Identification of a New Variant of PUF60 Gene

CANCER DIAGNOSIS & PROGNOSIS 1: 213-219 (2021) doi : 10.21873/cdp.10029

Identification of a New Variant of PUF60 Gene: Case Presentation and Literature Review DANIELA OANA TOADER 1,2* , RADU URSU 1, NICOLAE BACALBASA 1,3* , DRAGOS CRETOIU 1, LUCIAN G. POP 2, IRINA BALESCU 4, FLORENTINA GHERGHICEANU 5, FLORENTINA FURTUNESCU 6, DANIEL RADAVOI 7,8 and VIORICA RADOI 1

1Department of Obstetrics and Ginecology, "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania; 2Department of Obstetrics and Ginecology, National Institute of Mother and Child Care-Alessandrescu Rusescu, Bucharest, Romania; 3Department of Visceral Surgery, Center of Excellence in Translational Medicine, "Fundeni" Clinical Institute, Bucharest, Romania; 4Department of Surgery, "Ponderas" Academic Hospital, Bucharest, Romania; 5Department of Marketing and Medical Technology, "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania; 6Department of Public Health and Management University of Medicine and Pharmacy "Carol Davila", Bucharest, Romania; 7Department of Urology, "Prof. Dr. Th. Burghele" Clinical Hospital, Bucharest, Romania; 8Department of Urology, "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania

Abstract. Background/Aim: The aim of the study was to whole exome sequence, which described a variant of report the case of a 5-month-old boy with a complex prenatal unknown clinical significance (VUS, class 3 variant) in the and neonatal symptomatology diagnosed with a "de novo" PUF60 gene. We are mindful that changing the classification pathogenic variant of PUF60 gene. Case Report: Our of a variant of unknown significance is challenging and hospital, undertook the antenatal and postnatal care of a 27- requires supporting and robust criteria. Considering clinical year-old pregnant lady. This was her second baby with a symptomatology produced by the pathogenic mutation in the previously healthy boy. During her routine first-trimester PUF gene, the identified c.1640A>G variant may be anomaly scan, increased nuchal translucency was noticed. categorized as likely pathogenic. Conclusion: Our case adds Multiple anomalies were seen throughout her subsequent new insights on the pathology and the underlying process antenatal visits. This triggered a sequence of tests, involved in the PUF60 variant spectrum disorders. It also examinations and differential diagnosis. The final diagnosis highlights the limits of current prenatal tests. was made at 5 months postpartum following the result of the Latest advances in genetic tests such as microarrays, whole- genome sequence (WGS) and whole-exome sequence (WES) This article is freely accessible online. have caused a massive identification of different genes involved in neuromuscular and learning deficiency *These Authors contributed equally to this study. conditions. In prenatal life increased nuchal translucency (NT) is associated with increased risk of aneuploidy and fetal Correspondence to: Lucian G. Pop, "Carol Davila" University of defects. A fetus with a NT between 3.0 and 3.4 mm has a 7% Medicine and Pharmacy, 37 Dionisie Lupu Street, 020021 risk of aneuploidy. A normal karyotype does not rule out Bucharest, Romania. E-mail: [email protected] adverse pregnancy outcome as single-gene disorders are not Key Words: Exome sequencing, PUF60, gene variant, proteins, routinely assessed prenatally (1). Verheij syndrome is caused mutations. by a deletion involving chromosome 8q24.3 encompassing commonly two deleted genes, scribbled planar cell polarity ©2021 International Institute of Anticancer Resarch protein (SCRIB and poly(U)-binding-splicing factor (PUF60) www.iiar-anticancer.org and a phenotype including colobomata, microcephaly,

213 CANCER DIAGNOSIS & PROGNOSIS 1: 213-219 (2021)

Figure 1. (A) Increased nuchal translucency. (B) Persistent left umbilical vein. (C) Left hydronephrosis and absent right kidney.

developmental delay, short stature, craniofacial, cardiac and Case Report renal defects (2). Later on, it was discovered that not only deletions on chromosome 8 are causing Verheij syndrome, A 27-year-old secundipara women (with a previous healthy but also different variants of the PUF60 gene. At least for the baby), underwent a routine first-trimester scan, revealing moment, it can be considered as a contiguous gene syndrome increased nuchal translucency of 3.1 mm with normal with apparently unrelated clinical anomalies but underlying morphology and no other signs of anomaly. A chorionic chromosomal rearrangement involving contiguous dosage- villous sample with a subsequent Comparative Genomic sensitive genes. So far, there is an extremely limited number Hybridization Single Nucleotide Polymorphism (CGH SNP)- of patients diagnosed with the aforementioned syndrome (3, microarray analysis was performed at 12 weeks gestation, 4). Herein we report the case of a 5 months old boy with a giving a normal result. Anomaly scan and fetal complex prenatal and neonatal symptomatology diagnosed echocardiography were performed at 22 weeks depicting with a " de novo " pathogenic variant of PUF 60 gene that has persistent right umbilical vein, ductus venosus agenesis and never been published before. Prenatal ultrasound sings and empty right renal fossa (Figure 1). Unfortunately, the tests were suggestive of a genetic disorder. chorionic villus sampling (CVS) was not stored by the Written informed consent to perform genetic testing and laboratory so the option of amniocentesis and further genetic further studies were obtained from the family using a form testing was discussed with parents but declined as thwy felt approved by the Ethics Committee of our Department in line reassured with the CVS normal result. Nevertheless, the with the principles of the Declaration of Helsinki and any possibility of other genetic conditions, particularly Noonan other applicable local ethical and legal requirements. syndrome was mentioned as its association of increased The study was registered by the Institutional Review nuchal fold and ductus venosus agenesis is well known (5, 6). Board and Ethical Committee of National Institute of Mother Subsequent scans were performed at 28, 32, 35, and 37 weeks. and Child Care- Alessandrescu Rusescu. At 32 weeks, the foetus developed hydronephrosis on the left

214 Toader et al : Identification of a New Variant of PUF 60 Gene

Figure 2. PUF60 gene diagram (above) indicating mutations found in our case.

Table I. Phenotype of PUF60 patient.

Facial features Cardiac features Eye anomalies Skeletal features Kidney features

Proeminent forehead ASD Single eye coloboma Short stature Left kidney agenesis Long philtrum Bicuspid aortic valve Vertebral fusion C 4-C 5 Hydronephrosis Wide nasal bridge Thin upper lip Square face Short neck

ASD: Atrial sept defect.

Table II . Characteristics of the patient with Verhij syndrome started in prenatal life at 12 weeks of gestation.

MA CRL\NT Ultrasound Ultrasound Outcome Postnatal Sequenging Inheritance Current findings findings findings results situation second 3rd trimester trimester

27 Y 55.9/3.1 mm Absent DV Left 2,450 g/ Dysmorphic PUF60 De novo 16 months PRUV Hydronephrosis 37 weeks features, c1604G>cp. old, mild Absent right IUGR Apgar score-8 bicuspid aortic (Arg535Pro) neurological Kidney valve and atrial underdevelopment, septal defect and assisted walking, hearing deficiency, coloboma, small coloboma ASD, hearing deficiency, visual impairment

MA: Maternal age; CRL: crown rump length; NT: nuchal translucency; DV: ductus venosus; IUGR: intrauterine growth restriction; ASD: atrial septal defect.

kidney and intrauterine growth restriction. Delivery was examinations showed dysmorphic features of the baby, down- expedited at 37 weeks due to growth restriction with abnormal slanting palpebral fissures, long philtrum, thin upper lip, left blood flow on the umbilical and middle cerebral artery. A kidney agenesis, right kidney hydronephrosis, bicuspid aortic 2,450 g (4 centile) baby boy was delivered through Cesarean valve and atrial septal defect and hearing deficiency. Section Apgar score-8, length 47 cm (4 centile), head At two months of age the baby was diagnosed with circumference 32 cm (2 centile). Postnatal multiple clinical Coloboma of the retina and iris at the right eye, whole vertebral

215 CANCER DIAGNOSIS & PROGNOSIS 1: 213-219 (2021)

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216 Toader et al : Identification of a New Variant of PUF 60 Gene

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1 pregnancy revealed multiple anomalies but the second

217 CANCER DIAGNOSIS & PROGNOSIS 1: 213-219 (2021)

invasive test was declined by patient and pregnancy been picked up at an early gestational age, enabling parents progressed to 37 weeks when she delivered a growth-restricted to make an informed decision regarding current pregnancy. and dysmorphic fetus. Reviewing past literature on Medline and PubMed (Table III) we found a small number of articles, Conclusion cases or case series presentations, encompassing 18 cases of de novo heterozygous PUF60 variants with patients age Association between increased nuchal translucency and poor ranging from 2 to 16 years old (2, 7, 8, 9). Our case is number pregnancy outcome has been widely documented (11). This 19. Looking into the previously published papers and our case, includes stillbirth, chromosomal abnormality, congenital it appears that several findings are common in patients with malformation, and genetic syndrome (1). Up to date, there are Verheij syndrome such as short stature, developmental delay, over 100 genetic syndromes associated with increased NT heart defects, kidney and heart anomalies. Dauber et al. and as our case highlights that array CGH has its limitations reported several cases with symptoms such as joint laxity, and the importance of genetic evaluation in establishing a feeding problems, microcephaly, high palate, comprehensive testing algorithm for pregnancies with fetal bronchopulmonary dysplasia, cleft palat, seizures, hypoplastic ultrasound abnormalities is further stressed out, in order to corpus callosum. We observed other similarities: a high allow many more syndromes to be identified. proportion of cases had abnormal segmentation of the vertebrae, which occurred at different levels of the spine (7). Conflicts of Interest Although coloboma and microphthalmia are congenital eye malformations, the genetic cause remains unknow in over 80% The Authors have no conflicts of interest to declare regarding this of cases. Based on analyzed data in Table III, prenatal study. predictions on phenotype based on the position of specific variants are not possible at this stage. Several genes are more Authors’ Contributions often involved in these anomalies as was noticed in the article DOT, RU, DC, LGP performed investigation and follow-up of the published by Haug et al. (10). Both CHD7 and PUF 60 are patient; NB, IB, VR reviewed literature data; FG,FF,DR, DC among this, hence the initial supposition that these babies prepared the draft of the manuscript; LGP, DOT reviewed the final might have CHARGE syndrome. version of the manuscript. To the best of our knowledge, this is the first case of Verheij syndrome involving the heterozygous c.1604G>C/p.Arg535Pro References variant of unknown clinical significance (VUS, class 3 variant) in the PUF60 gene (Poly-U-Binding Splicing Factor, OMIM 1 Yang X, Huang LY, Pan M , Xu LL, Zhen L, Han J and Li DZ: #604819) on chromosome 8q24.3. Firstly, the obstetrician Exome sequencing improves genetic diagnosis of fetal increased raised suspicion of Noonan syndrome at 22 weeks of nuchal translucency. Prenat Diagn 40(11) : 1426-1431, 2020. pregnancy (PTPN11). Secondly, clinical postpartum and PMID: 32668055. DOI: 10.1002/pd.5789 referral diagnosis for WES was CHARGE syndrome. The 2 El Chehadeh S, Kerstjens-Frederikse WS, Thevenon J, Kuentz P, Bruel AL, Thauvin -Robinet C, Bensignor C, Dollfus H, Laugel WES result – did not confirm the referral diagnosis and V, Rivière JB, Duffourd Y, Bonnet C, Robert MP, Isaiko R, revealed the presence of a missense substitution. Considering Straub M, Creuzot-Garcher C, Calvas P, Chassaing N, Loeys B, the patient's phenotype and the medical symptomatology of the Reyniers E, Vandeweyer G, Kooy F, Hančárová M, Havlovicová syndrome generated by pathogenic mutations in the PUF60 M, Prchalová D, Sedláček Z, Gilissen C, Pfundt R, Wassink - gene, the identified c.1640A>G variant may be reclassified as Ruiter JSK and Faivre L: Dominant variants in the splicing factor a class 4 (Likely Pathogenic) mutation. PUF60 cause a recognizable syndrome with intellectual disability, 25(1) The findings of a heterozygous de novo nonsense change heart defects and short stature. Eur J Hum Genet : 43-51, 2016. PMID: 27804958. DOI: 10.1038/ejhg.2016.133 of PUF60 in our patient further supports haploinsufficiency as 3 Verheij JB, de Munnik SA, Dijkhuizen T, de Leeuw N, Olde the underlying mechanism. Loss-of-function variants are Weghuis D, van den Hoek GJ, Rijlaarsdam RS, Thomasse YE, predicted to result in abnormal splicing of targeted genes. Both Dikkers FG, Marcelis CL and van Ravenswaaij -Arts CM: An 8.35 Verheij syndrome and PUF60-related disorder display diverse Mb overlapping interstitial deletion of 8q24 in two patients with phenotypes, suggesting that dysregulated targeted genes due coloboma, congenital heart defect, limb abnormalities, psychomotor to the PUF60 deficiency may account for various phenotypes. retardation and convulsions. Eur J Med Genet 52(5) : 353-357, 2009. Whether WES should be performed in cases with PMID: 19464398. DOI: 10.1016/j.ejmg.2009.05.006 4 Wells C, Spaggiari E, Malan V, Stirnemann JJ, Attie-Bitach T, Ville increased NT (above 99%) and normal microarray is a matter Y, Vekemans M, Bessieres B and Romana S: First fetal case of the of debate. We believe that if trio exome sequencing (parents 8q24.3 contiguous genes syndrome. Am J Med Genet A 170 A( 1) : and fetal exome) was performed after the normal result of 239-242, 2016. PMID: 26437074. DOI: 10.1002/ajmg.a.37411 the array testing in a fetus with multiple abnormalities and 5 Moaddab A, Tonni G, Grisolia G, Bonasoni MP, Araujo Júnior E, increased NT, the pathogenic PUF60 variants would have Rolo LC, Prefumo F, de la Fuente S, Sepulveda W, Ayres N and

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Ruano R: Predicting outcome in 259 fetuses with agenesis of ductus 9 Xu Q, Li CY, Wang Y, Li HP, Wu BB, Jiang YH and Xu X: Role venosus – a multicenter experience and systematic review of the of PUF60 gene in Verheij syndrome: a case report of the first literature (.). J Matern Fetal Neonatal Med 29(22) : 3606-3614, Chinese Han patient with a de novo pathogenic variant and 2016. PMID: 26809266. DOI: 10.3109/14767058.2016.1144743 review of the literature. BMC Med Genomics 11(1) : 92, 2018. 6 Volpe P, Marasini M, Caruso G, Lituania M, Marzullo A, Volpe G PMID: 30352594. DOI: 10.1186/s12920-018-0421-3 and Gentile M: Prenatal diagnosis of ductus venosus agenesis and 10 Haug P, Koller S, Maggi J, Lang E, Feil S, Wlodarczyk A, Bähr its association with cytogenetic/congenital anomalies. Prenat Diagn L, Steindl K, Rohrbach M, Gerth-Kahlert C and Berger W: Whole 22(11) : 995-1000, 2002. PMID: 12424763. DOI: 10.1002/pd.456 exome sequencing in coloboma/microphthalmia: identification of 7 Dauber A, Golzio C, Guenot C, Jodelka FM, Kibaek M, novel and recurrent variants in seven genes . Genes (Basel) 12(1) : Kjaergaard S, Leheup B, Martinet D, Nowaczyk MJ, Rosenfeld 65, 2021. PMID: 33418956. DOI: 10.3390/genes12010065 JA, Zeesman S, Zunich J, Beckmann JS, Hirschhorn JN, 11 Grande M, Jansen FA, Blumenfeld YJ, Fisher A, Odibo AO, Haak Hastings ML, Jacquemont S and Katsanis N: SCRIB and PUF60 MC and Borrell A: Genomic microarray in fetuses with increased are primary drivers of the multisystemic phenotypes of the nuchal translucency and normal karyotype: a systematic review 8q24.3 copy-number variant. Am J Hum Genet 93(5) : 798-811, and meta-analysis. Ultrasound Obstet Gynecol 46(6) : 650-658, 2013. PMID: 24140112. DOI: 10.1016/j.ajhg.2013.09.010 2015. PMID: 25900824. DOI: 10.1002/uog.14880 8 Low KJ, Ansari M, Abou Jamra R, Clarke A, El Chehadeh S, FitzPatrick DR, Greenslade M, Henderson A, Hurst J, Keller K, Kuentz P, Prescott T, Roessler F, Selmer KK, Schneider MC, Stewart F, Tatton -Brown K, Thevenon J, Vigeland MD, Vogt J, Willems M, Zonana J, Study DD and Smithson SF: PUF60 variants cause a syndrome of ID, short stature, microcephaly, coloboma, craniofacial, cardiac, renal and spinal features. Eur J Received March 21, 2021 Hum Genet 25(5) : 552-559, 2017. PMID: 28327570. DOI: Revised April 25, 2021 10.1038/ejhg.2017.27 Accepted May 11, 2021

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