Identification of the DNA methylation signature of Mowat-Wilson syndrome

Mowat-Wilson syndrome (MOWS) is a rare congenital disease caused by haploinsufficiency of ZEB2, encoding a transcription factor required for neurodevelopment. MOWS is characterized by intellectual disability, epilepsy, typical facial phenotype and other anomalies, such as short stature, Hirschsprung disease, brain and heart defects. Despite some recognizable features, MOWS rarity and phenotypic variability may complicate its diagnosis, particularly in the neonatal period. In order to define a novel diagnostic biomarker for MOWS, we determined the genome-wide DNA methylation profile of DNA samples from 29 individuals with confirmed clinical and molecular diagnosis. Through multidimensional scaling and hierarchical clustering analysis, we identified and validated a DNA methylation signature involving 296 differentially methylated probes as part of the broader MOWS DNA methylation profile. The prevalence of hypomethylated CpG sites agrees with the main role of ZEB2 as a transcriptional repressor, while differential methylation within the ZEB2 locus supports the previously proposed autoregulation ability. Correlation studies compared the MOWS cohort with 56 previously described DNA methylation profiles of other neurodevelopmental disorders, further validating the specificity of this biomarker. In conclusion, MOWS DNA methylation signature is highly sensitive and reproducible, providing a useful tool to facilitate diagnosis.

Supplementary Results: Clinical and molecular findings in our cohort (see end of section for additional references) Individual 1 (not published before) is an 8-year-old female of European ancestry.She is the only affected child in the family born at term from an uneventful pregnancy, during pregnancy which the mother received teratogenic drugs.The proband has typical MOWS facial gestalt, microcephaly, triangular face, wide nasal bridge, bulbous nasal tip, broad flared eyebrows, deep-set eyes, pointed chin, bilateral coloboma, small iris, convergent strabismus, cupped ears with thick lobules, and brittle sparse hair.She presented global developmental delay with sitting at 2 years and walking at around 3 years.She manifested generalized epilepsy from the age of 8 months, not well controlled after treatment with several antiepileptic drugs.She is not verbal and has profound intellectual disability.She has no cardiac anomalies (systolic murmur 2/6) and no gastrointestinal problems.She has a happy demeanor, hand flapping, and stereotypic movements.Chromosomal microarray testing did not reveal deleterious copy number variations; trio exome sequencing identified a heterozygous de novo variant in ZEB2: NM_014795.4:c.2083C>T,p.(Arg695*).This variant, expected to undergo nonsense-mediated mRNA decay or to result in a truncated protein, was classified as pathogenic according to the recommendations of the American College of Genetics and Genomics/Association of Molecular Pathology (ACMG/AMP; Richards et al., 2015 [25], criteria PVS1, PS2, PM2, PP4).
Individual 2 (not published before) is a 22-year-old female, born from an uneventful pregnancy with spontaneous delivery at 41 gestational weeks.She showed normal neonatal and motor development.The language was impaired, with a tested total IQ of 78 (WPPSI scale).She never had seizures and the EEG was normal.Brain MRI showed hypoplasia of corpus callosum and bilateral gliotic areas in the peritrigonal area.At 5 years of age her measurements were weight 20 kg (50 th centile), height 110 cm (50 th centile), head circumference 51.5 cm (75 th centile).In addition, she presented with mild scoliosis, frequent upper airways infections, and she underwent adenoidectomy.She showed some dysmorphic features, including supernumerary nipples, widely spaced teeth, median flare of the eyebrows, wide nasal bridge and prominent columella.Array CGH testing was normal; trio exome sequencing identified a heterozygous de novo missense variant in ZEB2: NM_014795.4:c.3160C>G;p.(Pro1054Ala).This variant occurs within the critical C-terminal zinc finger region, known to harbor other pathogenic missense variants (Ghoumid et al., 2013 [18]), and was classified as likely pathogenic (ACMG/AMP criteria PS2, PM1, PM2, PP1, PP4).Standing with support at 24 months.Independent walking at 3 years.Absent language.He was operated for bowel obstruction due to Hirschsprung's disease, confirmed by biopsy, and underwent cardiac surgery for PDA, ventricular defect, ostium secundum atrial septal defect.He underwent cochlear implantation for bilateral sensorineural hearing loss.He had seizures with onset at the age of one year, initially in the course of fever and then in apyrexia.Valgus osteotomy surgery of the calcaneus was performed at 16 years old.Brain MRI examinations showed hypoplasia of the splenium of the corpus callosum with reduction of the corpus callosum also in its posterior third.Molecular analysis MLPA and Sanger sequencing) of the ZEB2 gene revealed de novo heterozygous variant NM_014795.4:c.2254dup,p.(Thr752Asnfs*4), classified as pathogenic (ACMG/AMP criteria PVS1, PS2, PM2, PP4).
Individual 4 (described as patient #2 in Cecconi et al., 2008 [49], #6 in Garavelli et al., 2009 [48] ) is currently a 20-year-old female.She is the sister of individual #14.At 3 days of life she was diagnosed with congenital heart disease with patent foramen ovale, multiple ventricular septal defects, aortic valve stenosis, aortic coarctation, pulmonic valve stenosis for which she underwent corrective surgery at 27 days of life.Her clinical conditions worsened as a result of pulmonary infectious complications, cardiocirculatory arrest, cerebral hypoxic suffering resulting in flaccid paraplegia and neurological bladder.At the age of 11 months, she underwent an operation of partial correction of the scoliotic and lordotic deformity and of the pelvic obliquity with instrumented posterior vertebral arthrodesis with anchorage to the sacrum.She has a typical MOWS facial gestalt.She has global developmental delay with no speech.Molecular analysis of the ZEB2 gene gave the following result: de novo heterozygous nonsense variant NM_014795.4:c.310C>T,p.(Gln104*), classified as pathogenic (ACMG/AMP criteria PVS1, PS2, PM2, PP1, PP4).The same variant was found in the affected sister, but not in the healthy parents, compatible with germinal mosaicism.
Individual 5 (described as patient #4 in Garavelli et al., 2009 [48] ) is a 13-year-old female.She has typical MOWS facial gestalt.She had a diagnosis of congenital heart disease (patent ductus arteriosus, ventricular septal defect and aortic coarctation) She presented with psychomotor developmental delay with independent walking at 3 years and 7 months, first words at 3 years and 6 months and moderate intellectual disability.She was diagnosed with epilepsy with onset of focal clonic seizures at 2 years and 3 months and absence seizures at 7 years.She is under ophthalmological follow-up for strabismus and astigmatism and she has pes planus and genu valgus.Molecular analysis of the ZEB2 gene gave the following result: de novo heterozygous frameshift variant NM_014795.4:c.1052del,p.(Gly351Valfs*19), classified as pathogenic (ACMG/AMP criteria PVS1, PS2, PM2, PP4).
Individual 7 (described as patient #1 in Garavelli et al., 2005 [50], #12 in Garavelli et al., 2009 [48] ) is a 24year-old male.He underwent surgery at age 50 days for Hirschsprung's disease.He had hypospadias and underwent surgery for right cryptorchidism.He has a typical MOWS facial gestalt.Psychomotor development: sitting at 36 months.He has not achieved independent walking.Absent language.He has a severe intellectual disability.He had seizures with onset at the age of 13 months, initially in the course of fever and then in apyrexia.He has epilepsy with focal seizures with secondary generalization.Pediatric neurologists diagnosed complex partial epilepsy and absence seizures.At the age of 13 years, pediatric nephrologists diagnosed kidney stones.He was diagnosed with chronic iron deficiency anemia and presented episodes of intestinal subocclusion.Molecular analysis of the ZEB2 gene gave the following result: de novo heterozygous frameshift variant NM_014795.4:c.901del,p.(Leu301Cysfs*37), classified as pathogenic (ACMG/AMP criteria PVS1, PS2, PM2, PP4). ) is a 22-year-old male with a typical MOWS-evoking facial phenotype.He has congenital heart disease: patent ductus arteriosus, atrial septal defect, pulmonary valve and peripheral stenosis and aortic valve stenosis.Also, he has webbed penis and skeletal involvement: pectus carinatum, scoliosis, genu valgus, pes planus calcaneovalgus.He has sensorineural hearing loss and intellectual disability.He has epilepsy with onset at 3 years, seizures begin during sleepiness and wakes staring, has focal seizures with secondary generalization.Brain MRI showed corpus callosum hypoplasia, lateral ventricles enlargement and hippocampal malrotation.Molecular analysis of the gene ZEB2 demonstrated the de novo heterozygous nonsense variant NM_014795.4:c.625C>T,p.(Gln209*), classified as pathogenic (ACMG/AMP criteria PVS1, PS2, PM2, PP4).
Individual 9 (described as patient #14 in Garavelli et al., 2009 [48] At the age of 3 months she underwent cardiac surgery for patent Botallo duct.She has hypotonia, motor delay with acquisition of sitting at 5 years, first words at 7 years; she is unable to walk and has a limited language of about ten words, with severe intellectual disability.She had seizures with an onset at 18 months and a previous diagnosis of myoclonic epilepsy, with a tendency to generalize and improve over the years.Brain MRI demonstrated agenesis of the corpus callosum, dilatation of the temporal horns of the lateral ventricles, and small heterotopic subependymal gray matter nodule along the superior contour of the body of the left lateral ventricle.Molecular analysis of the gene ZEB2 demonstrated the de novo heterozygous nonsense variant NM_014795.4:c.2083C>T,p.(Arg695*), classified as pathogenic (ACMG/AMP criteria PVS1, PS2, PM2, PP4). ) is a 18-year-old male.His facial features are compatible with those of the MOWS; he also has superior pectus carinatum/inferior pectus excavatum.Psychomotor development: first words at 18 months, he acquired the independent walking at 2 years.He had seizures with onset at the age of one year, initially during fever and then, at the age of 6 years, in apyrexia.He has had focal seizures with secondary generalization and then, since age 8, absence seizures.Brain MRI demonstrated corpus callosum hypoplasia and hippocampal malrotation.He had recurrent otitis media and was diagnosed with conductive hearing loss.Molecular analysis of the gene ZEB2 demonstrated the de novo heterozygous nonsense variant NM_014795.4:c.2701C>T,p.(Gln901*), classified as pathogenic (ACMG/AMP criteria PVS1, PS2, PM2, PP4). ) is a 12-year-old male.In the first month of his life and then at the age of 4 years he underwent cardiac surgery for congenital heart disease with pulmonary atresia, ventricular septal defect and bicuspid aortic valve.He has typical MOWS facial gestalt.Psychomotor development: he acquired the sitting position at 9 months and independent walking at 2 years, he has language delay.He was diagnosed with grade 2 hypospadias and was operated on for bilateral cryptorchidism.He had his first seizure at the age of 4 years with focal seizure with secondary generalization.Brain MRI showed corpus callosum agenesis, hippocampal malrotation and large basal ganglia.Molecular analysis of the gene ZEB2 demonstrated the de novo heterozygous frameshift variant NM_014795.4:c.2718del,p.(Ala907Leufs*23), classified as pathogenic (ACMG/AMP criteria PVS1, PS2, PM2, PP4). ) is a 22year-old male.He has typical MOWS facial gestalt.Psychomotor development: he acquired the sitting position at 12 months and independent walking at 23 months, he has language delay.He underwent surgery for hypospadias with penoscrotal meatus and severe chorda penis.He has scoliosis and hallux valgus.At the age of 9 years he presented the 1st convulsive episode in apyrexia with generalized crises.Brain MRI showed ectopia of the cerebellar tonsils below the foramen magnum (Chiari 1 malformation) and corpus callosum thinned posteriorly.Molecular analysis of the gene ZEB2 demonstrated the de novo heterozygous nonsense variant NM_014795.4:c.2180T>A,p.(Leu727*), classified as pathogenic (ACMG/AMP criteria PVS1, PS2, PM2, PP4). ) is a 21year-old female.She is the sister of individual #2.She has a typical MOWS facial gestalt.She had delayed psychomotor development with independent walking at two and a half years, first words at 5 years.She had seizures with onset at the age of 18 months, initially in the course of fever and then in apyrexia, with absence, myoclonic and tonic-clonic seizures.Brain MRI examinations showed agenesis of the corpus callosum.Molecular analysis of the ZEB2 gene gave the following result: de novo heterozygous nonsense variant NM_014795.4:c.310C>T,p.(Gln104*), classified as pathogenic (ACMG/AMP criteria PVS1, PS2, PM2, PP1, PP4).The same variant was found in the affected sister, but not in the healthy parents, compatible with germinal mosaicism.) is a 19-year-old girl of Italian origin with typical MOWS facial gestalt and some EEG anomalies (activation of spikes and waves during sleep).No epileptic seizures have been reported so far.MRI examinations of the brain showed a complete corpus callosum agenesis, lateral ventricles enlargement, hippocampal malrotation, reduction of white matter thickness and large basal ganglia.The patient has a mild intellectual disability and was able to pronounce her first words at 18 months.Currently she can construct simple sentences of 3 to 4 words.She was able to sit without support at 10 months and walked without help at 20 months of age.She has constipation, but no cardiac, urogenital, dental, or skeletal anomalies.Gene sequencing of ZEB2 identified a de novo heterozygous single base pair deletion in exon 9, NM_014795.4:c.3031del,p.(Ser1011Alafs*64), which is predicted to escape nonsense-mediated decay (https://nmdprediction.shinyapps.io/nmdescpredictor/) and result in a truncated protein missing the C-terminal zinc finger cluster, CZF.The variant was classified as pathogenic (ACMG/AMP criteria PS2, PM1, PM2, PM4, PP4).
Individual 16 (described as patient #28 in Garavelli et al., 2017 [8] and Ivanovski et al., 2018 [4]) is a female who died suddenly at the age of 10 years and 10 months.She had the typical MOWS facial features.She had a congenital heart disease, atrial septal defect.Psychomotor development: she acquired the sitting position at 24 months and independent walking at 3 years, she had language delay.She had epilepsy with onset of myoclonic seizures at 9 months.She had recurrent otitis media and was in ophthalmological follow-up for strabismus and myopia.Brain MRI demonstrated corpus callosum hypoplasia and hippocampal malrotation.Molecular analysis of the ZEB2 gene gave the following result: de novo heterozygous frameshift variant NM_014795.4:c.1631_1635dup,p.(Asp546Leufs*11), classified as pathogenic (ACMG/AMP criteria PVS1, PS2, PM2, PP4). ) is a 14-year-old girl with typical MOWS facial gestalt.She has pes planus and genu valgus.Psychomotor development: she acquired the sitting position at 10 months and independent walking at 3 years, she spoke her first words at age 2 and had speech delay.She has epilepsy with onset of focal seizures and absence seizures at 2 years and 6 months.Brain MRI demonstrated complete agenesis of corpus callosum and hippocampal malrotation.Molecular analysis of the ZEB2 gene gave the following result: de novo heterozygous frameshift variant NM_014795.4:c.2682del,p.(Leu894Phefs*36), classified as pathogenic (ACMG/AMP criteria PVS1, PS2, PM2, PP4).
Individual 19 (described as patient #27 in Garavelli et al., 2017 [8] and Ivanovski et al., 2018 [4]) is a 14-year-old boy with typical MOWS facial gestalt.He acquired sitting by age 2, had not acquired independent walking at the age of 8 years, and speech was absent.He has a moderate-severe intellectual disability.He had onset of epilepsy at the age of 3 with generalized seizures.He was diagnosed with patent ductus arteriosus, pyloric stenosis, Hirschsprung disease and cryptorchidism.He is in in ophthalmological follow-up for strabismus, myopia and astigmatism.Brain MRI showed partial agenesis of corpus callosum, ventricular temporal horn enlargement and hippocampal malrotation.Molecular analysis of the ZEB2 gene gave the following result: de novo heterozygous nonsense variant NM_014795.4:c.817del,p.(Leu273*), classified as pathogenic (ACMG/AMP criteria PVS1, PS2, PM2, PP4). ) is a 20-year-old girl with typical MOWS facial gestalt.Echocardiography demonstrated an atrial septal defect, while renal ultrasonography showed bilateral pyelic dilatation.She acquired sitting by age 12 months, acquired independent walking after 5 years, first words at 6 years.She had onset of epilepsy at the age of 6 years, with focal clonic seizures.She was in orthopedic follow-up for bilateral pes planus calcaneovalgus and in ophthalmological follow-up for strabismus.Brain MRI showed complete agenesis of corpus callosum, ventricular temporal horn enlargement and hippocampal malrotation.Molecular analysis of the ZEB2 gene gave the following result: de novo heterozygous nonsense variant NM_014795.4:c.310C>T,p.(Gln104*), classified as pathogenic (ACMG/AMP criteria PVS1, PS2, PM2, PP4). ) is a 23-year-old girl with typical MOWS facial gestalt.At 10 months of age, she underwent cardiac surgery for atrial septal defect, ventricular septal defect, and patent ductus arteriosus.At 40 days of life, the first convulsive crisis in apyrexia appeared, which was followed by other convulsive episodes.A diagnosis of drugresistant epilepsy with complex partial seizures with secondary generalization and absence seizures was made.Psychomotor development: she acquired sitting position at 12 months, acquired independent walking at 2 years and 6 months, absent speech.Brain MRI showed complete agenesis of corpus callosum and lateral ventricles enlargement.Molecular analysis of the ZEB2 gene gave the following result: de novo heterozygous frameshift variant NM_014795.4:c.650_653dup,p.(Gly219Profs*21), classified as pathogenic (ACMG/AMP criteria PVS1, PS2, PM2, PP4).
Individual 23 (described as patient #87 in Ivanovski et al., 2018 [4], #1 in Ricci et al., 2021 [9]) is an 8-year-old male of Ukrainian nationality with typical MOWS facial gestalt.The psychomotor development delay (head control acquired at 1 year) and the presence of typical features prompted a neurological and genetic evaluation in Italy at 1 year and 4 months, ultimately leading to genetic testing that confirmed the clinical suspicion.At the time of the first evaluation, the proband also displayed obstinate constipation and renal involvement.Moreover, he underwent surgery for cryptorchidism.The echocardiogram ruled out the presence of cardiac anomalies.the age of 4 he presented with secondarily generalized focal seizures for which he initiated treatment with valproic acid, with partial benefit.Currently, the child has no speech and his motor condition has deteriorated due to the impossibility to access rehabilitative treatments in his home country.During the past three years he has lost the ability to maintain an upright position, which he had previously acquired.Molecular analysis of the ZEB2 gene gave the following result: heterozygous frameshift variant NM_014795.4:c.715del,p.(Glu239Argfs*23), classified as pathogenic (ACMG/AMP criteria PVS1, PM2, PP4).Parents were unavailable for segregation analysis.
Individual 24 (described as patient #89 in Ivanovski et al., 2018 [4]) is a 10-year-old male with typical MOWS facial gestalt.Echocardiography demonstrated patent ductus arteriosus, muscular interventricular septal defect and pulmonary valve dysplasia.At the age of 4 months he underwent decompressive craniotomy and bilateral fronto-orbital cranioplasty for craniostenosis (trigonocephaly).At the age of 1 year and 6 months he underwent surgery for hypospadias.Psychomotor development: he acquired the sitting position at 9 months, acquired independent walking at 16 months and presented language delay.At the age of 3 years and 6 months, he began having febrile seizure episodes.Molecular analysis of the ZEB2 gene gave the following result: de novo heterozygous frameshift variant NM_014795.4:c.1578_1579delinsA,p.(Asp527Thrfs*17), classified as pathogenic (ACMG/AMP criteria PVS1, PS2, PM2, PP4).
Individual 25 (not published before) is a 12-year-old female who has typical MOWS facial features.Agenesis of the corpus callosum and enlarged lateral ventricles were diagnosed prenatally and confirmed after birth.She presented with epilepsy at 12 months of age, consistent with neurodevelopmental delay.She had two episodes of febrile seizures and subsequently developed tonic-clonic seizures during sleep; she is currently being treated with valproate and levetiracetam and had no further episodes since then.She started walking at 3 years of age, showing ataxic and broad-based gait, flat support with bilateral valgopronation, valgus of the first ray on the left, both knees kept flexed for a few degrees in all phases of the step.Neurodevelopmental delay became more pronounced during the second year of life (Griffiths score 33 at 2 years, 23 at 5 years).At 5 years she was diagnosed with dorsal lumbar scoliosis, treated with Milwaukee brace.No relevant visual/hearing impairment was noted.Molecular analysis of the ZEB2 gene gave the following result: de novo heterozygous nonsense variant NM_014795.4:c.2357dup,p.(Leu786Phefs*9), classified as pathogenic (ACMG/AMP criteria PVS1, PS2, PM2). ) is a 20-year-old girl with typical MOWS facial gestalt..She was diagnosed with complex congenital heart disease: pulmonary artery sling with tracheal stenosis, pulmonary valvular and peripheral stenosis, patent ductus arteriosus.She was also diagnosed with Hirschsprung disease and spleen agenesis.Psychomotor development: he acquired the sitting position at 17 months, acquired independent walking at the age of 6 years and presented language delay.She had onset of epilepsy at the age of 2 years and 7 months with myoclonic epilepsy with sleep seizures.She was in orthopedic follow-up for bilateral pes planus calcaneovalgus and in ophthalmological follow-up for strabismus, myopia and astigmatism.Brain MRI showed hypoplasia of the splenium of the corpus callosum, ventricular temporal horn enlargement and hippocampal malrotation.Molecular analysis of the ZEB2 gene gave the following result: de novo heterozygous nonsense variant NM_014795.4:c.696C>G,p.(Tyr232*), classified as pathogenic (ACMG/AMP criteria PVS1, PS2, PM2, PP4).

Supplementary figure 2 ;
Mowat-Wilson (MOWS; ZEB2) episignature discovery + validation cohort both added as training.(A) Euclidean hierarchical clustering heatmap, each column represent one MOWS case or control, each row represents one probe selected for this episignature.It shows a clear separation between the cases in red and controls in blue.(B) Multidimensional scaling (MDS) plot shows segregation of MOWS cases and controls.(C) Support Vector Machine (SVM) classifier model.Model was trained using the selected MOWS episignature probes, 75% of controls and 75% of other neurodevelopmental disorder samples (blue).The remaining 25% controls and 25% of other disorder samples were used for testing (grey).Plot shows that the MOWS samples all have methylation variant pathogenicity (MVP) scores close to 1.