.

BACKGROUND: Pallister-Killian syndrome (PKS) is a rare genetic disorder caused by mosaic tetrasomy of 12p with wide neurological involvement. Intellectual disability, developmental delay, behavioral problems, epilepsy, sleep disturbances, and brain malformations have been described in most individuals, with a broad phenotypic spectrum. This observational study, conducted through brain MRI scan analysis on a cohort of patients with genetically confirmed PKS, aims to systematically investigate the neuroradiological features of this syndrome and identify the possible existence of a typical pattern. Moreover, a literature review differentiating the different types of neuroimaging data was conducted for comparison with our population. RESULTS: Thirty-one individuals were enrolled (17 females/14 males; age range 0.1-17.5 years old at first MRI). An experienced pediatric neuroradiologist reviewed brain MRIs, blindly to clinical data. Brain abnormalities were observed in all but one individual (compared to the 34% frequency found in the literature review). Corpus callosum abnormalities were found in 20/30 (67%) patients: 6 had callosal hypoplasia; 8 had global hypoplasia with hypoplastic splenium; 4 had only hypoplastic splenium; and 2 had a thin corpus callosum. Cerebral hypoplasia/atrophy was found in 23/31 (74%) and ventriculomegaly in 20/31 (65%). Other frequent features were the enlargement of the cisterna magna in 15/30 (50%) and polymicrogyria in 14/29 (48%). Conversely, the frequency of the latter was found to be 4% from the literature review. Notably, in our population, polymicrogyria was in the perisylvian area in all 14 cases, and it was bilateral in 10/14. CONCLUSIONS: Brain abnormalities are very common in PKS and occur much more frequently than previously reported. Bilateral perisylvian polymicrogyria was a main aspect of our population. Our findings provide an additional tool for early diagnosis.Further studies to investigate the possible correlations with both genotype and phenotype may help to define the etiopathogenesis of the neurologic phenotype of this syndrome.

A novel center-based deep contrastive metric learning method for the detection of polymicrogyria in pediatric brain MRI.

Polymicrogyria (PMG) is a disorder of cortical organization mainly seen in children, which can be associated with seizures, developmental delay and motor weakness. PMG is typically diagnosed on magnetic resonance imaging (MRI) but some cases can be challenging to detect even for experienced radiologists. In this study, we create an open pediatric MRI dataset (PPMR) containing both PMG and control cases from the Children's Hospital of Eastern Ontario (CHEO), Ottawa, Canada. The differences between PMG and control MRIs are subtle and the true distribution of the features of the disease is unknown. This makes automatic detection of potential PMG cases in MRI difficult. To enable the automatic detection of potential PMG cases, we propose an anomaly detection method based on a novel center-based deep contrastive metric learning loss function (cDCM). Despite working with a small and imbalanced dataset our method achieves 88.07% recall at 71.86% precision. This will facilitate a computer-aided tool for radiologists to select potential PMG MRIs. To the best of our knowledge, our research is the first to apply machine learning techniques to identify PMG solely from MRI. Our code is available at: https://github.com/RichardChangCA/Deep-Contrastive-Metric-Learning-Method-to-Detect-Polymicrogyria-in-Pediatric-Brain-MRI. Our pediatric MRI dataset is available at: https://www.kaggle.com/datasets/lingfengzhang/pediatric-polymicrogyria-mri-dataset.

Cortical malformation adjacent to a large pial arteriovenous fistula.

The coexistence of an arteriovenous fistula (AVF) and neuronal migration abnormalities is a rare phenomenon. The underlying pathophysiology responsible for these anomalies remains elusive. Neuronal architectural irregularities arise from complex neuronal formation, migration and organisation dysfunctions. Isolated cases of these associations are rarely described in the literature. Here, we present an unusual case involving the coexistence of a pial AVF and a pachygyria-polymicrogyria complex in an early childhood boy. We have provided a detailed description of the neuroimaging characteristics and the therapeutic embolisation in this case, along with follow-up. Additionally, we conduct a comprehensive review of potential hypotheses about the association, referencing prior case reports. The presence of an aberrant blood supply or deviant venous drainage from the developing cortex may contribute to a variety of neuronal migration anomalies.

An unusual presentation of de novo RAC3 variation in prenatal diagnosis.

Pathogenic variants in RAC3 cause a neurodevelopmental disorder with brain malformations and craniofacial dysmorphism, called NEDBAF. This gene encodes a small GTPase, which plays a critical role in neurogenesis and neuronal migration. We report a 31 weeks of gestation fetus with triventricular dilatation, and temporal and perisylvian polymicrogyria, without cerebellar, brainstem, or callosal anomalies. Trio whole exome sequencing identified a RAC3 (NM_005052.3, GRCh38) probably pathogenic de novo variant c.276 T>A p.(Asn92Lys). Eighteen patients harboring 13 different and essentially de novo missense RAC3 variants were previously reported. All the patients presented with corpus callosum malformations. Gyration disorders, ventriculomegaly (VM), and brainstem and cerebellar malformations have frequently been described. The only previous prenatal case associated with RAC3 variant presented with complex brain malformations, mainly consisting of midline and posterior fossa anomalies. We report the second prenatal case of NEDBAF presenting an undescribed pattern of cerebral anomalies, including VM and polymicrogyria, without callosal, cerebellar, or brainstem malformations. All neuroimaging data were reviewed to clarify the spectrum of cerebral malformations.

Navigated transcranial magnetic stimulation to measure motor evoked potentials in a child with hemispheric polymicrogyria and focal epilepsy.

Malformations of cortical development such as polymicrogyria can cause medically refractory epilepsy. Epilepsy surgery (hemispherotomy) can be a good treatment option. In recent years, navigated transcranial magnetic stimulation (nTMS), a noninvasive brain mapping technique, has been used to localize the eloquent cortex for presurgical evaluation of patients with epilepsy. In the present case study, neurophysiological markers of the primary motor cortex (M1), including resting motor threshold (rMT), motor evoked potentials (MEPs), and silent period (SP), were assessed in both hands of a right-handed 10-year-old girl with a history of epilepsy and right hemispheric polymicrogyria. Bilateral MEPs with short latencies were elicited from the contralesional side. The average MEP amplitude and the latency for the patient's paretic and non-paretic hands differed significantly. We conclude that nTMS is a safe and tolerable procedure that can be used for presurgical evaluation in children with intractable epilepsy.

Cilostazol treats transient heart failure caused by ATP1A3 variant-associated polymicrogyria.

BACKGROUND: Some patients with ATP1A3 variant-associated polymicrogyria have recurrent transient heart failure. However, effective treatment for the transient cardiac condition remains to be elucidated. CASE REPORT: The patient started experiencing focal motor onset seizures in 12 h after birth, revealing bilateral diffuse polymicrogyria. The patient also experienced transient bradycardia (sinus bradycardia) attacks from 15 days old. Echocardiography revealed a reduced ejection fraction; however, no obvious electrocorticogram or electroencephalogram abnormalities were observed during the attacks. Initially, the attacks occurred in clusters daily. They later decreased in frequency, occurring at monthly intervals. Repeated episodes of transient bradycardia attacks and polymicrogyria indicated possible ATP1A3 gene abnormality and genetic testing revealed a novel heterozygous ATP1A3 variant (NM_152296: exon22:c.2977_2982del:p.(Glu993_Ile994del)), which was not found in the patient's parents. Cilostazol was administered at 3 months old for recurrent transient bradycardia attacks. Cilostazol significantly shortened the duration of bradycardia episodes and prolonged the interval between attacks. Cilostazol also effectively treats transient symptomatic bradycardia. CONCLUSION: Cilostazol could be a treatment option for recurrent transient bradycardia attacks associated with ATP1A3 gene abnormalities and polymicrogyria.

Prenatal diagnosis of microcephaly with simplified gyral pattern: series of eight cases.

Microcephaly with simplified gyral pattern (MSG) is an intrinsic genetic central nervous system disorder, characterized by microcephaly (a reduction of brain volume) and a simplified gyral pattern (a reduced number of gyri and shallow sulci associated with normal cortical thickness and neuroanatomical architecture), related to a reduced number of neuronal progenitors in the germinal matrix. We report the first prenatal series of MSG and define the prenatal imaging pattern, which should inform diagnosis and guide prenatal counseling in cases of fetal microcephaly. In this single-center retrospective study of fetuses with MSG, we assessed features on ultrasound and magnetic resonance imaging (MRI), as well as genetic and neuropathological/postnatal data. We included eight patients who had been referred following observation of microcephaly. Ultrasound examination confirmed microcephaly, with a mean growth delay in head circumference of 3.4 weeks, associated with both a lack of gyration and a lack of opercularization of the Sylvian fissure and without any extracephalic anomaly. Fetal brain MRI confirmed lack of gyration with normal cortical thickness and normal intensity of the white matter in all cases. These MRI features led to exclusion of migration/corticogenesis disorders (lissencephaly/polymicrogyria), instead suggesting MSG. The posterior fossa was normal in seven of the eight cases. The corpus callosum was thin in four cases, hypoplastic in two and dysgenetic in two. In four cases, the pregnancy was terminated. The diagnosis of MSG was confirmed from neuropathological and postnatal MRI data. MSG was associated with a genetic diagnosis of RTTN (n = 1) and ASPM (n = 2) biallelic variants in three of the six cases in which genetic work-up was performed. Mild or moderate intellectual deficit with speech delay was present in the three surviving children who were at least 5 years of age at their last examination, without seizures. In conclusion, in the presence of isolated fetal microcephaly with lack of gyration on ultrasound, fetal cerebral MRI is key to diagnosing MSG, which, in the majority of cases, affects the supratentorial space exclusively, and to ruling out other cortical malformations that show a similar sonographic pattern. In addition to imaging, genetic assessment may guide prenatal counseling, since the prenatal prognosis of MSG is different from that of both diffuse polymicrogyria and lissencephaly. © 2023 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Functional dysregulation of the auditory cortex in bilateral perisylvian polymicrogyria: Multiparametric case analysis of the absent speech phenotype.

The absence of speech is a clinical phenotype seen across neurodevelopmental syndromes, offering insights for neural language models. We present a case of bilateral perisylvian polymicrogyria (BPP) and complete absence of speech with considerable language comprehension and production difficulties. We extensively characterized the auditory speech perception and production circuitry by employing a multimodal neuroimaging approach. Results showed extensive cortical thickening in motor and auditory-language regions. The auditory cortex lacked sensitivity to speech stimuli despite relatively preserved thalamic projections yet had no intrinsic functional organization. Subcortical structures implicated in early stages of processing exhibited heightened sensitivity to speech. The arcuate fasciculus, a suggested marker of language in BPP, showed similar volume and integrity to a healthy control. The frontal aslant tract, linked to oromotor function, was partially reconstructed. These findings highlight the importance of assessing the auditory cortex beyond speech production structures to understand absent speech in BPP. Despite profound cortical alterations, the intrinsic motor network and motor-speech pathways remained largely intact. This case underscores the need for comprehensive phenotyping using multiple MRI modalities to uncover causes of severe disruption in language development.

Automatic Quantification of Normal Brain Gyrification Patterns and Changes in Fetuses with Polymicrogyria and Lissencephaly Based on MRI.

BACKGROUND AND PURPOSE: The current imaging assessment of fetal brain gyrification is performed qualitatively and subjectively using sonography and MR imaging. A few previous studies have suggested methods for quantification of fetal gyrification based on 3D reconstructed MR imaging, which requires unique data and is time-consuming. In this study, we aimed to develop an automatic pipeline for gyrification assessment based on routinely acquired fetal 2D MR imaging data, to quantify normal changes with gestation, and to measure differences in fetuses with lissencephaly and polymicrogyria compared with controls. MATERIALS AND METHODS: We included coronal T2-weighted MR imaging data of 162 fetuses retrospectively collected from 2 clinical sites: 134 controls, 12 with lissencephaly, 13 with polymicrogyria, and 3 with suspected lissencephaly based on sonography, yet with normal MR imaging diagnoses. Following brain segmentation, 5 gyrification parameters were calculated separately for each hemisphere on the basis of the area and ratio between the contours of the cerebrum and its convex hull. Seven machine learning classifiers were evaluated to differentiate control fetuses and fetuses with lissencephaly or polymicrogyria. RESULTS: In control fetuses, all parameters changed significantly with gestational age (P < .05). Compared with controls, fetuses with lissencephaly showed significant reductions in all gyrification parameters (P ≤ .02). Similarly, significant reductions were detected for fetuses with polymicrogyria in several parameters (P ≤ .001). The 3 suspected fetuses showed normal gyrification values, supporting the MR imaging diagnosis. An XGBoost-linear algorithm achieved the best results for classification between fetuses with lissencephaly and control fetuses (n = 32), with an area under the curve of 0.90 and a recall of 0.83. Similarly, a random forest classifier showed the best performance for classification of fetuses with polymicrogyria and control fetuses (n = 33), with an area under the curve of 0.84 and a recall of 0.62. CONCLUSIONS: This study presents a pipeline for automatic quantification of fetal brain gyrification and provides normal developmental curves from a large cohort. Our method significantly differentiated fetuses with lissencephaly and polymicrogyria, demonstrating lower gyrification values. The method can aid radiologic assessment, highlight fetuses at risk, and may improve early identification of fetuses with cortical malformations.

Polymicrogyria in a patient after twin-twin transfusion syndrome.

This case report presents a patient with a monochorionic twin pregnancy, development of twin-twin transfusion-syndrome (TTTS) and polymicrogyria (PMG) of one fetus. Due to TTTS grade 3, fetoscopic laser ablation was performed at gestational week 16+1. Sonographic follow-up showed a cortical malformation of the right parietal lobe in the former donor, which was identified as PMG by MRI scans. We describe the course of the pregnancy, as well as the clinical, especially neurological, development of the child over 3 years. This case report documents the power of neuroplasticity, leading to comparably good neurological outcome in an extensive, likely acquired cortical malformation. Further, it emphasises the importance of a thorough prenatal imaging characterisation of malformations of cortical development for optimal prenatal counselling of these cases.

Polymicrogyria: An Unusual Case of Secondary Mania.

BACKGROUND: Secondary mania refers to a manic episode that arises during a medical illness other than bipolar disorder or in response to a drug or medication. As the psychopathological features of secondary mania resemble those of mania due to bipolar disorder, misdiagnosis is frequent. PURPOSE AND BASIC PROCEDURES: We present the case of a 20-year-old woman who developed a manic episode with psychotic symptoms, in whom polymicrogyria, a malformation of the cortical development with abnormal electroencephalographic activity, was documented. After initiating antiepileptic management, the affective symptoms completely subsided. MAIN FINDINGS: To date, no specific recommendations are available concerning when to perform advanced studies in patients with a manic episode; however, as our case shows, these are much needed. PRINCIPAL CONCLUSION: Because the treatment of secondary conditions largely depends on finding the underlying cause, patients with a new-onset mania should undergo a thorough assessment for secondary causes.

Variants in PTEN Are Associated With a Diverse Spectrum of Cortical Dysplasia.

BACKGROUND: Inactivating mutations in PTEN are among the most common causes of megalencephaly. Activating mutations in other nodes of the PI3K/AKT/MTOR signaling pathway are recognized as a frequent cause of cortical brain malformations. Only recently has PTEN been associated with cortical malformations, and analyses of their prognostic significance have been limited. METHODS: Retrospective neuroimaging analysis and detailed chart review were conducted on 20 participants identified with pathogenic or likely pathogenic mutations in PTEN and a cortical brain malformation present on brain magnetic resonance imaging. RESULTS: Neuroimaging analysis revealed four main cerebral phenotypes-hemimegalencephaly, focal cortical dysplasia, polymicrogyria (PMG), and a less severe category, termed "macrocephaly with complicated gyral pattern" (MCG). Although a high proportion of participants (90%) had neurodevelopmental findings on presentation, outcomes varied and were favorable in over half of participants. Consistent with prior work, 39% of participants had autism spectrum disorder and 19% of participants with either pure-PMG or pure-MCG phenotypes had epilepsy. Megalencephaly and systemic overgrowth were common, but other systemic features of PTEN-hamartoma tumor syndrome were absent in over one-third of participants. CONCLUSIONS: A spectrum of cortical dysplasias is present in individuals with inactivating mutations in PTEN. Future studies are needed to clarify the prognostic significance of each cerebral phenotype, but overall, we conclude that despite a high burden of neurodevelopmental disease, long-term outcomes may be favorable. Germline testing for PTEN mutations should be considered in cases of megalencephaly and cortical brain malformations even in the absence of other findings, including cognitive impairment.

Exome Sequencing and the Identification of New Genes and Shared Mechanisms in Polymicrogyria.

Importance: Polymicrogyria is the most commonly diagnosed cortical malformation and is associated with neurodevelopmental sequelae including epilepsy, motor abnormalities, and cognitive deficits. Polymicrogyria frequently co-occurs with other brain malformations or as part of syndromic diseases. Past studies of polymicrogyria have defined heterogeneous genetic and nongenetic causes but have explained only a small fraction of cases. Objective: To survey germline genetic causes of polymicrogyria in a large cohort and to consider novel polymicrogyria gene associations. Design, Setting, and Participants: This genetic association study analyzed panel sequencing and exome sequencing of accrued DNA samples from a retrospective cohort of families with members with polymicrogyria. Samples were accrued over more than 20 years (1994 to 2020), and sequencing occurred in 2 stages: panel sequencing (June 2015 to January 2016) and whole-exome sequencing (September 2019 to March 2020). Individuals seen at multiple clinical sites for neurological complaints found to have polymicrogyria on neuroimaging, then referred to the research team by evaluating clinicians, were included in the study. Targeted next-generation sequencing and/or exome sequencing were performed on probands (and available parents and siblings) from 284 families with individuals who had isolated polymicrogyria or polymicrogyria as part of a clinical syndrome and no genetic diagnosis at time of referral from clinic, with sequencing from 275 families passing quality control. Main Outcomes and Measures: The number of families in whom genetic sequencing yielded a molecular diagnosis that explained the polymicrogyria in the family. Secondarily, the relative frequency of different genetic causes of polymicrogyria and whether specific genetic causes were associated with co-occurring head size changes were also analyzed. Results: In 32.7% (90 of 275) of polymicrogyria-affected families, genetic variants were identified that provided satisfactory molecular explanations. Known genes most frequently implicated by polymicrogyria-associated variants in this cohort were PIK3R2, TUBB2B, COL4A1, and SCN3A. Six candidate novel polymicrogyria genes were identified or confirmed: de novo missense variants in PANX1, QRICH1, and SCN2A and compound heterozygous variants in TMEM161B, KIF26A, and MAN2C1, each with consistent genotype-phenotype relationships in multiple families. Conclusions and Relevance: This study's findings reveal a higher than previously recognized rate of identifiable genetic causes, specifically of channelopathies, in individuals with polymicrogyria and support the utility of exome sequencing for families affected with polymicrogyria.

Cobblestone-like brain malformation with a new bi-allelic ADGRG1 (GPR-56) mutation: Fetal imaging-pathology correlation.

BACKGROUND AND PURPOSE: Autosomal recessive cobblestone-like cortical malformation of the brain, with no eye or muscle involvement, has been reported in patients with biallelic mutations in ADGRG1 (formerly GPR56) and in other brain surface defects (eg, variants in COL3A1). We reported the intra-uterine brain MRI (iu-MRI), post-mortem MRI (pm-MRI), and neuropathology findings of a new ADGRG1 mutation in a fetus at early gestation. Imaging findings were compared with those of the sibling harboring the same mutation, to provide insights about the evolving morphology of such malformation. METHODS: A 21-week fetus underwent iu-MRI for a suspected cortical anomaly on ultrasound. After the MRI results, the termination of the pregnancy was carried out. A pm-MRI scan and autopsy were performed. A neuropathology-imaging correlation was achieved. The 5-year old sibling affected by developmental impairment also underwent a brain MRI. Both subjects underwent a genetic investigation. RESULTS: Two patterns of abnormality of the cerebral surface were identified on both fetal MRI: one at the vertex resembling a cobblestone-cortex due to neuronal overmigration into the subarchnoid space and the other in the occipital areas resembling polymicrogyria. These details closely matched the neuropathology findings. MRI findings of the sibling consisted of typical ADGRG1/GPR56-related brain findings showing a polymicrogyric-like cortex, also reported as bilateral frontal-parietal polymicrogyria. A flattened pons and small cerebellar vermis were present in both cases. Genetic testing demonstrated a novel homozygous variant c.1484T>C in the c gene in both cases. CONCLUSION: Our findings provide further evidence of the overlap of ADGRG1/GPR56-related brain dysgenesis with cobblestone-like cortical malformation of the brain.

Unilateral perirolandic polymicrogyria with ipsilateral brainstem hypoplasia and compensatory contralateral hyperplasia.

Polymicrogyria (PMG) is a malformation of cortical development that occurs mostly in the perisylvian region bilaterally (60-70%), most often presenting with epilepsy. Unilateral cases are much rarer with hemiparesis being the predominant symptom. We report a case of a 71-year-old man with right perirolandic PMG with ipsilateral hypoplasia and contralateral hyperplasia of the brainstem, with only non-progressive left-sided mild spastic hemiparesis. This imaging pattern is thought to occur due to the normal process of withdrawal of the axons of the corticospinal tract (CST) connected to aberrant cortex, possibly with compensatory contralateral CST hyperplasia. However, the majority of cases is additionally present with epilepsy. We believe it is worthwhile to investigate imaging patterns of PMG with symptoms' correlation, particularly with the help of techniques such as advanced brain imaging to assist in the study of cortical development along with adaptive somatotopic organization of the cerebral cortex in MCD with possible clinical applications.

Germline homozygous missense DEPDC5 variants cause severe refractory early-onset epilepsy, macrocephaly and bilateral polymicrogyria.

DEPDC5 (DEP Domain-Containing Protein 5) encodes an inhibitory component of the mammalian target of rapamycin (mTOR) pathway and is commonly implicated in sporadic and familial focal epilepsies, both non-lesional and in association with focal cortical dysplasia. Germline pathogenic variants are typically heterozygous and inactivating. We describe a novel phenotype caused by germline biallelic missense variants in DEPDC5. Cases were identified clinically. Available records, including magnetic resonance imaging and electroencephalography, were reviewed. Genetic testing was performed by whole exome and whole-genome sequencing and cascade screening. In addition, immunohistochemistry was performed on skin biopsy. The phenotype was identified in nine children, eight of which are described in detail herein. Six of the children were of Irish Traveller, two of Tunisian and one of Lebanese origin. The Irish Traveller children shared the same DEPDC5 germline homozygous missense variant (p.Thr337Arg), whereas the Lebanese and Tunisian children shared a different germline homozygous variant (p.Arg806Cys). Consistent phenotypic features included extensive bilateral polymicrogyria, congenital macrocephaly and early-onset refractory epilepsy, in keeping with other mTOR-opathies. Eye and cardiac involvement and severe neutropenia were also observed in one or more patients. Five of the children died in infancy or childhood; the other four are currently aged between 5 months and 6 years. Skin biopsy immunohistochemistry was supportive of hyperactivation of the mTOR pathway. The clinical, histopathological and genetic evidence supports a causal role for the homozygous DEPDC5 variants, expanding our understanding of the biology of this gene.

COL4A1 mutation-related disorder presenting as fetal intracranial bleeding, hydrocephalus, and polymicrogyria.

Fetal intracranial hemorrhage represents a rare event with an estimated prevalence of 1:10 000 pregnancies. We report a patient diagnosed prenatally with intracranial hemorrhage and ventriculomegaly carrying a novel, previously unreported, likely pathogenic variant in COL4A1. At the gestational age of 27 weeks, dilation of lateral ventricles was detected during a routine prenatal ultrasound scan, confirmed by prenatal MRI at 30 + 3 weeks of gestation. Prenatal examinations included amniocentesis with conventional G-band karyotyping and arrayCGH, and maternal testing for TORCH and parvovirus B19 infections. Virtual gene panel based on whole-exome sequencing data was performed postnatally. At the age of 2.5 months, the patient manifested epileptic seizures that remain difficult to control. Postnatal MRI showed partial thalamic fusion and polymicrogyria, in addition to severe enlargement of lateral ventricles, multiple deposits of hemosiderin in cerebral and cerebellar hemispheres, and thin optic nerve and chiasma. Virtual gene panel based on whole-exome sequencing data led to a detection of a de novo previously unreported in-frame deletion NM_001845.5:c.4688_4711del in COL4A1 located in the highly conserved NC1 domain initiating collagen helix assembly. The presented case lies one a more severe end of the COL4A1 mutation-related disease spectrum, manifesting as fetal intracranial bleeding, malformation of cortical development, drug-resistant epilepsy, and developmental delay.