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Phenotypic expansion of EGP5-related Vici syndrome: 15 Dutch patients carrying a founder variant

Open AccessPublished:November 11, 2022DOI:https://doi.org/10.1016/j.ejpn.2022.11.003

      Highlights

      • Not all patients with EPG5 bi-allelic variants fulfill all clinical criteria for Vici syndrome.
      • Production of residual EPG5 protein leads to an attenuated phenotype of Vici syndrome.
      • There is substantial clinical variability between patients with biallelic EPG5 variants.
      • Suggestion to rename Vici syndrome into ‘EPG5-related neurodevelopmental disorder’.

      Abstract

      Vici syndrome (OMIM 242840) is a very rare autosomal recessive multisystem disorder first described in 1988. In 2013, bi-allelic loss-of-function mutations in EPG5 were reported to cause Vici syndrome. Five principal diagnostic features of Vici syndrome have been proposed: agenesis of the corpus callosum, cataracts, cardiomyopathy, hypopigmentation, and combined immunodeficiency. We identified 15 patients carrying a homozygous founder missense variant in EPG5 who all exhibit a less severe clinical phenotype than classic Vici syndrome. All 15 show typical brain abnormalities on MRI. The homozygous founder variant in EPG5 they carry results in a shorter in-frame transcript and truncated, but likely still residual, EPG5 protein. We speculate that the residual EPG5 protein explains their attenuated phenotype, which is consistent with two previous observations that low expression of EPG5 can lead to an attenuated Vici syndrome phenotype. We propose renaming this condition EPG5-related neurodevelopmental disorder to emphasize the clinical variability of patients with bi-allelic mutations in EPG5.

      Keywords

      1. Introduction

      Vici syndrome (OMIM 242840) is a very rare autosomal recessive multisystem disorder first described by Vici et al. in 1988 [
      • Dionisi Vici C.
      • Sabetta G.
      • Gambarara M.
      • Vigevano F.
      • Bertini E.
      • Boldrini R.
      • et al.
      Agenesis of the corpus callosum, combined immunodeficiency, bilateral cataract, and hypopigmentation in two brothers.
      ]. Since then, more than 80 patients have been reported with various neurodevelopmental features [
      • Abidi K.T.
      • Kamal N.M.
      • Bakkar A.A.
      • Almarri S.
      • Abdullah R.
      • Alsufyani M.
      • et al.
      Vici syndrome with pathogenic homozygous EPG5 gene mutation: a case report and literature review.
      ,
      • Alzahrani A.
      • Alghamdi A.A.
      • Waggass R.
      A Saudi infant with Vici syndrome: case report and literature review.
      ,
      • Byrne S.
      • Jansen L.
      • U-King-Im J.-M.
      • Siddiqui A.
      • Lidov H.G.W.
      • Bodi I.
      • et al.
      EPG5-related Vici syndrome: a paradigm of neurodevelopmental disorders with defective autophagy.
      ]. While five principal diagnostic features of Vici syndrome have been proposed (agenesis of the corpus callosum, cataracts, cardiomyopathy, hypopigmentation, and combined immunodeficiency) [
      • Byrne S.
      • Jansen L.
      • U-King-Im J.-M.
      • Siddiqui A.
      • Lidov H.G.W.
      • Bodi I.
      • et al.
      EPG5-related Vici syndrome: a paradigm of neurodevelopmental disorders with defective autophagy.
      ], three additional findings – profound developmental delay, acquired microcephaly, and failure to thrive – are consistently associated with the main diagnostic features [
      • Byrne S.
      • Dionisi-Vici C.
      • Smith L.
      • Gautel M.
      • Jungbluth H.
      Vici syndrome: a review.
      ]. Most patients with Vici syndrome have a severely reduced lifespan, with a median survival of 42 months, and most die of consequences of cardiomyopathy and/or infections [
      • Byrne S.
      • Dionisi-Vici C.
      • Smith L.
      • Gautel M.
      • Jungbluth H.
      Vici syndrome: a review.
      ].
      In 2013, bi-allelic mutations in EPG5 were reported to cause Vici syndrome [
      • Cullup T.
      • Kho A.L.
      • Dionisi-Vici C.
      • Brandmeier B.
      • Smith F.
      • Urry Z.
      • et al.
      Recessive mutations in EPG5 cause Vici syndrome, a multisystem disorder with defective autophagy.
      ]. While most of the EPG5 mutations identified in Vici syndrome appear to cause null alleles, a few missense mutations have been identified, and these also seemingly lead to a loss of EPG5 function [
      • Byrne S.
      • Dionisi-Vici C.
      • Smith L.
      • Gautel M.
      • Jungbluth H.
      Vici syndrome: a review.
      ]. To date (updated Oct 2021), 56 different mutations have been described throughout the entire EPG5 coding sequence, but clear genotype–phenotype correlations have not been established [
      • Byrne S.
      • Jansen L.
      • U-King-Im J.-M.
      • Siddiqui A.
      • Lidov H.G.W.
      • Bodi I.
      • et al.
      EPG5-related Vici syndrome: a paradigm of neurodevelopmental disorders with defective autophagy.
      ,
      • Cullup T.
      • Kho A.L.
      • Dionisi-Vici C.
      • Brandmeier B.
      • Smith F.
      • Urry Z.
      • et al.
      Recessive mutations in EPG5 cause Vici syndrome, a multisystem disorder with defective autophagy.
      ,
      • Ehmke N.
      • Parvaneh N.
      • Krawitz P.
      • Ashrafi M.-R.
      • Karimi P.
      • Mehdizadeh M.
      • et al.
      First description of a patient with Vici syndrome due to a mutation affecting the penultimate exon of EPG5 and review of the literature.
      ,
      • Hori I.
      • Otomo T.
      • Nakashima M.
      • Miya F.
      • Negishi Y.
      • Shiraishi H.
      • et al.
      Defects in autophagosome-lysosome fusion underlie Vici syndrome, a neurodevelopmental disorder with multisystem involvement.
      ]. Most mutations are unique to individuals or families, with only five recurrent mutations reported [
      • Ehmke N.
      • Parvaneh N.
      • Krawitz P.
      • Ashrafi M.-R.
      • Karimi P.
      • Mehdizadeh M.
      • et al.
      First description of a patient with Vici syndrome due to a mutation affecting the penultimate exon of EPG5 and review of the literature.
      ,
      • Demiral E.
      • Sen A.
      • Esener Z.
      • Ceylaner S.
      • Tekedereli I.
      A rare mutation in the EPG5 gene causes Vici syndrome.
      ,
      • Hızal M.
      • Yeke B.
      • Yıldız Y.
      • Öztürk A.
      • Gürbüz B.B.
      • Coşkun T.
      Two cases of Vici syndrome presenting with corpus callosum agenesis, albinism, and severe developmental delay.
      ].
      Recently, two case reports were published in which two patients with a less severe phenotype were diagnosed with Vici syndrome following identification of bi-allelic EGP5 mutations [
      • Maillard C.
      • Cavallin M.
      • Piquand K.
      • Philbert M.
      • Bault J.P.
      • Millischer A.E.
      • et al.
      Prenatal and postnatal presentations of corpus callosum agenesis with polymicrogyria caused by EGP5 mutation.
      ,
      • Waldrop M.A.
      • Gumienny F.
      • Boue D.
      • de Los Reyes E.
      • Shell R.
      • Weiss R.B.
      • et al.
      Low-level expression of EPG5 leads to an attenuated Vici syndrome phenotype.
      ]. Neither patient fulfilled the clinical characteristics of Vici syndrome, and their diagnosis was not suspected until the results of exome sequencing were known. The first patient had agenesis of the corpus callosum but no hypopigmentation, immunodeficiency, or cardiomyopathy. He displayed delayed achievement of motor milestones because of severe hypotonia and generalized weakness but was able to stand at the age of 4 years 3 months and to speak a few words. He developed a status epilepticus once but had no further seizures after starting levetiracetam [
      • Waldrop M.A.
      • Gumienny F.
      • Boue D.
      • de Los Reyes E.
      • Shell R.
      • Weiss R.B.
      • et al.
      Low-level expression of EPG5 leads to an attenuated Vici syndrome phenotype.
      ]. The second patient also presented with agenesis of the corpus callosum without cardiomyopathy or immunodeficiency. She did not have cataracts or hypopigmentation but did have fair hair. At age 2 years 5 months, she showed peripheral hypotonia, absence of tendon reflexes, and low truncal tone. No information about her cognitive development was provided. In addition to agenesis of the corpus callosum, frontoparietal polymicrogyria, and pontocerebellar atrophy were also described on her MRI [
      • Maillard C.
      • Cavallin M.
      • Piquand K.
      • Philbert M.
      • Bault J.P.
      • Millischer A.E.
      • et al.
      Prenatal and postnatal presentations of corpus callosum agenesis with polymicrogyria caused by EGP5 mutation.
      ]. These two reports demonstrate that mutations in EPG5 can lead to conditions with a wider and less severe clinical spectrum than ‘classic’ Vici syndrome. We therefore propose renaming this disorder EPG5-related neurodevelopmental disorder (EPG5-related NDD), with Vici-type EPG5-related NDD representing the severe end of the spectrum. Waldrop et al. [
      • Waldrop M.A.
      • Gumienny F.
      • Boue D.
      • de Los Reyes E.
      • Shell R.
      • Weiss R.B.
      • et al.
      Low-level expression of EPG5 leads to an attenuated Vici syndrome phenotype.
      ] showed alternative splicing to a transcript without exon 17 that very likely leads to low-level expression of EPG5 protein in their case, and they assume that this probably leads to an attenuated phenotype of EPG5-related NDD. In this paper, we further illustrate the phenotypic variation and describe the typical brain abnormalities associated with EPG5-related NDD in 15 patients with a homozygous founder missense variant in EPG5.

      2. Material and methods

      2.1 Patient collection

      The first patients described in this study were identified in 1986 during clinical investigations carried out at the outpatient department of the University Medical Center (UMC) Utrecht and the UMC Groningen in the Netherlands. Based on their birthplace, the homogeneous clinical picture, and the unique MRI images of the patients, genealogical research was performed, and this linked nine patients to a common ancestor pair ten generations earlier (Fig. 1). Over the years, six additional patients presenting with the same phenotype were identified, all with ancestral links to the same narrowly defined geographical area (these cases are not included in the pedigree as their direct link to the common ancestor was not investigated). As the affected family originates from a small town and individuals could identify themselves or others in the pedigree, to protect privacy, the carrier parents and their affected children are represented by an individual of unknown gender in the pedigree and unaffected sibs are not included. The genomic DNA and Epstein-Barr Virus (EBV) lymphoblast cell lines were collected via the respective genetic diagnostic centers of the two UMCs.
      Fig. 1
      Fig. 1Pedigree of a large consanguineous Dutch family with severe structural brain abnormalities with six distantly related parents, a common ancestor pair, and 9/15 affected children.

      2.2 Ethics statement

      The tests were performed in line with the original diagnostic request. Moreover, when the blood samples were taken, parents were asked whether they had objections to the use of their DNA and their children's DNA for future studies. If parents had objections, this was indicated on the original “Request for DNA test” form, and the DNA was not used. Written consent for publication of photographs was obtained from the parents of the patient in Fig. 2.
      Fig. 2
      Fig. 2Portrait photographs of one of the patients at different ages (newborn, age 7, and age 15). No major dysmorphic features were seen, although a thin upper lip and full cheeks can be noted. There was no hypopigmentation of skin or hair.

      2.3 Whole exome sequencing

      Whole exome sequencing (WES) was performed in two patients. Sequence data analysis and variant calling using an in-house pipeline was performed as described in Nibbeling et al. [
      • Nibbeling E.A.R.
      • Duarri A.
      • Verschuuren-Bemelmans C.C.
      • Fokkens M.R.
      • Karjalainen J.M.
      • Smeets C.J.L.M.
      • et al.
      Exome sequencing and network analysis identifies shared mechanisms underlying spinocerebellar ataxia.
      ] Sanger sequencing was used to validate the c.4862G > A variant in exon 28 of EPG5 using the following primers: Forward 5′-GGATCCTGGGCTACACACTG-3′ and Reverse 5′-CCTCCTCTACCCATGGTGAC-3’. Splice site prediction was performed using the tool Splice Site Prediction by Neuronal Network (https://www.fruitfly.org/seq_tools/splice.html).

      2.4 Cell culture, RNA isolation, and cDNA synthesis

      EBV-immortalized lymphocytes were cultured in Dulbecco's modified Eagle's medium, high glucose supplemented with 10% fetal bovine serum and 5% penicillin (100 U/ml), streptomycin (100 mg/ml), and glutamate (100 mg/ml). The cultures were maintained at 37 °C in an atmosphere of 10% CO2. After 48 h, the cells were harvested and RNA isolated using the Trizol method. cDNA was synthesized according to the manufacturer's protocol (GE Healthcare). A non-quantitative RT-PCR was subsequently performed using standard conditions and the following primers: For (EPG5) 5’ – CGGTAAGAAATGCCAAGGAG-3′ and Rev (EPG5) 5′-TCTGAATCCCAGGTGTAGGC-3′ and For (GAPDH) 5′-TCCCTGAGCTGAACGGGAAG-3′ and Rev (GAPDH) 5′-GGAGGAGTGGGTGTCGCTGT-3’. PCR products were analyzed by gel electrophoresis and Sanger sequencing.

      2.5 Magnetic resonance imaging

      MRI scans were made at different hospitals using various protocols and available for 8 patients for assessment (5 females, 3 males). Protocols comprised a sagittal T1 or T2, a transverse T2 and inversion recovery, coronal T1 and/or T2 weighted imaging sequences, and, in some patients FLAIR and DTI. MRI scans were made at different ages, ranging from 3 days to 13 years.

      3. Results

      3.1 Clinical phenotype

      All patients or their parents or grandparents originate from a small isolated fisherman's village in the Netherlands that was an island until it was connected to the mainland in 1939. Because of strong religious beliefs, the village is still isolated and thus constitutes a consanguineous population. The 15 patients (6 boys and 9 girls, Table 1) are offspring of 11 parental couples who are said to have ancestral roots on the island, and we could make a pedigree for 6 of these parental couples and their 9 affected children (Fig. 1). In four families, two affected children were born. All the children were born at term after uneventful pregnancies. In four children, agenesis of the corpus callosum was seen on routine ultrasound examinations during pregnancy. None of the families opted for invasive genetic prenatal diagnosis. Most children came to the clinic within the first 6 months of life because of developmental delay, failure to thrive, hypotonia, and poor eye contact (Table 1). Failure to thrive was most clear in an inability to gain weight, and weight gain returned to normal after introduction of gastric or nasal tube feeding. To date, 13 patients have died, with a median age of death of 13 years (range 20 months–23 years). In all cases, the cause of death was respiratory insufficiency due to pneumonia rather than immunodeficiency. Detailed clinical information for each patient can be found in the Supplementary Table.
      Table 1Clinical features of our patients (n = 15) compared to patients with the Vici type EPG5-related NDD (based on review articles of Byrne et al., 2016) [
      • Byrne S.
      • Jansen L.
      • U-King-Im J.-M.
      • Siddiqui A.
      • Lidov H.G.W.
      • Bodi I.
      • et al.
      EPG5-related Vici syndrome: a paradigm of neurodevelopmental disorders with defective autophagy.
      ,
      • Byrne S.
      • Dionisi-Vici C.
      • Smith L.
      • Gautel M.
      • Jungbluth H.
      Vici syndrome: a review.
      ]. See supplementary Table for detailed description per patient.
      Our patients (n = 15)Vici type EPG5-related NDD (based on Byrne et al. 4,5) (n = 50)
      DemographicsGender6 male24 male
      9 female26 female
      Pregnancy and birthIn 3 pregnancies reduced fetal movements reported.In some pregnancies reduced fetal movements reported, no further data.
      All births at term
      No arthrogryposis6/50 (12%) arthrogryposis
      Deceased13/15 (87%)No data reported – highly likely all deceased
      Failure to thriveBirthweight normalBirthweight normal
      Weight droppingWeight dropping < -2,5 SD
      Normal weight and growth with gastric tube feeding<10% normal body weight with gastric tube feeding
      Age at deathMedian age of death 13 years (range 20 mo–23 yrs)Median age of death 42 mo (range 1 mo–102 mo)
      Central Nervous SystemSeizures15/15 (100%)20/34 (59%)
      Age of OnsetFirst year of life 4/9 (44%)Onset always in first year of life (100%)
      After first year of life 5/9 (56%)
      (Range 13 mo–17 years)
      typesWest syndrome (hypsarrhythmia), multifocal seizures, therapy resistantEarly-onset epileptic encephalopathies with burst-suppresion
      Focal seizures, good control with therapyHypsarrythmia
      Mostly difficult to control
      Microcephaly5/14 primary, non-progressive microcephaly (36%)28/31 (90%) rapidly primary progressive microcephaly in the first year of life
      Developmental delay15/15 profoundProfound (50/50)
      Developmental milestones7 Laughing,No head control in most
      1 speak few words4/50 able to roll over, but lost this skill
      3 babblingNo indepent sitting
      Most have Head control
      Some clap hands, play simple games
      No independent sitting
      Brisk reflexes10/14 brisk,Around a third (33%) brisk
      9 spasticity lower extremities, increasing with age
      MuscleMyopathy1/8 yes (structural abnormal muscle biopsy)6/7 evidence of myopathy on EMG/nerve conduction studies
      17 patients with skeletal myopathy based on muscle biopsy
      Hypotonia15/15 profound, onset from birth, axial hypotonia increasing with ageAll profound, associated generalized weakness, paucity of movement
      NervesNeuropathy0/10 noReflexes reduced or absent in most cases
      Low reflexes
      SkinHypopigmentation (skin/eyes/hair)0/1550/50 (100%)
      No albinism. Relative to familial and ethnic background
      EyesCataracts0/1329/38 (76%)
      at birth or evolving over first months of life
      Optic nerve hypoplasia/atrophy11/14 optic hypoplasia11/38 (29%) optic atrophy
      2/14 optic atrophyno hypoplasia reported
      14/38 (37%) retinal changes
      5/38 (13%) ocular albinism
      Vision15/15 abnormal vision, light-dark perception only8/10 (80%) abnormal vision
      HearingSensorineural hearing loss4/12 (33%)6/9 (67%)
      HeartCardiomyopathy (dilated or hypertrophic)0/1328/34 (82%) hypertrophic and/or dilated cardiomyopathy, presenting in first year of life, progressive
      (all screened yearly)
      Minor congenital heart defects0/1010/50 (20%) persistent foramen ovale and atrial septal defects
      Immune systemCombined immunodeficiency0/938/38 (100%)
      In 11 patients a combined immunological defect with both T and B cells affected
      1 patient severe combined immunodeficiency
      Respiratory infections11/11 yes, recurrent and common infectionsAll, recurrent and unusual infections
      (due to hypotonia and insufficient cough)(based on immunodeficiency)
      BloodNo haematological abnormalities (0/11)24/50 (48%) patients haematological abnormalities (anaemia, leucopaenia)
      Other
      Skeletal5/15 (33%) developed scoliosisNot reported
      2 patients had surgical reconstruction
      Sleep abnormalities5/8 (63%)Reported once (central sleep apnoea)
      sleep apnoea (central and obstructive), frequent wakening, saturation dips
      Good response to nasal high-flow oxygen therapy
      Feeding11/11 gastrostomy feedingMost required gastrostomy feeding
      Precocious puberty5/15 (33%)Not reported
      MRI abnormalitiesAgenesis corpus callosum15/1538/38
      Deep frontal fissure8/8Not reported
      Hypoplasia white matter8/818/18
      Smaller mesencephalon and pons8/818/18 pontine hypoplasia
      Delayed myelination8/818/18
      The clinical characteristics of the 15 patients are quite distinct from those reported for patients with Vici-type EPG5-related NDD (Table 1). Since the phenotypes of our patients are very similar, we grouped their symptoms (Table 1). None of the 15 patients had immunodeficiency, cardiomyopathy, hypopigmentation, or cataracts.
      We did observe neurological features consistent with Vici-type EPG5-related NDD, including severe developmental delay, agenesis of the corpus callosum, failure to thrive before the start of tube feeding, and hypotonia. A third of the patients (5/15) had non-progressive primary microcephaly (head circumference range −3.8SD to -2SD), but most patients were normocephalic (head circumference range -2SD to +1.0SD). All patients suffered from epilepsy, ranging from mild generalized epilepsy to severe West syndrome evolving into refractory epilepsy. Age of onset of epilepsy ranged from 1 month to 17 years of age, with about half the patients developing epilepsy in the first year of life. They exhibited no or minimal language development. All patients had optic hypoplasia or pale optic disks. Their vision was limited, with only perception of light and dark in most patients. Most were evaluated by an ophthalmologist. Patients were evaluated yearly by a cardiologist, and at least twice a year by a pediatric neurologist and a pediatrician. Laboratory blood tests for thyroid function, immune status (complete blood count, IgG, IgA, IgM, T-cells, CD3, CD4, CD8 and B-cells CD19 and NK-cells CD16/56), and serum creatine kinase were carried out at least yearly, starting from 2013, with exception of 2020 (the first year of the SARS-CoV-2 pandemic).
      As they aged, most patients became more hypotonic, with low reflexes but no signs of areflexia. Electromyography was performed in only two patients, with normal results. Slowly elevating serum creatine kinase (maximum 1200 IU/L) was detected with aging in some patients, which may suggest the presence of a myopathy. For most patients, no additional tests, e.g. muscle biopsy or MRI of the muscles, were performed, so a myopathy could not be confirmed. In one individual, a myopathy was diagnosed later in life after a muscle biopsy, and 60% of patients (9/15) developed hypertonia in the lower limbs and Babinski reflexes (Table 1). Five out of 8 patients developed sleep apnea, and 5 out of 15 developed scoliosis. Five of our patients developed precocious puberty. None of our patients had hematologic abnormalities. In Fig. 2 one of the patients is depicted at different ages. There is no hypopigmentation of skin or hair. She has a thin upper lip and full cheeks, but no major dysmorphic features were seen. (Fig. 2).

      3.2 Radiological abnormalities

      In all 8 patients, the MRI scan showed the same morphological congenital abnormalities, consisting of corpus callosum agenesis, a deep frontal fissure, limited white matter volume leading to indentation of the dorsal insula into the posterior horns of the lateral ventricles, and a smaller mesencephalon and pons. These findings are indicated in MRI scans of 3 representative patients shown in Fig. 3, Fig. 4 and described in more detail in the figure legends. The morphological findings are present from birth, and do not develop or change over time, as is demonstrated by the MRI images of a 7 month old female patient and a 9 week old male patient, shown in Fig. 3, and of a female patient scanned at 3 days and again at 13 years, shown in Fig. 4. In the patient scanned at 3 days, shown in Fig. 4A, no visible T1 hyperintense myelin signal was seen in the posterior limb of the internal capsule (PLIC). In the patient scanned at 9 weeks, normal myelination was present in the PLIC, but no age expected dorsal extension was seen into the optic radiation, as shown on the Inversion Recovery (IR) in Fig. 3L. This was also found in the other children scanned at comparable age. In the 7 month old patient, the IR shown in Fig. 3H, shows myelination extending slightly in the direction of the optic radiation, however no age-expected myelination was seen in other cerebral areas, making the pattern more compatible with that seen at 3 months.
      Fig. 3
      Fig. 3MRI images of two patients. A–H: Images of a 7-month-old female patient with typical findings of (1) corpus callosum agenesis (A, C, G, H) accompanied by (2) symmetric bilateral medial position of the hippocampi with cranial extension of the collateral sulcus (G, arrow), (3) a symmetric deep fissure in the frontal lobes, extending from the frontal horn to the frontal pole, lined by normal-appearing cortex (E, F, arrows, (4) dysgyria in the cingulate gyrus with an adjacent lateral deep sulcus (D, G, arrows), (5) restricted cerebral white matter volume, leading to bulging of the dorsal insula into the lateral ventricle (C, H, arrows, (6) delayed cerebral myelination, limited to the posterior limb of the internal capsule (PLIC), shown on transverse inversion recovery (H, open arrow), slightly more advanced to the optic radiation compared to the 9-week-old child shown in L, and (7) a small mesencephalon and pons (A, B). No clear colpocephaly (wide dorsal atria of the lateral ventricles) is present, as seen in children with only corpus callosum agenesis. IL: Images of a 9-week-old male patient with similar morphological features to those seen in Figure AH, with age conforming myelination in the PLIC.
      Fig. 4
      Fig. 4Transverse and sagittal planes of the same female patient at age 3 days (A–C) and 13 years (D–F) showing similar morphological findings to those in the patients in . At 13 years, limited white matter volume is seen on the transverse T2 (D), comparable to that seen on the transverse T1 at age 3 days (A). Similar to the patient shown in C, this is most pronounced at the dorsal insulae, which bulge into the lateral ventricles (white arrows). On the transverse T1, the expected age-related small area of hyperintense myelination in the PLIC is not seen (white open arrow). On the transverse T2 at the age of 13 years, the myelination of the cerebral white matter appears to conform with age.

      3.3 Whole exome sequencing

      In all patients, WES identified only one shared novel homozygous missense variant, c.4862G > A, p.Arg1621Gln in exon 28 of EPG5 (NM_020964.2) (Fig. 5A and B), which segregated with the disease phenotype in the family, as assessed in four parent–patient pairs and their unaffected sibs (when available). Variant c.4862G > A, p.Arg1621Gln is not reported in the nomAD database (accessed October 2021). Notably, variant c.4862G > A, p.Arg1621Gln is in silico-predicted to induce a novel acceptor splice site that leads to an in-frame deletion of 18 amino acids in exon 28 of EPG5.
      Fig. 5
      Fig. 5A) Schematic representation of EPG5, comprising of 44 exons, which encodes for a 2579 amino acid long protein. Whole exome sequencing in all 15 of our patients identified missense variant c.4862G > A, p.Arg1621Gln in exon 28 (indicated by a dashed line). B) Electropherograms showing the presence of a homozygous c.4862G > A, p.Arg1621Gln missense variant in the gDNA of patients compared to healthy heterozygous carriers and a non-carrier. C) Samples of heterozygous carriers (±) (lanes 1–3) showed a shorter (∗ = mutant) and a longer (∗∗ = wildtype) transcript, whereas the patient sample (−/−) (lane 4) showed only the shorter (∗ = mutant) EPG5 transcript. No product was detected in the negative control (lane 5). The size of the GAPDH amplicon was similar in all samples. D) Electropherograms showing the sequence of the boundaries between exons 27 and 28 of EPG5 in the wildtype/longer (top panel) and mutant/shorter EPG5 transcript (bottom panel). E) Alignment of the wildtype and mutant transcripts revealed an in-frame 18 amino acid deletion (in red) in EPG5.
      Given that almost all reported Vici-typeEPG5-related NDD mutations cause null alleles and/or affect mRNA splicing, we investigated whether the c.4862G > A, p.Arg1621Gln variant also affects mRNA splicing. We extracted RNA from EBV-immortalized lymphocytes from a patient, mother, and two unaffected sibs (all heterozygous carriers of the c.4862G > A, p.Arg1621Gln variant). One short amplicon was observed in the patient's sample, whereas the mother and the two unaffected sibs showed a longer amplicon in addition to the shorter amplicon (Fig. 5C). Sanger sequencing confirmed an in-frame deletion of 18 amino acids in exon 28 of EPG5 in the shorter amplicon and confirmed the complete wildtype sequence in the longer amplicon (Fig. 5D and E). This atypical splicing event seemed quite complete given the complete absence of the (longer) wildtype amplicon in the patient sample. These data confirmed that thec.4862G > A, p.Arg1621Gln variant causes aberrant splicing of EPG5. Based on the American College of Medical Genetics and Genomics criteria, the c.4862G > A, p.Arg1621Gln variant was classified as pathogenic (PS3) [
      • Richards S.
      • Aziz N.
      • Bale S.
      • Bick D.
      • Das S.
      • Gastier-Foster J.
      • et al.
      Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of medical genetics and genomics and the association for molecular pathology.
      ].

      4. Discussion

      This is the first study to describe 15 patients with EPG5-related NDD linked by a common founder. All patients exhibit a phenotype different than the Vici-type EPG5-related NDD phenotype reported by Byrne et al. [
      • Byrne S.
      • Jansen L.
      • U-King-Im J.-M.
      • Siddiqui A.
      • Lidov H.G.W.
      • Bodi I.
      • et al.
      EPG5-related Vici syndrome: a paradigm of neurodevelopmental disorders with defective autophagy.
      ,
      • Byrne S.
      • Dionisi-Vici C.
      • Smith L.
      • Gautel M.
      • Jungbluth H.
      Vici syndrome: a review.
      ] None of our patients exhibited immunodeficiency, cardiac abnormalities, hypopigmentation, or cataracts. Nor did they develop these symptoms later in life. However, additional features of Vici-type EPG5-related NDD were present, including severe developmental delay, epilepsy/seizures, and failure to thrive. In addition, all patients showed complete absence of the corpus callosum, a key feature of Vici-type EPG5-related NDD. This work adds robustly to the phenotypic description of EPG5-related NDD. Following two previously published case reports from Maillard et al. [
      • Maillard C.
      • Cavallin M.
      • Piquand K.
      • Philbert M.
      • Bault J.P.
      • Millischer A.E.
      • et al.
      Prenatal and postnatal presentations of corpus callosum agenesis with polymicrogyria caused by EGP5 mutation.
      ] and Waldrop et al. [
      • Waldrop M.A.
      • Gumienny F.
      • Boue D.
      • de Los Reyes E.
      • Shell R.
      • Weiss R.B.
      • et al.
      Low-level expression of EPG5 leads to an attenuated Vici syndrome phenotype.
      ], this work is the third reported example of patients with a less severe clinical phenotype associated with a molecular diagnosis of EPG5-related NDD. Most mutations in EPG5 are unique to cases, making genotype–phenotype correlation difficult. Byrne et al. first suggested a correlation in two cases with a less severe phenotype and identical missense mutations [
      • Byrne S.
      • Jansen L.
      • U-King-Im J.-M.
      • Siddiqui A.
      • Lidov H.G.W.
      • Bodi I.
      • et al.
      EPG5-related Vici syndrome: a paradigm of neurodevelopmental disorders with defective autophagy.
      ]. Our study adds new evidence of a strong phenotype–genotype correlation. On MRI, typical morphological findings were seen in our patients, and these appear to be present from birth, as described in Fig. 3, Fig. 4. The persisting limited cerebral white matter volume, suggesting hypoplasia and not atrophy, is most pronounced in the area between the dorsal insula and the lateral ventricles. As this is where the optic radiation and the fronto-occipital fascicle and arcuate fibers are located, which are part of the associative bundles important for speech, the restricted local white matter volume might hypothetically contribute to the absence of speech and the visual impairment in these patients [
      • Catani M.
      • Mesulam M.
      The arcuate fasciculus and the disconnection theme in language and aphasia: history and current state.
      ,
      • Jellison B.J.
      • Field A.S.
      • Medow J.
      • Lazar M.
      • Salamat M.S.
      • Alexander A.L.
      Diffusion tensor imaging of cerebral white matter: a pictorial review of physics, fiber tract anatomy, and tumor imaging patterns.
      ,
      • Makris N.
      • Kennedy D.N.
      • McInerney S.
      • Sorensen A.G.
      • Wang R.
      • Caviness V.S.
      • et al.
      Segmentation of subcomponents within the superior longitudinal fascicle in humans: a quantitative, in vivo, DT-MRI study.
      ].
      Furthermore, the MRI findings in these patients suggest that the development of cerebral myelination is abnormal: it lags behind that of age-matched peers starting in the neonatal period, stagnates thereafter, and possibly increases and normalizes with advancing age. Given this observation, we speculate that EPG5 could play a role in the formation of cerebral white matter and the process of myelination. Notably, EPG5 mRNA is quite ubiquitously expressed throughout different tissues and in different brain regions and nerves (as reported in the GTEx database). Maillard et al. [
      • Maillard C.
      • Cavallin M.
      • Piquand K.
      • Philbert M.
      • Bault J.P.
      • Millischer A.E.
      • et al.
      Prenatal and postnatal presentations of corpus callosum agenesis with polymicrogyria caused by EGP5 mutation.
      ] describe brainstem atrophy in their patient. However, we suggest this is hypoplasia because it is present from birth and not an ongoing process, as seen in our patient scanned at 3 days and 13 years (Fig. 4).
      All patients carried a novel homozygous c.4862G > A, p.Arg1621Gln variant in EPG5 that leads to an in-frame deletion of 18 amino acids in exon 28 of EPG5 due to atypical splicing. We advocate that this shorter in-frame transcript likely produces a shorter EPG5 protein with residual function. Although thisstill needs to be proven, it could explain the attenuated EPG5-related NDD phenotype seen in our patients compared to Vici-type EPG5-related NDD.
      Similar results were reported by Waldrop et al. [
      • Waldrop M.A.
      • Gumienny F.
      • Boue D.
      • de Los Reyes E.
      • Shell R.
      • Weiss R.B.
      • et al.
      Low-level expression of EPG5 leads to an attenuated Vici syndrome phenotype.
      ], who describe an intronic EPG5 variant leading to aberrant splicing of ∼77% the EPG5 transcripts and an attenuated phenotype of EPG5-related NDD. Additionally, variants affecting different isoforms may explain the variability in phenotype in EPG5-related NDD [
      • Kane M.S.
      • Vilboux T.
      • Wolfe L.A.
      • Lee P.R.
      • Wang Y.
      • Huddleston K.C.
      • et al.
      Aberrant splicing induced by the most common EPG5 mutation in an individual with Vici syndrome.
      ]. The patients presented in our work underscore the link between residual mutant EPG5 protein and an attenuated EPG5-related NDD phenotype that lacks the most life-threatening features of Vici-type EPG5-related NDD, such as cardiomyopathy and immunodeficiency, and consequently results in a significantly longer lifespan (median 13 years (156 months)). The most common cause of death in Vici-type EPG5-related NDD patients was cardiorespiratory failure in the context of respiratory tract infection and/or immunodeficiency and a progressive cardiomyopathy [
      • Byrne S.
      • Jansen L.
      • U-King-Im J.-M.
      • Siddiqui A.
      • Lidov H.G.W.
      • Bodi I.
      • et al.
      EPG5-related Vici syndrome: a paradigm of neurodevelopmental disorders with defective autophagy.
      ]. Notably, previously reported patients carrying the missense variant p.Gln336Arg, which induces aberrant splicing of EPG5, also had a slightly longer lifespan (median survival 78 months) than patients carrying null alleles of EPG5 (median lifespan 42 months) [
      • Byrne S.
      • Jansen L.
      • U-King-Im J.-M.
      • Siddiqui A.
      • Lidov H.G.W.
      • Bodi I.
      • et al.
      EPG5-related Vici syndrome: a paradigm of neurodevelopmental disorders with defective autophagy.
      ]. However, this extended lifespan is still short compared to the patients presented in our work, who can survive into their teens and even twenties. The cause of death in our deceased patients was in all cases respiratory insufficiency due to recurrent and chronic lung infections, which were not caused by immunodeficiency, as in Vici-type EPG5-related NDD, but due to decreased cough efficacy with retention of secretions, as is seen in many children with neurocognitive and motor impairment. The longer lifespan might also explain why all of our patients develop epilepsy, as compared to around 65% in Vici-type EPG5-related NDD, since the age of onset of seizures in half (5/9) of our patients is after the first year of life (range 1 month–17 years). All our patients showed optic hypoplasia or pale optic disks at ophthalmological examination, which has so far only rarely been reported [
      • Byrne S.
      • Jansen L.
      • U-King-Im J.-M.
      • Siddiqui A.
      • Lidov H.G.W.
      • Bodi I.
      • et al.
      EPG5-related Vici syndrome: a paradigm of neurodevelopmental disorders with defective autophagy.
      ]. More than 60% of our patients suffered from sleep apnea, which was suspected to be a combination of obstructive sleep apnea (because of increasing hypotonia) and central sleep apnea. Treatment with nasal high-flow oxygen therapy improved their sleep greatly. Occurrence of sleep apnea has thus far only been reported once in EPG5-related NDD but is important to detect [
      • Byrne S.
      • Dionisi-Vici C.
      • Smith L.
      • Gautel M.
      • Jungbluth H.
      Vici syndrome: a review.
      ]. Whereas the occurrence of scoliosis and sleep apnea in our patients might also be caused by their severe neurocognitive impairment, regardless of the underlying cause, this is not the case for the precocious puberty seen in five of our patients. Whether this is a feature of EPG5-related NDD needs to be explored.
      In conclusion, we describe 15 patients, all linked to a common founder, with an attenuated form of EPG5-related NDD caused by a homozygous missense variant in EPG5 that results in an atypical splicing event, a shorter in-frame transcript and very likely in residual mutant EPG5 protein. We and others have now shown that bi-allelic variants in EPG5 leading to low expression of EPG5 can lead to a less severe clinical spectrum than that seen in Vici-type EPG5-related NDD. We have also shown there is a genotype–phenotype correlation in EPG5-related NDD. We hypothesize that this correlation is based on the level of residual function of EPG5 protein. To emphasize this phenotypic variability, we propose referring to this diagnosis in the future as EPG5-related neurodevelopmental disorder (EPG5-related NDD) with a phenotypic continuum ranging from less severe (of the five principle diagnostic features, patients only show corpus callosum agenesis) to severe (patients show the classic Vici phenotype of all five principle diagnostic features) and using the term Vici-type EPG5-related NDD for classic Vici patients only.
      EPG5-related NDD is associated with phenotypical features that differ from Vici-type EPG5-related NDD. The distinctive imaging features we report have not been comprehensively described in previous reports, but their consistent presence in our patients suggests they may be part of the spectrum of EPG5-related NDD, including Vici-type EPG5-related NDD. When seen, these features might alert physicians to consider the possibility of an EPG5-related NDD. With the expanding use of WES techniques in diagnosis, it is to be expected that an even broader clinical range of patients with EPG5-related NDD will be found. Therefore, future studies on the expression levels of EPG5 are necessary to further define genotype–phenotype correlations that will improve diagnostic counseling.

      Funding

      This work was supported by a Rosalind Franklin Fellowship by the University of Groningen , Groningen, the Netherlands awarded to DSV.

      Acknowledgments

      The authors would like to thank the patients and family members for participating in this study, Cleo van Diemen and Jelkje Bergsma of the Genome Facility of the Department of Genetics, University Medical Center Groningen, Groningen, the Netherlands for performing the exome sequencing, Danielle Boerrigter for technical assistance, and Kate Mc Intyre for critically reading the manuscript. Special acknowledgment goes to Robert Hofstra, head of the clinical genetics department in Rotterdam, and a great inspiration, who passed away last year.

      Appendix A. Supplementary data

      The following is the Supplementary data to this article:
      All clinical data on the 15 individual patients. Features of Vici-type EPG5-related NDD are listed, and compared with our clinical data.

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