Further Evidence for RFWD3 Gene Causing Fanconi Anemia Complementation Group W: Detailed Clinical Report of the Second Case in the Literature

Abstract

Introduction

Fanconi anemia (FA) is a heterogeneous genetic disorder that is characterized by progressive bone marrow failure, congenital malformations, predisposition to malignancy, and short stature. The RFWD3 gene was recently associated with FA complementation group W, and only 1 patient is reported in the literature so far.

Case Presentation

Here, we report the second patient, a 10-year-old male, who has failure to thrive, central nervous system abnormalities, bilateral radial ray defects, urogenital anomalies, facial dysmorphism, and thrombocytopenia. The patient was suspected to have FA according to the aforementioned findings, and the homozygous c.1501C>T variant in the RFWD3 gene was detected by whole-exome sequencing. The diepoxybutane test and mitomycin C-induced peripheral blood cultures revealed 0.46 and 0.90 chromosomal breaks, respectively.

Conclusion

In this article, clinical findings of the second patient with FA complementation group W are discussed in detail, aiming to expand the clinical and molecular spectrums of the disease.

Established Facts

• •Fanconi anemia (FA) is a rare, multisystem disorder which is characterized by bone marrow failure, congenital malformations, predisposition to malignancy, and short stature.
• •FA is a genetically heterogenous disease with more than twenty related phenotypes and genes identified so far.
• •The RFWD3 gene was recently related to FA complementation group W, and only 1 patient is reported in the literature so far.

Novel Insights

• •This is the second case of FA complementation group W.
• •The patient we report had both classical and rare findings (anal atresia and development delay) of FA.

Introduction

Fanconi anemia (FA) is a rare, multisystem disorder that is seen approximately in 1 in 136,000 newborns every year. FA is characterized by bone marrow failure, congenital malformations, predisposition to malignancy, and short stature. Congenital anomalies include bilateral radial ray deformities, abnormal skin pigmentation, genitourinary malformations, heart defects, central nervous system anomalies, and facial dysmorphic features such as triangular face shape, micrognathia, and midface hypoplasia.

FA is a heterogenous disorder caused by the pathogenic variants of DNA repair genes, specifically the DNA interstrand cross-link (ICL) repair system, generating chromosomal instability. Therefore, defects of this system cause high sensitivity of chromosomes to ICL-inducing agents such as diepoxybutane (DEB) and mitomycin C (MMC), leading to multiple chromosome breaks and tri-/quadriradial chromosomal formation [García-de-Teresa et al., 2020]. Although most of the genes playing causative role in the FA are inherited in an autosomal recessive manner, X-linked, autosomal dominant inheritance is reported as well [Fiesco-Roa et al., 2019]. There are 22 FA genes reported so far that are responsible for encoding critical proteins [Rageul and Kim, 2020]. Pathogenic frameshift and missense variants of the RFWD3 gene have been recently identified in a patient with classical findings of FA such as growth retardation, congenital malformations, and bone marrow failure. Animal models and cellular functional analysis of the relative gene supported the FA phenotype, and authors suggested RFWD3 as the responsible gene of FA complementation group W [Knies et al., 2017]. Here, we report the clinical, radiological, and genetic findings of the second patient of RFWD3-associated FA.

Case Presentation

Medical history of the patient was obtained from the parents and from the medical recordings. Genetic testing and radiological evaluation were performed. Informed consent was obtained from the parents of the patient. The patient was male and 10 years and 8 months old and was the second child of a 35-year-old mother and a 39-year-old father who had 2nd-degree consanguineous marriage, and he had a healthy 17-year-old brother. It was learned that he was born at 38 weeks after a normal pregnancy, by cesarean section due to fetal distress, with a weight of 1,510 g (−5.7 SD). Anal atresia, bilateral undescended testis, and a bilateral absence of thumb were detected at birth. Anorectoplasty was performed on the 2nd postpartum day. Afterward, the patient did not go to regular follow-up anywhere as he applied to different health institutions at different times, including our hospital.

From the patient’s previous epicrisis and reports, it was learned that a ventriculoperitoneal shunt operation was performed at the age of 16 months in another health institution for hydrocephalus. Other recorded findings at that period were neuromotor developmental delay, short stature, bilateral optic disc hypoplasia, agenesis of the left external auditory canal, bilateral undescended testis, and micropenis.

In the abdominal ultrasonography (USG) performed at the age of 3 years, no appearance of testicular tissue was detected in the bilateral scrotum and in the abdomen. With the human chorionic gonadotropin stimulation test, the level of stimulated total testosterone was found to be 25 times higher than the baseline. At 6 years of age, free T4 was 1.01 ng/dL and TSH was 21.65 uIU/mL, so levothyroxine treatment was started.

Neuromotor development, especially expressive language, was severely delayed compared to his peers. He could not walk unassisted until the age of 3 years, and he could only use few (3–4) syllables at the age of 10 years.

In his skeletal survey at the age 10 years 8 months, bilateral radial ray deformities (radius and thumb aplasia) (shown in Fig. 1a) and hypoplastic ulna (shown in Fig. 1b) were detected. Both proximal femoral heads were superiorly dislocated and hypoplastic (shown in Fig. 1d). Dextroscoliosis of the cervical spine (shown in Fig. 1e) and eversion of right tibiotalar joint were noted (shown in Fig. 1c).

Fig. 1.

Skeletal survey images of the patient. a Radial ray deformity of the left arm. b Short and hypoplastic ulna. c Eversion of right tibiotalar joint. d Superiorly dislocated hypoplastic femoral heads. e Dextroscoliosis of the cervical spine indicated with a white arrow.

In scrotal and urinary USG performed at the age 10 years 8 months, undescended testes with a size of 5 × 4 × 9 mm in the proximal right inguinal canal and 6 × 3 × 7 mm in the proximal left inguinal canal were detected. A 43 × 17-mm ectopic kidney was detected in the right latero-inferior part of the umbilicus, and agenesis of the left kidney was reported.

In the physical assessment performed at the age of 10 years 8 months, his weight was 14 kg (−6.15 SD), his height was 95 cm (−7.2 SD), and head circumference was 52 cm (−1.7 SD). His sclerae were pale. Triangular face, hypertelorism, short palpebral fissures, and strabismus in the left eye were detected (shown in Fig. 2a). He had a wide and flat nasal ridge, tubular nose, microstomia, and micrognathia; his right ear was 43 mm (<3p) and low set, and he had grade 3 microtia in the left ear (shown in Fig. 2b). He had a high-arched palate and bifid uvula. Bilateral thumb agenesis and hypoplasia of the forearm were noted, and bilateral radius agenesis was suspected by palpation. Cutaneous, partial syndactyly of the right 2nd–4th fingers, cutaneous and partial syndactyly of the left 2nd–3rd fingers, and camptodactyly of all existing fingers were noted. Also, the 2nd left finger was hypoplastic. There was a 1.5-cm length difference detected (left 52.5 cm and right 54 cm) between the two lower extremities. Also talipes valgus deformity was noted in the right foot.

Fig. 2.

Patient at 10 years of age. a Triangular face, esotropia of the left eye, wide and flat nasal ridge, microstomia, low set ears, radial ray deformities, and tibiotalar eversion of the right foot. b Microtia of the left ear and midface hypoplasia.

Genomic DNA of the patient was isolated according to the manufacturer’s protocol. The Nextera Rapid Capture Expanded Exome kit and NextSeq 550 (Illumina Inc, San Diego, CA, USA) were used for the whole-exome sequencing. The data had an average mean target coverage of 40×, and Burrows-Wheeler aligner 0.7.12 was used for mapping sequences to the reference genome (human genome 19). Single-nucleotide polymorphisms (SNPs) and insertions and deletions were calculated by using the Genome Analysis Toolkit (GATK) v3.3. Data analysis was performed by VariantStudio 2.2.1 (Illumina). All annotated variants were analyzed using databases including the SNP database (dbSNP142; https://www.ncbi.nlm.nih.gov/snp/?cmd=search), GnomAD (https://gnomad.broadinstitute.org/), VarSome (https://varsome.com/), Franklin by Genoox (https://franklin.genoox.com/clinical-db/home), Sorting Intolerant From Tolerant (SIFT; http://sift.jcvi.org/), Polymorphism Phenotyping version 2 (Polyphen-2; http://genetics.bwh.harvard.edu/pph2/), MutationTaster (http://www.mutationtaster.org/), and Combined Annotation Dependent Depletion (CADD; https://cadd.gs.washington.edu/) and were used as in silico prediction tools. The mutation residue was modeled by the 3D modeling platform PyMOL (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.), and binding energy changes have been predicted with the MCSM platform (https://biosig.lab.uq.edu.au/mcsm/). Variants located at exons and canonical splice sites (+/− 2 bp) were screened initially. Nonsynonymous, in/dels causing frameshift with minor allele frequency less than 0.01 in GnomAD were filtered. Because of the consanguinity among the parents of the proband, priority was given to the biallelic variants consistent with autosomal recessive inheritance. Since the patient was suspect to have the FA phenotype, FA-related OMIM IDs were screened as well. Detected variants were classified according to the ACMG criteria of sequence variant classification [Richards et al., 2015]. Conventional karyotype analysis (GTG banding), DEB, and MMC induction tests were performed with the lymphocyte culture of the heparinized peripheral blood. Conventional cytogenetic analysis revealed a normal male karyotype. According to the clinical findings, the patient was suspected to have FA, and analysis of DEB and MMC-induced cultures revealed 0.46 (NR: 0–0.08) and 0.90 (NR: 0–0.14) chromosomal break/cell, respectively (shown in Fig. 3b). Whole-exome sequencing was performed due to heterogenous nature of the disease, and a rare missense variant (ENST00000361070.9:c.1501C>T:p.Arg501Cys) in the RFWD3 gene was detected in a homozygous state (shown in Fig. 3a). It was previously reported in dbSNP yet not found in the ClinVar Database. Allele frequency of this variant was reported as ƒ = 0.0000159 in GnomAD exomes. In silico tools such as MutationTaster, SIFT, PolyPhen, and CADD predicted the deleterious effects of the variant. Binding energy change of the R501C mutation in RFWD3 protein (PMD: 6CVZ) chain A was highly destabilizing (PΔΔG −2.1 kcal/mol). Therefore, this variant may alter DNA-protein or protein-protein binding affinity of the RFWD3 protein. 3D modeling of the mutation residue of the protein is shown in Figure 3e. VarSome and Franklin Genoox platforms classified the p.Arg501Cys variant as a variant of uncertain significance. Parental segregation analyses were performed and confirmed the parents as carriers (shown in Fig. 3c, d).

Fig. 3.

Genetic analysis of the patient and the parents. a Homozygous p.Arg501Cys variant in the RFWD3 gene detected in the patient. b MMC-induced cultures demonstrated chromosomal tri-, tetra-, and polyradial formations detected at the metaphase of the peripheral blood culture of the patient indicated with blue arrows. c Heterozygous p.Arg501Cys variant in the RFWD3 gene detected in the mother. d Heterozygous p.Arg501Cys variant in the RFWD3 gene detected in the father. e 3D modeling of the mutation residue Arg501 of the protein is shown with a red arrow.

Discussion

FA is a genomic instability disorder characterized by bone marrow failure, microcephaly, predisposition to malignancy, short stature, and congenital malformations such as bilateral radial ray deformities, abnormal skin pigmentation, genitourinary malformations, heart defects, central nervous system anomalies, and facial dysmorphic features. It is a heterogenous disease with 22 underlying genes that have a prefix of FANC, identified so far [McReynolds et al., 2020]. Most of the genes are important components of the DNA ICL repair system, and pathogenic variants of these genes are responsible for the pleitropic nature of the disease [Cheung and Taniguchi, 2017].

The RFWD3 gene has recently been linked to FA. It encodes the RFWD3 protein consisting of the RING finger domain that acts as an E3 ubiquitin (Ub) ligase, a coiled-coiled domain acting as a protein-protein interaction module with three WD40 repeats. It is shown that the RFWD3 protein appears to have a role in the FA/BRCA pathway by ubiquitination of replication It is a protein which plays a critical function in homologous recombination [Inano et al., 2017]. RFWD3 knockout mice showed embryonic lethality and had a reduced life cycle similar to the murine models with classical Fanconi genes [Feeney et al., 2017; Morgan and Crawford, 2021].

There has been only a single case with the RFWD3 pathogenic variant reported previously [Knies et al., 2017]. This is the second report of the RFWD3 variant causing FA, validating RFWD3 as an FA gene.

The first case was a 12-year-old female with the cardinal findings of FA. She was born preterm, and her weight was small for gestational age. She had duodenal atresia, midface hypoplasia, bilateral radial ray malformations. Her weight, height, and head circumference were all below the third percentile. Growth hormone deficiency was detected, and growth hormone replacement therapy was started. Despite the fact of detecting white matter and posterior fossa abnormalities in her cranial MRI, her neuromotor development was compatible to her peers. In her abdominal USG, a hypoplastic left kidney and polysplenia were detected. Peripheral blood count of the patient was normal, but cytopenia and reduced hematopoiesis were detected in her bone marrow. Knies et al. [2017] reported the novel c.205_206dupCC (p.L69Pfs*12) and c.1916T>A (p.I639K) variants in the RFWD3 gene by whole-exome sequencing of the isolated DNA of the fibroblast tissue. They have suggested that the c.205_206dupCC variant generated a null allele resulted by a nonsense-mediated decay mechanism and c.1916T>A missense variant-impaired relocalization of the RFWD protein to the chromatin, causing abnormal physical interaction with replication protein A. By comparing our case with the previous case where RFWD3 was identified for a new FA subtype, we noticed that our patient had neuromotor developmental delay, gross motor delay, and the absence of speech that is not commonly expected (∼10%) in other FA subtypes as well. Also, his peripheral blood count showed severe thrombocytopenia in repetitive counts (46,000/μL, 52,000/μL, 58,000/μL). In his current complete blood count, isolated cytopenia was noted (Hgb: 12 g/dL, htc: 35.2%, erythrocyte count: 4.05 × 10⁶/μL, leukocyte count: 6.08 × 10³/μL, absolute neutrophil count: 2.33 × 10³/μL, absolute neutrophil count: 3.17 × 10³/μL, absolute eosinophil count: 0.02 × 10³/μL, absolute basophile count: 0.02 × 10³/μL, and thrombocyte count: 64 × 10³/μL). Our patient and the one reported previously both had structural renal abnormalities, but our patient also had anal atresia which is also one of the rare findings (<%5) of the syndrome. The clinical findings of our case and the case previously reported by Knies et al., 2017, are summarized in Table 1.

Table 1.

Summarized clinical data of the RFWD-related FA patients

	Our case	Knies et al. [2017]
Prenatal and/or postnatal short stature	+	+
Abnormal skin pigmentation	−	−
Skeletal malformations	+	+
Microcephaly	+	+
Facial dysmorphism	+	+
Ophthalmic anomalies	+	−
Genitourinary system anomalies	+	+
Endocrine disorders	+	+
Gastrointestinal system disorders	+	+
Cytopenia	+	+

+, the feature is present; −, the feature is absent.

The variant we detected was located at the WD40 domain of the RFWD protein. WD40 repeat domains are B-propeller domains that have a function in protein-protein interaction in multiprotein complexes. There have been several comprehensive studies for establishing genotype-phenotype correlation in more common subtypes of FA, but since there are not many patients reported with FANCW, further functional analyses remain as a necessity. Despite the fact that this rare variant is classified as a “variant of uncertain significance” according to the ACMG criteria and as one of the limitations, further functional analysis (Western/immune blotting) could not be performed; high correlation of clinical findings with the syndrome and highly destabilizing effect showed in silico programming suggest this variant as an underlying cause.

In conclusion, we identified the second patient with RFWD3-related FA. The patient manifested the cardinal findings of the FA including radial ray anomalies, microcephaly, failure to thrive, neuromotor delay, renal malformations, hypothyroidism, and increased chromosomal breakage. In addition to that, gonadal dysgenesis and anal atresia were noted. Since the patient we identified is the probably second patient in the literature with RFWD3 gene, we believe that gene-disease association is further enlightened and the clinical spectrum is expanded, yet still reporting of additional patients with variants in RFWD3 gene is needed to understand clinical variability of this rare subtype.

Statement of Ethics

Written informed consent for genetic analysis and publication of clinical reports and photographs was obtained from the patient’s parents. Ethical approval was not required for this study in accordance with local/national guidelines.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

No specific funding was received for this study.

Author Contributions

Sinem Kocagil was involved in diagnosis of the patient and wrote the manuscript. İkbal Şafak was involved in collection of the data. Can Aydın was involved in the management of the patient and clinical evaluation. Elif Saraç performed the genetic test and was involved in interpretation of the results. Sevilhan Artan was involved in interpretation of the genetic test results and helped writing the manuscript. Birgül Kırel was involved in the management of the patient and supervised the findings of this work.

Funding Statement

No specific funding was received for this study.

Data Availability Statement

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.