Disease: Dystrophic epidermolysis bullosa (DEB) pruriginosa *Note: A novel vwf variant was found in this patient with these other variants in COL7A1 gene

Patient(s): 17 YO Korean male

Variant: VWF NM_000552.5: c.3310C>T p. (R1101W) Located in exon 26

Hadn't been reported previously Variant found to be inherited in trans from family analysis

According to the paper, they have classified with with ACMG guidelines as likely pathogenic.

Reasoning: Variant absent in the Korea Ref Genome Database and Allele Freq = 0.00086% in gnomAD (Evidence: PM2)

Variant detected in trans with another well-known pathologic variant (c.5797C>T) (Evidence: PM3)

Multiple missense variants are known to be pathogenic in RDEB (Evidence: PP2)

Various computational in silico predictive programs reported the variant as pathogenic (Evidence PP3)

Disease: Von-willebrand Disease (Type 2A)

Patient: 50 yo female

Variant: VWF NM_000552.5 c:4232_4249del p.(Val411_Ile416del), Exon 28, heterozygous variant

According to this paper, ACMG-AMP guidelines for interpreting this variant resulted in classification of likely pathogenic

Phenotypes: increased bruising, fatigue, recurrent sinusitis, menorrhagia, neutropenia, anaemia. Reduction in high-molecular-weight multimers

Family: segregation analysis showed two affected family members had the variant and two unaffected family members did not have variant.

Note: Patient also has diagnosed Acute Myeloid Leukaemia (AML) NM_000546.6(TP53):c704A>G p.(Asn235Ser), listed as VUS and found by NGS

Disease: Von-willebrand Disorder Type 3

Patient: 26 yo, female

Variant: VWF NM_000552.5 c:997+118 T>G g.(6073501 A>C), homozygous, intronic

Phenotypes: No detectable VWF in plasma, early onset bleeding complications, epistaxis, easy bruising, bleeding following injury, menorrhagia, iron-deficient anemia

Note: underwent prophylaxis replacement therapy, on-demand antihemorrhagic treatments, oral contraceptives, and replacement therapy.

Family: not mentioned

Predictions:

VEP SpliceAI tool predicted variant likely deleterious (delta score 0.95)

Used Polyphen-2 and SIFT which determined pathogenic likelihood.

Neural Network Splicing, Alternative Splice Site Predictor, plug-in MaxEnt(For 5' donor site) of Human Splicing Finder all concur this variant can create a new donor splice site in intron 8. Contains premature stop codon and susceptible to NMD.

Functional work:

qRT-PCR performed to identify levels of VWF in IP-derived endothelial cells.

histochemical immunostaining for IP-derived endothelial cells confirm no VWF production, only a residual amount present. Suggests leaky mutation.

performed RNA sequencing to assess co-regulated gene networks

Disease: N/A, variant present in F12 gene

Patient: 36 yo, Female, Saudi descent

Variant:F12 NC_000005.9:g.176,830,269 G>A; p.Gly506Asp Homozygous mutation, exon 12 Located in peptidase S1 domain of F12

Family:

Consanguineous family history (parents first-degree cousins)

No family history of bleeding or thrombosis

Phenotypes:

Significantly high activated partial thromboplastin time

No history of bleeding during deliveries or tooth extractions

No history of thrombosis or skin manifestations

On no medications, physical examination unremarkable

Factor assays and VWF tests within normal ranges except Factor XII (Severely deficient)

variant is proposed to be deleterious but there is insufficient evidence to support this claim.

Disease: Von Willebrand Disease (VWD) Type 2A

Patient: 31 yo, Female

Variant1: VWF NC_000012.12: c.875-5T>Gdel, p.(Ser292_Glu333delinsLys) Causes complete exon 8 skipping

Variant2: VWF NM_000552.5: c.813C>G, p.(Tyr271*)

Phenotypes: History of bleeding (epistaxis, uncontrollable by conventional hemostatic treatment), Easy bruising, gum bleeding, excessive menstrual bleeding, mild decrease in plasma VWF:Ag, severe impairment in VWF function, VWF:Ab/VWF:Ag ratio decreased, VWF:CB/VWF:Ag ratio decreased, FVIII:C lvs slighly below normal range

Family: Son had bleeding diathesis and spontaneous epistaxis (less severe than proband), normal parents

In silico data available: SpliceAI delta score of 0.51 for loss of splice acceptor caused by variant 1

Alamut showed small to moderate effects of the variant on normal splicing of VWF

NetGene2 showed weak strength of 3' splice sites in exon 8

SpliceAid2 showed TIA-1 and TIAL 1, which bind to U-rich motifs and facilitate 5' splice site recognition where destroyed in the mutated sequence

Disease: Von-willebrand Disorder, type 1

Patient 1 Variant: VWF NM_000552.5: c.4135C>T p.(Arg1379Cys) Exon 28

Family: History not mentioned

Patient 1 phenotype: near normal VWF:Ag and WVF:RCo levels RIPA within normal range Platelet VWF levels were normal normal pattern for multimeric analysis of plasma Slightly reduced VWF levels

In silico predictions available:

I-Mutant 3.0 value = -1.36 PYMOL prediction = substitution of ARG 1379 with a cysteine results in the loss of hydrogen bonds with Lys1407 and Lys1408, predicted change in secondary structure of A1 domain