Open Access

Identification of a New Variant of PUF60 Gene: Case Presentation and Literature Review


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

Cancer Diagnosis & Prognosis Jul-Aug; 1(3): 213-219 DOI: 10.21873/cdp.10029
Received 21 March 2021 | Revised 25 May 2024 | Accepted 11 July 2021
Corresponding author
Lucian G. Pop, "Carol Davila" University of Medicine and Pharmacy, 37 Dionisie Lupu Street, 020021 Bucharest, Romania.


Background/Aim: The aim of the study was to report the case of a 5-month-old boy with a complex prenatal and neonatal symptomatology diagnosed with a "de novo" pathogenic variant of PUF60 gene. Case Report: Our hospital, undertook the antenatal and postnatal care of a 27-year-old pregnant lady. This was her second baby with a previously healthy boy. During her routine first-trimester anomaly scan, increased nuchal translucency was noticed. Multiple anomalies were seen throughout her subsequent antenatal visits. This triggered a sequence of tests, examinations and differential diagnosis. The final diagnosis was made at 5 months postpartum following the result of the whole exome sequence, which described a variant of unknown clinical significance (VUS, class 3 variant) in the PUF60 gene. We are mindful that changing the classification of a variant of unknown significance is challenging and requires supporting and robust criteria. Considering clinical symptomatology produced by the pathogenic mutation in the PUF gene, the identified c.1640A>G variant may be categorized as likely pathogenic. Conclusion: Our case adds new insights on the pathology and the underlying process involved in the PUF60 variant spectrum disorders. It also highlights the limits of current prenatal tests.
Keywords: Exome sequencing, PUF60, gene variant, proteins, mutations

Latest advances in genetic tests such as microarrays, whole-genome sequence (WGS) and whole-exome sequence (WES) have caused a massive identification of different genes involved in neuromuscular and learning deficiency conditions. In prenatal life increased nuchal translucency (NT) is associated with increased risk of aneuploidy and fetal defects. A fetus with a NT between 3.0 and 3.4 mm has a 7% risk of aneuploidy. A normal karyotype does not rule out adverse pregnancy outcome as single-gene disorders are not routinely assessed prenatally (1). Verheij syndrome is caused by a deletion involving chromosome 8q24.3 encompassing commonly two deleted genes, scribbled planar cell polarity protein (SCRIB and poly(U)-binding-splicing factor (PUF60) and a phenotype including colobomata, microcephaly, developmental delay, short stature, craniofacial, cardiac and renal defects (2). Later on, it was discovered that not only deletions on chromosome 8 are causing Verheij syndrome, but also different variants of the PUF60 gene. At least for the moment, it can be considered as a contiguous gene syndrome with apparently unrelated clinical anomalies but underlying chromosomal rearrangement involving contiguous dosage-sensitive genes. So far, there is an extremely limited number of patients diagnosed with the aforementioned syndrome (3,4). Herein we report the case of a 5 months old boy with a complex prenatal and neonatal symptomatology diagnosed with a "de novo" pathogenic variant of PUF 60 gene that has never been published before. Prenatal ultrasound sings and tests were suggestive of a genetic disorder.

Written informed consent to perform genetic testing and further studies were obtained from the family using a form approved by the Ethics Committee of our Department in line with the principles of the Declaration of Helsinki and any other applicable local ethical and legal requirements.

The study was registered by the Institutional Review Board and Ethical Committee of National Institute of Mother and Child Care- Alessandrescu Rusescu.

Case Report

A 27-year-old secundipara women (with a previous healthy baby), underwent a routine first-trimester scan, revealing increased nuchal translucency of 3.1 mm with normal morphology and no other signs of anomaly. A chorionic villous sample with a subsequent Comparative Genomic Hybridization Single Nucleotide Polymorphism (CGH SNP)-microarray analysis was performed at 12 weeks gestation, giving a normal result. Anomaly scan and fetal echocardiography were performed at 22 weeks depicting persistent right umbilical vein, ductus venosus agenesis and empty right renal fossa (Figure 1). Unfortunately, the chorionic villus sampling (CVS) was not stored by the laboratory so the option of amniocentesis and further genetic testing was discussed with parents but declined as thwy felt reassured with the CVS normal result. Nevertheless, the possibility of other genetic conditions, particularly Noonan syndrome was mentioned as its association of increased nuchal fold and ductus venosus agenesis is well known (5,6). Subsequent scans were performed at 28, 32, 35, and 37 weeks. At 32 weeks, the foetus developed hydronephrosis on the left kidney and intrauterine growth restriction. Delivery was expedited at 37 weeks due to growth restriction with abnormal blood flow on the umbilical and middle cerebral artery. A 2,450 g (4 centile) baby boy was delivered through Cesarean Section Apgar score-8, length 47 cm (4 centile), head circumference 32 cm (2 centile). Postnatal multiple clinical examinations showed dysmorphic features of the baby, down-slanting palpebral fissures, long philtrum, thin upper lip, left kidney agenesis, right kidney hydronephrosis, bicuspid aortic valve and atrial septal defect and hearing deficiency.

At two months of age the baby was diagnosed with Coloboma of the retina and iris at the right eye, whole vertebral body fusion of cervical 4-5 (C4-5), motor and neuronal delay (Table I). It is possible that subtle findings were not apparent at such a young age or were overlooked during the examination. Upon a clinical genetic examination, it was concluded that prenatal and postnatal findings are suggestive of CHARGHE syndrome. Whole exomique sequencing (WES) study was performed on a Illumina platform revealed the presence of the heterozygous c.1604G>C/p.Arg535Pro variant of unknown clinical significance (VUS, class 3 variant) in the PUF60 gene (Poly-U-Binding Splicing Factor, Online Mendelian Inheritance in Man - OMIM #604819) on chromosome 8q24, consistent with the genetic diagnosis of Verheij syndrome (Figure 2). The complete exome dataset was evaluated for variants clinically relevant to the above-mentioned phenotype and the coverage for Chromodomain Helicase DNA Binding Protein 7 CHD7 (associated with Charge syndrome) was 99.9%. The reputed pathogenic variant identified was confirmed by direct Sanger sequencing and a segregation analysis was carried out. Parental analysis for known mutations was performed by Sanger analysis and results were negative. The pathogenic variants in the PUF60 gene are documented to cause the autosomal dominant Verheij syndrome (OMIM #615583), a disorder with a complex but highly variable symptomatology with onset at birth which includes growth retardation, global developmental delay, eye abnormalities, renal agenesis. We are aware of the papers published by Low, Dauber and El Chehadeh (2,7,8). They report cases of PUF60 variants including deletions on chromosome 8q24.3 and missense mutations. The gene mentioned above, has never been described in the literature before. In light of these result, parental genetic analysis was performed but did not reveal any gene disorders. Hence, the conclusion that this was a "de novo" single gene disorder.


PUF60 encodes a nucleic acid-binding protein, which through interaction with other proteins such as SF3B4, regulates pre-RNA splicing and transcription. The current report presents the case of a male baby who was diagnosed with Verheij syndrome at 5 months of age. At 12 weeks of pregnancy, CVS was performed due to an increase NT above 99% (Table II). According to our hospital protocol for genetic testing, the CVS sample first underwent rapid aneuploidy testing using quantitative fluorescent polymerase chain reaction (QF-PCR). When QF-PCR detected triploidy, trisomy 21, 18, 13 or monosomy X, no additional testing is done. Cases showing normal QF-PCR results are being further tested with chromosomal microarray analysis. Microarray result came back normal arr(1-22)x2,(xy)x1 (CMA) (CytoScan750 K array, Affymetrix, Santa Clara, CA, USA). Unfortunately, CVS sample was not stored. Further assessment during pregnancy revealed multiple anomalies but the second invasive test was declined by patient and pregnancy progressed to 37 weeks when she delivered a growth-restricted and dysmorphic fetus. Reviewing past literature on Medline and PubMed (Table III) we found a small number of articles, cases or case series presentations, encompassing 18 cases of de novo heterozygous PUF60 variants with patients age ranging from 2 to 16 years old (2,7,8,9). Our case is number 19. Looking into the previously published papers and our case, it appears that several findings are common in patients with Verheij syndrome such as short stature, developmental delay, heart defects, kidney and heart anomalies. Dauber et al. reported several cases with symptoms such as joint laxity, feeding problems, microcephaly, high palate, bronchopulmonary dysplasia, cleft palat, seizures, hypoplastic corpus callosum. We observed other similarities: a high proportion of cases had abnormal segmentation of the vertebrae, which occurred at different levels of the spine (7). Although coloboma and microphthalmia are congenital eye malformations, the genetic cause remains unknow in over 80% of cases. Based on analyzed data in Table III, prenatal predictions on phenotype based on the position of specific variants are not possible at this stage. Several genes are more often involved in these anomalies as was noticed in the article published by Haug et al. (10). Both CHD7 and PUF 60 are among this, hence the initial supposition that these babies might have CHARGE syndrome.

To the best of our knowledge, this is the first case of Verheij syndrome involving the heterozygous c.1604G>C/p.Arg535Pro variant of unknown clinical significance (VUS, class 3 variant) in the PUF60 gene (Poly-U-Binding Splicing Factor, OMIM #604819) on chromosome 8q24.3. Firstly, the obstetrician raised suspicion of Noonan syndrome at 22 weeks of pregnancy (PTPN11). Secondly, clinical postpartum and referral diagnosis for WES was CHARGE syndrome. The WES result – did not confirm the referral diagnosis and revealed the presence of a missense substitution. Considering the patient's phenotype and the medical symptomatology of the syndrome generated by pathogenic mutations in the PUF60 gene, the identified c.1640A>G variant may be reclassified as a class 4 (Likely Pathogenic) mutation.

The findings of a heterozygous de novo nonsense change of PUF60 in our patient further supports haploinsufficiency as the underlying mechanism. Loss-of-function variants are predicted to result in abnormal splicing of targeted genes. Both Verheij syndrome and PUF60-related disorder display diverse phenotypes, suggesting that dysregulated targeted genes due to the PUF60 deficiency may account for various phenotypes.

Whether WES should be performed in cases with increased NT (above 99%) and normal microarray is a matter of debate. We believe that if trio exome sequencing (parents and fetal exome) was performed after the normal result of the array testing in a fetus with multiple abnormalities and increased NT, the pathogenic PUF60 variants would have been picked up at an early gestational age, enabling parents to make an informed decision regarding current pregnancy.


Association between increased nuchal translucency and poor pregnancy outcome has been widely documented (11). This includes stillbirth, chromosomal abnormality, congenital malformation, and genetic syndrome (1). Up to date, there are over 100 genetic syndromes associated with increased NT and as our case highlights that array CGH has its limitations and the importance of genetic evaluation in establishing a comprehensive testing algorithm for pregnancies with fetal ultrasound abnormalities is further stressed out, in order to allow many more syndromes to be identified.

Conflicts of Interest

The Authors have no conflicts of interest to declare regarding this study.

Authors’ Contributions

DOT, RU, DC, LGP performed investigation and follow-up of the patient; NB, IB, VR reviewed literature data; FG,FF,DR, DC prepared the draft of the manuscript; LGP, DOT reviewed the final version of the manuscript.


1 Yang X Huang LY Pan M Xu LL Zhen L Han J & Li DZ Exome sequencing improves genetic diagnosis of fetal increased nuchal translucency. Prenat Diagn. 40(11) 1426 - 1431 2020. PMID: 32668055. DOI: 10.1002/pd.5789
2 El Chehadeh S Kerstjens-Frederikse WS Thevenon J Kuentz P Bruel AL Thauvin-Robinet C Bensignor C Dollfus H Laugel V Rivière JB Duffourd Y Bonnet C Robert MP Isaiko R Straub M Creuzot-Garcher C Calvas P Chassaing N Loeys B Reyniers E Vandeweyer G Kooy F Hančárová M Havlovicová M Prchalová D Sedláček Z Gilissen C Pfundt R Wassink-Ruiter JSK & Faivre L Dominant variants in the splicing factor PUF60 cause a recognizable syndrome with intellectual disability, heart defects and short stature. Eur J Hum Genet. 25(1) 43 - 51 2016. PMID: 27804958. DOI: 10.1038/ejhg.2016.133
3 Verheij JB de Munnik SA Dijkhuizen T de Leeuw N Olde Weghuis D van den Hoek GJ Rijlaarsdam RS Thomasse YE Dikkers FG Marcelis CL & van Ravenswaaij-Arts CM An 8.35 Mb overlapping interstitial deletion of 8q24 in two patients with coloboma, congenital heart defect, limb abnormalities, psychomotor retardation and convulsions. Eur J Med Genet. 52(5) 353 - 357 2009. PMID: 19464398. DOI: 10.1016/j.ejmg.2009.05.006
4 Wells C Spaggiari E Malan V Stirnemann JJ Attie-Bitach T Ville Y Vekemans M Bessieres B & Romana S First fetal case of the 8q24.3 contiguous genes syndrome. Am J Med Genet A. 170A(1) 239 - 242 2016. PMID: 26437074. DOI: 10.1002/ajmg.a.37411
5 Moaddab A Tonni G Grisolia G Bonasoni MP Araujo Júnior E Rolo LC Prefumo F de la Fuente S Sepulveda W Ayres N & Ruano R Predicting outcome in 259 fetuses with agenesis of ductus venosus – a multicenter experience and systematic review of the literature (.). J Matern Fetal Neonatal Med. 29(22) 3606 - 3614 2016. PMID: 26809266. DOI: 10.3109/14767058.2016.1144743
6 Volpe P Marasini M Caruso G Lituania M Marzullo A Volpe G & Gentile M Prenatal diagnosis of ductus venosus agenesis and its association with cytogenetic/congenital anomalies. Prenat Diagn. 22(11) 995 - 1000 2002. PMID: 12424763. DOI: 10.1002/pd.456
7 Dauber A Golzio C Guenot C Jodelka FM Kibaek M Kjaergaard S Leheup B Martinet D Nowaczyk MJ Rosenfeld JA Zeesman S Zunich J Beckmann JS Hirschhorn JN Hastings ML Jacquemont S & Katsanis N SCRIB and PUF60 are primary drivers of the multisystemic phenotypes of the 8q24.3 copy-number variant. Am J Hum Genet. 93(5) 798 - 811 2013. PMID: 24140112. DOI: 10.1016/j.ajhg.2013.09.010
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 & Smithson SF PUF60 variants cause a syndrome of ID, short stature, microcephaly, coloboma, craniofacial, cardiac, renal and spinal features. Eur J Hum Genet. 25(5) 552 - 559 2017. PMID: 28327570. DOI: 10.1038/ejhg.2017.27
9 Xu Q Li CY Wang Y Li HP Wu BB Jiang YH & Xu X Role of PUF60 gene in Verheij syndrome: a case report of the first Chinese Han patient with a de novo pathogenic variant and review of the literature. BMC Med Genomics. 11(1) 92 2018. PMID: 30352594. DOI: 10.1186/s12920-018-0421-3
10 Haug P Koller S Maggi J Lang E Feil S Wlodarczyk A Bähr L Steindl K Rohrbach M Gerth-Kahlert C & Berger W Whole exome sequencing in coloboma/microphthalmia: identification of novel and recurrent variants in seven genes. Genes (Basel). 12(1) 65 2021. PMID: 33418956. DOI: 10.3390/genes12010065
11 Grande M Jansen FA Blumenfeld YJ Fisher A Odibo AO Haak MC & Borrell A Genomic microarray in fetuses with increased nuchal translucency and normal karyotype: a systematic review and meta-analysis. Ultrasound Obstet Gynecol. 46(6) 650 - 658 2015. PMID: 25900824. DOI: 10.1002/uog.14880