1 INTRODUCTION

Ras-associated protein 1B (RAP1B) is a member of the RAS family of small G proteins and regulates multiple signalling pathways involved in cell proliferation, cell adhesion, growth, and differentiation.¹ RAP1B is highly homologous (95% amino acid identity) to RAP1A, and although the two isoforms have been shown to have different functions,^{1, 2} their mechanism of action is similar to that of the canonical RAS GTPase.^{1, 2} Both act as molecular switches that activate downstream effectors when bound to GTP. The low intrinsic RAP1B GTPase activity can be stimulated by specific GTPase activating proteins (GAPs) resulting in negative regulation of the signalling pathways downstream.³ Amino acid residues critical for RAP1B GTPase activity include Gly12 of the P-loop that binds the beta phosphate of GTP/GDP; Thr35 of the switch I region that interacts with the gamma phosphate of GTP and the magnesium ion co-factor; and amino acids 57-61 of the switch II region (Asp-X-X-Gly-Gln) that is necessary for GTP hydrolysis and the interaction with cognate GAPs. Like other members of the RAS family, RAP1B is a conditional oncoprotein¹ and somatic, activating RAP1B variants have been identified in various human tumours.⁴ However, a role for germline RAP1B variants in human genetic disease has remained elusive.

A de novo NM_001010942.2(RAP1B):c.451A>G, p.(Lys151Glu) variant was identified in an individual with a Kabuki-like phenotype, without reported haematological abnormalities,⁵ and more recently, de novo RAP1B variants were identified in individuals with congenital thrombocytopenia, intellectual disability and congenital malformations.^{6, 7} Specifically, three de novo missense variants, NM_001010942.2(RAP1B):c.35G>T, (p.(Gly12Val),⁶ NM_001010942.2(RAP1B):c.178G>C, p.(Gly60Arg)⁶ and NM_001010942.2(RAP1B):c.176C>G, p.(Ala59Gly)⁷ were identified in three independent cases of syndromic thrombocytopenia. Other features were more heterogeneous.^{6, 7}

The Clinical Genome Resource (ClinGen)⁸ classifies the gene-disease association evidence between RAP1B and syndromic constitutional thrombocytopenia as limited,⁸ while there is no OMIM entry for this phenotype and RAP1B. In order to assess the evidence for a causative role of RAP1B variants in thrombocytopenia, we searched our Biodatabank for heterozygous variants in RAP1B and identified two individuals featuring thrombocytopenia and other syndromic features. Here, we describe the clinical and genetic features of these individuals and discuss the evidence supporting a causative role for RAP1B variants in syndromic thrombocytopenia.

2 METHODS

Diagnostic exome sequencing was performed at CENTOGENE GmbH as described previously.⁹ In short, the SureSelect Human All Exon kit (Agilent, Santa Clara, CA) or the Twist library preparation EF V.1.0 kit (Twist Bioscience, San Francisco, USA) was used for the enrichment, and a HiSeq4000 (Illumina, San Francisco, 2018) or NovaSeq 6000 (Illumina, San Diego, 2022), for the sequencing with the 150 paired-end protocol to yield at least 20× depth of coverage for >98% of the target region. An in-house bioinformatics pipeline, including read alignment to human genome reference (GRCh37/hg19), variant calling and variant annotation with publicly available databases, was used.⁹ All disease-causing variants reported in HGMD®, ClinVar or CENTOGENE's Biodatabank, and all variants with minor allele frequency (MAF <1%) in gnomAD database were considered. Evaluation was focused on coding exons along with flanking ±20 intronic bases. All identified variants were evaluated with respect to their pathogenicity and causality and classified following the ACMG/AMP guidelines¹⁰ and ClinGen Sequence Variant Interpretation recommendations (reference https://clinicalgenome.org/working-groups/sequence-variant-interpretation/). All relevant variants related to the phenotype of the patient were reported.

CENTOGENE's Biodatabank, which contains exome/genome sequencing data from over 136 000 individuals were searched to identify additional cases with relevant RAP1B mono-allelic variants. All rare variants in the RAP1B gene (<1% minor allele frequency) with good quality,¹¹ and predicted moderate/high impact on protein function were evaluated. For phenotypic prioritisation, the HPO thrombocytopenia (HP:0001873) was used. Informed consent was obtained for both cases.

3 RESULTS

The first patient (Case 1) is a female of 16 years of age at last examination referred from Finland (Table 1). The patient was born to non-consanguineous parents at 38 weeks of pregnancy with elective caesarean section. Increased nuchal translucency was detected during the first trimester screening and karyotype analysis of amniotic fluid cells was normal (46, XX). Ultrasound screenings during pregnancy showed multiple congenital abnormalities including unilateral multicystic kidney dysplasia, pyelectasis, cardiomegaly, ductus venosus agenesis, umbilical vein aneurysm and mild ventriculomegaly. The foetal brain MRI showed slightly abnormal ventricular morphology with mild ventriculomegaly. The Apgar score was 9/9. Birth weight was 2.92 kg (−1.5 SD), height 48.4 cm (−1.2 SD), and head circumference 32.2 cm (−2.4 SD). After birth, the patient required assisted ventilation (in the very early stages) and tube feeding (>4 months).

The postnatal echocardiogram showed pulmonary stenosis, ventricular hypertrophy, mild dilatation of the ascending thoracic aorta, as well as dilatation of the pulmonary trunk and pulmonary arteries. Coil occlusion of the patent ductus arteriosus (PDA) was performed at 6 months of age. At that time, the pressure gradient for pulmonary valve stenosis (by catheter) was 10 mmHg and the pulmonary stenosis has remained mild during follow-up. The prenatal diagnosis of unilateral multicystic kidney dysplasia was also confirmed postnatally.

The patient also presented with severe thrombocytopenia (thrombocytes 6 × 10^9/l; reference range 140–290 × 10^9/l) and clinically had petechiae and bruises on the skin soon after birth. Leukocyte counts were normal on the first 2 days of life, but the leucocyte and lymphocyte counts have been repeatedly low since the third day of life. Immunoglobulin levels (IgG, IgM, IgA) have been normal. She has had occasional mild iron deficiency anaemia. She has had some upper respiratory tract infections. Despite the very severe thrombocytopenia (platelet count 5–16 × 10^9/l), she has not had any major haemorrhages. A bone marrow biopsy at 4 months of age did not reveal the aetiology of the cytopenias. Desogestrel has been used to prevent menorrhagia and tranexamic acid to treat (rarely occurring) nose bleeds. The patient had hypertension that has been treated with metoprolol since 5 years of age.

Her motor and language development have been delayed. She was diagnosed with mild intellectual disability (ID) based on neuropsychological evaluation just before 6 years of age. Ophthalmologic examination showed bilateral and large retinal colobomas (affecting the optic discs and the inferior fundi), bilateral iris colobomas, with bilateral focal lens opacities, and nystagmus.

The patient also presented with height below average for her age. Weight has been +27%–34% as compared with the average weight for her height. She has also been noted to have dental crowding and pseudo hyper-parathyroidism. Facial dysmorphism is also present (Table 1).

Exome sequencing of the patient and parents revealed a heterozygous de novo missense variant in the exon 2 of RAP1B NM_001010942.2:c.35G>A, p.(Gly12Glu) that mapped to GTP-binding domain. We then searched CENTOGENE's Biodatabank for similar cases with heterozygous variants in RAP1B and identified an additional patient (Table 1).

The second patient (Case 2) was a 5-year-old male without a definite clinical diagnosis. The phenotype is summarised in Table 1 and included delayed neurological development (poor visual contact, irritability, poor communication), facial dysmorphism (downward palpebral fissure, anteverted ears) and thrombocytopenia. Other abnormalities included hydronephrosis, unilateral cryptorchidism and vesicoureteral reflux. Unfortunately, a more detailed phenotype was not available. He was born to non-consanguineous parents. Exome sequencing revealed a heterozygous missense variant in RAP1B: NM_001010942.2(RAP1B):c.178G>A, p.(Gly60Arg) mapping to the switch II region that is essential for the interaction with GAPs.³

An additional search in the DECIPHER¹² (v11.21) genomic web browser identified a female patient with intellectual disability and a heterozygous variant also likely to affect the switch II region, NM_001010942.2(RAP1B):c.179G>T, p.(Gly60Val), and described as likely pathogenic. No haematological alterations were reported (patient 323 709).

4 DISCUSSION

In this report, we describe the clinical and genetic characteristics of two patients with heterozygous variants in RAP1B, one of them confirmed as de novo. The two newly identified patients add to the clinical spectrum associated with RAP1B variants and increases the number of individuals reported to have thrombocytopenia to five. Nonetheless, the gene-disease association remains limited, as per the ClinGen guidelines.⁸

In the five individuals with thrombocytopenia, heterozygous missense variants all affecting amino acid residues likely to be critical for RAP1B GTPase activity were identified. In two cases, the Gly12 position of the P-loop was affected. These variants are highly likely to inactivate RAP1B GTPase activity,³ leading to constitutively active GTP-bound RAP1B.³ The three other variants (Table 1) map to the switch II region that is involved in the interaction between RAP1B and cognate GAPs,³ and are likely to render RAP1B resistant to GAP-dependent regulation, resulting in increased levels of the active RAP1B-GTP isoform.¹³ Thus, the variants associated with thrombocytopenia cluster into two hotspots with highly similar functional impact. In contrast, the c.451A>G, p.(Lys151G) variant, first reported in 2015 in an individual without described haematological problems, is located in another region of the protein and was shown to be unable to rescue the jaw defects observed in rap1 mutant zebrafish.⁵

Active GTP-bound RAP1B leads to expression of integrins which are the main platelet receptors. When tissue damage occurs, RAP1B is activated and results in platelet aggregation at the site of damage.¹⁴ However, how constitutive RAP1B activation leads to the syndromic characteristics in the patients with specific variants in RAP1B is not known. In mouse models, homozygous deletion of Rap1b or inactivation of pathways downstream of Rap1 have been shown to cause alterations in both platelet form and function, resulting in extended bleeding times.^13-15 Consistent with the idea that RAP1B activation causes thrombocytopenia, severe combined immune deficient (SCID) mice with homozygous knock-out of Rasa3, that encodes a Rap1b GAP a negative regulator of Rap1b, exhibited thoracic and peritoneal haemorrhages, megakaryocytic dysplasia and severe thrombocytopenia. Despite these intriguing observations, there are still a considerable number of gaps in our understanding of the role of RAP1B in the signalling pathways that regulate platelet differentiation and production. Experimental cellular, organoid and/or animal models targeting these specific variants would help further our understanding of the aetiology and genetics of syndromic thrombocytopenia. Furthermore, it might be useful to compare the effect of the identified RAP1B variants with the corresponding variants in RAP1A. In most tissues, RAP1B is expressed at much lower (approximately 10-fold) levels than RAP1A. It is possible that loss of RAP1A GTPase activity, and the consequent constitutive activation of GTP-bound RAP1A-dependent signalling, results in a more severe, even lethal, phenotype.

5 CONCLUSION

In the current work, we report two new patients with rare variants in RAP1B leading to syndromic thrombocytopenia, thus expanding the clinical and genetic spectrum of this ultra-rare disease.

AUTHOR CONTRIBUTIONS

Luba M. Pardo, Aida M. Bertoli-Avella: Conceptualization, data curation and formal analysis, as well as writing of the original draft. Ruxandra Aanicai, Emir Zonic, Susan Zielske: Analysis of laboratory data, data curation, variant classification, and reporting. Anna H. Hakonen: Clinical data. Peter Bauer: Review and editing of the manuscript. All authors reviewed and approved the manuscript.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.