Phenotypic and genetic characterizations of the Milan cohort of von Willebrand disease type 2

Abstract

von Willebrand disease (VWD) type 2 is caused by qualitative abnormalities of von Willebrand factor (VWF). This study aimed to determine the genotypic and phenotypic characterizations of a large VWD type 2 cohort from Milan. We included 321 patients (54% female) within 148 unrelated families from 1995 to 2021. Patients were fully characterized using laboratory phenotypic tests, and the genotypic diagnosis was confirmed by target genetic analysis using Sanger sequencing. Patients were diagnosed with type 2A (n = 98; 48 families), 2B (n = 85; 38 families), 2M (n = 112; 50 families), or 2N (n = 26; 12 families). Eighty-two unique VWF variants, including 8 novel variants, were found. The potential pathogenic effect of novel variants was assessed by in silico analysis. Most patients were heterozygous for a single variant (n = 259; 81%), whereas 37 cases (11%) had 2 variants (4 homozygous, 9 in trans, and 24 in cis). Twenty-five patients (8%) had ≥3 variants, mainly as a result of gene conversions. Among the 82 distinct variants identified, 5 different types, including missense (n = 64), gene conversion (n = 10), synonymous (n = 1), deletion (n = 4), and splice (n = 3), were observed. The results from this large cohort showed that VWD type 2 is invariably due to variants that do not prevent the synthesis of the protein, and a vast majority of patients (88%) had missense variants. Given the complexity of type 2 diagnosis and the necessity of performing several phenotypic tests, genetic analysis for patients suspected of having type 2 is beneficial to establish the correct diagnosis.

Graphical abstract

Figure 1.

Variants identified in 321 patients with VWD type 2. Eighty-two unique variants, including 8 novel variants, were found. A vast majority (78%) of identified variants were missense substitutions, although other types, such as gene conversions (12%), synonymous variants (1%), splice variants (4%), and deletions (5%), were also observed. Nevertheless, in dominant VWD types (2A, 2B, and 2M), all variants identified always led to the synthesis of mutated VWF. Most patients were heterozygotes for a single variant (n = 259; 81%), whereas 37 patients (11%) had 2 variants: 4 (1%) were homozygotes, and 9 (3%) were in trans and 24 (7%) in cis position. Twenty-five patients (8%) had ≥3 variants, mainly as a result of gene conversion. #Six distinct gene conversions in type 2B NY were determined, and p.Pro1266Leu was the core variant.

Figure 2.

Number of different variants types found in the VWD type 2 Milan cohort. Eighty-two unique variants, including 8 novel variants, were identified. The most frequent type of variant identified was missense substitution (n = 64; 78%), followed by gene conversions (n = 10; 12%), synonymous variants (n = 1; 1.5%), splice variants (n = 3; 3.5%), and deletions (n = 4, 5%). Of 4 different small deletions detected, 1 was out of frame and was found in type 2A(IIC) (c.1092_1093delTC/c.1583A>G, p.Asp366Leufs*16/p.Asn528Ser), and 3 were in frame, 1 in type 2A(IIA) (c.4606_4611delCACGTC, p.His1536_Val1537del) and 2 in type 2M (c.4222_4224delAAG, p.Lys1408del and c.3831_3833delCCT, p.Asp1277_Leu1278delinsGlu).

Figure 3.

Frequency spectrum of 321 patients diagnosed with VWD type 2 in the Milan cohort study. Type 2M was the most common type (35%), followed by 2A (31%), 2B (26%), and 2N (8%). In type 2M, 41% of patients had variants at the VWF A1 domain, with a platelet-binding defect (classical 2M), whereas 26% had variants at the A3 domain, with a collagen-binding defect (2MCB). Almost one third of type 2M patients (33%), with variants in the A1 domain, shared a similar phenotype between type 2M and 2A, and they were classified as 2M/2A. In type 2A, 65% of patients had variants located at the A2 domain, with an enhanced susceptibility for ADAMTS-13, and were classified as 2A(IIA), with an exceptional case in the A1 domain. The remaining 33% were located at the D3 domain, with multimerization defects, and were classified as 2A(IIE). Patients with a diagnosis of type 2A(IIC) or 2A(IIH) made up only 2% of the type 2A cohorts. Most patients with 2B (75%) had variants at the A1 domain, with a platelet-binding defect (classical 2B), and the rest (25%) had variants at the D3-A1 junction, classified as 2B NY. Patients with 2N were mainly compound heterozygotes for 2N/type 1 or 3 (46%), and only 16% were homozygotes for the 2N variant. Type 2N carriers accounted for 38% of the 2N population.