Expanding the phenotype of UPF3B‐related disorder: Case reports and literature review

UPF3B encodes the Regulator of nonsense transcripts 3B protein, a core‐member of the nonsense‐mediated mRNA decay pathway, protecting the cells from the potentially deleterious actions of transcripts with premature termination codons. Hemizygous variants in the UPF3B gene cause a spectrum of neuropsychiatric issues including intellectual disability, autism spectrum disorder, attention deficit hyperactivity disorder, and schizophrenia/childhood‐onset schizophrenia (COS). The number of patients reported to date is very limited, often lacking an extensive phenotypical and neuroradiological description of this ultra‐rare syndrome. Here we report three subjects harboring UPF3B variants, presenting with variable clinical pictures, including cognitive impairment, central hypotonia, and syndromic features. Patients 1 and 2 harbored novel UPF3B variants—the p.(Lys207*) and p.(Asp429Serfs*27) ones, respectively—while the p.(Arg225Lysfs*229) variant, identified in Patient 3, was already reported in the literature. Novel features in our patients are represented by microcephaly, midface hypoplasia, and brain malformations. Then, we reviewed pertinent literature and compared previously reported subjects to our cases, providing possible insights into genotype–phenotype correlations in this emerging condition. Overall, the detailed phenotypic description of three patients carrying UPF3B variants is useful not only to expand the genotypic and phenotypic spectrum of UPF3B‐related disorders, but also to ameliorate the clinical management of affected individuals.

a proper regulation of translation efficiency (Kunz et al., 2006).It is known that targets of NMD, like ARHGAP24, are involved in the regulation of neuronal growth and in neurodevelopmental issues (Nguyen et al., 2012).
The NMD machinery comprises the conserved UP-Frameshift proteins UPF1, UPF2, and UPF3.UPF3 exists as two paralogs (A and B), differentially expressed depending on cell type and developmental stage, believed to regulate NMD activity based on cellular requirements (Bufton et al., 2022).UPF3B's N-terminus comprises a conserved "RNA recognition motif-like" domain (RRM-L), interacting with a domain of UPF2.The RRM-L domain is followed by a "poorly characterized" middle domain, with two coiled-coil-like regions (CCL-1 and CCL-2), and by a C-terminal exon-junction complex binding motif.
The middle domain has been implicated in translation termination.
Recent evidence supports the important role of the middle domain, given its interactions with release factors, NMD substrate mRNAs, ribosomal subunits, and the MIF4GIII domain of UPF2 (Bufton et al., 2022).Though UPF3B is a key factor for mRNA surveillance and eukaryotic gene expression regulation, its precise mode of action in NMD still remains elusive (Bufton et al., 2022).Recent findings also indicate that UPF3B is a crucial regulator of brain development: Jolly et al. (2013) demonstrated on murine models that Upf3b expression is developmentally regulated, that loss of Upf3b-NMD promotes the proliferation and reduces the differentiation of neural progenitor cells, and that loss of Upf3b-NMD in primary hippocampal neurons affects neurite growth.In this study, we report three patients with UPF3B variants with diverse clinical features, widening the phenotypic spectrum of this ultra-rare syndrome.We also reviewed pertinent literature.

| METHODS
The study was conducted in accordance with the Declaration of Helsinki and approved by the local Institutional Ethics Committees.The patients were enrolled at Istituto Giannina Gaslini, Genoa, (Italy) Christiana Care, Newark, Delaware (USA), and Turku University Hospital, Turku (Finland) and clinically evaluated by pediatric geneticists and neurologists.
Informed consent was obtained from the parents, DNA was analyzed by comparative genome hybridization (CGH)-array, using the Human Genome CGH Microarray 4Â 180 K Kit, probe design 086332 (Agilent Technologies, Santa Clara, CA, USA), according to the manufacturer's instructions.The Agilent platform is an oligonucleotidebased microarray with an average resolution of about 25 kb to detect copy number variations and loss of heterozygosity (LOH) of 4 Mb.
Raw data were analyzed using the Genomic Workbench 7.0.40software (Agilent).Altered chromosomal regions and breakpoints and LOH events were detected using ADM-1 (threshold 10) with 0.5 Mb window size to reduce false positives.For aberration detection, the diploid peak centralization algorithm and the legacy centralization algorithm were applied to set the most common ploidy to zero.This is needed to ensure that the zero point reflects the most common ploidy state.Chromosome positions were determined using GRCh37/hg19 (UCSC Genome Browser).
Informed consent was obtained from the parents, trio-WES [AUTHOR: Please define (SDS, WES) in the first occurrence if necessary.]was performed in the family on genomic DNA extracted from peripheral blood.Agilent Sure Select QXT Clinical Research Exome (Agilent Technologies, Santa Clara, CA, USA) was used and Sequencing data were processed with in-house software for the GATK Best Practices pipeline for WES variant analysis execution.After filtering for allele frequency (≤0.01% in public databases, including GnomAD v2.1.1;https://gnomad.broadinstitute.org/),candidate variants were screened according to family segregation, conservation (GERP score), and predicted impact on protein function through in silico tools (including SIFT, PolyPhen-2, Mutation Taster).The variants were eventually validated through Sanger sequencing.

| Patient 1
A boy aged 3 years and 4 months is an only child of a nonconsanguineous couple of European origin.Family history is not remarkable.
During pregnancy, a tetralogy of Fallot was identified by ultrasound (US).Fetal brain magnetic resonance imaging (MRI) showed hypoplasia/dysgenesis of the corpus callosum, olfactory bulb agenesis, and a developmental venous anomaly in the left parietal lobe (Figure 1d [a-d]).The child was born at 41 weeks and 4 days by vacuum delivery.
Birth weight was 3770 g, and APGAR scored 8 and 9. Other parameters at birth are not available.The child showed normal adaptation to extrauterine life.Cardiac US confirmed the prenatal diagnosis of tetralogy of Fallot.Brain MRI at birth and 2-year follow-up confirmed corpus callosum dysgenesis, anterior commissure hypoplasia, olfactory bulbs agenesis, choroidal plexus cysts, bilateral incomplete hippocampal rotation, and a left parietal developmental venous anomaly ).Computed tomography highlighted choanal stenosis while the inner ear strictures were normal.CGH-array on blood's lymphocytes from the child did not identify any significant alteration.
In the following months, the child showed signs of developmental delay: evaluation with Griffith's scale at 12 months resulted with a developmental age of 5 months and 21 days.Only at the age of 1 year and 2 months did he started to take objects and feet to the mouth, but was not able to sit autonomously.His parents also reported chewing and swallowing difficulties, for which logopedic rehabilitation was started.At 1 year and 4 months axial hypotonia and limb stiffness were evident.Electroencephalography (EEG) was consistent with low voltage activity, without clear epileptic anomalies.Visual evoked potentials were altered, associated with evidence of mild blurring of optic disc.Some facial dysmorphisms can be appreciated like broad forehead, sparse eyebrows, low nasal bridge, bulbous nose tip, mandibular hypoplasia with retrognathia (Figure 1a).Exome sequencing (ES) was performed as previously described (Scala et al., 2022) and led to the identification of a de novo variant, c.619A > T, p.(Lys207*) in the UPF3B gene.This STOP variant, absent in the general population, is not reported in ClinVar.It involves a conserved amino acid and is predicted to have functional consequences on the protein.According to the American College of Medical Genetics and Genomics (ACMG) this variant is classified as likely pathogenic.

| Patient 2
Patient 2 is a 7-year-old boy of nonconsanguineous European parents.
He is the youngest of the three children (older siblings are girls).Family history is unremarkable.He was born at term from an uneventful pregnancy with normal height and weight measurements.Head circumference (HC) at birth was À2.0 SDS.Other parameters at birth were not available.During the first months of life the child showed global muscular hypotonia and developmental and language delay.He started walking at the age of 20 months.Now the child produces simple sentences, with pronunciation defects.The child does not have an autism spectrum disorder (ASD) diagnosis.He however shows features of trichotillomania, and motor stereotypies, like shaking his hands on the side when excited.Global hypotonia and gross motor delay are persistent, in addition to dysarthria and feeding difficulties (but normal oral motor function).Brain MRI at the age of 2 years was normal.The child has presented with benign sleep myoclonias since early childhood, every night while falling asleep, together with bruxism.Treatment with valproate was stopped due to poor response.EEG at age 6 showed bilateral spikes, with no bursts.The boy shows some dysmorphisms like triangular, hypotonic face, midface hypoplasia, long philtrum, small open mouth with tented upper lip, small chin, and protruding ears (Figure 1b).He also has a slender build and poor muscle bulk.Cardiac evaluation (ECG, US) performed at 6 years of age was normal.The last clinical evaluation at the age of 7 years old highlighted an HC of À3 SDS, so a diagnosis of microcephaly was made.CGH-array and analysis for Fragile-X syndrome revealed no significant alterations.

| Patient 3
Patient 3 is a now 26-year-old male, son of a nonconsanguineous couple from America.No relevant family history was reported.The child was born at 32 gestational weeks, after a pregnancy characterized by maternal hypertension.Weight at birth was 3400 g.Other parameters at birth were not available.The newborn was noticed with congenital hypotonia.A diagnosis of cerebral palsy was made.In the following months, global developmental delay was evident.At 12 months he was noticed to be floppy and could not sit autonomously.He also had significant feeding problems, including refusal to take liquids.Feeding therapy was recommended with increasing acceptance of texture food, cup drinking, and a more typical motor pattern for spoon feeding.
Currently, he is nonverbal and has a diagnosis of intellectual disability (ID).Motor stereotypies and autistic features are recognizable, including sounds and gestures mimicry, and refusal to eat solids.Seizures began approximately in the patient's early 20s characterized by shaking tremors, followed by confusion and sleepiness, with no drooling or incontinence.Frequency is around one to two events/week.
One episode lasting 10-60 s was reported of focal impaired awareness seizure with stiffness, eyes rolled back, incontinence, without tongue bite, probably precipitated by concurrent sinus infection.
Treatment with Levetiracetam and Lamotrigine was started with clinical response.Brain MRI at the age of 26 showed mild cerebellar atrophy.
The patient shows narrow face and slightly elongated head, occipital prominence, upturned nose, short philtrum, and shortened and grayish upper central incisors.Other relevant findings include bilateral hip subluxation, flexible planovalgus feet, dysplastic hips, bilateral coxa valga deformity, and mild subluxation of the left femoral head.The patient is waiting for complete cardiac and renal evaluations.CGH-array did not reveal any significant alteration.Trio WES analysis detected the maternally inherited c.674_677del, p.

| DISCUSSION
Loss of function mutations in UPF3B are associated with "intellectual developmental disorder, X-linked syndromic 14" (OMIM 300676).
Variants in this gene were first described in 2007 in four families with syndromic (suspected Lujan-Fryns syndrome), and nonsyndromic X-linked ID and ASD (Tarpey et al., 2007).Since then more patients have been studied with next generation sequencing (NGS) techniques and more genetic defects in UPF3B have been reported (Table 1; Chérot et al., 2018;Hu et al., 2016;Soden et al., 2014;Tzschach et al., 2015;Zhang et al., 2015).Although 21 variants have been identified in UPF3B according to the Human Gene Mutation Database (HGMD), the spectrum of clinical features of affected individuals remains insufficiently defined.To date, a very limited number of patients and families have been reported with full clinical/ instrumental descriptions (Laumonnier et al., 2010;Lynch et al., 2012;Tejada et al., 2019;Xu et al., 2013).
Cardiac and renal issues have occasionally been reported: renal dysplasia was detected in two brothers (Lynch et al., 2012;Tarpey et al., 2007).Patent ductus arteriosus in one patient (Lynch et al., 2012), and multiple atrial septum defects in another were recorded (Lovrecic et al., 2018).Non-neurological features may be underrated in UPF3B individuals.However available data are still limited to infer a clear relationship between UPF3B variants and these clinical issues: other genetic/epigenetic or environmental factors may be contributing.
Since then, other pathogenic variants have been signed in ClinVar (Table 1).The majority of variants are represented by missense and frameshift variants.Patients with truncating variants show variable features; on the other hand, missense variants seem associated with milder phenotypes (patients described as "not overtly dysmorphic", Bufton et al., 2022).However, only a limited number of UPF3B patients have been reported, and no precise genotype-phenotype correlation has been inferred.Also copy number variants (CNVs), involving the locus of UPF3B on chromosome X, found by microarray techniques, have been described (Deka et al., 2021).Some hypotheses have been made about phenotypic heterogeneity: while varying UPF3A protein stability appears to be one of the candidate modifying factors, other genes and proteins might also be contributing (Jolly et al., 2013;Lynch et al., 2012;Nguyen et al., 2012).To what extent the clinical expressivity of UPF3B mutations across neuropsychiatric conditions is mediated by the variable level of the rescue UPF3Amediated NMD is yet to be fully investigated (Lynch et al., 2012;Tarpey et al., 2007).Additionally, the precise molecular mechanisms underlying neurodevelopmental defects still remain elusive (Bufton et al., 2022).
In our cohort, two patients harbored novel hemizygous variants in UPF3B and Patient 3 had an already reported one.They variably presented with neurodevelopmental impairment, and syndromic features.   .Other novel features in our cases included microcephaly, midface hypoplasia, long philtrum, and small mouth presented by Patient 2. The identification of microcephaly is especially interesting, since most UPF3B patients mostly present macrocephaly instead (Deka et al., 2021).This suggests that the differential diagnosis of UPF3B-related disorder needs to include other neurodevelopmental conditions with microcephaly, such as MFSD2A-and EEF1A2-related disorders (Carvill et al., 2020;Scala et al., 2020).Based on current knowledge, it is difficult to establish clear genotype-phenotypes correlations and future studies will be helpful in this regard.
As for neuroradiological anomalies Patient 1 showed corpus callosum dysgenesis, anterior commissure agenesis, olfactory bulbs agenesis, and bilateral incomplete hippocampal rotation.Patient 3 had mild cerebellar atrophy, not previously reported in affected individuals.Knowledge about neuroradiological features in UPF3B patients is limited, as brain MRIs are available only in a very small number of the patients described in the literature.Dysgenesis of the corpus callosum (Tarpey et al., 2007), dilatation of Virchow Robin spaces (Chérot et al., 2018), and hydrocephaly (Laumonnier et al., 2010) were respectively reported in three different works (Table 1).Lynch et al. (2012) reported two brothers with mild, not specific brain anomalies: a small pineal cyst in one, and prominent subarachnoid spaces in the other.To date, a clear correlation between UPF3B variants and brain anomalies is difficult to infer.
It is interesting to notice that congenital heart and brain anomalies are well-known features of other syndromes caused by genes involved in the same pathway as UPF3B, the NMD pathway, such as SMG8 (Alzahrani-Kuwahara syndrome, OMIM 619268) and SMG9 (Heart and brain malformation syndrome, OMIM 616920).SMG8 and SMG9 encodes for components of the SMG1-SMG8-SMG9 complex, which is activated by the UPF2-UPF3B complex (Bufton et al., 2022;Zhu et al., 2019).Even though SMG8 and SMG9-related disorders are recessive, we cannot exclude possible consequences of UPF3B variants on these downstream target genes of the common pathway, with possible similar phenotypic features.
The pathophysiology and clinical consequences of UPF3B variants are still poorly understood.Further cases, along with functional studies, are needed to better understand the pathophysiology of UPF3B-related disorders, possible involvement of the gene in clinical phenotypes, along with clearer genotype-phenotype correlations.

| CONCLUSIONS
We reported three patients harboring pathogenic variants in UPF3B and presenting with complex neurodevelopmental phenotypes and some previously unreported features, suggesting an expansion of the phenotypic spectrum of this rare condition.Two variants were novel, while one was already described.Atypical features (especially brain and cardiac anomalies) should not be considered as exclusion criteria.
We suggest that cardiac and brain evaluation should be performed in all carriers of UPF3B variants.Despite the growing evidence of a pathogenic role of UPF3B variants the phenotypic spectrum remains to be fully elucidated.An expanded cohort of patients and more in-depth studies on UPF3B protein functions will be crucial to better understand the pathomechanism behind the syndrome and to infer more precise genotype-phenotype correlations.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author (M.S.) upon request.

ORCID
Ferruccio Romano https://orcid.org/0009-0005-3933-0597 Clinical exome analysis identified the hemizygous variant c.1285_1286del, p.(Asp429Serfs*27) in the UPF3B gene (Figure1c).It is a small de novo deletion, occurring in exon 10, resulting in a premature truncation of the protein.It is classified as likely pathogenic.It is a novel variant, absent in the general population.Exome analysis also identified the c.2215G > A variant of uncertain significance in the KMT2D gene, inherited from his healthy father.

F
I G U R E 1 (a) Facial appearance of Patient 1: Broad forehead, sparse eyebrows, low nasal bridge, bulbous nose tip, mandibular hypoplasia, retrognathia.(b) Facial appearance of Patient 2: Triangular, hypotonic face, small mouth with tented upper lip, small chin, and protruding ears.(c) Schematic representation of protein UPF3B domain architecture; the first variants described in affected individuals are signed in black (Tarpey et al., 2007), while the three identified in our patients are highlighted in red; CCL, coiled-coil like; EJC BM, exon-junction complex binding motif; RRM-L, RNA recognition motif-like domain.Modified from Bufton et al., 2022; (d) Neuroimaging features of Patient 1. Fetal brain magnetic resonance imaging performed at 34 gestational weeks (a-d) and 2 years of age (e-h) showing callosal hypoplasia and dysgenesis (thick arrows), olfactory bulb agenesis (arrowheads), a left parietal developmental anomaly (thin arrows), and bilateral incomplete hippocampal rotation (dashed arrows).Note the persistence of cavum vergae et septi pellucidi (asterisk).
Patients 1 and 3 also had brain malformations.Patient 1 had ID, choanal atresia, neuroradiological findings, and tetralogy of Fallot.WES analysis led to the identification of a new variant in the UPF3B gene (c.619A> T).In addition, he showed developmental delay, persistent T A B L E 1 UPF3B variants and associated clinical phenotypes.

F
.R. drafted the article, revised literature, and contributed to data analysis.M.H., P.P., A.R.P., J.R.P., and M.D. collected clinical and genetic data.M.F.F.contributed to literature revision and collected genetic data.M.T. and E.H. performed genetic analysis and collected genetic data.Ms.S. reviewed brain MRI scans and analyzed the neuroimaging spectrum.F.Z, F.F., and V.C. supervised the work and critically revised the article.M.S. conceived the study, supervised the work, and revised the article.ACKNOWLEDGMENTS One of the authors of this publication is a member of the European Reference Network on Rare Congenital Malformations and Rare Intellectual Disability ERN-ITHACA.[EU Framework Partnership Agreement ID: 3HP-HP-FPA ERN-01-2016/739516].The authors aknowledge Yeraldin Chiquinquira Castillo De Spelorzi and all the staff of Genomics Facility, Istituto Italiano di Tecnologia (IIT), Genova, Italy for their precious contribution to genetic analyses.Open access funding provided by BIBLIOSAN.