De novo missense variants in the RAP1B gene identified in two patients with syndromic thrombocytopenia

We present two independent cases of syndromic thrombocytopenia with multiple malformations, microcephaly, learning difficulties, dysmorphism and other features. Exome sequencing identified two novel de novo heterozygous variants in these patients, c.35G>T p.(Gly12Val) and c.178G>C p.(Gly60Arg), in the RAP1B gene (NM_001010942.2). These variants have not been described previously as germline variants, however functional studies in literature strongly suggest a clinical implication of these two activating hot spot positions. We hypothesize that pathogenic missense variants in the RAP1B gene cause congenital syndromic thrombocytopenia with a spectrum of associated malformations and dysmorphism, possibly through a gain of function mechanism.


| INTRODUCTION
Hereditary thrombocytopenia (HT) is a heterogeneous group of genetic disorders characterized by a low platelet count causing impaired hemostasis. Until today, about 40 genes have been associated with HT. 1 Patients with Kabuki syndrome (KS, MIM#147920/MIM#300867) present with intellectual disability, postnatal growth deficiency, organ malformations, skeletal anomalies and other features. 2 Typical facial dysmorphism include long palpebral fissures with eversion of the Jan Hendrik Niemann and Chen Du contributed equally to this study. lateral third of the lower eyelid, large ears and persistent fetal fingertip pads. 2 Hematological symptoms, mainly idiopathic thrombocytopenic purpura (ITP), occur in up to 20% of all KS cases. 3,4 Recently, pathogenic variants in the RAP1A and RAP1B genes have been identified to cause KS with a less pronounced phenotype, designated Kabuki-like syndrome. 5

| MATERIALS AND METHODS
Whole exome sequencing (WES) was performed as trio analysis in case 1 and as singleton analysis in case 2 (see Supplemental Methods).
The two cases in this study were matched using GeneMatcher. 6 3 | CLINICAL DESCRIPTION Case 1 is a 36-year-old female patient of non-consanguineous parents presenting with unclear pancytopenia, multiple congenital malformations, mild intellectual disability, endocrine disorders, dysmorphism and other features ( Figure 1A-D). The family history was negative for comparable diseases. Her parents and two brothers were unaffected.
The patient was born at term by caesarian section due to transverse presentation. Thrombocytopenia with 30 to 50 × 1000/μL platelets, with normal erythropoiesis and granulopoiesis was documented for the first time at the age of 14 months. Later, leucopenia with lymphopenia and anemia as well as splenomegaly developed. She showed easy skin bruising without muscle or joint bleeding.
The patient presented with feeding difficulties and failure to thrive in infancy. She developed postnatal microcephaly, developmental delay (sitting up at 18 months, crawling at 2 years, walking at 2 years 9 months), a reduced muscle tone and learning difficulties. Dysmorphism ( Figure 1A) included a preauricular tag, upslanting palpebral fissures, a flat mid face, scarce eyebrows, low-set posteriorly rotated ears, hypoplastic primary and permanent teeth, brachydactyly and lack of pubic and axillary hair. Her skin was thin and dry with multiple nevi and hematomas, and she had broken nails due to nail biting.
She showed short stature with growth hormone deficiency and obesity (BMI 34.2 kg/m 2 at age 36 years). Pubarche and menarche did not set in while thelarche did occur.
Congenital malformations included congenital hip dysplasia, unilateral cystic renal hypoplasia, a obstructive hydroureter and a complex structural abnormality of the brain affecting the supratentorial ventricle system including aplasia of anterior parts of the septum Growth parameters are normal (weight on the mean, height +1 SD) except postnatal microcephaly with OFC −2.5 SD. Brain MRI performed at 13 years old revealed two nodular heterotopias of the right ventricle and hypoplasia of the cerebellar vermis ( Figure 2). Ophthalmological examination showed hypermetropia and astigmatism.
The thrombocytopenia was responsible for minor to mild hemorrhage with a Buchanan score around 1 to 2. 7 The patient usually presents with petechiae and bruises without mucosal or internal bleeding. Flow cytometry revealed no deficit in platelet glycoproteins (GPIIb/IIIa, GPIb).
Standard karyotype, array-CGH, sequencing of genes involved in known RASopathies and telomere length study were normal.
A comparison of the patients' features is given in Table S1. A list of the patients' features in HPO terms is given in Table S2.  Figure S1A). In case 2, prioritization using the deep phenotyping tool PhenoTips 8 (see Table S2 for HPO terms used, and Table S3 containing  Both RAP1B variants have not been described as germline variants in public databases. The amino acid positions 12 and 60 both involve a glycine residue and are highly conserved among species (phyloP: 9.48 for p.Gly12 and 9.53 for p.Gly60, Figure S2). Both variants are predicted to be disease causing and in silico structural predictions argue for a deleterious effect on the basis of conformational changes of the GTPase domain ( Figure S3). Furthermore, since both variants occur in domains sharing significant sequence homology in RAS-related genes (codons 12-13 and 59-61) and frequently involved in the malignant activation of RAS oncogenes, 10 we hypothesize that these variants contribute to the presented phenotypes through a gain of function mechanism.

| DISCUSSION
We identified de novo variants in the RAP1B gene (c.35G>T p. (Gly12Val) and c.178G>C p.(Gly60Arg)) in two unrelated patients with thrombocytopenia, microcephaly, learning difficulties, renal malformations, structural anomalies of the brain and other features.
RAP1B is a member of the RAS superfamily of small GTPases, which are involved in many cellular processes. Murine skeletal development, 11 rat diabetic nephropathy, 12 rat neuronal development, 13 and murine neutrophil migration 14 are regulated by RAP1B-dependent pathways.
Studies have shown a link between RAP1B activation and platelet function in human and mice. [15][16][17] Thrombocytopenia due to defective thrombocyte production has been described in Rap1a/b-double knockout mice. 17 Interestingly, a somatic p.Gly12Arg variant has been described in myelodysplastic syndrome indicating that RAP1B contributes to the pathophysiology of myeloid disorders. 18 Furthermore, RAP1B was identified to act as a key modulator of lymphocyte recruitment during immune reaction. 19 These reports indicate a possible causative link between pathogenic variants in RAP1B and thrombocytopenia and pancytopenia, respectively, bleeding diathesis and recurrent infections. Future investigations may determine whether bleeding diathesis including easy bruising is merely caused by a reduced thrombocyte count or additionally by defective platelet function.
There is no major overlap between the symptoms of our patients and the international consensus diagnostic criteria for KS. 2 Therefore, we propose a RAP1B-associated phenotype distinct from KS. Notably, the previously described variants p.Arg163Thr in the RAP1A gene and p.Leu151Glu in the RAP1B gene in patients with Kabuki and Kabukilike syndromes both resulted in a loss of function and decreased downstream BRAF activation/MEK/ERK signaling. 5 Moreover, no hematological findings were reported in these patients by the authors.
There is strong evidence that the p.Gly12Val and p.Gly60Arg variants in the RAP1B gene lead into a dysregulation of the downstream pathway. Both substitutions have been described previously as dominant constitutively active in RAS-related proteins. 10,[20][21][22] The variant p.(Gly12Val) is located within the phosphate binding loop (L1) of the catalytic RAP1B domain. 23 Almost every amino acid change at codon 12 of RAS-related proteins has been shown to alter the protein function. 20 Especially a valine at this position seems to have a strong dominant activating effect in transfected cells. 20 It has been shown that this activation occurs through impacted GTPase activity, which in turn leads to a relative surplus of active GTP. 22 The variant p.(Gly60Arg) is located in the GTPase domain. In the active form of the protein, this residue is linked by a hydrogen bond to the gamma-phosphate of the nucleoside triphosphate. 24 The same substitution in let-60 ras gene causes hallmarks of gain-of-function RAS mutations in Caenorhabditis elegans. 20,21 Missense variants involving flanking residue 61 in RASrelated proteins are also responsible for dominant activation of the pathway, by altered GTPase activity. 25 Therefore, we conclude that the RAP1B variants identified contribute to the presented phenotypes through dysregulation of the MEK/ERK pathway.
In summary, we hypothesize that germline gain-of-function variants in the RAP1B gene are causative for congenital syndromic thrombocytopenia. Further functional studies and identification of more affected patients may elucidate the clinical variability of RAP1Bassociated syndromes.