Congenital macrothrombocytopenias manifest with reduced platelet count and high mean platelet volume (MPV). This phenotype can arise from pathogenic variants located in genes encoding components of the platelet cytoskeleton or microtubular system, such as TUBB1, 1 which has been linked to macrothrombocytopenia since 2009. 2, 3 Furthermore, although traditionally associated with autosomal recessive Glanzmann thrombasthenia (GT), ITGA2B and ITGB3 have also been linked to an autosomal dominant (AD) form of macrothrombocytopenia with mucocutaneous bleeding, 4 referred to as platelet-type 16 bleeding disorder (BDPLT16) for ITGA2B heterozygous variants and platelet-type 24 bleeding disorder (BDPLT24) for ITGB3 heterozygous variants. 5-8 Genetic analysis has become essential for the molecular characterization of patients with inherited platelet disorders (IPD) due to their genetic heterogeneity and phenotypic overlap. We present the case of a 50-year-old female proband with macrothrombocytopenia and lifelong mucocutaneous bleeding (International Society on Thrombosis and Haemostasis bleeding assessment tool ISTH-BAT = 11) who was recruited for a research project aimed at unravelling the molecular basis of rare inherited primary haemostasis disorders by whole-exome sequencing (WES). 9 Written informed consent was provided and the study was approved by the research ethics committee of Hospital Universitari Vall d'Hebron in accordance with the guidelines of the Declaration of Helsinki. To identify the molecular cause of the proband's phenotype, WES was performed and variants were filtered as previously described. 9 The application of WES to the proband (II. 5) resulted in the identification of two heterozygous candidate variants, in TUBB1 and ITGA2B, correlating with the macrothrombocytopenia and bleeding phenotype (Figure 1A). In TUBB1, we identified the variant NM₀30773. 4: c. 35del, p. (Cys12LeufsTer12) located in exon 1, with a frequency of 0. 0038% in the total population of the gnomAD database (available at: https: //gnomad. broadinstitute. org/; accessed 9 Jan 2026). It has been previously described in patients with macrothrombocytopenia and is classified as pathogenic according to the American College of Medical Genetics and Genomics (ACMG) guidelines. 3, 10, 11 This variant introduces a premature termination codon in the N-terminal GTPase domain and removes the intermediate and C-terminal domains required for microtubule-associated protein binding. 10 In ITGA2B, we identified the novel NM₀00419. 5: c. 998+1G>A variant, which is classified as likely pathogenic according to the ACMG guidelines and is not reported in the gnomAD database. This variant is predicted to disrupt normal messenger ribonucleic acid (mRNA) processing by the SpliceAI algorithm (Available at: https: //spliceailookup. broadinstitute. org/; accessed 9 Jan 2026). Notably, a different change in the same position (c. 998+1G>C) has been reported in patients with GT and is classified as pathogenic by the ClinGen Platelet Disorders Expert Panel (available at: https: //erepo. clinicalgenome. org/evrepo/ui/interpretation/aff8f164-2aa9-4376-b011-420535e3a386; accessed 9 Jan 2026), supporting the deleterious impact of variants at this splice site, although it does not constitute evidence of pathogenicity in the context of BDPLT16. Direct ribonucleic acid (RNA) sequencing was performed to unravel the effect of the ITGA2B: c. 998+1G>A variant on splicing. Total RNA from leucocytes and platelets of the proband and two controls was isolated from 10 mL of EDTA-anticoagulated blood, following a centrifugation-based protocol (Supporting information). 12, 13 The purity of platelet preparations was assessed with the Sysmex XN-1000, confirming minimal leucocyte contamination (usual range between 0. 002 and 0. 78 leucocytes per 1000 platelets). cDNA was synthesized using the high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) and the region of interest was amplified using custom primers located in exons 9 and 13. PCR products were fragmented and sequenced using next-generation sequencing (NGS) on a MiSeq platform (Illumina, San Diego, CA, USA). The analysis from FASTQ files was performed using the CLC Genomic Workbench v25. 0. 1 software (QIAGEN, Hilden, Germany) mapping trimmed reads to the ITGA2B reference transcript (NM₀00419. 5). Amplification by reverse transcription polymerase chain reaction (RT-PCR) of the ITGA2B segment spanning exons 9–13 yielded the anticipated 425 bp product, as well as a weaker, smaller band of ~280 bp observed in the proband only (Figure 1B). NGS revealed the presence of three aberrant transcripts, not present in controls, in both platelet and leucocyte mRNA from the patient: (1) a transcript lacking the 53 bp of exon 11, which generates a frameshift: r. 946₉98del, p. (Met316GlufsTer3), ≈1% sequencing reads; (2) a transcript lacking the last 4 bp of exon 10 and skipping exon 11: r. 942₉98del, p. (Glu315Gly333del), ≈1% sequencing reads; (3) a transcript skipping exon 11 along with a partial deletion of exon 12 corresponding to the 278 bp band observed in the agarose gel (Figure 1B, C): r. 946₁092del, p. (Met316Gln364del), ≈2. 6% sequencing reads (Figure 1C). Although NGS is not inherently quantitative, it provides a relative representation of transcript abundance. 12 In this case, alternative transcripts account for only a minor fraction of the total sequencing reads (C variant (available at: https: //erepo. clinicalgenome. org/evrepo/ui/interpretation/aff8f164-2aa9-4376-b011-420535e3a386; accessed 9 Jan 2026). The affected region lies within the β-propeller domain of αIIb, which is essential for fibrinogen binding and for proper assembly of the αIIbβ3 integrin complex, potentially impairing its surface expression and/or function. 14 To clarify the individual contribution of each variant to the proband's phenotype, phenotypic assessment through platelet aggregation and flow cytometry evaluation of CD41/CD61 (Supporting information) and segregation studies by Sanger sequencing were performed on 14 family members. None of the family members presented solely the TUBB1 variant, but two (II. 4 and III. 6) were found to carry both variants (Table 1; Figure 1A). They were the only individuals, besides the proband, presenting with mild macrothrombocytopenia. Both patients showed reduced platelet aggregation with epinephrine, but whereas the proband had severe mucocutaneous bleeding, they only experienced sporadic ecchymoses. F: 40 years), suggesting a possible sex and age-dependent effect. Four patients (I. 1, II. 2, II. 3, III. 2) showed impaired aggregation with different agonists (Table 1). Surprisingly, patient I. 1 showed the classic aggregation pattern of GT, although sequencing of the entire ITGA2B gene revealed no additional variants, and the three younger carriers (III. 3, III. 5 and III. 8) displayed normal platelet aggregation results. In addition, the proband and all carriers of the ITGA2B variant showed a reduction of relative fluorescence intensity (RFI) of CD41 (and when assessed also CD61) compared to healthy controls (Table 1), consistent with previous reports on heterozygous ITGA2B variants. 6 Platelet count and MPV were normal among the five family members who did not carry any variant. Of these non-affected family members, only one (III. 4) had sporadic epistaxis and increased platelet aggregation in response to a low dose of ristocetin (Table 1; Figure 1A). Moreover, platelet CD41 and CD61 expression, measured in two of these patients, was compatible with controls (Table 1). In summary, through family-based segregation, flow cytometry studies and transcript-level analysis, we characterized the contribution of two candidate variants in a proband with suspected IPD. This integrative approach allowed us to dissect the genotype–phenotype correlations within the family and to expand the current understanding of the molecular basis of TUBB1- and ITGA2B-related disorders. Our findings suggest that, in this family, macrothrombocytopenia is primarily attributable to the TUBB1 variant, as none of the exclusively heterozygous carriers of the ITGA2B presented with macrothrombocytopenia. Conversely, the haemorrhagic phenotype appears to be linked to the ITGA2B variant, as reported in previous studies5-7 and supported by RNA sequencing demonstrating a pathogenic splicing mechanism and by flow cytometry showing reduced CD41/CD61 expression in all carriers. Nevertheless, as functional studies assessing PAC-1 binding were not performed, an additional qualitative defect of CD41/CD61, reported in other BDPLT16 patients, 6 could not be excluded. Notably, more severe bleeding was observed in older female compared to younger female or male patients. This could be caused by female-specific physiological processes such as menstruation and childbirth, which place additional stress on haemostasis and can unmask a latent bleeding tendency that might otherwise remain unnoticed unless triggered by trauma. In fact, carriers of the ITGA2B variant who were male or young female that had not yet undergone menarche remained asymptomatic or experienced only sporadic ecchymosis. Consistent with a previous report, 6 considerable variability in platelet aggregation was also observed among individuals heterozygous for the ITGA2B variant. In summary, these results confirm the implication of the ITGA2B variant in the bleeding phenotype, but do not provide evidence for a major role in macrothrombocytopenia in this family. Several cases of oligogenic inheritance have been described in patients with IPD, including a reported French family carrying candidate variants in ITGB3 and TUBB1. 15 To our knowledge, we report the first family with co-occurring variants in ITGA2B and TUBB1 and the first description of BDPLT16 caused by a splicing variant in ITGA2B which does not correlate with macrothrombocytopenia in an extensive family. The integrative strategy combining WES, family-based segregation studies and RNA sequencing enabled clarification of the contribution of both variants and helped to establish genotype–phenotype correlations. This case highlights how a seemingly straightforward phenotype may actually result from the combined effect of variants in distinct genes, underscoring the importance of analysing all relevant genes that may contribute to the clinical presentation of IPD. C. Altisent, N. Fernández-Mosteirín, M. R. Aguinaco and O. Benítez have recruited the patients and reported all the clinical data presented in this study. P. Bandini, N. Comes, L. Ramírez, C. Lera and N. González performed the molecular analyses. P. Bandini, N. Borràs, F. Vidal and I. Corrales identified and validated the candidate variants. L. Martin-Fernandez, N. Borràs, F. Vidal and I. Corrales provided support in the analysis and organization of the results. P. Bandini wrote the manuscript. All authors revised the final version of the manuscript. This study was supported by the Spanish Ministry of the Economy and Competitiveness (MINECO, Ministerio de Economía y Competitividad) —Instituto de Salud Carlos III (ISCIII) (PI18/01492, PI23/01672). CIBERCV is an initiative of ISCIII, co-financed by the European Regional Development Fund (ERDF), ‘A way to build Europe’. We are grateful for the kind collaboration of the participating patients and their families, the Fundació Privada Catalana de l'Hemofília and the Real Fundación Victoria Eugenia. The authors declare no competing financial interests. This study was approved by the research ethics committee of Hospital Universitari Vall d'Hebron and was conducted according to the Declaration of Helsinki. All patients provided written informed consent for the study. The data that support the findings of this study are available from the corresponding author upon reasonable request. Data S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
Bandini et al. (Wed,) studied this question.