Platelet function and filamin A expression in two families with novel FLNA gene mutations associated with periventricular nodular heterotopia and panlobular emphysema

Abstract Pathogenic variants of the X‐linked FLNA gene encoding filamin A protein have been associated with a wide spectrum of symptoms, including the recently described pulmonary phenotype with childhood‐onset panlobular emphysema. We describe three female patients from two families with novel heterozygous FLNA variants c.5837_2del and c.508C > T. Analysis of immunofluorescence of peripheral blood smears and platelet function was performed for all patients. FLNA‐negative platelets were observed, suggesting that these variants result in the loss of a functional protein product. All three patients also had periventricular nodular heterotopia and panlobular emphysema. However, they had considerably milder symptoms and later age of onset than in the previously reported cases. Therefore, patients with pathogenic FLNA variants should be studied actively for lung involvement even in the absence of pronounced respiratory symptoms. Conversely, any patient with unexplained panlobular emphysema should be analyzed for pathogenic FLNA variants. We also suggest that immunofluorescence analysis is a useful tool for investigating the pathogenicity of novel FLNA variants.


| INTRODUCTION
The X-linked FLNA gene (OMIM 300017) encodes filamin A, a Factin-binding cytoplasmic phosphoprotein involved in cytoskeletal remodeling of cell morphology and migration. Pathogenic variants of the FLNA gene cause a wide spectrum of rare diseases called filaminopathies A. Heterozygous truncating variants primarily result in periventricular nodular heterotopia (PVNH), a brain disorder in which neuronal migration to the cortex is disrupted during early fetal development (OMIM #300049). Additional characteristics of FLNArelated PVNH include megacisterna magna, cardiovascular malformations, epilepsy and Ehlers-Danlos-like collagen defects. It is mostly encountered in heterozygous females, as truncating variants are usually lethal in males (Parrini et al., 2006;Sheen et al., 2005).
Abnormal platelet morphology and function have been described in several patients with FLNA-associated disease and Nurden et al. have suggested that macrothrombocytopenia may even present as the primary phenotype (Nurden et al., 2011). As blood platelets are easily available for isolation, their structural and functional analysis may provide useful data on the pathogenicity of novel FLNA variants.
Ieda et al. have suggested immunofluorescence staining of peripheral blood smears focusing on platelets as a convenient method for identifying patients with FLNA variants (Ieda et al., 2018). FLNA is expressed in platelets and its pathogenic variants alter platelet production from the bone marrow megakaryocytes, leading to reduced number and abnormal size of platelets (Rosa et al., 2019). Lopez et al. have also suggested that deficient expression or function of filamin A might lead to altered Ca 2+ homeostasis in platelets and, subsequently, functional abnormalities (Lopez et al., 2018).
We describe here two novel FLNA variants associated with periventricular heterotopia and pulmonary emphysema, with respective alterations observed in platelet function and FLNA expression. The index patient has one 15-year-old daughter (2-2), who carries both of the FLNA variants detected in her mother. She was diagnosed with asthma in early childhood and has been suffering from exercise-induced dyspnea responding poorly to medication but has otherwise been doing well. Diagnostic work-up again revealed panlobular emphysema, mild joint hypermobility, and PVNH. collagen under flow conditions. The PFA is a platelet function screening test with a high sensitivity (Koscielny et al., 2004). Platelet impedance aggregometry in whole blood by using Multiplate ® analyzer (Roche) was also performed (Moenen et al., 2019). In the Multiplate ® assay, platelet function can be further characterized using different F I G U R E 2 Contrast enhanced coronal chest CT (left) and X-ray (right) images with panlobular emphysema in patient 1-1 F I G U R E 3 Contrast enhanced coronal chest CT (left) and X-ray (right) images with marked panlobular emphysema in patient 2-1. There is abnormal hyperlucency of both lungs with paucity of the peripheral lung vessels (arrows) agonists. Hirudin-anticoagulated blood (25 μg/ml) was run with the following agonists and concentrations: ristocetin 1.2 mg/ml, 0.6 mg/ml, and 0.3 mg/ml, ADP 6.5 μM, ADP 2.0 μM arachidonic acid (ASPI) 0.5 mM, thrombin receptor activating peptide (TRAP) 32 μM and collagen 3.2 μg/ml. In addition, ristocetin at concentrations 1.2, 0.6 and 0.3 mg/ml were added to 3.2% citrated blood as well, per our routine protocols. Only the results of ristocetin 1.2 mg/ml (RISTOH), ADP 6.5 mM, ASPI 0.5 mM, TRAP 32 μM in Hirudin-anticoagulated blood are routinely reported, the other abovementioned assay results are reported only if abnormal. We chose these assays for platelet function analyses since they are performed routinely in our laboratory.
They are analyzed in whole blood, facilitating sample preparation and improving workflow.
In order to screen for coagulation defects as well, a panel from 3.2% Na-citrated plasma was performed, including prothrombin time Bleeding symptoms were assessed with ISTH/SSC bleeding assessment tool (bleedingscore.certe.nl).
Platelet interaction with collagen under blood flow was most sensitive in detecting platelet abnormalities in these patients by providing prolonged closure times under high shear rate flow conditions. Collagen/epinephrine results were grossly abnormal in patient 1-1. Collagen/ADP assessment was abnormal in patient 1-1 and borderline abnormal in patient 2-2. The mild thrombocytopenia is insufficient to explain the PFA findings, but instead a platelet adhesion and aggregation defect on collagen is likely, even in the presence of co-activation with epinephrine or ADP and shear forces. In contrast, Multiplate aggregation dissociated with the PFA findings, providing essentially normal results, with tight duplicate values. However, in patient 1-1 ristocetin response was diminished at low concentration but normal at the highest ristocetin concentration, suggesting an impaired interaction with platelet glycoprotein Ibα with von Willebrand factor to trigger the signaling.
Coagulation panel was essentially normal in all, with minor inconsequential findings (prolonged PT with marginally low fibrinogen) in patient 1-1.

| DISCUSSION
In this case series, we were able to prove with immunofluorescence analysis that heterozygous variants c.5837_2del and c.508C > T cause loss of functional filamin A protein in platelets.
Childhood-onset emphysematous lung disease has only recently been added to the spectrum of FLNA-associated phenotypes. The clinical phenotype has been variable, but several cases requiring lung transplantation have been reported (Burrage et al., 2017). Pelizzo et al., 2020 also recently reported a case where mesenchymal stromal cell infusions were successfully used as a rescue therapy in a child with life-threatening respiratory syndrome associated with a pathogenic FLNA variant. FLNA-associated lung disease has typically been diagnosed in the neonatal period or in early childhood (Sasaki et al., 2019). In our patient cohort, one patient was diagnosed with panlobular emphysema in adulthood and two patients in their teens, further broadening the phenotypic spectrum of pathogenic FLNA variants. Computed tomography was required to obtain the correct diagnosis, and in two cases it was only triggered by the available results of the genetic analysis. Pulmonary emphysema is extremely rare in children and even young adults. The most prevalent forms of pulmonary emphysema are smoking-related centrilobular and paraseptal emphysema. In contrast, panlobular emphysema is typically associated with alpha-1-antitrypsin deficiency (Smith et al., 2014).
Majority of patients with FLNA-associated pulmonary disease have had PVNH and/or aortic dilatation (Burrage et al., 2017;Sasaki et al., 2019). On the other hand, in a cohort of 114 patients with FLNA-associated PVNH described by Chen et al., 2018 21 patients (18.4%) were diagnosed with thoracic aortic aneurysm. Six of them required surgical repair and two additional patients, who did not fulfill the clinical criteria for surgical correction, nevertheless died of spontaneous aortic rupture. Indeed, the importance of screening must be emphasized for all patients with pathogenic FLNA variants both for aortic dilatation and interstitial pulmonary disease.
In our case series, pathogenic loss-of-function FLNA variants were associated with abnormal platelet interactions under high shear rate blood flow conditions, compatible with the platelet cytoskeletal defects altering adhesive functions toward standardized collagen. As to the modest bleeding tendency and quantity of FLNA-negative platelets, the severity of the overall clinical phenotype makes skewed X-inactivation an unlikely explanation. It is likely that FLNA-negative megakaryocytes produce abnormal platelets with shortened lifespan, as suggested by Begonja et al., 2011. Majority of the circulating platelets would therefore have wild-type DNA.
Truncating FLNA variants have been reported by Berrou et al. to cause abnormal platelet responses to collagen impairing aggregation, secretion, thrombus formation and GPVI signaling (Berrou et al., 2013). Yet none of our patients exhibited abnormal response to collagen in the Multiplate assay, all having some expression of FLNA.  Immunofluorescence analysis of peripheral blood smears seems to be a useful tool in demonstrating the absent filamin in the novel variants.

ACKNOWLEDGMENTS
We thank the patients and their families for their participation in this study. This study was supported by research grants from HUS Diagnostic Center, Helsinki University Hospital.

CONFLICT OF INTEREST
None declared.

AUTHOR CONTRIBUTIONS
LMT, EL, TH, RL, and MP were involved in study design. LMT was involved in collection of clinical data, drafting, and submission of manuscript. SK was involved in immunofluorescence analyses. TH was involved in platelet functional analyses. KV was involved in radiographic images and their interpretation. LMT, SK, EL, TH, KV, RL, and MP were involved in critical review and revision of manuscript.

FUNDING INFORMATION
The authors have not declared a specific grant for this research from any funding agency in the public, commercial, or not-for-profit sectors.

PATIENT CONSENT FOR PUBLICATION
Obtained.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ORCID
Laura M. Tanner https://orcid.org/0000-0003-4451-2238 F I G U R E 4 Immunofluorescence staining of peripheral blood of patients 1-1 (a) and 2-2 (b). FLNA-negative platelets only have the beta1-tubulin positive green control stain whereas platelets with both the beta1-tubulin positive green control stain and the orange signal positive for filamin A can be seen as yellow